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+{"text": "Objective. To obtain pilot data on the endometrial histology of Depomedroxyprogesterone acetate  usersexperiencing breakthrough bleeding (BTB) versus users with amenorrhea. To compare the endometrial histology of patients whoused DMPA continuously for 3\u201312 months versus those who used it for 13 months or more. Methods. Cross-sectional study. Endometrialbiopsy was obtained fromall consenting patients who used DMPA for at least 3 months. Patients were divided into thosewith BTB in the last 3 months versus those with amenorrhea for at least 3 months. Histology results and duration of therapy werecompared. Results. The proportion of women with chronic endometritis, uterine polyps, atrophic, proliferative, or progesteronedominantendometrium did not differ between those DMPA users with BTB versus those with amenorrhea. Duration of therapydid not correlate with symptoms of BTB or endometrial histology. Chronic endometritis was the most common histologic finding and occurred more often in women experiencing BTB (35% versus 15%) (RR 1.62 CI 0.91\u20132.87). Moreover, 45% ofwomen with BTB had received DMPA for more than 12 months. Conclusions. BTB was more common than previously reported in women using DMPA for more than 12 months. Chronic endometritis, which may indicate an underlying infectious or intracavitary anatomic etiology, has not been previously reported as a frequent finding in DMPA users, and may be related to ethnic or other sociodemographic characteristics of our patient population. Further study to elucidate the etiology of chronic endometritisin these patients is warranted. Depomedroxyprogesterone acetate (DMPA or Depo-Provera)is a long-acting injectable contraceptive, approved for use in the United States in 1992 . DMPA hThis is a cross-sectional study. Based on previous studies, wepredicted that 75% of amenorrheic DMPA users would havea progesterone-dominant or atrophic endometrium comparedto only 25% of patients with BTB on DMPA , 13.BasAll patients presenting to the outpatient gynecologyclinic who used DMPA for at least 3 months were askedto participate in the study. We recruited the first 20 patientswith BTB and the first 20 patients with amenorrhea onDMPA. This study was approved by the Institutional ReviewBoard at the Medical University of South Carolina.Inclusion criteria were the ability to give informed consentand use of Depo-Provera continuously for at least 3months. BTB was defined as unscheduled uterine bleedingin the last 3 months. Amenorrhea was defined as no uterinebleeding for at least 3 months. Patients were excludedif they had other possible causes of vaginal bleeding: usingother hormone medications in the last 3 months, intrauterinedevice use, and active cervical dysplasia greater than lowgradesquamous intraepithelial neoplasia. Patients were alsoexcluded if they had an absolute or relative contraindicationto endometrial biopsy including pelvic inflammatory disease,diabetes, active cervical infection, blood dyscrasias, and pregnancy.N gonorrhoea and Chlamydia trachomatis within the last year were confirmed to be negative prior to theprocedure. A wet prep was prepared to determine the presenceof yeast vaginitis or bacterial vaginosis (BV). BV wasdiagnosed using Amsel's criteria (14). An endometrial biopsywas performed, using a 3 mm Unimar Pipelle .Informed consent was obtained by the physician performingthe biopsy (AT). The duration of uninterruptedDMPA use, in months, and the patient's age, race, gravidity,and parity were recorded. The patient's pap smear andcervical tests for A pathologist, who was blinded to the patient's clinicalsymptoms, interpreted the endometrial biopsies. Chronicendometritis was defined as greater than or equal to 2 plasmacells in the endometrial sample. Treatment regimens for BTBwere not investigated in this study.Chi-square statistic and 2-tailed Fisher's exact test wereused to compare the proportions of patients with various endometrialhistologies in each group.n = 20) are presented in n = 20) are presented in The biopsy results of patients experiencing BTB on DMPA(n = 19) with women who received DMPA continuously for 13 months or longer (n = 21) in P = .49. Also, there was no difference in the incidenceof BV in patients with chronic endometritis (4/10)versus those without chronic endometritis (8/30) P = .45, but these data are limited by sample size. We did not collectinformation on body mass index (BMI).Because the DMPA package insert shows a significantdecrease in BTB at the 1-year mark, we also compared women who used DMPA continuously for 3\u201312 months  and 3 women with amenorrhea on DMPA (15%).Chronic endometritis was not reported in previous studies ofDMPA patients and may reflect ethnic or other demographicfactors of our study population, as BTB on DMPA has beencorrelated with ethnicity . ChroniOur findings suggest that antimicrobial or antiinflammatorytherapy may be the most effective to treat BTB in thesepatients. This new avenue of therapy for BTB in DMPA usersneeds to be investigated in future studies. The treatment ofBTB on DMPA is based on assumptions of the endometrialhistology, and many of the recommendations are from dataof Norplant users with BTB , 10, 22The package insert for DMPA reports that the level ofmedroxyprogesterone acetate (MPA) should be 1\u20137 ng/mL at3 weeks after the DMPA injection and < 100 pg/mL at 120\u2013200 days postinjection. A study of Thai patients, whose averageBMI was 22, found that estradiol and progesterone levelswere not correlated with the presence or duration of BTBin DMPA users . The seIt has been shown that adolescents who transition fromcombined oral contraceptives to DMPA have less BTB thanthose who begin DMPA without prior hormone exposure. It is pOur pilot study was limited by the small number of patients.Our pretest power analysis, based on commonly appliedassumptions regarding the endometrial histology ofDMPA users, was inaccurate. We were surprised to find thatless than 25% of our patients had evidence of a progesteronedominantendometrium and so many of the patients hadevidence of chronic endometritis. We would require additional134 patients to render a statistically valid evaluationof chronic endometritis. Finally, the cross sectional designof the study limited analysis of how the endometrial liningchanges with time in DMPA users.In summary, this study found that DMPA users can exhibitseveral endometrial histologic diagnoses, and the tissuefindings do not correlate easily with the patient's symptomsor duration of medication use. Chronic endometritiswas the most common histologic diagnosis in womenexperiencing BTB and has not previously been reported.Chronic endometritis likely reflects an underlying infectiousor anatomic abnormality which would completely changethe management of BTB in these patients, and will requirefurther evaluation.We believe further studies to elucidate theetiology of BTB in DMPA users are indicated."}
+{"text": "A new and better order to make scholarship available for free to all is emerging through deposit mandates like those adopted by Harvard, MIT, and Kansas. The faculty of Arts and Sciences of Harvard University is committed to disseminating the fruits of its research and scholarship as widely as possible.\u201d\u201cWhy would university faculty choose to place their scholarship on electronic archives for a world-wide audience? Many US universities have adopted such mandates for public access to faculty research, perhaps most notably Harvard This spring, the Association of Public and Land-Grant Universities, the Association of American Universities, the Association of Research Libraries, and the Coalition for Networked Information sent a document entitled \u201cThe Research University's Role in the Dissemination of Research and Scholarship,\u201d The last 25 years have been an age of disorder, not an unusual beginning for a revolution. Stewart Brand's declaration at the dawn of the digital age that \u201cinformation wants to be free\u201d foretold the porous electronic world that scholarship has come to inhabit. In the 25 years since Brand uttered those words, scholarly works have grown increasingly free. That which, prior to the digital age, could be found only within the covers of the scholarly journal, first emerged from those covers as electronic replacements for working papers. Unlike the mimeographed and later photocopied versions of papers, the new electronic versions could be circulated without cost and, even after hundreds of reproductions, remain readable.Soon, the informal digital circulation of working papers was followed by Web posting. Those far beyond the author's mailing list could get copies of the work. The first stirrings of the arXiv occurred in August 1991 and rapidly grew as a means of facilitating sharing of physics article preprints and post-prints. Other disciplines\u2014funding agencies, national libraries, and universities\u2014copied this innovation. The Directory of Open Access Repositories A diligent electronic search for most any article or manuscript today will produce the item itself or some version of it. However, what one finds often will reflect the disorderly nature of this age. Unfortunately, many of the hits will be accessible only if one has a subscription to the journal, is part of an institutional community that has a subscription, or is willing and able to pay for the manuscript on an ad hoc basis. Many researchers find that these hurdles inhibit their research. Surveying 2,157 US scientists in 2007, Stephen Hansen of the American Association for the Advancement of Science found that 29% of respondents said that their own research had been affected by difficulties in gaining access to or disseminating copyrighted scientific literature \u201cDifficulties with obtaining access to or disseminating scientific literature\u201d may mean that specific articles could not be found, that a version \u201cof record\u201d could not be found, or that multiple versions of an article were found, leaving the researcher unable to determine which version properly might be cited. Sources that are not curated and/or associated with stable URLs can be found one day and then vanish the next.And the opportunity cost of blocking access to potentially valuable information increases as understanding of science grows. Those who already suffer from what Robert Merton dubbed \u201cthe Matthew effect\u201d While serving as head of the National Institutes of Health (NIH), Elias Zerhouni observed that \u201cwe have no one place where the integration of the information can be used as a powerful hypothesis generator\u201d The only solution that gives science the maximum chance for advancement is one that ensures that all science findings are available to all researchers. \u201cAvailable\u201d does not permit permanent subscription or price barriers to stand between the researcher and scientific findings. When potentially important works that may bear on one's research number in the tens of thousands, \u201cavailable\u201d means that crawlers with sophisticated artificial intelligence must also have full access to help sort through the mass.Public access mandates from funding agencies and foundations like NIH and the Wellcome Trust are part of the solution, but not all of it. While deposit mandates should be universally adopted by funders, such agencies support only a fraction of the work that is published in scholarly journals. Large portions of important work in most fields originate beyond US borders. Most work outside the physical and biological sciences is not funded by grants external to the university and will not be touched by such mandates. Given that important problems are seldom bounded by a single discipline's research, access to the non-science scholarly literature is potentially important to all researchers.The most effective method of ensuring that the majority of important work is available is by replicating across the academy university public-access mandates like those of Harvard, MIT, and Kansas throughout the world. Most works originate with university-affiliated faculty or have co-authors who are faculty members. Deposit of articles in the form in which they were published in a journal requires permission of journals that require that authors provide exclusive copyright to them. In the Harvard policy, the faculty member grants a \u201cnonexclusive, irrevocable, paid up, world-wide license to exercise any and all rights under copyright\u201d to Harvard College Note that I use the term \u201cpublic access\u201d rather than \u201copen access.\u201d Fortunately, open-access journals like those of BMC and PLoS have found a way to make open access work. Unfortunately, most of the scholarly literature journals depend on the subscription model and feel threatened by immediate open access to the material they publish. While open access is the desired goal in the long term, the same logic that compelled PubMed Central to design itself as \u201cpublic access\u201d\u2014with up to a year's embargo permitted to protect the subscription base of journals\u2014compels me to support public access as an interim measure. Public access permits the possibility of brief embargoes at the request of the journal of publication, in contrast to open access, which requires that access to full text and databases, without permission restrictions, occur immediately.Journals opposing open access often claim that it will take away the funding needed for the refereeing process. Clearly the refereeing process must be supported. I know of no rigorous evidence that even very brief embargo periods before making articles publicly available cause scientific journal subscriptions to decline; therefore, I believe that public access has little impact on subscription revenue and is thus fully consistent with ensuring that refereeing of the literature continues.An explicit tradeoff between having access to all scholarly journal articles after no more than one year's delay is preferable to running even a small risk that immediate access would damage the refereeing process. In the long run, it will be incumbent on any journal insisting that access be delayed to produce evidence that the harm done to science by delayed access is less than the harm that would be done to science if immediate access were provided. As more and more scholarly journals change their practices and permit immediate posting on publicly accessible Web sites, it will be increasingly difficult to defend the position that short embargo periods cause harm to journals.In this period of great financial stress for universities, the question of the cost of maintaining public-access repositories must be addressed. Fortunately, most US research universities already have operating repositories in which public-access\u2013mandated collections may be placed. For the few institutions that do not, repository software is available for free The future of all libraries is digital. Most collection access is now through electronic means. To argue that maintaining a digital archive of faculty scholarly articles will be too expensive is essentially to argue that the university will be unable to maintain a viable library resource in the future.Not many taxpayers know what university faculty are doing. In fact, not many university administrators or even other faculty know what research their colleagues are performing. This veil over faculty research may contribute to the 20-year trend of declining real per-student subsidy from states to their institutions of higher education. The decline in real state support is especially pronounced at research universities.University public-deposit mandates will enhance the ability of universities to demonstrate faculty research productivity to the citizens of their states and to their donors. Imagine the massive collection of research that universities will accumulate after five years of mandated deposits. Further imagine alerting the public and donor community to the ability to search university X's repository to discover what local faculty findings exist on any subject. The results of such a search\u2014on subjects ranging from stem cells to menopause and hair loss\u2014would be impressive. Suddenly the invisible campus becomes a place populated by individuals researching topics relevant to the average citizen. Legislators who complain about faculty productivity would find their arguments more difficult to sustain. Donors and potential donors might even alter their gift-giving based on such searches.As a careful observer of scholarly communications, I'm convinced that the public goods aspect of faculty research will ultimately compel public access to it. Public goods have the characteristic that use of them by one individual does not diminish their value to others. In fact, the knowledge presented through scholarship generally becomes more valuable as it is shared more widely and becomes a building block upon which further scientific advances may occur.Faculty members can accelerate the process. We can persuade colleagues on our own campuses to pass public-access mandates like those at Harvard, MIT, and Kansas. We can speed up what otherwise might be a 20-year process and make it happen in three or four. We can urge Congress to expand the NIH mandate to all federal funding agencies It is impossible to know how much more rapidly scientific progress will occur if all the scholarly literature becomes accessible. What we each know is the frustrations we've experienced in our own research because of access difficulties. It is within the power of the university faculty in this country to remove these roadblocks. Supporting adoption of a public-access deposit mandate on your campus is an effort most worthy of the involvement of dedicated scientists."}
+{"text": "Compound 1, pyridothienopyridopyrimidin-6-one, exhibits reasonably high fluorescence quantum yields in all solvents studied (0.20 \u2264 \u03a6F \u2264 0.30), while for compound 2, 3-[(p-methoxyphenyl)ethynyl]pyridothienopyridopyrimidin-6-one, the values are much lower (0.01 \u2264 \u03a6F \u2264 0.05). The interaction of these compounds with salmon sperm DNA was studied using spectroscopic methods, allowing the determination of intrinsic binding constants, Ki = (8.7 \u00b1 0.9) \u00d7 103 M-1 for compound 1 and Ki = (5.9 \u00b1 0.6) \u00d7 103 M-1 for 2, and binding site sizes of n = 11 \u00b1 3 and n = 7 \u00b1 2 base pairs, respectively. Compound 2 is the most intercalative compound in salmon sperm DNA (35%), while for compound 1 only 11% of the molecules are intercalated. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment.Fluorescence properties of two new potential antitumoral tetracyclic thieno[3,2- Liposomes are among technological delivery developments for chemotherapeutic drugs in the treatment of cancer. This technique can potentially overcome many common pharmacologic problems, such as those involving solubility, pharmacokinetics, in vivo stability and toxicity -3. Liposb]pyridine derivatives 1 and 2  were recorded. The solutions were left several hours to stabilize.Salmon sperm DNA from Invitrogen  and compounds stock solutions were prepared in 10 mM Tris-HCl buffer (pH = 7.4), with 1 mM EDTA. The DNA concentration in number of bases was determined from the molar absorption coefficient, \u03b5 = 6600 Mt 260 nm . FluoresDipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC), from Sigma-Aldrich , and dioctadecyldimethylammonium bromide (DODAB), from Tokyo Kasei , were used as received. Liposomes were prepared by the ethanolic injection method, previously used for the preparation of Egg-PC and DPPC liposomes -15 and Dr = 0.95 thiophene derivative of the same type, a benzothienopyridopyrimidone pyridine derivatives was studied using spectroscopic methods. Compound 2 was shown to be the most intercalative compound in salmon sperm DNA (35%). The binding to DNA grooves seems to be the main type of interaction with the nucleic acid. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment. Our data thus suggest that both potential antitumoral compounds may be transported in liposomes for drug delivery applications.The interaction with DNA of two new potential antitumoral fluorescent planar thieno[3,2-DLS: dynamic light scattering; DODAB: dioctadecyldimethylammonium bromide; DPPC: dipalmitoyl phosphatidylcholine; DTS: Dispersion Technology Software; Egg-PC: egg yolk phosphatidylcholine; GUV: giant unilamellar vesicles; SV: small unilamellar vesicles.The authors declare that they have no competing interests.EMSC conceived the study, was responsible for its coordination and for the interpretation of results, and drafted the manuscript. MSDC carried out the liposome preparation and the fluorescence studies in liposomes. AROR carried out the experimental studies of the compounds interaction with DNA. RCC carried out the synthesis, purification and characterization of the new compounds. MJRPQ supervised the organic synthesis and participated in the draft of the manuscript. All authors read and approved the final manuscript."}
+{"text": "Erylus discophorus were screened for their capacity to produce bioactive compounds against a panel of human pathogens (Staphylococcus aureus wild type and methicillin-resistant S. aureus (MRSA), Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumanii, Candida albicans and Aspergillus fumigatus), fish pathogen (Aliivibrio fischeri) and environmentally relevant bacteria (Vibrio harveyi). The sponges were collected in Berlengas Islands, Portugal. Of the 212 isolated heterotrophic bacteria belonging to Alpha- and Gammaproteobacteria, Actinobacteria and Firmicutes, 31% produced antimicrobial metabolites. Bioactivity was found against both Gram positive and Gram negative and clinically and environmentally relevant target microorganisms. Bioactivity was found mainly against B. subtilis and some bioactivity against S. aureus MRSA, V. harveyi and A. fisheri. No antifungal activity was detected. The three most bioactive genera were Pseudovibrio (47.0%), Vibrio (22.7%) and Bacillus (7.6%). Other less bioactive genera were Labrenzia, Acinetobacter, Microbulbifer, Pseudomonas, Gordonia, Microbacterium, Micrococcus and Mycobacterium, Paenibacillus and Staphylococcus. The search of polyketide I synthases (PKS-I) and nonribosomal peptide synthetases (NRPSs) genes in 59 of the bioactive bacteria suggested the presence of PKS-I in 12 strains, NRPS in 3 strains and both genes in 3 strains. Our results show the potential of the bacterial community associated with Erylus discophorus sponges as producers of bioactive compounds.Heterotrophic bacteria associated with two specimens of the marine sponge  These values can exceed seawater concentrations by two to three orders of magnitude Due to their physico-chemical properties, which rarely exceed the biological tolerance limits, the oceans provide a safe environment for most living organisms Natural bioactive compounds have been used since the beginning of traditional medicine et al. in vitro. This is hardly the case with sponges. As the sponge bioactivity may in fact be due to their microbiome, these organisms became the subject of many works.The true origin of many of these bioactive molecules is uncertain. The production of secondary metabolites could be due to the cooperation between sponges and symbionts, only to the symbionts or to the sponges Secondary metabolites possess complex structures and involve unusual biochemistry. Two families of enzymes, the polyketide synthases (PKS) and the nonribosomal peptide synthetases (NRPS), are of particular importance in the production of various secondary metabolites many of which are important drugs e.g. Schirmer et al. The discovery of new biosynthetic pathways encoding genes of secondary metabolites opens the possibility of heterologous production and the genetic manipulation of the gene cluster to obtain new natural products Erylus , are well known as producers of saponins and other oligoglycosides Clostridium perfringensErylus deficiens Topsent, 1927 from the Mediterranean sea showed antibacterial activity against the marine bacteria Alteromonas luteo-violacea and 8 terrestrial bacteria  Bacillus subtilis, Escherichia coli and Candida albicans was observed in the extracts of Erylus lendenfeldi Sollas, 1888 collected in the Red Sea Erylus formosus Sollas, 1886 contained sufficiently high concentrations of erylosides to protect itself against predatory fishes, bacterial settlement, and fouling Erylus as well as from different geographic locations E. discophorus Schmidt, 1862, was reported to have haloperoxidase with iodo- and bromoperoxidases activities Sponges belonging to the genus E. discophorus collected in Berlengas Islands against a panel of pathogenic and environmental microorganisms. Antimicrobial bioactivity was detected in 31% of the 212 isolated heterotrophic bacteria from two specimens of E. discophorus and the presence of PKS-I and NRPSs genes was detected in several isolates showing the biotechnological potential of these bacteria.In this study we aimed to assess the bioactive potential of bacteria isolated from the marine sponge E. discophorus (named Berg01 and Berg02) that were sampled nearby, at 15 m by scuba diving in the Berlengas Islands a protected area of UNESCO located off the W coast of Portugal . The authors thank Reserva Natural das Berlengas (Instituto da Conserva\u00e7\u00e3o da Natureza e da Biodiversidade \u2013ICNB) for the sponges samples harvesting permission. Voucher samples of the sponges were preserved in 90% ethanol for taxonomic identification and deposited in the Biology Department's zoological collection of the University of the Azores (DBUA.Por). Specimens were identified from the analysis of general external and internal morphological characters, i.e. shape, type, size and arrangement of skeletal structures (spicules) following the Systema Porifera classification system The 212 bacteria (here designated test bacteria) under study were isolated from two specimens of the sponge \u22121 to 10\u22126) of the homogenized from the sponges in heterotrophic media. After isolation in pure culture, bacteria were cultivated in Marine Broth  in the dark at 26\u00b0C. Their taxonomic identification was based on the analysis of the 16S rRNA gene by direct colony PCR or with template DNA extracted by the boiling method. A loop full of bacterial culture was resuspended in 200 \u00b5l of distilled deionized H2O and subjected for 10 min to 100\u00b0C, cooled on ice and the extracted DNA amplified with the universal primers 27f and 1492r The bacterial isolation was performed inoculating serial dilutions , Staphylococcus aureus MRSA, Acinetobacter baumannii and Candida albicans were tested while in the Duetz assays Pseudomonas aeruginosa PAO1, Acinetobacter baumannii, Vibrio harveyi (CECT 525), Aliivibrio fischeri (CECT 524), Bacillus subtilis (ATCC6633), Staphylococcus aureus wild type Smith strain, Staphylococcus aureus MRSA, Candida albicans, Aspergillus fumigatus (ATCC46645) and (\u0394\u03b1kuBKU80) were used.The isolated E. discophorus. The isolates were initially fermented in 96-deep well Duetz plates Janus and Duetz plates.The search for bioactivity was performed with the 212 bacteria isolated from 2HPO4; 0.2 g CaCl2; 0.75 g KCL; 23.4 g NaCl; 7 g MgSO4; 1 g Mannitol; 1 g Yeast extract; 1 g Peptone; 16 g Agar; 1 ml Hutner's basal salts 2O), R2A (218263 - Difco) and saline  R2A and Starch Agar (Difco).This double-faced plated assay optimized by Fundaci\u00f3n MEDINA Janus plates the test bacteria were inoculated employing a replication procedure previously described Janus plates were incubated at 37\u00b0C for 20\u201324 h. A search of inhibition zones indicative of antibacterial or antifungal activity was then carried out in vitro screening assay was performed against a panel of Gram positive bacteria (Bacillus subtilis (ATCC6633) and Staphylococcus aureus MRSA), Gram negative bacteria (Acinetobacter baumannii) and the yeast Candida albicans. All target organisms were pre-inoculated and grown at 28\u00b0C, 220 r.p.m. for 12 h and 70% of humidity. Pre-inoculum and inoculum medium used to grow A. baumannii and B. subtilis was Luria Broth (LB) . For C. albicans the pre-inoculum medium was Sabouraud Dextrose Broth (SDB) and the inoculum medium was YNBD S. aureus MRSA was Brain Heart Infusion (BHI). Inocula absorbance for all target microorganisms was adjusted to a final 600 nm optical density (OD) of 0.4 before being placed in the Janus plates. Non-inoculated medium was used as negative control.On the top layer of the Tests of growth interference of the target microorganisms with saline media were performed in advance to rule out possible interferences.et al. Janus plates, half saline concentration of the media were also tested. The inoculated Duetz plates were incubated (1 mL) for 5 days at 28\u00b0C, 220 r.p.m. and 70% of humidity. Bacterial broth were then subjected to an organic extraction with 800 \u00b5l acetone and 40 \u00b5l DMSO per well. The plates were incubated for 1 h at 220 r.p.m. and then transferred to a vacuum centrifuge (GeneVac HT-24) in order to reduce the final volume to 400 \u00b5l . The supernatants of the extracts solutions were transferred to 96-deep well plates. The organic extracts were then assayed against the Gram-negative Pseudomonas aeruginosa PAO1, Acinetobacter baumannii, Vibrio harveyi (CECT 525) and Aliivibrio fischeri (CECT 524); and the Gram-positive Bacillus subtilis (ATCC6633), Staphylococcus aureus wild type Smith strain and Staphylococcus aureus MRSA. Antifungal assays were also prepared against the yeast Candida albicans and Aspergillus fumigatus (ATCC46645) and (\u0394\u03b1kuBKU80). Unless specified negative controls were always uncultivated culture medium.MPs were used to carry out microfermentations in 96-deep well plates (here designated by Duetz system assay) following the approach described by Duetz Acinetobacter baumannii, S. aureus (Smith), Pseudomas aeruginosa PAO1 and Candida albicans, overnight cultures grown in liquid Luria Broth (Miller's media) at 37\u00b0C and 220 r.p.m. were measured at an absorbance of 612 nm for A. baumannii and S. aureus (Smith) and 600 nm for P. aeruginosa. The inocula in LB media were adjusted to an OD of 5\u00d7105 for A. baumannii and to a cell concentration of 2.5\u00d7105 CFU/ml for S. aureus (Smith) and 5\u00d7105 CFU/ml for P. aeruginosa. A suspension of C. albicans in medium RPMI  was adjusted to 0.250 at 660 nm. This suspension was then diluted 1/10.For the screening assays against A. baumannii were, as positive controls, 0.3125, 0.625, 12.5 and 25 \u00b5g.mL\u22121 rifampicin and, as negative controls, 7.8125, 15.625, 31.25 and 62.5 \u00b5g.mL\u22121 amphotericin B. In the case of P. aeruginosa the positive controls were 1.9625, 3.925, 7.85 and 15.7 \u00b5g.mL\u22121 ciprofloxacin and the negative controls were 7.8125, 15.625, 31.25 and 62.5 \u00b5g.mL\u22121 amphotericin B. In the assays performed with S. aureus (Smith) the positive controls used were serially diluted between 0.5\u00d710\u22127 to 5 mg.mL\u22121 penicillin G and with C. albicans the antibiotics used were as positive controls 7.8125, 15.625, 31.25 and 62.5 \u00b5g.mL\u22121 amphotericin B and as negative controls 0.039, 0.078, 0.156 and 0.312 mg.mL\u22121 penicillin G.For the liquid screens, Nunc plates (LTC-330) were filled as follows: 10 \u00b5l of each extract plus 90 \u00b5l of inoculum in 80 wells; 10 \u00b5l of a series of antibiotic concentrations in the range of the minimum inhibitory concentration plus 90 \u00b5l of inoculum (positive and negative controls) in 8 wells; 100 \u00b5l (90 \u00b5l of LB media +10 \u00b5l DMSO 20%) as blank control in 4 wells; and 100 \u00b5l of the inoculum for the assessment of the total microorganism growth. The antibiotics used against The plates were incubated at 37\u00b0C for 20 h under humid conditions. The absorbances were measured at 612 nm in a Tecan ULTRAEVOLUCION before and after incubation. To confirm the results, 30 \u00b5l of a 0.02% resazurin stock solution was added to each well (100 \u00b5l) and incubated for 2 h. Changes in colour from blue (growth inhibition corresponding to detection of bioactivity) to pink (no growth inhibition corresponding to no detection of bioactivity) and fluorescence readings radiated from the resazurin were measured in a Perkin Elmer VICTOR2 multi-function fluorometer. All ODs and fluorescence measurements were analysed using the Genedata Screener software.Aspergillus fumigatus ATCC46645 and \u0394\u03b1kuBKU80, cultures of both A. fumigatus were prepared in medium RPMI with 0.002% resazurin from a stock spore suspension in Tween saline buffer to a final concentration of 2.5\u00d7104 conidia/ml as described by Monteiro et al. \u22121 amphotericin B. The plates were incubated for 30 h at 37\u00b0C and, then, the fluorescence was recorded in a Perkin Elmer VICTOR22\u2122 Multi-function fluorometer.For the screening assays against Staphylococcus aureus MRSA, an overnight culture of S. aureus MRSA in 10 ml of liquid Brain Heart Infusion (BHI) medium was incubated at 37\u00b0C and 220 r.p.m. The OD of the culture measured at 660 nm was adjusted to 0.2 and used to inoculate BHI agar medium (3 ml of the adjusted inoculum per 100 ml of medium) which was then distributed (30 ml) in OmnyTray plates. Disposable 96 pin trays were used to generate 96 wells in each of the BHI agar plates in which, subsequently, 10 \u00b5l of each extract in each well were dispensed. Alternatively extracts were distributed in BHI agar plates without wells. Ten \u00b5l of 0.5 mg.mL\u22121 kanamycin was used as positive control. Plates were then incubated at 37\u00b0C for 20 h and zones of inhibition (ZOI) were measured. Any extract producing a visibly discernible ZOI, regardless of zone quality, was considered to be positive.For the screening assays against Bacillus subtilis (ATCC 6633), a culture of B. subtilis was prepared using 1 ml of spore suspension/1 L medium (23 g/L of Nutrient Agar and 2 g/L of yeast extract) that had been previously sonicated for 3 min. The assay was performed in a similar way to the one used against S. aureus MRSA and the positive control was 150 \u00b5g/ml tunicamycin.For the screening assays against Vibrio harveyi CECT 525 and Aliivibrio fischeri CECT 524 were carried out in a cell density optimized agar assay. Ten ml of Luminous Medium V. harveyi and A. fischeri, incubated at 25\u00b0C, 220 r.p.m. for 12 h and the optical density adjusted to 0.3 for V. harveyi and 0.4 for A. fischeri at 600 nm and, subsequently, both diluted by 1/10. The assay was performed in a similar way to the one used against S. aureus MRSA and the positive control consisted on 0.256 mg/ml chloramphenicol. The cultures were incubated at 25\u00b0C for 24 h. Inhibition was detected with the presence of non-phosphorescent halos in a dark-room.Screening assays against The molecular analysis of the genes PKS-I and NRPS involved in the production of secondary metabolites was investigated in the 59 of the bioactive bacteria. Genomic DNA was extracted with an E.Z.N.A. bacterial KIT from OMEGA. Specific degenerated primers MDPQQRf and HGTGTr Erylus discophorus collected in Berlengas (Berg01 and Berg02) allowed the isolation of a large number of heterotrophic bacteria (212 isolates) of which 31% (66 isolates) showed bioactivity. Of the screened bacteria for bioactivity and based on the 16S rDNA gene analysis, 57% (n\u200a=\u200a120) were Alphaproteobacteria, 21% (n\u200a=\u200a45) Gammaproteobacteria, 16% (n\u200a=\u200a34) Actinobacteria and 6% (n\u200a=\u200a13) Firmicutes.The oceans, an almost endless source of microbial diversity, are the habitat of organisms such as sponges that harbour a large microbial diversity with important biosynthetic potential due to their secondary metabolites profiles Bacillus subtilis (87.9%) and at a lower percentage against Staphylococcus aureus MRSA (10.6%), Aliivibrio fischeri (9.1%) and Vibrio harveyi (6.1%). Bacteria with bioactivity against both B. subtilis and S. aureus MRSA represented 9.1% and against both A. fischeri and V. harveyi accounted for 4.6%. No bioactivity was observed against P. aeroginosa, A. baumannii, S. aureus wild type, C. albicans and A. fumigatus.Bioactivity was observed against one or more of the target microorganisms tested. The majority was active against The taxonomic affiliation of all bioactive isolates is provided in Janus and Duetz systems. Thirty two isolates of Alphaproteobacteria (48.5%) belonging to the genera Pseudobrivio and Labrenzia were bioactive against B. subtilis, S. aureus MRSA and A. fischeri. Eighteen isolates of Gammaproteobacteria (27.3%) belonging to the genera Vibrio, Acinetobacter, Microbulbifer and Pseudomonas were active against B. subtilis, V. harveyi and A. fischeri. Eight isolates of Actinobacteria (12.1%) belonging to the genera Gordonia, Microbacterium, Micrococcus and Mycobacterium were active against B. subtilis and S. aureus MRSA. Eight isolates of Firmicutes (12.1%) belonging to the genera Bacillus, Paenibacillus, Sporosarcina and Staphylococcus were active against B. subtilis, V. harveyi and A. fischeri. Pseudovibrio (47.0%), Vibrio (22.7%) and Bacillus (7.6%) are the three most bioactive genera of all the bioactive isolates. No activity was observed in isolates affiliated to Ruegeria, Rhodobacter, Erythrobacter, Martelella, Nautella, Photobacterium, Thalassomonas, Rhodococcus and Dietzia.Alphaproteobacteria followed by the Gammaproteobacteria, Actinobacteria and Firmicutes. However, if the analysis is made based on the number of bioactive isolates relative to the total number of bacteria in each group, Firmicutes are the most bioactive (61.5%) followed by Gammaproteobacteria (40%), Alphaproteobacteria (26.7%) and Actinobacteria (23.5%). Bioactivity results obtained with marine bacteria and sponge associated bacteria are somehow different. Regarding marine bacteria in general, most of the new marine bacterial compounds from 1997 to 2008 were originated from Actinobacteria (40%), Cyanobacteria (33%), Proteobacteria (12%) and Firmicutes and Bacteroidetes (5% each) Actinobacteria (46.66%), Proteobacteria (23.33%), Firmicutes (11.66%), Cyanobacteria (8.33%), Verrucomicrobia (5%) and others (5%) Actinobacteria may be biased due to their extensive study in the production of antibiotic compounds since 50% of known microbial antibiotics are derived from these bacteria The group with the higher number of isolates demonstrating bioactivity was the Several of the obtained bioactive genera are well known producers of metabolites with antimicrobial properties but others are less known. Furthermore, many of the examples referred to below are from sponge associated bacterial isolates.PseudovibrioAlphaproteobacterium related to Pseudovibrio denitrificans, displayed a weak and unstable antimicrobial activity, which was easily lost during cultivation LabrenziaA suite of antimicrobial compounds with spectra of different antimicrobial activity was observed in Vibrio sp. and sponge extracts was observed against BacillusVibrionaceaeMarine vibrios have been reported as a rich source of novel biologically active metabolites Pseudomonas species have been reported Pseudomonas spp. as potential source for medically relevant bioactive substances were revised by Isnansetyo and Kamei Bioactive metabolites produced by marine Acinetobacter sp. from saltpans of Ribandar, Goa was observed Acinetobacter from the ascidian Stomozoa murrayiMicrobulbifer produce anticancer antibiotics (pelagiomicins) Microbulbifer was obtained by Penesyan et al. The production of antibacterial compounds by a halotolerant Bacillus spp. from terrestrial origin are well known sources of antimicrobial compounds Bacillus spp. have also been reported Bacillus have revealed its potential as a source of antibiotic-like compounds Paenibacillus sp., P. elgii, P. polymyxa and P. koreensis has been observed et al. Staphylococcus bacteria isolated from four species of sponges.Antimicrobial activity of the Gram negative strains Micrococcus strains possessing antimicrobial activity were described by Bultel-Ponc\u00e9 et al. et al. et al. Microbacterium species Gordonia was described as being capable of degrading xenobiotics, environmental pollutants, or otherwise slowly biodegradable natural polymers as well as to transform or synthesize possibly useful compounds Mycobacterium genus which is well known for its infectivity.against B. subtilis, S. aureus MRSA, V. harveyi and A. fischeri was MB. In saline R2A bioactivity was observed against both S. aureus MRSA and V. harveyi, and in saline Starch and MF against A. fischeri. In solid media assays, higher numbers of inhibitions were observed in saline Starch followed by MF and saline R2A. No inhibitions were found in MA medium when used as culture medium in the Janus system. The non-saline R2A medium was the only one where no bioactive compounds were produced in both systems.The most efficient liquid medium for the production of bioactive compounds Janus system, after the initial growth of the sponge isolates, the medium, containing diffused secondary metabolites is put in contact with the target organism to assess inhibitory responses. The Janus system allowed the assessment of a higher number of bioactive positive hits (84%) when compared to the Duetz system (16%). However, the Duetz results are more reliable than the ones obtained with the Janus plates due to the overlap of inhibition halos in the latter. Moreover, due to bacterial swarming and gliding, results in the Janus system were obtained after 3 days of incubation, whereas longer incubation times were employed in the liquid microfermentations. Bacterial swarming and gliding also interfered with the visualization of results in other antimicrobial studies The screening of the 212 bacteria was performed using two different plate systems. The Duetz system is a miniaturized fermentation system that allows carrying out a high number of microfermentations with a lot less effort than the effort required to carry out the fermentations in tubes/flasks. Bacteria are fermented in liquid media, crude extracts are obtained and assayed for bioactivity against target organisms. However, in the double-faced diffusion assay Janus plates assay a microbial culturing system, the production of secondary metabolites is possibly continued along the entire assay at least for some bacteria able to grow at 37\u00b0C (incubation temperature of the target microorganisms). Thus some bacteria might be able to produce bioactive compounds in a continuous way for longer while in direct contact with target microorganisms Janus assays where the cultivation of the 95 bacteria was performed in the surface of agar without barriers. Therefore, there might be competition for nutrients, growth inhibition due to metabolites produced by the neighbours or even the synergic or antagonistic interaction of two or more bacteria in the production of secondary bioactive metabolites. Thus, this system is not as \u201cclean\u201d as the individual fermentations in Duetz. These aspects can also have some repercussion in the number of hits that were obtained with the two approachesIt is well known that microbial secondary metabolite production is highly-dependent on the fermentation conditions The search for PKS-I and NRPS genes in 59 of the bioactive bacteria revealed that 30.5% (n\u200a=\u200a18) of the bacteria amplified one or two of the genes for secondary metabolites. The presence of PKS-I was observed in 12 strains, NRPS in 3 strains and both PKS-I and NRPS in 3 strains . The nonPseudovibrio (n\u200a=\u200a10). These genes were already reported in Pseudovibrio strains isolated from a Irciniidae sponge The majority of the bioactive bacteria that amplified PKS-I and NRPS genes belongs to the genus Vibrio, only 3 bacteria amplified the PKS-I gene and none the NRPS gene. A recent review on the family Vibrionaceae suggests that only NRPS or hybrid PKS-NRPS genes were amplified Gordonia (n\u200a=\u200a2), Bacillus (n\u200a=\u200a2) and Pseudomonas (n\u200a=\u200a1) amplified the genes NRPS and PKS in lower numbers.Although a high number of the bioactive bacteria belongs to the genus Actinobacteria of the genera Streptomyces, Mycobacterium, Corynebacterium, MicromonosporaGordoniaPKS and NRPS genes have been extensively studied in Pseudomonas, Vibrio and BacillusA wide range of bacterial groups were tested for the presence of the genes PKS and NRPS and they were found among other genera in Erylus strains that are, thus, the most promising ones for future work.These results confirm the production of secondary active metabolites by some Erylus discophorus and open the way for further studies.The complex bacterial communities in marine sponges play a considerable ecological role in several aspects of the biology of these organisms, namely by the production of secondary metabolites fundamental for sponge protection against other organisms. These communities have thus a great biotechnological importance in the search for new and more effective pharmaceutical drugs needed for the treatment of severe human diseases such as cancer, microbial infections and inflammatory processes. Our results evidenced the bioactive potential of the heterotrophic bacterial community of the sponge"}
+{"text": "The type and pattern of organisms that cause ocular infection changes over time. Moreover, the causative organisms have developed increased drug resistance. Therefore, the aim of this study was to determine the prevalent bacterial agents of eye discharge and their drug susceptibility patterns to commonly used antimicrobial agents.A retrospective study was conducted at Gondar University Hospital, Northwest Ethiopia from September, 2009 to August, 2012. Culture and drug susceptibility test results of patients who had eye infections were taken for analysis. Eye discharge samples were cultured on MacConkey agar, blood agar and chocolate agar plates. A standard biochemical procedure was used for full identification of bacterial isolates. Antimicrobial susceptibility tests were done on Mueller-Hinton agar by using disk diffusion method. Data was entered and analyzed by using SPSS version 16 software.S. aureus (21%). Within the age group of 1\u00a0day-2\u00a0years old, (66.1%) of bacteria were isolated. Most of the bacterial isolates were resistance to ampicilin (71%), amoxicilin (62.9%), erythromycin (43.5%), gentamicin (45.2%), penicillin (71%), trimethoprim-sulphamethoxazole (58.1%), and tetracycline (64.6%) while Ceftriaxon and Ciprofloxacin showed (75.8%) and (80%) susceptibility respectively. From the total bacterial isolates, (87.1%) were showed multi drug resistance (MDR) to two or more drugs.A total of 102 eye discharges were submitted for microbiological evaluation, of which (60.8%) had bacterial growth. The most frequently isolated bacterial isolates were gram-positive bacteria (74.2%). The predominant bacterial species isolated was Coagulase-negative staphylococci (27.4%) followed by The prevalence of bacterial isolates in eye discharge was high in the study area and majority of isolates were gram-positive bacteria. Most of the bacterial isolates were resistant to frequently used antimicrobials. Therefore, drug susceptibility test is necessary before prescribing any antimicrobials. Eye is one of the sense organ which is important throughout our life. The awareness given to eye health and cleanliness is vital due to many factors. Dust, high temperature, microorganisms and other factors can lead to various eye diseases which can lead to blindness. The clinical signs and symptoms of inflammation of the eyes, in the presence of mucous pus are frequently caused by bacteria, the formation of pus increase, conjunctival hyperemia and lid edema ,2.Bacteria causes eye disease because of their virulence and host's condensed fighting from various factors such as socio-economic status, individual hygiene, lifestyle, nutrition, inheritance, physiology, and age . Eye mayPseudomonas aeruginosa, Proteus spp, Haemophilus aegyptius, Neisseria gonorrhoeae, Moraxella spp such as Moraxella catarrhalis, Moraxella lacunata, Streptococcus pyogenes, and Staphylococcus aureus[. The microbial etiology and drug susceptibility as well as resistance profile may differ with geographic location according to the restricted inhabitants [Bacterial conjunctivitis is an inflammatory condition of the conjunctiva that results from infection due to one or more bacterial species. Most cases of acute bacterial conjunctivitis pointed eye are common and can affect both sexes and all age groups . The comus aureus. The micabitants .Bacterial eye infection needs instant institution of treatment. Treatment of bacterial eye infections may engross empirical treatment with topical ophthalmic broad-spectrum antibiotic formulations that become a prevailing practice among ophthalmologists and general practitioners. These jointly with irrational use of drugs, availability of antibiotics without prescription, have led to the development of resistance to commonly used antibiotics. Thus, the current trends in the etiology of bacteria that cause eye infections and their susceptibilities must be updated to make a rational choice of initial antibiotic therapy. The aim of this study was to determine bacterial isolates and drug susceptibility patterns of eye discharge at Gondar University Hospital.A retrospective study was conducted at Gondar University Hospital, Northwest Ethiopia, from which procedures  were carried out from September 2009 to August 2012. This University Hospital provides inpatient and outpatient services for more than 5 million inhabitants surrounding it.The study participants were all patients\u2019 who were clinically diagnosed with ocular infections and those who provide eye discharge sample at Gondar University Hospital during the study period. Socio-demographic and laboratory results which contain different bacterial isolates and drug susceptibility patterns of patients who had eye discharges were collected from the University Hospital Microbiology Laboratory unit registration books by using standard data collection format.2) atmosphere was for the chocolate agar. Pure isolates of bacterial pathogen were preliminary characterized by colony morphology, gram-stain, and catalase test. A standard biochemical procedure was used for full identification of gram- positive and gram negative bacteria.According to the standard operation procedures, eye discharge samples were collected by using sterile cotton swabs moisturized with normal saline solution and cultured on MacConkey agar, 5% Sheep's blood agar and chocolate agar plates. This was before the instillation of antimicrobial or steroidal eye drops for treatment. The isolation of bacteria was done by incubating the agar plates at temperature of 37\u00b0C for 24 and 48hs. Aerobic atmospheric condition was maintained for the MacConkey agar and blood agar, while 10% carbon dioxide  [E. coli ATCC 25922,S. aureus ATCC 25923 and Pseudomonas aeruginosa (ATCC 27853), were used for quality control for antimicrobial susceptibility tests [Antimicrobial susceptibility testing was performed for bacterial isolates by using agar diffusion method described by Bauer (oxoide) . The antty tests .Statistical analysis was performed using SPSS version 16 software. The proportion of isolated bacteria with patient\u2019s demographic information; and susceptibility to commonly used antibiotics was compared by using the Pearson Chi-square test. P-value\u2009\u2264\u20090.05 was considered as statistically significant.Ethical clearance was obtained from the Institutional Ethical Review Board of University of Gondar.A total of 102 patients who gave eye discharge sample to bacteriological analysis were enrolled.Of all, 65 (63.7%) were males and 37 (36.3%) were females. The mean age of the study subjects was 8.5\u00a0years, ranges from 1\u00a0day of life to 73\u00a0years old. Bacterial isolation in both sexes  and various age groups  was not showed statistically significant.S. aureus 13 (21%)  bacterial isolates were identified. The most frequently isolated bacterial isolates were gram-positive 46 (74.2%). The predominant bacterial species isolated was Coagulase-negative staphylococci (CONS) 17 (27.4%) followed by . aureus and k. pneumoniae accounts 7(17.1%) each  bacteria were isolated. Of these Coagulase-negative staphylococci accounts 11 (26.8%); and both SMost of the bacterial isolates were resistant to ampicilin (71%), amoxicilin (62.9%), erythromycin 43.5%), gentamicin (45.2%), penicillin (71%), trimethoprim-sulphamethoxazole (58.1%), and tetracycline (64.6%). Ceftriaxon and Ciprofloxacin showed 75.8% and 80% susceptibility respectively . This finding is in agreement with previous study [S. aureus followed by S. Pneumoniae. This may be due to the difference in climate and geographical variations in different countries. Other isolates included S. pneumoniae (11.3%), S. pyogene (14.5%), E. coli (8.1%), Klebsella spps (14.5%), and non lactose fermentor gram negative rods (3.2%). These results are consistent with the study by Kasper et al.,[In this study, the overall prevalence of bacterial eye infection was 60.8%. Similar findings have been reported in previous study conducted in Ethiopia, (54.2%)  and otheus study . Howeverus study , the prer et al.,. Forty 4r et al., or fungir et al.,.The majority of the bacterial isolates, (66.1%) were from patients in the age range of less than two years of life. Susceptibility to infection is increased in babies because they are at a greater risk after their maternal immunity has been disappeared and before their own immunity system had matured . In addiStaphylococcus aureus isolates to antimicrobials used showed the highest sensitivity to ciprofloxacin with percentage (84.6%) followed by ceftriaxone with percentage (76.9%) while the proportion was less sensitive to ampicilin with percentage(23.1%), penicillin and trimethoprim-sulphamethoxazole, (30.8%) each. This result is consistent with the previously studies [S. aureus strains produce pencillinase and alternative penicillin binding proteins (PBP-2A) helps the organisms to become resistant to most beta lactam antibiotics [Commonly used antibiotics in a study area were; tetracycline, erythromycin, chloramphenicol, gentamicin, ciprofloxacin, Trimethoprim-sulphamethoxazole, penicillin, ceftriaxone, norflaxocin and amoxicillin. However, in the present study, different bacterial species had high level of resistance pattern to different antimicrobial agents. For example, Coagulase-negative staphylococci showed high level of resistance to ampicilin (76.5%), amoxicilin (64.7%), erythromycin and tetracycline each (64.7%), gentamicin (58.8%), penicillin and trimethoprim-sulphamethoxazole each (70.6%). This is in agreement with the previously studies . The sen studies . It is wibiotics .In this study, most of bacterial isolates have shown high resistance to ampicilin (71%), penicillin (71%), amoxicilin (62.9%), tetracycline (64.6%), trimethoprim-sulphamethoxazole (58.1%), and erythromycin (43.5%). Similar findings have been reported in Iran  and in APrevalence of multidrug resistance (MDR) to two or more of bacterial isolates to the commonly prescribed antimicrobials was observed in 87.1% of the isolates. This is in agreement with the previous studies ,28. HoweS. aureus. Most of the bacterial isolates were resistant to commonly used antimicrobials. Therefore, drug susceptibility test is essential before prescribing any antimicrobials.The prevalence of bacterial isolates in eye discharge was high in the study area and majority of isolates were gram-positive bacteria. The predominant isolates were Coagulase-negative staphylococci and Due to the nature of the study, eye diagnosis is not clearly indicated and it is difficult to show whether the patients who underwent culture may have had chronic conjunctivitis and/keratitis and may have been treated earlier. Some of the bacterial isolates were reported as non-lactose fermenting gram negative rods and CN Staphylococci which are not specific. Moreover, there was no data about Chlamydia, Viral and other fungal eye infections.The authors declare that they have no competing interests.DM: participated in conception and design of the study, data collection and analysis, interpretation of the findings, reviewed the manuscript. YW: Participated in conception and design of the study, data analysis and interpretations of the findings, reviewed the manuscript. FM: participated in conception and design of the study, interpretation of the findings, reviewed the manuscript. TN: Participated in conception and design of the study, data collection, reviewed the manuscript. GF: Participated in the design of the study, analysis and interpretations of the findings, drafting the manuscript and write up. All authors reviewed and approved the final manuscript."}
+{"text": "Nanowire-based field-effect transistors (FETs) have demonstrated considerable promise for a new generation of chemical and biological sensors. Indium arsenide (InAs), by virtue of its high electron mobility and intrinsic surface accumulation layer of electrons, holds properties beneficial for creating high performance sensors that can be used in applications such as point-of-care testing for patients diagnosed with chronic diseases. Here, we propose devices based on a parallel configuration of InAs nanowires and investigate sensor responses from measurements of conductance over time and FET characteristics. The devices were tested in controlled concentrations of vapour containing acetic acid, 2-butanone and methanol. After adsorption of analyte molecules, trends in the transient current and transfer curves are correlated with the nature of the surface interaction. Specifically, we observed proportionality between acetic acid concentration and relative conductance change, off current and surface charge density extracted from subthreshold behaviour. We suggest the origin of the sensing response to acetic acid as a two-part, reversible acid-base and redox reaction between acetic acid, InAs and its native oxide that forms slow, donor-like states at the nanowire surface. We further describe a simple model that is able to distinguish the occurrence of physical versus chemical adsorption by comparing the values of the extracted surface charge density. These studies demonstrate that InAs nanowires can produce a multitude of sensor responses for the purpose of developing next generation, multi-dimensional sensor applications. The ability to detect the identity and quantity of certain volatile organic compounds like ethanol or acetic acid finds key applications in areas such as process control , environSemiconductors are, by virtue of their energy band structure, highly suited for this function. It is well known that the interaction between the surface of a semiconductor and adsorbed chemical species produces a change in the local band structure, whether electrostatically via the charge of adsorbed molecules or directly by altering the distribution of allowed electron states at the surface ,15. By u2O, NO and NO2 \u2212) by dissociation of the hydrogen from the hydroxyl group results in a population of negatively-charged species on the NW channel. These act as repulsive scattering centres to greatly decrease conductance [III and AsV, with the +5 oxidation state being less stable, reduction is possible by electron transfer from acetate. The rate of these redox reactions contributes to the time dependence of the current transient. Upon removal of acetic acid vapour from the chamber, ductance . Thereaf2 and vapour. To facilitate comparison, the data have been normalized by the baseline values taken at the start of exposure and are given in terms of a relative change of conductance (i.e., 2, a larger number of active surface sites will be replenished through the desorption of hydrogen. This agrees with our observations of the current transient in the initial exposure and when a longer time was taken between consecutive exposures (see In ures see . In the From our analysis in previous sections, we have identified the relation of some device parameters to the type and extent of analyte interaction with the NW surface see . To utilHowever, the present study is limited in both the concentration range and complexity of the analytes tested. In real-word applications, such as the detection of breath biomarkers in patients, the analyte is a mixture of species, and competing adsorptive events come into play. Here, especially, having a multitude of sensor responses that are selective of different phenomena is key to obtaining a better understanding of the unknown. The result is a multi-dimensional dataset, which can be interpreted using the aid of machine-learning and feature-reduction techniques ,45,46. TThrough sensing experiments performed with glacial acetic acid, methanol and 2-butanone, we demonstrated the capability of InAs multi-NWFETs to distinguish between adsorptive events of both a physical and a chemical nature. Specifically, we found that differential values of surface charge density ("}
+{"text": "Arabidopsis serine/threonine/tyrosine protein kinase (STYK) and its role in seed oil accumulation; the role of Arabidopsis OLE1, a major seed OB protein has also been elucidated. In vitro kinase assay followed by mass spectrometry identifies residue that are phosphorylated by STYK. Further, co-expression of OLE1 and STYK in yeast cells increases the cellular lipid levels and reduces the total lipid when OLE1 was replaced with OLE1T166A. Moreover, in vivo experiments with OB isolated from wild-type and styk knock-out lines show the ability of STYK to phosphorylate distinct OB proteins. OLE1T166A mutant and Arabidopsis styk mutant demonstrate the significant reduction of its substrate phosphorylation. styk mutant line significantly reduces the amount of total seed oil as compared to wild-type seeds. Together, our results provide the evidences that Arabidopsis At2G24360 (STYK) is phosphorylating oil body proteins and the phosphorylation regulates the oil content in Arabidopsis seeds.Protein phosphorylation is an important post-translational modification that can regulate the protein function. The current knowledge on the phosphorylation status of plant oil body (OB) proteins is inadequate. This present study identifies the distinct physiological substrates of  Seed OBs contain a variable number of proteins, depending on the oleaginous plants and their tissues3. The major seed OB proteins oleosin, caleosin and steroleosin have been shown to play an important role in regulating the OB structure and lipid accumulation4. There are other minor proteins in OBs that are reported to be involved in solute transport, protein synthesis and vesicular transport3. The proteomics data for the purified seed OBs from more than 18 species, including nine crop plants provide a detailed insight into the nature of the proteins belonging to the organelle and the diversity of OB functions1. However, there has been no dedicated study to report the different phosphoproteins of the seed OBs.In plants, oil body (OB) proteins are highly conserved and are grouped into structural proteins or enzymes6. Recent studies have indicated that post-translational modifications (PTM) of OB proteins play a role in these interactions. Specifically, the ubiquitinylation of oleosin and caleosin in germinating sesame seeds has been proposed to play a role in the interaction of OBs with glyoxysomes which is needed for breaking down the lipids to release free fatty acids to provide energy7. Similarly, the phosphorylation of oleosin has been suggested to help in the interaction of OBs with lipases and other enzymes of lipogenic pathway5. Previously, we have shown that peanut oleosin 3, although a structural protein, acts as a bifunctional enzyme with both monoacylglycerol acyltransferase and phospholipase activities8. Furthermore, a recent report has demonstrated the role of the proteasome in the degradation of ubiquitinylated oleosins9; oleosin degradation is required for lipid mobilization which further highlights the role of PTM in regulating the function of seed OB proteins.Neutral lipids that are stored in the OBs break down following seed imbibition and germination to provide energy and carbon skeletons to support the growth of seedling10. Although there are many in silico phosphorylation sites predicted for caleosin, there are only two reports showing its partial phosphorylation11. OLE5 and OLE2 from Arabidopsis thaliana have been recently shown to be phosphorylated during lipid droplet degradation9. However, the protein kinase phosphorylating these proteins has not been reported. Previous studies from our laboratory have identified a non-mitogen activated dual specificity STY protein kinase from Arachis hypogaea (AhSTYK) that is involved in cold, salt stress and seed development. The transcript of AhSTYK was also shown to be increased in the mid-cotyledonary stage of seed development indicating the possibility that AhSTYK may be involved in signal transduction mechanisms related to storage of metabolites13. Furthermore, our earlier studies have also identified a Mn2+ dependent dual-specificity kinase (AtSTYK), a homolog of AhSTYK in Arabidopsis15. However, the physiological substrate for these protein kinases has not been identified despite its physiological significance in abiotic stress and seed development. Interestingly, we have recently shown that AhOLE3 is phosphorylated by AhSTYK in vitro and that the phosphorylation regulates the bifunctional activity of AhOLE316. Nonetheless, the in vivo phosphorylation of AhOLE3 was not shown. These studies demonstrate the current need for the identification of phosphorylated proteins and their corresponding kinases in plant seed OBs of Arabidopsis.The current knowledge on the phosphorylation status of plant seed OB proteins is vastly inadequate. There is only one report showing the phosphorylation of steroleosinArabidopsis phosphorylated OB proteins in seeds and their corresponding protein kinases. Using a combination of radiometric and proteomic approaches, we have identified the different phosphorylated OB proteins from Arabidopsis seeds. Arabidopsis has fifty-seven distinct STY protein kinases17. However, both our in vitro experiments in yeast and in vivo experiments in yeast and Arabidopsis demonstrated that the AtSTYK (AT2G24360) is one of the major protein kinases phosphorylating these OB proteins and which in turn affects the seed oil content. Seed OBs contain a variable number of proteins depending on the oleaginous plants and their tissues. Oleosins are the most abundant OB proteins that control OB structure and lipid accumulation18. Although, we show various OB protein phosphorylation, the role of OLE1 in regulating the lipid content and its effect upon phosphorylation has been demonstrated in detail.The present study is directed to identify the role of Arabidopsis STYK, a standard in vitro kinase assay was performed using E. coli-expressed and purified GST-STYK . The mutant protein was expressed in E. coli and the purified protein was subjected to in vitro kinase assay. Interestingly, the phosphorylation of the mutant protein OLE1T166A by STYK was completely abolished . The lipid droplets (LD) of yeast cells are structurally and functionally related to those of plant and animal cells. Hence, yeast is an ideal model to study the lipid droplet associated function of these proteins. To study the effect of phospho-OLE1 on its cellular function, both STYK and OLE1 were overexpressed as an N-terminal Hisels Fig.\u00a0. Furtherels Fig.\u00a0. Interesels Fig.\u00a0. On the sed Fig.\u00a0. FurtherTYK Fig.\u00a0. The phoTYK Fig.\u00a0. Taken tin vitro phosphorylation and to check the phosphorylation status of OLE1 in vivo, experiments were conducted using A. thaliana seed oil body (OB) proteins. The phosphorylation status of Arabidopsis OB proteins was investigated using [32P]orthophosphoric acid-imbibed seeds. The isolated OB proteins from the radio-labeled seeds were resolved onto a 15% SDS-PAGE gel and subjected to phosphorimaging analysis. Interestingly, the labeling experiment revealed approximately five distinct proteins in the range of 5\u201370\u2009kDa . The major identified proteins were ubiquitin and polyubiquitin fragments. Furthermore, the proteins with high MASCOT scores and sequence coverage that were identified in the clustered band are listed in Table\u00a0To validate the kDa Fig.\u00a0. The disin vitro kinase assay was performed using Ni-NTA affinity purified CAL4 PLFEKLEK, is the most phosphorylated site, followed by Thr-39, represented by the peptide NKDGIVYPSE(T)PFQGFR, and Ser-75, represented by the peptide GF(S)PIWFPIEVK . CK2 has been shown to phosphorylate lipid droplet proteins in yeast19. STYK was used as a source of protein kinase in the control reaction. Phosphorimaging analyses of the SDS-PAGE-separated kinase assay product showed that OLE1 and CAL4 were significantly phosphorylated by STYK  is responsible for the phosphorylation of OB proteins in Arabidopsis, T-DNA insertion mutants of STYK were obtained from ABRC. The site of T-DNA insertions in styk1 (SALK_105195), styk2 (SALK_120808), and styk3 (SALK_144442) mutants was given in the pictorial representation . Specifically, the Km values of STYK for OLE1 and CAL4 were 0.9659\u2009\u00b5M and 0.9972\u2009\u00b5M, respectively, demonstrating that STYK prefers OLE1 and CAL4 equally as substrates, although OLE1 has a slightly greater affinity for STYK. Furthermore, OLE1 is specifically phosphorylated at Thr-166 in the residue DXDXT, a haloacid dehalogenase motif that is specific for phosphatase activity. Similarly, CAL4 is phosphorylated at three different sites, Thr-39, Ser-75, and Ser-177, by STYK. Furthermore, the importance of OLE1(T166) phosphorylation site was demonstrated by using site directed mutation studies on T166 residue. Kinase assay clearly showed the lack of phosphorylation in the OLE1T166A mutant by STYK.In this present study, we hypothesized that AtSTYK, the homolog of AhSTYK, could phosphorylate the OB proteins of 22. Therefore, we studied the effect OLE1 and its phosphorylation on the cellular lipid biosynthesis. The co-expression of OLE1 and STYK in yeast cells led to an increase in the cellular total lipid levels. The increase in the lipid levels was significantly higher than the cells overexpressing OLE1 alone. Interestingly, co-expression of OLE1 and STYK in yeast cells increased the lipid droplets. However, the effect was significantly reduced when STYK was co-expressed with OLE1T166A. This further confirmed our in vitro observation that phosphorylation of OLE1 does play a role in modulating the lipid levels in vivo.Heterologous expression of plant OB protein oleosin in yeast cells has been shown to increase the cellular nonpolar lipid levelsA. thaliana seeds with radicles. The mass spectrometric experiment revealed that the four distinctly separated phosphoproteins in the SDS-PAGE gel were identified as oleosin 1, oleosin 2, caleosin 4 and 12\u2009S seed storage protein CRU3. In contrast, the major proteins that were identified in the 5th band (clustered phosphoproteins) were polyubiquitin, 2\u2009S seed storage protein 3 (AT2S3), 12\u2009S seed storage protein CRU3, putative uncharacterized protein At2g40765 and a mitochondrial import receptor subunit TOM6 homolog. Previous studies have identified phosphorylation occurring in two of the identified proteins: oleosin 2 and CRU3. Specifically, a serine residue in the peptide \u201c13HFQFQ(S)PPYEGGR23\u201d from oleosin 2 is phosphorylated24. Further, a recent report demonstrated the phosphorylation of a proteolyzed fragment of oleosin 29. Similarly, CRU3 is also phosphorylated at the residue Tyr-406 with the peptide \u201cYNMNANEIL(Y)PCTGGQGR\u201d . 12\u2009S seed storage proteins are the most abundant and phosphorylated proteins in Arabidopsis seeds. These studies further supported our proteomic approach and the phosphoprotein identification in OBs.Most proteome studies on OBs were carried out on mature, dry seeds, presumably giving an impression of OBs as quiescent organelles. Therefore, in the current study, we used 36\u2009h imbibed in vitro and the microscopic evidences using yeast as a model system were further validated by assessing the OB phosphorylation status of the confirmed styk mutant. In accordance with our previous observation, the phosphorylation of most of the OB proteins was significantly reduced in the styk mutant as compared to wild-type OBs. This result further confirms STYK as a major protein kinase phosphorylating the OB proteins oleosin 1, oleosin 2, caleosin 4, and 12S seed storage protein CRU3. Interestingly, the microarray analyses of other STYK also supported by showing no significant alteration in its expression levels to compensate the styk (At2g24360) knock-out effect. We further studied the role of STYK mediated OB phosphorylation in Arabidopsis seed lipid accumulation. The seeds of the styk mutant accumulated 10\u201312% less TAG compared to WT, validating the role of STYK phosphorylation in cellular lipid accumulation.The 26. Studies in B. napus have shown that there exists a significant negative correlation between oilbody size and oil content27. Furthermore, B. napus with higher OB organelle to cell area ratio has been shown to have high oil content compared to the seeds with lower ratio. STYK by phosphorylating oleosin 1 could possibly prevent the coalescence of small oilbodies to form large droplets and increase the OB organelle to cell area ratio by dispersing them which ultimately increases the oil content. Furthermore, OLE1 has a \u201cDXDXT\u201d haloacid dehalogenase/phosphatase motif, STYK phosphorylation could modulate the enzyme activity to increase the seed oil content. Future studies would be directed to address how oleosin 1 phosphorylation affects the lipid levels in the Arabidopsis seeds.The oilbody structural proteins plays an important role in regulating the morphology of oilbodies which in turn is correlated with the oil content and fatty acid compositions of seedsArabidopsis and their corresponding protein kinase. The role of phosphorylation in the modulation of the structural function of oleosin 1, a major OB protein, is further discussed. Here, we also report the role of STYK mediated OB phosphorylation in affecting the seed oil content in Arabidopsis.In conclusion, the present study identified the different phosphorylated OB proteins of 32P]ATP , [32P]orthophosphoric acid  and [14C]acetate (51\u2009mCi/mmol) were obtained from Bhabha Atomic Research Centre (BARC), Mumbai, India. Phos-tag\u2122 acrylamide was purchased from Wako Pure Chemical Industries, Ltd., Japan. Restriction endonucleases and Pfu polymerase were from New England Biolabs. The plasmid miniprep kit, agarose gel elution kit, PCR purification kit, nickel-nitrilotriacetic acid (Ni2+-NTA) matrix, and RNeasy plant minikit were purchased from Qiagen. HCS LipidTOX Red neutral lipid stain and BODIPY\u00ae 493/503  were from Life Technologies. Casein kinase 2 was from New England Biolabs (#P6010S) Oligonucleotide primers, anti-His6 tag monoclonal antibody, and all other reagents were obtained from Sigma-Aldrich. An enhanced chemiluminescence kit (ECL) was obtained from Perkin Elmer. The At4g25140 (OLE1) clone, At1g70670 (CAL4) clone, Col-0 and styk knock-out lines were obtained from the Arabidopsis Biological Resource Center (ABRC). Yeast strains were purchased from the EUROSCARF collection center.ATP , and the incubation was carried out at 30\u2009\u00b0C for 30\u2009min. The reaction was stopped by the addition of SDS-PAGE gel loading buffer. Phosphorylated products were separated by 15% (w/v) SDS-PAGE, and the labeled proteins were detected using a phosphorimager.The in vitro kinase assay was performed using STYK as the enzyme and OLE1 as the substrate. The kinase assay mixture consisted of 50\u2009mM Tris-HCl (pH 7.5), 10\u2009mM MnCl2, 1\u2009\u00b5g of STYK and 2\u2009\u00b5g of OLE1 in the presence of 100\u2009\u00b5M ATP, and the incubation was carried out at 30\u2009\u00b0C for 30\u2009min. The reaction was stopped by the addition of SDS-PAGE loading buffer. Phosphorylated products were separated by 10% (w/v) SDS-PAGE with 25\u2009\u00b5M Phostag. The phosphorylated OLE1 proteins had a mobility shift that was detected in silver staining. Phosphorylated OLE1 was excised from the gel and subjected to in-gel trypsin digestion30.An Digested peptides were reconstituted in 20\u2009\u03bcL of 2% acetonitrile (ACN) with 0.1% formic acid, and 6\u2009\u03bcL of the same was injected onto an Agilent zorbax SB300 C18 column . The mobile phase consisted of buffer A (0.1% formic acid) and buffer B (80% acetonitrile 0.1% formic acid) . A linear gradient elution (11% buffer B to 100% buffer B) was done for 75\u2009min to separate the phosphopeptides. The HPLC system was coupled online to a high-resolution LTQ Orbitrap mass spectrometer (Thermo Scientific). The mass spectrometer was operated in the data-dependent mode, in which a full-scan MS (from m/z 350\u20135000) was followed by 20 MS/MS scans of the most abundant ions using collision-induced dissociation (CID). Standard phosphopeptide mix (25 fmoles) spiked into a 250 fmol BSA digest was also analyzed to check the performance of the instrument. A minimum of two highly confident peptides were used as a prerequisite to identify the proteins.Arabidopsis database that was downloaded from NCBI, with no redundant entries using the SEQUEST algorithm on Proteome Discoverer . The peptide precursor mass tolerance was set to 10 ppm, and the peptide fragment mass tolerance was set to 0.8\u2009Da. Search criteria included a dynamic modification of +79.996\u2009Da on normal phosphorylated serine, threonine, and tyrosine residues, +15.9949\u2009Da on oxidized methionine and a static modification of +57.0214\u2009Da on cysteine residues. Searches were performed with full tryptic digests and a maximum of two missed cleavages were allowed on peptides that were analyzed by the sequence database. False discovery rates (FDR) were set to 1% for each analysis. The number of unique phosphopeptides and non-phosphopeptides was manually counted and compared. Phosphorylation site localization from CID mass spectra was determined by PhosphoRS scores31. For phosphopeptides with inconclusive phosphorylation sites, the one with highest phosphoRS score was selected for further data interpretation.The raw files obtained from LTQ-Orbitrap were searched directly against the Yeast cells used in this study  glucose. For in vivo labeling, cells at A600 0.2 were transferred to fresh induction medium containing 2% (w/v) galactose and 0.2 \u00b5Ci/mL [14C]acetate and grown for an additional 24\u2009h until the cells reached the stationary phase. Cells (A600\u2009=\u200925) were harvested by centrifugation, and lipids were extracted using chloroform/methanol . Individual phospholipids were separated by two-dimensional thin-layer chromatography on silica gel 60 using chloroform/methanol/25% ammonia  as a first-dimension solvent and chloroform/methanol/acetone/acetic acid/water  as the solvent for a second dimension8. Neutral lipids were separated by one-dimensional TLC using petroleum ether:diethylether:acetic acid  as the solvent system. The labeled lipids were visualized by a phosphorimager. The spots corresponding to labeled lipids were scraped off to determine the radioactivity by liquid scintillation counting in a Microbeta 2 microplate counter (Perkin Elmer) using OptiPhase Supermix scintillation cocktail.The 34.The total lipid content was extracted from yeast cells expressing the different constructs . The extracted lipids were converted to total fatty acid methyl ester (FAMEs) and analyzed as described previouslyOLE1 or vector control was performed using Huygens software. (Z section series projected to a 3D image) with the deconvoluted images from five different fields, with approximately 25 cells per field. The quantification of fluorescence was performed using image J software.All the yeast microscopic pictures were captured by confocal microscopy using a Leica SP8 laser-scanning confocal microscope. The samples were viewed using a 100\u00d7 oil immersion objective lens. For lipid droplet analysis, yeast cells were grown in synthetic media to stationary phase. The cells were washed with PBS and stained with BODIPY 493/503 (1\u2009\u00b5g/mL) for 30\u2009min at room temperature. Excess stain was removed by washing the cells with PBS. The cells were resuspended in 50% glycerol and viewed under a microscope. Comparison of lipid droplet in yeast cells overexpressing Arabidopsis wild-type (Col-0) and styk knock-out line  plants were grown vertically on soil and on half-strength Murashige and Skoog medium (supplemented with 0.5% (w/v) sucrose and solidified with 0.7% agar) after 2 days of stratification at 4 \u00b0C. The plants were grown at 23\u2009\u00b0C under a 16\u2009h\u2009day (140 \u00b5mol m\u22122 sec\u22121) and 8\u2009h night regime.To identify the T-DNA insertions in the SALK_105195, SALK_120808, and SALK_144442 lines, the T-DNA borders of these SALK lines were amplified using the T-DNA left border and the corresponding gene-specific primers. Homozygous lines were identified by PCR using genomic DNA as a template from wild-type and SALK line leaves to confirm the disruption of the gene. The true knock-out lines were further confirmed by PCR using cDNA as a template. Ubiquitin was used as an internal control. RNA isolation was performed using a Qiagen RNeasy Plant minikit as per the manufacturer\u2019s protocol with DNase treatment. The RNA concentration and purity were determined at an optical density ratio of 260/280 using the NanoDrop\u00ae ND-1000 spectrophotometer. cDNA was prepared using a high-capacity cDNA reverse transcription kit (Applied Biosystems).et al.4, with the following modifications. Approximately 200\u2009mg of mature seed was ground in 1\u2009mL of ice-cold Buffer A , 10\u2009mM KCl, 1\u2009mM EDTA, 1\u2009mM MgCl2, 5\u2009mM \u03b2-mercaptoethanol and 1\u2009mM phenylmethanesulfonyl fluoride (PMSF)) using a mortar and pestle. The crude homogenate was centrifuged at 1200\u2009\u00d7\u2009g for 15\u2009min to separate the unlysed cells and debris. The collected supernatant was layered with 1\u2009mL of cold Buffer B (Buffer A containing 0.4\u2009M sucrose instead of 0.6\u2009M sucrose). The sample was centrifuged at 100,000\u2009\u00d7\u2009g (Beckman Optima MAX-XP tabletop ultracentrifuge with TLS 55 rotor) for 30\u2009min. The top hydrophobic layer was carefully removed using a spatula and resuspended in Buffer C (Buffer A with 2\u2009M NaCl) using a 2\u2009mL glass dounce homogenizer. The suspension was added to 1\u2009mL of Buffer D (Buffer B with 2\u2009M NaCl) and centrifuged at 100,000\u2009\u00d7\u2009g for 30\u2009min. The top layer was resuspended with a glass homogenizer in 1\u2009mL of Buffer A, over layered with 1\u2009mL of Buffer B and centrifuged at 100,000\u2009\u00d7\u2009g for 30\u2009min. The procedure was repeated, and the final oil body layer was resuspended in Buffer E (50\u2009mM Tris-HCl (pH 7.5), 10\u2009mM KCl, 1\u2009mM EDTA, 1\u2009mM MgCl2, 5\u2009mM \u03b2-mercaptoethanol, and 1\u2009mM PMSF).Oil bodies were isolated according to the method of Tzen styk seeds were incubated in a microfuge tube containing MilliQ water and 500 \u00b5Ci of [32P]orthophosphoric acid for 4\u2009h and allowed to germinate on moistened paper (two layers of Whatman #2 filter paper soaked with 500 \u00b5Ci of [32P]orthophosphoric acids in 5\u2009mL of MilliQ water). The petri dishes were incubated at 23\u2009\u00b0C in an 8-h-dark/16-h-light cycle for 36\u2009h . The seeds were collected, washed with MilliQ water three times, and used to isolate oil bodies. The oil bodies were delipidated using diethyl ether, and the proteins were separated on a 15% SDS-PAGE. The phosphorylated proteins were identified by a phosphorimager (Typhoon FLA 9000). Phospho peptides from Col-0 OB proteins were identified as previously described.Col-0 and styk 1 and 2 lines was used to prepare cDNA followed by cRNA generation. Whole genome micro array was performed as described previously. The microarray data was submitted to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94596 and assigned GSE94596 as the accession number. Gene expression profiling of all the STYK genes in Arabidopsis were analyzed using equal amount of total RNA and the amount of cDNA was normalized using endogenous actin primer and the expression profile was determined by using the respective STYK gene specific primers.Briefly, radicle emerged seeds were used to isolate total RNA and 500 ng of total RNA from both WT and styk mutant lines were extracted and analyzed as described in the Kansas Lipidomics Research Center (KLRC) protocol35. Ten plants of Col-0 and the styk lines were grown under controlled growth conditions. Seeds were pooled from ten plants and the lipid extraction was performed as four independent replicates for each plant line. Lipid extracts were dried completely and used for mass spectrometric analysis in the KLRC mass spectrometric facility. TAG analysis, was performed by continuous infusion into an ESI source on a triple quadrupole mass spectrometer . TAG molecular species were detected as [M\u2009+\u2009NH4]+ ions by a series of neutral loss scan targeted for C16-20 fatty acid losses. TAG data were produced by subtracting each background spectrum using ABI Analyst software and quantified using internal standards. Data obtained from four independent replicates for each line were averaged and were subjected to Student\u2019s t-test at the P\u2009<\u20090.05 and P\u2009<\u20090.01 level to determine the statistical significance.Seed lipids from Col-0 and the t test. Each experiment was repeated at least three times, and at least 125 cells were scored per experiment to quantify the microscopic data. Significance was determined at *p\u2009<\u20090.05; and **p\u2009<\u20090.01.The experimental data are shown as the mean\u2009\u00b1\u2009S.E., and data were analyzed using Student\u2019s Arabidopsis Genome Initiative under the following accession numbers: OLE1 (At4g25140), corresponded to the S3 gene as reported in the Kim et al.36, CAL4 (At1g70670), and AtSTYK (At2g24360). Arabidopsis mutants used in this article can be found in the Arabidopsis Genome Initiative under the following accession numbers: styk1 (SALK_105195), styk2 (SALK_120808) and styk3 (SALK_144442). styk knock-out lines and WT microarray data deposited at the https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94596, accession number: GSE94596.Sequence data from this article can be found in the The author who is responsible for the distribution of materials that are integral to the findings that are presented in this article is Ram Rajasekharan .Supplementary Information"}
+{"text": "Monocytes have been recently subdivided into three subsets: classical (CD14++CD16\u2212), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets, but phenotypic and functional abnormalities of the three monocyte subsets in HIV-1 infection have not been fully characterized, especially in acute HIV-1 infection (AHI). In the study, we explored the dynamic changes of monocyte subsets and their surface markers, and the association between monocyte subsets and the IFN-\u03b3, interleukin (IL)-4, IL-17, and TNF-\u03b1 producing CD4+ T cells in acute and chronic HIV-1-infected patients. We found that, in the acute HIV-1-infected individuals, the frequency of the intermediate CD14++CD16+ monocyte subsets, the CD163 density and HLA-DR density on intermediate CD14++CD16+ monocytes, and plasma soluble form of CD163 (sCD163) were significantly higher than that in healthy controls. Intermediate CD14++CD16+ monocyte subsets and their HLA-DR expression levels were inversely correlated with the CD4+ T cell counts, and the intermediate CD14++CD16+ monocytes were positively correlated with plasma sCD163. In contrast to the non-classical CD14+CD16++ and classical CD14++CD16\u2212 monocyte subsets, the frequency of the intermediate CD14++CD16+ monocytes was positively associated with the frequency of IFN-\u03b3 and IL-4 producing CD4+ T cells in HIV-1-infected patients. Taken together, our observations provide new insight into the roles of the monocyte subsets in HIV pathogenesis, particularly during AHI, and our findings may be helpful for the treatment of HIV-related immune activation. The interactions between the virus and the immune system during acute HIV-1 infection (AHI) determine the viral load set point and other critical events. Monocytes can act as regulators of the immune system, and monocytes abnormalities are responsible for the hyperactivity observed in HIV-1-infected patients . PhenotyIdentification of the three subsets within monocytes populations has put monocyte heterogeneity into sharp focus. The perturbation of the three monocyte subsets has been examined in various diseases including bacterial infection, viral infection, and autoimmune disease \u20135. AnalyDepending on the cytokine environment, na\u00efve CD4+ T cells differentiate into T helper (Th) 1, Th2, Th17, and other lineages, each lineage has distinct biological functions. In immune thrombocytopenia, CD16+ monocytes promoted Th1 development, which in turn negatively regulated interleukin (IL)-17 and Treg induction . In rheuFrom 2007 to 2012, 484 acute HIV-1-infected cases among 5,800 men who have sex with men (MSM) were identified by our team . We founAll the participants provided written informed consent for their information, and clinical samples were stored and used for research. This study and all relevant experiments have been approved by the Beijing You\u2019an Hospital Research Ethics Committee and informed consent was provided according to the declaration of Helsinki. The methods were carried out in accordance with approved guidelines and regulations.Thirty-seven homosexual men with acute HIV-1 infection (AHI) were enrolled in the study, these patients were recruited from an HIV-1-negative high risk MSM cohort who were screened every 3\u2009months for HIV-1 infection at Beijing You\u2019an Hospital. The progression of early HIV-1 infection can be depicted as six discrete stages as proposed by Fiebig et al. . These aCryopreserved peripheral blood mononuclear cells (PBMCs) were used, and cell viability was evaluated in cell surface and intracellular cytokine staining experiment. Cryopreserved PBMCs were thawed in RPMI 1640 medium , washed with PBS containing 1% BSA, and then incubated at room temperature for 20\u2009min with the cells viability marker fixable viability stain 510 .Monocyte phenotypic analysis was performed after staining with anti-CD14-FITC (eBioscience), anti-CD16-PE (eBioscience), anti-HLA-DR-PerCP-Cyanine5.5 (eBioscience), and anti-CD163-APC . Intracellular IFN-\u03b3, IL-4, IL-17, and TNF-\u03b1 staining was performed after stimulating with a leukocyte activation cocktail with BD GolgiPlug  for 5\u2009h, the leukocyte activation cocktail contained PMA, ionomycin, and brefeldin A. Cells were initially stained with anti-CD4-PE-Cy7 , cells were then fixed and permeablized with Cytofix/Cytoperm according to the manufacturer\u2019s instructions , followed by staining with anti-IL-4-PE (eBioscience), anti-IL-17-APC (eBioscience), anti-IFN-\u03b3-eFluor 450 (eBioscience), anti-TNF-\u03b1-PerCP-Cyanine5.5 .All expression analyses were performed by flow cytometry using BD FACSCanto\u2122 II with Diva software . Forward scatter and side scatter light gating were used to exclude cell debris from the analysis. Forward height and forward area were used to exclude doublet cells. The final analysis was performed using the Flowjo 10.0.7 software .m2000 system  according to manufacturers\u2019 instruction, and the sensitivity of detection was 40 copies per milliliter.The CD4+ T-cell count was determined by using anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE monoclonal antibodies (BD Biosciences). Analysis was then carried out using a BD FACSCanto\u2122 II Flow cytometry  system. HIV-1 viral load tests were done by using an automated real-time PCR-based Quantification of soluble CD163 (sCD163) was determined by using a commercial ELISA Kit  according to the manufacturer\u2019s instruction without modification.t-test, or non-parametric tests. All reported p values were two sided and considered significant at *p\u2009<\u20090.05 or **p\u2009<\u20090.01. The association was evaluated by Spearman\u2019s correlation test. All data were analyzed using SPSS 21.0 statistical software .Statistical analysis was performed by using an ANOVA test, Student\u2019s p\u2009<\u20090.01) and higher than those of chronic HIV-1-infected patients without cART (p\u2009<\u20090.01).Thirty-seven homosexual men with acute HIV-1 infection (AHI), 31 CHI&ART-, 63 chronic HIV-1-infected patients with undetectable viral load after cART (CHI&ART+), and 42 HC were enrolled in the study. The information of numbers, ages, viral loads, cell counts, and CD4+/CD8+ ratio is presented in Table Based on CD14 and CD16 expression, monocytes are divided into three subsets: classical (CD14++CD16\u2212), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets. The gating strategy is shown in Figure To characterize the phenotypic changes of the three monocyte subsets, we analyzed the expression pattern of plasma sCD163 and the following surface markers: scavenger receptors CD163 Figures A\u2013C and aFigure The gating strategy of the IFN-\u03b3, IL-4, IL-17, and TNF-\u03b1 producing CD4+ T cells is shown in Figure Next, correlation analyses are performed between monocyte subsets and the IFN-\u03b3, IL-4, IL-17, and TNF-\u03b1 producing CD4+ T cells in HIV-1-infected patients. The frequency of the non-classical monocyte CD14+CD16++ subsets is positively associated with the percentage of IFN-\u03b3 Figure A and TNFIn the study, we evaluated the dynamic changes of monocyte subsets and their surface markers in acute and chronic HIV-1-infected individuals, we also analyzed the association between monocyte subsets and Th cell differentiation. We found that in the acute HIV-1-infected individuals, the frequency of the intermediate CD14++CD16+ monocyte subsets, the HLA-DR density and CD163 density on CD14++CD16+ monocytes, and plasma CD163 were significantly higher than those in HC, but lower than those in chronic HIV-1-infected individuals. Intermediate CD14++CD16+ monocytes and their HLA-DR expression on intermediate CD14++CD16+ monocytes were inversely correlated with the CD4 cell counts, and the intermediate CD14++CD16+ monocytes were positively correlated with plasma sCD163. The frequency of IL-4 producing CD4+ T cells is negatively associated with CD4 cell counts as well as with the CD4/CD8 ratio. In contrast to non-classical CD14+CD16++ and classical CD14++CD16\u2212 monocyte subsets, the frequency of the intermediate CD14++CD16+ monocytes was positively associated with the frequency of IFN-\u03b3 and IL-4 producing CD4+ T cells in HIV-1-infected patients. The intermediate CD14++CD16+ monocyte subsets were positively associated with IFN-\u03b3 (CHI&ART\u2212 group) and IL-4 (AHI and CHI&ART\u2212 groups) producing CD4+ T cells. There was no association between the monocyte subsets and T helper cell responses in CHI&ART+. Intermediate monocyte subsets, surface CD163 density on intermediate monocytes, and plasma sCD163 levels in CHI&ART+ still higher than those in healthy control, abnormalities of the three monocyte subsets still existed despite suppression of HIV after cART.Our findings indicated that monocytes were heterogeneous with subset-specific phenotypes and functions during HIV-1 infection, intermediate CD14++CD16+ monocytes were correlated with disease progression in acute and chronic HIV-1-infected individuals, and the three monocyte subsets played different roles in Th cell differentiation in HIV-1-infected patients. The abnormalities of three monocyte subsets may be related to chronic immune activation despite suppression of HIV after cART.Based on the expression of the CD14 and CD16 antigen, human blood monocytes were initially classified into two subsets: CD14+CD16\u2212 and CD14+CD16+ monocyte subsets . RecentlBurdo et al. showed that sCD163 levels were positively correlated with CD14+CD16+ monocytes . CD14+CDin vitro PBMC stimulation culture system was used in this study, in which PBMCs were stimulated with PMA/ionomycin. Direct contact of monocytes and T cells is important for T cell differentiation  producing cells and an increased percentage of IL-4 (Th2) producing cells  as well ntiation , and theIn summary, we evaluated the perturbations of monocyte subsets and their association with Th cell differentiation in acute and chronic HIV-1-infected individuals. Our observations provide new insight into the roles of monocyte subsets in HIV pathogenesis, particularly during AHI, and our findings may be helpful for the treatment of HIV-related immune activation.BS, LL, and HW conceived the study, designed the experiments, and analyzed the data. PC, XZ, HX, and GZ performed the experiments; BS, TZ, WX, LD, LS, and YF contributed to reagents and materials; and PC, BS, LL, and HW wrote the article. 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": "Candida parapsilosis sensu stricto isolates. The aim of this study is to develop and perform an initial validation of an alternative protocol for the reliable and accurate microsatellite genotyping of C. parapsilosis sensu stricto isolates using high-throughput multiplex PCR. To achieve this, the results obtained using the new protocol were compared to the ones obtained using a previously described reference method. To that end, diagnostic accuracy, informativeness and discrimination parameters were estimated.Analysis of polymorphic microsatellite markers (STR) is a helpful genotyping technique to differentiate Our results showed good concordance between both methods (Kappa index: 0.920), leading to a high sensitivity  (0.991\u20131)) and specificity  (0.772\u20131)) after the validation of the new protocol. Moreover, the electropherograms profiles obtained with the new PCR scheme showed a high signal to noise ratio (SNR).C. parapsilosis sensu stricto, with direct clinical applications. Besides, the new protocol represents a shortening the hands-on time, reducing the sample manipulation (dismissing the possibility of cross-contamination), maintaining the quality of the results (when compared to the ones obtained with the reference method), and helping to the standardization and simplification of the genotyping scheme.The new multiplex protocol is valuable for the differentiation of  Candida parapsilosis could be considered as a genetically related species complex which includes C. parapsilosis sensu stricto, Candida metapsilosis and Candida orthopsilosis [C. parapsilosis sensu stricto the most commonly isolated. However, C. parapsilosis sensu stricto is not a homogeneous species, and therefore, accurate and reliable typing methods are necessary for a better knowledge of this species [In 2005, Tavanti et al. proposed that the fungus psilosis , being C species \u20133. These species .C. parapsilosis isolated from different clinical sources, such as blood or catheters, and from medical or surgical wards [C. parapsilosis sensu stricto.Last decades technological innovations allowed the extensive use of microsatellites or Single Sequence Repeats (SSR) in plant and eukaryote genetics studies, using different genotyping approaches ranging from low to high throughput ones, not only in genetic research but also with interesting applications in clinical practice. In fact, microsatellite typing has been described to study genetic relatedness among colonizing and infective strains from diverse geographical locations or even the relatedness of al wards , 4, 5. CCandida parapsilosis sensu stricto isolates using high-throughput multiplex PCR.This issue aroused an explosion of alternative protocols to standard procedures giving a large number of genotyping schemes but with no extensive validation by comparing them to the existing ones \u20138. This C. parapsilosis sensu stricto blood isolates retrospectively collected during 2010 to 2015 using a convenience sample of 33 patients suffering from invasive candidemia during their hospital stay at La Fe University Hospital . In addition to those clinical isolates, C. parapsilosis sensu stricto ATCC 22019, and ATCC MYA-4646 (CDC317) obtained from the American Type Culture Collection  were used as positive controls. Moreover, C. orthopsilosis ATCC 96139, C. metapsilosis ATCC 96143, Candida albicans ATCC 90028, Candida africana ATCC MYA-2669, Candida dubliniensis NCPF 3949, Candida glabrata ATCC 90030, Candida bracarensis NCYC 3133, Candida nivariensis CBS 9984, Candida tropicalis NCPF 311, Candida krusei ATCC 6258, Candida guilliermondii NCPF 3099 and Lodderomyces elongiosporus ISA1308  were included as subrogate negative controls to assess the specificity of the multiplex PCR protocol. Besides, PCR grade water  was used as negative control for all PCR tests performed.Thirty-three SADH gene using a conventional RFLP-PCR protocol, as previously described [All isolates were plated onto Sabouraud dextrose agar  and incubated at 37\u00a0\u00b0C for 24\u00a0h. Presumptive identification was performed considering colony morphology and color on ChromID Candida  and Candida chromogenic agar  agars and subsequently confirmed using the API 32C (bioM\u00e9rieux) auxanogram according to manufacturers. DNA was extracted using the UltraClean\u00ae Microbial DNA Isolation Kit  following the recommendations of the manufacturers. Definitive identification was reached either by amplification of a short portion of the escribed , 9 or byThis step was only performed using the control strains and each prepared multiplex reaction was tested under different primer concentrations (ranging from 0.2 to 0.5\u00a0\u03bcM) to ensure the best PCR performance. After this step, another multiplex reaction was tested at different final DNA concentrations (ranging from 10 to 30\u00a0ng) to establish the best amount of yeast genomic DNA needed. Finally, the addition of bovine serum albumin   to the PCR mastermix to enhance the reaction efficacy was evaluated.Based on previous reports of polymerase inefficiency of prominent slippage phenomena under certain conditions , 11, we C. parapsilosis sensu stricto were genotyped using CP1, CP4, CP6 and B5 microsatellite markers previously described by Sabino et al. [2, 0.4\u00a0\u03bcM of each primer, and 0.2\u00a0mM deoxynucleoside triphosphates (dNTPs) and the amplification touchdown PCR protocol was performed in a C 1000TM Thermal Cycler .All Briefly, PCR protocol had two differentiated phases. In the first step, annealing temperature of 60\u00a0\u00b0C gradually decreased 0.35\u00a0\u00b0C per cycle until it reached 55\u00a0\u00b0C. The second phase was the same as the latter except for three minor modifications, which were a slight increase in the number of cycles from 14 to 19), a fixed annealing temperature of 55\u00a0\u00b0C . Additionally, a bootstrap test was implemented for evaluating the robustness of the results [The identification of similarities between genotypes was achieved by the constructions of a minimum spanning tree using R statistical software (v.3.1.0). Besides, to represent the relationship between all the  results .Besides, we also calculated other parameters of each microsatellite marker considered in this study which are linked to the microsatellite informativeness content and their discrimination power such as the polymorphic information content (PIC), the Simpson index, the heterozygosity and the entropy , 14.Finally, an estimation of sensibility, specificity, and the Kappa index was performed to estimate not only the diagnostic characteristics of each microsatellite detection protocol but also the agreement among the results obtained after the microsatellite amplification using each compared PCR protocol. All the statistical procedures were performed using the Stata(R) and R statistical software (v. 12 and 3.1.0 respectively). The associations between categorical variables were studied using a chi-squared test or Fisher\u2019s exact test when necessary.C. parapsilosis sensu stricto strains from different repositories or collections to fulfill the objectives of the study. Although some of the strains used for validation came from a clinical origin, no processing of primary samples was made during the experimental work and therefore, the need for ethics approval and consent to participate was unnecessary according to the Spanish Biomedical Research Law and other European Union regulations. However, a formal approval was asked to the Ethical and Research Committee of the University of the Basque Country to ensure that all the issue research was in accordance with the legal and ethical requests prior to its beginning .This study does not involve human participants, human data or human tissue. The authors solely used Despite the several approaches implemented along with the literature to establish a successful microsatellite based genotyping scheme, we focused on the optimization and restructuring of the original PCR protocol proposed by Sabino and coworkers  convertiC. parapsilosis sensu stricto microsatellite genotyping protocols published along the literature compared to the one proposed in our study. The optimization and redesign strategy mentioned earlier implied the evaluation and subsequent election of the two cornerstones of the PCR reaction: the polymerase and primer concentration.Table\u00a0Table\u00a0Furthermore, we found that there were false positive results (non-specific bands) when we used the KAPA2G and Takara mastermixes. Besides, based on our findings, among all the concentrations tested, we found that the 0.4\u00a0\u03bcM final concentration of each allele primer pair lead to the best PCR results. Using this primer concentration, all PCR products obtained by the multiplex protocol showed the same intensity.L. elongiosporus) included in this study. Besides, the ATCC 22019 positive control strain gave the same profile described in the literature after microsatellite fragment analysis , restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST), have been described for s typing , 18, 19.uishable . In receisolates , 20. Howisolates . Therefoisolates , 22.Until recently, microsatellite genotyping is a rather time-consuming technique, because every microsatellite marker must be processed alone. Up to our knowledge, no multiplex PCR protocol following the original scheme proposed by Sabino et al.  has beenC. parapsilosis sensu stricto genotyping protocols. The most reliable explanation of the observed results is that the three mastermixes tested had different polymerases in their composition, being the AmpliTaq Gold\u00ae the most suitable one to carry out C. parapsilosis sensu stricto microsatellite genotyping using this multiplex PCR scheme.Based on our results, there are several crucial points to consider before getting satisfactory results, being the appropriate polymerase election the most important one when a multiplex PCR protocol is used. Although all the PCR mastermixes tested in our work were explicitly fabricated to operate under their best conditions using multiplex PCR protocol, KAPA2G and Takara mastermixes, showed lack of specificity, conditioning their future use in multiplex PCR based A total of 35 samples were genotyped to validate the utility of our method in contrast to the one described by Sabino and coworkers. Though our results were concordant with those published previously, we could see slight differences in the estimation of the Simpson index and the observed heterozygosis among Sabino\u2019s original data and ours, probably explained because of the differences in the total sample number of strains analyzed in each work.Finally, the high-quality profiles of the electropherograms obtained using the new multiplex protocol are due to the adoption of a touchdown PCR strategy which improves the profile analysis and prevents misclassification. In a recent review, such schemes are described as a suitable option to increase the specificity of the obtained PCR products without losing sensitivity .There are some limitations in our study such as the small number of strains analyzed in this study and the fact that all of them were isolated from the same clinical source (blood). This issue has probably an impact on the precision of the confidence intervals and the generalization of our informativeness parameters estimates. However, the consistency of our results with those published in the literature suggesting that the possibility of bias is rare.Despite these limitations, our validation results support that the new protocol seems to be as accurate and reliable as the original one. However, it represents a significant decrease in the turnaround time necessary to get accurate genotyping results compared to other approaches published along with the literature. The main disadvantage the new protocol is that it is slightly more expensive than the original technique in case we use primers labeled with different fluorophores. This limitation could be overcome by using the same fluorophore for those primers targeting loci that have very different sizes (such as CP6 and B5), decreasing the total cost of the technique and increasing its cost-effectiveness if the researchers decide to adopt the latter option.C. parapsilosis sensu stricto isolates, with direct applications to clinical practice and infection control procedures . Besides, our protocol helps the standardization and simplification of the existing microsatellite typing systems, improving the quality of data, the sample hands-on time and lab turnaround time to get accurate genotyping results for further clinical or infection control epidemiological studies.In conclusion, this new protocol is a valuable tool for the differentiation of"}
+{"text": "Breastfeeding is considered the gold standard for infants\u2019 nutrition, as mother\u2019s own milk (MOM) provides nutritional and bioactive factors functional to optimal development. Early life microbiome is one of the main contributors to short and long-term infant health status, with the gut microbiota (GM) being the most studied ecosystem. Some human milk (HM) bioactive factors, such as HM prebiotic carbohydrates that select for beneficial bacteria, and the specific human milk microbiota (HMM) are emerging as early mediators in the relationship between the development of GM in early life and clinical outcomes. The beneficial role of HM becomes even more crucial for preterm infants, who are exposed to significant risks of severe infection in early life as well as to adverse short and long-term outcomes. When MOM is unavailable or insufficient, donor human milk (DHM) constitutes the optimal nutritional choice. However, little is known about the specific effect of DHM on preterm GM and its potential functional implication on HMM. The purpose of this narrative review is to summarize recent findings on HMM origin and composition and discuss the role of HMM on infant health and development, with a specific focus on preterm infants. Nutrition in early life plays a key role in shaping an infant\u2019s future health. Specifically, human milk (HM) is known to exert a series of beneficial effects for infants, including improved neurological, immunological, and metabolic outcomes. Breastfeeding is considered the gold standard for infants\u2019 nutrition, as mother\u2019s own milk (MOM) provides all the nutritional factors required for optimal infant development. In addition to its nutritional content, MOM constitutes \u201cnature\u2019s first functional food\u201d , as it hThe gut microbiota (GM) constitutes the most studied ecosystem in infants: Many factors, such as gestational age, mode of delivery, antibiotics, type of nutrition, and social environment, impact on the composition of the infant\u2019 early GM. Among these factors, some HM bioactive factors, such as HM prebiotic non-digestible carbohydrates ) that select for beneficial bacteria, and the specific HM microbiota, are emerging as early mediators in the relationship between the development of GM in early life and short and long-term health outcomes. In this review we will summarize recent findings on HMM origin and composition and discuss the role of HMM on infant health and development, with a specific focus on preterm infants.Until recently, HM was considered a sterile fluid. Although there existed reports of viable bacteria in HM from healthy women, these bacteria were thought to be contaminants from the skin or other environmental sources . The lon5 to 1 \u00d7 107 bacteria daily for an infant receiving 800 mL/day of HM) [At present, several studies support the idea that HM harbours a complex microbial community, which is a source of a huge number of viable commensal, mutualistic, or potentially probiotic bacteria for developing infants and their gut (estimated as 1 \u00d7 10y of HM) . Streptococcus and Staphylococcus being the most frequently isolated and abundant bacterial groups, together with skin-derived or environmental contaminants . However, well-known intestinal probiotic bacteria  had been isolated as well.The first characterizations of HMM in healthy women were based on culture-dependent techniques ,17, whosThe development of culture-independent techniques, such as quantitative polymerase chain reaction (qPCR) and, later on, next generation sequencing (NGS), mostly based on 16S rRNA gene, has allowed the characterization of the composition and diversity of HMM in deep detail, and has documented a huge number of bacteria in breast milk and a high variability in HMM composition. Indeed, microbiome research has experienced an unprecedented rate of data productivity in the last decade. While allowing for an everyday-increasing amount of evidences on microbiome\u2013human dynamics and interactions, such an amount of studies and results have also highlighted considerable limitations in data obtained from the molecular approaches. For instance, NGS studies provide only a sense of relative abundance because actual bacterial counting is lost during amplification. Another limitation of molecular methods is the so called \u201cviability bias\u201d, i.e., the detection of bacterial DNA does not mean the corresponding microorganism is alive in the original sample. Moreover, different methods of DNA extraction and processing are known to have an impact on the results and their reproducibility . NonetheStreptococcus and Staphylococcus appeared to be the predominant genera in HM independently from the geographic location of the study and from the selected technique (qPCR or NGS). Culture independent studies have also allowed the detection in HM of obligate anaerobic, gut-associated genera, such as Bacteroides, Blautia, Dorea, and Faecalibacterium [Staphylococcus, Streptococcus, Bacteroides, Faecalibacterium, Ruminococcus, Lactobacillus, and Propionibacterium [Staphylococcus, Streptococcus, Serratia, Pseudomonas, Corynebacterium, Ralstonia, Propionibacterium, Sphingomonas, and uncultured members of Bradyrhizobiaceae) microbial genera [Staphylococcus, Streptococcus, and Propionibacterium were the only three genera reported as predominant in both these studies, suggesting that the concept of \u201ccore microbiota\u201d was probably dependent on the geographic location of the study, and on the method used for HM collection, storage, and analysis. The predominance of Streptococcus and Staphylococcus genera in HM was confirmed also in the study by Lackey et al. [Fitzstevens et al.  recentlyacterium . Single acterium ) or ninel genera . Staphyly et al. , who anaStaphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Propionibacterium acnes, Enterococcus faecalis, Bifidobacterium breve, Escherichia coli, Streptococcus sanguinis, Lactobacillus gasseri, and Salmonella enterica. The breast and HM microbiota shared 49% of the species of their repertoire with the gut, 30% with the vagina, 28% with the urinary tract, 28% with the respiratory tract, and 21% with the oral cavity. Approximately 300 bacterial species were found only in the breast and HM microbiota and not in other human microbial niches, suggesting the existence of a breast and HM microbiota with distinctive features.The very recent work by Togo et al.  summarizVariation in HMM composition among women and populations could be related to several factors, including but not limited to length of gestation, delivery mode, time postpartum, and maternal factors such as diet, intake of specific nutrients , and use of antibiotics/probiotics. In addition, it is unclear how much of the geographical variability in HMM composition is truly related to actual differences among studied populations or rather to differences in the setting and procedure of milk collection, storage, and analysis. However, it should be noted that recent large-scale studies have documented that infants\u2019 GM and their mothers\u2019 HMM are more similar within than across cohorts, suggesting that the characteristics of both GM and HMM could be tailored not only to the infants\u2019 and mothers\u2019 lifestyle but also to specific environmental settings . The origin of bacteria in HM is not well established, but growing literature suggests that the genesis of HMM is a complex phenomenon. Two different routes, which are probably not mutually exclusive, have been proposed for HMM establishment: microbes can colonise HM through surface skin contamination and retrograde flow  during breastfeeding or, alternatively, they might translocate to HM through a more speculative gut-mammary route, the so-called \u201centero-mammary pathway\u201d.Streptococcus and Staphylococcus being the most abundant [Streptococcus and Rothia. The changes in HMM following the beginning of breastfeeding were associated with an increase in the abundance of beneficial bacteria, such as Bifidobacterium, in the infants\u2019 faeces and to a reduction of Pseudomonas in oral samples. Biagi et al. examined HMM, infant\u2019s oral as well as gut microbiota in a cohort of healthy term infants and their mothers. According to their data, the infant\u2019s mouth, which is the transition point for HM to reach the gut, could play a relevant role in shaping the characteristics of both HMM and the infant\u2019s gut microbiota . The exiabundant . These oabundant . The infOn the other side, according to the entero-mammary pathway theory , maternaPutting these observations together, HMM may be considered as a dynamic crossroad of different and inter-related bacterial communities. However, HMM assembly is far from clear and mother\u2013infant microbial dynamics during breastfeeding need further investigation because of the critical role played by the microbial colonization of the infant\u2019s gut in immune system education and development.In the complex scenario of the HMM assembly, it is likely that also maternal , infant , and environmental factors  interact in influencing HMM composition. Several studies have explored environmental determinants of microbial composition and diversity in HMM, among which geographical and lifestyles differences have been shown to explain a certain degree of interindividual variation ,31,32. IOther environmental factors that could shape HMM are exposure to disinfection agents , exposurLactobacillus spp., Bifidobacterium spp.) vary among cohorts and studies [Other factors associated with an overall variation in the HMM structure include mode of delivery ,32,34,36 studies ,32,34,36 studies .Bifidobacterium at later stages of lactation, though such differences have not been consistently observed across studies [Differences in the composition of HMM among colostrum, transition, and mature milk have been reported by some authors, with an increased abundance of typical oral inhabitants in transition and mature milk, and higher counts of  studies ,38,39.Bifidobacterium spp. in milk samples from term-delivering compared to preterm-delivering mothers. As for the degree of premature delivery, a progressive increase in total bacterial count appears to be associated with increasing length of gestation [The influence of length of gestation has also been investigated, with higher counts of estation . In the estation ,28, examIt is likely that milk components other than HMM, such as HMOs, milk fatty acids, hormones, immune cells, and antibodies, could modulate the milk \u2018\u2018microenvironment\u2019\u2019, possibly affecting the composition of its microbial community ,32,40. Although HMM studies have mainly focused on bacteria, HM harbours and may also vehiculate yeasts or viruses. Pannaraj et al. examined the relationship in the virome composition between HM and infant stools: the milk virome showed a higher diversity compared to the infant gut virome, and both showed distinctive features compared to the adult sites\u2019 virome. Although differences in viral microbiota composition between HM and stools were substantial, a significant number of viruses was shared between the two ecosystems, with a large proportion of bacteriophages transmitted from the mother to the infant through breastfeeding, which could contribute actively at modifying HMM bacterial composition . To dateHuman breast milk is considered the gold standard for infant nutrition. Beyond its nutritional benefits, breastfeeding is known to reduce respiratory and gastrointestinal infections in early life and decrease the risk of non-communicable diseases such as atopy, diabetes, obesity, and inflammatory bowel disease. As for preterm infants, exclusive HM feeding is also associated with improved neurodevelopmental outcomes and reduced risk of NEC ,43,44. Bifidobacterium [Bifidobacterium, Lactobacillus, Enterococcus, and Staphylococcus [Among bioactive factors, which could modulate clinical outcomes, HMOs and HMM appear to exert a synergic action in shaping the infant\u2019s GM ,13. HMOsacterium \u2014the mostacterium . Microbeacterium ,47. Indelococcus ,49,50. Ilococcus ,52: hostlococcus . It alsolococcus . Furtherlococcus . In earllococcus . Pretermlococcus . NEC doelococcus ,58. The lococcus ,60. A relococcus .Beyond the unique set of clinical factors that predispose preterm infants to gut dysbiosis, infant feeding has a huge impact in shaping the GM. Several studies have documented substantial differences in the composition of GM for preterm infants fed MOM, donor HM (DHM), and formula, with MOM leading to the highest microbial diversity compared to both DHM and formula , as wellLactobacillus and Bifidobacterium species isolated from HM appear to play a beneficial role in the infant\u2019s health [Lactobacillus or Bifidobacterium spp. to be used as probiotics. Some Lactobacillus and Bifidobacterium strains [Streptococcus and Staphylococcus spp. in the faeces of breast-fed infants, as well as in their mothers\u2019 milk microbiota, might call for a possible biological role for these bacteria during the infant\u2019s microbiota assembly [Thus, it can be speculated that both HM and HMM may act as early mediators between the development of GM in early life and health outcomes. s health . These b strains  have demassembly . Lactobacillus [Beyond the seeding effect on infant gut of HM beneficial bacteria, the presence of bacterial DNA, including DNA derived from dead bacterial cells, may also play a role in the infant immune development. Ward et al.  analysedbacillus . TherefoExclusive HM feeding is of utmost importance for preterm infants, as MOM-feeding is linked to a reduction in the incidence of life-threatening diseases, such as NEC and LOS, as well as to an improvement in preterm infants\u2019 neurodevelopment . HM feedThe beneficial role of HM in infants\u2019 feeding becomes even more crucial for preterm infants born with a very low birth weight , whose sBifidobacterium in the infants\u2019 faeces collected after actual breastfeeding had started. Taken together, these evidences suggest that latching, the act of suckling and/or skin-to-skin contact, may contribute to the protective effect of breastfeeding. Specifically, the infant\u2019s latching to the mother\u2019s breast might constitute an independent factor helping the health-promoting assembly of the infant gut microbiome, and thus an early initiation of breastfeeding should be encouraged and promoted among mothers of preterm infants.However, providing an exclusive HM diet to preterm infants presents a variety of challenges related to prematurity itself and to hospitalization. For preterm infants, the term \u201cexclusive HM feeding\u201d covers a range of feeding practices other than direct breastfeeding, such as the use of fresh vs. frozen expressed breast milk given by bottle or tube feeding, the addition of HM fortifiers, and a variable duration of exclusive HM feeding. Some of these interventions  might affect the nutritional and non-nutritional components of HM. The results of the CHILD study reported a reduced risk of wheezing, asthma, and high BMI among breastfed children . These aWhen MOM is unavailable or insufficient, the latest recommendations identify DHM, provided by a qualified HM bank, as the optimal alternative or supplement to MOM . In ordeEven if some analogies are documented between DHM and MOM in terms of the composition of GM in preterm infants, little is known about the specific effect of DHM on preterm GM and its potential functional implications. Some authors postulate that preterm infants fed DHM or formula are devoid of contact with HMM , and forNo specific evaluation of potential probiotic properties of DHM in its current form is available at present. It appears reasonable that, given the pasteurization process underwent by DHM, no viable beneficial bacteria would be retrieved in pasteurized DHM. However, according to recent evidence, the probiotic effect of beneficial microbes does not rely necessarily on their viability, but their interaction with the host might be based on the capacity of human cells to recognise specific bacterial components or products, giving rise to responses that commonly involve the mucosa-associated lymphoid tissue and, therefore, the immune system. For this reason, the term \u201cpara-probiotics\u201d or \u201cghost probiotics\u201d has been proposed for \u201cnon-viable (more often heat-inactivated) microbial cells (intact or broken) or crude cell extracts , which, when administered  in adequate amounts, confer a benefit on the human or animal consumer\u201d. Ghost probiotics might act through several mechanisms, including the modulation of various steps of the inflammatory and immune responses; given their potentially higher safety compared to standard viable microbial cells, a potential application for para-probiotics has been recently suggested also for preterm infants .In analogy with ghost probiotics , one can speculate that DHM processed through Holder Pasteurization (HoP), despite not retaining intact microbial cells, might harbour a ghost microbiota, whose clinical significance has not been yet investigated. The exact role of this putative \u201cghost microbiota\u201d in DHM still needs to be described.Further studies are required to evaluate in detail the characteristics and biological role of HMM. It is of paramount importance to first understand what constitutes a healthy reference for HMM, as well as what are the determinants of milk microbial community structure. Such increased knowledge would shed light on how this important source of microbes impacts on infant health, and how the process might be positively influenced by clinical practice in preterm infants. More targeted studies are warranted to define the mechanism by which the HMM impacts immune and gut development in the infant host. Indeed, it appears that this field of research, merging microbial ecology and clinical practice, has reached a point in which observational studies should be gradually replaced by mechanistic approaches, which would be able to provide a finer comprehension on microbes\u2013mother\u2013infant interaction during this crucial period of human life. Knowledge that goes beyond the community profile and starts to provide functional insights into HMM, as well as other non-viable probiotic components of HM, may provide new opportunities to improve health or modify disease outcomes."}
+{"text": "In Ethiopia, the burden of malaria during pregnancy remains a public health problem. Having a good malaria knowledge leads to practicing the prevention of malaria and seeking a health care. Researches regarding pregnant women\u2019s knowledge on malaria in Ethiopia is limited. So the aim of this study was to assess malaria knowledge and its associated factors among pregnant woman, 2018.An institutional-basedcross-sectional study was conducted in Adis Zemen Hospital. Data were collected using pre-tested, an interviewer-administered structured questionnaire among 236 mothers. Women\u2019s knowledge on malaria was measured using six malaria-related questions . The collected data were entered using Epidata version 3.1 and exported to SPSS version 20 for analysis. Bivariate and multivariate logistic regressions were computed to identify predictor variables at 95% confidence interval. Variables having P value of <0.05 were considered as predictor variables of malaria knowledge.A total of 235 pregnant women participated which makes the response rate 99.6%. One hundred seventy two pregnant women (73.2%) of mothers had good knowledge on malaria.Women who were from urban , had better family monthly income , attended education  were more knowledgeable.Majority of participants had good knowledge on malaria. Educational status, household monthly income and residence werepredictors of malaria knowledge. Increasing women\u2019s knowledge especially for those who are from rural, have no education, and have low monthly income is still needed. Malaria is a life-threatening disease caused by parasites that is transmitted to people through the bites of infected female Anopheles mosquitoes . Around Despite the incidence of malaria and deaths due to malaria from 1995 to 2015 decreased in Ethiopia, the morbidity and mortality of the population due to malaria is still a major health problem that needs to be solved timely . As malaAdditionally; malaria causes significant economic losses, and can decrease the gross domestic product (GDP) by as much as 1.3% in countries with high levels of transmission . In the Moreover; malaria infection during pregnancy remains a preventable cause of maternal mortality and morbidity globally, including in Ethiopia . AccordiEvery year in Sub-Saharan Africa there are about 25 million pregnancies which are at risk for malaria infection, 15. In Studies done in Sub-Saharan Africa including Ethiopia showed that women\u2019s knowledge regarding malaria remains low \u201323. StudCross-sectional studies were done in Ethiopia especially in Shahsango , Bonke , and TepDifferent articles revealed that knowledge of pregnant women on malaria is influenced by socio-demographic characteristics like education status, occupation, residence, ownership of television or radio, religion, ethnicity, age, and family monthly income\u201323.As there is a high burden of malaria, low knowledge of malaria and studies done in Ethiopia are limited; this study was conducted in Adis Zemen primary hospital to assess knowledge on malaria and its associated factors of among pregnant woman. So this study will be helpful in guiding policymakers and concerned bodies and will be used as baseline information for other investigators.o07\u201937o47\u2019E/12.117oN 37.783oE and an elevation of 1975 meters above sea level. The town is divided into three kebelles .This institutional-based cross-sectional study was carried out in Adis Zemen primary hospital from May1-30, 2018. Addis Zemen primary hospital is found in Adis Zemen town which is an administrative town of Libo Kemkem Wereda. Libo Kemkem Wereda is one of the wereda which found in South Gondar Zone of Amhara regional state. It is located90 kilometers far from Bahirdar  and it is 656 kilometers far from Addis Ababain the north direction. Addis zemen has a latitude and longitude of 12According to 2018 Adis Zemen town health statistics report, the estimated total population is 45, 125 of whom 22, 260 (49.3%) are men and 22, 865(50.7%) are women. The total number of women in the reproductive age group (15\u201349 years) is 14, 843 which accounts for 32.9% of the total town population. The town has one district hospital, one health center and two private clinics. Adis Zemen Hospital established in 2015 with a total of 91 staffs and currently, the hospital has a total of 236 staff.All pregnant women who attended antenatal clinics of the Adis Zemen Hospital were the source of population and all pregnant women who attended antenatal clinics of the Adis Zemen Hospital during the study period were the study population. All pregnant women who attended antenatal clinics of the Adis Zemen Hospital during the study period and who were voluntary to participate were included in the study whereas; all pregnant women who attended antenatal clinics of the Adis Zemen Hospital for the second time during the study period were excluded from the study.2p(1-p)//d2 where; n is the required sample size, Za/2 is the value of standard score at 95% confidence interval, p is the expected proportion of knowledge, and d2 is marginal error. And the following assumptions were used inorder to calculate the required sample size; 17.7% population proportion of malaria knowledge [th value was found to be 2.3 (539/236) and every 2nd woman was interviewed.The required sample size was calculated using single population proportion formula;n = (Z\u03b1/2)nowledge , 95% conFor the purpose of data collection, interviewer-administered questionnaire was adopted from differentliteratures. The questionnaire was prepared originally in English which had three parts like socio-demographic, and knowledge and utilization parts. The questionnaire was translated to the local language, Amharic for the purpose of data collection and it was translated back to English again for consistency. Before the actual data collection, pre-test was made on 5% of the total sample size of the respondent\u2019s in Addis Zemen health centre. The data were collected via face to face interview by two diploma holder midwives under the guidance of one BSc midwife supervisor before the women receiving the care in waiting room. Two days of training about data collection procedures and research ethics was given for data collectors and supervisors.The data collection process was closely supervised on a daily basis and prompt feedback was given timely. Regular manual check-up for completeness and consistency was made.The dependent variable of this study was women\u2019s knowledge (poor/good knowledge) and the independent variables of this study were socio-demographic characteristics like age, religion, ethnicity, residence, occupation, marital status, monthly income, educational status, and means of communication.Knowledge on malaria; was assessed by using 5 malaria knowledge related questions. Questions used to assess the knowledge were; 1) what is the causes of malaria?2) what are the sign and symptoms of malaria? 3) What is the mode of transmission of malaria? 4) what is the complication of malaria on pregnancy? 5) what are the prevention mechanism of malaria?. The first question  had only 1 correct answer wheras the rest had multiples answer. Each multiples answer which was correct were considered as one point and coded 1 whereas incorrect answers were coded 0. Finally women\u2019s knowledge on malaria was masured based on 22 points of 5 questions and dichotomized in to two;- Good Knowledge- those who scored more than 60% of correct response for Knowledge related questions [uestions .- Poor Knowledge\u2014those who scored less than 60% of correct response for Knowledge related questions [uestions .The collected data were coded and entered into epidata software version 3.1 and exported to SPSS V-20 for analysis. The collected data were presented by frequency and percentage using tables, bar and pie charts. Mean and standard deviation was computed for numerical variables. To see the association between dependent and independent variables, binary and multivariate logistic regressions were used at 95% confidence interval. To control confounding factors, variable having a P value of<0.25 in binary logistic regression were transferred into multivariate logistic regression. After controlling confoundings, variables which had a P value of<0.05 were treated as predictor variables of knowledge. The direction and strength of association were determined based on adjusted odds ratio.The ethical clearance of this study was approved by an instituttional review board of Debre Tabor University. Before data collection, informed verbal consent was obtained from every respondent. Participants were informed about the purpose of study and their full right not to be interviewed at all or at any time. Participants were also informed that there was no direct benefit they gain in participating in this research. Confidentiality of participants was ensured through by keeping the information confidential, not including address and name of the respondents.From a total of the required 236 respondents, two hundred thirty-five mothers participated which made the response rate 99.6%. One hundred thirty-two mothers (56.2%) were in the age group of 25\u201334. All of the respondents (100%) were Amhara and the mean age of the participants was 28.1 years (SD \u00b14.8 years). One hundred sixty-six mothers (70.6%) were from urban and most of the participants (95.3%) were married. More than three-fourths of participants (81.3%) were orthodox Christian and around one-sixth of participants (15.7) were governmental employee. One fourth 59(25.1%) of participants couldn\u2019t able to read and write. Most of participants 214 (91.1%) had at least one type of means of communication. Of them who had at least one type of communication, 211 (89.9%) of respondents had mobile Of all a total of 235 subjects, 217 (91.6%) of the participants mentioned fever as a symptom of malaria and headache was mentioned by 183 (81%) of women. All of participants 235 (100%) said malaria can be transmitted through mosquito biting whereas 8 (3.4%) women said that malaria can be transmitted through direct contact. Two hundred four (86.8%) of participants listed abortion as a complication of malaria on pregnancy whereas, 156 (66.4%) of women listed stillbirth. When women asked to list the prevention mechanism of malaria all of the participants (100%) listed using ITN whereas, aminority of participants (1.3%) listed taking medicine as prevention mechanism of malaria The and multivariate logistic regression. First eight variables were tested in binary logistic regression. Variables which had a P value of <0.25 were transferred to multivariate logistic regression to control the confounding variables. Educational status, income, and residence were significantly associated with women\u2019s knowledge on malaria. Women who were from urban were more knowledgeable than women who were from rural . Mothers who had family monthly income of 101\u2013150 US dollars were more knowledgeable than mothers had family monthly income of 50 US dollars or less . Participants who attended primary education were more knowledgeable than who could not able to read and write  In this hospital-based cross-sectional study, we assessed women\u2019s knowledge on malaria and associated factors among mothers attending antenatal clinics of Adis Zemen primary hospital and we found that 172 pregnant women (73.2%) had good knowledge on malaria. Assessing pregnant women\u2019s knowledge on malaria and associated factors is very helpful for policymakers and stakeholders in planning maternal and child health care services. The figure of this study is lower than studies done in Nigeria (83.9%) . This diThe finding of this study is comparable to studies done in Nigeria (71.5%)  and EthiAfter controlling confounding factors in multivariate logistic regression; educational status, monthly income, and residence were found to be significantly associated with women\u2019s knowledge on malaria.This finding revealed that living in the urban parts of the country increase the level of knowledge on malaria. Women who were from urban were 2.4 times more likely knowledgeable than women who were from rural. The association may be explained by women who are from urban may be more exposed for information like mass media and other health-related meeting than rural. This finding is supported by studies done in Nigeria  and BurkThis finding indicated that monthly income was positively associated with women\u2019s knowedge on malaria. Women who had family monthly income of 101\u2013150 US dollars were 3.4 times more likely knowledgeable than mothers who had family monthly income of 50 US dollars or less. Mothers who had family monthly income of 151or more US dollars were2.7 times more likely knowledgeable than mothers had family monthly income of 50 US dollars or less. This association may be due to the fact that mothers who have better income may easily access information regarding malaria. In addition to this, the association may be due to the fact that women who have better income may visit health institutions while they are sick and may get information regarding to malaria. This is similar to studies done in Nigeria  and EthiThe other most important variable which was significantly associated with knowledge on malaria was women\u2019s educational status. According to this study, educated women were more knwoledgeable than those who were not educated. Participants who attended primary education were 1.8 times more likely knowledgeable than who could not able to read and write respectively. Participants who attended college and above were 2.3 times more likely knowledgeable than who could not able to read and write.The finding of this study is similar to studies done inUganda , CamerooThe current study was conducted among mothers who had antenatal visit and there might be a limitation of knowledge difference between women who had one ANC visit and woman who had three or four ANC visit. Since this study was a hospital based study design, the true figure of women\u2019s knowledge on malaria might not be studied. Another limitation of this study was participants\u2019 feeling about preconception care was not studied.Pregnant women\u2019s knowledge on malaria is relatively high in Addis zemen hospital when it compares to most other similar studies. In this study, women\u2019s educational status, income, and residence were predictor variables knowledge on malaria.This study confirmed that having a high educational status, having better income, being from urban lead to having good knowledge. Despite women\u2019s knowledge is relatively high in Adis Zemen, increasing women\u2019s knowledge about malaria via health education especially for those who are from rural, have no education, and have low month incomeis needed.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file."}
+{"text": "Malaria is a life threatening disease caused by the Plasmodium parasite, transmitted through the bites of infected female anopheles' mosquitoes. According to the latest WHO data published in 2017, malaria deaths in Cameroon reached 9.161 deaths accounting for 4.14% of total deaths. The age adjusted death rate is 29.11 per 100,000 and Cameroon is ranked the 30th in the world with a high prevalence of malaria. The aim of this study was therefore, to access the knowledge of the modes of transmission and prevention of malaria among pregnant women attending Antenatal Clinic (ANC) at the Nkwen Health Center, Bamenda.This was a cross-sectional hospital based survey study. The researchers recruited 51 eligible women in the Nkwen Health Centre and used a validated and pre-tested questionnaires to collect data. Collected data were entered into Excel and analysed using descriptive statistics and the results presented in tables and figures.Sixty four percent of the women have basic knowledge about the mode of malaria transmission. Thirty six percent of the women had little knowledge about malaria transmission modes and the possible dangers of the disease.Slightly above 50% of pregnant women have basic knowledge on the modes of malaria transmission. Lack of knowledge regarding the modes of malaria transmission can be one of the reasons why there is still quite a high level of malaria prevalence among pregnant women attending ANC at the Nkwen Health Center, Bamenda. There is therefore, a need to educate women on malaria transmission modes. Plasmodium which can be transmitted by the bite of a female anopheles mosquito, congenitally or through exposure of infected blood products [Malaria is a mosquito-borne infectious disease affecting humans and other animals caused by parasitic protozoans (a group of single-celled microorganisms) belonging to the plasmodium type . Malariaproducts . Accordiproducts . The danproducts . Malariaproducts .In Cameroon, malaria is the leading cause of hospital visits and deaths with children and pregnant women being the most affected. Everyone in Cameroon is considered at high risk of contracting malaria . AccordiStudy design, study area and population: this study was a cross-sectional hospital-based survey carried out at the PMI Medicalized Health Center Nkwen in Mile 2 Bamenda which is found in the Northwest Region of Cameroon. The PMI Medicalized health center consists the following unit: emergency units, gynecological unit, medical and surgical unit, diabetic unit, and ANC and postnatal unit. This study was carried out specifically at the ANC clinic during the visiting hours.Inclusion criterion: this study involved all pregnant women who came for ANC at the Nkwen Health Centre, Bamenda during the study period (1-21 March 2018) and who gave their consent either in written or verbal forms.Exclusion criterion: the study excluded pregnant women who came for ANC but never gave their consent to participate.Sample size and Sampling: the sample size was exhaustive. A total of 78 pregnant women came for ANC during the study period (1-21 March 2018). However, out of these number, only 51 pregnant women gave their consent to take part in the study and were administered questionnaires.Administration questionnaires: questionnaires were administered among consented participants in the health center who agreed to participate in the study. The questionnaires captured data on socio-demographic characteristics, and level of the women's awareness on the malaria transmission modes.Data collection: this was a health center based survey where all participants who consented were interviewed using a structured questionnaire filled at the health center. Prior to the use of the questionnaires in the study by participants, a total of 5 questionnaires were pretested among pregnant women attending ANC at the Saint Louis Clinic in Bamenda with the aim of revising poorly structured questions, estimating the average time required to fill the questionnaire and by so doing validating the use of the questionnaire in study. It was estimated that, each questionnaire could be administered for 20-30 minutes after the pretest. A total of 51 questionnaires were administered to the pregnant women attending ANC at the the Nkwen Health Centre for a period of 3 weeks to assess their level of awareness and knowledge on malaria transmission modes. Knowledge on malaria transmission modes was tested through 8 questions. Each correct response was scored as 1 and 0 for a wrong response. The knowledge scores for an individual was calculated and summed up to give a total knowledge score on 8. A score between 0-3 was classified as poor, 4-6 as good and 6-8 as excellent for knowledge malaria transmission modes. This was adapted from a study conducted by Mazigo HD et al. [Data analyses: data were entered into Excel. The data were verified for completion and incomplete entries were deleted, cleaned and analysed. Descriptive analysis was carried out by calculating the mean and frequencies of different variables. Results were presented in the form of frequency tables and chartsEthical and administrative consideration: ethical clearance was obtained from the Institutional Review Board of the Cameroon Baptist Convention Health Services. Administrative clearance was obtained from the Regional Delegation of Public Health for North West Region Cameroon. Participants had the study protocol carefully explained to them and participation was voluntary. Written and verbal informed consent was obtained from all participants.Socio-demography characteristics of respondents: the majority of the women (88%) were between the ages 15-30 years (adolescents and young women), while 12% of the women fell between the ages 36-40 years old. The study also revealed that a greater percentage (64.7%) of the women were not married compared to others. The table also reveals that 58.2% of the women were within the 3rd trimester compared to the others who were within the 1st and 2nd trimester of their pregnancies. We can also deduce from the table that 29% of the women have had more than one pregnancy compared to the other 41.7% who were pregnant for their first time (rst time .Distribution of respondents by level of education: there was a greater percentage (52.92%) of the women who had had at least high school level of education as compared to the others (47.08%) who hadn't attained a high school level of education  have experience malaria at least once during their pregnancy as compared to a few (29.1%) who hadn't had malaria during their pregnancy  of the pregnant women know exactly the main mode of malaria transmission which is through mosquito bites. This also supports the findings of a similar study carried out in Buea Health District, Cameroon by Helen et al.  which stet al.  9 years et al.[The study also display that 70.8% of the pregnant women have had malaria at least once during their pregnancy which ties in with the fact that pregnant women are 3 times more likely to suffer from malaria infection and have a higher mortality rate as compared to their non-pregnant counterpart . Also a et al. in CamerLimitation of the study: the researchers faced some challenges during this research; the study had a small sample size and was a facility-based research. The limited scope didnt give room for extensive research and pronuncement. Furthermore, studying self-reported knowledge is itself a limitation because one cannot rely completely on the information provided by the participants because of recall and social desirability biases. Despite these shortcomings, this study provides relevant information in the context of malaria in pregnancy in the Nkwen Health Center, Bamenda.Despite concerted efforts and measures taken such as continuous education on malaria, distribution of LITNs and subsidized malaria treatment just to name a few, malaria continues to be one of the killing diseases even among pregnant women in the Nkwen neighborhood. There is need for continues sensitization as this will go a long way to reduce its prevalence since many people will be educated and will be more aware of their environment. This paper reveals that slightly above 50% of pregnant women have basic knowledge on the modes of malaria transmission. There is lack knowledge regarding malaria transmission modes which accounts for the continues high prevalence rate of malaria among pregnant women attending ANC at the Nkwen Health Center, Bamenda. There is therefore, a need to educate women on malaria transmission modes. This paper recommends further studies on malaria as it still remains the number one killer disease that claims more life in Cameroon and the world over. Also, these researchers challenge the state of Cameroon to embrace and take the education of the public on malaria prevention very serious. The government should adopt policies that will ensure clean environments to curb the spread of the diseases in the country.Malaria is more prevalent among pregnant women than their non-pregnant counterpart as a result of their weaken immune system;Malaria still remains the number one leading cause of death among pregnant women and children in the world;The dangers posed by malaria during pregnancy are not limited to the mother but have an adverse effects on the unborn baby which cannot by any means be taken for granted.The study reveals that slightly above 50% of pregnant women attending ANC at the Nkwen Health Center, Bamenda had basic knowledge on malaria. There is need for admonishment of the women on the dangers of malaria and how to avoid being infected;This study will serve as a reference document for government, policy makers and stakeholders in decision making;The article lays a path to guide them in the implementation of projects regarding the curbing of malaria in the country and region as a whole.The authors declare no competing interests."}
+{"text": "Adequate knowledge of malaria prevention and control can help in reducing the growing burden of malaria among vulnerable groups, particularly pregnant women and children aged under 5\u00a0years living in malaria endemic settings. Similar studies have been conducted but with less focus on these vulnerable groups. This study assessed knowledge of malaria prevention and control among the pregnant women and non-pregnant mothers of children aged under 5\u00a0years in Ibadan, Oyo State, South West Nigeria.In this cross sectional study, data on socio-demographic, clinical and knowledge on malaria prevention was collected using interviewer administered questionnaires from consenting study participants attending Adeoyo maternity hospital between May and November 2016. Data was described using percentages and compared across the two maternal groups in the study population. Knowledge scoring from collected data was computed using the variables on causes, symptoms and prevention of malaria and thereafter dichotomised. Multivariate analyses were used to assess the interactive effect of socio demographic and clinical characteristics with malaria knowledge. Level of statistical significance was set at p\u2009<\u20090.05.Of the 1373 women in the study, 59.6% (818) were pregnant women while 40.4% (555) were mothers of children aged under 5\u00a0years. The respondents mean age was 29\u00a0years\u2009\u00b1\u20095.2. A considerable proportion of both the pregnant women  and the non-pregnant mothers of children aged under 5\u00a0years  did not have correct knowledge on malaria prevention measures based on our assessment threshold (p\u2009<\u20090.001). Having a tertiary level education was associated with better knowledge on malaria . Multivariate analyses showed that marital status, educational attainment, gravidity, and HIV status were significantly associated with knowledge of malaria prevention and control.The findings indicate that socio-demographic factors such as marital and educational status greatly influence knowledge on malaria prevention and control measures. Key health stakeholders and authorities need to implement strategies and direct resources to improve the knowledge of mothers on malaria prevention and control. This would stem the tides of malaria related deaths among pregnant women and children aged under 5\u00a0years. Malaria is a major public health problem in ninety-one countries world-wide with sub-Sahara Africa bearing 80% of the disease burden . MalariaEvidence from malaria knowledge, attitudes, and practices (KAP) studies reported that misconceptions on malaria transmission and risk factors still exist with adverse impact on malaria control programmes , 12. FinPrior to data collection, ethical approval was obtained from the Oyo state ministry of health ethics committee (IRB AD13/479/1035) in Nigeria and from the biomedical research ethics committee (BREC- BE199/16) of the University of KwaZulu-Natal, South Africa. Study participants voluntarily signed written informed consent forms without any incentives. They consented because they believed their responses would contribute to increased effective control of malaria. The participants were also assured of confidentiality. The data collection tool was translated to both Yoruba, which is the dominant local language, and English language.Using a cross sectional study design, this survey was conducted between May and November 2016. The study recruitment site was the Adeoyo Maternity Hospital located in Ibadan North East-Oyo state, Nigeria. The elevation of the study area lies between 64 and 414\u00a0mm  status, gravidity status, blood group; and questions assessing the participants\u2019 awareness and extent of knowledge on malaria symptoms, prevention and management.Overall knowledge score was computed by aggregating the knowledge related variables (1) awareness of malaria (2) knowledge of cause of malaria (3) knowledge of breeding sites for mosquito (4) knowledge of three or more symptoms of malaria (5) knowledge of when malaria mosquito feeds (correct knowledge when at night), and (6) knowledge of malaria prevention knowledge  and environmental sanitation). The knowledge variables were recoded to binary level such that respondents with correct option in the knowledge variables were coded 1 while not having correct knowledge was coded 0. Knowledge score was computed as the sum of the six knowledge variables, with 0 as the least possible score and 6 as highest possible score. Increasing score indicated better malaria knowledge. Subsequently, the median of the composite score was used as the cut-off to classify knowledge level as either poor or good. Individuals who scored less than the median of knowledge score were categorized as having poor knowledge while scoring within the exact median cut off and above were classified as having good malaria knowledge.Categorical variables were presented as numbers and percentages; numerical variables were presented as means and standard deviation to describe the study population by their socio demographic and clinical characteristics. To assess the level of relationship and interaction between malaria knowledge score and the respondents\u2019 socio demographic and clinical characteristics, analytical statistics involving Chi square and analysis of variance was carried out. Multivariate linear analysis was further performed to determine predictors of malaria knowledge. Level of statistical significance was set at p\u2009<\u20090.05. Analyses were performed using Statistical Package for the Social Sciences software (SPSS) version 25, Chicago, IL.Table\u00a0With regards to the clinical characteristics of respondents, about a third of the pregnant women were primegravida (33.6%) while the rest were multigravidae (66.4%). There were about 1.5% of pregnant women and 1.4% among the non-pregnant mothers of children aged under 5\u00a0years who self-reported that they HIV positive. Also, 24.8% and 18.9% of the pregnant women and non-pregnant mothers of children aged under 5\u00a0years did not know their HIV sero-status, respectively. With regards to the blood group of the respondents blood group \u2018AB\u2019 was less common . Conversely, the predominant genotype was \u2018AA\u2019 (pregnant women: 70.4% vs non-pregnant mothers of children aged under 5\u00a0years: 65.9%) followed by \u2018AS\u2019 (pregnant women: 23.3% vs non-pregnant mothers of children aged under 5\u00a0years: 22%), \u2018AC\u2019 (pregnant women: 5% vs non-pregnant mothers of children aged under 5\u00a0years: 8.8%) and \u2018SS\u2019 (pregnant women: 1.2% vs non-pregnant mothers of children aged under 5\u00a0years: 3.2%).Table\u00a0There was no significant difference in malaria knowledge score between pregnant women and non-pregnant mothers of children aged under 5\u00a0years in the study Table\u00a0. There wTable\u00a0In the multivariate linear regression analysis to examine the predictors of malaria knowledge, socio-demographic factors including marital status, education, gravidity status and the clinical factor HIV status remained significant with malaria knowledge Table\u00a0.Table\u00a05MNigeria contributes the highest morbidity and mortality rates to the global burden of malaria, accounting for 25% of the global malaria cases and about 24% of global malaria-related deaths . Thus, tIn relation to the knowledge on malaria symptoms and preventive measures by respondents in this study, about 60% of pregnant women and 46% of non-pregnant mothers of young children did not have correct knowledge on malaria prevention. Further, there were 26% of pregnant mothers and 31% of the non-pregnant mothers of young children who correctly reported more than 3 clinical symptoms of malaria. Similar studies conducted in rural South West Nigeria , North CLevel of knowledge on malaria was associated with; socio-demographic factors such as marital status, education and clinical factors like gravidity and HIV status of the mothers. Good malaria knowledge was associated with higher level of educational status of the women. In previous studies, educational status has been linked with good health awareness and health-seeking behaviour for the child , 25, andAlthough the study investigated the knowledge of malaria prevention and control, and sought to find the socio-demographic and some clinical factors associated with malaria knowledge this study did not investigate the programmatic factors that may influence the knowledge of the respondents on malaria and would like to recommend this for future studies. Limitations of this study include recall bias on account of information provided by the respondents. Since the study population was hospital-based, another bias related to the limitation of this study is selection bias because this hospital based study population could have been more knowledgeable than similar population if recruited from the community. Though these limitations, this study has implications for control programmes given the findings, which highlights the knowledge gaps requiring urgent interventions targeted at mothers.This study has demonstrated that pregnant women and mothers of children under 5\u00a0years are aware of malaria, but still lack comprehensive knowledge about the disease. Many mothers know some important symptoms of malaria such as fever, cold and headache. There was also some level of misconception about malaria, which needs to be totally debunked by intensifying education about malaria among mothers who are either pregnant and or caring for young ones who are more vulnerable to malaria disease. Education as a socio-demographic factor was an important predictor knowledge of malaria among mothers and so government policies should be geared towards improving citizens \u2018educational statuses in order to reduce the burden of the disease in the country, especially among the most vulnerable population. Mothers need to be educated about the importance of a better health-seeking behaviour and awareness about their health status. Nigeria\u2019s malaria strategic plan should to ensure that the knowledge cleft on malaria prevention and treatment needs to be addressed. This insight will help the policy makers to implement continuous strategic intervention including health awareness and educational programs to attain 2030 malaria goals."}
+{"text": "Aim: The aim of this study was to analyze the expressed profiles of miRNAs in plasma, platelets, and platelet-derived microvesicles (PMVs) obtained from experimental induced atherosclerosis animal model and to investigate the effect of EPC transplantation on these profiles.Methods: Seventeen selected circulating miRNAs  were individually analyzed in plasma, platelets, and PMVs isolated from peripheral blood of hypertensive-hyperlipidemic hamsters treated or not with endothelial progenitor cells (EPCs), and of healthy hamsters taken as control group.Results: Comparative with control group, in hypertension associated with hyperlipidemia the investigated miRNA expression profiles were different: (i) in plasma, the levels of all investigated miRNAs were significantly increased, the highest enhances being noticed for miR-21,-146a,-221,-143,-34a, and miR-204; (ii) in platelets, the expressions of almost all miRNAs were significantly elevated, remarkable for miR-126,-146a,-223,-222, and miR-214, while levels of miR-143, miR-10a, and miR-145 were significantly reduced; (iii) in PMVs, numerous miRNAs were found to have significantly increased levels, especially miR-222,-221,-210, and miR-34a, whereas expressions of various miRNAs as miR-223,-214,-146a,-143,-10a, and miR-145 were significantly decreased. The treatment with EPCs had the following reverse effects: (i) in plasma, significantly reduced the expression of miR-26b,-143,-34a,-204, and miR-214; (ii) in platelets, significantly decreased the levels of almost investigated miRNAs, with remarkably diminishing for miR-126 and miR-221; and (iii) in PMVs, significantly lowered the expression of some miRNAs, with considerably reductions for miR-222,-221,-210, and miR-19a, while the level of miR-214 was found elevated.Conclusions: The present study revealed that miRNAs have differential expression profiles in plasma, platelets, and PMVs under hypertension associated with hyperlipidemia conditions. The different miRNA profile in PMVs compared with platelets indicated an active mechanism of selective packing of miRNAs into PMVs from maternal cells; various miRNAs such as miR-19a,-21,-126,-26b,-92a,-155,-204,-210,-221,-222, and\u221234a delivered by PMVs may contribute to enrichment of circulating plasma miRNA expression. In addition, our study showed that the EPC-based therapy can regulate the expressions of investigated miRNAs into the three sources. These results provide novel information that could help in finding potential targets for the development of new therapeutic strategies in the cardiovascular disease. Atherosclerosis is a chronic immune-inflammatory disorder that integrates multiple cell types and a diverse set of inflammatory mediators . SeveralThe discovery of miRNAs and their role in the regulation of gene expression in humans is one of the most exciting scientific discoveries in the last years. The miRNAs are highly conserved, single-stranded non-coding RNA molecules, with 22 nt in length, that exert post-transcriptional effects on gene expression by promoting the degradation of mRNA target and/or inhibiting mRNA translation. Gene regulation modulated by miRNAs is involved in almost biological processes in mammals and has important roles in health and disease states . MoreoveMostly miRNAs in the circulation have been shown associated with microparticles, exosomes, lipoproteins, or protein complexes , 9. Micrde novo, platelets contain mRNA, YRNA fragments and premature miRNAs that were inherited from megakaryocytes. Some miRNAs, such as miR-126 and miR-223, have an important role in regulating platelet reactivity. In this sense, it has been demonstrated that miR-223 is involved in aggregation and thrombus formation, targeting the P2Y12, a purinergic receptor known to intensify aggregation induced by all known platelet agonists , can activate platelets, that further can interact with ECs of inflamed arteries and stimulate generation of pro-inflammatory PMVs . Althougagonists , 21. Morlatelets .Endothelial progenitor cells (EPCs) have generated a significant attention as potential novel diagnostic/prognostic biomarkers for vascular integrity and also for use in therapeutic clinical approaches . FurtherThe research in platelets and PMV field has grown in recent years, and in the near future due to the technologies arrive at the horizon will probable advance and will take a new position in therapy of cardiovascular disease. Consequently, the aim of this study was to analyze the differentially expressed profiles of miRNAs in plasma, platelets, and PMVs obtained from experimental induced atherosclerosis animal model and to investigate the effect of EPC transplantation on these profiles.As our purpose was to investigate the miRNA profiles with key role in the CVD development in plasma, platelets, and PMVs obtained in peripheral blood collected from hypertensive-hyperlipidemic hamster , we individually quantified several miRNAs employing hybridization probes (miRNA TaqMan assays) in each investigated sample. Furthermore, we explored the changes induced by EPC transplantation on profiles of these miRNAs in plasma, platelets, and PMVs from HH group injected with EPCs (HH-EPCs group). The experimental animal models were characterized previously by our group in different papers, illustrating the plasma profile for glucose, cholesterol, triglyceride concentrations, and also the blood pressure and heart rate \u201331. MoreFirst of all, the 17 selected circulating miRNAs  were individually quantified in plasma obtained from each animal in HH and HH-EPCs groups and compared to those in plasma collected from healthy hamsters taken as control group (C group).*p \u2264 0.05, #p \u2264 0.05, Results showed that almost all selected miRNAs had levels significantly increased in the plasma of HH hamsters compared to those quantified in plasma collected from hamsters in C group  in the blood . ConsequIn view of these findings, we selected a subset of 17 miRNAs related to CVD  and we analyzed their distribution in plasma, platelets, and PMVs isolated from atherosclerotic animal model compared to control group, and also we investigated the effect of EPCs treatment on these profiles.In our study, we showed that in plasma from hypertensive-hyperlipidemic hamsters, all investigated miRNAs were augmented compared to values obtained in healthy animals, and the highest levels were noticed for miR-21, miR-146a, miR-221, miR-143, miR-34a, miR-204. These results are in agreement with several studies that indicated significant increases in levels of: (i) miR-21 and miR-210 in serum samples from patients with peripheral arterial disease, specifically atherosclerosis obliterans ; (ii) miIn order to explore whether circulating plasma miRNAs reported as biomarkers for CVD could be associated with miRNA carried by microvesicles released from activated platelets we evaluated the same miRNA profile in plasma and also in PMVs and platelets, their maternal cells. Our findings showed that PMVs isolated from hypertensive-hyperlipidemic hamsters contain increased levels of miR-19a,-21,-126,-26b,-92a,-155,-204, and a remarkably augmentation for miR-222,-221,-210, and-34a. The same miRNAs have been found elevated also in platelets from hypertensive-hyperlipidemic hamsters. Notable, rise of miR-126 expression was much higher in platelets than in PMVs. Thus, PMVs by their miRNA content may contribute to the enhance of circulating miRNA expression in plasma of experimental induced atherosclerosis model, mainly of miR-19a,-21,-126,-26b,-92a,-155,-204,-210,-221,-222, and-34a levels. Our results are supported by other studies from literature. Therefore, it has been shown that levels for miR-92a, miR-21, and miR-126 in MVs were augmented in patients with CAD, suggesting that MVs may contribute to the regulation of circulating miRNA expression . MoreoveMoreover, expressions of miR-143,-10a, and-145 were reduced both in PMVs and platelets comparative with sample from control hamsters suggesting that perhaps PMVs do not have an influence on these miRNA levels in plasma of hypertensive-hyperlipidemic animals. Interestingly, although the expressions of miRNA-146a,-223, and-214 were significantly greater in platelets, in PMVs were decreased. The differences in the miRNA profile in platelets and PMVs indicate that platelets may have a selective mechanism to packing miRNAs into PMVs. Furthermore, there results indicated that at high levels of circulating miRNA-146a,-223, and-214 in hypertension associated with hyperlipidemia conditions may contribute other circulating cells, but not platelets. In literature, was reported that miR-223, as well miR-126, are highly expressed in platelets in patients with CVD .In our previous study we demonstrated that transplantation of EPCs in the experimental induced atherosclerosis animal model reduced platelet activation, modulated their pro-inflammatory and antithrombogenic properties  and had In conclusion, our findings bring new information concerning PMV involvement in the regulation of the miRNA profile in plasma, in hypertension associated with hyperlipidemia. Numerous miRNAs, such as miR-19a,-21,-126,-26b,-92a,-155,-204,-210,-221,-222, and-34a delivered by PMVs may contribute to augmentation of circulating plasma miRNA expression. Additionally, our present data have shown that the EPC-based therapy modified the expressions of these miRNAs in the three compartments. The miRNAs contained by PMVs can be internalized by the different recipient cells involved in atherosclerosis development, e.g., macrophages, ECs, leukocytes, vascular SMCs, and hepotacytes, modulating the gene expression and regulating their functions. Although, further studies are needed to elucidate the exact role of specific platelet miRNAs in vascular biology and atherosclerosis progress and involved signaling pathways, our data bring answers which may help to develop the forthcoming therapeutic applications for cardiovascular disease.n = 30) divided in three equal groups: (1) C (control hamsters), age-matched normal healthy animals which received an ordinary hamster diet; (2) HH (simultaneously hypertensive-hyperlipidemic hamsters), fed with ordinary diet enriched with 3% cholesterol, 15% butter and 8% NaCl, for 4 months; (3) HH-EPCs (HH hamsters treated with EPCs), animals that received 1 x 105 EPCs (isolated from C group) in a volume of 300 \u03bcl, in a dose per month, via the retro-orbital venous sinus injection, during 4 months of diet specific to the HH hamster, to prevent atherosclerosis process; , using the specific antibodies for VEGF-R2 (KDR or Flk-1) and CD34. The CD34 and VEGF-R2 are markers for late or mature EPCs, that are found more than early EPCs , in the peripheral circulation of adults , platelets and PMVs isolated from peripheral blood of C, HH, and HH-EPCs hamsters were measured by miRNA TaqMan assay.First of all, total RNA from plasma was obtained using miRNeasy Serum/Plasma Kit , while total RNA of platelets and PMVs was isolated using miRNeasy Micro Kit  according to the manufacturer's guidelines. In order to isolate plasma circulating miRNAs, the blood samples were centrifuged at 1,900 x g , 10 min at 4\u00b0C to remove blood cells and platelets from plasma, and the resulting supernatant was centrifuged at 16,000 x g, 10 min at 4\u00b0C to remove additional cellular nucleic acids attached to cell debris. The latter centrifugation step significantly reduced the amount of cellular or genomic DNA and RNA in samples. At the end of the procedure, the RNAs were eluted from the silica columns with 15 \u03bcl of RNase-free water, and after concentration measurement using the NanoDrop 2000c spectrophotometer, were kept at \u221280\u00b0C until examinations. In the next step, the reverse-transcription was achieved using TaqMan MicroRNA ReverseTranscription Kit in combination with TaqMan-Gene Expression Master Mix and miRNA specific stem-loop primers of miRNA TaqMan Assays on a Veriti real-time PCR system . The miRNA specific stem-loop primers used were hsa-miR-19a-3p (ID:000395), hsa-miR-21-5p (ID: 000397), hsa-miR-126-3p (ID:002228), hsa-miR-146a-5p (ID:000468), hsa-miR-223-3p (ID: 002295), hsa-miR- 26b-5p (ID: 000407), hsa-miR-92a-3p (ID:000431), hsa-miR-222-3p (ID: 002276), hsa-miR-221-3p (ID:000524), hsa-miR-143-3p (ID:002249), hsa-miR-10a-5p (ID:000387), hsa-miR-145-5p (ID:002278), hsa-miR-155-5p (ID: 002623), hsa-miR-204-5p (ID:000508), hsa-miR-210-3p (ID: 000512), hsa-miR-34a-5p (ID:000426), hsa-miR-214-3p (ID: 002306).\u2212\u0394\u0394Cq) values represent the normalized miRNA expression (2\u2212\u0394Cq) in the test sample divided to the normalized miRNA expression (2\u2212\u0394Cq) in the C sample. Normalized expression of a specific human miRNA is given relative to that of exogenously added cel-miR-39 (\u0394Cq = Cq hsa-miRNA\u2013Cq cel-miR-39). In order to obtain a distribution of positive values, data were expressed as log-transformed values of multiplied individual 2\u2212\u0394Cq values.The VIIA7 Software v1.2 (Applied Biosystems) with the automatic quantification cycle (Cq) setting was used to analyze the data. MiRNAs levels in plasma isolated from HH and HH-EPC hamsters were expressed relative to those obtained in C plasma with normalization to an endogenous control . The miRNAs with Cq values over 35 were omitted from further analysis. Fold change values of circulating miRNAs analyzed in the pooled plasma of HH and HH-EPC hamsters were expressed relatively to plasma C group. Fold change (2\u2212\u0394\u0394Cq (\u0394Cq = Cq hsa-miRNA - Cq U6)  and quantified as 2- Cq U6) . The Cq P \u2264 0.05 values.For the quantification and comparison of the data, One-Way ANOVA method and Bartlett's test in GraphPad Prism 7.02 programme were applied. The statistically significant differences between the groups were calculated, and represented as All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by Ethics Committees from Institute of Cellular Biology and Pathology Nicolae Simionescu.NA, AC, and AG contributed to conception and design. NA, AC, MN, IC, AV, and AP performed the experiments. NA, AC, MN, GT, and AG contributed to data analysis, and interpretation. NA, AC, and AG wrote the paper. AG provided a critical revision of the manuscript for important intellectual content. NA, AC, MN, IC, AV, AP, GT, and AG agrees to be accountable for all aspects of work ensuring integrity and accuracy.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": "Scientific Reports 10.1038/s41598-018-25119-y, published online 27 April 2018Correction to: A supplementary file, \u201cExample of a dual-task trial for the Feedback group\u201d was omitted from the original version of this Article. This has been corrected in the HTML version of the Article; the PDF version was correct at time of publication."}
+{"text": "The clinical use of cytotoxic agents is plagued by systemic toxicity. We report a novel approach that seeks to design a \u201ccombi-molecule\u201d to behave as an alkylating agent on its own and to undergo acid-catalyzed conversion to two bioactive species at a pH range akin to that of a tumor microenvironment: an AL530 prototype was synthesized and we studied its ability to release a chlorambucil analogue (CBL-A) plus a potent mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor (PD98059) at different pHs in buffered solutions, plasma and tumors. Its potency was compared in vitro with CBL+PD98059 (SRB assay) and in vivo in a xenograft model. Its target modulation was studied by western blotting and immunohistochemistry. AL530 released PD98059+CBL-A at mild acidic pH and in vitro was fivefold more potent than CBL and three-to-fivefold more potent than CBL+PD98059. In vivo it released high levels of PD98059 in tumors with a tumor/plasma ratio of five. It induced \u03b3-H2AX phosphorylation and blocked pErk1,2, indirectly indicating its ability to damage DNA and modulate MEK. It induced significant tumor delay and less toxicity at unachievable doses for CBL and CBL+PD98059. We demonstrated the feasibility of a pH-labile combi-molecule capable of delivering high MEK inhibitor concentration in tumors, damaging DNA therein, and inducing tumor growth delay. In advanced stages, the clinical management of solid tumors is primarily based on the combination of potent cytotoxic agents. The therapeutic activity of these regimens is often hampered by severe toxicity and has acquired resistance in refractory tumors. In order to improve the therapeutic index, efforts are now directed at combinations of cytotoxic agents with targeted therapy. In this context, over the past decade, we developed a novel approach termed \u201ccombi-targeting\u201d that seeks to design molecules termed \u201ccombi-molecules\u201d to block receptor tyrosine kinase-mediated signaling while carrying a cytotoxic DNA-damaging moiety ,4,5,6,7.The strategy to target MEK and DNA in this combi-molecule was inspired by common knowledge that tumor microenvironment and stromal tissues drive the growth of solid tumors, predominantly through the secretion of growth factors. When tumors overexpress receptors responding to these growth factors, their progression is accelerated by both uncontrolled proliferation and invasion. Signaling mediated by these receptors converges toward the activation of MEK, which is at the cross-roads of the Ras-mitogen-activated protein kinase (MAPK) and stress response pathways. Activation of many growth factor receptors  leads to gene expression through the MAP kinase pathway in which the protein kinases MEK1,2 (henceforth referred to as MEK) play a major role . Along wThe drug design approach described herein is also influenced by the hypoxic conditions of the tumor microenvironment. Indeed, in advanced stages, tumors contain hypoxic areas and undergo hypermetabolism that increases acid production, thereby lowering the pH of the microenvironment ,15,16. W8S and the aniline mustard 12S are outlined in 8S under basic conditions to produce 1, which was deprotected under basic conditions to give acid 2. Coupling of acid 2 with the 4-amino aniline mustard 12S gave 3, the nitro group of which was catalytically reduced to give AL530 in good yield. Various observations showed that AL530 to be acid- and temperature-sensitive. Therefore, we sought to verify whether its hydrolysis could occur at a pH range akin to that of the tumor microenvironment. To ascertain the kinetics of its stability under acidic and variable temperatures, a rigorous pH and variable temperature study was performed as follows.The approach we chose to study was to design a DNA-damaging aniline mustard with a masked MEK inhibitor. The latter was \u201cprogramed\u201d to be released under the mild acidity of the tumor microenvironment, thereby locally sensitizing tumors to the cytotoxic effects of the released CBL-A. Our molecular design was inspired by previous work reported by Atwell et al.  who, in To study the pH and temperature influence on the kinetics of AL530 hydrolysis into its two drug components, we used a LC\u2013UV instrument to determine the rate of formation (k) of PD98059 under these different conditions: pH ranging from 5.7 to 7 and temperatures of 37 to 42 \u00b0C. As shown in As depicted in The interest in using higher temperatures than physiological conditions (37 \u00b0C) was driven by various housekeeping works and medical care in our institution using hyperthermic intraperitoneal chemotherapies ,21. IndeIn order to establish that AL530 can inhibit cell growth, a panel of solid tumor cell lines of different origins  and at a relatively high concentration of AL530 (25 \u00b5M). This 25 \u00b5M dose was selected to maximize the probability of releasing free PD98059. As shown in The observed in vitro cellular and molecular mechanisms of action of AL530 strongly suggest that it possesses all the characteristics required to become the lead prototype of the approach under study. While these characteristics were evidenced in vitro, the pharmacokinetics and pharmacodynamics study of AL530 in vivo remained the ultimate test for the feasibility of the approach. Prior to starting a human xenograft model, a preliminary in vivo biodistribution study was conducted in the 4T1 breast syngeneic mouse model that allows to form large tumors in vivo in a very short time. In a second instance, we selected from the panel of cancer cell lines of the study, the human CAL33 head and neck cancer cells to perform a xenograft model, on the basis of its aggressive growth in vivo.4T1 cell (murine)-bearing mice were given AL530 i.p., and plasma and tumors were collected after 3 h and analyzed by HPLC. As depicted in 0\u201324h-PD98059 (tumor)/AUC0\u201324h (plasma) ratio was consistent with an approximately fivefold more abundant release of PD98059 from AL530 in the tumors than in the plasma. Overall the tumor/plasma ratio of intact AL530 was 1.6 and that for PD98059, the ratio was 5, which is again consistent with a preferential tumor distribution of AL530 and its derived primary metabolite PD98059.To further ascertain these results, a full pharmacokinetics study involving analyses at various time points was performed with CAL33-bearing mice. The results, which are summarized in In vitro assays showed that AL530 was capable of blocking MEK after a long exposure time. Given the preferential tumor distribution of AL530 and the higher concentration of PD98059 within tumors, we analyzed its ability to modulate its two targets in vivo: DNA for CBL-A or intact AL530 and MEK for PD98059. This was studied using \u03b3-H2AX A immunosThe dose tolerance study of AL530 was based on known values for CBL, used as a positive control. While CBL showed signs of acute toxicity at 11 mg/kg both as a single drug or in combination with PD98059, AL530 could be tolerated at 25 and 50 mg/kg . Higher p < 0.01 and p < 0.0001, respectively). By contrast, combinations of CBL with PD98059 or CBL alone did not show significant tumor delay in the model. Strikingly, AL530 showed a better tolerance in vivo than CBL and its combination with PD98059. The combi-molecule was tolerated at 25 and 50 mg/kg, two doses that were not achievable by CBL alone or in combination. At doses as low as 11 mg/kg, more than 15% weight loss was observed in the animals 13 days after treatment (B).The in vivo efficacy A,C and tOver the past two decades, resistance to DNA alkylating drugs has been primarily associated with DNA repair enzyme levels and activity. Advances in molecular biology led to the elucidation of the role of upstream cell signaling in the modulation of DNA repair activity. This progress inspired therapeutic interventions directed at receptor- or stress-mediated signaling to sensitize refractory tumors to DNA interactive drugs and radiation. In previous work, we and others ,22, showAL530 is a combi-molecule with three compartments: (a) one masking the release of the kinase inhibitor, (b) another containing the anchimeric assistance engine designed to release the masked inhibitor under controlled conditions and (c) an aniline mustard carrier that is a precursor of another molecule (CBL-A) targeted to DNA. Thus, AL530 is a novel, first-in-class aniline mustard \u201cprogramed\u201d to generate an inhibitor of MEK, or more simply an aniline mustard designed to be a MEK inhibitor strictly under hydrolytic conditions favored by the tumor microenvironment. Following its successful design and synthesis, we sought to demonstrate first that it was indeed a prodrug of PD98059.Our results unequivocally demonstrated that AL530 was capable of generating PD98059 plus a mustard-carrying metabolite in a pH- and temperature-dependent fashion. The pH rate profile suggests that protonation of the tetrahedral intermediate can be proposed at the rate determining step of the cleavage process. The results show that AL530 behaves as a prodrug of PD98059 at physiological pH and temperature ranges. Indeed, previous reports confirmed using specific probes that the pH of the microenvironment could be as low as 5.5 ,15,16.The study of the temperature-dependent cleavage of AL530 was performed in vitro. The purpose of the evaluation of temperature superior to 37 \u00b0C (40 and 42 \u00b0C) was to investigate whether this molecule could also be used for thermotherapy. Currently, hyperthermic intraperitoneal chemotherapies (HIPECs) are used in the clinical management of ovarian cancer. It consists of directly delivering highly concentrated and stably heated platinum-based chemotherapy treatments at 42 \u00b0C to the abdomen of the patient ,21. As sAL530, being an aniline mustard, means that its growth inhibitory potency was compared to that of the clinical analogue CBL under standard conditions in a monolayer culture. Moreover, continuous exposure studies showed AL530 to be fivefold more potent than CBL as well as two-to-threefold more potent than equimolar combinations of CBL + PD98059. This indicated that perhaps AL530 exerted its antitumor activities by a mechanism different from that of CBL alone. Given that, in the cell culture medium, the rate of hydrolysis of AL530 was found to be significantly slower (data not shown), the molecule probably owes its superior activity to a faster cleavage in the intracellular milieu. This concurs with the dissection of its mechanism of action that showed its ability to block Erk1,2 phosphorylation in CAL33 cells under a four-day continuous exposure assay. These observations were prima facie evidence that the hydrolysis of AL530 was milieu-dependent, thus warranting further investigation in vivo.Given the dependence of AL530 cleavage on pH, plasma and tumor pharmacokinetics were the most appropriate models for studying its mechanism of action. The first striking observation was that the pharmacological half-life for AL530 was twice as long in tumors than in plasma. Interestingly, it was four times longer for PD98059 in tumor tissues, suggesting that the inhibition of MEK could be sustained even after AL530 has been cleared from the tumors. All pharmacokinetic parameters were remarkably consistent with a preferential distribution of AL530 and PD98059 in the tumors. The areas under the curves (AUCs), which represent exposure at all time points showed a tumor/plasma ratio, respectively, of 1.6 for AL530 and of 5 for PD98059. Since this was consistent with strong tumor distribution, we dissected the mechanism of action of AL530 in immunocompromised mice 13 days after drug administration. High staining for \u03b3-H2AX suggested the presence of DNA damage, and decreased staining for pErk1,2 indicated the inhibition of phosphorylation in vivo. Taken together, the results were consistent with the in vitro observation and further support the feasibility of developing a molecule capable of massively accumulating in the tumor tissue and releasing its bioactive kinase inhibitor therein, despite its large size (MW = 6872 g/mol).One of the premises of the combi-targeting concept is that by imprinting multiple mechanisms of action within a single molecule, the pharmacotoxicology of multiple mechanisms associated with multiple drugs will be reduced to that of a single agent. This postulate is supported by the results obtained herein, which showed AL530 to be much better tolerated than CBL alone or in combination with PD98059. A maximum tolerated dose could not be defined due to the insolubility of the drug at high doses. Nevertheless, significant tumor delay was observed at 25 and 50 mg/kg, indicating that further strategies for enhancing solubility may lead to the full-blown potency of the approach in vivo.1H NMR spectra were recorded on a Varian 300 or 400 MHz spectrometer. Chemical shifts are given as \u03b4 values in parts per million (ppm) and are referenced to the residual solvent proton peak. Mass spectrometry was also performed on a Finnigan LC QDUO spectrometer by the McGill University Mass Spectroscopy Center using electrospray ionization (ESI) mode. Data are reported as m/z (intensity relative to base peak = 100). All chemicals were purchased from Sigma\u2013Aldrich  or Alfa Aesar . The compound SL02548 was purchased from Sinova .2 (6 mL) for 1 h (compound 8S), the mixture was then evaporated and thoroughly vacuum-dried to obtain a brown solid. PD98059  was dissolved in dry CH2Cl2 (200 mL) with Et3N  at 0 \u00b0C, and the acyl chloride formed was dissolved in THF (130 mL) and added dropwise to the cold solution via a canula. The reaction mixture was heated at 50 \u00b0C under argon for three days. It was then evaporated and the resulting crude material was resuspended in CH2Cl2 and extracted with brine. The organic layer was dried over magnesium sulphate, filtered and evaporated. The orange crude product was purified by silica gel chromatography (CH2Cl2 100% to CH2Cl2/MeOH 8/2) to give compound  1.25 , 1.74 , 3.87 , 3.97 , 6.39 , 6.70 , 7.37 , 7.50 , 7.64 , 7.64 , 7.84 , 7.95 , 8.06 , 8.22 , 8.29 .The 2-(5-(Methoxycarbonyl)-2-nitrophenyl)-2-methylpropanoic acid linker, compound 7S , was refluxed in neat SOClcompound  as a pur1  was hydrolyzed in MeOH (50 mL) with an aqueous solution of LiOH 2N (25 mL) added dropwise. The mixture was stirred at room temperature during 18 h and the reaction mixture was then acidified with an aqueous solution of HCl 1N to pH = 2. The precipitate formed was extracted with ethyl acetate; the organic layer was washed with water and brine, dried over magnesium sulphate, filtered and evaporated. The yellow solid, not further purified, gave compound 2 .1H NMR  1.24 , 1.67 , 3.76 , 6.89 , 7.02 , 7.29 , 7.45 , 7.58 , 7.72 , 7.98 , 8.15 , 8.27 , 8.42 .The ester 2  in dry N,N-dimethylformamide(DMF)/acetonitrile (ACN) (3/4 mL) was treated with 1,l\u2019-carbonyldiimidale  at room temperature for 30 min and then cooled to 0 \u00b0C and treated with a solution of aniline mustard 12S  in ACN (7 mL) with Et3N . The reaction mixture was warmed to room temperature and stirred under argon. After 18 h, the DMF was azeotroped with heptane, and the dark oil obtained was resuspended in ethyl acetate and extracted with water. The organic layer was dried over magnesium sulfate, filtered and evaporated. The dark orange oil obtained was purified by silica gel chromatography (CH2Cl2/EtOAc 8/2 to 7/3) to give compound 3 in the form of a pure orange powder . 1H NMR  1.63 , 3.63 , 3.72 , 3.86 , 6.43 , 6.62 , 7.04 , 7.19 , 7.27 , 7.36 , 7.42 , 7.51 , 7.62 , 7.75 , 7.78 , 8.08 , 8.12 .A stirred solution of compound 3  in MeOH (90 mL) was hydrogenated for 3 h over Pd-C 10% (450 mg) at room temperature. The solution was filtered through celite, and the solvent was evaporated. The crude material was purified by silica gel chromatography (CH2Cl2 /EtOAc 6/4 to 4/6) to give AL530 as a pure light yellow powder . 1H NMR  1.45 , 3.65 , 3.75 , 5.16 , 6.44 , 6.65 , 6.71 , 7.24 , 7.29 , 7.43 , 7.51 , 7.62 , 7.79 , 8.06 , 8.92 , 9.61 . HRMS: m/z calcd for C37H36O5N4Cl2\u00b7H+ 687.21355; found 687.21174 (\u0394 = \u22121.81 ppm).A solution of the nitro compound DU145 (prostate carcinoma), A427 (lung carcinoma) and MDA-MB-468 (breast carcinoma) were obtained from the American Type Culture Collection.HN  cells wCAL33 and CAL166 cells . 2 in a cell incubator. Cells were cultured in Dulbecco\u2019s modified Eagle medium (DMEM)containing 10% fetal bovine serum (heat-decomplemented for HN cells) supplemented with 2 mmol/L L-glutamine and 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid HEPES (10 mmol/L) for DU145 and A427.All cell lines were maintained in an exponential growth phase in monolayer culture at 37 \u00b0C in a humidified atmosphere of 5% COv/v).The combi-molecules AL530 and PD98059 were synthesized in the Cancer Drug Research Laboratory . Chlorambucil (CBL) was purchased from Sigma\u2013Aldrich. All drugs were dissolved in DMSO to obtain a solution at 5 mM for AL530 and PD98059 and 100 mM for CBL. Drug dilutions were carried out under sterile conditions in the culture medium where the DMSO concentration never exceeded 1%  incubated at 37, 40 or 42 \u00b0C for 96 h to study AL530 cleavage.Solutions of 25 \u00b5M of AL530 were used in this series of experiments. These solutions (600 \u00b5L) were prepared using a Tris\u2013base buffer (50 mM) at a pH ranging from 7 to 5.5. Each solution contained a maximum of 0.5% DMSO (2O at 0.8 mL/min on a Waters LC\u2013UV system. Results are expressed as means \u00b1 standard error of the mean (SEM) and represent at least three independent experiments. The retention times of AL530 and PD98059 were, respectively, 10.5 and 5.1 min. The rates of hydrolysis of the AL530 in PD98059 were determined by measuring the areas of peaks observed in the HPLC chromatograms per time point as per each experimental condition.Every 24 h, 100 \u00b5L of each solution were collected from each incubated sample and then extracted for analysis by HPLC. After extraction, 20 \u00b5L of the organic phase were injected and compounds were eluted using an isocratic gradient of 60% acetonitrile and 40% H3 were divided into groups of 3, including vehicle control . AL530 was administered i.p. (100 mg/kg) and after 3 h post-administration, the mice were sacrificed and their tumors and plasma collected. Samples were processed for analysis as described in the HPLC sample preparation section.Experiments in the mouse 4T1 models were performed in female Balb/c mice  strictly in accordance with the protocol #4934 approved by the Facility Animal Care Committee (FACC), McGill University. Briefly, mice were implanted subcutaneously with 4T1 mammary tumor cells suspended in 200 \u03bcL of phosphate buffered saline (PBS). Mice with a tumor size greater than 500 mmFemale Swiss athymic nude mice , 4\u20135 weeks old, were housed in filter-capped cages kept in a sterile facility and maintained in accordance with the FELASA  standards. Following a two-week quarantine, mice were included in the protocols. The Claudius Regaud Institute animal ethics committee\u2019s approval was obtained for the animal model and the study protocols.6 cells into both mouse flanks. When the tumors reached an average volume of 200 mm3 (day 4 post-implantation), the mice were pooled and randomly assigned to control or treated groups. Mice of the treated groups were i.p. injected with the following different drugs and doses: AL530 = 50 or 25 mg/kg, CBL = 11 mg/kg, PD98059 = 9.7 mg/kg, equimolar combination of CBL + PD98059. The vehicle  and drugs  were administered daily for 12 days. Two perpendicular diameters of tumors were daily measured by the same investigator and caliper square. Tumor volume was calculated according to the following formula: V (mm3) = d2 (mm2)\u00b7D(mm)/2, where d and D, are the smallest and the largest tumor diameters, respectively. In each group, the relative tumor volume was expressed as a Vt/V0 ratio where Vt is the mean tumor volume on a given day during the treatment, and V0 is the mean tumor volume at the beginning of the treatment. The maximum body weight loss and/or tumor volume tolerated by the ethics committee and the protocol were, respectively, 20% of initial weight and 2000 mm3. When tumor volume reached 1000 mm3, mice were inspected twice daily and sacrificed before the occurrence of poor health manifestations. At the end of the experiment, following anesthetization, the mice were sacrificed by total blood collection via cardiac puncture using heparinized syringes and collection tubes. Then, the tumors in each flank of the mice were removed and one tumor was fixed in formalin for pathological and immunohistochemical analysis and the other was frozen at \u221280 \u00b0C for the pharmacokinetics studies.Xenografts were established by subcutaneous injection of 10 \u00d7 106 cells). After one week and when tumor size reached 1000 mm3, mice were treated with the vehicle or 50 mg/kg i.p. of AL530 and sacrificed. Plasma and tumor tissue were collected at 10 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 4 h, 8 h and 24 h. Blood was collected in heparinized tubes, and plasma was separated from blood samples after centrifugation at 1500\u00d7 g for 10 min at 4 \u00b0C. Tissues were immediately frozen at \u221280 \u00b0C until analysis. The data obtained by LC\u2013UV were analyzed with the software NONMEM to generate the pharmacokinetics parameters.Pharmacokinetic studies were carried out by determining AL530 concentrations in plasma and tumors of mice having received a single dose of AL530 . Three mice per time point were implanted in both flanks with CAL33 cells  and stored at \u221220 \u00b0C for two hours for decantation in two phases (organic/aqueous). The resulting supernatant (organic phase) was collected and filtered using 0.2 \u00b5m of Nylon membrane  and dried, using a SpeedVac\u00ae Concentrator  at room temperature.Plasma samples were precipitated with acetonitrile (two volumes of acetonitrile for one volume of plasma), vortexed (30 s), centrifuged at 4 \u00b0C . The supernatant was collected and cleaned through an HPLC filter (0.2 \u00b5m of Nylon), and the tube was stored at \u221220 \u00b0C for two hours leading to the separation of the two phases. The upper acetonitrile phase was transferred into a new evaporation tube to be dried using a SpeedVac\u00ae Concentrator.When frozen tumor tissue samples were thawed, they were immediately cut into small pieces with a scalpel, introduced into a 2 mL tube with steel balls and ground with a QIAGEN Tissue Lyser II system (3 min). This tissue homogenate was added to 250 \u00b5L of denaturing buffer , vigorously vortexed for 30 min at room temperature before adding two volumes of acetonitrile. The mixture underwent a new vortex cycle for 1 min, followed by an 8 min sonication in an ultrasonic cleaner bath and centrifuged  before being injected for LC\u2013UV analysis as described above (UV detection at 254 nm). Results are representative of at least two independent experiments.Prior to HPLC analysis, the dried samples were resuspended in 100 \u00b5L of acetonitrile, sonicated for 8 min in an ultrasonic cleaner bath and centrifuged at 4 \u00b0C  were plated in the culture medium in 100 mm culture dishes. On day 2, for total and phosphorylated Erk1,2 analysis, cells were treated with either the vehicle or with AL530 at a concentration of 25 \u03bcg/mL for 48 h. The medium and treatment were changed every 24 h and the cells were harvested and lysed in a RIPA lysis buffer . A total of 70 \u03bcg of proteins of the cleared lysates were separated onto a 12.5% SDS-PAGE gel, blotted onto PVDF membranes  and incubated with specific antibodies. Detection was performed using peroxidase-conjugated secondary antibodies (Bio-Rad) and an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech). The blots were scanned and analyzed with a Molecular Dynamics densitometer and ImageQuant software. Results are representative of at least two independent experiments.On day 1, CAL33 cells (2 \u00d7 10The immunohistochemistry analyses were performed by the Department of Anatomic Pathology at the Claudius Regaud Institute. Tumors were collected from mice in control and treated groups. Thick sections of 4 \u00b5m of formalin-fixed and paraffin-embedded tumors from CAL33 cell xenografts were prepared and stained with antibodies against \u03b3-H2AX and pErk1,2. Mouse data represent two independent experiments. Anti-phospho \u03b3-H2AX and pErk1,2 antibodies were obtained from Santa Cruz.p < 0.05.Data are expressed as mean \u00b1 S.E.M. Student\u2019s two-sided t-test or Mann\u2013Whitney test was used to compare values. Differences were considered statistically significant at This study demonstrated the feasibility of a complex combi-molecule designed to mask a kinase inhibitor and to release it in a pH range similar to the tumor microenvironment. We also demonstrated that the pharmacokinetics and distribution of the combi-molecule was markedly favorable to higher tumor than plasma concentration of its hydrolytic metabolites. Thus, as depicted by the model proposed in"}
+{"text": "Non-alcoholic steatohepatitis (NASH) is an increasing cause of chronic liver disease characterized by steatosis, inflammation, and fibrosis which can lead to cirrhosis, hepatocellular carcinoma, and mortality. Quantitative, noninvasive methods for characterizing the pathophysiology of NASH at both the preclinical and clinical level are sorely needed. We report here a multiparametric magnetic resonance imaging (MRI) protocol with the fibrogenesis probe Gd-Hyd to characterize fibrotic disease activity and steatosis in a common mouse model of NASH. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with advanced fibrosis. Mice fed normal chow and CDAHFD underwent MRI after 2, 6, 10 and 14\u00a0weeks to measure liver T1, T2*, fat fraction, and dynamic T1-weighted Gd-Hyd enhanced imaging of the liver. Steatosis, inflammation, and fibrosis were then quantified by histology. NASH and fibrosis developed quickly in CDAHFD fed mice with strong correlation between morphometric steatosis quantification and liver fat estimated by MRI (r\u2009=\u20090.90). Sirius red histology and collagen quantification confirmed increasing fibrosis over time (r\u2009=\u20090.82). Though baseline T1 and T2* measurements did not correlate with fibrosis, Gd-Hyd signal enhancement provided a measure of the extent of active fibrotic disease progression and correlated strongly with lysyl oxidase expression. Gd-Hyd MRI accurately detects fibrogenesis in a mouse model of NASH with advanced fibrosis and can be combined with other MR measures, like fat imaging, to more accurately assess disease burden. Between 20\u201330% of adults in the western world are now estimated to have NAFLD3, and between 5\u20136% of those patients with NAFLD will develop non-alcoholic steatohepatitis (NASH)4 in which substantial liver injury and inflammation are present5. While patients with NAFLD have good long-term prognosis, with no increased liver related morbidity or mortality6, those with NASH have increased risk of cirrhosis, hepatocellular carcinoma and liver or cardiovascular related mortality7. The financial burden of NAFLD and NASH healthcare management is currently estimated to cost in excess of $100 billion in the USA alone8. There is therefore a need to identify those NAFLD patients who are at risk of developing NASH and cirrhosis so as to better manage patient healthcare through improved lifestyle, exercise and diet10. In addition, a large number of new therapies have entered clinical trials12, however there have been a number of failures pointing to the challenge of designing safe, effective therapies for this complex disease13.Nonalcoholic fatty liver disease (NAFLD) is fast becoming one of the most prominent causes of liver disease15. Preclinically, there is also a need for better tools to allow longitudinal, noninvasive, quantitative characterization of NASH disease models, where historically treatment response has been assessed ex vivo. Disease progression in animal models is heterogeneous and thus at the time of treatment initiation some animals may have severe disease while others have mild disease, making it challenging to assess treatment effects. Multiparametric imaging can potentially provide a three dimensional assessment of the entire liver and can be performed prior to, and at multiple timepoints following treatment to assess the time course and the effect of the therapeutic intervention.There remains an unmet need for improved diagnostics to better stratify patients into clinical trials and to accurately monitor response to therapy17 and the only current way to distinguish hepatic steatosis (intracellular fat content in\u2009>\u20095% hepatocytes) from steatohepatitis 18. Biopsy however is an imperfect method with appreciable sampling error, high inter-observer variability, and risk of complications20. Transient elastography (TE)21 and magnetic resonance elastography (MRE)22 both reliably detect moderate and advanced liver fibrosis, but these methods cannot reliably distinguish simple steatosis from NASH23. Combining three-dimensional MRE (3D-MRE) with magnetic resonance imaging proton density fat fraction (MRI-PDFF) has shown more promise predicting NASH disease activity by the non-alcoholic fatty liver disease activity score (NAS)24. Recently, a multiparametric MRI algorithm, called LiverMultiScan, that assesses liver fat, T2, and iron-corrected T1 was shown to be superior to liver stiffness for stratifying patients with simple steatosis from those with NASH, but LiverMultiScan could not accurately stage fibrosis25.Liver biopsy is the gold standard for NASH diagnosis35. More recently, we have developed the gadolinium-based probe Gd-Hyd to allow for MR imaging of the allysine concentration in tissue which is responsible for the cross-linking and stabilization of collagen proteins during scar tissue formation38.For the last several years, our group has been developing molecular MRI to quantify the collagen deposition that occurs during scar tissue formationThe choline-deficient, L-amino acid defined, high fat diet (CDAHFD) mouse model is increasingly used as a preclinical model to test novel NASH therapies. The model is characterized by hepatic steatosis that gives way to increasing fibrosis with duration on diet, as well as a persistent inflammatory component. The goal of this study was to combine Gd-Hyd imaging of fibrogenesis with other MR measurements to create a multiparametric MRI exam to non-invasively assess the natural history of disease progression and resolution in the mouse CDAHFD model. The ultimate aim of this work is to develop a comprehensive MRI protocol to quantify steatosis and fibrogenesis in NASH patients without the need for biopsy.36. Gd-Hyd was shown to bind to allysine containing proteins and to allysine rich porcine aorta (Kd\u2009=\u2009650\u00a0\u03bcM), and was used to detect lung fibrogenesis in a bleomycin induced mouse model36 and liver fibrogenesis in CCl4 and CDAFHD models38. The gadolinium core provides MR signal enhancement on allysine binding (relaxivity\u2009=\u200916.2\u00a0mM\u22121\u00a0s\u22121 at 1.4\u00a0T when bound to protein vs 4.1\u00a0mM\u22121\u00a0s-1 when unbound).Gd-Hyd Fig.\u00a0 is a wat39, and approved by the MGH Institutional Animal Care and Use Committee. A total of 70 mice were used in this study and randomized to each study group. To induce NASH, 6-week old, male C57BL/6 mice  were fed a CDAHFD consisting of 60\u00a0kcal% fat and 0.1% methionine by weight as previously described40. Three groups of mice were studied: Group 1 mice were fed normal chow for 2 (n\u2009=\u20096), 6 (n\u2009=\u20096), 10 (n\u2009=\u20099) or 14\u00a0weeks (n\u2009=\u20095); Group 2 mice were fed CDAHFD for 2 (n\u2009=\u20096), 6 (n\u2009=\u20096), 10 (n\u2009=\u200912) or 14\u00a0weeks (n\u2009=\u200912); and Group 3 mice were fed CDAHFD for 10\u00a0weeks followed by normal chow for 4\u00a0weeks (n\u2009=\u20098). No animals were excluded from the study.All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and in compliance with the ARRIVE guidelinesAnimals were anesthetized with isoflurane (1\u20132%) and placed in a specially designed cradle with body temperature maintained at 37\u00a0\u00b0C. Anesthesia was adjusted to maintain a respiration rate of 60\u2009\u00b1\u20095 breaths per minute. The tail vein was cannulated for intravenous (i.v.) delivery of the contrast agent while the animal was positioned in the scanner. Imaging was performed at 9.4\u00a0T using a small bore animal scanner with a custom-built volume coil. Mice were imaged with a dose of 200\u00a0\u03bcmol/kg of Gd-Hyd.41, two-point Dixon sequence42, and 2D T1 weighted dynamic contrast enhanced (DCE) imaging) were first acquired, then a bolus (maximum volume 100 \u03bcL) of Gd-Hyd was administered i.v. and imaging performed for a period of 30\u00a0min post injection. Following the imaging session, animals were euthanized (45\u00a0min post injection), and liver and other tissues were removed for further analysis.A series of baseline images (T1- and T2*-mapping sequenceshttps://surfer.nmr.mgh.harvard.edu/fswiki/FreeviewGuide). Blood vessels were excluded from the liver ROI via the signal threshold method. The mean and standard deviation of the MR signal over an ROI was then calculated. The signal to noise ratio (SNR) for liver and muscle ROIs were established using SNRliver\u2009=\u2009mean(SIliver)/stdevnoise, and SNRmuscle\u2009=\u2009mean(SImuscle)/stdevnoise.T1-mapping sequence: Rapid Acquisition with Relaxation Enhancement (RARE) inversion recovery (IR), TR/TE\u2009=\u20095000/7.27\u00a0ms, matrix\u2009=\u2009195\u2009\u00d7\u2009195, FOV\u2009=\u20092.5\u2009\u00d7\u20092.5\u00a0cm, 9 inversion times of 0, 300, 550, 700, 850, 1500, 3000, 5000, and 7000\u00a0ms, single slice (1\u00a0mm), RARE factor 16. T1 was quantified from a three parameter fit of the dependence of liver signal intensity (SI) on inversion time (TI). T2*-mapping sequence: Multi-echo gradient-echo, TR\u2009=\u20091500\u00a0ms, matrix\u2009=\u2009128\u2009\u00d7\u2009128, flip angle\u2009=\u200990\u00b0, FOV\u2009=\u20092.56\u2009\u00d7\u20092.56\u00a0cm, 2 averages, 12 echo times of 2.62, 5.17, 7.72, 10.27, 12.82, 15.38, 17.93, 20.48, 23.03, 25.58, 28.13, 30.68\u00a0ms. T2* maps were generated from exponential fitting of the signal intensity as a function of the gradient-echo time (TE). Two-point Dixon sequence: Conventional gradient echo sequence with TR\u2009=\u2009500\u00a0ms, TE (in-phase)\u2009=\u20091.06\u00a0ms and TE (out-of-phase)\u2009=\u20091.41\u00a0ms, FOV\u2009=\u20092.4\u2009\u00d7\u20092.4\u00a0cm, matrix\u2009=\u200996\u2009\u00d7\u200996, 18\u2009\u00d7\u20091\u00a0mm slices, flip angle\u2009=\u200930\u00b0. 2D T1 weighted dynamic contrast enhanced (DCE) images were acquired prior to and at 5, 15 and 25\u00a0min following intravenous administration of probes. TR/TE\u2009=\u200941/2.5\u00a0ms, temporal resolution\u2009=\u20092.64\u00a0ms, number of repetitions\u2009=\u200954, matrix\u2009=\u200964\u2009\u00d7\u200964, flip angle\u2009=\u200960\u00b0 and FOV\u2009=\u20094.385\u2009\u00d7\u20092.644\u00a0cm. For DCE analysis region of interests (ROIs) were segmented using FreeView  was morphometrically quantified on whole slide scanned Sirius red sections with image processing software  according to our established protocol46. Hepatic lipid vacuolization (LV) was calculated from whole slide scanned H-E sections using ImageJ.Formalin-fixed samples were embedded in paraffin, cut into 5\u00a0\u00b5m-thick sections and stained with hematoxylin\u2013eosin (H-E) and Sirius red according to standard procedures. The grading of steatosis, ballooning47, and was expressed as amounts per wet weight of tissue.Hydroxyproline in tissue was quantified by HPLC analysis using a reported methodp\u2009<\u20090.05 considered as significant.All data are shown as mean\u2009\u00b1\u2009SEM. Differences between two groups were tested with unpaired Student\u2019s t-Test, and differences among more than two groups were tested with one-way analysis of variance (ANOVA) followed by Tukey\u2019s post-hoc test with 48. However, fibrosis development is weak in traditional animal models of obesity and fatty liver disease, like high fat or western diets49. We therefore chose to use a CDAHFD for these studies as NASH and fibrosis develop quickly in mice fed CDAHFD.The main objective of this study was to image fibrogenesis as fibrosis stage is the only histological feature of disease that is associated with worse outcomes in NASHMale C57BL/6 mice were fed control or CDAHFD diet and subsets of animals were imaged and sacrificed after 2, 6, 10, and 14\u00a0weeks. Steatosis developed quickly . LV continued to increase in the CDAHFD groups up to 6\u00a0weeks (30.91\u2009\u00b1\u20091.11%) followed by a steady decrease at the 10 (17.28\u2009\u00b1\u20090.82%) and 14 (14.10\u2009\u00b1\u20091.35%) week time-points. By comparison, LV for mice on a normal diet remained consistently below 4% for the whole 14\u00a0weeks , but increased steadily over time compared to control animals, which remained relatively constant, and were greatest at week 14 . Switching mice that had been fed CDAHFD for 10\u00a0weeks to normal chow for 4\u00a0weeks caused a significant decrease in collagen levels compared to those animals that received CDAHFD for the entire 14\u00a0weeks. CPA decreased to 12.34\u2009\u00b1\u20090.85% and hydroxyproline levels dropped to 634\u2009\u00b1\u200932\u00a0\u03bcg/g by week 14. However, contrary to steatosis, collagen levels did not reduce down to the levels seen for those animals on a normal diet for 14\u00a0weeks.Quantitative analysis of hydroxyproline (Hyp) was used as an additional measure of the total amount of collagen in tissue Fig.\u00a0d. Hydroxp\u2009=\u20090.0002). Prolonged exposure to CDAHFD led to an increase in T1 value over time, with measurements at 14\u00a0weeks reaching similar levels to those of mice on the normal diet . Switching mice fed a CDAHFD for 10\u00a0weeks to normal chow for 4\u00a0weeks increased the liver T1 value even further compared to those mice fed CDAHFD for the entire 14\u00a0weeks, with no significant difference between mice on diet withdrawal and mice on normal chow . Baseline T1 values did not provide a reliable method to distinguish the extent of fibrosis in the CDAHFD model  at 2\u00a0weeks was significantly reduced compared to the liver T1 of mice on normal diet . Switching mice fed a CDAHFD for 10\u00a0weeks to normal chow for 4\u00a0weeks led to a small but significant increase in liver T2* signal compared to mice fed CDAHFD for the entire 14\u00a0weeks (p\u2009=\u20090.0005), with little difference between mice on diet withdrawal and mice on normal chow . Baseline T2* measurements also did not provide a reliable method to distinguish the extent of fibrosis in the CDAHFD model  differences between the fibrotic CDAHFD and control livers for the allysine-targeted agent Gd-Hyd would become visible. Since the kinetics of NASH liver washout with Gd-Hyd was unknown, T1 weighted DCE measurements were performed prior to, and at multiple time points out to 30\u00a0min post injection.Based on the results from our previous CClliver/SImuscle , and \u0394LMR\u2009=\u2009LMRpost\u2009\u2212\u2009LMRpre. The AUC characterized for the \u0394LMR vs time on diet  in liver pre- and post-injection was determined and the percentage increase in SNR (%\u0394SNR) from baseline (pre injection) calculated. After Gd-Hyd injection, the CDAHFD mice and the controls showed distinct differences in liver signal enhancement over the time course of the imaging. Liver signal was greatest at 5\u00a0min post injection and decreased at 15 and 25\u00a0min post Gd-Hyd injection. The area under curve (AUC) for the DCE-derived change in signal was also calculated and plotted as a function of time on diet  and Loxl1 at week 14   Fig.\u00a0. CompareTo determine whether existing non-invasive MRI measures like the Dixon method or T1 or T2* relaxation times could report on steatosis and fibrosis, we correlated these measurements with histological estimates of steatosis assessed by % lipid vacuolization (% LV) and fibrosis by the collagen proportional area (CPA), Fig.\u00a050. We reasoned that non-contrast MR imaging and molecular MR imaging of allysine could represent a direct and objective measure of NASH fibrosis.Biopsy, the current gold standard for NASH diagnosis, is an invasive procedure with inherent risks and not a practical solution for monitoring disease progression or response to therapy54. Here, we report for the first time a longitudinal, pathological characterization of the CDAHFD mouse model with respect to non-invasive imaging. We demonstrate that multiparametric MR assessment of the liver can reliably monitor changes in key pathological features like steatosis and fibrosis. Our results suggest that multiparametric MRI could not only be a useful tool for following disease progression in human patients in order to determine when to start treatment but also for monitoring the response to intervention.While no animal model represents all aspects of human NASH, the CDAHFD model has become increasingly common for investigating novel therapeutics over the last few years given its ability to generate a robust fibrotic phenotypeMice fed CDAHFD diet showed an immediate increase in steatosis with fat content build up within the vescicles of the hepatocytes leading to macrovesiculation and disruption of hepatocyte function. Over time, loss of hepatocytes in combination with increased myofibroblast activation and recruitment of immune cells leads to an overall decrease in total steatosis, increased inflammation and advanced fibrosis as observed in the human disease. Consistently, MR imaging (Dixon method) distinguishes this decrease in steatosis over time, with measurements of fat content in liver correlating with morphometric histologic methods (Fig.\u00a025. T2* shows little dependence on the extent of either steatosis or fibrosis over time in this model, and is not indicative of the extent of disease (Fig.\u00a0T1 times are shorter for mice fed CDAHFD compared to those that received normal chow, but increased over time. In fact, after 14\u00a0weeks there is little difference between the control and CDAHFD cohorts suggesting T1 shortening is sensitive to the fat content of tissue (Fig.\u00a0ase Fig.\u00a0e,f.4 disease model, with a highly significant increase in MR signal in the fibrotic liver tissue compared to control animals, that tracked with hydroxyproline concentration36. In this NASH model of advanced fibrosis, Gd-Hyd uptake is significantly increased in the livers of mice fed CDAHFD as compared to mice fed normal diet, but there is no direct correlation between Gd-Hyd liver signal enhancement and the extent of fibrosis. CPA and hydroxyproline levels are at a maximum at week 14 whereas the maximum Gd-Hyd signal enhancement is at week 10. Gd-Hyd signal enhancement did show a strong correlation with Lox expression, which also peaked at week 10 and decreased at 14\u00a0weeks. In the previous 12\u00a0week CCl4 study, Gd-Hyd signal enhancement similarly tracked with Lox gene expression with both Gd-Hyd signal and Lox expression maximally elevated at the 12\u00a0week timepoint36. A decrease in Lox expression would lead to a reduction in the amount of allysine formed, and the amount of Gd-Hyd bound to collagen. This might indicate that fibrogenesis has started to decrease leading to disease stabilization in the mouse CDAHFD model by 10\u00a0weeks but not the mouse CCl4 model by 12\u00a0weeks. Small decreases in Acta2 expression (Fig.\u00a0Col1a1 expression (Fig.\u00a0Timp1 expression, a marker of fibrogenesis, between weeks 10 and 14 (Fig.\u00a036. Importantly, even though Gd-Hyd imaging decreased from weeks 10 to 14 as the disease stabilized, a further reduction was seen when animals fed CDHAFD for 10\u00a0weeks were switched to normal chow, suggesting that Gd-Hyd is a sensitive method for detecting reduced fibrogenesis during disease resolution. Importantly, additional decreases in Acta2, Col1a1, and Timp1 expression were also noted on diet withdrawal.The MRI probe Gd-Hyd which targets allysine was previously shown to be effective and specific for identifying liver fibrogenesis in a 12\u00a0week CCl57. MRE is very effective at detecting advanced fibrosis in patients59, however its ability to monitor treatment response in clinical trials is still to be established59. A multi-parametric MRI approach utilizing a fibroisis/fibrogenesis specific probe is therefore most likely to give an accurate treatment assessment.The development of novel NASH treatments is restricted by the need for biopsy to monitor treatment response. There is therefore a need for noninvasive objective and quantitative biomarkers of treatment response. MRI would be an ideal modality for a number of reasons: it is non-invasive and provides whole liver coverage with the ability to detect and quantify disease heterogeneity; it does not involve ionizing radiation making it safe for repeated imaging which is important in a slow progressing disease where patients may be followed for decades; MRI readily scales from mouse to human such that MRI protocols can be developed preclinically and then translated to clinical trials. MRI is increasingly used in NASH drug development, and MRI measurements of liver fat and relaxation times are highly reproducible and repeatable36. They also showed that Gd-Hyd could be used to image liver fibrogenesis in the mouse CCl4 model as well as the resolution in liver fibrosis when CCl4 was withdrawn. Zhou et al. used Gd-Hyd to measure hepatic fibrogenesis in the rat CDAHFD model and showed that Gd-Hyd enhanced MRI was superior to collagen-targeted molecular MRI, to magnetic resonance elastography, and to native T1 in measuring response to treatment with either elafibrinor or diet change26. Ferreira et al. recently showed using different transgenic mouse models of parasitic infection that Gd-Hyd enhanced MRI was sensitive to fibrogenesis but not to the presence of inflammation60.This study builds upon prior work with Gd-Hyd enhanced MRI. Chen et al. showed in the bleomycin injured mouse model of pulmonary fibrosis that Gd-Hyd imaging of allysine reflected fibrogenesis and that Gd-Hyd could distinguish active fibrogenesis from stable scar, and also showed that Gd-Hyd could monitor treatment response with the pan lysyl oxidase inhibitor beta-aminopropionitrile61 or MR elastography62 or emerging molecularly targeted approaches64 may be considered for noninvasive staging of disease. In very advanced disease, altered liver perfusion may also need to be considered for quantitative molecular imaging.Gd-Hyd enhanced MRI is expected to be a good noninvasive measure of the rate of fibrosis, however fibrogenesis may not necessarily correlate with disease stage, as was shown here. Especially at very advanced stages of disease, the rate of fibrogenesis may not be reflective of overall fibrotic burden and other methods such as ultrasoundBaseline non-contrast fat MR imaging followed by contrast Gd-Hyd enhanced MRI represent a promising multi-parametric approach for the non-invasive detection and staging of steatosis and fibrogenesis, and for monitoring treatment response for patients with NASH. Gd-Hyd is a derivative of the stable, clinical contrast agent gadoterate and is well suited for clinical translation. Target localization with Gd-Hyd is fast after injection and the contrast agent was previously shown to be rapidly and completely eliminated into the urine. Moreover, the imaging performed here utilized standard T1-weighted protocols that are available on commercial clinical scanners. While additional pre-clinical safety studies are required before commencing human studies, the results presented here indicate that these translational studies are warranted."}
+{"text": "Tnfa and Il1a at the BBB. Our findings suggest that blocking PDGF-CC counteracts fundamental aspects of endothelial cell activation and disruption of the BBB by decreasing Tnfa and Il1a expression. We also demonstrate that both PDGF-CC and its receptor PDGFR\u03b1 were upregulated in MS lesions indicating that blocking PDGF-CC may be considered a novel treatment for MS.Disruption of blood\u2013brain barrier (BBB) integrity is a feature of various neurological disorders. Here we found that the BBB is differently affected during the preclinical, progression and remission phase of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We have identified an upregulation of pro-inflammatory and pro-angiogenic factors in the BBB transcriptome and down-regulation of endothelial tight junction members coinciding with elevated BBB leakage specifically during the progression phase. These changes were antagonized by blocking PDGFR\u03b1 signaling with the small tyrosine kinase inhibitor imatinib. Moreover, targeting the PDGFR\u03b1 ligand PDGF-CC using a neutralizing antibody, facilitated recovery of BBB integrity and improvement of EAE symptoms. Intracerebroventricular injection of PDGF-CC induced upregulation, whereas blocking PDGF-CC during EAE led to downregulation of  MS, an inflammatory demyelinating disease characterized by multifocal CNS lesions, represents the leading cause of non\u2010traumatic disability among young adults in Europe and the USA19. Currently available immunomodulatory therapies have a modest effect on disease progression and may even be associated with a risk for developing an opportunistic infection such as progressive multifocal leukoencephalopathy (PML)26. Hence, there is a need for developing novel treatments for MS, preferably acting on molecular targets expressed exclusively at the BBB and not on immune cells, which should decrease the risk for adverse complications.The blood\u2013brain barrier (BBB) with counterparts found in the spinal cord  and retina , represents a dynamic interface between the central nervous system (CNS) and the peripheral immune system. The anatomical basis of this barrier are tight junctions between endothelial cells (ECs). ECs are supported by pericytes and both vascular cell types are surrounded by the basement membrane comprised of extracellular matrix components (ECM). Astrocyte endfeet ensheath blood vessels as well as neuronal synapses. The association of these neuronal cells, vascular cells, and ECM components is termed the neurovascular unit, which is highly responsive to both physiological and pathological changes while tending to maintain regular function and integrity. In vivo demonstration of gadolinium uptake54. For example, TNF-\u03b1 and IFN-\u03b3 promote both adhesion and migration of leucocytes across the BBB by influencing the expression of various chemokines such as CCL2 to CCL528. This leads to endothelial cell activation and subsequent disruption of BBB integrity, resulting in influx of immune cells into the CNS53. In addition, disturbed balance between pro- and anti-angiogenic chemokines contributes to formation of dysfunctional blood vessels17.It is believed that in MS, activated T cells, macrophages and brain resident microglia produce cytokines and chemokines that mediate upregulation of respective receptors and adhesion molecules at the BBB36. The formal identity of perivascular PDGFR\u03b1 expressing cells presumed responsible for mediating BBB opening and onset of vascular leakage is still under investigation but suggested to be a distinct arteriolar population of GFAP-positive astrocytes57 or potentially fibroblast-like cells61. PDGF-CC, a ligand of PDGFR\u03b1, is a member of the PDGF family consisting of a growth factor domain (GFD) and a N-terminal CUB domain40. The latter domain has to be proteolytically removed by tissue plasminogen activator (tPA)23 in order to release the GFD dimer and subsequently allow the PDGF-CC ligand to activate its receptor23. Inhibition of PDGFR\u03b1 signaling with the small tyrosine kinase inhibitor imatinib has been shown to reduce BBB dysfunction, as demonstrated in animal models of neuroinflammatory and neurodegenerative diseases such as MS2 and amyotrophic lateral sclerosis (ALS)37, respectively, but also in acute conditions such as stroke57, spinal cord injury (SCI)1, traumatic brain injury (TBI)58 and seizures25. Moreover, a recently performed phase II randomized trial in patients with acute ischemic stroke treated with intravenous thrombolysis showed that imatinib significantly improves neurological outcome in comparison to patients treated with tPA only62.Recent studies have shown that the CNS displays a common injury response mechanism involving activation of platelet-derived growth factor receptor \u03b1 (PDGFR\u03b1) signaling, leading to BBB dysfunction and increased vascular permeability55, this model allows investigation of distinguishable disease phases representing preclinical, progression as well as remission properties15. Accordingly, we were able to analyse the disease phase-specific impact on the BBB transcriptome and phenotype and found that blocking PDGFR\u03b1 leads to downregulation of transcripts important for endothelial cell activation and immune cell transmigration, correlating with restored BBB integrity specifically during disease progression. In order to further investigate the therapeutic potential of PDGFR\u03b1 modulation in autoimmune neuroinflammation, we utilized a neutralizing PDGF-CC antibody38 in a transgenic mouse line expressing PDGF-CC with a humanized growth factor domain (PDGF-CChum)66. We could observe that inhibiting PDGFR\u03b1 signaling by blocking its ligand PDGF-CC indeed preserves BBB integrity, reduces immune cell infiltration and consequently ameliorates EAE. Our data strongly indicate the possibility for establishing a novel treatment specifically targeting disruption of BBB integrity during disease progression.The present study is based on yet unreported systematic profiling of transcriptional and phenotypic changes at the BBB during myelin oligodendrocyte glycoprotein (MOG)- induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Along with many shared features of the human pathologyAll experiments in this study were approved and performed in accordance with the guidelines from the Swedish National Board for Laboratory Animals and the European Union Directive (2010/63/EU) under ethical permits approved by the North Stockholm Animal Ethics Committee.tm3633Arte mice were originally generated by Taconic  and are referred to as PDGF-CChum throughout the manuscript. PDGF-CChum mice have been described elsewhere66. In short, to exchange the mouse with the human PDGFC, six mutations were introduced into the mouse Pdgfc sequence. The generation of Pdgfc\u2013deficient mice has been described previously18. C57BL/6N Pdgfc/\u2212+ and littermate wildtype controls were used in experiments.Animals were housed in the animal facility at the Karolinska Institute  or at the animal facility at Karolinska Hospital\u00a0 in a climate-controlled environment in polystyrene cages containing aspen wood shavings with free access to standard rodent chow and water with regulated 12-h light/dark cycles. The C57BL/6NTac-PdgfcEscherichia coli and purified to homogeneity by chelate chromatography7. The purified protein, dissolved in 6\u00a0M urea, was dialyzed against PBS to obtain a physiological preparation that was stored at \u2212 80\u00a0\u00b0C. Mice at the age of 10\u00a0weeks were anaesthetized with isoflurane  and injected subcutaneously in the tail base in order to induce EAE with a 100\u00a0\u00b5l inoculum containing 50\u00a0\u00b5g MOG in PBS, emulsified 1:1 with complete Freund\u2019s adjuvant . Mice were intraperitoneally (i.p.) injected twice with 100\u00a0ng pertussis toxin , once on the day of immunization and once 2\u00a0days post-immunization (p.i.).The N- terminal amino acids 1\u2013125 of myelin oligodendrocyte glycoprotein (MOG) were expressed in P\u2009<\u20090.05, **P\u2009<\u20090.01 and ***P\u2009<\u20090.001).Mice were monitored daily for clinical signs of EAE, from day 7 p.i. until they were sacrificed as follows: score 0\u2009=\u2009no clinical signs of EAE; 1\u2009=\u2009tail paralysis; 2\u2009=\u2009hind leg paraparesis or hemiparesis; 3\u2009=\u2009hind leg paralysis or hemiparalysis and 4\u2009=\u2009tetraplegy or moribund. Statistics were calculated using Kruskal\u2013Wallis test with Dunn's correction for multiple testing  6B3 or BM4 (IgG mAb control). mAb 6B3 has been described previously38. For imatinib treatment, C57BL/6N wildtype mice were oral gavage fed with steel gavage needles with a daily dose of 250\u00a0mg/kg/mouse imatinib or phosphate buffered saline (PBS) as control delivered as a morning (1/3) and evening (2/3) dose 8\u00a0h later, starting 2\u00a0days p.i. Imatinib tablets  were crushed into a fine powder, solubilized in sterile PBS, vortexed and incubated at 37\u00a0\u00b0C (water bath) for 5\u00a0min. Insoluble components were spun down in a table microcentrifuge  for 2\u00a0min. The supernatant was used for oral gavage immediately.To neutralize PDGF-CC in vivo during EAE, PDGF-CC2.5\u00a0mg tetramethylrhodamine-conjugated 70\u00a0kDa dextran  dissolved in PBS (0.1\u00a0ml) was intravenously infused at the preclinical, progression and remission phase, respectively. The tracer was allowed to circulate for 2\u00a0h and thereafter the animals were thoroughly perfused with Hanks\u2019 balanced salt solution (HBSS) followed by 4% paraformaldehyde (PFA). Spinal cords were immediately removed and post-fixed for 4\u00a0h in PFA and thereafter cryo-protected in 20% sucrose at 4\u00a0\u00b0C over night (o/n). The spinal cords were then imaged using a Zeiss Lumar V12 stereo microscope and an Axiocam MRm digital camera  was used to capture the dorsal view of the entire spinal cords. Epi-fluorescence of the entire dorsal surface was then measured using Image J64 .To obtain sections from different segments, spinal cords were first cut in 7\u00a0mm long pieces, rostral to caudal. Cryosections of 12\u00a0\u03bcm thickness from the different spinal cord segments were obtained and immunofluorescence was performed as follows: Sections were air-dried for 30\u00a0min before permeabilization in PBS/0.2% Triton X-100 for 10\u00a0min. Staining against rabbit anti-human ADAMTS9, rabbit anti-mouse CXCL10 (Bioss), monoclonal anti-mouse occludin, monoclonal anti-mouse ZO-1, rabbit anti-mouse claudin-5  and rabbit anti-mouse p65 NF\u03baB (RnD Systems), required antigen retrieval with citrate buffer pH 6.0 (Dako) by warming for 20\u00a0min in a steamer device . Staining against goat anti-mouse CD31 (RnD systems), rabbit anti-mouse collagen IV , goat anti-mouse aquaporin 4 , rabbit anti-mouse ETS related gene , rabbit anti-mouse Ki67 (Abcam), goat anti-mouse podocalyxin , rabbit anti-mouse CD3 (Abcam), rat anti-mouse CD45 (BD Pharmingen), rat anti-mouse CD68 (Biorad) and rabbit anti-mouse Iba-1 (Wako) did not require antigen retrieval. Blocking was performed in PBS/10%FBS (blocking solution) and primary antibodies diluted in blocking solution were applied o/n at 4\u00a0\u00b0C. The antibody signal was visualized using Alexa-Fluor conjugated secondary antibodies (Invitrogen). DAPI  was included in the last PBS wash to visualize the nuclei.10. After deparaffinization in x-tra-solv, sections were transferred to 99.5% ethanol. Endogenous peroxidase was blocked by incubation in methanol with 0.02% H2O2 for 30\u00a0min at room temperature (RT) and rehydration to distilled water followed via a 99.5%, 90%, 70%, and 50% ethanol series. Staining against rabbit anti-mouse occludin  and mouse anti-human PDGF-CC mAb 6B3  required retrieval with high pH  by warming for 1\u00a0h in a steaming device . Sections were incubated in 10% FBS in PBS 30\u00a0min prior to incubation with primary antibody on 4\u00a0\u00b0C, o/n. After washing in PBS, sections were incubated with Alexa-Fluor conjugated secondary antibody. DAPI was included in the last PBS wash to visualize the nuclei. All images were acquired with a Zeiss LSM700 confocal microscope and the ZEN 2009 software . Representative images shown are 2D renderings of 12\u00a0\u00b5m thick z-stacks from the lumbar spinal cord region. For all quantifications of antibody immunoreactivity done in this study, images were acquired using the same settings (within each experiment). For quantification of intensity the number of pixels above a set threshold was determined and the quantifications were performed using ImageJ64. For occludin, claudin-5, ZO-1, CD45, CD3, CD68 and Iba-1 evaluation, ten adjacent fields of vision were measured. Whole spinal cord portions were measured to evaluate dextran extravasation. For evaluation of endothelial cell proliferation (Ki67/ERG1 co-staining), all double-positive nuclei were manually counted. Quantifications of immunofluorescence and immunohistochemical stainings, as well as dextran leakage, were calculated using Student\u00b4s unpaired t-test or one-way ANOVA with Fisher\u00b4s LSD . Results are depicted as average\u2009\u00b1\u2009S.E.M.For immunohistochemical stainings , paraffin-embedded sections of the spinal cord or brain were treated as previously describedPDGFR\u03b1 staining in human brain samples was performed on the Ventana Discovery Autostainer System  using PDGFR\u03b1 antibody  in Discovery antibody diluent . Extensive antigen retrieval was performed with Discovery Cell Conditioning buffer 1 . Antibodies were incubated for 1\u00a0h at room temperature. Detection was performed using the OmniMap DAB anti-rabbit kit  with 30\u00a0min incubation of the secondary antibody at room temperature. Representative images shown are from inflammatory demyelinating/remyelinating lesions and normal appearing parenchymal white matter in MS brain parenchyma.2. HE and Kluever images were acquired with a Zeiss Axio Observer Z1 inverted microscope and the ZEN 2009 software . The inflammatory index (I.I.) and demyelination score (DM) were determined from the number and size of demyelinated lesions of each animal on an average of ten complete spinal cord cross-sections as previously described55.Hematoxylin&eosin (HE) and Luxol Fast Blue (Kluever) were stained according to standard protocol to assess tissue inflammation and demyelination, respectivelyhum mice i.p. injected with anti-PDGF-CC (6B3) or BM4 (IgG control) antibodies were sacrificed during the progression phase. The mice were anesthetized with isoflurane and thereafter perfused with HBSS. Spinal cords were subsequently rapidly dissected out and placed into ice-cold HBSS. The rest of the protocol was performed as described except for the antibody and magnetic beads for pulling out the endothelial vessel fragments9. We used biotin rat anti-mouse CD31 (BD Biosciences) together with magnetic beads . The purity of vascular fragments was analyzed by real-time qPCR. Markers for endothelial cells , pericytes (Pdgfrb), astrocyte endfeet (Aqp4), neurons (Dlg4), microglia (Iba1) and immune cells  were used to assess the percentage of the distinct cell populations in the vascular fragments. The mean expression level of Cldn5 was set to 10. The analysis revealed high enrichment of endothelial cells (5.8\u20137.5 fold for Pecam1 and tenfold for Cldn5), followed by pericytes (0.7\u20131-fold) and astrocyte endfeet (0.1\u20130.78 fold). The vascular fragments were depleted of neurons (0.002\u20130.005 fold), microglia (0.003\u20130.11 fold) and showed neglectable contamination from immune cells  that was similar for EAE and naive vascular fragments . Confirmation of RNA quality was assessed using the Agilent 2100 Bioanalyzer 2. Total RNA was subsequently either used for expression array analysis or cDNA generation for qPCR analysis. cDNA was prepared using the iScript kit .Total RNA was extracted from both the wash and the eluate fraction using the RNeasy kit  and the QIAcube (Qiagen) including on column DNA-digestion for fully automated sample preparationRpl19. Primers used in this study are described in Supplementary Table P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001 and ****P\u2009<\u20090.0001) was used.Real-time quantitative PCR was performed using KAPA SYBR FAST qPCR Kit Master Mix (2x) Universal (KAPA Biosystems) in Rotor-Gene Q (Qiagen) Real-Time PCR thermal cycler according to the manufacturers\u2019 instructions. Expression levels were normalized to the expression of 2. 250 nanograms of total RNA from each sample were used to generate amplified and biotinylated sense-strand cDNA from the entire expressed genome according to the Ambion WT Expression Kit (P/N 4425209 Rev C 09/2009) and Affymetrix GeneChip WT Terminal Labeling and Hybridization User Manual . GeneChip ST Arrays (GeneChip Gene 2.0 ST Array) were hybridized for 16\u00a0h in a 45\u00a0\u00b0C incubator, rotated at 60\u00a0rpm. According to the GeneChip Expression Wash, Stain and Scan Manual  the arrays were then washed and stained using the Fluidics Station 450 and finally scanned using the GeneChip Scanner 3000 7G.The procedure and data analysis were done as inhttp://www.affymetrix.com) using the robust multi-array average (RMA) method first suggested by Li and Wong in 200130. Subsequent analysis of the gene expression data was carried out in the freely available statistical computing language R (http://www.r-project.org) using packages available from the Bioconductor project (www.bioconductor.org). In order to search for the differentially expressed genes between X and the Y groups an empirical Bayes moderated t-test was then applied51, using the \u2018limma\u2019 package. To address the problem with multiple testing, the p-values were adjusted using the method of Benjamini and Hochberg.The raw data was normalized in the free software Expression Console provided by Affymetrix . The molecules in this data set were associated with a canonical pathway in Ingenuity\u2019s knowledge base. The significance of the association between the data set and the canonical pathway was measured in 2 ways: (1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed. (2) Fisher\u2019s exact test was used to calculate a P-value determining the probability that the association between the genes in the data set and canonical pathway is by chance alone.Molecules from the data set that met the\u2009log2 fold change >1\u00a0cut\u00a0off\u00a0(i.e. at least twofold up- or downregulated where a positive log2 fold change indicate an upregulated gene) and an adjusted The full EAE and healthy na\u00efve microarray data sets of vascular fragments presented in this publication have been deposited to the GEO expression omnibus database with the accession number GSE150562 and GSE157604, respectively.40. For vascular fragment isolation and subsequent qPCR analysis, mice were perfused with HBSS 4\u00a0h after ICV injection and the left hemisphere was immediately processed for vascular fragment isolation and quantitative real-time PCR as described above for spinal cords. Statistics were calculated using Student\u00b4s unpaired t-test .Wildtype female C57BL/6N mice were anesthetized with isoflurane, placed on a stereotactic frame, and thereafter injected with either 4\u00a0\u00b5l of active PDGF-CC core protein (3\u00a0\u00b5M) or PBS into the left lateral ventricle  using an automatic injection pump over 7\u00a0min . The recombinant core domain of PDGF-CC was produced in baculo-virus infected insect Sf9 cells and purified as describedN,N-dimethylformamide (Sigma-Aldrich) and centrifuged for 45\u00a0min at 25,000 rcf . The supernatants were collected and quantitation of EB extravasation was performed as described65. EB levels in each brain were determined from the formula: (A620 nm\u2009\u2212\u2009((A500 nm\u2009+\u2009A740 nm) \u2215 2)) \u2215 mg wet weight. One-way ANOVA with Fisher\u00b4s LSD  was used.For BBB permeability analysis active PDGF-CC core protein (3\u00a0\u03bcM) or PBS as control was ICV injected into the dorsal 3rd ventricle  in mice pre-treated with either imatinib (or PBS as control) or 6B3 antibody (or BM4 as control) 2-5\u00a0h before ICV injection. Directly after ICV injection mice were intravenously injected with 0.1\u00a0ml of 4% EB dye (Sigma-Aldrich). 2\u00a0h later, animals were perfused with HBSS for 8\u00a0min and the brains were removed and photographed with a Samsung s4mini mobile phone . Each brain was then homogenized in For enzyme-linked immunosorbent assay (ELISA) active PDGF-CC core protein (3\u00a0\u03bcM) or PBS as control was ICV injected into the left lateral ventricle  in wildtype C57BL/6N female mice. Eight hours later, cerebrospinal fluid (CSF) was harvested from cisterna magna with a fine glass microcapillary followed by euthanasia of the mice. The CSF was immediately frozen and kept at \u2212\u00a080\u00a0\u00b0C.t-test .CSF harvested 8\u00a0h after PDGF-CC or PBS ICV injection or at the EAE progression phase was subjected to ELISA according to the manufacturer\u2019s protocol . Statistics were calculated using Student\u00b4s unpaired Immunohistochemical staining of PDGF-CC and PDGFR\u03b1 was performed on brain samples from 12 MS cases supplied by the Multiple Sclerosis Society Tissue Bank, funded by the Multiple Sclerosis Society of Great Britain and Northern Ireland, registered charity 207,495. All participants gave prospective premortem written informed consent for their brains to be banked and used for research. Besides normal appearing white matter (NAWM), the sample collection comprised different lesion phenotypes (active (AL), chronic active (CAL), chronic (CL) and remyelinated lesions (RL).All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All animal experiments in this study were approved and performed in accordance with the guidelines from the Swedish National Board for Laboratory Animals and the European Union Directive (2010/63/EU) under ethical permits approved by the North Stockholm Animal Ethics Committee. All experiments involving human material in this study were approved by the Regional Ethics Committee in Stockholm (No. 2012/1417\u201331/1).The Multiple Sclerosis Society Tissue Bank at the Imperial College London has been approved as a Research Tissue Bank by the Wales Research Ethics Committee (Ref. No. 08/MRE09/31\u2009+\u20095). All participants gave prospective premortem written informed consent for their brains to be banked and used for research. All procedures performed involving human participants were in line with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.2. Here we extend our study focusing on the consequences of blocking PDGFR\u03b1 signaling at the neurovascular unit (NVU) in mouse EAE utilizing imatinib as well as a neutralizing antibody against the PDGFR\u03b1 ligand PDGF-CC. For this we first performed a genome wide analysis of the BBB transcriptome on endothelial cell enriched vascular fragments isolated from the spinal cords of: i) MOG-immunized, imatinib-treated and ii) MOG-immunized, PBS-treated C57BL/6N mice sacrificed during the preclinical, progression and remission phase of EAE, respectively. As control, we used vascular fragments isolated from na\u00efve (non-immunized) littermates. The purity of the vascular fragments subjected to transcriptome analyses was assessed using cell type specific markers and found to be highly enriched in endothelial cells with minor enrichment of pericytes, while devoid of contaminating non-vascular cells such as neurons, microglia and immune cells ), we detected 59 transcripts that were differentially expressed in the preclinical  and leucocyte adhesion  as well as chemokines Ccl4 and Ccl3l3  hydrolyzing lipase Lpl as well as its co-activator Apoc2, and Fabp5, involved in fatty acid uptake, were specifically upregulated during the remission phase  Fig.\u00a0 and leucCcl2, Ccl5, Ccl7, Cxcl2, Cxcl10, Ccr2 or Cxcr4  injections 2\u00a0h after treating C57BL/6N na\u00efve mice with imatinib or PBS, respectively. Indeed, evaluation of Evans Blue (EB) extravasation revealed BBB protection by imatinib - analysis of the tight junction (TJ) components occludin, claudin-5 and ZO-1 in spinal cords of mice injected with 70\u00a0kDa dextran. During the progression phase, all TJ markers exhibited a discontinuous and aberrant expression pattern Fig.\u00a0 and quanjp1 Fig.\u00a0N. In conjp1 Fig.\u00a0. During jp1 Fig.\u00a0. This wajp1 Fig.\u00a0N.61 we aimed to investigate whether interfering with the PDGF-CC/PDGFR\u03b1 axis is responsible for the imatinib-mediated preservation of BBB integrity in EAE by analyzing the effect of genetically reducing PDGF-CC levels utilizing Pdgfc deficient mice and neutralizing PDGF-CC monoclonal antibodies (mAb), respectively, in autoimmune neuroinflammation. Since Pdgfc-/- mice on C57BL/6 background show multiple congenital defects in the CNS we decided to use heterozygous mice24. Pdgfc+/- mice showed amelioration of disease symptoms compared to wild type littermate controls . For the antibody experiments, we utilized PDGF-CChum mice, which have a partially humanized PDGF-CC growth factor domain66. PDGF-CChum mice were used in order to allow in vivo antibody blocking with a murine anti-human PDGF-CC mAb. The mice were injected i.p twice weekly with 15\u00a0mg/kg anti-PDGF-CC mAb 6B3 or control IgG (BM4) from day 2 post MOG-immunization until the end of the experiment. Notably, in contrast to the control group, PDGF-CChum mice injected with mAb 6B3 exhibited less severe EAE course from the progression phase until the end of the experiment  in mice  Fig.\u00a0L. The PDTnfa and Il1a at the BBB, which in turn preserves the integrity of the BBB and ameliorates the disease. In addition, upregulation of PDGF-CC and its receptor PDGFR\u03b1 in MS indicates relevance of these targets in the disease treatment.Taken together, our data show that blocking PDGF-CC in MS-like neuroinflammation decreases endothelial cell activation and downregulates 52. By systematic gene profiling of ex vivo isolated endothelial cell enriched vascular fragments from three subsequent disease phases of EAE , we have generated a unique BBB transcriptome database with correlation to structural and functional changes at the BBB. The uncovering of disease phase-related transcriptional differences at the BBB presents a novel source of potential candidate genes that can be targeted at different phases of MS. Combined with data from our previous study2, the present study strongly suggests modulation of PDGFR\u03b1 signaling as a novel therapeutic approach to restore BBB integrity during the progression phase of EAE, which in turn attenuates further disease exacerbation. As initially reported2 and now functionally demonstrated, blocking PDGFR\u03b1 with the small tyrosine kinase inhibitor imatinib or its ligand PDGF-CC with neutralizing antibodies, respectively, resulted in EAE amelioration and a better-preserved BBB.Loss of BBB integrity and subsequent recruitment of immune cells into the CNS parenchyma is a hallmark of MS and its animal model, EAE57, and similar protective effect as seen with imatinib was observed upon specifically blocking PDGF-CC in EAE-induced mice. Restoration of BBB function and integrity by blocking PDGF-CC is likely based on downregulation of the endothelial cell activators TNF-\u03b1 and IL-1\u03b1 since Tnfa and Il1a expression were specifically increased in CNS vascular fragment isolates upon PDGF-CC ICV injections and correspondingly found to be downregulated in MOG-immunized, PDGF-CChum mice treated with the PDGF-CC blocking mAb. Indeed, TNF-\u03b1 and IL-1 have been shown to potentiate BBB disruption in vivo and endothelial cell permeability in vitro47.Based on transcriptome and pathway analyses of endothelial cell enriched vascular fragments of the BBB we here show that blocking PDGFR\u03b1 in EAE with imatinib resulted in downregulation of multiple signaling pathways critical for endothelial cell activation, immune cell transmigration and angiogenesis during disease progression. Amelioration of the clinical symptoms in the imatinib-treated group was associated with reduced TJ degradation and better preservation of BBB function and integrity. PDGF-CC is the prevailing PDGFR\u03b1 ligand regulating BBB integrity39. Endothelial IL-1R1 has an effect on both adhesion and transmigration of leucocytes across the BBB and recruitment of additional leucocytes45. TNF-\u03b1 and IFN\u03b3 modulate the expression of a wide variety of chemokines, cytokines and CAMs for example CXCL8, CXCL9, CXCL10, CX3CL1, CCL2, CCL3, CCL4 and CCL564, promoting both adhesion of leucocytes to endothelial cells and migration of leucocytes across the BBB endothelial cells through upregulation of ICAM-1 and VCAM-164 as well as E- and P-selectins63. We found downregulation of Ccl2, Ccl3, Ccl4, Cxcl9, Cxcl10, Icam1, Sele and Selp in vessel isolates of imatinib-treated mice during disease progression. Thus, our results suggest that the pathway initiated by the PDGF-CC/PDGFR\u03b1 signaling in the NVU potentially regulates the expression of TNF-\u03b1 and IL-1\u03b1 during EAE, and through these factors increase endothelial cell activation and ultimately disruption of BBB integrity. The presumed endothelial source of TNF-\u03b1 and IL-1\u03b1 expression in our expression analysis is intriguing and may represent an autocrine mode of action.Endothelial cells can be activated by proinflammatory cytokines such as TNF-\u03b1, IL-6, IL-1 and IFN\u03b3, which facilitates the recruitment and attachment of circulating leucocytes to the vessel wall. For example, knock down of IL-1R1 in endothelial cells delays EAE onset and severity44.Upregulation of proinflammatory markers in endothelial cells in EAE is in line with a recent publication based on a different approach to endothelial cell isolation and high sensitivity RNA sequencing instead of microarray-based analysis, strengthening the concept of BBB dysfunction and an altered BBB immune profile contributing to CNS diseases4. In addition, in MS, an increased tPA expression in neurons and in perivascular inflammatory cells has been documented, and high tPA activity in the circulation has been shown to correlate with disease progression16. Similarly, data from animal studies show correlation of plasma tPA levels with the clinical signs of EAE48. tPA activity is also increased in inflammatory lesions in the EAE model60 and tPA-/- mice show a delayed onset of EAE; however, symptoms are more severe42. The delayed onset can possibly be explained by decreased BBB leakiness in the acute phase due to lack of tPA-mediated PDGF-CC activation, whereas the more severe delayed symptoms can be explained by less regenerative capacity in the absence of tPA. Fibrin is known to limit axonal regeneration20 and genetic deficiency of the endogenous tPA inhibitor PAI-1, leading to increased tPA activity, resulted in higher fibrinolysis capacity, accompanied by less axonal damage and brain inflammation3. This is in line with our data since we show that imatinib and mAb 6B3 are most effective during the EAE progression phase.Upregulation of PDGF-CC in the vascular bed of MS lesions was accompanied by PDGF-CC\u2009+\u2009infiltrating immune cells. Of note, we also detected upregulation of PDGFR\u03b1 around the vessels in MS lesions in contrast to NAWM. This suggests that the PDGF-CC/PDGFR\u03b1 axis also plays an important role in MS. Activation of PDGF-CC/PDGFR\u03b1 signaling during MS is potentially due to tPA-mediated activation of latent PDGF-CC in the CNS. Indeed, elevated tPA levels in CSF have been reported in MS patients33. On the other hand, PMS disease course is characterized by less extensive inflammation behind a nearly intact BBB27. While expression profiles of the brain vasculature during the progression phase of EAE exhibit similarities with pathological changes at the BBB during disease relapse in RRMS55, it is tempting to speculate analogy between the remission phase of EAE and PMS. In this sense, the remission phase of EAE was characterized by: (i) downregulation of proinflammatory and proangiogenic cytokines at the BBB comparing to the progression phase, (ii) partially restored BBB including upregulation of Cldn1, which was previously shown to facilitate resealing of the TJ of the compromised BBB in EAE46 and (iii) an upregulation of various transcripts involved in lipid and cholesterol metabolism, especially those important for fatty acid and cholesterol transport. Interestingly, simvastatin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor used for treating hyperlipidaemia, was associated with overall reduced brain atrophy in secondary progressive MS (SPMS)13. Increased levels of plasma cholesterol and triglyceride-rich lipoproteins (TGRL) have been associated with cerebrovascular inflammation, vascular dementia and Alzheimer\u00b4s disease35 and increased TGRL lipolysis were shown to disturb the BBB and induce lipid droplets in astrocytes34. The accumulation of lipid droplets was associated with upregulation of the proinflammatory NF\u03baB pathway and secretion of proinflammatory cytokines. Moreover, glial lipid droplets and reactive oxygen species (ROS) formation attributable to mitochondrial dysfunction were shown to promote neurodegeneration41. Thus, our data indicating increased cholesterol uptake and esterification, as well as increased lipid uptake during the remission phase of EAE, may implicate accumulation of lipid droplets at the BBB. Thus, a thorough investigation of lipid and cholesterol metabolism during the remission phase of EAE may contribute to further elucidation of MS pathology.Current knowledge suggests differences in pathological mechanisms underlying early and late stages of MS, which are commonly manifested as relapsing\u2013remitting MS (RRMS) and progressive MS (PMS) clinical course, respectively. Thus early stages of MS are associated with profound BBB leakage, as demonstrated by contrast enhancement on magnet resonance (MR) images, which enables entry of circulating inflammatory cells into the CNS26, imatinib exhibit much less severe side effects and is used for treating cancer since many years43. In blood, imatinib is strongly bound to plasma proteins, e.g. albumin and alpha-1-acid glycoprotein and will gain access to the CNS parenchyma by co-leakage through a permeable BBB8. Imatinib will however also be transported out from CNS via the P-glycoprotein efflux system, probably explaining the higher doses needed for CNS implications62. Recently, a phase II clinical trial (NCT03674099) to test safety and efficacy of imatinib treatment in RRMS was initiated. In addition to the PDGFR\u03b1 antagonist imatinib, the anti-PDGF-CC mAb 6B3 represents a prospective therapy for brain disorders characterized by a leaky BBB. Blocking PDGF-CC with mAb 6B3 could provide an even more superior treatment for MS than imatinib, since a mAb likely excludes off-target effects, and thus reduces potential adverse side effects. Importantly, in a long-term toxicology study in mice with mAb 6B3 we could not observe any adverse side effects66.Understanding molecular mechanisms controlling BBB function and integrity in health and disease is a prerequisite for developing novel therapeutic strategies. Unlike currently available MS treatments such as natalizumab, a humanized antibody against the cell adhesion molecule \u03b14-integrin which is associated with developing PML, an opportunistic infection caused by the John Cunningham (JC) virus37, spinal cord injury (SCI)1, traumatic brain injury (TBI)58 and seizures25. We have also shown that targeting PDGFR\u03b1 signalling with imatinib reduces stroke volume and BBB disruption after MCAO in mice57. Interestingly, we could detect an extensive overlap of differentially expressed transcripts and disease-promoting pathways at the BBB between the data sets from progression phase of EAE and 24\u00a0h post MCAO, as well as with published datasets from various other CNS disease models44, which points to a common injury response elicited at the BBB in the respective disease models.Besides EAE, beneficial therapeutic effects of imatinib treatment were also demonstrated in animal models of other neuropathologies associated with a compromised BBB such as amyotrophic lateral sclerosis (ALS)In summary, we here show that blocking either PDGFR\u03b1, or its ligand PDGF-CC, leads to amelioration of a MS-like neuroinflammation through restoration of BBB function and integrity. Since PDGF-CC/PDGFR\u03b1 signaling-related disruption of the BBB and neurovascular dysfunction are common features of several neuropathologies, agents blocking this pathway are likely to attain larger therapeutic range than currently indicated.Supplementary Information 1.Supplementary Information 2."}
+{"text": "Biobased binders were synthesized by batch miniemulsion polymerization of 2-octyl acrylate and isobornyl methacrylate monomers using a phosphate polymerizable surfactant (Sipomer PAM200) that lead to the formation of phosphate functionalized latexes. Upon the direct application of such binders on steel, the functionalized polymer particles were able to interact with steel, creating a thin phosphatization layer between the metal and the polymer and avoiding flash rust. The in situ incorporation of the CeO2 nanoparticles during the polymerization process led to their homogeneous distribution in the final polymer film, which produced outstanding anticorrosion performance according to the Electrochemical Impedance Spectroscopy measurements. In fact, steel substrates coated with the hybrid polymer film (30\u201340 \u00b5m thick) showed high barrier corrosion resistance after 41 days (~1000 h) of immersion in NaCl water solution and active inhibition capabilities thanks to the presence of the CeO2 nanoparticles. This work opens the door to the fabrication of sustainable hybrid anticorrosion waterborne coatings.CeO Nowadays, mild steel is one of the most important materials in construction, industry and transportation because of its versatility and good mechanical properties. Nevertheless, the main drawback of steel is its susceptibility to deterioration by corrosion, which causes dramatic economic losses . Such barrier capabilities are mainly provided by the polymer matrix in organic coatings, where solvent-based polymers are the most popular among the commercial ones. However, due to the more and more demanding environmental regulations on the emission of volatile organic compounds (VOC), the coating market is moving towards the use of waterborne coatings.Waterborne coatings are based on polymer latexes, and even if they are an excellent environmentally friendly alternative to solvent-based coatings, their main drawback is the inherent higher hydrophilicity of the formed films due to the presence of surfactants and salts (needed for the synthesis of the latex). Films cast from waterborne latexes have shown to present higher permeability to water than the ones cast from solvent-based systems ,4,5. ThiNevertheless, an important challenge while designing an environmentally friendly waterborne coating is the replacement of oil-based monomers by biobased ones to reduce the overall carbon footprint of the final product, while maintaining or improving its performance. In fact, the market demand of biobased paints and coatings has constantly increased in the last years ,13. The 2) [2) [Apart from the barrier properties, anticorrosion properties of coatings can also be improved by adding corrosion inhibitors. Most chemical inhibitors reduce the rate of corrosion forming a passive adsorption layer on the metal surface . Chromat2) ,31,32,332) [2)  and zinc2) [2) ,36) have2) [2) ,38,39,402) [2) ,42.2 nanoparticles as inhibitor have been successfully synthesized and assessed for the production of direct to metal sustainable coatings.In this work, and for the first time, a high biobased content waterborne anticorrosion binder containing a phosphatizing agent and CeO\u00ae PAM200 (Solvay) were used as received. Octadecyl acrylate  was used as co-stabilizer during the miniemulsion polymerization. A solution of CeO2 nanoparticles (NANO BYK 3812) was kindly supplied by ALTANA . In order to obtain the pure nanoparticles, the solvent was evaporated in an oven at 60 \u00b0C for 48 h. The resulting crystals were grinded before their use. Distilled water  was used in all reactions. Sodium bicarbonate  and ammonium hydroxide solution  were used to adjust pH values. Steel substrates (medium carbon steel with 0.5% of C) were purchased from URDURI ACEROS. UniClean 251  was used as a degreasing agent for the steel substrates. HCl 1 M solution  was used in the cleaning treatment of the steel substrates. High purity NaCl  was used for the preparation of a 3.5 wt% solution for the corrosion test.IBOMA  and 2-OA  monomers were used as supplied. The thermal initiator azobisisobutyronitrile  and the polymerizable surfactant Sipomer2, named Bioacrylic and CeO2-Bioacrylic, respectively) were synthesized by batch miniemulsion polymerization. The used recipe is shown in 2) powder. This mixture was stirred magnetically for 5 min. The aqueous phase was obtained by dissolving the Sipomer\u00ae PAM200 in water and adjusting the pH to 7 adding ammonium hydroxide dropwise. Both phases were mixed for 5 min under magnetic agitation and then sonified for 20 min using a Branson 450 w. During sonication, the flask was immersed in an ice bath to avoid overheating. The miniemulsion was later charged into a 0.5 L glass jacketed reactor fitted with a reflux condenser, a sampling device, a N2 inlet and a stirrer rotating at 150 rpm. The temperature was controlled by an automatic control system . After reaching the desired temperature (70 \u00b0C), a shot of AIBN initiator was added. The reaction was carried out for three hours.Two different latexes  was used to measure the z-average diameter of the miniemulsion droplets and final polymer particles. The morphology of the final latex particles and of the cryosectioned films was analyzed by Transmission Electron Microscopy (TEM) in a TECNAI G2 20 TWIN (FEI) operating at an accelerating voltage of 200 kV in a bright field image mode. Polymer particles and films were observed without any staining.For water uptake experiments, the films were formed by casting the latexes (around 1.5 g) onto round silicone molds and drying them at 24 \u00b1 2 \u00b0C and 50 \u00b1 5% relative humidity during 6 days until a constant weight was achieved. These films were carefully peeled from the silicone mold and immersed in distilled water during fourteen days. Films were withdrawn from the water container at 24 h intervals, they were smoothly dried and quickly weighed.For the water static contact angle and EIS measurements, 90 \u00b5m wet thick films were cast onto steel plates. Steel substrates were degreased with UniClean 251 solution at 70 \u00b0C in a shaking bath for 5 min, followed by 1 min pickling in HCl solution (1:1). Then, the waterborne latexes were uniformly applied on the steel substrates using a quadruple film applicator (Khushbooscientific). Films were dried at a relative humidity of 60% and a temperature of 23 \u00b0C for 24 h using a humidity chamber (ESPEC SH-641 benchtop type). The contact angle of the film\u2013air interface was measured in a Contact Angle System OCA  equipment, taking an average value from 20 measurements.2. Although OCP was monitored continuously with time, it was interrupted to carry out EIS measurements (once per hour). The frequency range was from 100 kHz to 10 mHz, obtaining 10 points per decade. Frequency scans were carried out by applying \u00b1 10 mV sinusoidal wave perturbation versus OCP.A potentiostat  was used to evaluate the corrosion behaviour of the systems by electrochemical measurements: open circuit potential (OCP) and EIS. The following three electrodes configuration was used: Ag/AgCl saturated with KCl as reference electrode, platinum mesh as a counter electrode and coated steel specimens as working electrode. OCP and EIS tests were conducted in 3.5 wt% NaCl solution at room temperature at least by triplicate, using an area of 1 cmTg of \u221244 \u00b0C and 150 \u00b0C, respectively. For coating formulations, the Tg of the used polymer should be below the application temperature  in order to form a coherent film, but at the same time it should be high enough to produce good mechanical properties and avoid problems such as dirt pick up and blocking. The Tg of the polymers used for coatings is usually around 10\u201315 \u00b0C. Therefore, the 2-OA/IBOMA ratio used in this work was 58.5/41.5 wt% in order to obtain a copolymer with a Tg of 10 \u00b0C. Badia et al. [\u00ae, an allyl polyglucoside maleic acid ester functional monomer, in combination with 2-OA, IBOMA and butyl acrylate . To the best of our knowledge, this is the first time that 2-OA and IBOMA are used as sole monomers in a coating formulation.In this work, high biobased content latexes were produced using 2-OA and IBOMA as monomers. 2-OA is a soft monomer whereas IBOMA is a hard one; their homopolymers have a a et al.  synthesi2, less than 5% coagulum was obtained in the sample with 1 wbm % of CeO2, which can be attributed to a certain incompatibility between the CeO2 nanoparticles and the Sipomer PAM200, as observed previously with ZnO by Chimenti et al. [i et al. .2 nanoparticles (a) and the cryosections of the film produced by casting such latex (b).2 nanoparticles did not aggregate during the polymerization but migrated to the surface of the polymer particles as in Pickering stabilized latexes. The lack of aggregation of the individual CeO2 nanoparticles was also proved by XRD of the hybrid CeO2-Bioacrylic films, by which an average CeO2 nanoparticle size of 6.8 nm, close to the original CeO2 size [2 nanoparticles do disperse well in the monomer mixture (see 2 in 2-OA/IBOMA) as they do in a mixture of MMA/BA/AA [2 nanoparticles agglomerated , a single nanoparticle aggregate per polymer particle was found at the edge of the polymer particles [2 nanoparticles were initially well dispersed inside the MMA/BA/AA droplets, but as polymerization proceeded, they aggregated due to the incompatibility between the formed copolymer and the modified CeO2 nanoparticle surface, leading to the formation of a single larger CeO2 nanoparticle aggregate [The morphology of the hybrid polymer particles and the film shown in articles ,46,47,482 nanoparticles (which was not disclosed by ALTANA), made the CeO2 nanoparticles to migrate to the monomer droplet/aqueous phase interface (polymer particle/aqueous phase after polymerization). This surfactant\u2013nanoparticle interaction could be the reason for the decreased stabilization capability of the surfactant, causing the formation of some coagulum in this polymerization. Anyway, good quality clear films were obtained after casting Bioacrylic and CeO2-Bioacrylic latexes in silicone molds, even if the hybrid one had a slight yellowish color due to the presence of CeO2 nanoparticles  did not have a significant detrimental effect on the hydrophobic properties of the films.Hydrophobic films were obtained, providing low water uptake values and high contact angle compared to values reported in the literature for acrylic latexes stabilized by polymerizable surfactant (18% of water uptake in 14 days and 75\u00b0 contact angle for an MMA/BA acrylic binder ). The hy2 nanoparticles, intact films (both neat and hybrid) with similar thickness (30\u201340 \u00b5m) were evaluated by EIS. 9\u20131010 \u03a9cm2 (|Z|0.01Hz). It indicates an excellent barrier protection compared to acrylic waterborne coating without [2 during exposure to NaCl electrolytes [0.01Hz in more than one order of magnitude, reaching a value around 108 \u03a9cm2. The better performance of the hybrid coating can be justified by the physical blocking effect to the electrolyte diffusion thanks to the CeO2 nanoparticles [2 nanoparticles located in the surface of the polymer particles.In order to study the effect of the CeO without  and with without  or to eptrolytes ,51, wherarticles ,52, takiarticles . Therefo2 nanoparticles [2-Bioacrylic film showed an exponential increase of the potential from \u22120.52/\u22120.51 V (11/12 h) up to \u22120.02 V (94 h), which is in agreement with the potential trend observed for a waterborne acrylic coating doped with CeO2 nanoparticles acting as corrosion inhibitor [2 nanoparticles grafted with ferrocene, where the formation of a passive layer provided less negative  OCP values over a large period of time (30 days) [In order to confirm such hypothesis, an artificial defect has been created by laser on both films. The aim was to reach the metal/film interface in order to explore the inhibition capabilities of CeOarticles . Just imarticles , and it nhibitor . The qua30 days) . Finally2 as a corrosion inhibitor. Therefore, a tailored analysis of the EIS diagrams was done for the different times of interest  for each time, it can be observed that |Z|0.01Hz was 6 \u00d7 105 \u03a9cm2 after 1 h of exposure. It was decreasing slightly to around 105 \u03a9cm2 after 11 h, indicating that the corrosion process was taking place up to then. However, this trend was drastically changed when a sudden increase of |Z|0.01Hz occurred at 12 h\u2014impedance reaching 106 \u03a9cm2 and 6 \u00d7 106 \u03a9cm2 at 12 and 13 h, respectively. A steady state value was maintained above 5 \u00d7 106 \u03a9cm2 from 13 h until 94 h of exposure, thanks to the corrosion inhibition capabilities of CeO2 nanoparticles. This is in agreement with the delay of the corrosion onset shown on coatings formulated with CeO2 nanoparticles having an artificial defect: the corrosion activity is limited to the vicinity of the defective area according to the results obtained by localized electrochemical techniques [5 \u03a9cm2), indicating that most probably the corrosion inhibition was exhausted and the metal/film interface on the artificial defect was not protected anymore.Apparently, the completely different behavior shown by both films, in terms of OCP, might be attributed to the role of CeOchniques . Finally5 \u03a9cm2) at low frequency (|Z|0.01Hz) compared to the hybrid film (6 \u00d7 105 \u03a9cm2), which also decreased to values below 105 \u03a9cm2 after 11 h of exposure. In contrast to the CeO2-Bioacrylic film, the impedance value slightly varied until a minimum was reached at 57 h (7 \u00d7 104 \u03a9cm2) due to the absence of any corrosion inhibition into the film. The slight |Z|0.01Hz increase (3 \u00d7 105 \u03a9cm2) observed after 65 h may be related to the presence of corrosion products that are blocking the pinhole/damage that was created with the artificial defect. It can be observed that |Z|0.01Hz remains in a very narrow range (7 \u00d7 104/3 \u00d7 105 \u03a9cm2) during the entire test, and it confirms that no protection can be achieved in the absence of CeO2 nanoparticles in the film formulation. Indeed, a |Z|0.01Hz value of 9 \u00d7 104 \u03a9cm2 was obtained after 95 h of exposure, indicating the absence of protection in the interface. Therefore, these results confirm that homogeneously distributed CeO2 nanoparticles are required into the film to provide an efficient corrosion protection of the metal/film interface.On the other hand, 2 biobased acrylic binders were synthesized by miniemulsion polymerization. CeO2 nanoparticles interacted with the phosphate moieties of the surfactant and migrated to the interphase leading to polymer particles with Pickering morphology. Thus, CeO2 nanoparticles did not aggregate and were well distributed in the surface of the polymer particles. The biobased acrylic copolymer, together with the phosphate surfmer used in the synthesis of the polymer particles, produced films with low water uptake and high contact angle to water. EIS results also showed enhanced barrier properties of both films, independently of the presence of CeO2 nanoparticles. However, the long-term durability of the intact hybrid film was higher than the neat one. This can be attributed to the corrosion inhibition capabilities of CeO2 nanoparticles, according to the results obtained when both types of films had an artificial defect. EIS diagrams showed an increase of the impedance modulus in one order of magnitude (|Z|0.01Hz went from 6 \u00d7 105 to 6 \u00d7 106 \u03a9cm2 after 13 h) thanks to key role of CeO2 nanoparticles. In fact, the neat film did not show any protection of the interface in the presence of an artificial defect.Novel waterborne hybrid CeO"}
+{"text": "Development of deep-learning models for intermolecular noncovalent (NC) interactions between proteins and ligands has great potential in the chemical and pharmaceutical tasks, including structure\u2013activity relationship and drug design. It still remains an open question how to convert the three-dimensional, structural information of a protein\u2013ligand complex into a graph representation in the graph neural networks (GNNs). It is also difficult to know whether a trained GNN model learns the NC interactions properly. Herein, we propose a GNN architecture that learns two distinct graphs\u2014one for the intramolecular covalent bonds in a protein and a ligand, and the other for the intermolecular NC interactions between the protein and the ligand\u2014separately by the corresponding covalent and NC convolutional layers. The graph separation has some advantages, such as independent evaluation on the contribution of each convolutional step to the prediction of dissociation constants, and facile analysis of graph-building strategies for the NC interactions. In addition to its prediction performance that is comparable to that of a state-of-the art model, the analysis with an explainability strategy of layer-wise relevance propagation shows that our model successfully predicts the important characteristics of the NC interactions, especially in the aspect of hydrogen bonding, in the chemical interpretation of protein\u2013ligand binding. Human-curated heuristics and descriptors have been used for decades in cheminformatics including early machine learning methods, and it is the representation learning of three-dimensional molecules that is one of the recent endeavors of deep-learning chemistry. The direct learning of molecular structures for the prediction of target properties, without prior assessment on the structures or quantum-chemical calculations, has been enabled by the remarkable discriminative ability of deep neural networks (DNNs)6. Compared with the physics-based computational methods for calculating molecular properties, the deep-learning approach offers a fast, but still powerful, option for estimating diverse characteristics of molecules through the data-driven discovery of molecular patterns8.The approach of deep learning has recently been adopted to the chemistry discipline for tackling diverse chemical tasks, such as prediction of physicochemical properties, protein\u2013ligand interactions, and retrosynthetic analysis10. The molecular graphs contain the structural information on molecules in two-dimensional (2D) space, with atoms as vertices and bonds as edges in the graphs. In the GNN, the neurons in a layer are connected to their graph neighborhoods, and layer stacking generates broader local structures in molecules. Many GNN models have been developed for the prediction of molecular energies8, physical properties12, protein interactions14, and biochemical functions16. Since the pioneering report by Baskin et al.17 on the utilization of molecular graphs for the prediction of physicochemical properties of hydrocarbons and other molecules, Duvenaud et al.11 and Kearnes et al.15 have examined the GNN approaches on the prediction of molecular properties, and the GNN architecture has further been refined to message-passing neural networks (MPNNs) that outperform other machine-learning methods based on molecular fingerprints7. Moreover, the expansion of the GNN architecture into the 3D space for modeling the actual molecular structures has recently been explored, and the efficacy of the GNN approach on the problems requiring 3D molecular structures has been proven18.The recent rise of graph neural networks (GNNs) has upscaled deep-learning capability in chemistry with the easy handling of molecules as molecular graphs, which are defined by two sets of vertices and edges23. The essence of DNN models for deep-learning scoring compared to the force field energy functions and scoring functions is the appropriate database to learn molecular patterns and their relationship to binding affinity, and they are strongly linked to prediction performance. The PDBbind database25 is the most widely used dataset, which is a curation of 3D protein\u2013ligand structures obtained from X-ray crystallography and multidimensional NMR techniques with complementary binding affinities, for training DNN models on prediction of the binding constant from a complex structure. Many DNN models were developed based on the PDBbind database and can be classified into two categories: convolutional neural networks (CNNs) with voxelized images and GNNs working on graph representations of complexes.One of the focused fields of deep learning in chemistry is the replacement of the scoring function on the structure-based drug design with data-driven DNN models27. Protein\u2013ligand complexes were transferred into the angstrom-level voxel grid and used for training the CNN models. Meanwhile, the GNN models14, which focused on the interpretation of molecular bonding  as graph edges, was utilized after the success of CNN models by incorporating noncovalent (NC) interactions as graph edges in molecular graphs, which play significant roles in the programmed formation of 3D molecular structures of biomolecules  and polymers and their dynamics30. In the GNN models, NC connectivity was utilized in conjunction with covalent connectivity for post-refinement of atomic features after the convolution with covalent-bond connectivity. Gaussian decay functions have been used to mimic decreased influences from distant atoms, or multiple kernels for distance bins have been adopted for simulating NC interactions31. These approaches enrich the graphic representation of molecules by adding topological information and acquiring the shape-awareness, which has only been feasible in the CNNs. In addition to the decay simulation, an approximation of the entire atomic contribution to a smaller subset was widely utilized22. Due to the extremely large number of atoms in the protein\u2013ligand complex compared to other molecules in molecular property datasets, training the complex data is challenging for both CNN and GNN models. By limiting the protein atoms into a spatial neighborhood of the ligand molecule, the complex can be greatly reduced into smaller sizes and trained efficiently without losing important interactions. Owing to the aforementioned advanced approaches, the GNN models became a competitive option for developing deep-learning scoring models with a direct interpretation of molecular structures.Rapid development of high-performance and deeply-stacked CNN models in computer science has dramatically raised the prediction performance of binding constants through enhanced pattern recognition of the 3D molecular images[C] and CV[NC] layers, respectively), and evaluate the significance of NC convolution. There have been reports on the incorporation of NC connectivity in GNN models, but in strict combination with covalent connectivity. Here, we apply the covalent and NC connectivity separately to investigate the importance of each convolution layer, which has not been explored. In extreme cases, only CV[NC] is used, without any CV[C] layers, and compared to other models. Moreover, we investigate the optimal cropping strategy for downsizing the protein\u2013ligand structure and efficient training. Based on the findings, we further investigate the explanations for the predictions of the trained model, i.e., how the trained model predicts for the first time from the given input data in the protein\u2013ligand complexation problem. By performing decomposition-based, layer-wise relevance propagation (LRP)33 on behalf of explainable AI36 and visualizing the obtained atomic contribution for the prediction of the protein\u2013ligand complex, we explore the relationship between machine-predicted NC interactions and knowledge-based NC interactions from the molecular structures.In this paper, we propose a GNN architecture, denoted as InteractionNet, that directly learns molecular graphs without any physical parameters, wherein the NC interactions are encoded as graphs along with the bonded adjacency that models covalent interactions. We utilize the PDBbind dataset for evaluation of the concept and examine the model performance on predicting the binding constant from a complex structure. Specifically, we divide the convolutional layers in InteractionNet into two, the covalent and NC convolution layers (CV[C] and [NC]), similar to the PotentialNet reported by Feinberg et al.13[C] and [NC] are defined by the combination of molecular graphs for a protein and a ligand but with different connectivity strategies. The covalent adjacency matrix, [C], consists of the bond connectivity in the protein and the ligand, and is constructed by a disjoint union of the protein and the ligand graphs, maintaining the bond connectivity only within each molecule. The NC adjacency matrix, [NC], defined by a graph having full connectivity between the vertices of the protein and the ligand graphs, contains all the possible edges between the protein and the ligand but not within the same molecule  layer, and fully-connected (FC) layers. The node-embedding layers update the atomic feature matrix [C] and CV[NC] layers receive the node-embedded 13, we separate the graph convolution layers utilizing the two adjacency matrices, [C] and [NC], one-by-one at each layer  as our prediction target because the protein\u2013ligand binding is governed primarily by the NC interactions, not covalent bonds, which is important in investigating the efficacy of the proposed architecture. We conducted a 20-fold-cross-validated experiment on the refined set of the PDBbind v2018 dataset25, consisting of 4186 complexes and their experimental Kd values.We examined the efficacy of the CVIn the data preprocessing for model training, we cropped the protein structure for faster training and less memory consumption. The binding pockets of the proteins in the refined PDBbind set contained a maximum of 418 atoms, which was almost 16 times larger than the ligands that had only 26 atoms at maximum. We thought that the interactions between a protein and a ligand could be simulated with a smaller subset of atoms in the protein because the number of atoms that participated in the protein\u2013ligand binding is much less than the maximum value. The appropriate cropping strategy, without any loss in performance, is also highly important for efficient training, considering the exponential increase in the memory consumption of the training data. We utilized the spatial atom filtering for simplification of protein structures, which excluded the atoms of a protein distant from a ligand by the range cutoff. In detail, the shortest distance of a protein atom to the ligand atoms was measured, and the protein atom was excluded if the distance exceeded the predefined range cutoff. By spatial cropping with the range cutoff, we obtained a subset of the protein structures, similar to the shape of the van der Waals surface of the ligand but with a much larger radius, and used the subset for the generation of the molecular graphs.[C-NC] by changing the cutoff with 1-\u00c5 increment. The number of atoms included in the cropped complex increased linearly with respect to the cutoff, while the data size for the training dataset increased exponentially . The performance of InteractionNet[C-NC] was measured to be slightly higher than InteractionNet[NC], but the difference was not significant in statistical analysis (p\u2009=\u20090.450). These results indicated that the interactions between a protein and a ligand could be simulated accurately, even with a single CV[NC] layer, without any help from previous covalent-refinement steps. We compared the performance of InteractionNet with that of PotentialNet, which was the state-of-the-art GNN model for prediction of protein\u2013ligand affinity. For the comparison, we built an in-house PotentialNet model according to the original paper13. In detail, we replaced the CV[C] and CV[NC] layers of InteractionNet with the stages 1 and 2 of PotentialNet, respectively, and trained the in-house PotentialNet model with the same dataset used in InteractionNet. The averaged RMSE value for the test set was measured to be 1.343 in the case of the PotentialNet, which was comparable to that of InteractionNet .The root-mean-square error (RMSE) results from the cross-validation experiments, based on the 5-\u00c5 filtering of protein structures, confirmed that the CVhs Table . InteracKd values revealed a high correlation with the experimental Kd in a linear relationship  that modeled the noncovalent (NC) interactions and discussed the in-depth analysis of the model combined with the explainability technique for understanding deep-learning prediction. In the graph-based deep-learning models, there has been less attention to the NC interactions compared with the bonded interactions because of the ambiguity of NC connectivity. InteractionNet, presented herein, showed satisfactory predictive-ability for predicting the dissociation constant with RMSE of 1.321 on the PDBbind v2018 dataset. The NC convolution layers enhanced InteractionNet\u2019s prediction accuracy, even without the utilization of the traditional bonded connectivity. We further demonstrated that InteractionNet successfully captured the important NC interactions between a protein and a ligand from a given complex through posthoc LRP analysis. The visualization of the atomic contributions showed a strong correlation with the actual hydrogen bonds in the complex. In the case of the ligand that had multiple hydrogen-bond donors and acceptors, the positive atomic contributions were observed only on the atoms participating in the actual hydrogen bonds. We believe that our model would widen the applicable tasks of the chemical, deep-learning models to the problems beyond the bonded interactions within a single molecule and also provide a meaningful explanation for the prediction, enabling the real-world applications that require prediction evidence and reliability.25. We used the refined set from the provided dataset, consisting of 4462 protein\u2013ligand complexes with their experimentally measured Kd values. Initially, all protein\u2013ligand data were loaded by RDKit 2019.09.237 and Openbabel 3.0.038, and inspected for improper conformation. During the inspection process, the molecules that failed for loading were excluded from the training dataset for further tensorization. We also excluded the complexes that had the interatomic distance below 1\u00a0\u00c5 and/or the atomic collisions in the provided 3D molecular structures. Our inspection on the sanity of the molecular structure was performed to avoid any misguided training of the model with chemically abnormal data. The noncovalent adjacency relied heavily on the distances between the atoms in the complex, and the model without the sanity check would lead to inappropriate conclusions. After inspection, 4186 protein\u2013ligand complexes were obtained (see the Supporting Information for entire list of the PDB codes). The protein structure was cropped by retrieving the atoms of a protein within the range cutoff , and the size of the protein\u2013ligand complex structure was reduced for faster training. Only heavy atoms were considered in the entire preparation. Atomic features for building the feature matrix are listed in Table We employed the PDBbind v2018 dataset for the evaluation target of our InteractionNet models39 on Python 3.6.9. The training was controlled by learning-rate scheduling, early-stopping techniques, and gradient norm scaling. The learning rate was initially set to 0.00015 and lessened by a factor of 0.75 when the validation loss did not decrease within the previous 200 epochs, and the termination proceeded when the loss stopped decreasing for the previous 400 epochs. To avoid gradient exploding, a clipping parameter of 0.5 was used for gradient norm scaling. For the loss function, mean-squared-error (MSE) was used and optimized by the Adam optimizer40. The list of hyperparameters explored is described in Table https://github.com/blackmints/InteractionNet).All models were implemented by using TensorFlow 2.0.0[C] and CV[NC] layers, based on the guideline described elsewhere33. Obtained relevance for the atomic feature was reduced to the atomic contribution by summation across features. The graph-embedding layers were omitted for the relevance calculation, because the graph-embedding layers only redistributed the relevance between features, not between atoms, resulting in the same atomic contribution before and after redistribution. For the parameters \u03b5 and \u03b3, 0.25 was used for all LRP-\u03b5, and 100 was used for all LRP-\u03b3 layers. The cross-validation trial that was most similar to the average in root-mean-squared-error (RMSE) was used for LRP analysis, and the LRP examples were chosen from the test set of the trial, which were predicted accurately, for comparison with knowledge-based analysis. Three-dimensional visualization of the molecular structure was obtained by Mol*41, and the expected hydrogen bonds and hydrophobic contacts were determined by the rules RCSB PDB use45.We performed the LRP as a post-modeling explainability method. Three LRP rules, LRP-0, LRP-\u03b5, and LRP-\u03b3, were used for the calculation of relevance on each layer from the trained model. We adopted the LRP-0 for the output layer, LRP-\u03b5 for the FC layers, and the LRP-\u03b3 for the CVSupplementary Information."}
+{"text": "Emerging evidence suggests close domestic proximity of livestock and humans may lead to microbiological contamination of hands, objects, food and water supplies within domestic environments, adversely impacting public health. However, evidence quantifying the relationship between livestock, domestic animals, humans and microbiological contamination of household stored water remains limited.This longitudinal study aimed to examine the relationship between domestic contact with livestock and domestic animals on microbiological contamination of household Point-of-Use (POU) stored drinking water in rural Kenya and assess the influence of choice of faecal indicator on such associations.E. coli and intestinal enterococci. Livestock-related risk factors for water contamination were examined through multinomial regression, controlling for confounders.A survey was performed in 234 households in Siaya county, Kenya, to observe presence of livestock  and domestic animals  in household compounds, alongside other risk factors for contamination of POU stored drinking water such as sanitation, storage conditions and hygiene practices. Samples from water sources  and from POU storage containers were tested for E. coli) for households where goats were observed, and/or where poultry roosted in proximity to stored household water . Presence of a poultry coop was also associated with elevated intestinal enterococci densities . Associations between contamination and livestock risk factors were thus similar for both bacteria groups, but E. coli counts declined more rapidly following collection from surface waters than enterococci counts (p\u00a0=\u00a00.024).Rainwater was the main POU water source and was found to be highly susceptible to contamination. Multivariate analysis showed greater risk of gross (>100\u00a0CFU/100\u00a0mL) water contamination (with The presence of livestock (particularly goats) and poultry within household compounds increases POU water contamination risk, suggesting the need for improved interventions to address cross-contamination within rural domestic settings. Within Siaya county, more effective community education is needed to raise awareness of POU water quality protection, particularly of rainwater.  \u2022Poultry and goats are risk factors for household stored water contamination.\u2022E. coli contamination.Poultry are risk factors for both enterococci and \u2022E. coli.Attenuation of enterococci in household stored water is lower than for \u2022Residual free chlorine is mostly too low to prevent stored water recontamination. The proportion of population using safely managed drinking water services increased from 61 to 71 percent between 2000 and 2017 . AccordiE. coli and intestinal enterococci) to assess the health risks associated with drinking-water consumption. The presence of E. coli is associated with faecal contamination, and the WHO guidelines recommend the total absence of E. coli per 100\u00a0mL of drinking water. Presence of intestinal enterococci also indicates faecal contamination, but these microorganisms may persist longer in marine waters and be carried further than E. coli in the environment .This study aimed to add to this evidence-base by assessing the association between domestic contact with livestock and the microbial contamination of household POU stored water in Siaya County (Kenya), controlling for known risk factors for such contamination. As an auxiliary objective, the study also investigated whether apparent associations between POU water contamination and livestock are dependent on the choice of indicator bacteria group  and research took place in ten villages  on the s2.2We conducted a longitudinal observational study of livestock-related risk factors for contamination of POU water with faecal indicator bacteria. Eligible study participants were adult members of households participating in the ongoing PBASS study, with children aged 6\u201359 months as the cohort at greatest risk of diarrhoeal disease. The sample size was powered to detect differential proportions of contaminated drinking-water using preliminary effect size estimates from Ghana , in the absence of evidence from Siaya. In Ghana, approximately 70% of water samples were contaminated in non-cattle keeping households, 90% were contaminated in cattle-keeping households, and the proportion of contamination variance in cattle-keeping households explained by other covariates was estimated at 0.3. Within the study population, 55% of households owned cattle . Based o33.1Eligible households were randomly selected from lists of those participating in the PBASS study. After seeking informed consent, questionnaire interviews were conducted in the Dholuo language with adult respondents during an initial  and follow-up sampling visit . The initial and follow-up visits were intended to coincide with wet and dry seasons, respectively. To assess domestic contact with animals, interviewers observed the presence of livestock , dogs and cats in the compound during interview and observed evidence of animal presence in the home . Compounds were designated as fenced areas surrounding homes. Interviewers also assessed whether drinking-water containers were accessible to animals and where poultry coops were observed, asked whether poultry were permanently confined in such coops. To measure other known risk factors for stored water contamination, water storage characteristics (e.g. whether containers were covered) were observed  and resp3.2in situ for free residual chlorine using SenSafe Water Check test strips, capable of detecting 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.2, 1.5, 2.0, 2.6, 4.0, and >6.0\u00a0mg/L. The method is approved by the US Environmental Protection Agency (ITS Method 99-003) , observing how the respondent collected water. Samples were also tested  99-003) . On each3.3E. coli and intestinal enterococci.All sample bottles were kept in a cooled container (Approx. 4\u00a0\u00b0C) and transported within 4\u00a0h to the Kenya Medical Research Institute (KEMRI) laboratories in Kisian, Kisumu. Samples were either processed immediately or kept in the fridge (4\u00a0\u00b0C) and processed within 24\u00a0h. Microbiological water quality was assessed via faecal indicator bacteria (FIB), namely Escherichia coli and total coliforms, and ISO 7899-2:2000 for Intestinal enterococci) and was performed using membrane filtration (Enumeration of FIB followed ISO standard methods (ISO 9308-1:2014 for ltration . As piloE. coli detection: The filters were placed onto coliform chromogenic agar (CCE) agar (Difco\u00ae) in \u00d8 55\u00a0mm petri plates (Fisher\u00ae). Plates were then incubated upside down for 24\u00a0\u00b1\u00a02\u00a0h at 37.0\u00a0\u00b1\u00a00.5\u00a0\u00b0C. Colonies that showed shades of dark-blue to violet were counted as E. coli, while those that appeared pink to red-coloured were recorded as presumptive coliforms  that were not E. coli (ISO 9308-1:2014)  .Intestinal enterococci detection: The filters were placed onto Slanetz and Bartley agar (Oxoid\u00ae) in \u00d8 55\u00a0mm petri plates (Fisher\u00ae). Plates were then incubated upside down for 48\u00a0\u00b1\u00a02\u00a0h at 37.0\u00a0\u00b1\u00a00.5\u00a0\u00b0C. Colonies (raised) coloured red, maroon or pink were counted as presumptive intestinal enterococci (ISO 7899/2:2000) .From the four volumes  filtered for each sample, the plate used for enumeration was based on the highest countable volume (100\u00a0mL) that was not TNTC. All FIB results were expressed as colony-forming units (CFU) per 100\u00a0mL.3.4in-situ station observations with satellite-based precipitation estimates. The 5\u00a0km\u00a0\u00d7\u00a05\u00a0km CHIRPS grid cells were overlaid on study village boundaries using ArcGIS 10.4.1 . For each fieldwork date between March 12, 2018 and February 18, 2019, average daily rainfall over the entire study area was determined as the area-weighted average of overlapping grid cell values.Given reported associations between water supply contamination and rainfall , daily r3.5E. coli and intestinal enterococci were used in the main statistical analyses, since they are WHO's chosen indicator microorganisms for assessing microbiological drinking water quality. Water source types with small sample sizes were grouped prior to analysis as follows: \u2018Piped\u2019 , \u2018Well/Spring\u2019 , \u2018Surface Water\u2019 . Boreholes and harvested rainwater were included in analysis without grouping. Kruskal-Wallis statistical analyses were performed to test for significant differences in E. coli and enterococci levels between the five source type groups .Statistical analysis was undertaken in IBM SPSS v25.0 and Stata v16 .Only E. E. coli and 20,200\u00a0CFU/100\u00a0ml for intestinal enterococci). Logged POU FIB counts and the change in counts between source and POU were then plotted as strip charts for surface and piped water supplies, the only source types with sufficient data for such visualization. A Wilcoxon signed rank test for matched pairs was used to test for differences in attenuation for E. coli versus enterococci.To visualise bacterial contamination data, bacteria counts were logged, first replacing left-censored values with 0.5 and right-censored with values immediately above the upper limit of detection , medium (10\u201399\u00a0CFU/100\u00a0ml) or high (\u2265100\u00a0CFU/100\u00a0ml) faecal contamination. Following examination of cross-tabulations of FIB levels versus risk factors, univariate multinomial regression was used to examine the relationship between each risk factor and contamination. Robust regression in Stata v16  was used44.1Most stored drinking-water was harvested rainwater, corresponding to 93.1% and 43.6% of samples from visit 1 and 2, respectively. As average daily rainfall fell between visit 1 (7.749\u00a0mm/day) and visit 2 (2.354\u00a0mm/day), use of non-rainwater sources increased significantly , as did the range of source types used (six in visit 1 versus nine in visit 2). It was not possible to collect \u2018paired\u2019 source samples to compare with household water in 218 households during visit 1 (and 100 households during visit 2), because either rainwater had been harvested directly into storage vessels, or there was no longer harvested rainwater available. Across both visits, respondents were unsure of the exact source of stored water on 46 occasions, which also prevented \u2018paired\u2019 source samples being collected. Nine sources had run dry during visit 2, so could not be sampled. Thirty-nine (47%) of 83 water source samples were collected within 2 days of POU sampling, but 34 (41%) were collected on dates more than a week apart. Thirty-eight (46%) of source samples were collected prior to POU samples, 33 (40%) afterwards, and 12 (14%) on the same day.4.2Most household survey respondents were the household head's spouse (73.5%), the person responsible for domestic water management (81.6%), and female (83.2%). To store drinking-water, 60.3% of households used a small container (\u226420\u00a0L); 31.3% used a large container (>20\u00a0L and\u00a0\u2264\u00a0100\u00a0L) and 7.5% used a water tank (>100\u00a0L). 98.1% of these containers/tanks were located inside the household. 52.8% self-reported that they did not perform any water treatment. Of those that did, 6.7% boiled their water; 18.5% used chlorination; 5.0% added coagulant; 27.2% strained water through a piece of cloth and just 0.6% filtered water. 74.8% of households had a pit latrine with slab and 5.8% had a ventilated improved pit latrine (VIP), both constituting improved sanitation. However, 16.4% of households practiced open defecation (OD) and 4.5% had a pit latrine without slab, constituting unimproved sanitation. Interviewers observed poultry in 90.5% of household compounds; cattle in 53.7%; dogs in 48.5%; cats in 45.9%; and goats in 35.1%. 65.7% of drinking-water vessels were accessible to domesticated or wild animals.4.3E. coli and intestinal enterococci) by source of POU stored water.Summary statistics for FIB levels according to the source of stored water can be found in supplementary material (SM1). E. coli (Md\u00a0=\u00a017.50\u00a0CFU/100\u00a0mL) for all source types with the exception of water kiosks. E. coli and intestinal enterococci) by water source type .Overall detected levels of intestinal enterococci (Md\u00a0=\u00a069\u00a0CFU/100\u00a0mL) were statistically (p\u00a0<\u00a00.001) above those of E. coli compliance with the WHO guideline value (\u20180\u2019 or not detectable in 100\u00a0ml). Supplemental material SM3 shows percentages of samples by FIB health risks categories  during tE. coli and enterococci in 100\u00a0mL of POU water, respectively and met WHO (2011) and (EU (1998) guidelines. High faecal contamination levels were observed in POU water, with 33% of samples for E. coli and 48% for intestinal enterococci constituting a \u2018moderate risk\u2019 to population health, with 23% for E. coli and intestinal enterococci a \u2018high\u2019 or \u2018very high\u2019 risk (Only 20% (46/229) and 6% (13/229) of samples from visit 2 were negative for the presence of gh\u2019 risk .Strip tests indicated six of 50 samples (12%) reportedly treated using home chlorination contained a free chlorine residual of\u00a0\u2265\u00a00.2\u00a0ppm, whilst 12 (24%) contained no detectable free chlorine residual. Two of 59 (3.4%) piped water samples contained a free chlorine residual of\u00a0\u2265\u00a00.2\u00a0ppm, with 23 (39.0%) containing no detectable free chlorine residual.4.4E. coli and intestinal enterococci contamination levels in POU stored drinking water are cross-tabulated against risk factors associated with animal contact. Supplementary Material Table SM4 contains similar information, but includes all risk factors controlled for in regression analysis of POU water contamination.E. coli, samples from households where coops were used to confine poultry had the highest percentage of samples containing >100\u00a0CFU/100\u00a0mL of intestinal enterococci (52.0%). In all such cases, poultry were allowed to roam free for some time.In contrast to E. coli only when poultry spent the night in proximity to stored water   and where poultry reportedly spent the night near stored water . Both risk factors remained significant in models adjusted for confounders . For the unadjusted model of E. coli, the risk of water being grossly contaminated was significantly higher for households in which storage containers were accessible to animals  (E. coli contamination in unadjusted models. Absence of a lid/cover on water storage vessels increased the risk of high E. coli contamination (SM5).In unadjusted multinomial regression analysis, observations of domestic animal contacts were associated with medium contamination with =\u00a00.044) , Table 5=\u00a00.001) , but notFor the unadjusted model of intestinal enterococci, the risk of water being grossly contaminated was significantly greater for households where either cats  and/or poultry  were observed, and/or when storage containers were accessible to animals . These variables were no longer significant in a preliminary multivariate model, however. Also, the presence of a coop to confine poultry was associated with greater risk of high contamination . Furthermore, this risk factor remained significant in the adjusted model  . Of the 4.5E. coli. However, POU samples originating from piped versus surface water sources had similar median levels of FIB. Since initial E. coli levels in surface source waters were greater than enterococci levels in such waters, E. coli counts thus declined more rapidly following water collection than enterococci counts, as shown by a Wilcoxon signed rank test (p\u00a0=\u00a00.024). As mentioned previously, 84% of surface water sources underwent treatment when compared to 24% of piped sources.Paired sample analysis , Fig. 7 5E. coli. This study thus meets calls for greater evidence on domestic exposure to contamination from livestock based on direct observations of livestock presence, as opposed to reported livestock ownership  also found evidence via MST molecular techniques of faecal contamination from ruminants  on children's hands and on household floors in Dhaka, Bangladesh. In Kakamega county (Kenya), Bacteroides MST marker present in stored drinking-water within 64% of participating households but not in source waters, suggesting post-collection ruminant faecal contamination. However, only 4% of the households visited in their study owned goats compared to 47% owning cattle. During the study design phase of our project, we hypothesized that cattle ownership was a likely risk factor for POU water contamination, based on the fact that cattle ownership has been shown to increase E. coli contamination of POU water in Ghana and Bangladesh  were present within household compounds.After controlling for other known risk factors such as inadequate sanitation, our findings suggests that the presence of poultry in household compounds (as indicated by poultry coops) is associated with high POU water contamination. The presence of goats was also associated with POU water contamination with wnership . Harvestwnership , rainwatwnership  is also ngladesh . HoweverE. coli outnumber intestinal enterococci in human faeces . In domestic animals, Harris et al. (2016) found E. coli concentrations to be 0.7, 3.0 and 2.0 Log10 higher than intestinal enterococci in chicken, cow and goat faeces, respectively. In our study, median enterococci levels in surface water samples were lower than E. coli levels  was generally consistent with findings of previous studies, suggesting plausible bacterial contamination patterns. For example, high cumulative rainfall preceding sampling increased risk of stored water contamination with both FIB in our study, and a Rwandan study  similarl Ecuador . However Ecuador , low-incFollowing systematic review recommendations , we meas5.1Cryptosporidium has been implicated in child diarrhoea in Siaya County  for this , which received funding from the 10.13039/501100000265UK Medical Research Council/10.13039/501100002992Department for International Development via a 10.13039/100016270Global Challenges Research Fund foundation grant (Ref.: MR/P024920/1). The study sponsors had no role in the subsequent execution of the study. This UK funded award is part of the EDCTP2 programme supported by the European Union.This research is a contribution to the OneHealthWater project  and the Kenya Medical Research Institute .http://doi.org/10.5255/UKDA-SN-854302. The datasets on precipitation used and analysed in this study are available from the CHIRPS website at http://chg.geog.ucsb.edu/data/chirps/.The datasets on water samples collected and household questionnaires used in the study are available from the corresponding author on reasonable request and are available in the UK Data Archive repository at Diogo Trajano Gomes da Silva (DTGS), Joseph Okotto-Okotto, Jim Wright (JW) and Thumbi Mwangi designed the study. Emmah Kwoba and Peggy Wanza undertook data capture and archiving. Oscar Mito and Frederick Ade undertook laboratory work. Weiyu Yu (WY) undertook spatial data capture and archiving. DTGS, JW and WY undertook visualization of the published work. DTGS and JW analysed data from the study. James Ebdon (JE) supervised the study. DTGS, JE and JW drafted the manuscript with the support of the other authors.The authors declare no conflict of interest."}
+{"text": "To evaluate the potential benefits of the Magnetic Resonance-guided high intensity Focused Ultrasound (MRgFUS) introduction in the clinical practice, for the treatment of uterine fibroids, in comparison with the standard \u201cconservative\u201d procedures, devoted to women who wish to preserve their uterus or enhance fertility: myomectomy and uterine artery embolization (UAE).A Health Technology Assessment was conducted, assuming the payer\u2019s perspective . The nine EUnetHTA Core Model dimensions were deeply investigated, by means of i) a literature review; ii) the implementation of health economics tools , to define MRgFUS economic and organizational sustainability, and iii) administration of specific questionnaires filled by uterine fibroids\u2019 experts, to gather their perceptions on the three possible conservative approaches .p-value<\u20090.001), and in patients\u2019 productivity loss . From an economic point of view, the Italian NHS would present an economic saving of \u2212\u20096.42%. A positive organizational and equity impact emerged regarding the capability to treat a larger number of women, thus performing, on average, 131.852 additional DRGs.Literature revealed that MRgFUS would generate several benefits, from a safety and an efficacy profile, with significant improvement in symptoms relief. Advantages emerged concerning the patients\u2019 perspective, thus leading to a decrease both in the length of hospital stay (Results suggest that MRgFUS could be considered an advantageous technological alternative to adopt within the target population affected by uterine fibroids, demonstrating its economic and organisational feasibility and sustainability, with consequent social benefits.The online version contains supplementary material available at 10.1186/s13561-022-00367-x. Magnetic Resonance-guided high intensity Focused Ultrasound (MRgFUS) is an emerging not-invasive procedure that applies the energy deriving from ultrasound to targeted soft tissues, within the human body, to heat and destroy diseased or damaged tissues, through ablation.MRgFUS has been approved for use and is currently employed to treat uterine fibroids (UFs), worldwide. Since it was first approved in the U.S. by the Food and Drug Administration in 2004, MRgFUS has represented a new approach for UF treatment \u20133, broadUFs are common in women of reproductive age, affecting on average 13.80% of the female population in Italy . The \u201cstClinical studies demonstrate that MRgFUS is a safe and effective treatment for symptomatic uterine fibroids \u20138, with At present, there still exists a debate due to uncertainty about the optimum management of\u00a0UFs. In particular, in Italy, no consensus exists with regard to the introduction of MRgFUS in the clinical practice, due to a paucity of real-world data, as well as the hospital consequences related to its acquisition, concerning not only economic aspects, efficacy or safety, but also other implications that have acquired importance over the time, such as organizational, equity and social domains, since not only cost-effectiveness assessment  is imporTherefore, an in-depth study is needed, particularly in the Italian and in European setting, to produce real-world evidence concerning this peculiar topic, and evaluating all the comparative alternative technologies, useful to solve the UF clinical problem, focusing the attention on the not-invasive approaches, devoted to women who wish to preserve their uterus or enhance fertility.Moving on from these premises, the present study aims at evaluating, with a multidimensional approach, the benefits concerning the introduction of MRgFUS in Italy, in comparison with other conservative and widely diffused procedures, such as myomectomy (standard surgery) or Uterine Artery Embolization (UAE), whose specific managements are very different in terms of activities, outcomes and costs.The coverage of this knowledge gap could be useful to support the evidence-based decision-making and reimbursement process, both at hospital and at National level, not only in the Italian context, but also in other European National Healthcare Services.The present study was structured as a Health Technology Assessment (HTA) analysis and it was conducted assuming the Italian NHS point of view, focusing on the comparation between the three different conservative technologies available for the treatment of uterine fibroids, devoted to child-bearing age women, who wish to preserve their uterus . Data refer to the year 2018.Due to the multidimensional and multidisciplinary nature of HTA, several aspects were considered, as stated within the EUnetHTA Core Model : i) geneFor the assessment of the above dimensions, data were gathered, according to a mixed-method approach, a relevant methodology used for healthcare services research, to give a more comprehensive understanding of complex interventions , 17. TheBefore starting the assessment, the PICO approach for the literature validation, in terms of \u201cPatients\u201d, \u201cIntervention\u201d, \u201cComparator\u201d and \u201cOutcomes\u201d, was identified and discussed before setting the specific search strategy, for the HTA report. Literature evidence came from the systematic search of literature databases  up to December 2019. Search terms were: \u201cmagnetic resonance-guided (high intensity) focused ultrasound\u201d, \u201cMRgFUS\u201d, \u201cMRgHIFU\u201d, \u201cuterine fibroids\u201d, \u201cUAE\u201d, \u201cmyomectomy\u201d, \u201csymptoms relief\u201d. Papers with the following characteristics, were collected and considered: manuscripts including adult women >\u200918), describing uterine fibroids symptoms, treated with magnetic resonance-guided (high intensity) focused ultrasound or with surgical approach (laparoscopy or laparotomy), and uterine fibroid embolization, in the same study. The level of evidence of the studies included in the analysis was evaluated, according to the Oxford Centre for Evidence-Based Medicine Table. Papers meeting the abovementioned inclusion criteria were consequently included and synthetized according to a PRISMA flow diagram [, describLiterature was then used, to retrieve evidence-based information regarding the safety and the efficacy profiles of the different methodologies used in the clinical practice for the conservative treatment of patients with UFs.For the deployment of the economic, organizational, and social dimensions, real-life data derived from the anonymous administrative and accounting flows, by cost center provided by the management control of Niguarda Hospital , referring to the UF fibroids\u2019 management, collected from January to December 2016. The anonymous data collection was verified, approved, and validated by the Healthcare Directorate of Niguarda Hospital.According to the above, 224 patients\u2019 clinical pathways, derived from the observation of 224 administrative records , were analyzed and economically valorized in an anonymous and aggregated manner. All the UF procedures referring to adult women undergone to surgical or interventional treatment for the removal of a maximum of three uterine fibroids, smaller than 10\u2009cm, were considered and were economically valorized.At first, through the implementation of an Activity Based Costing (ABC) approach , all theThe economic evaluation of the process was integrated with a budget impact analysis  assumingBesides the healthcare evolution up to 12-months after surgery, the impact related to a second surgical/interventional procedure was examined, considering a different failure-rate for each technology, derived from the observation of the 224 administrative records used for the assessment of the economic dimension. A failure-rate equal to 9.78, 7.61 and 11.22%, for surgery, UAE and MRgFUS respectively, was considered.Real-world data information, derived from the above administrative databases, were also used for the deployment of both the organizational and the social domains. On the one hand, the organizational impact was detected, in terms of organizational advantages related to the release of the operating room (OR) occupancy hours, since the innovative technology does not need to be implemented within that setting, as happened for the reference comparators (UAE and surgery). On the other hand, the social impact was assessed through the economic quantification of the patients\u2019 productivity losses to solve their health needs, according to the different length of stay related to the three procedures under assessment.In conclusion, a specific questionnaire was administered through structured interviews for the assessment of the ethical (in terms of accessibility to care), social and legal domains. Thus, the questionnaire was filled in by eleven experts in the treatment of uterine fibroids, who gave their comparative perceptions to the three technologies under assessment, according to an evaluation scale ranging from \u2212\u20093 (less performant technology) to +\u20093 (most performant technology) . This waOn the other hand, the social dimension required the professionals\u2019 perceptions with regard to i) Ability of the technology to protect the patients\u2019 autonomy; ii) Protection of human rights; iii) The use of technology guarantees the social values and the willingness to pay of the patient; iv) Protection of persons of a legally protected status; v) Ability of the technology to protect the patients\u2019 religion; vi) Impact of the procedure on the social costs; vii) Patients and citizens can have a good level of understanding of technology; viii) Impact of the procedure on the patient\u2019s perceived quality of life; ix) Impact of the procedure on the care giver\u2019s life and perception; and x) Recovery rate.In conclusion, the legal domain explored the following items: i) Permission level of technology; ii) Need for inclusion of the technology in registry; iii) Fulfillment of the safety requirements; iv) Infringement of intellectual property rights; v) The need to regulate the acquisition of technology; vi) The legislation covers the regulation of technology for all categories of patients; vii) The user manual of the technology is complete.p-value), thus using the one-way ANOVA. All the analyses were performed using the Statistical Package for Social Science of IBM SPSS (Version 22).Focusing on statistical methods, data were first analyzed, considering descriptive statistics. Differences among technologies  were evaluated, according to a significance level lower than 0.05  studThe implementation of both the Cochrane risk-of-bias tool for randomized trials and the Newcastle-Ottawa Scale, declared that none of the studies were at critical risk, due to selection bias, missing data and results. Thus, the risk of bias was not high. The control group was determined and the outcomes measurement proved to be relevant and replicable.As previously mentioned, literature evidence was accordingly utilized to define the safety and the efficacy profiles of the three technologies under assessment.p\u00a0<\u20090.001); fewer patients in MRgFUS arm experienced infections, hemorrhages requiring infusion, unintended major surgeries, and life-threatening events [Focusing on the safety domain, from an evidence-based point of view, the 92.5% of patients treated with UAE presented abdominal pain and bloating, fever, and vomiting, whereas patients treated with MRgFUS presented less post-treatment symptoms , 27. Focg events , 31.Focusing on the efficacy profile, the \u201csymptoms relief\u201d was considered the primary outcome of all the UF treatment procedures and was assessed by means of the UFS-QOL, that is a validated scale to rate disease-specific symptoms and health-related quality of life questionnaire for UF . Literatp-value\u2009=\u20090.024) and UAE .The average costs related to the different clinical pathways have been estimated, considering the pre-operative and post-operative costs . In this view, the surgical procedure absorbed more economic resources, than both MRgFUS , 1,996,495 patients emerged to be potentially treated for UFs, in the national context.After having defined the costs related to each treatment procedure, a BIA was conducted, taking into consideration the potential Italian population eligible to UF treatments. Thus, the number of women suffering from UFs was defined, based on the disease prevalence rate, equal to 13.80%, presented in the literature evidence . ConsideAccording to the above, the Italian NHS would benefit of economic advantages from the adoption of MRgFUS, ranging from 6.42 to 7.04% Table\u00a0, for theIt is important to notice that this saving could be reinvested for the implementation of additional DRGs related to the treatment of uterine fibroids. Considering the economic saving equal to \u20ac421,343,642.50, Italian NHS would be able to perform 131.852 additional DRGs, for UFs treatment and cure.From a quantitative point of view, MRgFUS would also generate significant advantages related to the release of the operating room (OR) occupancy hours, since the innovative technology does not need to be implemented within that setting, as happened for the reference comparators (UAE and surgery), even if the innovative technology takes more than 3\u2009h. Based on the number of patients potentially treated for uterine fibroids and evaluating the duration of a single intervention equal to 110\u2009min for the surgical procedure and to 98\u2009min for UAE, the OR time saving was evaluated by comparing the previously mentioned baseline scenario and the real-life scenario, achieving an organizational OR saving, equal to \u2212\u200916.00% of time Table\u00a0.Table 2p-value <\u20090.001) and 1.28\u2009days for UAE .In addition, from the analysis of the administrative databases related to the patient\u2019s length of stay, it emerged that MRgFUS required on average 1.16\u2009days spent in hospital after the procedure, compared with 4.03\u2009days for surgery , when compared with surgery, and a saving of 20.37% , when compared with UAE.The implementation of MRgFUS could bring significant advantages, concerning the social aspects. On the one hand, literature , 27 repoTable\u00a0p-value >\u20090.05). MRgFUS is not always accessible on local level, since not all the hospitals have, to date, acquired the innovative large size equipment useful for the implementation of guided-ultrasound procedures. However, the adoption of MRgFUS could generate health migration phenomena . In particular, the choice of providing a larger number of technological alternatives is relevant for the increase in the number of procedures and extension of the catchment area of the reference, thus indicating how the innovative technology could increase, in the future, healthcare migration and mobility.From an equity perspective, it emerged that UAE achieved the better perception, and should be considered the preferable technology, followed by MRgFUS, albeit not statistically differences occurred between alternatives , regarding an increase in the quality of life perceived by the patients themselves , and by their families , as well as a faster return to regular working activities .As for the quantitative analysis of the social dimension, professionals agreed that MRgFUS would be the preferable solution . In addition, concerning the two minimally invasive techniques, the professionals involved, declared the completeness and integrity of the user manuals.The legal impact examination reported no statistically significant differences among technologies considering the indication of use, for all the surgical/interventional procedures, and for all the categories of patients  are increasingly used to treat symptomatic fibroids. Indeed, the selection of the proper methods to be preceded by a thorough analysis of the case, patient\u2019s age, tumor location and related symptoms.In this view, MRgFUS may constitute an alternative solution for\u00a0patients who meet the qualification criteria and deny other methods, which also facilitates the use of other treatment options in case the procedure is ineffective, with important organizational, economic, and social benefits, even if further randomized studies are necessary, to confirm the above information.In conclusions, the healthcare services may consider the evidence provided by the present study as an opportunity to differentiate UFs patients\u2019 procedures, guaranteeing a personalized clinical pathway and different alternatives, thus becoming more efficient and effective.Additional file 1.Additional file 2."}
+{"text": "HMGS1, HMGCR, IDI1, SQLE, MSMO1, SREBF1, and SOAT1 was up-regulated by CDPs exposure. Accordingly, metabolites of the mevalonate pathway were accumulated in cultures treated with CDPs. This finding further suggests that the metabolism of cholesterol is crucial for the occurrence of CC, and the blockade of the sterol synthesis as an anti-proliferative mechanism of the bacterial CDPs, represents a reasonable chemotherapeutic drug target to explore. Our transcriptomic study supports the anti-neoplastic effects of bacterial CDPs in HeLa cells shown previously, providing new insights into the transduction signals, transcription factors and metabolic pathways, such as mevalonate and cholesterol that are impacted by the CDPs and highlights its potential as anti-neoplastic drugs.The incidence of human cervix adenocarcinoma (CC) caused by papillomavirus genome integration into the host chromosome is the third most common cancer among women. Bacterial cyclodipeptides (CDPs) exert cytotoxic effects in human cervical cancer HeLa cells, primarily by blocking the PI3K/Akt/mTOR pathway, but downstream responses comprising gene expression remain unstudied. Seeking to understand the cytotoxic and anti-proliferative effects of CDPs in HeLa cells, a global RNA-Seq analysis was performed. This strategy permitted the identification of 151 differentially expressed genes (DEGs), which were either up- or down-regulated in response to CDPs exposure. Database analysis, including Gene Ontology (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed differential gene expression on cancer transduction signals, and metabolic pathways, for which, expression profiles were modified by the CDPs exposure. Bioinformatics confirmed the impact of CDPs in the differential expression of genes from signal transduction pathways such as PI3K-Akt, mTOR, FoxO, Wnt, MAPK, P53, TGF-\u03b2, Notch, apoptosis, EMT, and CSC. Additionally, the CDPs exposure modified the expression of cancer-related transcription factors involved in the regulation of processes such as epigenetics, DNA splicing, and damage response. Interestingly, transcriptomic analysis revealed the participation of genes of the mevalonate and cholesterol biosynthesis pathways; in agreement with this observation, total cholesterol diminished, confirming the blockage of the cholesterol synthesis by the exposure of HeLa cells to CDPs. Interestingly, the expression of some genes of the mevalonate and cholesterol synthesis such as  Human cervical cancer (CC) is the third leading cause of cancer death among women around the world, along with breast, lung and colorectal cancers  and the The PI3K/Akt/mTOR signaling pathway is involved in several biological processes such as cell survival, apoptosis, and tumor development and progression \u20139. In thSearching for novel and natural molecules with anticancer activity is always in progress, as natural molecules are considered more target-specific than their synthetic counterparts. Specifically, bacterial cyclodipeptides (CDPs) have been proposed as compounds with strong pharmaceutical potential for cancer treatment . CDPs haPseudomonas aeruginosa PAO1 strain composed mostly of cyclo (l-Pro- l-Tyr), cyclo , and cyclo (l-Pro- l-Phe), suppress the proliferation of human adenocarcinoma HeLa and CaCo-2 cell lines , fetal bovine serum , and trypsin solution (Sigma Life Science). Cyclodipeptides were obtained from escribed . CDPs we2 to confluency. Cells were then trypsin-treated, counted using a hemocytometer chamber, and used for subsequent assays. Cell cultures and other procedures were performed in class II biological safety cabinets. For RNA-Seq and RT-qPCR analysis, HeLa cells were incubated in DMEM complete medium containing the CDPs mixture at 0.01 mg/mL for 15\u00a0min and 4\u00a0h, along the untreated controls. Afterwards, cells were pelleted by centrifugation for total RNA extraction.The HeLa human cancer cell line was obtained from the American Type Culture Collection , which contained mutated the H-Ras oncogene and low-level expression of the P53 tumor suppressor protein. Cells were cultured in complete media . Cell culture media were changed twice a week and maintained at 37\u00b0C under 80% humidity, and incubated in an atmosphere of 5% COhttps://www.ncbi.nlm.nih.gov/sra).Total RNA was isolated using TRIzol reagent (Thermo-Fisher Scientific). Total RNA was treated with DNase (Thermo Fischer Scientific). cDNA libraries were generated using the Illumina TruSeq RNA Sample Preparation Kit according to the manufacturer\u2019s instructions. Transcriptome sequencing was conducted using NextSeq 500 System . A configuration for pair-end reads with a 75 bp read length was\u00a0used. RNA-seq Massive sequencing was carried out in the\u00a0\u201cUnidad Universitaria de Secuenciaci\u00f3n Masiva y Bioinform\u00e1tica\u201d (UUSMB), at the Instituto de Biotecnolog\u00eda, UNAM, Cuernavaca, Mor., M\u00e9xico. The Sequence Read Archive (SRA) were loaded in the Bioproject ID PRJNA725963  of raw reads was performed using FASTQC software and contamination and adapter removal was carried out using in-house Perl scripts designed by the UUSMB. Because adaptor sequence was present at the three-prime end of some reads, these were further trimmed using CUTADAPT version 0.9.5 with a minimum overlap of two and a minimum length of thirty two . Reads w 2.3.4.3 . Sam and 2.3.4.3  and BEDT 2.3.4.3 . Read co 2.3.4.3 . DEG ana 2.3.4.3 . Pairwisage in R , and theage in R .Functional Gene Ontology (COG) and Pathway enrichment analysis of the co-modified DEGs were conducted using bioinformatic tools with automated interpretation of genomic data, which perform statistical and network analysis on biological hierarchical vocabularies: the PANTHER classification , KEGG rehttp://www.string-db.org/) database web-server application was used to predict whether gene-encoded proteins interacted with each other. A PPI network was constructed for the DEGs identified in the current study. The minimum required interaction score parameters were set at the medium confidence level  was utilized to obtain the first cDNA strand by using Superscript II Reverse Transcriptase (Thermo Fischer Scientific) and oligo-dT primer in the reaction volume of 30 \u03bcl for 3 \u03bcg of RNA material, according to the manufacturer\u2019s instructions. RT-qPCR was performed on a LightCycler Nano  and the amplification was carried out using 75\u2009ng cDNA for each reaction based on the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) as fluorescent probe. After an initial denaturation step of 10\u00a0min at 95\u00b0C, the product was routinely examined using a dissociation curve, and the amount of transcript was compared with the relative Ct method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal reference control. The 2HMGS1, HMGCR, SREBF1, IDI1, MSMO1, SQLE, ACAT1, and RHOA genes, semi-quantitative RT-PCR was carried out using 50\u2009ng cDNA obtained with ImProm II-Reverse Transcriptase reagent kit  and amplified using PCR Platinum Super Mix High Fidelity (Invitrogen). PCR was performed using the Bio Rad T100TM Thermal Cycler at 94\u00b0C for 3\u00a0min, followed by cycles of 15 sec at 94\u00b0C, 15 sec at 54\u00b0, 60\u00b0 or 64\u00b0C and 15 sec at 72\u00b0C; samples were taken at different cycles for DNA quantitation. The products were examined using amplification curves, the amounts of transcript were obtained at 20 cycles of the exponential amplification curve and expressed as relative units using the Image J software. Oligonucleotides sequences utilized are shown in For the mRNA expression of the For cholesterol and mevalonate metabolites analysis, cells-free supernatants of HeLa cultures grown as described above were used. 10 mL of cell-free supernatants and cells-pellets were lyophilized and separately extracted with 1 mL methanol. After filtering the samples were dried and dissolved in 200 \u00b5L ethanol/methanol (1:1). Cholesterol was determined by spectrophotometry analyzer at 505 nm as described by the provider using the Cholesterol SL-234-60 determination Kit (Sekisui Diagnostics). Metabolites of mevalonate pathway were determined on cell-free supernatant samples by quantitation of organic acids accumulated, such as 3-hydroxyhex-4-enoic acid by GC-MS as described by Pitt et\u00a0al. , with soP values \u2265 0.95 were used to identify the mRNAs expression that were significantly different between untreated and CDPs-exposed HeLa cells. These differentially expressed mRNAs were identified through fold change filtering. For correlation analysis, data obtained from RNAseq were analyzed by correlation analysis of response variables (treatments) vs data of expression intensity for each DEG (cases) using the STATISTICA software . Other data were statistically analyzed using GraphPad Prism 6.0 software (GraphPad Software).False discovery rate (FDR) filtering and Gene expression profiles from HeLa cells were determined at a dose of 0.1 mg/mL of CDPs for 15\u00a0min and 4\u00a0h exposure times, conditions previously established to induce apoptosis and differential protein expression profiles , 29. AftP values \u2264 0.05 for all samples and selected with a minimum detection threshold of 10 counts for each gene in all samples. The number of genes below these thresholds of detection was 10,127. A scatter plot illustrating different expression is shown  and a second group strongly associated with up-regulated genes at a longer time of CDPs exposure (4\u00a0h). Also, a smaller number of DEGs with a down-expression profile was observed , ShinyGO v0.61 GO terms (http://bioinformatics.sdstate.edu/go/), Reactome FIVIz (https://reactome.org/tools/reactome-fiviz), STRING-db v.10 (https://string-db.org), Pathway Commons tool (https://www.pathwaycommons.org), and the KEGG pathway mapping (https://www.genome.jp/kegg/mapper.html), clustered the DEGs into two major categories: biological processes and biological pathways.Analysis of gene expression patterns using RNA-Seq revealed several biological processes and biological pathways that could be targeted when HeLa cells are exposed to bacterial CDPs. Gene Ontology analysis of the 151 DEGs using bioinformatics platforms, including the PANTHER classification System (P \u2264 0.05), including cellular component organization or biogenesis (21-14.9%), cellular processes (61-44%), localization (15-10.6%), biological regulation (44-31.2%), response to stimulus (19-13.5%), signaling (16-11.3%), developmental processes , rhythmic processes (1-0.70%), multicellular organismal processes (10-7.1%), locomotion (2-1.4%), biological adhesion (1-0.7%), metabolic processes (35-24.8%), growth (1-0.7%), cell population proliferation (2-1.4%), and immune system processes (1-0.7%), although there exist others. The biological pathways (P \u2264 0.05) included insulin/IGF/MAP kinase cascade (1-1.8%), P38/MAPK (2-3.5%), interleukins (1-1.8%), EGF receptor (2-3.5%), PI3K kinase(1-1.8%), PDGF (2-3.5%), Notch (2-3.5%), Cadherin (1-1.8%), P53 (1-1.8%), Gonadotropin-releasing hormone receptor pathway (5-8.8%), Hedgehog (2-3.5%), TGF-beta (1-1.8%), FGF (1-1.8%), FAS signaling pathways (1-1.8%), Gonadotropin-releasing hormone receptor pathway (5-8.8%), Angiogenesis (2-3.5%), Cholesterol biosynthesis (2-3.5%), as well as other pathways that are involved in inflammation (2-3.5%), oxidative stress response (4-7.2%), apoptosis (2-3.6%), cytoskeleton (1-1.8%), and T and B cell growth/activation (2-3.6%).The present study identified 15 biological processes  and functional enrichment analyses, using an established computational ChEA3 TF tool that offers a better understanding of gene regulatory networks. TF analysis revealed that from the 1632 TFs identified, 151 DEGs have TF/target\u2013gene associations and the more enriched genes are related with a neoplastic phenotype S2. The YOD1, RASAL2, MSMO1, MAFF, BCL2, DUSP8, DDIT3, IDI1, GADD45A, HMGCR, LRRR2, EGR3, BCL6, ATRX, FOSB, FOS, SGK1, GAS1, FZD4, FRAT2, SMAD6, BTG2, and COL6A1 , clearly grouped the DEGs into three correlation groups: i) a group of 31 DEGs with modified expression in correlation with the CDPs exposure in a short time period (15\u00a0min); ii) a second group of 95 DEGs associated with the CDPs exposure at the longer exposure time period (4\u00a0h); and finally, iii) a third group of DEGs that more closely resembled the control (HeLa cells without treatment) Table 3,ATRX, BCL6, EGR3, FOS, COL6A1, and ANKDR12), which expression was modified in the top DEG-enriched ranking score, to verify their relative mRNA expression in HeLa cells through RT-qPCR , MSMO1 (encoding for sterol-C4-methyl oxidase), SOAT1/ACAT1 (encoding for cholesterol acyltransferase), and SREBF1 (encoding for sterol regulatory element-1 transcription factor), belonging to the sterols synthesis pathway, also increased by the CDPs treatment . These findings were further confirmed when mervastatin (statin) was used as inhibitor of the HMGCR enzyme and with the combination of statin and CDPs addition; in this case the mevalonate metabolites accumulation was synergistic , cyclo (l-Pro- lPhe), and cyclo , whose ability to arrest the cell cycle at the G0-G1, transition has already been implicated in the inhibition of the proliferation of human HeLa cell line. The mechanism of bacterial CDPs on inhibition of proliferation of the HeLa cells model involves the inhibition of phosphorylation of protein-kinases, such as Akt-Ser473, mTOR-Ser2448, and p70S6K-Thr389, mediated by the mTORC1 and mTORC2 complexes  that directly impact cell proliferation have a high potential for cancer treatment. The bacterium In the cancer context, evidences indicate that cancer progression is related to dysregulation in the expression of a plethora of molecules, including TFs, representing approximately 20% of known oncogenes associated with the development and maintenance of cancer . The rolOur RNA-sequencing data identified 151 genes that were differentially expressed by effect of the CDPs-exposure, including 141 up-regulated and 10 down-regulated genes. The function of these DEGs was annotated and enriched by databases, such as the ChEA3 TF tool, ShinyGO, Common Pathways, and KEGG. The top 151 DEG-enriched were further investigated by bioinformatics analysis in the KEGG database in order to study in deep the molecular elements associated with the cytotoxic effect caused by the CDPs exposure on HeLa cell cultures Table 2.ATRX, BCL6, EGR3, COL6A1, ANKDR12, HMGA2, which are key members of AP-1 transcription factor such as FOS, FOSB, and MAFF. Thus, our findings show that the expression of transcripts in HeLa cell cultures were significantly modified by CDPs-exposure. With regards to functional Gene Ontology and pathways analyses, an interesting result was the prevalence of protein-kinases belonging to cancer-related signaling transduction pathways. We identified three uniquely up-regulated genes of the PI3K/Akt/mTOR signaling pathway, including FNIP2, SGK1, and BCL2; and the up-regulated genes of the Wnt pathway, including APC, HLTF, MYH7B, and WDR37. Meanwhile in the MAPK signaling pathway, four exclusive genes were up-regulated, DDIT3, DUSP1, DUSP8, and GADD45A    and the  (BCL-2) , the dua (BCL-2) , were alEGR3 and FOS, which are components of intracellular signal transduction pathways   (GADD45B)  are expressed following a wide range of external stimuli, to modify the mechanisms of regulation involved in cell proliferation. The PRGs are classically grouped in two types: immediate early response genes (IEGs), and delayed primary response genes (DPRGs). The mRNA of IEGs can appear in cells within 5 to 60\u00a0min after the stimulus, whereas the DPRGs are induced later, around 4\u00a0h after the stimulus . Typicald DUSP8) , 64. Theas CHOP) , the groGADD45B) , and thexecution . Prolongxecution , 69.The major signaling pathways affected by CDPs 5, also FAM135A, MTRNR2L2, PSD3, HMGA2, LIN7A, and MYH7B; as well as circRNAs i.e., ANKRD12 (recruitment of histone deacetylases), APC (involved in tumorigenesis), and PSD3 , and MTRNR2L10; from the lncRNA type, KTN1 (an anti-sense RNA involved in tumorigenesis and EMT). The TFs most commonly identified are related to fundamental processes of DNA modifications, which have been associated with cancer, chromatin remodeling, neurodegenerative diseases, and epigenetics, i.e., ATRX, ASCL2, ATOH8, BCL6, BTG2, C11orf65, CCDC148, CCDC88A, CDC6, CENPE, CEP170-2, CHD9, CLK4, CWC22, DGCR8, FOXP2, HFM1, HIST1H4K, HLTF, ID2, KIF20B, KBTBD8, LMX1B, LINGO1, MAFF, NAA16, PHF1, PPIG, RFX1-2, SLF1, STK36, YOD1, ZFY, and ZNF236 , are positively regulated by bacterial CDPs on HeLa cell cultures  can be found below: Conception and design: JC-G. Development of methodology: PL-M, LH-P, JG-C, EM-C, and LM-A. Analysis and interpretation of data: PL-M, JG-C, JL-B, AG-G, and JC-G. Writing, review, and/or revision of the manuscript: PL-M, JL-B, AG-G, and JC-G. Administrative, technical, or material support: JL-B, AG-G, and JC-G. Study supervision: AG-G and JC-G. All authors read and approved the final manuscript.This study received funding from the Consejo Nacional de Ciencia y Tecnologia (CONACYT-M\u00e9xico) grant No 256119, Marcos Moshinsky Fundation grant 2014-16, Universidad Michoacana de San Nicol\u00e1s de Hidalgo grant CIC-2.14, and DGAPA PAPIIT UNAM Grant No 209420.\u00a0PL-M and EM-C were awarded with a CONACYT Postdoctoral Fellowship. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit 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.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": "MLL (or KMT2A) gene, which confer highly dismal prognoses on current combination chemotherapeutic regimens. Hence, more adequate therapeutic strategies are urgently needed. To expedite clinical transition of potentially effective therapeutics, we here applied a drug repurposing approach by performing in vitro drug screens of (mostly) clinically approved drugs on a variety of human ALL cell line models. Out of 3685 compounds tested, the alkaloid drug Camptothecin (CPT) and its derivatives 10-Hydroxycamtothecin (10-HCPT) and 7-Ethyl-10-hydroxycamtothecin (SN-38: the active metabolite of the drug Irinotecan) appeared most effective at very low nanomolar concentrations in all ALL cell lines, including models of MLL-rearranged ALL (n = 3). Although the observed in vitro anti-leukemic effects of Camptothecin and its derivatives certainly were not specific to MLL-rearranged ALL, we decided to further focus on this highly aggressive type of leukemia. Given that Irinotecan (the pro-drug of SN-38) has been increasingly used for the treatment of various pediatric solid tumors, we specifically chose this agent for further pre-clinical evaluation in pediatric MLL-rearranged ALL. Interestingly, shortly after engraftment, Irinotecan completely blocked leukemia expansion in mouse xenografts of a pediatric MLL-rearranged ALL cell line, as well as in two patient-derived xenograft (PDX) models of MLL-rearranged infant ALL. Also, from a more clinically relevant perspective, Irinotecan monotherapy was able to induce sustainable disease remissions in MLL-rearranged ALL xenotransplanted mice burdened with advanced leukemia. Taken together, our data demonstrate that Irinotecan exerts highly potent anti-leukemia effects against pediatric MLL-rearranged ALL, and likely against other, more favorable subtypes of childhood ALL as well.Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) remains one of the most aggressive types of childhood hematologic malignancy. The majority (~80%) of infant ALL cases are characterized by chromosomal translocations involving the  Mixed Lineage Leukemia  gene, and this patient group in particular fares significantly worse with EFS rates of 30\u201340% [Acute Lymphoblastic Leukemia (ALL) in infants (children <1 year of age) is an aggressive hematologic malignancy characterized by a poor prognosis, with 5-year event-free survival (EFS) rates of ~50% ,3. In coMLL translocations give rise to chimeric MLL fusion proteins, which induce inappropriate histone modifications by recruitment of the histone methyl transferase DOT1L [MLL translocations [MLL-rearranged ALL. So far, this has led to the discovery of inhibitors against DOT1L , DNA methyltransferases , BET family proteins  and histone deacetylases (HDACs)  as potential drug candidates [se DOT1L ,7. This ocations ,10,11,12ndidates ,15,16,17MLL-rearranged ALL, which have been characterized and approved for other human diseases and thus expediting their potential transition into clinic [Although these epigenetic-based drugs do show promising results, the transition from preclinical studies towards clinical applications unfortunately remains an elaborate and time-consuming process. Therefore, awaiting clinical evaluation of these inhibitors, we decided to adopt a drug repurposing approach using drug library screening of >3500 FDA-approved and off-patent therapeutic agents. Using this strategy, we aimed to identify effective therapeutics against o clinic .MLL-rearranged ALL. In fact, we here demonstrate that irinotecan monotherapy successfully induced disease remission in xenograft mouse models of MLL-rearranged ALL.In this study we identified the camptothecin-derivative 7-Ethyl-10-hydroxycamptothecin (SN-38), and in particular its pro-drug irinotecan (Camptosar), as highly effective agents against MLL-rearranged ALL cell lines SEM (MLL-AF4+) and KOPN8 (MLL-ENL+), the BCP-ALL cell lines REH (TEL-AML1+), 697 (E2A-PBX+) and Sup-B15 (BCR-ABL+), and the T-ALL cell line Jurkat (pseudodiploid) were obtained from the German Collection of Microorganisms and Cell Cultures . The MLL-rearranged ALL cell line RS4;11 (MLL-AF4+) was obtained from The Global Biosource Center . Leukemia cell lines were cultured in RPMI-1640 with GlutaMAX, 10% Fetal Calf Serum, 100 IU/mL penicillin, 100 IU/mL streptomycin and 0.125 \u00b5g/mL amphotericin B  at 37 \u00b0C under 5% CO2 atmosphere. Regular DNA fingerprinting and mycoplasma testing were performed to ensure cell line integrity. Primary samples derived from MLL-rearranged infant ALL patients were obtained at the Sophia Children\u2019s Hospital  as part of the international INTERFANT treatment protocol. Informed consent was obtained according to the Declaration of Helsinki. The study was approved by the Ethics Committee of all collaborating institutions and registered with the National Cancer Institute  and with the European Clinical Trials database (EudraCT 2005-004599-19) (24-11-2005). Processing of samples occurred as described previously [The We purchased the drug libraries \u2018Spectrum Collection\u2019  and \u2018Prestwick library\u2019 , and the anti-neoplastic \u2018Sequoia library\u2019 . DMSO dissolved stocks were diluted in non-supplemented RPMI, and cytotoxicity was assessed using MTT/MTS assays (DMSO concentration <0.5%). Data were normalized to vehicle control, and cell viability measures of 100% or more were deemed completely viable and unaffected by drug treatment. Heatmaps were generated using Gene-E software .Cell proliferation was tracked by propidium-iodide exclusion on a MACSQuant flow cytometer (Miltenyi). Apoptosis was assessed using flow cytometry with the PE Annexin-V Apoptosis Detection Kit . Flow cytometry data were analyzed using FlowJo software . For whole cell lysates, cells were washed with PBS and lysed on ice in RIPA buffer supplemented with protease and phosphatase inhibitors. MTS and MTT dose\u2013response data were acquired in duplicate and presented as mean +/\u2212 s.e.m. (cell lines) or mean +/\u2212 SD (patient samples).Protein lysates were resolved on pre-cast SDS-polyacrylamide gels  and transferred to nitrocellulose membranes using the Transblot Turbo Transfer System . Membranes were blocked with 5% BSA or skim milk in TBS and probed with primary antibodies against PARP, (phospho-)Chk2, (phospho-)H2AX  or \u03b2-actin , and subsequently with fluorophore-conjugated secondary antibodies. Images were acquired using the Odyssey imaging system .+ cell counts in peripheral blood samples. Vehicle or irinotecan (40 mg/kg) treatment was administered intraperitoneally three times per week. Mice were humanely euthanized and tissue samples were acquired for further analysis. Statistical tests were performed in GraphPad Prism. Spleen weight differences were assessed using non-parametric tests . Differences in leukemic cell infiltration from FACS data were assessed using parametric tests .Animal experiments were performed according to Dutch legislation and approved (10-12-2014) by the Erasmus MC Animal Ethical Committee, Rotterdam, The Netherlands (EMC3389). Briefly, immunodeficient NSG mice were transplanted with SEM-SLIEW or patient-derived leukemic cells, and leukemia progression was assessed through intra-vital imaging or human CD45MLL-rearranged ALL cell lines as well as non-MLL B-cell precursor (BCP) ALL cell lines. While the majority of drugs did not affect the leukemia cell lines tested, approximately 12% of the compounds inhibited MLL-rearranged ALL cell viability by at least 20%, for both the Spectrum , suggesting that most of these drugs are not specifically targeting MLL-rearranged ALL  at a drug concentration of 1 \u00b5M on various Spectrum a and PreSpectrum b libraringed ALL c,d. More, RS4;11 ; top. Sior KOPN8 ; bottom.MLL-rearranged ALL cell lines compared to the BCP-ALL cell lines, which was probably due to the BCP-ALL cell line REH being non-responsive to glucocorticoids as it lacks detectable expression of the glucocorticoid receptor (GR). Nonetheless, we tested several of the corticosteroid drugs on the MLL-rearranged ALL cell lines SEM and KOPN8 but found none to be more potent or effective than prednisolone  was screened in the same setup at 1 \u00b5M drug concentration. This drug screen yielded a substantial number of effective compounds, i.e., 52 compounds with >75% inhibition of iability e; left. MLL-rearranged ALL and BCP-ALL cell lines for the top five drugs from the Spectrum and Prestwick libraries combined, and the top five drugs from the Sequoia library at a 10 nM drug concentration. Among these drugs, gemcitabine appeared to be the only chemotherapeutic more specifically targeting MLL-rearranged ALL cells compared to BCP-ALL cells. Although no other MLL-rearranged ALL specific agents could be identified, the top compounds in all three drug libraries appeared to be camptothecin and its derivatives 10-hydroxycamptothecin (10-HCPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and topotecan  induces DNA single strand breaks to release DNA supercoiling-induced torsional stress  SN-38 biMLL-rearranged ALL xenograft mouse model [n = 19) were xenotransplanted with our MLL-rearranged ALL luciferase reporter cell line SEM-SLIEW and equally allocated over vehicle (n = 10) and irinotecan  treatment groups, ensuring comparable leukemia burden before treatment (MLL-rearranged ALL patients often occur early during treatment [n = 6) of the irinotecan-treated mice were sacrificed at day 28 and used to investigate leukemic infiltration of tissues, while the remaining n = 3 irinotecan-treated mice were kept alive without any further treatment (from here on referred to as the treatment follow-up group)  A; right,p group) B, indicap group) C.n = 6), irinotecan (n = 6) and follow-up (n = 3) groups, relevant tissues were extracted and further analyzed. In addition, we included two healthy, non-transplanted and untreated NSG mice as basal controls. As expected, vehicle-treated control mice characteristically presented with splenomegaly, while irinotecan-treated mice, both in the initial treatment and the follow-up group, had significantly lower spleen weights (p < 0.0001), resembling the spleens of healthy control mice  already displayed more substantial engraftment levels at treatment initiation, with 7\u201344% human CD45+ leukemic cells. Mice were randomly allocated to either the vehicle control group  or the irinotecan treatment group . The treatment regimen was comparable to the cell line xenografts. Regardless of the PDX model, in less than two weeks after treatment initiation a substantial decrease in human chimerism was observed in the irinotecan treatment mice, while an exponentially increased accumulation of human CD45+ leukemic cells was observed in the vehicle groups. After 10 doses of irinotecan, mice were in complete remission (<0.2%), while the leukemic burden in control mice was 38\u201383%  models with leukemic cells from two s 38\u201383% A. Both vs 38\u201383% B. In lins 38\u201383% C.+ leukemic cells in the bone marrow and spleen at the end of treatment, which could act as a cell reservoir inducing leukemia relapse.In parallel to the curative set-up, we also established a separate treatment follow-up arm from both PDX models  to monitor for relapse of the disease, similar to the treatment arms in the cell line model. Irinotecan treatment was stopped after 10 dosages, and the irinotecan-treated mice were kept alive in the treatment follow-up group, whereas the control mice were sacrificed. Here, in contrast to the cell line model, the irinotecan-treated mice of the follow-up group relapsed after 4 weeks following the last irinotecan administration A. The miMLL-rearranged infant ALL is an aggressive hematologic malignancy with a poor prognosis, which urgently requires improved therapeutic strategies [MLL-rearranged infant ALL. Our data, combined with the fact that irinotecan is a well-characterized anti-neoplastic drug, strongly advocate for clinical evaluations of irinotecan for the treatment of MLL-rearranged infant ALL.rategies ,2. In thMLL-rearranged ALL with drug libraries of this magnitude. However, earlier drug discovery attempts involving FDA-approved drug screens have resulted in the identification of leukemia-subtype specific inhibitors against for example AML1-ETO-positive acute myeloid leukemia (AML) and NOTCH1-mutated T-cell ALL [Although the rationale behind drug repurposing through screening of FDA-approved drug libraries in itself is not innovative, it has not previously been reported for cell ALL ,26.MLL-rearranged ALL cell lines, as well as other ALL subtypes, with SN-38 as the most potent inhibitor of ALL cell viability. The mechanism of action for this drug class involves the generation of DNA/TOP1/drug complexes, which lead to collisions with either DNA synthesis- or transcriptional elongation-related replication forks, thereby inducing double-strand DNA breaks [50 values for infant ALL patient samples compared to the ALL cell line models, which might be explained by the limited division capacity of patient-derived leukemic cells in vitro and the high proliferation rate of immortalized cell lines. Still, nanomolar SN-38 concentrations could effectively inhibit primary leukemia cells in vitro, even in the absence of leukemic cell proliferation. The DNA breaks induced by SN-38 activate DNA damage response pathways, including the cell cycle regulating ATM-Chk2 pathway, and this is accompanied by the formation of phosphorylated H2AX (\u03b3-H2AX) foci [Our drug library screens resulted in the identification of the camptothecin-derived drug class of TOP1 inhibitors, which effectively inhibited A breaks ,28. We oAX) foci ,30. We cMLL-rearranged ALL xenograft models, where the SN-38 pro-drug irinotecan effectively inhibited leukemia development. Moreover, since the leukemia is usually fully disseminated in patients at diagnosis, we started administering irinotecan to mice with advanced leukemia to mimic the start of treatment in a clinical setting. In all mice, progressive leukemia drastically regressed within days, with no detectable residual leukemic cells at the endpoint of the experiment in cell line xenografts, and minimal residual disease levels in PDX mice. These very low levels of residual human leukemic cells most likely represented the relapse-initiating cells in the PDX follow-up treatment arm. Prolonging the treatment should help to eradicate this, as it has been previously described that dosage regimen and duration is pivotal for the efficacy of camptothecin-derivatives [MLL-rearranged acute leukemia sample, and reported TOP1 inhibitor topotecan effectively inhibited ALL in seven out of eight PDX models [MLL-rearranged BCP-ALL samples with IC50 values in the order of 10\u2013100 nM [MLL-rearranged infant ALL has stagnated over the last decades and novel treatment rationales are urgently required; the results from the present study, particularly in the context of the clinical data on irinotecan treatment in literature, suggest that TOP1 inhibitors may indeed be a valuable addition to infant ALL treatment protocols.The anti-leukemic effects of SN-38 could be validated in ivatives , and theivatives ,35,36,370\u2013100 nM . Moreove0\u2013100 nM ,42,43,44MLL-rearranged ALL, and possibly other high-risk types of childhood leukemia.Advantageously, irinotecan has been on the market for two decades already, and the amount of available information and clinical data is substantial. Besides the extensive use in adult patients for the treatment of several solid tumors, irinotecan has been an important component of pediatric sarcoma therapy for the past 15 years, and earlier clinical experiences and challenges have recently been reviewed, including important considerations for dosage regimens . AdditioMLL-rearranged ALL both in vitro and in vivo [Moreover, whereas irinotecan/SN-38 monotherapy already shows promise, combination therapy with HDAC inhibitors has been shown to synergistically inhibit other malignancies ,49,50,51 in vivo , this opMLL-rearranged infant ALL.In summary, the data presented here strongly advocate for the implementation of irinotecan (or comparable compounds) in the treatment of"}
+{"text": "The polymer-derived SiC fibers are mainly used as reinforcing materials for ceramic matrix composites (CMCs) because of their excellent mechanical properties at high temperature. However, decomposition reactions such as release of SiO and CO gases and the formation of pores proceed above 1400 \u00b0C because of impurities introduced during the curing process. In this study, polycrystalline SiC fibers were fabricated by applying iodine-curing method and using controlled pyrolysis conditions to investigate crystallization and densification behavior. Oxygen and iodine impurities in amorphous SiC fibers were reduced without pores by diffusion and release to the fiber surface depending on the pyrolysis time. In addition, the reduction of the impurity content had a positive effect on the densification and crystallization of polymer-derived SiC fibers without a sintering aid above the sintering temperature. Consequently, dense Si-Al-C-O polycrystalline fibers containing \u03b2-SiC crystal grains of 50~100 nm were easily fabricated through the blending method and controlled pyrolysis conditions. Silicon carbide (SiC) fiber with excellent oxidation resistance, high tensile strength, elastic modulus at high temperature is mainly used as a reinforcing material for ceramic matrix composites (CMCs) ,2,3. SiCThe curing methods essential for conversion from polymer fiber to ceramic fiber are classified into oxidation curing method ,9,10, elThe EB curing method was developed to manufacture high-heat resistant SiC fibers through the curing process without oxygen. The EB-cured PCS fibers are relatively easily converted into polycrystalline SiC fibers above 1500 \u00b0C through the formation of radical bonds such as Si-Si and Si-C under a strong electron beam. However, the EB curing method is expensive and difficult to access in various industries because the EB irradiation equipment is very sophisticated and high power.Among CVC methods, the iodine curing method induces the oxidation reaction of PCS at 120 \u00b0C or less, so it has the advantage of being applicable to low-melting point or low-molecular weight PCS. However, the amorphous SiC fiber fabricated by the iodine curing method contains iodine impurity as a cross-linking accelerator and oxygen impurity as a cross-linking agent. As a result, this amorphous SiC fiber not only has lower high-temperature heat resistance but is also difficult to convert to polycrystalline SiC fibers.xCy and SiO2 phase and are decomposed into SiO and CO gases at high temperatures (\u22651300 \u00b0C) [As mentioned above, it is known that amorphous SiC fibers fabricated by oxidation curing and CVC methods contain oxygen impurity in the vicinity of 10% . In amor1300 \u00b0C) ,19. ConsPCS containing various organometallic compounds have been synthesized to fabricate polycrystalline SiC fibers by inhibiting the decomposition of oxygen impurities during the conversion from amorphous SiC to crystalline SiC ,21. In pTherefore, in this work, amorphous SiC fibers without sintering aid were fabricated using the iodine curing method and controlled pyrolysis conditions, and then pyrolyzed at 1600 and 1800 \u00b0C to investigate the crystallization behavior of the polymer-derived SiC fibers. As a result, the amorphous SiC fibers prepared through the novel process of impurities control were converted into dense SiC polycrystalline fiber without additives such as sintering aids. In addition, Al-added PCS was easily prepared by the solution blend method, and dense Si-Al-C-O polycrystalline fibers were successfully fabricated by applying the crystallization behavior of SiC fibers investigated in this study.w) of 3327, number average molecular weight (Mn) of 1565, and melting point of 185 \u00b0C, respectively, was purchased from ToBeMTech Co., Ltd. . Iodine (extra pure 99.0%) as a cross-linking accelerator was purchased from Samchun pure chemical Co., Ltd. . Aluminum acetylacetonate (anhydrous 99.0%) and toluene (anhydrous 99.7%) were purchased from Sigma-Aldrich Inc. .Polycarbosilane (PCS) having weight average molecular weight , as shown in PCS was melted at 180\u2013190 \u00b0C for 3 h in NThe PCS solution was prepared using toluene, and aluminum acetylacetonate in an amount of 1, 3, and 5 wt% based on the weight of the PCS was added and stirred at room temperature for 6\u201312 h. The Al-added PCS solutions were vacuum-dried in the range of 150\u2013180 \u00b0C. During the melt-spinning process, the unreacted Al source causes the problem in reducing the diameter of the fiber and making it continuous. Therefore, the Al-added PCS bulk was again dissolved using toluene, and then filtered using a filter paper with a 0.08 \u03bcm particle. The Al-added PCS solution prepared by removing the unreacted Al source was vacuum-dried under the same conditions, and the modified PCSs were named PCS-Al1, PCS-Al3, and PCS-Al5, respectively. The amorphous and polycrystalline SiC fibers containing Al content fabricated using PCS-Al precursor in the same method as described above.\u22121. The PCS and modified PCS were pulverized into powder and measured in attenuated total reflection (ATR) mode by 16-times scan.Fourier-transform infrared spectroscopy  analysis was performed in the range of 450\u20134000 cmThermogravimetric analysis  was conducted at the heating rate of 10 \u00b0C/min up to 1600 \u00b0C to confirm the decomposition behavior and ceramic yield. PCS of 10.0 mg was placed in an alumina crucible and analyzed after stabilization for 30 min.The morphology and element distribution of polymer-derived SiC fibers were observed by field emission-scanning electron microscopy . Pt coating was performed using an ion coater for 70 s. Energy dispersive spectroscopy (EDS) was measured by repeating 5\u201310 times after pulverizing polymer-derived SiC fibers into fine powder. The standard deviation for the element content measurements of Si, C, O, and I were 4.81%, 3.95%, 2.06%, and 0.01%, respectively.X-ray diffraction analysis  in the range of 20\u201380\u00b0 was conducted to analyze the phase and crystallinity of the amorphous and polycrystalline SiC fibers. The measurement was carried out by scanning at 8\u00b0 per minute in continuous mode.The microstructural analysis of SiC-polycrystalline fibers was carried out by transmission electron microscopy . TEM analysis samples were prepared using focused ion beam .xCy phase.As shown in 2, and free carbon generated in the core region, but also filled the micropores formed in the decomposition temperature region by sintering as shown in For this reason, the polymer-derived SiC fibers fabricated below sintering temperature (at 1600 \u00b0C) showed dense core region and porous rim region despite the control of impurity contents due to the lower sintering temperature and residual impurities. On the other hand, above the sintering temperature (at 1800 \u00b0C), the polymer-derived SiC fiber not only induced SiC crystal growth by reacting SiO gas, SiOPCS with aluminum source were prepared through the blend method, which is a method for easily reacting PCS with organometallic compounds. Al-added PCS fibers were heat-treated with iodine at a weight ratio of 1:1 in the same manner as described above. Then, polycrystalline Si-Al-C-O fibers were fabricated by pyrolyzing at 1400 and 1800 \u00b0C applying an impurity control process. In this work, the crystallization behavior of polymer-derived SiC fibers was investigated by applying controlled pyrolysis conditions without sintering aid. It was confirmed that oxygen and iodine impurities gradually decreased without defects by long-time pyrolysis conditions of the fiber to which iodine curing was applied. Additionally, SiC fibers fabricated at 1800 \u00b0C exhibited the release of SiO and CO gases to the surface and densification behavior from the inside during crystal growth of \u03b2-SiC. On the other hand, the polycrystalline SiC fiber fabricated at 1600 \u00b0C was not densified despite the use of amorphous SiC fibers with low-oxygen content. This meant that a specific temperature above the sintering temperature was required as well as a reduction in the content of oxygen impurities for dense SiC-polycrystalline fibers. As a result, the polycrystalline Si-Al-C-O fibers prepared using controlled pyrolysis conditions had a dense structure including fine crystal grains of 50\u2013100 nm."}
+{"text": "Efficient conversion of light from short wavelengths to longer wavelengths using color conversion layers (CCLs) underpins the successful operation of numerous contemporary display and lighting technologies. Inorganic quantum dots, based on CdSe or InP, for example, have received much attention in this context, however, suffer from instability and toxic cadmium or phosphine chemistry. Organic nanoparticles (NPs), though less often studied, are capable of very competitive performance, including outstanding stability and water-processability. Surfactants, which are critical in stabilizing many types of nano-structures, have not yet been used extensively in organic NPs. Here we show the utility of surfactants in the synthesis and processing of organic NPs by thoroughly characterizing the effect of ionic and non-ionic surfactants on the properties of fluorescent organic NPs. Using this information, we identify surfactant processing conditions that result in nearly 100 % conversion of organic fluorophores into sub-micrometer particles, or nano-dots, with outstanding performance as CCLs. Such water dispersions are environmentally benign and efficiently convert light. They can be used for a range of fluorophores covering a full spectral gamut, with excellent color purity, including full-width at half-maximum (FWHM) values as low as 21\u2009nm. Compared to inorganic (InP) reference CCLs, the organic nano-dot based CCLs show superior color conversion efficiency and substantially improved long-term stability. Compared to inorganic nanoparticles, organic nanoparticles aren\u2019t as well understood. Here the authors explore the use of surfactants to prepare organic semiconductor nanoparticles with outstanding photophysical properties. Salient features like facile synthesis2, easy processability3, and excellent biocompatibility4 give them tremendous potential in a wide range of applications including drug delivery5, biosensing6, optical sensing7, cell imaging8, and so-on, where their inorganic counterparts have been ineffective or incompatible due to their toxicity, non-degradability or physical properties. A wide range of approaches to synthesize organic NPs have been developed; for instance, emulsification9, nano-precipitation10, and solvent evaporation11 are examples of successful approaches that have been reported by many different research groups.Fluorescent organic NPs have attracted tremendous attention worldwide in recent years due to their unique optical and chemical properties that differ from their bulk state13, however, the use of surfactants in organic NP synthesis has so far been sporadic. Among the most frequently used approaches to synthesize organic NPs is the re-precipitation technique, which involves dissolving organic fluorophores in a solvent, and injecting this solution into a non-solvent to yield nano-crystalline particles16. Despite impressive NP growth dynamics, this method suffers from poor uniformity and low yields of small particles. As with inorganic NPs, the use of surfactants can greatly improve the uniformity and yield of organic NPs by reducing their surface energy and making NPs the most thermodynamically favorable state. Some reports have used surfactants in the synthesis of organic NPs; for example, cholesterol NPs have been prepared using bis(2-ethylhexyl)sodium sulfosuccinate, polyethylene glycol tert-octylphenyl ether (Triton X-100), and cetyltrimethylammonium bromide as surfactants17. In another study, NPs of fluorescent organic materials like 2-(anthracene-9-yl)-9,9-dioctyl-fluorene and 2-(anthracene-9-yl)-spirobifluorene were synthesized by semi-continuous emulsion polymerization of MMA and a polymerizable surfactant sodium 3--2-hydroxypropane-1-sulfonate18. Aggregation-induced emission-based fluorescent organic nanoparticles were also prepared in another study using a commercial surfactant namely, poly(ethyleneglycol)-block-poly(propyleneglycol)-block-poly(ethyleneglycol) diacrylate (F127) for cell imaging purposes19. Due to concentration quenching21 effects observed in many fluorescent molecules, NPs based on molecular fluorophores have not been investigated as often as inorganic NPs, and systematic, quantitative analysis of the effects of surfactants on organic NP properties such as yield, morphology, and photophysical properties, as well as the influence of surfactant properties, such as CMC, is currently lacking.Surface active agents, such as oleate salts and alkyl phosphines, are used extensively in the synthesis of inorganic NPs like Ag, Cu, PbS, ZnS, etc. both in aqueous and non-aqueous dispersions22, organic light-emitting transistors (OLETs)23, and color generation devices as both active materials and color conversion layers (CCLs). Although light-emitting molecules and polymers are well known in these fields, NPs of fluorescent organic materials are comparatively less explored.Fluorescent and phosphorescent organic dyes have immense commercial importance in light emission applications. In recent years, they have evolved to suit new high-tech applications such as organic light-emitting diodes (OLEDs)26 has been used in CCL fabrication to produce white light in the recent past. Apart from phosphors, many luminescent materials like inorganic quantum dots (QDs)29, perovskites31, and organic dyes34 have been incorporated as CCLs in recent reports. Organic dyes, despite showing reasonable performances as CCLs, suffer greatly from aggregation resulting in emission quenching35 and poor photochemical stability36. Inorganic QDs are also very promising candidates for constructing CCLs as they exhibit good photoluminescence quantum yield (PLQY), good color purity, reasonably good stability, and better light scattering effects. However, inorganic QDs are highly sensitive to moisture and temperature37. They also involve undesirable synthetic procedures involving toxic heavy metals like arsenic, cadmium, lead, or spontaneously flammable, malodorous reagents like phosphines. In contrast, fluorescent carbon dots and carbonized polymer dots have shown practical importance in white LEDs42. Similarly, organic NPs also offer the possibility to fabricate CCLs with enhanced photochemical stability, moisture stability, and high uniformity using facile, environmentally-conscientious, synthetic methods.Despite being relatively inconspicuous in the literature, organic NPs offer several key advantages compared to other materials, which has motivated us to study their application in optoelectronic devices. We believe that the study of organic NPs in optoelectronics will open new possibilities in the field of light-emitting diodes (LEDs) and displays, especially as CCLs. A variety of phosphor-based materialsIn this work, we explore the use of both ionic and non-ionic surfactants to synthesize uniform, water-based dispersions of NPs (which we will refer to as nano-dots) of fluorescent organic materials at very high yields. We include a comprehensive study of the effects that surfactants have on the properties of the fluorescent organic nano-dots, which, to our knowledge, has not been reported before. Moreover, we demonstrate the application of the nano-dots as CCLs, which result in high color conversion efficiencies, good photochemical stability, and excellent repeatability. We believe that CCLs prepared using fluorescent organic nano-dots will have wide applicability in light-emitting devices due to their water-processability and exceptional combination of properties compared to conventional CCL materials; they have the potential to give many types of light-emitting devices substantial improvements in stability and performance.43 are amphiphilic organic compounds, which contain both hydrophilic and hydrophobic groups. In aqueous solutions, their hydrophobic tails aggregate and self-assemble into cores, while hydrophilic heads remain in contact with the aqueous phase, minimizing interaction between the hydrophobic tails and the polar solution. Such micelle structures spontaneously self-assemble above a certain critical concentration, called the critical micelle concentration (CMC), which is unique to each surfactant. In this study, we have explored the synthesis of organic nano-dots using both ionic and non-ionic surfactants at concentrations below and above the CMC of surfactants to develop an understanding of how surfactants influence the size of organic nano-dots and utilized these nano-dots to make CCLs for LEDs. Nano-dots are formed when a solution of fluorophore rapidly precipitates as it is mixed with a non-solvent (water). If this happens in the presence of a surfactant above its CMC, micelles are able to encapsulate the fluorophore particles as they precipitate, through interaction between the non-polar surfactant tails and non-polar fluorophore molecules. The presence of surfactant molecules is expected to minimize the surface energy of small particles, whereas in the absence of surfactant, the surface energy is minimized when large fluorophore particles are formed; thus small particles (nano-dots) are formed when the precipitation occurs in the presence of a surfactant above its CMC. Since the formation of nano-dots involves a physical change in the state of the fluorophore , most of the properties of the fluorophore are not significantly changed, though the absorption and emission spectra of such nano-dot dispersions fall between those of the bulk, solid-state phase and true solution phase of the fluorophore, as can be seen in the photoluminescence (PL) spectra of nano-dots.Surfactants44 -4,6-dicyanobenzene, 2,4,5,6-tetrakis(9H-carbazol-9-yl) isophthalonitrile), CzDABNA45 phenyl)-5,9-dihydro-5,9-diaza-13b-boranaphthoanthracen-7-amine), and 4tBuMB46 phenyl)-5,5-difluoro-10-(2-methoxyphenyl)-5H-4l4,5l4-dipyrrolodiazaborinine)  emitter Ttrz-DI phenyl)-10,15-dihydro-5H-diindolocarbazole) was synthesized and purified  Fig.\u00a0.Fig. 1Sc47 represents the ionic surfactant and Triton X-100 represents non-ionic surfactant having (CMC~0.22\u20130.24\u2009mM)48.A number of studies have reported using surfactants to synthesize organic nano-dots, however, to our knowledge, a systematic, experimental investigation of the influence of surfactants on the properties of fluorescent organic nano-dots is currently lacking. This motivated us to carry out a comprehensive investigation of the effects of surfactants on the properties of organic nano-dots. We used both ionic and non-ionic surfactants for the synthesis of nano-dots where tetra-butyl ammonium oleate (TBA oleate) (CMC~0.45\u2009mM)Concentrations ranging from 0.2\u2009mM to 10\u2009mM of these surfactants were used to synthesize the nano-dots, in order to characterize relationships between surfactant concentration, particle size, and yield of the fluorophore as sub-micron particles. Comparing the PL spectra of the bulk solutions, nano-dot dispersions, and bulk films showed significant differences in peak positions due to the variation in particle sizes. In the solution state, fluorophores exist as isolated molecules, whereas in the bulk film state, fluorophore molecules are aggregated into a bulk, solid structure with dimensions much larger than the size of the molecule. However, in the nano-dot dispersions, the particle size tends to be in micro to nanometer scale with a finite number of molecules in each particle, falling somewhere between bulk solid and solution states. PL data of nano-dot dispersions show peak positions in-between solution and film states as shown in Fig.\u00a049, and likely suffer from the inefficient conversion of valuable fluorophore materials into sub-micron particles.The influence of surfactant concentration on the size and shape of fluorophore particles was initially inspected by optical microscopy Fig.\u00a0\u201311; surfND dispersions prepared with different surfactant concentrations were studied by fluorimetry. In order to prepare samples for PL measurement, dispersions were filtered through 200\u2009nm PTFE filters to remove any particles larger than 200\u2009nm in diameter. As shown in Fig.\u00a0We investigated the yield of Ttrz-DI nano-dots with diameters below a certain threshold  using both ionic and non-ionic surfactants. The yield was calculated spectrophotometrically as described in the methods section. The ionic surfactant TBA oleate exhibited a superior yield of Ttrz-DI nano-dots compared to Triton X-100, and the yield of the particles using 6\u2009mM concentration of surfactants was relatively higher than other concentrations. These data show that particles of <200\u2009nm size were synthesized with yields of 61.73% (Triton X-100) and 96.3% (TBA oleate) Fig.\u00a0. This daN-methylpyrrolidone (NMP), and N-methyl imidazole were used for the nano-dot synthesis. A slight increase in yield and PLQY was obtained in the case of 1,3-dioxalane and NMP however, the average particle size was found to be the lowest when the solvent medium was THF . Organic nano-dot dispersions were mixed with PVA solutions and processed to make uniform films. A blue LED with 400\u2009nm emission wavelength was used Fig.\u00a0 to excit51. To make a direct comparison, the molar concentration of QDs in the CCL film, host polymer concentration, and film casting procedure were kept identical to the nano-dot films.To perform a comparative study, we prepared CCLs using the fluorophores described in previous experiments, as well as a high-performance, green TADF emitter, 4CzIPN, which has a PLQY of over 95%. A LED with 400\u2009nm emission wavelength was used as a primary light source simulating the type of light source used in commercial\u00a0lighting or display applications. The CCE was calculated and compared to a reference color conversion filter of inorganic green fluorescent QDs  dispersed in a poly(methyl methacrylate) (PMMA) host, which is representative of the current state-of-the-art in inorganic color conversion filtersCompared to the reference QD filter, which converted 21.0% of the incident blue light at 400\u2009nm into green light of 527\u2009nm, 4tBuMB and 4CzIPN showed similar color conversion efficiency, converting it to red or green light with 27.8% or 19.3% efficiency, respectively. Since the intrinsic PLQY of Ttrz-DI (PLQY~ 70%) is less than 4CzIPN, 4CzIPN showed higher color conversion efficiency than Ttrz-DI under similar conditions , it demonstrates that the organic nano-dots can be used to make CCLs even with blue emission. Since boron-containing fluorophores tend to show narrow FWHMs, nano-dots based on materials like CzDABNA show great promise as emissive layers in display applicationsIn order to quantify the advantages of CCLs based on NDs, we have performed a study comparing the performance and efficiency of nano-dot-based CCLs vs bulk organic fluorophores. Organic fluorophores were dissolved in a PMMA matrix with the same molar concentration as used in the nano-dot CCLs. These results are summarized in Supplementary Table\u00a053. Boron-containing nano-dots like TNAP and CzDABNA with narrow FWHM and high color purity (obtained from CIE coordinates) could find merit as potential candidates in display application since their color gamut occupies 105.96% area compared to NTSC color space in the CIE coordinates. Whereas, nano-dots of TADF materials like Ttrz-DI and 4CzIPN with broader FWHM show 71.05% and 76.91% area compared with NTSC color space, would be applicable as CCLs in white LEDs for future lighting purposes. Table\u00a0Supplementary Fig.\u00a0The stability of CCLs is a primary concern, as CCLs must withstand thousands of hours of continuous use in order to make marketable displays or light sources, and many emerging emissive materials, such as inorganic QDs, despite having excellent photophysical properties, cannot be used commercially owing to instability. Therefore, the photo-stability of the nano-dot films was quantified under accelerated aging conditions in which the filters were exposed to constant UV radiation for 120\u2009hours while their CCE with excitation at 400\u2009nm was measured periodically. Compared with the bulk Ttrz-DI film, the nano-dot film of this material showed a much slower decay rate under UV radiation  producing optimal nano-dot dispersions with sizes ranging from 120 to 165\u2009nm. The resulting water-based nano-dot dispersions were easily prepared and could be processed into neat films or dispersed in carrier polymers such as PVA. Such films showed outstanding color conversion characteristics with excellent air and photo-stability as well as high color purity (FWHM from 21.1 to 87.4\u2009nm). The organic nano-dot CCLs showed superior CCE and stability compared with state-of-the-art InP-based QDs with similar PLQY. Among these nano-dots, boron-containing TNAP allowed 105.96% area coverage in the color gamut (CIE-1931) compared with the standard NTSC color space; revealing their potential applicability in modern display devices. Whereas, nano-dots based on TADF materials with broader emission spectra exemplify merits as efficient white LEDs. CCLs made from these nano-dots are also potentially cost-effective compared to their inorganic counterparts due to their superior stability, similar synthetic cost, and reduced environmental/safety concerns associated with water-based processing. By choosing appropriate fluorophores, we show that CCLs based on organic nano-dots prepared by this method have superior characteristics and potential as environmentally friendly replacements for conventional CCL materials, with the potential to greatly impact the futures of display and white light-emitting devices.TBA oleate was synthesized by reacting tetrabutylammonium hydroxide with oleic acid. In all, 10\u2009mL of 1.0\u2009M tetrabutylammonium hydroxide in methanol solution was taken in a round bottom flask followed by the addition of 10\u2009mL methanol. Oleic acid was added to the flask dropwise and the reaction was allowed to proceed for 1\u2009hour at room temperature with constant stirring. After the completion of the reaction, the product was separated by washing with diethyl ether to remove unreacted oleic acid. The product was further washed with ether and subjected to drying under vacuum for 5\u2009hours at room temperatureFirst,\u00a01\u2009g of PVA was dissolved in deionized water to make a 10 weight percentage stock solution. Then, 0.3\u2009g of a\u00a01\u2009mM Ttrz-DI nano-dot dispersion was measured and added into 2.7\u2009g of the\u00a010% PVA solution in water. The mixture was sonicated for 10\u2009mins to make the dispersion uniform. Next, 3\u2009mL of the mixture was drop-cast uniformly on a 2.5\u2009cm\u2009\u00d7\u20092.5\u2009cm plastic tray and the solvent was evaporated by heating at 60 \u00b0C for 4\u2009hours in a convection oven. After solvent evaporation, the film was taken off using tweezers and the thickness of the film was found to be 160\u2009\u00b5m measured using digital Vernier calipers.PMMA  was dissolved in 5.0\u2009mL chloroform to make a 10 weight percentage stock solution. To 2.7\u2009g of this solution was added 0.30\u2009g of a 1\u2009mM InP QD dispersion in chloroform. The mixture was sonicated for 10\u2009minutes to achieve uniform dispersion. 3\u2009mL of the mixture was then drop-cast uniformly onto a 2.5\u2009cm\u2009\u00d7\u20092.5\u2009cm glass substrate and the solvent was evaporated by heating on a hot plate at 40 \u00b0C for 3\u2009hours. The thickness of the film was 120\u2009\u00b5m measured using digital Vernier calipers.A series of solutions of each fluorophore in THF was prepared with a known concentration of each fluorophore and suitable optical density (in the range of 0.05\u20132 absorbance units) and the absorbance was recorded. These data were used to prepare a calibration curve (absorbance vs concentration) for each fluorophore in THF based on the Beer-Lambert law where concentration and absorbance are linearly proportional. Concentrations of nano-dots in aqueous dispersions (after passing through 0.2\u2009\u00b5m and 0.45\u2009\u00b5m filters) were determined spectrophotometrically by carefully diluting the aqueous dispersions by a known ratio in a large excess of THF using a micropipette, yielding homogenous THF solutions with optical density suitable for UV-vis. UV-Vis spectra of these dilute solutions were then compared to the calibration curve to back-calculate the molar concentration of each fluorophore in the filtered, aqueous dispersions. The yield calculation was based on the number of moles of the particles which passed through a filter (0.2\u2009\u00b5m and 0.45\u2009\u00b5m) relative to the total moles of fluorophore initially dissolved in THF prior to anti-solvent precipitation.Supplementary InformationPeer Review File"}
+{"text": "This investigation of morphology-wetting links was performed using a biomimetic approach. Three natural leaves\u2019 surfaces were studied: two bamboo varieties and Ginkgo Biloba. Multiscale surface topographies were analyzed by SEM observations, FFT, and Gaussian filtering. A PDMS replicating protocol of natural surfaces was proposed in order to study the purely morphological contribution to wetting. High static contact angles, close to 135 Controlling the wetting properties of surfaces is an important issue for many applications. For instance, highly hydrophobic surfaces may induce anti-icing , self-clrphology . Since trphology  and in trphology ,23,24,25rphology .Theoretical approaches of hierarchical surface wetting have been developed by coupling Wenzel and Cassie\u2013Baxter\u2019s models as a function of scale . In suchThese amazing properties of natural surfaces are at the basis of the biomimetic approach currently under development both in academia and in the industry. The manufacturing of such hierarchical surfaces inspired from nature is performed through material removal processes such as photolithography  or laserIn this work, three natural surfaces of plant leaves are studied: two bamboo leaves and the Gingko Biloba leaf. A specific PDMS replication protocole is proposed. At first, the topography of the surfaces is analyzed through a coupled approach: SEM observations and spatial filtering analysis from optical interferometric measurements. Second, wetting properties analysis of surfaces is performed through static and dynamical contact angles measurements. Topographical and wetting analyses are both performed on natural leaves and on replicas. Finally, using a hierarchical approach, the relationship between multiscale topography and wetting behavior is proposed.Three natural surfaces of plant leaves are studied: two different varieties of bamboo and the Ginkgo Biloba. The Ginkgo Biloba leaf has been chosen for its well-kown water-repellent properties as studied by Neinhuis and Barthlott . Bamboo Phyllostachys Aureosulcata Aureocaulis  Sylgard 184 reference in a 10:1 weight ratio with the curing agent ,42. To mTo obtain the positive replica, the same protocol is used with the negative replica previously made as the original surface. However, to limit PDMS/PDMS adhesion, a coating of a few nanometers of gold is made with a vacuum sputtering metallizer. This step is traditionally carried out with a fluorine treatment ,43. HoweThe different steps of this PDMS-replication protocol are presented in the The morphologies were observed with a scanning electron microscope (MIRA3 Tescan). These observations were carried out with a secondary electron detector to reveal the topographic contrast.The various patterns constituting the topography of the surfaces are thus described. The in-plane contributions of the patterns (width and periodicity) can be measured precisely.The characterization of the morphology by SEM was completed using a quantitative analysis based on optical inteferometric measurements. These measurements were carried out in VSI mode , with green light (515 nm wavelength). The analyses of interferometric measurements were performed with the MountainsMap software  using the ISO-25178 roughness standard .The first filtering method involves Fast Fourier Transform (FFT). This method is used to characterize the range of periodical structures. FFT is performed on an experimental profile a and proThe second filtering process involves Gaussian filters . Gaussiaobtained . The firobtained b. The seobtained c. For a To overcome the difficulty of topographic analysis of natural leaves due to their hierarchical structure, two spatial filtering analysis methodologies are proposed. They enable the description of each topographical scale separately:In addition, classical roughness parameters are measured on the raw interferometric images as well as on the surfaces obtained by Gaussian filtering. The parameters calculated from the interferometric measurements are arithmetic mean height Sa , root meSa Sa=1r [The meter, r . These tThe measurement of the The wettability of the surfaces was quantified using a DSA 30 goniometer (Kruss). Static apparent contact angle measurements ,46 were Dynamic measurements were performed by injection/absorption method. From a 3 \u03bcL droplet, the volume increases by 7 \u03bcL with an injection velocity of 10 \u03bcL/min. Then, the 10 \u03bcL droplet is absorbed with same velocity as for injection. The base diameter of the droplet is measured during the variation of volume, and it is used to detect advancing and receding of the droplet. The apparent contact angle is measured with the tangent-2 method to obtain advancing contact angle When the texture is anisotropic, the anisotropy of the wetting properties was quantified by measuring the contact angles in two orthogonal directions: longitudinal direction (observation perpendicular to the grooves) and transversal direction .Morphological and topographical characterizations of the Phyllostachys and Sasa leaves are presented, respectively, in The textures of both bamboo types can be classified into three categories: ridge lines, bumps, and epicuticular wax. The main ridge line is the extension of the stem of the leaf. This main ridge line is visible in The ridge lines are covered with bumps. Two different bumps are identified: oriented bumps and simple bumps. Oriented bumps are observable in A last roughness scale is observable on the bamboo leaves and corresponds to the presence of an epicuticular wax. This wax is superimposed on the textures already presented and adds a nanometric scale to these surfaces. This wax can be seen in The orders of magnitude of the dimensions of these objects are summarized in the Both bamboo species possess the same pattern. However, the dimensions of these topographies are different between Phyllostachys and Sasa. The period of the third-type of ridge line measures 160 \u03bcm on the Phyllostachys, while it is evaluated at 300 \u03bcm on the Sasa. The oriented bumps are almost three times larger on the Sasa, and they are much more numerous. These differences could lead to differences in wetting properties. In addition, ridge lines and oriented bumps bring an anistropy on the topographies. This anisotropy could lead to anisotropic wetting properties.The morphology and the topography of the ginkgo biloba leaf are presented in Two areas of different textures can be observed on SEM images and interferometric measurements. The first one, which is very predominant, is composed of bumps with a diameter of 15\u201320 \u03bcm. These bumps can be seen on SEM images f. The seIn addition, the structures of ginkgo biloba are covered by an epicuticular wax visible on the SEM images i. HoweveAmong the bumps, it is also possible to observe structures associated with the biological behavior of the ginkgo biloba leaf: the stomata. The stomata is a structure that ensures gas exchange between the leaf and its environment. These structures, which are observed on many plants, are shown in The texture of the PDMS replicas is presented in Leaves and replicas are also compared by interferometric measurements . On bambed bumps . These cOn ginkgo surfaces, only one micrometric pattern is present; consequently, no filtering approach is involved on this type of natural surface.The roughness parameters The results on  another . The difThe urfaces ( and 2)))Sa and SA last observation concerns the comparison of The wetting experiments carried out on plant surfaces show a high hydrophobicity of these surfaces . Both spThe positive replicas of these plants present static contact angles greater than 130\u2218 . In addil leaves . These dThe contact angles on the replicas compared to the flat PDMS indicate a very high texture contribution to the hydrophobicity of these surfaces . The staThe positive replicas present very close contact angles despite topographic differences. This observation does not ensure that the wetting configurations of these surfaces are identical. However considering the very low hysteresis values  also seems unlikely, with a predicted angle of 166v\u2019s work ,52. TherThe same approach is developed for the Sasa replica. The hypothesis of a pure Wenzel (configuration 6) wetting is also doubtful. The predicted contact angle is much lower than the experimental value. Configurations 4 and 5 also do not explain the experimentally measured contact angles. The predicted angles for configurations 1, 2, and 3 are much more consistent with the measured values. Among these three configurations, configuration 3 predicts a Wenzel-type wetting for bumps. However, for Phyllostachys surfaces, this configuration is not revelant for the hysteresis contact angle value. The probable configuration for Sasa surfaces is therefore among configurations 1 and 2.For the replica of Ginkgo Biloba, the pure Cassie\u2013Baxter state (configuration 1) predicts 144From the point of view of contact angles and hysteresis, Phyllostachys surfaces are wet in configuration 5 and Sasa surfaces are wet in configurations 1 or 2. These supposed configurations are consistent with the other observations on the experimental measurements. The experimental values show that the hysteresis is greater on the Sasa replica than on the Phyllostachys replica. According to Dubov\u2019s work ,52, the Another experimental observation is that texture anisotropy does not lead to anisotropy of the wettability properties. Texture anisotropy is mainly provided by the second type of ridge line and by the oriented bumps. The second type of ridge line does not contribute to the wetting: it does not bring anisotropy. Concerning the oriented bumps, in the case of Phyllostachys, it contributes less to the wetting than the simple bumps, which are much more numerous. The observation of an isotropy of the wetting properties is therefore not surprising. In the case of Sasa, configuration 1 depends a lot on the oriented bumps. However, configuration 2 depends on all of the bumps. Configuration 2 is much less anisotropic than configuration 1. Configuration 2 is therefore more relevant for the replica of Sasa, with regard to the spreading anisotropy.Thus, it is interesting to note that, despite the similarity of patterns on the bamboo topographies, the differences in dimensions result in two different wetting configurations.Natural surfaces provide very complex and often hierarchical topographies, which gives them remarkable wetting properties. These complex topographies as well as the chemistry of natural surfaces make the study of the topography-wetting link very complicated. In this study, the description of three plant topographies was carried out by coupling SEM observations and interferometric measurements. An FFT analysis allowed us to access the height of these textures. In addition, through Gaussian filtering approach, the roughness parameters associated with the different scales were determined.Using a PDMS replication process, these topographies were reproduced on surfaces for which the chemistry was controlled. The efficiency of this replication was proven by demonstrating that the micrometric structures of these replications were similar to those observed on the original leaves. However, it was also shown that the protocol is not effective in replicating the nanometric structure of these plants.An experimental study of the contact angles of the leaves and the replicas was carried out. The measured contact angles as well as the hysteresis showed that the plants studied presented high hydrophobic properties. These hydrophobic properties were well transferred to the PDMS, since 110The link between the topography and wetting properties of these surfaces was thus studied through a multi-scale approach considering several wetting configurations. The topographic parameters related to the hierarchical wetting model were determined on the one hand through This work could however be extended by realizing a nanometric texture superimposed on the replicated micrometric structures. This would make it possible to study the influence of the epicuticular wax that disappeared during the replication protocol. This nanometric scale could in particular make it possible to reach a superhydrophobic behavior on polymeric surfaces."}
+{"text": "In the eighth edition of their cancer classification system, the Union for International Cancer Control (UICC) defines contralateral hepatic artery invasion as T4, which is considered unresectable as it is a \u201clocally advanced\u201d tumour. However, in the last decade, several reports on hepatic artery resection (HAR) for perihilar cholangiocarcinoma (PHCC) have been published. The reported five-year survival rate after HAR is 16\u201338.5%. Alternative procedures for the treatment of HAR have also been reported. In this paper, we review HAR for PHCC, focusing on its history, diagnosis, procedures, and alternative procedures.Perihilar cholangiocarcinoma (PHCC) is one of the most intractable gastrointestinal malignancies. These tumours lie in the core section of the biliary tract. Patients who undergo curative surgery have a 40\u201350-month median survival time, and a five-year overall survival rate of 35\u201345%. Therefore, curative intent surgery can lead to long-term survival. PHCC sometimes invades the surrounding tissues, such as the portal vein, hepatic artery, perineural tissues around the hepatic artery, and hepatic parenchyma. Contralateral hepatic artery invasion is classed as T4, which is considered unresectable due to its \u201clocally advanced\u201d nature. Recently, several reports have been published on concomitant hepatic artery resection (HAR) for PHCC. The morbidity and mortality rates in these reports were similar to those non-HAR cases. The five-year survival rate after HAR was 16\u201338.5%. Alternative procedures for arterial portal shunting and non-vascular reconstruction (HAR) have also been reported. In this paper, we review HAR for PHCC, focusing on its history, diagnosis, procedures, and alternatives. HAR, undertaken by established biliary surgeons in selected patients with PHCC, can be feasible. Perihilar cholangiocarcinoma (PHCC), also known as a Klatskin tumour, is one of the most intractable gastrointestinal malignancies ,2. ThesePHCC sometimes invades the surrounding tissues, such as the portal vein, hepatic artery, perineural tissues around the hepatic artery, and hepatic parenchyma . As surgBefore 2000, when the use of portal vein embolisation (PVE) became widespread and a major hepatectomy could be performed safely, postoperative liver failure was a serious problem . SeveralIn this article, we review HAR for PHCC, focusing on its history, diagnosis, procedures, and alternatives.The right hepatic artery is closely associated with the posterior or anterior surface of the biliary confluence, and is often involved in tumours . PreviouThe bile duct wall in the hepatic hilum is very thin. As a result, the tumour easily invades the area outside of the bile duct, and spreads along the hepatic arterial plexus and other structures. Therefore, even if it does not directly invade the arterial wall structures, it is clinically in close proximity and requires HAR. In this case, patients with advanced perihilar cholangiocarcinoma, who required both HAR and PVR, are involved. A preoperative computed tomography (CT) reveals a tumour close to the right hepatic artery .Surgical results and outcomes improved after 2010. In 2010, Nagino et al.  reportedAPS was first described in 1989 by Sheli et al.  in the fWe have advocated APS as an alternative procedure for microvascular arterial anastomosis for HAR in hepatopancreatic biliary surgery . Suzuki From 2000 to 2007, we reported the results of APS in 18 patients with PHCC . We confPerforming the procedure for salvage after postoperative complications has also been reported by a few authors ,39. BassAnother alternative procedure after HAR is non-vascular reconstruction. Peng et al.  reportedIt is well known that arterial collateral flow from arteries around the liver, such as the right inferior diaphragmatic artery and/or adrenal gland artery, can provide intrahepatic arterial flow when native hepatic arterial flow is absent. Therefore, HAR without arterial reconstruction is a possible option in selected cases.PHCC with insufficient remnant liver volume, and highly advanced vascular invasion not amenable to reconstruction, are clearly considered unresectable. However, associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a potential option for effectively increasing remnant liver volume to allow resection. ALPPS achieves fast hypertrophy of the future liver remnant (74% after a median of 9 days), avoiding the risk of tumour progression compared with PVE . Howeverp = 0.001) [The ideal option for unresectable PHCC would be liver transplantation (LT). A few early studies failed to show promising results after LT for PHCC, with three- and five-year survival rates never exceeding 40% and 30%, respectively, and with very high tumour recurrence rates ,42,43. I= 0.001) . The his= 0.001) .One of the major causes of mortality after PHCC surgery is liver failure , againstRecent developments in operative procedures and perioperative management may have made radical resection of T4 PHCC with contralateral hepatic artery involvement more amenable. Hepatic resection with contralateral HAR for locally advanced PHCC is feasible in selected patients at high-volume centers."}
+{"text": "A receptor \u03b32 subunit Gabrg2Q390X mutation in patients, which presents more severe epileptic symptoms in female patients than male patients. However, the seizure onset mechanism during sleep still remains unknown. Our previous work has shown that the sleep-like state-dependent homeostatic synaptic potentiation can trigger epileptic spike\u2013wave discharges in one transgenic heterozygous +/Q390XGabrg2 knock-in mouse model.1 Here, using this heterozygous knock-in mouse model, we hypothesized that slow-wave oscillations themselves in vivo could trigger epileptic seizures. We found that epileptic spike\u2013wave discharges in heterozygous +/Q390XGabrg2 knock-in mice exhibited preferential incidence during non-rapid eye movement sleep period, accompanied by motor immobility/facial myoclonus/vibrissal twitching and more frequent spike\u2013wave discharge incidence appeared in female heterozygous knock-in mice than male heterozygous knock-in mice. Optogenetically induced slow-wave oscillations in vivo significantly increased epileptic spike\u2013wave discharge incidence in heterozygous +/Q390XGabrg2 knock-in mice with longer duration of non-rapid eye movement sleep or quiet\u2013wakeful states. Furthermore, suppression of slow-wave oscillation-related homeostatic synaptic potentiation by 4-(diethylamino)-benzaldehyde injection (i.p.) greatly attenuated spike\u2013wave discharge incidence in heterozygous knock-in mice, suggesting that slow-wave oscillations in vivo did trigger seizure activity in heterozygous knock-in mice. Meanwhile, sleep spindle generation in wild-type littermates and heterozygous +/Q390XGabrg2 knock-in mice involved the slow-wave oscillation-related homeostatic synaptic potentiation that also contributed to epileptic spike\u2013wave discharge generation in heterozygous +/Q390XGabrg2 knock-in mice. In addition, EEG spectral power of delta frequency (0.1\u20134\u2005Hz) during non-rapid eye movement sleep was significantly larger in female heterozygous +/Q390XGabrg2 knock-in mice than that in male heterozygous +/Q390XGabrg2 knock-in mice, which likely contributes to the gender difference in seizure incidence during non-rapid eye movement sleep/quiet\u2013wake states of human patients. Overall, all these results indicate that slow-wave oscillations in vivo trigger the seizure onset in heterozygous +/Q390XGabrg2 knock-in mice, preferentially during non-rapid eye movement sleep period and likely generate the sex difference in seizure incidence between male and female heterozygous +/Q390XGabrg2 knock-in mice.Sleep is the preferential period when epileptic spike\u2013wave discharges appear in human epileptic patients, including genetic epileptic seizures such as Dravet syndrome with multiple mutations including SCN1A mutation and GABA Dravet syndrome mouse model with +/Q390XGabrg2 mutation. Meanwhile, the suppression of slow-wave oscillation-related synaptic plasticity in vivo dramatically inhibits epileptic activity in the het +/Q390XGabrg2 mice, suggesting that slow-wave oscillations can trigger seizures during non-rapid eye movement sleep.Catron et al. report that increased slow-wave oscillations cause epileptic activity in one   However, there still remains lack of a clear mechanism for seizure onset/incidence modulation by day\u2013night cycle.Sleep is the brain state when cortical activity decreasesde novo genetic mutations identified in human patients, such as SCN1A and GABAergic receptor Gabrg2Q390X mutations.24-28 These mutations impair the GABAergic synaptic transmission/plasticity and seizures/epileptogenesis in animal models.26 Moreover, Dravet syndrome patients exhibit preferential incidence of seizures during sleep, rather than wake periods.29-31 With mouse models containing SCN1A mutation,25 GABAergic interneuron dysfunction may play a major role for seizure activity generation,32 while in the het +/Q390XGabrg2 KI mouse model, GABAergic synaptic transmission and plasticity are disrupted for seizure generation.33 However, recent treatment failure with stiripentol [enhancing \u03b13 subunit containing GABAergic receptor function35] in het +/Q390XGabrg2 KI mice36 suggests that there are more complicated epileptic mechanisms to explain seizure onset/treatment in the Dravet syndrome model, especially in day\u2013night cycle modulation. Even in these Dravet animal models, there is no mechanism to address day\u2013night cycle modulation of seizure incidence.37 In the same way, we still lack a mechanism for the sex difference in seizure activity in human epileptic patients,38 which may be linked to sex sleep/wake differences.39-43Dravet syndrome is a severe genetic seizure disorder with multiple +/Q390XGabrg2 KI mice by a sleep-related, state-dependent homeostatic synaptic potentiation mechanism.1 In this study, we examined if sleep SWOs themselves in vivo could causally trigger seizures in het +/Q390XGabrg2 KI mice, which might have implication for seizure onset mechanism and sex incidence difference in human epilepsy patients.Our recent work has found that artificially induced sleep-like slow-wave oscillations (SWOs) can trigger epileptic seizures in het 1 Briefly, both wild-type (wt) littermates and het +/Q390XGabrg2 KI mice [by crossing het KI mice with NpHR expressing mice #012334 (The Jackson Laboratory)45] [both male and female mice used] underwent brain surgery [anaesthesia 1\u20133% isoflurane (vol/vol)] to implant three EEG screw electrodes , one concentric bipolar tungsten electrode in somatosensory cortex  and one fibre optic cannula  for laser light delivery in vivo . The tungsten electrode tip was placed slightly deeper than the optic cannula depth in S1 cortex to ensure that all neurons recorded were controlled by the laser delivered (589\u223c680\u2005nm). Two EMG leads were inserted into neck trapezius muscles to monitor mouse motor activity. After surgery, mice were continuously monitored for recovery from anaesthesia and remained in the animal care facility for at least 1 week  before simultaneous EEG/EMG/multi-unit activity recordings (by persons blind to mouse genotypes). The tungsten electrode/optic cannula placement within S1 cortex was checked in mouse brains after euthanasia. All EEG , multi-unit activity (band filtered 300\u20132000\u2005Hz) and EMG (band filtered at 0.1\u2013400\u2005Hz) were collected  by using two multiClamp 700B amplifiers  and Clampex 10 software , and digitized at 20\u2005kHz using a Digidata 1440\u2005A.With all procedures in accordance with the guidelines set by the institutional animal care and use committee of Vanderbilt University Medical Center and to the National Institutes of Health Guide for Care and Use of Laboratory Mice, mouse EEG surgery  was performed as our previous methods and procedures that experimental mice do not suffer unnecessarily.in vivo or up\u2013down states  were induced by alternating laser delivery  and no laser delivery . Three to four hundred pA currents and 20\u2005ms were chosen to avoid kindling since kindling induction needs larger \u00b5A currents.47-49The laser [DPSS laser MGL-III-589\u201350 ] was delivered through an optic fibre cable connected to the implanted optic cannula, and the laser timing was controlled by Clampex 10 software. Intracortical stimulations of 300\u2013400\u2005pA (20\u2005ms) were applied to simulate neuronal up-states through the planted tungsten electrodes. SWOs ex vivo,1 4-(diethylamino)-benzaldehyde (DEAB) (blocking retinoid acid synthesis)50 was used as a drug to suppress SWO-induced homeostatic synaptic potentiation of excitatory currents in neurons in vivo. The DEAB was dissolved in dimethyl sulfoxide (DMSO)/saline and injected (i.p.) with dosages 100\u2005mg/kg body weight, based on its pharmacokinetical potency in vivo to inhibit aldehyde dehydrogenase in mice.52 Injections were given once per day for 5 days [at almost the same morning time (10:00\u201312:00 pm) in each day] consecutively for its cumulative treatment effect. Mouse EEG recordings were performed before DEAB injections and right after the 5th injection. Control mice were injected with vehicle DMSO/saline, and no epileptic SWDs were subsequently induced. The pre-DEAB and post-DEAB EEG/EMG activity was recorded for continuous 3\u2005h at the same circadian time of the day to avoid potential circadian variant effect on seizure incidence.Based on our previous study 53-55 by a blinded person. For transition epochs containing both wake and sleep states, we defined a state duration >5\u2005s as dominant. Any EEG/EMG activity artefacts associated with motor behaviours (video monitored) were removed from this analysis. EEG data will be down-sampled at 200\u2005Hz for further Matlab  multitaper power spectral analysis.56 For optogenetic and DEAB experiments in vivo, data were obtained from EEG recordings during light phase (mouse sleep period from 10:00 am through 4:00pm) but not during 24\u2005h recordings.Both male and female mice were acclimated to recording chambers (with food/water access) for 2 days before behaviours were recorded for a 24\u2005h period. Mouse sleep/wakeful states  were determined by EEG/EMG activity and video-recorded motor activity and analyzed by persons either blind to mouse genotypes or not-performing recordings. EMG activity and simultaneous video recordings were used to determine wake (continuous movement) or sleep (prolonged periods of no movement). All recorded EEG/EMG were scored as 10\u2005s epochs of awake (low EEG amplitudes with large EMG amplitudes), non-rapid eye movement (NREM) sleep (high EEG amplitudes and dominant frequency <4\u2005Hz), or rapid eye movement (REM) sleep (uniform/low amplitude EEG waveforms and dominant theta frequency 6\u201310\u2005Hz)57) were defined as trains (>1\u2005s) of rhythmic biphasic spikes, with a voltage amplitude at least twofold higher than baseline amplitude for REM sleep and wake periods.58 During NREM sleep, due to large delta EEG amplitudes (0.5\u20134\u2005Hz), bilateral synchronous SWDs  were defined as at least \u00d7 1.5 higher  than the preceding delta EEG amplitudes.60 However, these detected SWDs and SSWDs during NREM sleep or REM/wake period showed similar epileptic waveform patterns. In addition, unilateral SWD/SSWDs were defined as focal epilepsy. All atypical and typical absence epilepsy and GTCS started with SSWDs or SWDs, accompanied by characteristic motor behaviours. The behaviour associated with SWD/SSWD consisted of immobility, facial myoclonus, and vibrissal twitching. The SWD/SSWDs and animal epileptic behaviours were also checked and confirmed by persons blind to animal genotypes. Myoclonic seizures started with focal epileptic spikes in the mice, similar to A322D mice.61 Owing to infrequency of tonic\u2013clonic seizures in these mice, this study did not examine this type of seizures. The onset times of SWD or SSWDs were determined by their leading-edge points crossing (either upward or downward) the precedent EEG baseline and the offset time of SWD or SSWDs by the last epileptic spikes/waves\u2019 trailing points crossing the subsequent EEG baseline. Then the duration of SWDs/SSWDs were determined the time between the onset and offset time. Any EEG episodes with high-frequency activity and muscle artefacts associated with motor behaviours (video monitored) were removed from the power spectral analysis. With Clampfit , NREM power spectral density of sleep EEG was averaged all 0.1\u20134\u2005Hz range with continuous 30\u2005min EEG recordings (10\u2005s window) at the same circadian time of the recording day and portion of REM/wake EEG recordings was not used.39Mouse epileptic behaviours were simultaneously video-recorded with EEG recordings and Racine-scaled and analyzed by persons either blind to mouse genotypes or not-performing recordings. Bilateral synchronous SWDs (6\u201312\u2005Hz) and SSWD  and Matlab. The threshold to detect sleep spindles was set as the amplitudes that were at least 1.5 X the standard deviation larger than the preceding baseline amplitudes and whose durations were between 0.5 and 5\u2005s.63EEG data were downsized to a 200\u2005Hz sampling frequency for analyzing sleep spindle frequency (10\u201315\u2005Hz), based on AASM manualt-tests were used between wt and het mice or between pre- and post-groups with Excel or SigmaStat.All figures were prepared with Microsoft Excel, SigmaPlot, and Adobe Photoshop software. Power analyses were conducted with preliminary data to determine final sample sizes. Data were expressed as mean \u00b1 SEM (standard error of mean). Holm\u2013Sidak test and one-way ANOVA were performed data in figures with SigmaStat when necessary. Otherwise, student paired/unpaired Data of original EEG/EMG recordings will be available upon reasonable request.+/Q390XGabrg2 KI mice. As indicated in +/Q390XGabrg2 KI mice . In addition, compared with wt littermates, sleep in het +/Q390XGabrg2 KI mice seemed to be more fragmented  421.67 \u00b1 140.56 versus het  655.00 \u00b1 189.08, t-test P = 0.017; REM wt 184.11 \u00b1 61.37 versus het 363.58 \u00b1 104.95, t-test P = 0.029; wake wt 340.67 \u00b1 113.56 versus het 515.25 \u00b1 148.73, t-tests P = 0.03) and a trend of increased sleep-state transition .Using 24\u2005h continuous recordings after mouse habituation, mouse active or rest states, EEG patterns and epileptic SWD activity during sleep and wakeful period were examined from wt littermates and het agmented  with mor+/Q390XGabrg2 KI mice exhibited significantly more epileptic SWDs  95.11 \u00b1 31.70 versus het  3876 \u00b1 1119.5, t-test P = 0.0001; during REM wt 17.22 \u00b1 5.74 versus het 566 \u00b1 163.39, t-test P = 0.003; during wakeful period wt 15.33 \u00b1 5.1 versus het 1378.16 \u00b1 397.84, t-test P = 0.002] . Particularly, epileptic SWD incidence preferentially occurred during NREM sleep period  SWD# during NREM/REM/wake, one-way ANOVA P = 0.005 and NREM versus REM P = 0.017, NREM versus wake P = 0.025; SWD duration during NREM/REM/wake, one-way ANOVA P < 0.001 and NREM versus REM P = 0.00003, NREM versus wake P = 0.0003]. These results indicate that in this genetic epilepsy het +/Q390XGabrg2 KI mice, epileptic SWD preferential incidence during NREM sleep period is very similar to sleep preferential incidence of epileptic activity in human Dravet syndrome patients.Unlike EEG patterns in wt littermates, EEG activity in het tic SWDs  .In addition, we found significantly more SWD incidence and longer SWD duration in female het  KI mice   and intracortical stimulation   pre-SWO 40.40 \u00b1 5.78% versus post-SWO 59.60 \u00b1 5.33%, paired t-test P = 0.00001; het  pre-SWO 52.04 \u00b1 4.89% versus post-SWO 63.77 \u00b1 4.76%, paired t-test P = 0.007]. However, under the same conditions, REM sleep and wake duration were slightly decreased in wt and het +/Q390XGabrg2 KI mice (except REM sleep)  pre-SWO 35.53 \u00b1 5.71% versus post-SWO 26.62 \u00b1 5.29%, paired t-test P = 0.0003; het  pre-SWO 30.76 \u00b1 4.35% versus post-SWO 23.64 \u00b1 3.67%, paired t-test P = 0.075] . Correspondingly, following in vivo SWO induction, epileptic SWDs in het +/Q390XGabrg2 KI mice, but not wt littermates, significantly increased in both incidence number and duration (even reached status epilepticus in some het KI mice) pre-SWO 2.78 \u00b1 1.38 versus post-SWO 5.71 \u00b1 2.64, paired t-test P = 0.08; het  pre-SWO 114.86 \u00b1 15.70 versus post-SWO 197.38 \u00b1 13.31, paired t-test P = 0.00001] . These findings likely indicate that SWO induction in vivo can increase NREM sleep duration in all mice and trigger epileptic SWDs in het +/Q390XGabrg2 KI mice.EEG activity during NREM sleep exhibits slow-wave oscillations within delta-frequency range accompanied by simultaneous neuronal up- and down-state activity in the cerebral cortex. Thus, we examined whether induction of slow-wave oscillation  , 20\u2005ms)  inset. W KI mice    . However, following DEAB injections in vivo, epileptic SWDs in het +/Q390XGabrg2 KI mice significantly decreased in incident number and duration  pre-DEAB 7.85 \u00b1 0.86 versus post-DEAB 8.54 \u00b1 2.30, paired t-test P = 0.751; het  pre-DEAB 163.85 \u00b1 32.05 versus post-DEAB 47.03 \u00b1 7.63, paired t-test P = 0.007] . More interestingly, epileptic SWDs in het +/Q390XGabrg2 KI mice were significantly suppressed during NREM sleep but not REM sleep and wake period  pre-DEAB 129.59 \u00b1 16.35 versus post-DEAB 43.63 \u00b1 7.67, paired t-test P = 0.0003]     . In addition, both pre-DEAB and post-DEAB SWD incidence preferred NREM sleep period (both one-way ANOVA P < 0.00001). Together with the results of SWO induction in vivo, these results indicate that SWOs in vivo do causally trigger epileptic SWDs in het +/Q390XGabrg2 KI mice and SWOs may create brain states to trigger or facilitate epileptic SWD generation in het KI mice. However, SWOs seem not to influence myoclonic jerk generation in het +/Q390XGabrg2 KI mice.It is difficult to find a way to suppress SWOs duration , while iriggered  , suggesting that NREM sleep difference in delta-frequency power may generate the gender difference in epileptic SWD incidence during sleep in human Dravet syndrome patients.Since SWO induction  KI mice   only suppresses 70% of seizure incidence in het +/Q390XGabrg2 KI mice. Higher DEAB dosage administration may be needed for the therapeutic treatment with its maximal potency in vivo. However, this SWO-related mechanism might determine the seizure duration for status epilepticus evolution  and its subsequent effects on peripheral organs such as liver and heart (mitochondria),90 which can cause new seizure onset or exacerbate seizures in human patients with aldehyde hydrogenase mutations92 (comparable with acute DEAB action in vivo). Thus, we cannot exclude potential seizure-prone nonessential effects of DEAB in peripheral organs (if this happens), which might cause varied DEAB suppressing effects on epileptic activity in different het +/Q390XGabrg2 KI mice and is the limit to interpret the result of DEAB treatment in vivo in this study. Overall, our data show that the blocking aldehyde hydrogenase with DEAB in vivo (DEAB can be lipid-dissolved51) suppresses epileptic activity, indicating that the homeostatic synaptic plasticity does contribute to seizure incidence/onset mechanism in the het +/Q390XGabrg2 KI mouse model.However, due to half-life time of DEAB+/Q390XGabrg2 KI mice for one genetic generalized epilepsy such as Dravet syndrome due to Q390XGabrg2 mutation. This mechanism may contribute to chronic development of status epilepticus and likely determines the gender difference of seizure incidence and leads to the cognitive deficits in human genetic epilepsy patients.All together this study suggests a novel mechanism of SWO-triggered seizures in het"}
+{"text": "Background: increased pressure on healthcare systems and possible risk of nosocomial COVID-19 infection during pandemic urged many guidelines to severely restrict the number of operations. The aim of this study was to investigate the risk of COVID-19 infection and its complications in patients undergoing urgent or elective operations.Methods: a prospective observational cohort study was conducted in a tertiary surgical center and all patients with no preoperative history of COVID-19 undergoing elective or emergent surgeries were included in this investigation. chest computed tomography (CT) scan or polymerase chain reaction (PCR) test were performed on patients before and after surgery.183 patients who underwent an operation were enrolled in this study. In postoperative follow-up, 12 patients were positive for COVID-19 infection as identified by RT-PCR and non-contrasted chest CT scans. Regrettably, 2 individuals passed with one of these individuals dying as a direct result of COVID-19 infection. All the 12 cases of post-operative COVID-19 patients underwent elective surgeries.the gathered results indicate a need for the re-evaluation of the risks of operation during the COVID-19 pandemic. If operations are performed while observing protective and preventative protocols, the risk of post-operative nosocomial COVID-19 is significantly reduced. Hence, the consequences imposed on patients by the delay or cancellation of operations (most notably in cancer cases) may outweigh the risk of post-operative COVID-19 infections. \u2022If operations are performed, considering protocols, the risk of post-operative nosocomial COVID-19 are significantly reduced.\u2022The\u00a0risks of delay in operations (most notably in cancer cases) may outweigh the risk of post-operative COVID-19\u00a0infections.\u2022It depends on availability of resources to decide about performing elective surgical procedures during covid-19 outbreaks. The COVID-19 virus was first identified in the Wuhan region of China and progressed to spread to other nations over the successive months . As of M country .Admission of an unprecedentedly high number of patients to unprepared hospitals due to the COVID-19 pandemic resulted in increased pressure on healthcare systems ,6. The sThe discussed guidelines provided instructions for all aspects of the health care system including health care personnel, patients, transportation processes, operating rooms, and equipment use . Thus, gAs rapid diagnostic tests for COVID-19 have become widely available and the COVID-19 pandemic has continued, several surgical centers are applying screening systems for COVID-19 testing of patients before admission . Despite2https://ethics.research.ac.ir/ProposalCertificateEn.php?id=128698&Print=true&NoPrintHeader=true&NoPrintFooter=true&NoPrintPageBorder=true&LetterPrint=true.The main goal of this study is to evaluate the rate of postoperative COVID-19 infection and its resultant complications and mortality to provide a basis for surgical risk assessment, ensuring the delivery of the best medical care to patients a prospective observational study was conducted in a tertiary surgical center. Patients who underwent surgery from April to September 2020 was enrolled in this study. This period coincided with the peak of the COVID-19 pandemic in Iran when many non-urgent operations were canceled. This research is fully compliant with the STROCSS 2021 criteria [All patients over 18 years of age, while they had no evidence of COVID-19 infection undergoing elective or emergent surgeries in a tertiary hospital were included in this investigation. Patients suspected of COVID-19 infection based on preoperative chest computed tomography (CT) scan or positive polymerase chain reaction (PCR) test were excluded from the study. Moreover, patients with a positive COVID-19 test within 3 days after surgery were excluded due to the likelihood of the acquirement of this infection pre-operation. For enrolled patients, suspicious postoperative symptoms followed by a definitive diagnosis based on PCR testing and Chest CT scans 3\u201314 days post-surgery was considered as positive post-operative COVID-19 infections.Demographic information and comorbidities were recorded and tabulated. Based on surgical protocols, all patients underwent a non-contrasted chest CT scan but a reverse transcription polymerase chain reaction (RT-PCR) of the oropharynx and nasopharynx was only done for symptomatic patients. Information regarding the operation, including operation type, anesthesia, and operation duration were recorded. All included patients were followed up after surgery and were assessed every day during their hospitalization in addition to follow up appointments 2 and 6 weeks after discharge in person or by phone. During these appointments, they were asked about and examined for suspicious symptoms indicative of COVID-19 infection. While following up, if there was any suspicion for COVID-19 infection, a non-contrasted chest CT scan and RT-PCR test from a nasopharyngeal and oropharyngeal swab was completed.For patients with postoperative COVID-19 infection, the percentage of lung involvement, re-hospitalization, ICU admission due to COVID-19, and its complications and mortality were recorded.t-test was used to compare continuous data, whereas chi-square test and Fisher's exact test was used to compare discrete data.For statistical analysis, the application SPSS version 23 was used. Continuous data was reported as mean\u00a0\u00b1\u00a0standard deviation while discrete data was reported as percentages and frequencies. Independent 3After excluding patients with pre-operative COVID-19 infections and those with the mentioned exclusion criteria, a total of 183 COVID-19 negative patients who underwent an operation were enrolled in this study. 98 participants (53.6%) were female, and 85 participants (46.4%) were male. Furthermore, the mean age within the sample was 47.06\u00a0\u00b1\u00a016.54 years and the mean body mass index (BMI) of the participants was 25.79\u00a0\u00b1\u00a04.29. 76 patients (41%) were overweight, 24 (13.3%) were obese, 75 (41%) were within the normal BMI range, and 8 (4.47%) were underweight. The mean pre-operative hospital admission was 1.74\u00a0\u00b1\u00a02.28 days. The indication and type of surgeries included as; Head & neck cancer 20 (11%), Upper GI cancer 10 (5.5%), Hepatobiliary cancer 20 (11%), Breast cancer 25 (13.5%), Colorectal cancer 8 (4%), Gynecologic cancer 4 (2%), Cholecystectomy 38 (21%), Appendectomy 16 (11.5%), Liver transplant 1 (0.5%), Laparotomy due to peritonitis or obstruction 16 (8%), Tissue repair after traumatic lacerations 11 (5.3%) and Coronary artery bypass graft 14 (6.7%).Among the participants, 27 (17.5%) had diabetes mellitus, 40 (21.9%) had hypothyroidism, 32 (17.5%) had ischemic heart disease, 4 (2.2%) had renal disease, 56 (30.6%) had cancer, and 6 (3.3%) had respiratory disease. 80 patients (9.8%) underwent chemotherapy within three weeks pre-operation. 106 patients (57.9%) did not have any underlying conditions or comorbidities. Moreover, 132 patients underwent an elective operation, and 51 patients underwent a non-elective operation. During the pre-operation admission, it was noted that 1 patient had chronic myeloid leukemia (CML) and underwent an elective open splenectomy associated with a resultant prolonged hospital stay (44 days). Thus, this patient was excluded from the study .Table 1TIn the postoperative follow-up, 12 patients were positive for COVID-19 infection as identified by RT-PCR and non-contrasted chest CT scans. Regrettably, 2 individuals passed with one of these individuals dying as a direct result of COVID-19 infection. All the 12 cases of post-operative COVID-19 patients underwent elective surgeries and the mean age of patients in this group was 49.17\u00a0\u00b1\u00a013.69 years while those with no post-operative COVID-19 had a mean age of 46.99\u00a0\u00b1\u00a016.74 years, demonstrating no significant difference in age between the two groups. The mean operation time in the COVID-19 positive group was 200.75\u00a0\u00b1\u00a0116.25\u00a0min, while this value was 152.5\u00a0\u00b1\u00a0109.5\u00a0min in the COVID-19 negative group. Moreover, no significant difference was observed between the two groups in terms of BMI and sex distribution. The mean hospital stay duration before the operation was 1.55\u00a0\u00b1\u00a01.29 days in the COVID-19 positive group, while this value was 1.74\u00a0\u00b1\u00a02.28 days in the healthy group, once again demonstrating no significant difference between the two groups.However, operation time was significantly higher in elective surgeries. . Interestingly, the distribution of underlying diseases, such as diabetes, hyperthyroidism, ischemic heart disease, renal disorders, cancer, respiratory disorders, and recent chemotherapy and steroid consumption, did not show a significant difference between the post-operative COVID-19 positive and negative groups.4The COVID-19 pandemic significantly affected the efficacy of health care systems as well as the degree and type of care provided to patients all over the world . MoreoveThus far, some studies report a high risk of COVID-19 infection and its related morbidity and mortality in hospital settings which suggests the need for careful observation of patients and the use of appropriate guidelines . Thus faIn our medical center, operations were performed by complete observance of pre-operative, intraoperative, and postoperative protocols. All patients were screened for COVID-19 before operations, and symptomatic patients were tested using RT-PCR. Visiting the patients was only allowed by medical staff to prevent viral spread. All patients were obligated to wear face masks except those requiring oxygen supplements or patients with dyspnea and all healthcare providers always wore face masks and observed infection protection protocols. In our center, no routine screening for COVID-19 was done for healthcare providers, and only symptomatic healthcare staff were evaluated for COVID-19. To add, patients with COVID-19 were admitted to separate wards and operated on in specifically allocated operating rooms. Finally, based on hospital protocol, it was ensured that both pre-operative and postoperative hospital stay were reduced as much as possible.In a study by Axiotakis et al. study 9 cases out of 511 patients undergoing operation were infected with COVID-19. Prior to implementing pre-operative testing in their surgical center. Furthermore, they noted that the rate of COVID-19 infection has been reported to be related to Diabetes and ischemic heart disease .In our study, 12 patients became symptomatic and infected with COVID-19, 2 of whom passed, 1 as a direct result of COVID-19. Thus, the observed mortality rate was less than that observed in similar studies (8.3%). In the Aminian et al. study during the onset of the pandemic, it was reported that three patients developed COVID-19 after the operation with 2 of these individuals passing . In thisIn our study, the incidence of post-operative COVID-19 infection only occurred in patients undergoing elective surgeries. This may be due to the simpler procedures, shorter hospitalization time, and the limited number of patients in non-elective surgeries and can be explained based on the significant difference observed between elective and non-elective operation duration. Although the present study did not show any relationship between the pre-operative length of stay and COVID-19 infection, reduced viral exposure due to observing preventive protocols and reducing the length of hospital stay where possible is key in reducing the risk of COVID-19 and further studies are required to clarify and amend guidelines .One-third of the patients in the present study had cancer. Postponing elective but necessary surgeries for malignant diseases during the COVID-19 pandemic has resulted in increased mortality and morbidity due to metastatic and inoperable cases . ConsideThe limitations of this study include the small sample size and the resulting low number of COVID-19 cases, hence, reducing the study's power and generalizability. Furthermore, it must be noted that non-inclusion of asymptomatic COVID-19 patients who presented with negative RT-PCR and no abnormal findings in the chest CT scans is also a limitation. Furthermore, future investigations comparing the postoperative complications between patients with COVID-19 and healthy individuals should also be conducted and will help shed light on the risks and benefits involved in conducting operations during the pandemic for policy makers.Despite the mentioned limitations, the gathered results indicate a need for the re-evaluation of the risks of operation during the COVID-19 pandemic. If operations are performed while observing protective and preventative protocols, the risk of post-operative nosocomial COVID-19 are significantly reduced. Hence, the risks and the reduction in the quality of care provided and patient quality of life by the delay or cancellation of operations (most notably in cancer cases) may outweigh the risk of post-operative COVID-19 infections although a case-by-case analysis of the risks and comorbidities of each patient is likely required.Ethical approval was requested and obtained from the \u201cTehran university of medical sciences\u201d ethical committee.Tehran university of medical sciences.All authors contribute in this study equally.This study did not interfere with patients care but all patients was infirmed and consent was obtained.1.Name of the registry:2.Unique Identifying number or registration ID:3.Hyperlink to your specific registration (must be publicly accessible and will be checked):Mohammad Ashouri.Not commissioned, externally peer reviewed.The authors have no conflict of interest."}
+{"text": "Burkholderia pseudomallei. Although most global melioidosis cases are reported from tropical regions in Southeast Asia and northern Australia, there are multiple occurrences from sub-tropical regions, including the United States (U.S.). Most melioidosis cases reported from the continental U.S. are the result of acquiring the disease during travel to endemic regions or from contaminated imported materials. Only two human melioidosis cases from the continental U.S. have likely acquired B. pseudomallei directly from local environments and these cases lived only ~7 km from each other in rural Texas. In this study, we assessed the risk of acquiring melioidosis from the environment within the continental U.S. by surveying for B. pseudomallei in the environment in Texas where these two human melioidosis cases likely acquired their infections. We sampled the environment near the homes of the two cases and at additional sampling locations in surrounding counties in Texas that were selected based on ecological niche modeling. B. pseudomallei was not detected at the residences of these two cases or in the surrounding region. These negative data are important to demonstrate that B. pseudomallei is rare in the environment in the U.S. even at locations where locally acquired human cases likely have occurred, documenting the low risk of acquiring B. pseudomallei infection from the environment in the continental U.S.Melioidosis is an underreported human disease of tropical and sub-tropical regions caused by the saprophyte  Burkholderia pseudomallei is a Gram-negative saprophytic bacterium and the causative agent of the tropical and sub-tropical human disease, melioidosis [B. pseudomallei are usually acquired from the environment via contaminated soil or water with percutaneous inoculation and inhalation as the main routes of transmission [e.g., the Caribbean, Central/South America, Southeast Asia), 2) acquired within the CONUS from contaminated items imported from endemic regions, and 3) acquired directly from the local environment in the CONUS . Howironment , 28.B. pseudomallei is persisting in the environment in Texas, as the two patients acquired melioidosis 14 years apart and only ~7 km from each other [B. pseudomallei to persist in arid regions is poorly understood, although it has been speculated that a latent state may play a role in the persistence of B. pseudomallei in these situations, based upon a study from Western Australia that demonstrated limited genetic changes in B. pseudomallei in an arid region over a >50 year period [B. pseudomallei near the underground water table to migrate towards the surface from deeper soils. If a better understanding of how and where B. pseudomallei persists in the environment in arid regions, such as Texas, can be developed, then it could help mitigate future melioidosis cases, such as advising the treatment of well water [Although rare, it is possible that ch other , 14. Ther period . Additior period , 11, sugll water  or avoidll water .B. pseudomallei in the environment in the CONUS. Future efforts should focus on searching for B. pseudomallei in the environment in regions where it is predicted to likely occur within the CONUS  cluster of B. pseudomallei [To prepare the environmental scrapes for culturing, the 2 mL tubes containing the scrapes were first vortex at high speed for 1 minute and then sonicated for 5 minutes using a Branson sonicator bath set to 70W, 42kHz at room temperature. All samples were processed in the same way for the detection and isolation of escribed  with thel., 2019  except tl., 2019 . Brieflydomallei .S1 Appendix(PDF)Click here for additional data file.S1 FigTo construct the accessible area used as a model calibration area (pink), we used a ratio of 500 km around 28 recent environmental soil isolations starting in the 2000s obtained from . Locatio(TIF)Click here for additional data file.S2 FigModel interpretation in the purple areas would be highly inadvisable.(TIF)Click here for additional data file.S3 FigMaps show the continuous model output (upper panel) and the uncertainty is represented as the interquartile range (IQR) among bootstrap replicates (bottom panel). Gray represents areas of strict extrapolation automatically set to zero by the algorithm.(TIF)Click here for additional data file.S1 TablePCs included in the analyses are highlighted in gray.(PDF)Click here for additional data file.S2 TableOR = omission rates, pROC = partial receiving operating characteristic curve (ROC), AICc = Akaike Information Criterion corrected for sample size.(PDF)Click here for additional data file."}
+{"text": "The perception, processing, and recall of space- and context-related information has become a major topic in mammalian cognitive neurosciences, following several milestone discoveries: the location-specific activation of \u201cplace cells\u201d , the entThere are, however, major gaps in our knowledge. First, the mechanisms underlying long-term consolidation of spatial memories are largely unknown. Second, the physical substrate of remote memories and the mechanisms of their re-activation are elusive. Third, the functions and interactions of different brain areas in spatial cognition are highly under-studied. In order to answer these questions several groups go beyond the traditional focus on the hippocampal formation and include further areas which are involved in spatial information processing: medial and lateral entorhinal cortex, peri- and postrhinal cortex, frontal, prefrontal, parietal and somatosensory neocortex, ventral and dorsal striatum, thalamic nuclei, and other subcortical areas. This development is not at least fostered by technological progress in structural and functional connectomics and in long-term high-resolution recordings from awake, behaving animals.Sauer, Folschweiller, and Bartos  have nowIn this situation, Sauer and colleagues analyzed spatial tuning properties of neurons throughout the dorsal-ventral axis of the mPFC. Using implantable electrodes with multiple (64) linearly arranged contact sites they were able to record action potentials from differently located, identified neurons over prolonged periods . The behavioral setup consisted of a circular track, presented as virtual reality, while the animal was head-fixed and running on a wheel. This setting restricts the animal\u2019s behavior and allows fast switches between different \u201cenvironments,\u201d facilitating interpretation of data. There was also no reward or emotional load by aversive stimuli, such that the experiment merely addressed spatial information processing during the natural exploration behavior of the animals. With this approach, they made three important discoveries: (i) about one third of all neurons in the mPFC are active at specific locations ; (ii) changing the virtual environment rapidly changes neuronal tuning, a process called remapping which had, hitherto, only be observed in the hippocampus; and (iii) spatial tuning is stronger in dorsal than in ventral regions of the mPFC. This finding suggests that the dorsal mPFC is particularly important to convey spatial information to downstream motor networks, supporting well-adapted movements in the environment. At the same time, the functional gradient is exactly opposite to the reported distribution of neuronal connections from the hippocampus, raising the question of its origin. In any case, the unexpected topology of neuronal tuning suggests that spatial information arrives in the medial prefrontal cortex more independently from the hippocampus than previously thought Fig. . This noFinally, the findings by Sauer, Folschweiller, and Bartos remind us that we rely on precise structural and functional data from multiple brain regions. Combined with the increasingly powerful interventional methods this may enable us to generate concrete, causal models of information processing and behavioral control. This endeavor also requires a cross-species, comparative approach, especially with respect to the massive changes in size and structure of prefrontal areas during mammalian evolution. There is a lot of work ahead, and the tools are all there!"}
+{"text": "Anterolateral thigh perforator (ALTP) flap is considered a versatile flap for soft tissue reconstruction. Computed tomography angiography (CTA) is used for mapping perforator in abdominal-based reconstruction; however, it is less commonly used in ALTP due to its poor imaging efficacy. In this study, we introduced a novel CTA technique for preoperative localization and design of ALTP flap and evaluated its value in directing surgical reconstruction.Thirty-five patients with soft tissue defects were consecutively enrolled. Modified CTA procedures, such as sharp convolution kernel, ADMIRE iterative reconstruction, 80\u00a0kV tube voltage, high flow contrast agent and cinematic rendering image reconstruction, were used to map ALTPs. A total of 287 perforators (including 884 sub-branches) were determined, with a mean of 5 perforators per thigh (range 2\u201311). The ALTPs were mainly concentrated in the \u201chot zone\u201d  or the distal zone . Most perforators originated from the descending branch of the lateral circumflex femoral artery . Three perforator types, namely musculocutaneous , septocutaneous , and mixed pattern , were identified. The median pedicle length measured by two methods was 4.1\u00a0cm (range 0.7\u201320.3\u00a0cm) and 17.0\u00a0cm (range 4.7\u201333.9\u00a0cm), respectively, and the median diameter of the skin flap nourished by one perforator was 3.4\u00a0cm (IQR 2.1\u20135.7\u00a0cm). Twenty-eight ALTP flaps were obtained with the guidance of CTA, and 26 flaps survived after follow-up.The proposed CTA mapping technique is a useful tool for preoperative localization and design of ALTP flap. Sharp convolution kernel combined with ADMIRE iterative reconstruction improves the imaging efficacy.80\u00a0kV tube voltage with high flow contrast agent ensures the perforator enhancement.Cinematic rendering reconstruction model facilitates the visualization of perforator anatomic variations.Preoperative CTA mapping contributes to the localization and design of the ALTP flap. The anterolateral thigh perforator (ALTP) flap is a versatile flap and one of the most popular procedures for reconstructing three-dimensional defects in the extremities. Its major advantages are pliable skin paddle, long vascular pedicle, large soft tissue volume, and low donor site morbidity \u20134. The pSo far, several imaging modalities have been proposed to map the anatomic distribution of ALTPs, such as computed tomography angiography (CTA), magnetic resonance angiography, and Doppler ultrasound \u201312. CompIn 2016, Dappa et al.  first prIn this study, we introduced a novel CTA technique for preoperative localization and design of ALTP flap and evaluated its value in directing surgical reconstruction.Patients with soft tissue defects caused by trauma or tumor resection who underwent CTA between June 2020 and August 2021 were consecutively enrolled in this retrospective study. The exclusion criteria were: (1) patients with a history of abnormal renal function or allergic reaction to an iodinated contrast agent; (2) patients with acute vascular injury; (3) presence of clinically significant pathology in bilateral thigh ; and (4) insufficient imaging or presence of motion/metal artifacts which could affect image analysis.The study was approved by the institutional review board and was conducted in accordance with the Declaration of Helsinki. The informed consent was waived.A modified CTA of the lower extremities from the pelvis to below the knee was performed using the third-generation dual-source computed tomography scanner . The reticular position lines were drawn on patient\u2019s thigh prior to examination. Immediately before CTA acquisition, all subjects received sublingual nitroglycerin . The details of the CTA acquisition are shown in Table Iohexol  was injected via the median cubital vein at a flow rate of 6\u20137.5\u00a0ml/s for 15\u00a0s. Saline solution was immediately given at the same flow rate for 8\u00a0s. A \u201cmanual\u201d bolus-tracking technique was used, and the monitoring section was set at the middle segment of the descending branch of the lateral circumflex femoral artery (LCFA). The region of interest (ROI) was placed outside the body. Image acquisition was manually triggered with a delay of 3\u00a0s after the descending branch of LCFA appeared.The dose-length product was obtained from the scan protocol. The effective dose was calculated according to the product of the dose-length product and a conversion factor \u201ck\u201d .All images were transferred to an external workstation  for maximum intensity projection, multiplanar reformation, and CR. A CR reconstruction model was based on voxel data for segmentation. First, enhanced veins in the skin and superficial fascia were manually removed, and the LCFA and its perforators were displayed inside the deep fascia. Second, the emerging location of the perforator was determined in the axial plain and marked at the deep fascial level. Then, three types of volume reproduction images were used to display different organizational structures by adjusting the template. Ultimately, the emerging location of perforators was marked by projection onto the skin.The number, position, origin, course, caliber, and pedicle length of the perforator in reconstructed and axial images were determined and evaluated by two senior radiologists  in a consensus reading. As for the location distribution of perforators, the \u201chot zone\u201d was defined as the area covered by 5\u00a0cm radius from the midpoint of the A\u2013P line. The proximal and distal zones were defined as the proximal and distal areas of the \u201chot zone,\u201d respectively . The cou2). Measurement was performed three times for each target, and the mean attenuation and image noise were obtained. The contrast-to-noise ratio was calculated as the difference between the average attenuation of the target vessel and muscle divided by the image noise [Subjective image quality was assessed by two experienced observers  independently using a five-point scale : 1\u2009=\u2009pooge noise \u201323.The surgical planning for perforator flap harvesting was based on the preoperative localization of CTA. The skin paddle was designed with the dominant perforators at the center of the flap. The flap dissection was performed when the dominant perforator and its origin and pathway were determined by surface projection,p value\u2009<\u20090.05 indicated statistical significance.Statistical analyses were performed using the SPSS software package . Kolmogorov\u2013Smirnov analysis was used in assessing the normality distribution of data. All continuous variables were expressed as mean\u2009\u00b1\u2009standard deviation or median and interquartile range (IQR), whereas categorical variables were expressed as a number with a percentage. In addition, intraclass correlation coefficient was used for calculating interobserver agreement for image quality. A A total of 37 patients with soft tissue defects caused by trauma or tumor resection who underwent CTA were enrolled in this study. Two patients were excluded due to the presence of metal artifacts in the images. Finally, 35 patients  were included in the data analysis, 23 patients with bilateral thigh, and 12 with the unilateral thigh. The flow diagram for this study is shown in Fig.\u00a0Overall, 287 ATLPs of 35 patients were identified based on CTA results, with a mean of 5 perforators per thigh (range 2\u201311) . Eight percent 23/287) of the perforators originated from the oblique branch, six percent (17/287) from the ascending branch, four percent (11/287) from the transverse branch, and six percent (17/287) directly from the other main vessels. The descending branch mainly originated from the LCFA . Importantly, the anatomic variants were observed in seven cases with the descending branch directly from the common femoral artery (CFA), superficial femoral artery, or deep femoral artery. However, the majority of LCFA  originated directly from the deep femoral artery, followed by the CFA  and the external iliac artery . Regarding the course of perforators, 177 musculocutaneous perforators, 96 septocutaneous perforators, and 14 mixed patterns were found. Different origin variation types of the descending branch are shown in Fig.\u00a0/287 of tIn terms of the caliber, there were 198 perforators with a diameter of \u02c3 0.5\u00a0mm and 89 perforators with a diameter of\u2009\u2264\u20090.5\u00a0mm. Twenty-nine patients had more than one perforator with a caliber of \u02c30.5\u00a0mm in the \u201chot zone\u201d, and 4 patients only had small perforators in this area. The minimum lengths of perforators ranged from 0.7\u00a0cm to 20.3\u00a0cm , and the maximum length that could be dissected ranged from 4.7\u00a0cm to 33.9\u00a0cm . The median diameter of the skin flap nourished by each perforator was 3.4\u00a0cm (range 0.1\u00a0cm\u201414.7\u00a0cm). Representative images are shown in Fig.\u00a0p\u2009<\u20090.001). The mean contrast-to-noise ratio was 25.3\u2009\u00b1\u200910.6.All the images were good for post-processing and analysis. The median (IQR) score of subjective image quality assessment was 4 (4\u20134.5). The agreement between two observers was good . One case developed postoperative infection 2\u00a0days after surgery, and the other showed partial necrosis 5\u00a0days after surgery. Figure\u00a0The ALTP flap is the most popular choice for autologous soft tissue defect reconstruction because of its versatile design capability, increased pedicle length, and reduced donor site morbidity. The key to successful flap reconstruction is accurately predicting the location of ALTPs and determining their course through the skin. Many previous studies examined multiple imaging modalities \u201327, espeAppropriate CTA scan parameters and reconstruction models contribute to good imaging of the perforator. However, owing to limited imaging space, traditional CTA has great challenges in displaying peripheral perforator, even when the contrast agent is at its peak concentration . In thisWhen performing the CTA scan, the following steps are required: Firstly, all patients must receive sublingual nitroglycerin before CTA acquisition. Nitroglycerin dilates small peripheral arteries and increases the number of assessable branches . HoweverIndeed, due to the great anatomic variation of ALTPs , 36, theAnother important problem of CTA is accurately transferring the anatomic information to surgery guidance. Rozen et al.  first exThe present study has some limitations. First, this was a retrospective study with a small sample size, which may have restricted the description of anatomical variations. Second, the anatomical data of perforators observed during the operation were not analyzed and evaluated in detail. Yet, all the surgical flap harvests were successful with the guidance of preoperative CTA and most flaps (26/28) survived.This study shows that the proposed CTA mapping technique is a useful tool for the localization of ALTPs. With the guidance of preoperative CTA, surgeons could optimize flap design and harvesting."}
+{"text": "The rapid increase of obesity and associated diseases has become a major global health problem. Adipose tissues are critical for whole-body homeostasis. The gut microbiota has been recognized as a significant environmental factor in the maintenance of energy homeostasis and host immunity. A growing body of evidence suggests that the gut microbiota regulates host metabolism through a close cross-talk with adipose tissues. It modulates energy expenditure and alleviates obesity by promoting energy expenditure, but it also produces specific metabolites and structural components that may act as the central factors in the pathogenesis of inflammation, insulin resistance, and obesity. Understanding the relationship between gut microbiota and adipose tissues may provide potential intervention strategies to treat obesity and associated diseases. In this review, we focus on recent advances in the gut microbiota and its actions on adipose tissues and highlight the joint actions of the gut microbiota and adipose tissue with each other in the regulation of energy metabolism. Obesity, a chronic disease characterized by the excessive expansion of adipose tissues and associated low-grade inflammation, is the risk factor for various disorders, including insulin resistance (IR), metabolic syndrome, hypertension, type 2 diabetes mellitus (T2D), cardiovascular diseases, nonalcoholic fatty liver disease (NAFLD), cancers and mental diseases , 2. GenoAlthough obesity is characterized by excessive accumulation of adipose tissues, under physiological conditions, adipose tissues are critical for whole-body homeostasis, including lipid storage, thermoregulation, and secretion of adipokines to regulate energy balance, metabolism, and immune responses . AccordiBacteroidetes, one of the most abundant phyla in the gut  and many bioactive mediators such as hepatokine FGF21  in fat storage. Conventional mice have 42% more total body fat mass than germ-free (GF) mice (raised in the absence of any detectable microorganisms) . Adult GFirmicutes and fewer Bacteroidetes counts than normal-weight individuals -induced hypercholesterolemia and an increase in body weight (L. paracasei ssp paracasei F19 (F19) decreases lipid accumulation in mice fed with HFD by increasing Angptl4 and AMPK activity , can turn white adipocytes into beige adipocytes . SeveralMestdagh et\u00a0al. first showed that gut microbiota modulates the lipid metabolism in BAT. The absence of gut microbiota enhances BAT lipolysis and suppresses lipogenesis in GF mice, suggesting activated lipid catabolism in their BAT . In lineBacteroidetes, Proteobacteria, Tenericutes, Actinobacteria, Verrucomicrobia, and Cyanobacteria, while increasing Firmicutes, Deferribacteres, and Firmicutes versus Bacteroidetes ratios , family 7, and subfamily b in the liver. This sets off a metabolic process that integrates lipoprotein clarification in BAT and hepatic conversion of cholesterol to bile acids synthesis pathway, leading to increased hepatic synthesis and more fecal excretion of bile acids, which distinctly changes the gut microbiota composition and increases heat production .AMPK, an essential nutrient sensor for maintaining cellular energy status, plays important role in regulating glucose metabolism in various tissues. Very recently, Huang et\u00a0al. identified that intestinal AMPK\u03b11 stimulates thermogenesis by modulating anti-microbial peptide that manipulates gut microbiota and metabolites. These intestine-BAT communications through AMPK\u03b1- related signaling might partially be the underlying mechanism of beneficiary action of metformin on the intestine .via antibiotic treatment or in GF mice impairs the browning of WAT  and GF mice models, Krisko and colleagues have demonstrated that the gut microbiota seems disposable for both cold- and diet-induced thermogenesis and not required for the recruitment and activation of thermogenic tissues. The gut microbiota maintains blood glucose by regulating hepatic gluconeogenesis rather than adipose tissue in cold  in WAT and BAT (via vascular endothelia growth factor (VEGF) expression in WAT and the expression of Adrb3, Cidea and Ucp1 were significantly elevated in HFD-IF mice, which might due to \u03b2-AR-dependent thermogenesis (Intermittent fasting (IF) is known as an effective and useful strategy for weight loss and has multiple benefits for metabolism and health . Recent  acetate . Both la and BAT , 59. Con and BAT . Kim et\u00a0ogenesis .Erysipelotichaceae and Lactobacillaceae and decreases in other Firmicutes families, as well as increased levels of Bacteroidaceae and Verrucomicrobiaceae  and is known to ameliorate weight regain  also induces metabolic improvements and stimulates the browning within the subcutaneous and visceral adipose tissues . Change obiaceae . In a stsitivity , which m reduced . Parabact regain .Firmicutes decreases, whereas compared with insulin sensitive subjects, Bacteroidetes and Proteobacteria RA increase  in subcutaneous but not in visceral adipose tissue. Specifically, those bacteria belonging to the Ruminococcaceae family, are positively connected with plasma level of acetate, which is also associated with increased beige fat development and insulin sensitivity  of increase . Interessitivity . A clinisitivity . Very resitivity . HoweverIn summary, the gut microbiota plays an essential role in whole-body energy metabolism by regulating thermogenic programs in different fat depots. Some environmental and nutrition situations  could induce a shift of gut microbiota composition that stimulates browning of WAT and activity of BAT. However, the underlying mechanisms and pathways have not yet been fully identified.Chronic low-grade inflammation is a trademark of many metabolic diseases, such as obesity, T2D, and NAFLD , 69. DurRuminococcus gnavus or Bacteroides species, might prevail and certain \u2018anti-inflammatory\u2019 strains, such as Faecalibacterium prausnitzii, are less prevalent in obesity   . Faecalin humans . Oral adoduction . F. prauedicines . Parabac obesity . Xu et\u00a0afat mass .Firmicutes to Bacteroidetes ratio and promote a healthier metabolic phenotype, while saturated lipids aggravate WAT inflammation and promote a higher degree of obesity, which is partly attributed to distinct gut microbiota richness and diversity , bone morphogenetic protein (BMP)-4, BMP-7, dipeptidyl peptidase 4 (DPP-4), vaspin, apelin, and progranulin . At a syLeptin plays an important role in the regulation of food intake, satiety, appetite, and energy expenditure , 103. StClostridium genus and a lower relative abundance of Sutterella, and enhances the expressions of interferon-\u03b1 (TNF-\u03b1), mucin  during suckling period of rats, indicating a modulator role of leptin on the intestinal activation  in adipose tissues, but also increases mRNA level of adiponectin , activated AMPK signal, and upregulated anti-obesity effects . Yao et\u00a0ponectin . In addiponectin . Also, Mponectin .Proteobacteria phylum, Blautia and Roseburiagenus, and a higher proportion of the Enterococcus genus. Exogenous administration of adiponectin can increase the percentage of Tc TCR\u03b1\u03b2+ and NKT cells and decrease the NK cells in intraepithelial lymphocytes (IEL)  . Additioes (IEL) .FGF21 is a member of the FGF superfamily and an important metabolic regulator, which is produced by adipose tissue, liver, and skeletal muscle , 113. LiA. muciniphila, Lactobacillus) and regulate lipid metabolism through PPAR\u03b1/FGF21 pathway (Ketogenic diet (KD) or carbohydrate-restricted diet can shift the gut microbiota toward \u201cfavorable bacteria\u201d -dependent mechanisms , are extracted from gut microbial fermentation of foods that cannot be digested due to the lack of appropriate enzymes . SCFA caFirmicutes to Bacteroidetes ratio is connected to increased caecal concentrations of acetate and butyrate or acetate and propionate respectively  dependent manner. By inhibiting intracellular lipolysis (mainly through acetate and propionate) and increasing the lipid buffering capacity of adipose tissues (mainly by propionate), SCFA can lead to a reduction of lipid overflow and decrease of ectopic fat accumulation, thereby positively regulating insulin sensitivity  treatment, however, butyrate was not altered and, under cold exposure, ncreased . Butyrat tissues , 63. Butn of NPY . Cold min of NPY , indicatvia the nuclear Farnesoid X receptor (FXR) and the G protein-coupled membrane receptor 5 (GTR5), which are involved in numerous metabolic pathways in the host , 2-arachidonoyi-glycerol (2-AG), and the anabolic and catabolic enzymes for the endocannabinoids (The eCB system is comprised of endogenous lipids that trigger specific G protein-coupled receptors termed cannabinoid receptors 1 and 2 (CB1 and CB2). It has been demonstrated that the eCB system consists of more than 100 lipid mediators and 50 proteins, such as CB1, CB2, abinoids , 147. Thabinoids \u2013150. Theabinoids . Geruts abinoids . The actabinoids .N-oleoyl-ethanolamine (OEA) administration to mice in normal diet could shift Firmicutes/Bacteroides ratio, decrease Lactobacillus, and reduce intestinal cytokines expression (N-acyl ethanolamide (NAE) similar to AEA, could decrease body weight, food intake, and fat mass in HFD rats , National Key R&D Program of China (2020YFA0803604), and Natural Science Foundation of Hunan Province (2019JJ40410) to F.H.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": "Vitamin D has been known to be associated with asthma, particularly in children, while the evidence among adults is limited and inconclusive. This study aimed to investigate the association between serum, vitamin D concentrations, and the incidence of adult-onset asthma and also the modified effect caused by sleep patterns and genetic risks.A prospective cohort study with 307,872 participants aged between 37 and 73 years was conducted based on the UK Biobank, with a median follow-up of 12 years. The Cox proportional hazard model was applied to evaluate the association between vitamin D status and incident adult-onset asthma, and the modified effect was investigated by conducting stratified analysis according to sleep pattern score and genetic risk score, and subgroup analyses were performed by sex, age, BMI, and smoking status as well.p = 0.005). Moreover, stratification analysis demonstrated that the protective effect of vitamin D on asthma risk was modified by sleep patterns or genetic susceptibility, with the strongest protective effect being observed in the subpopulation with a moderate sleep pattern  and a moderate genetic risk . In subgroup analyses, the protective effect of optimal vitamin D levels was only significant among men, individuals younger than 60 years of age, overweight individuals, and current or previous smokers.Individuals with optimal vitamin D concentration were associated with 11.1% reduced risk of incident asthma compared to those participants with deficient vitamin D (HR = 0.889; 95% CI: 0.820\u20130.964; Increased serum vitamin D levels were associated with a lower risk of incident adult-onset asthma, and this association was modified by sleep patterns and genetic predisposition to some extent. As one of the most widespread, non-communicable, chronic disorders, asthma imposes a substantial global burden of disease due to lifelong treatment . In 2017Although vitamin D has been commonly recognized to regulate the immune response and inhibit a variety of inflammatory pathways , observaAs a typical chronic disease, the risk of asthma is closely related to lifestyle. Recent research studies indicate that improving sleep behaviors might slow the development and exacerbation of asthma , 9. MoreAdditionally, various studies provided evidence linking sleep behaviors to vitamin D metabolism. Researchers have reported that serum vitamin D concentrations may alter sleep behavior by affecting inflammatory substances . For exaTo address the shortcomings of existing research studies, this large prospective cohort study aimed to elucidate the relationship between serum vitamin D concentrations and the incidence of adult-onset asthma using the data from the UK biobank and examine the modification effect of sleep patterns and genetic risk on this relationship as well.The UK Biobank is initiated by the UK government to collect and store long-term human biological samples including physiological, pathological, and socioeconomic information of the sample subjects through standardized procedures. It has recruited more than half a million participants aged 37 to 73 years from across the UK between 2006 and 2010, and the UK Biobank website was opened to public registration in 2012. This prospective cohort study was conducted based on data from the UK Biobank. All participants provided electronically signed consent forms. The North-West Multicenter Research Ethics Committee approved the study. We submitted an application to the UK Biobank database and obtained access to some of the data (application number 84979).Our study's exclusion criteria are as follows: (1) participants with asthma at the baseline; (2) participants with missing data on the serum vitamin D concentration, sleep behavior, and genetic risk; (3) participants whose sex inferred from the relative intensity of markers on the X and Y chromosomes do not match to self-reported sex; and (4) non-white participants.The UK Biobank measured biochemical markers in blood samples from all 500,000 participants. Serum vitamin D concentration was measured by a clinical chemistry analyzer, Beckman Coulter DXI 800, using a direct competitive chemiluminescent immunoassay. The observed reportable range of serum vitamin D concentration was 10\u2013375 nmol/L. The UK Biobank website provides more detailed information on the measurements. Based on clinical guidelines  and prevThe sleep pattern score was established according to the previous studies \u201323. We dThe UK Biobank collected genetic data from 488,377 participants and determined it with two very similar genotyping arrays to capture genome-wide genetic variation, including short insertions and deletions (indels) and single nucleotide polymorphism (SNPs) . AccordiAsthma was defined with the International Classification Disease (ICD-10) code (J45) with all sources of asthma, including self-report, hospital admission data, primary care, and death register in the national hospital registers. Individuals were followed until the date of occurrence of asthma, the end of follow-up in December 2020, or death, whichever came first. Moreover, death registry records were used to ascertain the death.t-test, and the association of serum vitamin D  with the incidence of asthma was evaluated by the Cox proportional hazard model. All variables met the proportional hazards assumption, and the categorical variables were tested by Kaplan\u2013Meier estimation and the continuous variables by the Schoenfeld residual method. We then stratified the association between serum vitamin D and asthma incidence according to the overall sleep pattern score and genetic risk. A subgroup analysis based on sex, age, BMI, and smoking status was also conducted. Furthermore, the relative excess risk (RERI) and the attributable proportion (AP) were calculated by a SAS program to estimate the additive interaction effect between vitamin D and sleep patterns or genetic risk. In addition, the population-attributable fraction (PAF) was calculated to compare the changes in the incidence rate after altering a specific exposure. To ensure the stability of our results, we conducted sensitivity analysis from the following two aspects: (1) excluding participants with asthma within 3 years of enrollment to reduce the possibility of reverse causality; (2) performing multiple imputations for missing covariates, including BMI, income, education, smoking status, and vitamin D supplement.We used number (proportion) and mean (standard deviation) to describe categorical variables and normally distributed continuous variables of recruited individuals, respectively. The comparison between groups was evaluated by chi-square tests or student After excluding participants based on the criteria described in the method, a total of 307,872 participants were recruited for the main analysis , with anp = 0.005) in the full-adjusted model, including age, sex, race, BMI, income, education, smoking status, assessment center, and vitamin D supplements, which indicated the robust protective role of optimal vitamin D on preventing the incidence of asthma. Sensitivity analysis was conducted after excluding participants with asthma within 3 years and performing multiple imputations for missing covariates and yielded similar results (The association between serum vitamin D concentrations and the incidence of asthma was represented in  results .p < 0.001) lower risk of asthma. As age subgroup analysis manifested (p = 0.008) but not in those older than 60 years. The results of the BMI subgroup (p = 0.033). In the smoking subgroup analysis (p = 0.007), followed by previous smokers , which was not observed among never-smokers.Subgroup analyses based on sex indicated that the relationship between serum vitamin D levels and incident asthma was only statistically significant in men  but not nifested , optimalsubgroup  showed tanalysis , optimalp = 0.004).Next, we investigated whether the relationship between vitamin D levels with incident asthma was modified by sleep behaviors and genetic risk. The results showed that individuals with higher sleep pattern scores represented a healthier sleep pattern and had a significantly decreased incidence of asthma . A promiMeasures for the interaction of vitamin D concentrations with sleep patterns and genetic risk are shown in In our large prospective cohort based on the UK Biobank, a significant relationship between increased serum vitamin D levels and decreased risk of incident adult-onset asthma was revealed. These association was modified by sleep patterns and genetic predisposition and was relatively robust in subgroup populations with intermediate sleep patterns and intermediate genetic risks.The association between serum vitamin D concentrations and the risk of adult-onset asthma was scarce and currently inconclusive. Two cross-sectional studies of adults from Canada and the United States have indicated that serum vitamin D levels below 50 or 30 nmol/L were related to an increased odds ratio of current asthma , 26. In Surprisingly, in this study, we observed a protective role of serum vitamin D concentrations on the incidence of adult-onset asthma, particularly in men rather than women. A similar finding has been reported in a Norwegian HUNT study, which manifested that low vitamin D concentrations were related to the high risk of asthma only in men without allergic rhinitis . EpidemiFurthermore, the effect of sleep patterns and genetic risk on asthma risk might partially explain the uncertainty about the relationship between serum vitamin D concentrations and incident asthma. The protective effect of vitamin D was only seen in individuals who were not affected by sleep patterns and genetic predisposition, that is, those at the intermediate risk. The relatively small sample size of the healthy and poor sleep pattern groups could be one of the reasons. In addition, the overall sleep pattern had a complex interaction with vitamin D function and metabolism, as well as the risk of asthma. Poor sleep patterns, such as daytime sleepiness, might be associated with less outdoor activity and sun exposure time, which affected vitamin D synthesis of the skin ; insomniIn the stratified analysis of genetic susceptibility, although the protective effect of optimal vitamin D level was statistically significant only in the intermediate genetic risk group, the risk of asthma in each group showed a decreasing trend with the increase in the serum vitamin D level, indicating that the protective effect of vitamin D was limited in people with high or low genetic susceptibility to asthma. Numerous research studies identified a critical role of genetic susceptibility in asthma development . TherefoCompared with previous studies, this study has certain advantages, it is a prospective large sample size cohort study with a median follow-up of 12 years. In sensitivity analyses, we excluded patients with asthma developed within the first 3 years of enrolment, reducing the possibility of reverse causality. Certainly, several limitations are found in this study. First, the participants included were between 37 and 73 years of age and were restricted to European ancestry; thus, the results had the limitation of extrapolating them into adolescents, young adults, or other races. Second, because not all individuals participated in the follow-up and specific follow-up time was not reported, the evaluation of vitamin D level and sleep behavior were at the baseline and not longitudinally assessed throughout the study process. Hence, our study still could not prove a causal relationship between serum vitamin D levels and asthma.Our study discovered that optimal serum vitamin D concentrations were associated with a reduced risk of incident adult-onset asthma, which was significant in men, individuals younger than 60 years of age, overweight individuals, and current or previous smokers. Additionally, to some extent, this association was modified by sleep patterns or genetic predisposition. Collectively, our findings support the protective role of vitamin D on reduced risk of adult-onset asthma.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by North West Multicenter Research Ethics Committee (Application number 84979). The patients/participants provided their written informed consent to participate in this study.YZha developed the concept and provided data sources. PP is responsible for supervision and management. QC, YZhu, and GZ completed the methodology and writing. HL, DL, and JC performed the investigation and validation. All authors read and approved the final manuscript."}
+{"text": "Relational reasoning is a key component of fluid intelligence and an important predictor of academic achievement. Relational reasoning is commonly assessed using matrix completion tasks, in which participants see an incomplete matrix of items that vary on different dimensions and select a response that best completes the matrix based on the relations among items. Performance on such assessments increases dramatically across childhood into adulthood. However, despite widespread use, little is known about the strategies associated with good or poor matrix completion performance in childhood. This study examined the strategies children and adults use to solve matrix completion problems, how those strategies change with age, and whether children and adults adapt strategies to difficulty. We used eyetracking to infer matrix completion strategy use in 6- and 9-year-old children and adults. Across ages, scanning across matrix rows and columns predicted good overall performance, and quicker and higher rates of consulting potential answers predicted poor performance, indicating that optimal matrix completion strategies are similar across development. Indices of good strategy use increased across childhood. As problems increased in difficulty, children and adults increased their scanning of matrix rows and columns, and adults and 9-year-olds also shifted strategies to rely more on consulting potential answers. Adapting strategies to matrix difficulty, particularly increased scanning of rows and columns, was associated with good overall performance in both children and adults. These findings underscore the importance of both spontaneous and adaptive strategy use in individual differences in relational reasoning and its development. Children\u2019s ability to discover and utilize patterns between different objects and mental representations, a key component of fluid intelligence known as relational or inductive reasoning, improves dramatically across development , range: 6.02\u20136.96, 23 female), 9-year-olds , range: 8.93\u201310.07 (2 exact age unknown), 25 female), and college-aged adults , range: 17.90\u201330.72 (1 exact age unknown), 30 female). Eight additional 6-year-olds were recruited but not included in the final sample: three quit during the matrix completion task, four quit the study before the matrix completion task, and one had no valid eyetracking data. These age groups were selected based on prior research showing dramatic improvements, high variability, and likely strategy changes in matrix completion performance at 6 years of age and from 6 to 9 years of age .Supplementary Materials). These matrices were designed to instantiate the row- and column-wise processing strategies that have predicted adult performance in other matrix completion tasks and to systematically vary difficulty across matrix problems. Typical relations within the matrix included increasing/decreasing size, all different/same shapes, increasing/decreasing number of items, etc. All matrices used for child participants except the final problem had been normed in prior work with 100% accuracy in adults  assessment. These stimuli were obtained from the RAPM set used in Hayes et al.  Bors & . ChildreSupplementary Materials.All participants completed two practice items: one in which shapes were consistent within columns but differed across rows, and one in which shape and color were consistent within rows but differed across columns. Instructions and corrective feedback were given by the experimenter, followed by a repeatable practice trial without instructions. The final practice trial was repeated if participants selected the incorrect answer or needed additional practice with spacebar presses or mouse navigation. Trials were initiated by successfully fixating on a centralized cross for 500 ms or by an experimenter via keypress upon failing to detect fixation. All participants were instructed to press the spacebar when they knew the correct answer. Then, the matrix disappeared, and only the solution array remained, mirroring prior testing procedures in adults  and marginally correlated in 6-year-olds , indicating successful variation in matrix difficulty. For children, each set of eight problems contained three matrices with one relation, three matrices with two relations, and then two matrices with three relations, except for the final problem.Matrices were presented in sets of eight with increasing anticipated matrix difficulty, using either performance in prior samples for adults or the number of relations as a proxy for difficulty in children . Eyetracking data were captured with a Tobii X50 Eyetracker with 50 Hz sampling rate using Clearview software . AOIs were drawn around each item in the matrix (1\u20139) and the entire solution array (10). Response time was considered total detected fixation time on the defined AOIs.Supplementary Materials. In total, 4 trials from adults, 8 trials from 9-year-olds, and 43 trials from 6-year-olds were excluded due to poor data quality. Most excluded trials in 6-year-olds were clustered within 5 participants, and all significant correlations between strategy use and overall performance remained significant when excluding only these participants.Eyetracking data were pre-processed using the \u2018gazepath\u2019 package in R  than 9-year-olds (M = 31%), who in turn had a lower percentage of missing fixation data than 6-year-olds . This metric was included as a covariate to determine whether age differences in strategic indices were driven by systematic differences in available fixation data.We also calculated the percentage of detected fixation time on a trial by dividing the summed fixation time on AOIs by the full trial time. Thus, this metric includes saccades, missing data, and fixation outside of the matrix problem as non-valid data. Expectedly, adults had a lower percentage of missing fixation data  = 17.57, p < .001). Reported values are the number of detected toggles per second. Higher values on this index reflect response elimination , replicating prior work showing that children and adults take longer to respond on more difficult problems .All statistical analyses were conducted with R software  scored below chance (<12.5%), participants with poor performance were retained in initial analyses to capture potential changes in strategy use, as in Chen et al. (t(52.55) = 8.83, p < .001). Child groups exhibited unequal variance in accuracy according to Levene\u2019s test  = 16.75, p < .001), indicating that 9-year-olds had significantly less variance in accuracy than 6-year-olds. Notably, this restricted range could attenuate correlations between strategy use and performance in 9-year-olds, while the very low performance for some 6-year-olds could exaggerate correlations between strategy use and performance. Statistical differences between children and adults were not assessed because adults completed a different set of matrix problems.Descriptive statistics for performance, response time, and strategic eyetracking indices across all matrix problems for the full sample are provided in n et al. . Poor pen et al.  and thatn et al. . As expeSupplementary Materials.To preview the series of analyses: First, we tested whether the implementation of specific strategies increases across childhood via eyetracking indices. Second, we tested the relationship between strategic indices and overall performance, including the specificity of these indices in predicting trial accuracy. Third, we investigated whether age groups adapted strategy to increasing difficulty and whether strategy adaptation (or persistence) predicted better overall performance across age groups. This analytic strategy tests whether the strategies linked with good overall performance are also better at the trial level and on more difficult problems. Analyses of relationships between matrix completion strategy use and performance on Analysis-Synthesis, a separate fluid intelligence task, are included in SDs from group mean) for each index and removed these participants from each age group for the following analysis . Analyses with the full sample are included in Supplementary Materials and qualitatively mirror the results reported below.We performed a univariate outlier analysis  and integration  than 6-year-olds. Nine-year-olds had significantly longer times to first toggle to the response array , spent more time fixating on the matrix relative to the response array , and spent more time fixating on the initial rows and columns of the matrix relative to the latter rows and columns  compared with 6-year-olds. In contrast, the number of toggles, a metric of response elimination, was not different between child groups ; however, toggle rate, a measure of response elimination that corrects for differences in response time, was significantly lower in 9-year-olds than 6-year-olds . We reproduced these results including a covariate indexing the percentage of available eyetracking data, and differences between child groups remained significant for all strategic indices , suggesting that these results were not solely due to differences in data availability.Strategies associated with constructive matching increased from 6- to 9-year-olds. Nine-year-olds had significantly more trials with encoding , while weaker positive correlations were observed in 9-year-olds (p < .11). In contrast to prior work in children, the mean number of toggles per problem was not associated with performance. However, toggle rate correlated negatively with performance in adults and 6-year-olds (p\u2019s < .001), with a smaller negative correlation in 9-year-olds (p = .052). Time to first toggle and matrix time distribution positively correlated with performance across age groups . Proportion matrix time positively correlated with performance in adults and 6-year-olds, while weaker positive correlations were observed in 9-year-olds. All correlations are reported in Supplementary Materials). These results are inconsistent with the hypothesis that response elimination is especially beneficial for younger children. Strategic indices of good performance were qualitatively similar from childhood into adulthood: indices reflecting constructive matching were associated with better performance, and indices reflecting response elimination were associated with poor performance.Performance significantly positively correlated with the proportion of trials with encoding and integration in 6-year-olds and adults , we replicated our analyses with these participants excluded. Our aim in this follow-up analysis was to determine whether the large correlations observed in 6-year-olds reflected divergences between children who understood the matrix completion task and those who did not, rather than genuine correlations between strategy use and task performance. We observed highly convergent results with low-performing 6-year-olds excluded. Performance positively correlated with the proportion of trials with encoding , integration , time to first toggle , and matrix distribution time  and negatively correlated with toggle rate . Proportion matrix time was not significantly correlated with performance . Further analysis of errors for 6-year-olds scoring below chance suggested that these participants were not responding randomly; instead, these participants were more likely to respond with a duplicate item and less likely to select a response that contained a novel feature than expected by chance. Increased use of response elimination predicted a greater likelihood of selecting a duplicate answer .Given the high number of 6-year-olds with accuracy below chance . These findings generally mirror the aggregate task results, in which increased use of constructive matching was linked with increased probability of responding correctly across age groups. These results indicate some potential for specific strategic indices, particularly encoding, toggle rate, and greater proportion of fixation time on the matrix, for predicting correct responses at the trial level. However, the lack of consistent correlations suggests that predicting trial-level accuracy remains difficult with these somewhat coarse strategy indices. Some problems may not require systematic strategies and instead rely only on pattern completion to derive the correct answer, which may explain the lack of significant correlations in the 9-year-old group, who performed very well overall. Adults completed problems from Advanced Progressive Matrices, which involved a broader and more complex range of rules than the child matrices; some of these matrices may require different and more complex strategies than those derived from eyetracking.Next, we tested the specificity of these strategic indices for predicting correct responses at the trial level. We assessed relationships between strategic indices and trial accuracy by conducting separate multilevel logistic regression models for each strategic index correlated with aggregate task performance, with random intercepts for participants. Number of toggles was excluded because the index was not related to overall performance. All models included the matrix difficulty parameter as a covariate. Predictors of trial accuracy varied across age groups : Trial aTo determine whether children and adults adapted strategy to matrix difficulty, we conducted an item-level analysis in which each strategic index was averaged within trial across each age group. Then, the mean of each strategic index on that trial was regressed onto matrix difficulty. We include analysis of the number of toggles because this index is also informative for potential strategy changes in response to difficulty; utilization of pure constructive matching alone would not lead to an increased number of toggles with increased difficulty, as only one toggle to the response array would be necessary to locate the correct response after using constructive matching. Increased response elimination could be reflected in an increased number of toggles with increased difficulty. Thus, toggle rate could decrease due to longer response times on more difficult trials, reflecting more constructive matching, while the number of toggles may also increase, reflecting more response elimination.All age groups exhibited evidence of shifts in strategy in accordance with matrix difficulty . In 6-yeTo test whether adaptive strategy use predicted matrix completion performance, we conducted a series of multilevel models in which each strategic index on a trial was predicted by matrix difficulty within each age group, with random slopes for participants. We then extracted the random participant slopes as indices of adaptive strategy use. Values different from 0 indicate greater adaptation to difficulty. For example, higher values in adaptive encoding indicate a greater probability of encoding as matrix difficulty increases.p\u2019s < .051) and integration (p\u2019s < .066) with increasing difficulty. Increases in toggle rate correlated negatively with accuracy across all groups (p\u2019s < .05). Increases in the time to first toggle (p\u2019s < .02), proportion of relative matrix time (p\u2019s < .066), and matrix distribution time (p\u2019s < .05) generally positively correlated with performance across groups. The mean number of toggles on matrix problems was not significantly associated with accuracy. All correlations are reported in Across age groups, accuracy generally positively correlated with a greater probability of encoding , whereas older children are more likely to select partial relational matches or the correct answer  to first complete subgoals  .Materials, data, and analysis scripts for this manuscript are available on the project\u2019s Open Science Framework repository .Click here for additional data file."}
+{"text": "This article presents a numerical model of electromagnetic levitation melting and its experimental validation. Levitation melting uses the phenomenon of magnetic induction to float a melted, usually metallic, conductor in an electromagnetic field. With the appropriate configuration of the coil , the eddy currents induced in the molten batch interact with the coil magnetic field, which causes the melted metal to float without direct contact with any element of the heating system. Such a contactless process is very beneficial for melting very reactive metals  or metals with a high melting point . The main disadvantage of levitation melting is the low efficiency of the process. The goal of the authors is to develop, by means of a numerical simulation and optimization tools, a system for levitation melting with acceptable efficiency. To achieve this, it is necessary to develop a reliable and representative computational model. The proposed model includes an analysis of the electromagnetic field, with innovative modeling of the convective heat transport. Experimental validation of the model was performed using aluminum alloy, due to the lack of the need to use a protective atmosphere and the ease of measurements. The measurements included electrical values, the melted batch positions during levitation, the melting time, and the temperature distribution in its area. The verification showed that the compliance between the computational model and the simulation for the position of the batch was accurate to 2 mm ( Technological progress requires the use of more and more perfect materials. However, their use is often hindered by the price and technological difficulties associated with the processing of new materials. Typical examples of this type of material are refractory metals. Depending on the adopted definition, these include niobium, molybdenum, tantalum, tungsten, and rhenium, as well as titanium, vanadium, chromium, manganese, zirconium, ruthenium, rhodium, hafnium, osmium, and iridium ,2. TheseDue to its high strength and low density, titanium and its alloys are used in aviation and space technologies  , as wellBy its very nature, a process that allows melting without contact with the \u201cenvironment\u201d is melting using electromagnetic levitation ,35,36. MThe main issue with this type of melting is its generally low energy efficiency; therefore, improvement and optimization of this process are needed. Electromagnetic levitation melting takes place in an induction coil of a specific design  powered by a high-frequency source ,39,40. TThere are many benchmark problems available in the literature, such as the TEAM 35 and TEAM 36 benchmark problems. This benchmark has a good theoretical and measurement basis to compare results or consider different methodologies ,50. CompTemperature as a function of time,Batch position during levitation,Current frequency,Voltage,Current.The research presented in the paper relates to the preparation and validation of the simulation model. The inductor model is designed for electromagnetic levitation melting and is suitable for future optimization. The validation of the model is based on the physical inductor and the measurement station, which allows the following features to be obtained:The main novelties of the presented model are the relationships between the fluid and the air surrounding the molten batch. Air movements are related to the convection caused by the heating batch. Moreover, the model is asymmetric, so it represents the inductor available to us relatively accurately. Another representation of the actual process that has been considered is the change in the electrical parameters of the material with changes in temperature.In view of future optimizations, the simulation model must provide a compromise between reasonable consumption of computer memory/time and acceptable accuracy. This paper does not yet cover the optimization problem but is an entry point to it.Thermal camera (infrared)-PI 640 , , . We alsoriptions , (3), , , .Based on the results of the electromagnetic calculations, thermal analysis was performed . For thiThe surroundings of the batch  had a boThe electromagnetic losses were imported from the results of the electromagnetic calculations and converted to induction power  and 10)10). We ek\u2014thermal conductivity,T\u2014temperature,q\u2014volume density of the heat source,c\u2014specific heat capacity.The heating of the batch through eddy current induction also affected the system. The heat of the batch  also penp\u2014static pressure,g\u2014gravitational acceleration,I\u2014unit tensor,Equation  is the mk-In order to reduce the number of variables and close the system of equations, an appropriate turbulence model should be introduced. The two-equation model  introduc-\u03f5 model , the effk\u2014kinetic energy of turbulence,C\u2014empirical constant.The main advantage of the k-As for the thermal field analysis, the surroundings of the batch  had a booundings  as open,During the measurements, the batch levitated inside the inductor in a stable position during and after melting. The ruler visible in The vertical position of the batch during the measurements was 32 mm. For the simulated model, we searched for the position in which the batch levitated. The ascending force was calculated according to  and 21)21). For At position 17 mm, lowering the batch resulted in a decrease in the ascending force, and the batch fell out of the inductor. However, increasing the batch position increased the ascending force, and the batch started to rise. As a consequence, this point represented the unstable equilibrium point. At the 30-mm position, the lower position of the batch increased the ascending force, and the lower position of the batch decreased the ascending force. This implies that this position was a stable equilibrium point and, for this position, further calculations should be carried out . The calWe calculated the heating time of the batch in the inductor based on the records from the video and thermal cameras. We merged both recordings into one frame, as shown in As a result of the simulations, the melting temperature was obtained in 86 s. The distribution of heat on the batch surface is shown in The accuracy of the simulation calculations depended on the assumptions made to simplify the model, but for the numerical simulations, the accuracy also depended on the correctness of the model\u2019s discretization.Maxwell provides the possibility of different mesh strategies; the initial mesh prepared by the user is usually homogeneous and may consist of too few elements. To prevent this, an adaptive mesh has been introduced, which performs an analysis before starting the actual simulation. The user can specify the minimum number of mesh densifications that must be made, while the program estimates the error that the adopted mesh introduces into the result. In the case of the model under consideration, we set that at least two mesh compactions took place before the right mesh was determined. In the end, three densifications were performed.Regardless of the program\u2019s built-in mechanisms, we conducted an independence analysis. Since the purpose of the model was to simulate the temperature changes in the batch while it was in the levitation state, the independence analysis was carried out for two values: the Lorentz force acting on the batch  and the The need to prepare computational models is widespread among researchers, but there is no universal model that is both useful to specific situations and addresses all researchers\u2019 needs. The model presented here complements previous approaches by taking into account the changes in material properties when heating the material, the fluid dynamics in the air surrounding the batch, and the asymmetry of the inductor model.The previous computational model that we prepared  was modeThere are other similar models, but they differ in some aspects.In their work, Witteveen et al.  proposedRoyer et al.  preparedFurthermore, Kermanpur et al.  introducSassonker and Kuperman  proposedOther applications of numerical modeling related to electromagnetic levitation melting can be found in the literature, which are difficult to compare with the presented model because of their different purposes. The first subtype of such models involves convective flow inside the melted batch ,59,60. TThe summary of selected properties of simulation models found in the literature is in the The simulated melting time takes 86 s, which is 5 s faster than the median measured time.Batch levitation occurs at the 30 mm position, which is 2 mm lower compared to the measurements.We investigated the quality of the model by increasing the number of mesh elements and comparing the results. We state the solution quality as good.The computational time of the model is approximately 50 min, which is acceptable for future predictive uses.Parameters of the simulation model were introduced from the measurement station where the inductor was examined. Based on the geometry of the available inductor, we prepared a geometric model and a strongly coupled electromagnetic\u2013temperature computational model. The following conclusions were drawn:The purpose of the proposed simulation model is to perform calculations to examine the impact of the change in the geometry of the melting process. Moreover, the geometry of the inductor should be optimized to increase the efficiency of the melting."}
+{"text": "A primary colorectal cancer (CRC) tumor can contain heterogeneous cancer cells. As clones of cells with different properties metastasize to lymph nodes (LNs), they could show different morphologies. Cancer histologies in LNs of CRC remains to be described.Our study enrolled 318 consecutive patients with CRC who underwent primary tumor resection with lymph node dissection between January 2011 and June 2016. 119 (37.4%) patients who had metastatic LNs (mLNs) were finally included in this study. Cancer histologies in LNs were classified and compared with pathologically diagnosed differentiation in the primary lesion. The association between histologies in lymph node metastasis (LNM) and prognosis in patients with CRC was investigated.The histologies of the cancer cells in the mLNs were classified into four types: tubular, cribriform, poorly differentiated, and mucinous. Same degree of pathologically diagnosed differentiation in the primary tumor produced various histological types in LNM. In Kaplan\u2013Meier analysis, prognosis was worse in CRC patients with moderately differentiated adenocarcinoma who had at least some mLN also showing cribriform carcinoma than for those whose mLNs all showed tubular carcinoma.Histology in LNM from CRC might indicate the heterogeneity and malignant phenotype of the disease. Recognizing tumor heterogeneity is important in cancer therapy \u20133. In tuLymph node metastasis is a prognostic factor for patients with colorectal cancer (CRC). Adjuvant chemotherapy for stage III CRC is based on nodal metastasis and is standardized worldwide , being aIn the present study, we set out to detail the cancer histology in LNM from CRC, any similarity in the degree of differentiation between the LNM and the primary tumor, and the suitability of cancer histology in the mLNs for prognosticating the cancer-specific survival (CSS) of patients with node-positive CRC.th, 2021. All methods were performed in compliance with the Declaration of Helsinki, the guidelines for ethical principles for medical research involving human subjects, and the ethics guidelines of the National Hospital Organization Minami Wakayama Medical Center. Informed consent was obtained in the form of opt-out on the web page of the National Hospital Organization Minami Wakayama Medical Center.The study enrolled 318 consecutive patients with CRC who underwent colectomy, anterior resection, abdominoperineal resection, or Hartmann operation with lymphadenectomy between January 2011 and June 2016 at the National Hospital Organization Minami Wakayama Medical Center . The follow-up period was 5 years. The Ethics Committee of National Hospital Organization Minami Wakayama Medical Center approved the study (#2021\u201310). Medical data records were accessed for the purpose of this study after the ethics committee approval on January 24The hematoxylin and eosin stained colorectal and LN tissue sections were examined microscopically. All specimens were blindly reviewed twice by three individuals . Where discrepancies arose, these specimens were discussed to achieve a consensus. All pictures were acquired using an Olympus CX33  with NY1S adaptor , EOS X9 and EOS utility software program . All mLNs were assessed to evaluate the histological type.p value less than 0.05 was considered statistically significant. All calculations were performed using the JMP Pro software application .The Kaplan\u2013Meier method was used to estimate postoperative survival, and a log-rank test was used to determine statistical significance. A n = 74) or the rectum (n = 45). The number of patients with T stage: T2, T3, T4a and T4b are 4, 84, 30, and 1, respectively. Patients with stage III tumors received 5-fluorouracil\u2013based postoperative chemotherapy. Of 32 patients stage IV tumors, 6 (17.1%) patients underwent R0 resection. The average of the number of LNs harvested was 15.7. The average of the number of mLNs was 4.3.Of 318 patients with CRC, 119 (37.4%) patients who had LNM were finally included in this study. Using the Union for International Cancer Control TNM classification, staging was III for 87 patients and IV for 32 patients. Mean age in this cohort was 70 years (range: 34\u201389 years), there were 67 males and 52 females. The resected tumors were located in either the colon . Kaplan\u2013Meier plots of CSS times showed longer survival for patients with all mLNs showing tubular carcinoma than for those with all mLNs showing cribriform carcinoma or for those with at least some mLNs showing either tubular or cribriform carcinoma (p < 0.0001) . No survRecognition of tumor heterogeneity is important in diagnosing malignancy and in choosing treatments for cancer patients. Because a primary tumor can contain an intricate set of various cancer cell clones, distinguishing the differences is difficult. In colon cancer, LNM have been reported to be polyclonal at the genetic level , thus opThere are some questions whether genetic and histological heterogeneity can exist within a single LN. In our study, each mLN contained only one histologic cancer type, and there were patients with multiple mLNs including different histologic cancer types in each mLN. The report presented that a single mLN contained a mixture of genetically different subclones derived from the primary tumor . There iMorphology such as lumen formation is important for understanding the malignant potential of cancer cells \u201313. WellKRAS mutation has been reported to be significantly higher in cribriform carcinoma than in the well-formed glandular type [The presence of cribriform compared with tubular carcinoma in mLNs seems to suggest more malignancy. The frequency of lar type , and a hlar type . Cribriflar type . Some relar type ,23. In clar type . PrognosVarious cancer cell clones spread widely from the primary lesion. Speculation has been that \u201cmore malignant\u201d cancer cells metastasize faster and farther than \u201cless malignant\u201d cancer cells. The mLNs are the front line of metastasis. The phenotype of the cancer cells in mLNs might represent the malignant potential of CRC. In the present study, CSS was significantly longer for tub group than for cri group, suggesting that when the \u201cfrontline\u201d mLNs all show tubular carcinoma, the phenotype might be considered \u201cless malignant.\u201d In contrast, when \u201cat least some\u201d mLNs show cribriform carcinoma, the phenotype might be considered \u201cmore malignant.\u201dIn clinical practice, after resection of the primary tumor and regional lymph nodes, patients with stage III CRC typically receive adjuvant chemotherapy based on the status of LNM \u20137. If alAlthough there is limitation with a small number of cases, we describe for reference the association between prognosis of patients with pathologically diagnosed well differentiated adenocarcinoma in the primary tumor and histologies in LNM. Of 8 cases with well differentiated adenocarcinoma in the primary tumor, 3 cases (37.5%) with cribriform type in LNM did not survive for 5 years. Whereas, one of 5 cases (20%) with tubular type in LNM did not obtain 5-year survival. Prediction of prognosis by LNM histology may be possible even in cases with well differentiated adenocarcinoma in the primary tumor. We also present for reference prognosis in patients with poorly differentiated-type in LNM. All 6 cases with at least 1 mLN showing poorly differentiated-type did not survive for 5 years. They seemed to be worse prognosis than patients with cribriform type in mLN. Poorly differentiated-type in mLN may indicate more malignant property. Large-scale investigation may clarify the implications of the histology in LNM.Limitations of the present study include its small number of patients and retrospective nature. Further investigations, including larger retrospective studies and prospective clinical trials, are required to address the issues raised here. Moreover, a more detailed pathologic classification scheme for the histologic cancer types in the mLNs is needed to establish clinical applications.To summarize, we demonstrated that the cancer histology in LNM from CRC was varied and could be associated with prognosis. A better understanding of cancer histology in LNM from CRC might provide novel insights in cancer research."
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