diff --git "a/deduped/dedup_0458.jsonl" "b/deduped/dedup_0458.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0458.jsonl" @@ -0,0 +1,101 @@ +{"text": "The expression of the regulatory (RI and RII) and catalytic (C) subunits of cAMP-dependent protein kinase was found to depend on the growth-state in oestrogen-dependent DMBA-induced mammary adenocarcinomas as well as in uteri of the rat. Castration-induced atrophy of the oestrogen-dependent tissues was accompanied by a decrease of the concentration of regulatory subunits (RI and RII) relative to both the catalytic subunit (C) and total protein, decreasing the R/protein and R/C ratios. A hyperplastic burst caused by high-dose oestrogen-replacement treatment was associated with an increased level of RI and little change in RII and C levels. Only minor differences were noted for the expression of mRNA for the alpha and beta subtypes of RI, RII and C between rat uteri from castrated and oestrogen-treated animals, or between mammary tumours from normal and castrated animals. Expression of RI beta-mRNA was detected only in the uterus. Our findings provide an experimental correlate for the reported value of the parameter R/protein in human mammary cancer biopsies to predict prognosis and outcome of therapy. Due to the sensitivity of the R/protein ratio towards changes in extracellular protein content, we recommend the biologically more meaningful R/C ratio in further clinical evaluations of mammary tumour biopsies."} +{"text": "Oestrogen receptor beta (ER\u03b2) modulates ER\u03b1 activity; wild type ER\u03b2 (ER\u03b21) and its splice variants may therefore impact on hormone responsiveness of breast cancer. ER\u03b22/ER\u03b2cx acts as a dominant negative inhibitor of ER\u03b1 and expression of ER\u03b22 mRNA has been proposed as a candidate marker for outcome in primary breast cancer following adjuvant endocrine therapy. We therefore now assess ER\u03b22 protein by immunostaining and mRNA by quantitative RT-PCR in relation to treatment outcome.ER\u03b22-specific immunostaining was quantified in 141 primary breast cancer cases receiving adjuvant endocrine therapy, but no neoadjuvant therapy or adjuvant chemotherapy. The expression of mRNA for ER\u03b22/ER\u03b2cx was measured in 100 cases by quantitative RT-PCR. Statistical analysis of breast cancer relapse and breast cancer survival was performed using Kaplan Meier log-rank tests and Cox's univariate and multivariate survival analysis.High ER\u03b22 immunostaining (Allred score >5) and high ER\u03b22 mRNA levels were independently associated with significantly better outcome across the whole cohort, including both ER\u03b1 positive and negative cases (Log-Rank P < 0.05). However, only ER\u03b22 mRNA levels were significantly associated with better outcome in the ER\u03b1 + subgroup (Log-Rank P = 0.01) and this was independent of grade, size, nodal status and progesterone receptor status . High ER\u03b22 mRNA was also associated with better outcome in node negative cases (Log Rank P < 0.001).ER\u03b22 protein levels were greater in ER\u03b1 positive cases (T-test P = 0.00001), possibly explaining the association with better outcome. Levels of ER\u03b22 protein did not correlate ER\u03b22 mRNA levels, but 34% of cases had both high mRNA and protein and had a significantly better outcome (Log-Rank relapse P < 0.005).High ER\u03b22 protein levels were associated with ER\u03b1 expression. Although most cases with high ER\u03b22 mRNA had strong ER\u03b22 immunostaining, mRNA levels but not protein levels were independently predictive of outcome in tamoxifen-treated ER\u03b1 + tumours. Post-transcriptional control needs to be considered when assessing the biological or clinical importance of ER\u03b2 proteins. Oestrogen Receptor alpha (ER\u03b1) is an accepted prognostic marker in breast cancer and is used to plan adjuvant endocrine treatment (e.g. use of the anti-oestrogen tamoxifen). The majority of the breast cancers are positive for ER\u03b1 (ER\u03b1+), but not all patients with ER\u03b1+ cancer respond to endocrine therapy and many subsequently succumb to local relapse or metastasis. The failure of some breast cancers to respond to tamoxifen, currently the most common adjuvant endocrine treatment, is a major clinical problem and several resistance mechanisms have been elucidated ,2.ER\u03b2 and its splice variants are differentially expressed in a variety of normal tissues and cancers including breast ,4, but nFurther verification of ER\u03b2 variants, including ER\u03b22, as potential clinical markers is still required. Many previous studies make use of mRNA levels as a surrogate marker for ER\u03b2 protein expression and few have attempted to relate mRNA to protein levels. Other studies that do assess the expression of ER\u03b2 protein use techniques that rely on detection of N-terminal epitopes that are shared by most variants. A good proportion of studies also fail to take into account menopausal status, stage of the disease or the treatment given.We have previously identified ER\u03b22 mRNA levels as being more closely associated with treatment outcome than mRNA levels of ER\u03b21 or ER\u03b25 in a treWe were able to confirm a significant association of both high ER\u03b22 protein and high mRNA levels with good outcome, but ER\u03b22 protein levels were not a useful marker of outcome. Strong ER\u03b22 staining was associated with better outcome, but not independently of ER\u03b1. Although ER\u03b22 mRNA and protein levels did not correlate with each other, approximately one third of cases (34%) were seen to have both high mRNA and high protein levels; these had a significantly better outcome than the other cases, so ER\u03b22 protein may have a role in improved outcome for a subset of breast cancers.Patients undergoing treatment for invasive breast cancer during the period 1993 and 1999 at the Royal Liverpool University Hospital were identified from a database at the Cancer Tissue Bank Research Centre (CTBRC), University of Liverpool , but a similar lack of correlation was seen previously for ER\u03b21 . This is in contrast to ER\u03b1 in the same cohort [Pearson (%+) 0.30 P = 0.003; Spearman (Allred) 0.50 P = 1.0 \u00d7 10for ER\u03b21 . Due to High ER\u03b22 protein levels and high ER\u03b22 mRNA levels, when entered into a Cox multivariate model, were independently associated with better relapse-free survival in the whole cohort . In multivariate analysis of mRNA and protein in the ER\u03b1 + tamoxifen-treated cohort, only high ER\u03b22 mRNA levels were significantly associated with lower BCR (HR 0.28 CI 0.212\u20130.72 P = 0.008), but a trend remained for protein (HR 0.42 CI 0.15\u20131.19 P = 0.10). Similar results were obtained for analysis of BCS. This indicates that both mRNA and protein levels may contribute to the relationship of ER\u03b22 with improved outcome.Using the cut-points optimised for outcome analysis, the majority of cases (69%) with high ER\u03b22 mRNA levels also had high levels of ER\u03b22 protein. However, only a minority of cases (44%) with high ER\u03b22 protein were also classified as having high ER\u03b22 mRNA. Thus ER\u03b22 mRNA expression is frequently associated with expression of significant levels of ER\u03b22 protein, but ER\u03b22 protein expression is often dissociated from mRNA expression. Hence, there is a subset of cases (34%) with concomitant high ER\u03b22 mRNA and protein and another subset of cases (44%) in which high protein levels are not accompanied by high mRNA levels. The cases with both high ER\u03b22 protein and mRNA had a significantly better outcome than those with low levels of either mRNA or protein or both being related to some form of protein stabilization, or detection of inactive ER\u03b22. The disparity between protein and RNA expression for ER\u03b22 is even suggestive of an inverse relationship. Nevertheless a significant proportion of cancers (34%) had both high protein and high mRNA levels and these had a significantly better outcome than the remaining cases. This suggests that transcription of ER\u03b22 mRNA drives ER\u03b22 protein levels in some cases, and these cases do particularly well on tamoxifen treatment. It is perhaps unsurprising that previous studies of ER\u03b22 protein expression did not find significant associations between ER\u03b22 and outcome in ER\u03b1 + tamoxifen treated cases as these did not include measurement of ER\u03b22 mRNA levels. They were thus unable to distinguish between ERB2 protein associated with increased transcription and that possibly present due to some form of post-transcriptional control .Whilst our data would suggest that high ER\u03b22 levels could contribute to an improved outcome in a subgroup of patients, it provides further evidence that determination of ER\u03b22 protein by immunostaining is unlikely to provide the predictive test that is needed for better targeting of additional therapy in those women for whom adjuvant tamoxifen is not likely to be sufficient. The failure to link protein expression to outcome measures does not preclude the use of ER\u03b22 mRNA levels in a clinical setting. Low ER\u03b22 mRNA was significantly associated with worse outcomes in ER\u03b1 + tamoxifen-treated patients independently of other factors such as grade and nodal status. Larger trials to validate ER\u03b22 mRNA as a biomarker are needed and should be extended to alternative adjuvant endocrine therapies such as aromatase inhibitors.BCR = breast cancer relapse; BCS = breast cancer survival; CI = confidence interval; ER\u03b1 = oestrogen receptor alpha; ER\u03b2 = oestrogen receptor beta; HR = hazard ratio; MW = Mann-Whitney U test; %+ = percentage positive cells; PgR = progesterone receptor; qRTPCR = quantitative reverse-transcription PCR analysis; ROC = Receiver Operating Characteristic.The author(s) declare that they have no competing interests.RV performed qRTPCR analysis and immunostaining under the supervision of MD. RV and VA scored the immunostaining and MD and RV performed the statistical analysis. MD, DRS and CH participated in conception, design and coordination of the study. All authors helped to draft the manuscript and approved the final version.The pre-publication history for this paper can be accessed here:"} +{"text": "S-Adenosylmethionine (SAM) serves as a methyl donor in biological transmethylation reactions. S-Adenosylhomocysteine (SAH) is the product as well as the inhibitor of transmethylations and the ratio SAM/SAH is regarded as the measure of methylating capacity (\u201cmethylation index\u201d). We present a rapid and sensitive LC\u2013MS/MS method for SAM and SAH determination in mice tissues. The method is based on chromatographic separation on a Hypercarb column filled with porous graphitic carbon stationary phase. Sufficient retention of SAM and SAH on the chromatographic packing allows simple sample preparation protocol avoiding solid phase extraction step. No significant matrix effects were observed by analysing the tissue extracts on LC\u2013MS/MS. The intra-assay precision was less than 9%, the inter-assay precision was less than 13% and the accuracy was in the range 98\u2013105% for both compounds. Stability of both metabolites during sample preparation and storage of tissue samples was studied: the SAM/SAH ratio in liver samples dropped by 34% and 48% after incubation of the tissues at 4\u00a0\u00b0C for 5\u00a0min and at 25\u00a0\u00b0C for 2\u00a0min, respectively. Storage of liver tissues at \u221280\u00a0\u00b0C for 2 months resulted in decrease of SAM/SAH ratio by 40%. These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues. S-Adenosylmethionine is synthesized from methionine and ATP in a reaction catalysed by methionine adenosyltransferase. SAM is providing methyl group moieties in several dozens of transmethylation reactions of crucial biological importance During the past decade, animal models of several disorders of homocysteine metabolism became available for studying the pathogenesis of the respective enzyme deficiencies 18 columns without the use of ion-pairing additive in the mobile phase, SPE is primarily required in the LC\u2013MS/MS applications to separate both analytes from salts and other early eluting matrix components that can lead to ion-suppression on mass spectrometer.Various methods for the analysis of SAM and SAH have been published. These include LC methods with UV detection utilizing ion-pairing Other authors used penta-fluorinated column for LC\u2013MS/MS analysis of SAM and SAH in mouse embryos Our goal was to develop a sensitive LC\u2013MS/MS method with minimal sample requirements and with sufficient sample throughput. Preferably, we tried to omit the SPE step, which demands usually higher sample and reagents volumes, prolongs labour time and increases the cost of the method. This was accomplished by the use of column filled with porous graphitic carbon (PGC) as a stationary phase, which retains both analytes and separates them from interfering compounds causing ion-suppression effects on mass spectrometer. In contrast to Burren et al. The methods for metabolite determinations in biological samples should be validated in detail focusing on stability of the metabolites in the sample matrix. A marked decrease of SAM and increase of SAH has been demonstrated in untreated plasma samples stored for 3\u00a0h at room temperature and for 1 month at \u221220\u00a0\u00b0C If we have to use some clich\u00e9 to describe the presented method, we would choose the attributes simple, cost effective and reproducible. This paper includes detailed sample preparation protocol with regard to demonstrated instability of SAM and SAH in tissue samples, and presents an original chromatographic separation of SAM and SAH utilizing simple mobile phases without ion-pairing reagents.22.12H3]-SAM was purchased from CDN Isotopes , [13C5]-SAH was obtained from Dr. Herman J. ten Brink . SAM p-toluenesulfonate salt for preparation of SAM standards as well as all other chemicals was obtained from Sigma\u2013Aldrich .Acetonitrile (LC ultra-gradient grade) was purchased from J.T.Baker . The internal standard -SAM (m/z 402.3\u00a0\u2192\u00a0250.3) and [13C5]-SAH (m/z 390.3\u00a0\u2192\u00a0136.3) were monitored. The scan dwell time was set at 0.3\u00a0s for both the analytes and the internal standards. The optimized ion source parameters were: declustering potential: 42 and 40\u00a0V for SAM and SAH, respectively, collision energy: 25 and 27\u00a0V for SAM and SAH, respectively, entrance potential: 4.5\u00a0V, collision cell exit potential: 5.2 and 5.8\u00a0V for SAM and SAH, respectively, ionspray voltage: 4500\u00a0V and ionspray source temperature: 450\u00a0\u00b0C. The interface heater was set to 100\u00a0\u00b0C. The collision gas, curtain gas and ion source gas 1 and 2 (nebulizer gas and turbo gas) were set to 5, 14, 60 and 60\u00a0psi, respectively.The detection of the analytes was carried out using positive electrospray ionization technique and selected reaction monitoring mode. The precursor\u00a0\u2192\u00a0product transitions for SAM (2.3x), the peak area ratio was plotted versus the analyte concentration.Stock solutions of SAM and SAH were prepared by dissolving approximately 4\u00a0mg of each compound in 1000\u00a0\u03bcl of ice cold water. The concentration of SAM and SAH in stock solution was determined by UV absorption spectroscopy at 260\u00a0nm using molar extinction coefficient of 15400 2.4Cbs\u2212/\u2212) kindly provided by Dr. Jan P. Kraus, University of Colorado School of Medicine, USA.Male C57BL/6J mice, aged 3\u20134 months, were used for determination of SAM and SAH in tissues and for experiments described in the section \u201cMethod validation\u201d. Mice were maintained in a temperature- and light-controlled environment, they had free access to tap water and standard laboratory food. The experiments were approved by the Animal Care Committee of the 1st Faculty of Medicine. For the precision evaluation of the LC\u2013MS/MS determination in samples with high SAM and SAH concentrations we used extracts from livers of cystathionine-\u00df-synthase deficient mice . The homogenate was centrifuged 10\u00a0min at 7 000\u00a0\u00d7\u00a033.113C5]-SAH which is expensive and commercially not readily available. The residual tissue extract can be used for determination of additional metabolites, as described previously Protein precipitation is the simplest way of sample preparation for LC\u2013MS/MS analysis. There are pros and cons which should be considered when choosing this approach instead of some of the other extraction methods. The pros are speed, reduction of sample and standard volumes to minimum resulting thus in cost effectiveness. The major disadvantage is the complexity of the sample matrix which may lead to ion suppression effects and to possible loss of sensitivity. We have chosen this option of sample preparation especially because of the minimal sample requirement. Using our sample preparation protocol, we obtained 300\u2013900\u00a0\u03bcL of tissue extract. Only a small aliquot (30\u00a0\u03bcL) of the extract was spiked with labeled internal standards in order to minimize the consumption of the internal standard [Because of instability of SAM and SAH in tissues we performed all handlings with tissue specimens following the excision in liquid nitrogen frozen samples. Tissue powderizing is required to prevent the formation of rubbery nugget in Potter-Elvehjem homogenizer. The homogenization was performed in denaturing ice-cold perchloric acid solution, in order to prevent enzymatic conversions of SAM and SAH during homogenization and further procedures. Moreover, acidic pH of perchloric acid stabilizes SAM, as previously reported 3.2t0) result in poor ionization efficiency in the mass spectrometric detection. Some authors use ion-pairing additives in the mobile phase to improve the retention and also to increase the content of organic solvent in the mobile phase, however, these additives may cause loss of sensitivity on mass spectrometer 18 stationary phases t0. Because the retention factor of SAH was too high (more than 15\u00a0min) at acetonitrile concentrations lower than 20% in the mobile phase, we finally adopted a gradient elution to keep the retention of both metabolites in optimal range. After optimization of chromatographic conditions, the retention times of SAM and SAH were 5.9 and 7.8\u00a0min, respectively. The total chromatographic run time including column regeneration was 15\u00a0min. Typical chromatogram of mouse liver extract is shown in S-Adenosylmethionine is a polar compound and as such it is poorly retained on standard reversed-phase columns. High water content in the mobile phase and the presence of interfering substances close to the hold-up time of the calibration curve (n\u00a0=\u00a05) was y\u00a0=\u00a00.0643(\u00b10.0018)x\u00a0+\u00a00.0025(\u00b10.0018) (correlation coefficient r\u00a0=\u00a00.999) for SAM and y\u00a0=\u00a00.0434(\u00b10.0009)x\u00a0\u2212\u00a00.0088(\u00b10.0043) (correlation coefficient r\u00a0=\u00a00.999) for SAH.The calibration curves were linear in the range 1.25\u2013320\u00a0\u03bcM for both SAM and SAH. The calibration curve equation is The limit of detection determined in standards diluted in 0.4\u00a0M PCA was 7.5\u00a0nM for SAM and 15\u00a0nM for SAH.3.3.2Simplicity of the sample preparation protocol, which consists of protein deproteination and sample neutralization without the SPE step, brings in return the risk of increased matrix effects. We have evaluated the ion suppression effects by two methods.n\u00a0=\u00a05) showed enhancement of the SAM signal in spiked tissue extracts versus standards prepared in water by 9%(\u00b14), whereas spiking of the tissue extracts with SAH resulted in small ionization suppression by 4%(\u00b12). If analyte/I.S. ratios were used for the calculations as described above, the mean differences between data obtained from analysis of spiked tissue extracts and standard samples were lower than 2% for both analytes, showing that the matrix effects affecting the analyte signal were compensated by analogous enhancement or suppression of respective internal standards.Firstly, we compared the MRM signals in deproteinized tissue sample spiked with known SAM and SAH concentration (50\u00a0\u03bcM) to that of the analyte standards prepared in water. The comparison was done after subtracting the signals of endogenous SAM and SAH from the signals obtained in spiked sample. The results expressed as a mean(\u00b1standard deviation) of data obtained by replicate set of analyses the precision of the entire method, including sample preparation and LC\u2013MS/MS determination and (b) the precision of the LC\u2013MS/MS determination alone. The reason for this approach was to test whether the complex sample preparation protocol added significant imprecision to the LC\u2013MS/MS determination alone. Because of ethical reasons, we performed the precision determination with limited number of liver samples excised from male C57BL/6J mice for 2 and 5\u00a0min and at 4\u00a0\u00b0C for 5 and 15\u00a0min, respectively.The results are shown in 3.3.4.2To evaluate the changes of SAM, SAH and SAM/SAH ratio during storage of tissues at \u221280\u00a0\u00b0C we analyzed tissue aliquots stored for 2 and 6 months, respectively. The results given in 3.4The concentrations of SAM and SAH in mice tissues excised from male C57BL/6J mice, aged 3\u20134 months (see Section 3.5Presented method was used to determine tissue concentrations in mice tissues. The concentrations of SAM and SAH in tissue homogenates was in micromolar range whereas the limit of detection of our method is at least two magnitudes lower. The sensitivity of our method enables determination of SAM and SAH in whole blood extracts prepared by deproteinization of whole blood with equal volume of 0.6\u00a0M PCA, which was proved in pilot experiments. The whole blood concentrations of SAM and SAH are in the range of hundreds of micromoles, which is close to the detection limits of some published LC-UV methods, but still easily detectable using our LC\u2013MS/MS method. The concentrations of SAH in plasma are, however several dozens of nanomoles and are too low to be determined using our method without further optimalization. Lowering the detection limit may be possible for example by the use of one of the variety of SPE methods, as described for example in previous publication 4The presented method allows precise and sensitive determination of SAM and SAH. Limits of detection of presented method \u22127.5\u00a0nM for SAM and 15\u00a0nM for SAH \u2013 are at least one order of magnitude lower than in previously described LC-UV methods used for determination of SAM and SAH and are comparable with the sensitivity of published LC\u2013MS/MS methods.The method comprises special sample preparation protocol to prevent metabolite changes due to demonstrated limited stability of the analytes in tissue samples. The stability studies show that tissues samples should be snap frozen in liquid nitrogen immediately after excision, kept frozen at \u221280\u00a0\u00b0C and processed by the described protocol as soon as possible, preferably within a week."} +{"text": "The citation analysis of the research output of the German economic research institutes presented here is based on publications in peer-reviewed journals listed in the Social Science Citation Index for the 2000\u20132009 period. The novel feature of the paper is that a count data model quantifies the determinants of citation success and simulates their citation potential. Among the determinants of the number of cites the quality of the publication outlet exhibits a strong positive effect. The same effect has the number of the published pages, but journals with size limits also yield more cites. Field journals get less citations in comparison to general journals. Controlling for journal quality, the number of co-authors of a paper has no effect, but it is positive when co-authors are located outside the own institution. We find that the potential citations predicted by our best model lead to different rankings across the institutes than current citations indicating structural change. In the last decade, German economic research institutes have undergone remarkable changes initiated by the German Scientific Council journal articles published in 2000\u20132009 by staff members of the German economic research institutes. The analysis focuses on institutes that are members of the Leibniz Association (WGL), which includes the Kiel Institute for the World Economy (IfW), the ifo Institute for Economic Research (ifo), the Halle Institute for Economic Research (IWH), the Rhineland-Westphalian Institute for Economic Research (RWI), the Centre for European Economic Research (ZEW) and the German Institute for Economic Research (DIW Berlin). Our study also takes into account publications of the Hamburg World Economic Archive (HWWA), which was a member of the WGL until 2006. Part of the research is now being continued by the privately financed Hamburg World Economic Institute (HWWI).The publications used in this study are originally identified using the annual reports of the institutes or, if not available there, from their websites for the period from 2000 to 2009.EconLit which is used for rankings in the German scientific community following a recommendation by the German Economics Association (VfS).EconLit as a journal reference list will lead to distortions in the measurement of the research output in our context since it ignores multidisciplinarity in some of the institutes.In addition, the SSCI index guarantees a high standard of quality in academic journals through strict admission criteria and is characterized by its interdisciplinary nature.In order to carry out a clearly defined evaluation of the scientific impact of the institutes\u2019 publication output, our study only includes publications by the institutes\u2019 regular employees and its mid-term scholarship holders. Publications by so-called fellows and short-term visiting fellows who list an affiliation to the institutes in their publications are, as a rule, not taken into account. In general, visiting fellows provide research impulses for the scientific activities at the institutes, but their research is usually not conducted in the institutes. In case we are in doubt about the affiliation, we use the CV of the respective individual to identify the position of the researcher.In a related paper, Ketzler and Zimmermann have preFigure\u00a0Table\u00a0Other variables contained in Table\u00a0Remarkably, joint production of research is of great importance for the institutes. Almost three quarters (73\u00a0%) of all published papers have more than one author (Manyauthors).The average impact factor is 0.73 for the articles that are published in economics journals, while the total average in our data is 0.79. This reflects the known fact that the field of economics cites less than others in the social sciences like sociology and demography. For 2009, the year with the highest publication output, the average impact factor for articles published in economics journals is 1, indicating also a strong rise in average quality over time.The aim of this study is to provide a citation-based research evaluation. The pure analysis of the publication output using a variety of indicators does not give a complete picture of the institute\u2019s research performance. The quality of a published article is measured only unprecisely by the impact factor of the respective journals Oswald . For assTable\u00a0Of further interest are the number of citations per article. We obtain the second row in the Table\u00a0However, these results are biased since the analysis ignores the very different age distribution of the articles between institutes. We present below in \u201cThe distribution of all citations achieved in January 2011 for all papers published in 2000\u20132009 by all institutes classified by the journal\u2019s year of publication is recorded in Table\u00a0In order to identify the determinants of citation success, we specify an empirical model for the analysis. Since the dependent variable of interest is the number of cites per published article, a count data regression model seems to be an appropriate choice. The baseline model of count data is a Poisson regression, which imposes equality of the variance of the data to its mean, an assumption that often does not hold in real data. Typically, the variance in real applications is larger than the mean, which is called overdispersion, and leaves the Poisson model with too low standard errors. The negative binomial model is the most popular approach to deal with overdispersion. Due to the specific nature of the citation data, which is characterized still by a large number of papers with zero citations and a high degree of overdispersion we use a negative binomial regression model with robust standard errors.The dependent variable is the number of citations received in January 2011 by SSCI articles published by members of the institutes between 2000 and 2009. Our regression equation includes the number of pages for each article (Pages) as the length of an article is widely regarded as an indicator for scientific substance.The model also includes a number of dummy variables to capture additional characteristics of the respective scientific work. First, we relate the number of citations to different journal selection criteria that are used for the evaluation of research performance. Thereby, a number of issues are addressed. We analyze if articles published in general interest journals are significantly more cited than papers in so called field journals (Field). Given the interdisciplinary nature of the research at the institutes and the various disciplines covered by the SSCI, we classify the research output whether it is published in an economics journal (Economics) according to the SSCI index, or if the journal belongs to another sub-index. Second, we analyze if collaboration with other researchers, which is believed to produce higher research quality and hence impacts the quality of the published research. A dummy variable for co-authorship is included which indicates that a paper has more than one author (Manyauthors). As our study focuses on the research quality of research institutes, we investigate if co-authorship with researchers from other institutions like universities enhances the quality of an article (Divauthors).The overall scientific output of the institutes in peer-reviewed journals has increased remarkably since 2000. While some institutes made a greater contribution to this development, as indicated in Fig.\u00a0Using the detailed data for all articles in our sample, Table\u00a0The length of a paper, as well as the journal quality measured by the impact factor, has a significantly positive impact on the number of citations. As is indicated by the signs of the coefficients of the squared terms of the variables the positive effect of the impact factor is decreasing with paper size or impact factor. These results of the regressions are in line with the existing literature. We also find that a journal setting a page or word limit for an article experiences a positive impact on the quality of a published article (Limit).Our analysis provides no evidence that the publication of an article in an economics journal (according to the SSCI classification) exhibits a significant influence on the number of citations (Economics). In spite of the large share of non-economics research at the institutes, our finding does not support the general observation that different disciplines exhibit different citation standards. However, from the perspective of the economic research institutes, one should note that research results published in non-economics journals are equally acknowledged beyond the economics profession as is economics research in its field. This indicates that interdisciplinarity is well established at the institutes. It does not \u201churt\u201d to publish in the journals of other disciplines. But one may also argue that there is no \u201cadvantage\u201d to publish outside the own discipline, either because one receives lower attention among the others readers of those journals or the work submitted is of lower quality. Finally, the evaluation has to take into account that the journal impact factor included in the regression already covers some differences across the disciplines.We provide evidence that publications in field journals (Field) are significantly less cited than articles in journals that cover all branches of economics, although the journal impact factor included in the regression should have taken up already some effects. For the applied policy-oriented research of the institutes, field journals are the natural focus as well as a key element of the publication strategies. This strategy, however, leads to fewer citations than publications in general interest journals, which are to some extent associated with top journals. For the institutes it can be regarded as a success that research results\u2014as far as it is possible\u2014are published in general interest journals, which are very well acknowledged in the academic field, expressed through higher citation numbers. Publications in general interest journals are, in addition to the pure number of publications, an important tool for increasing the visibility of the institutes in the scientific community and should have an important part of the institute\u2019s publication strategy.Our results suggest that there is a difference in the quality of the research of the institutes whereas DIW Berlin serves as the reference category left out in the estimation. Publications from ZEW researchers are significantly more cited than articles published by researchers from DIW Berlin and all the other institutes controlled for all the other factors included in the regression. This citation success of ZEW researchers is shown through the high number of citations per article in Table\u00a0Interestingly, there is evidence of a publisher bias in this study. The estimated regression in Table\u00a0Collaboration of researchers through formal co-authorship is supposed to increase research quality compared to single author research. The empirical evidence is, however, mixed. Our findings are in accordance with Medoff who rejeOn the other hand, if we focus on collaborations among researchers from the institutes with colleagues from different institutions, co-authorship is statistically significant (Divauthors). From the perspective of the institutes, research quality increases through collaboration with external scholars. This result may reflect the general trend of the globalisation of research involving a strengthening of network activities that leads to an increased number of cooperations. Modern communication technologies facilitate a greater exchange of ideas that leads to improved research quality. However, we also note that the empirical evidence for cooperation with external researchers must be considered in light of the rising research performance at the institutes, as indicated in Fig.\u00a0This development was supported by intensifying the cooperation with universities that enabled the institutes to catch up state-of-the-art scientific methods that are publishable in peer-reviewed journals. The increasing number of joint appointments for example, expresses the increasing cooperation. Establishing networks with external researchers represent a key element in the strategy of the institutes. We also note that the analysis by Medoff as well As expected, the age of an article has a significant positive impact on the number of citations. Given the results of our empirical model in Table\u00a0At the level of institutions we focus on the evaluation of the research quality for a fixed time period. However, compared to the individual analysis, the scientific output of an institution is likely to change over time for a number of reasons that do not apply to the individual researcher. Publication output is among others influenced by the fluctuation of researchers or a new strategic focus of the institute. In fact, due to the requirements of the Leibniz Association, research output has increased since 2000, but differs across institutes. While the ZEW dominated the field at the beginning of our surveyed period, since 2004 a number of institutes have caught up with the development and show high publication growth rates including DIW Berlin which leads the field in 2009. The high number of SSCI publications since 2004 shown in Fig.\u00a0In order to establish a ranking for the total citation success of the institutes, we eliminate the time effect by using our regression model in Table\u00a0Table\u00a0The ranking is also changed if we use the market share among the predicted cites (third row), which suggests that DIW has 28\u00a0% of the cites, ZEW 27\u00a0%, ifo 16\u00a0%, IfW 12\u00a0%, RWI 9\u00a0%, HWWA 5\u00a0%, and IWH 3\u00a0%. The variation in the market shares of predicted compared to actual citation numbers underlines the rising scientific significance of DIW\u2019s publication output, which has not been realized yet. Our model estimates an increasing market share of citations for DIW Berlin by nearly four percentage points, which is the highest increase for all institutes. In contrast, ZEW\u2019s share in the number of all citations drops about the same amount. The publication output of DIW Berlin, which has continuously increased over the sample period, is very well reflected in the ranking of the simulated number of citations representing the total citation success of the institutes from 2000 to 2009.The relative numbers of citations that result from our simulation provides evidence that there is an overall improvement in the quality of the research at the institutes. On average papers authored by members of the economic research institutes can expect about 21 citations, 12\u00a0years after the paper is published. This is an increase by six citations compared to articles that are originally published in 2000 Table\u00a0. Table\u00a05The rankings using the relative citation numbers reveal differences in the publication strategy of the institutes. The high number of citations estimated for RWI\u2019s moderate research output is an indication that the publication strategy is primarily focused on quality. Compared to articles published in 2000, which received 15 citations on average RWI nearly doubles the citations according to our model prediction in Table\u00a0German research institutes have undergone a regime shift after 1998, when the German Scientific Council when co-authors are located outside the own institutes for instance in universities. We should note, however, that such results are achieved in a regression analysis that controls for journal quality by average SSCI impact factor.Further, we have made transparent the existence of employer and publisher bias among the cites of published articles of research staff from the German research institutes: If from the Centre for European Economic Research (ZEW), articles have significantly more cites than if from the German Institute for Economic Research (DIW Berlin) or one of the others. If published in a Springer journal, the articles receive more cites than with Blackwell, and if published with Sage, the articles receive significantly less cites than with Blackwell. The other publishers are not different from Blackwell.We further find that the calculated potentials from our regression model lead to quite different rankings across the institutes than the current cites are indicating. In the raw data, ZEW holds the leading position with 1,511 citations, followed by DIW Berlin and the ifo (753 citations). The simulated number assuming that all output would have been published in the base year 2000 is highest for DIW Berlin with 6,250 citations followed by the ZEW and the ifo indicating some structural change underway.Our study has also shown that rankings among the institutes are quite different if cites per articles is the major objective independent of the number of papers published, the structure of the research staff and other objectives like providing policy advice. While a high-quality strategy is valuable if the key object is research and feasible, if the institute is small, this is not a useful strategy in general. The request of research orientation and publication output formulated by the German Scientific Council (Wissenschaftsrat Scientometric analysis can gain substantially through measurement and quantification. However, the ideal concept is still under debate. While the inclusion of papers published in journals ranked by the SSCI is often used next to the average impact of the respective journals, these measures are also debatable. We have suggested here to use actual and potential cites and have demonstrated its usefulness for a particular example, the ranking of German economics research institutes. However, we think that the suggested approach is also applicable and has promise in other contexts of scientometrics."} +{"text": "Candida and Trichomonascus ciferrii, were detected on the skin and mucous membranes in the English full blood mares. Growth of yeasts was observed in more than half of the samples taken from mares that had foaled and approximately 46\u00a0% of non-conceiving and barren mares. The high prevalence of various yeast strains in the mouth, nostrils, and collateral groove may suggest widespread occurrence of the microflora in the breeding environment. The results obtained indicate that the yeasts isolated in this study may be components of the normal microflora of the skin and mucous membranes in horses. The analysis results do not indicate unambiguously that the isolated microflora affects reproduction in mares, although this cannot be excluded.The aim of the study was a quantitative and qualitative analysis of microflora, presentation of current data about prevalence of the microflora on the skin and mucous membranes, and determination of its possible effect on reproduction of English full blood horses bred in Poland. The material for analyses was sampled from the skin and mucous membranes (385 samples) of 55 English full blood mares. Taking into account reproduction traits, the mares were classified into three groups. Six yeast-like species, including five species from the genus Candida albicans (39.1\u00a0%), Geotrichum (8.7\u00a0%), Cr. neoformans (4.3\u00a0%), and other yeasts (13\u00a0%). In horses, pathogenic fungi are Candida albicans, Candida parapsilosis, Candida lusitaniae, Candida rugosa, Cr. neoformans, Hansenula anomala, Hansenula polymorpha, Rhodotorula minuta, Rhodotorula rubra, and Torulopsis candida [C. parapsilosis (21.7\u00a0%), Candida guilliermondi (8.4\u00a0%), Candida kefyr (6\u00a0%), and Candida albicans (4.8\u00a0%).Microorganisms are responsible for development of many diseases, which directly or indirectly affect reproductive performance in various animal species. Knowledge of the microflora of animal skin and mucous membranes is essential for better understanding of pathological processes. Few publications have described prevalence of normal fungal flora in the animal reproductive tract. Some fungi are potentially pathogenic. According to Garoussi et al. , in cows candida , and in albicans 9.1\u00a0%, GeAspergillus, Penicillium, Acremonium, Cladosporium, Mortierella, Aureobasidium pullulans, Zygomycetes, and Candida, might cause infections of the reproductive organs in cows and buffalos. Vaginal, uterine, and cervical inflammations caused by fungal infections have also been reported from horses [Previously, not much attention was paid to fungal infections of the reproductive tract in animals. However, extensive antibioticsand hormonal therapies have resulted in rising incidence of fungal infections in humans . Verma em horses , 7, cat m horses , and dogm horses .Candida) comprised only yeast-like opportunistic microorganisms that are regarded as invasive species by other authors, for example, C. albicans, Candida glabrata, Candida tropicalis, Candida krusei, C. guilliermondi, C. rugosa, and Trichomonascus ciferrii.The microflora present in the breeding environment of various groups of healthy horses was described by R\u00f3\u017ca\u0144ski et al. . The 13 Given the potential causative role of yeast-like fungi in endogenous infections, identification of fungal flora occurring on the skin and mucous membranes of English full blood horses will contribute to elucidation of the role of these microorganisms in various types of infections, including asymptomatic infections of reproductive organs. Furthermore, this investigation will be helpful in determination of the species composition of the physiological yeast-like microflora occurring on the skin and mucous membranes in English full blood horses.The aim of the study was a quantitative and qualitative analysis of microflora, presentation of current data about prevalence of the microflora on the skin and mucous membranes, and determination of its possible effect on reproduction of English full blood horses bred in Poland.The investigations were carried out on material sampled from 55 English full blood mares aged between 4 and 12. All the horses were kept in separate boxes, in two brick, metal-roofed stables in one of the equestrian centres in Poland.The mares were classified into three groups, where group I comprised 12 high-quality mares kept in the boxes together with their offspring in stable B. Group II consisted of mares that had not conceived after mating. These were 24 mares, which had previously had healthy foals, but during the last 2 seasons failed to conceive; they were kept in separate boxes in stable A. Group III was composed of 19 barren mares that had not foaled within three or more years and were excluded from reproduction (stable B). It should be stressed that the examinations involved all mares with reproductive disturbances. Mares that had foaled at an approximate time were randomly chosen for analyses from a bigger group of mares in the stud.Temperature was measured with an instant-read liquid thermometer. Relative humidity was assessed using the Assmann\u2019s aspiration psychrometer. The mean values of temperature and relative humidity were, respectively, 23\u00a0\u00b0C and 54\u00a0% outside, and 21\u00a0\u00b0C and 76\u00a0% inside the boxes.Smears from nostrils, mouth, ear, back, groin, vagina, and collateral groove were taken from each mare. Material obtained upon inoculation of 385 samples was analysed. The samples were collected once in the summer period (July).All the samples were inoculated on the Sabouraud medium; next, macroscopically homogenous colonies were obtained in selective cultures, which, after growing, were assayed with the API 20C AUX (bioM\u00e9rieux) biochemical tests. The ability of biofilm formation was also assayed.All the horses kept in the stables were fed in accordance with the current horse feeding standards providing mineral-vitamin supplements.The experimental horses received permanent zootechnical and veterinary care. All the animals underwent a prophylaxis programme of parasitic invasion control and vaccinations. All the horses were healthy during the investigations. None of them received any treatment before (at least 1\u00a0month earlier) or after the study (within minimum 1\u00a0month).The greatest abundance of yeast fungi was found in the smear samples from mouths, nostrils, and collateral grooves in all the mares. The samples collected from the vaginal mucous membrane and groin skin contained the smallest number of the microorganisms .Candida, were detected on the skin and mucous membranes of the experimental mares . The number of isolates obtained from other sites was smaller; they were detected in 21\u201364\u00a0% of the samples. The smallest number of strains was isolated from the groin (12.6\u00a0%) and vaginal (21\u00a0%) material.C. guiliermondii was most prevalent and constituted over 75\u00a0% of all microorganisms identified in the mares with conception failure.In group II, C. guiliermondii and C. tropicalis, which were the most prevalent yeasts in the group of the barren mares and constituted 52 and 26\u00a0%, respectively, of all the isolated yeasts . Interestingly, the microbial presence in the test material was not accompanied by any deleterious health effects in the animals. The representatives of the fungal microflora were natural components of the environmental flora [The greatest abundance of various yeast strains indentified in the mouth, nostrils, and collateral groove in the horses Tables\u00a0, 3, 4 maal flora .Table\u00a03FC. guiliermondii, which was the most common yeast in the mares from all the groups . Yet, it was twofold higher in mares with reproductive failure than in the foaling and barren mares. This may lead to a conclusion that a correlation between the presence of yeasts in the vagina and reproduction performance cannot be ruled out; 80\u00a0% of the microflora isolated from the vaginal mucosa of the mares with conception failure were Candida-associated fungal infections to be caused by several different species, for example C. guilliermondii or C. lusitaniae. Chengappa et al. [C. guilliermondii was one of the pathogenic yeast species. It was described as a causative agent of cervical, uterine, and vaginal infections in mares. The results obtained in this investigation and data provided by the literature cited imply a possible relation between C. guilliermondii occurrence and reproductive failure in English full blood mares.Pfaller and Diekema found 3\u2013a et al. reportedC. rugosa species. It should be mentioned that genetic differences may exist also within the C. guilliermondii species, depending on its occurrence site. This assumption, however, needs further investigations at the molecular level.Studies carried out by \u015alaska et al. explorinC. guiliermondii. It can be assumed that only C. guiliermondii may be indirectly associated with reproductive failure. C. tropicalis may be regarded as opportunistic microflora in mares, which is corroborated by its presence in the vaginas of mares that have foaled . However, from 16,8\u00a0% of mares from this group, there was isolated d Tables\u00a0. In contC. albicans isolated from non-conceiving mares and C. lusitaniae\u2014from non-conceiving and barren animals are regarded as the predominant etiological disease factors worldwide [Candida-associated fungal infections in humans were caused by several species, for example C. albicans, C. tropicalis C. guilliermondii, and C. lusitaniae. All these species were isolated from the skin and mucous membranes of the English full blood horses, which showed no signs of disease is baffling; therefore, further investigations should be undertaken to elucidate the reasons for the absence of clinical symptoms normally caused by the microflora in English full blood horses.An important trait of numerous pathogenic microorganisms is their ability of biofilm formation. However, none of the strains isolated and described in this paper exhibited such properties. The presence of the yeasts that are commonly regarded as pathogenic in mares were present in all the groups of mares, irrespective of their reproduction performance. C. albicans and C. lusitaniae did not occur in the mares that had foaled. However, since the yeasts were absent from the vaginal mucosa of the mares with conception failure and barren mares, they should not be associated with reproductive disorders. The most prevalent yeast, irrespective of reproductive performance, was C. guiliermondii. The growth of the yeasts isolated from mares\u2019 vaginas, compared to that of all the isolated species, was insignificant in all the groups of horses under study and ranged from 8 to 21\u00a0%. In mares that exhibited conception failure, it was almost threefold higher (21\u00a0%) than in the high-quality mares (8\u00a0%). C. tropicalis may be regarded as opportunistic flora in mares, as it was isolated from the vaginal mucosa of the mares that had foaled. T. ciferrii occurring on the skin and mucous membranes of the English full blood horses is an opportunistic rather than invasive fungus. The results obtained suggest that the yeasts isolated may constitute the normal microflora in horses\u2019 organisms, although their contribution to reproductive disorders cannot be excluded clearly.The present paper presents pioneer study results. Representatives of five microflora species of the genus"} +{"text": "L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-\u03b3 dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after Leishmania major. In this model, some strains (e.g. C57BL/6) are resistant due to a Th1 immune response promoting parasite killing. Conversely, other strains (e.g. BALB/c) are susceptible due to a nonprotective Th2 response. DCs are professional antigen-presenting cells that educate antigen-specific T cells. Improving the migration of DCs from the site of infection to the lymph nodes, where T cells reside, may improve the T cell response. JAM-C is a vascular adhesion molecule implicated in leukocyte migration in different inflammatory models. We found that JAM-C blockade with antibodies increases vascular permeability and consequently improves the migration of DCs to sites of infection and draining lymph nodes. This increased leukocyte migration boosted the induction of the Th1 response in resistant mice, while in susceptible mice the Th2 response was augmented. This led to disease improvement or exacerbation, respectively. Our results illustrate the key role of a vascular adhesion molecule in controlling leukocyte migration and the subsequent immune events in response to pathogen infections.Leishmaniasis is a parasitic disease transmitted to humans through sand fly bites. Clinical symptoms vary from self-healing cutaneous lesions to death. Cutaneous leishmaniasis is particularly studied in mice inoculated with Leishmania is an obligate intracellular parasite responsible for a wide spectrum of clinical manifestations, such as cutaneous, mucocutaneous or visceral leishmaniasis Leishmania major in the skin of humans or rodents, promastigotes are taken up by phagocytic cells L. major infection is associated with the production of IFN-\u03b3 by CD4+ Th1 lymphocytes L. major. At early stages of infection in C57BL/6 mice, resident dermal DCs phagocytose the parasites L. major.Dendritic cells (DCs) are professional antigen-presenting cells that play a key role in the induction of the adaptive immune reaction against M\u03b22 (Mac-1) and \u03b14\u03b21 (VLA-4), respectively Transendothelial migration of leukocytes from blood to the site of inflammation is a complex process controlled by adhesion molecules, such as PECAM-1, ICAM-2, ICAM-1, CD99, ESAM, or junctional adhesion molecules (JAMs) M\u03b22, an integrin found on neutrophils and monocytes, therefore increasing their adhesion on endothelial cells We previously described a monoclonal antibody raised against mouse JAM-C, namely H33 L. major infection. We first observed that JAM-C expression by vascular endothelial cells is down regulated after infection with L. major at a time window when inflamed endothelium modulates and redistributes its network of junctional proteins for leukocyte transmigration L. major infection in vivo increased vascular permeability and promoted leukocyte recruitment to the inflamed tissue, and DC migration to the draining lymph node. More importantly, sustained JAM-C blockade boosted the immune response in both resistant C57Bl/6 and susceptible BALB/c mice. On one hand, H33 treatment improved the IFN-\u03b3-dominated Th1 response in resistant animals, together with decreased lesion size and parasite burden. On the other hand, JAM-C blockade boosted the IL-4-dominated Th2 response in susceptible mice, resulting in disease exacerbation. Collectively, our results show that JAM-C blockade potentiates the immune responses to pathogen infections by improving leukocyte migration.In this report, we studied the involvement of JAM-C in leukocyte trafficking and the subsequent immune response against \u2212 CD31+ gp38\u2212) and LECs (CD45\u2212 CD31+ gp38+) were JAM-C positive as previously described for other organs L. major inoculation were all JAM-C negative and lymphatic endothelial cells (LECs) from the skin of mouse ears were analyzed by flow cytometry. In the steady state, BECs . In C57BL/6 mice, we found smaller lesions in H33-treated compared to control animals at both time points were used. In all experiments, C57BL/6 mice were infected in the ear dermis with 2\u00d7106 stationary phase L. major promastigotes in a volume of 10 \u00b5L. The disease outcome in BALB/c was followed after infection with 2\u00d7106 and 1\u00d7104 stationary phase L. major promastigotes in a volume of 10 \u00b5L.Female C57BL/6J and BALB/c mice were purchased from Charles River . Mice were bred in the P2 animal facility at the CMU, and used between 6\u20138 weeks of age. The ventral and dorsal sheets of mouse ears were split with forceps, and digested with 3 mg/mL collagenase type IV (Invitrogen) and 1 mg/mL DNAse type I (Sigma Aldrich) for 45 minutes at 37\u00b0C, filtered through a 70 \u00b5m gauge strainer (Becton Dickinson), and the cells labelled for FACS analysis. Fc receptors were blocked with the monoclonal antibody (mAb) 2.4G2 (Becton Dickinson). Cells were stained with the following reagents: Alexa Fluor 488-conjugated anti-mouse podoplanin (clone 8.1.1), PE-conjugated anti-mouse CD31 (clone 390), PE-Cy7-conjugated anti-mouse CD45 (clone 30-F11), all from affimetrix eBioscience. JAM-C was labelled with an affinity purified polyclonal anti-mouse JAM-C antibody raised in rabbit L. major in the ear dermis. Twenty-four hours post infection, mice were sacrificed and ears explanted. The ventral and dorsal sheets of the ears were separated with forceps, and transferred overnight in twelve well plates filled with RPMI-1640 medium supplemented with 10% fetal calf serum and antibiotics at 37\u00b0C. Over this period of time, the leukocytes that have been recruited to the infected ears spontaneously emigrated from the explants. Emigrated cells were then counted with a hemocytometer, and stained for FACS analysis. Fc receptors were blocked with the mAb 2.4G2. Cells were stained with the following reagents: Alexa Fluor 488-conjugated anti-mouse Ly6C , PercP-Cy5.5-conjugated anti-mouse Ly6G , PE-Cy7-conjugated anti-mouse CD11b , APC-Cy7-conjugated anti-mouse CD11c , and efluor 450-conjugated anti-mouse IA/IE . Cells were analyzed with a Gallios FACS machine (Beckman Coulter) and data processed with Kaluza software (Beckman Coulters). The number of cells per population was calculated by multiplying the total number of emigrating cells with the percentage of cells of interest.Mice were injected i.p. with the rat IgG2a anti-mouse JAM-C H33 or the rat IgG2a isotype control 2A3 (BioXCell), 200 \u00b5g/mice, 2 hours before inoculation of \u03b2 , efluor 450-conjugated anti-mouse CD11b , Brilliant Violet 785-conjugated anti-mouse CD8\u03b1 . Blood cells were stained with the following reagents: Alexa Fluor 488-conjugated anti-mouse Ly6C , PE-conjugated anti-mouse CD115 (clone AFS98), PercP-Cy5.5-conjugated anti-mouse Ly6G , PE-Cy7-conjugated anti-mouse CD4 , APC-conjugated anti-mouse NK1.1 , APC-Cy7-conjugated anti-mouse CD19 , efluor 450-conjugated anti-mouse CD11b , Brilliant Violet 785-conjugated anti-mouse CD8\u03b1 . Cells were analyzed with a Gallios FACS machine (Beckman Coulter) and the data were processed with Kaluza software (Beckman Coulter). The number of cells per population was calculated by multiplying the total number of cells with the percentage of cells of interest. The total number of cells was calculated using the number of Trucount beads analyzed by the flow cytometer.Mice were injected i.p. with the mAb H33 or the control mAb 2A3 (200 \u00b5g/mice). Mice were then sacrificed 24 hours after treatment to collect ears, blood and femurs. Ears were processed as described above. Femurs were flushed to extract bone marrow cells. Red blood cells from blood and bone marrow samples were lysed with Ammonium-Chloride-Potassium (ACK) lysis buffer. A fraction of each sample was used for FACS staining using BD Trucount tubes according to the manufacturer's instructions. Fc receptors were blocked with the mAb 2.4G2. Bone marrow cells were stained with the following reagents: Alexa Fluor 488-conjugated anti-mouse Ly6C , PE-conjugated anti-mouse CD115 , PercP-Cy5.5-conjugated anti-mouse Ly6G , PE-Cy7-conjugated anti-mouse F4/80 , APC-conjugated anti-mouse CD11c , APC-Cy7-conjugated anti-mouse TCR+ migratory DCs was calculated by multiplicating the total number of lymph node cells with the percentage of IA/IEhigh CD11c+ FITC+ DCs.Mice were injected i.p. with the mAb H33 or the control mAb 2A3 (200 \u00b5g/mice) 2 hours before FITC painting of mice ears. FITC (Sigma) was used at 5 mg/mL and dissolved in aceton: dibutyl phthalate . Twenty microliters were applied to each side of the ear. Eighteen hours after painting, the ear draining lymph node was harvested and digested with 3 mg/mL collagenase type IV (Invitrogen) and 1 mg/mL DNAse type I (Sigma) for 45\u2032 at 37\u00b0C, and filtered through a 70 \u00b5m gauge strainer (Becton Dickinson). The cells were counted with a hemocytometer, and labelled for FACS analysis. Fc receptors were blocked with the mAb 2.4G2. Cells were stained with the following reagents: APC-Cy7-conjugated anti-mouse CD11c, and efluor 450-conjugated anti-mouse IA/IE. Cells were analyzed with a Gallios FACS machine (Beckman Coulters) and data processed with Kaluza software (Beckman Coulters). The number of FITCMice were injected i.p. with the mAb H33 or the control mAb 2A3 (200 \u00b5g/mice). Twenty-four hours after injection, ears were embedded in Tissue-Tek OCK compound, frozen at \u221280\u00b0C, then cut (5 \u00b5m) with a cryostat. Fresh ear sections were fixed in cold acetone for 5 minutes, rehydrated in PBS for 10 minutes, and blocked with 10% normal donkey serum. CD31 was detected with an Alexa Fluor 647-conjugated rat anti mouse CD31 , while JAM-C was detected with a polyclonal anti-mouse JAM-C antibody raised in rabbit L. major inoculated i.d. in the ear. Five hours after infection, mice were killed, and the permeability of Evans blue in the ear documented by picturing each ear. Ears were then cut, weighted, split into dorsal and ventral sheets, and finally transferred into formamide for 2 days at room temperature to extract the Evans blue dye. The absorbance of the samples was measured at 620 nm and normalized to the weight of tissue.Mice were treated i.p. with the mAb H33 or the control mAb 2A3 (200 \u00b5g/mice) 2 hours before 100 \u00b5L of Evans blue (12 mg/mL) was injected i.v. and L. major inoculation in the ear dermis. Eight or 24 hours after infection, ears were homogenized on ice in a protease inhibitor cocktail using a polytron as tissue homogenizer. The expression of the chemokine CCL3 were measured in tissue homogenates with the BD CBA mouse Flex Set kit according to the manufacturer instructions. Beads were analyzed on a Cyan (Beckman Coulters) flow cytometer and data processed with the FCAP array software (Becton Dickinson).Mice were injected i.p. with H33 or the control mAb 2A3 (200 \u00b5g/mice) 2 hours before 2 for 72 hours in the presence of UV-irradiated L. major . Supernatant were collected and the levels of IL-4 and IFN-\u03b3 were measured by ELISA (eBioscience) or CBA (Becton Dickinson) according to the manufacturer instructions.The ear draining lymph nodes were digested with 3 mg/mL collagenase type IV (Invitrogen) and 1 mg/mL DNAse type I (Sigma) for 45\u2032 at 37\u00b0C, and filtered through a 70 \u00b5m gauge strainer (Becton Dickinson). The cells were counted with a hemacytometer and labelled for FACS analysis. Fc receptors were blocked with the mAb 2.4G2. Cells were stained for cell surface antigens with the following reagents: FITC-conjugated anti-mouse TCR\u03b2 , Brilliant Violet 421-conjugated anti-mouse CD8\u03b1 , Brilliant Violet 785-conjugated anti-mouse CD4 . Cells were analyzed with a Gallios FACS machine (Beckman Coulters) and the data were processed with Kaluza software (Beckman Coulters). The number of cells per population was calculated by multiplying the total number of lymph nodes cells with the percentage of cells of interest. For T cell restimulation, draining lymph nodes cells were incubated at 37\u00b0C under 5% COL. major in the ear dermis. Injections of mAbs (100 \u00b5g/mice) were repeated twice a week for twenty-one days. The evolution of the lesion was documented weekly with a picture of each ear, as well as the picture of a 1 cm scale. The camera was fixed on a support for the scale to be unchanged from one picture to the other. The pictures were analyzed with ImageJ software. Briefly, the picture of the 1 cm scale provides the number of pixels per 1 cm unit. Each lesion was then defined manually with the software, and the precise lesion area calculated using the number of pixels in the selected area. For parasite burden, the infected ears were explanted, weighted, and separated into two halves. Ear leaflets were enzymatically digested before tissue dissociation with a gentleMACS Octo Dissociator (Miltenyi Biotech). Ears homogenates, lymph nodes or spleens cells were serially diluted, and the parasite load estimated by limiting dilution assay as described Mice were injected i.p. with the mAb H33 or the control mAb 2A3 (200 \u00b5g/mice) 2 hours before inoculation of Data were analyzed with the GraphPad Prism statistics software. We used the Student's t-test for unpaired data for all experiments.Figure S1L. major infection do not express JAM-C.Leukocytes emigrating to the site of The expression of JAM-C by leukocytes emigrated from L. major infected ears was measured 24 hours post infection. CD11b+ Ly6C+ Ly6G+ represent neutrophils, CD11b+ Ly6C+ Ly6G\u2212 CD11c\u2212 IA\u2212 are monocytes, CD11b+ Ly6C+ Ly6G\u2212 CD11c+ IA+ are mo-DCs, CD11b+ Ly6C\u2212 Ly6G\u2212 CD11clow IA+ are dermal m\u03c6, and CD11b+ Ly6C\u2212 Ly6G\u2212 CD11chigh IA+ are dermal DCs. A representative histogram overlay of JAM-C expression is shown for each population, with JAM-C staining (black line), and isotype control staining (grey line). Data are representative of two separate experiments.(TIFF)Click here for additional data file.Figure S2JAM-C expression in ear endothelial cells does not decrease 24 hours after saline injection (A) JAM-C levels in endothelial cells populations of mouse ear. Ears were enzymatically digested and stained for FACS analysis. CD45\u2212 CD31+ gp38\u2212 cells represent blood endothelial cells (BECs), whereas CD45\u2212 CD31+ gp38+ cells are lymphatic endothelial cells (LECs). For each population a representative histogram overlay is shown with JAM-C in endothelial cells from na\u00efve ears (white filled), JAM-C in endothelial cells from saline injected ears (black filled), and the isotype control staining (grey filled). (B) The MFI of JAM-C in na\u00efve mouse ears (white bars) versus saline injected mouse ears (black bars) was measured in BECs and LECs. The Y-axis scale represents MFI normalized to the mean MFI of na\u00efve ears. Data represent the mean \u00b1 SEM of five mice per group pooled from two separate experiments.(TIFF)Click here for additional data file.Figure S3Control staining for JAM-C in ear endothelial cells. Ear sections were stained for Rabbit IgG control (green), CD31 (red). Nucleus was stained with DAPI (blue). Scale bars, 10 \u00b5m. This supporting information is related to (TIFF)Click here for additional data file.Figure S4Blocking JAM-C does not result in leukocyte emigration to tissue in the steady state. The number of neutrophils (A), monocytes (B), mo-DCs (C), dermal m\u03c6 (D), and dermal DCs (E) emigrating from ears was measured in H33-treated versus isotype control-treated mice 24 hours after antibody administration. Data represent the mean \u00b1 SEM of fifteen mice per group pooled from 3 separate experiments, and were analyzed by the unpaired Student's t test.(TIFF)Click here for additional data file.Figure S5Blocking JAM-C in the steady state does neither increase hematopoiesis nor leukocyte migration from bone marrow to the blood. Na\u00efve C57BL/6 mice were treated with H33 or isotype control antibody for 24 hours. The number of neutrophils (A), monocytes (B), DCs (C), T cells (D), eosinophils (E), and macrophages (F) from the bone marrow (BM); and B cells (G), CD4+ T cells (H), CD8+ T cells (I), neutrophils (J), monocytes (K), and NK cells (L) from blood in H33-treated (black bar) versus isotype control-treated mice (white bars) is shown. Data represent the mean \u00b1 SEM of five mice per group, and were analyzed by the unpaired Student's t test. Data are representative of three separate experiments.(TIFF)Click here for additional data file.Figure S6H33 antibody does neither decrease the parasite burden in infected ears, nor increase parasite dissemination to lymph nodes 48 hours p.i. (Raw data of). The parasite burden in infected ears (A) and draining lymph nodes (B) were measured 48 hours p.i. by LDA. Data represent the mean \u00b1 SEM of five mice per group from one representative experiment, and were analyzed by the unpaired Student's t test. These supporting informations are related to (TIFF)Click here for additional data file.Figure S7Blocking JAM-C increases the number of DCs migrating to the draining lymph node (Raw data of). The ear draining lymph nodes were harvested and stained for FACS analysis 18 hours after FITC-painting. The number of IAhigh CD11c+ FITC+ migratory DCs was counted. Data represent the mean \u00b1 SEM of six mice per group, and were analyzed by the unpaired Student's t test with *: p<0.05. This supporting information is related to (TIFF)Click here for additional data file.Figure S8Blocking JAM-C improves the Th1 cell response and favours healing in C57BL/6 mice (Raw data of). Mice were inoculated with L. major in the ear dermis and treated with H33 or the isotype control antibody for 3 weeks, twice a week. (A) The parasite burden in infected ears was measured by LDA 4 and 8 weeks p.i. Data represent mean \u00b1 SEM of ten mice per group for both time points. (B) Draining lymph node cells were restimulated for 72 hrs with UV-irradiated L. major and the secreted IFN-\u03b3 was measured. Data represent the mean \u00b1 SEM of mice from panel A. Data were analyzed by the unpaired Student's t test with *:p<0.05. These supporting informations are related to (TIFF)Click here for additional data file.Figure S9Blocking JAM-C boosts the Th2 cell response and worsens the disease in BALB/c mice (Raw data of). (A\u2013C) Mice were inoculated with 1\u00d7104 stationary phase L. major promastigotes in the ear dermis and treated with H33 or control antibody for 3 weeks. The parasite burden in infected ears (A), draining lymph nodes (B), and spleens (C) were measured by LDA. (D\u2013E) Draining lymph nodes cells were restimulated with UV-irradiated L. major for 72 hours, and the IL-4 (D) and IFN-\u03b3 (E) produced were measured. Data represent the mean \u00b1 SEM of 5 mice per group. Data were analyzed by the unpaired Student's t test with *:p<0.05, ***: p<0.001. n.d. not-detectable. These supporting informations are related to (TIFF)Click here for additional data file."} +{"text": "TP53 mutations in non-serous was much lower than in serous epithelial OC, whereas the prevalence of PIK3CA, PIK3R1, PTEN, CTNNB1, ARID1A, and KRAS was higher. We confirmed the high prevalence of FOXL2 and KRAS mutations in granulosa cell tumors and in mucinous tumors, respectively. We also identified POLE proofreading domain mutations in three endometrioid ovarian tumors. These results highlight mutational differences between serous and non-serous ovarian cancers, and further distinguish different non-serous subtypes.Epithelial ovarian cancer is a leading cause of death in gynecological cancers. While several systematic studies have revealed the mutation landscape of serous epithelial ovarian cancer, other non-serous subtypes of the disease have not been explored as extensively. Here we conduct exome sequencing of nine non-serous epithelial ovarian tumors (six endometrioid and three mucinous) and their corresponding normal DNA as well as a tumor-only granulosa cell sample. We integrated the exome data with targeted gene sequencing for 1,321 genes selected for their involvement in cancer from additional 28 non-serous ovarian tumors and compared our results to TCGA ovarian serous cystadenocarcinoma and uterine corpus endometrial carcinomas. Prevalence of These subtypes differ in terms of histopathology, morphology, and genomic alterations and are considered distinct diseases, which require careful diagnosis and specific therapies that are critical for successful treatment3. Ovarian granulosa cell tumors do not have epithelial origin but are the most common sex cord-stromal tumor and represent 5% of all ovarian malignancies4.Epithelial Ovarian Cancer (EOC) is a heterogeneous disease with five major histologic types5. Type I tumors are slow growing and encompass low-grade serous, low-grade endometrioid, mucinous and clear cell carcinomas characterized by mutations in KRAS, BRAF, and PIK3CA and by the absence of TP53 mutations6. Type II tumors are highly aggressive and encompass high-grade serous tumors as a typical representative, as well as high-grade endometrioid and undifferentiated carcinomas with disruption of TP537. Type II tumors have expression profiles that cluster separately from type I tumors and are characterized by genomic instability, extremely aggressive clinical progression and poor prognosis11.Based on molecular profiles, disease development and prognosis, the different histological subtypes can be hierarchically grouped into type I and type II22 and somatic alterations in high-grade serous ovarian carcinoma has recently emerged and included genes such as MYC and KRAS (gain) and PTEN, RB1, and NF1 (loss). Many of these larger gains and losses were observed in the majority of tumors, highlighting the genomic instability of high-grade serous ovarian carcinoma. In addition, analyses of complete genomes for sixteen low stage high grade serous EOC revealed frequent TP53 mutations, tetraploidy and homologous recombination repair defects26.A comprehensive view of the germline variation associated with cancer risk28. Most published studies, however, report germline or somatic targeted sequencing of a limited number of genes31, or exome studies with a limited number of samples32 tumors and performed targeted gene sequencing of exons in 1,321 genes for 28 additional tumor samples including the clear-cell subtype. We report mutational and copy number profiles indicating that non-serous ovarian cancer is genomically distinct from serous EOC and displays similarities to non-serous endometrial uterine cancer.Tumors were chosen by interrogating Moffitt tissue bank for cases that fulfilled the following criteria: a) non-serous histology; b) Stage I; and c) available fresh-frozen tissue; d) available matched normal blood or tissue. Whole exome sequencing was performed on 9 tumor/normal matched pairs, and one tumor only referred to as the ES (exome sequencing) cohort Table\u00a0. No caseGOLGA6L1 (q\u2009=\u20090.62), PTEN (q\u2009=\u20090.73), and OR2T33 (q\u2009=\u20090.76). No single confident mutated position was observed in more than three samples, and only PTEN stood out as a gene containing truncating mutations in three samples . Several COSMIC cancer genes were found to have various mutations in multiple samples, including PTEN, PIK3CA, BRCA2, and ATM . Only one sample (OV8) had a TP53 mutation .MutSigCV_1.4 was used to identify significantly mutated genes, but no genes were observed with significant q-value. Only three genes were observed with q-value <1: les Fig.\u00a0. MutatioTP53 mutation was observed. Additional cancer mutations were detected in OV7 (FOXL2 p.C134W) and OV9 (KRAS p.G12V) in our dataset, no mutations were observed, suggesting it may not be a common driver of mucinous tumors. Interestingly, for sample OV10, the KRAS variant was only called in the tumor/normal analysis Fig.\u00a0. We obsesis Fig.\u00a0 but not sis Fig.\u00a0. The varPIK3CA, a missense variant in ARID1A (p.Ala532Thr) observed in only 2/25 tumor and 0/100 normal reads, and an NF1 large insertion that introduces a stopgain. Sanger sequencing did not confirm those changes suggesting that they may have been incorrectly called or that only a small percentage of cells in the tumor carry that variant.GATK-based analysis of OV10 also detected a large insertion in 34. Only one clearly pathogenic variant, BRCA2:c.6944_6947delTAAA (p.Ile2315Lysfs) (OV8) variant and few variants of uncertain clinical significance were identified tumors. TP53 mutations were prevalent in endometrioid (6/14) and mucinous (3/4) subtypes but less frequent in clear cell (1/5) and absent in granulosa cell (0/5). We also observed the presence of FOXL2 p.C134W mutation in 4/5 (80%) of the additional granulosa cell samples. Although other FOXL2 mutations were observed in different samples, the p.C134W driver mutation was only observed in the granulosa cell subtype. KRAS mutations were observed in 3/4 (75%) of additional mucinous samples and 3/14 (21%) of additional endometrioid samples. ARID1A and CTNNB1 were mutated more frequently in endometrioid tumors , further suggesting these are important contributors to that tumor type31. ARID1A truncating mutations were also recurrently observed in 2/5 (40%) clear cell tumors.We found 11 unique 8 and endometrial cancers36 at genes known to be mutated in these diseases and of few other cancer genes. Serous endometrial also has a high TP53 mutation rate of 74%, as well as PIK3CA (36%), FBXW7 (26%), and PPP2R1A (23%). TP53 mutation rates were much lower in our non-serous OC samples: 10% in ES, and 36% in our TGS cohort. PIK3CA mutation rates were, on average, higher in our non-serous OC samples compared to serous cancers. We observed higher mutation rates in each of the following genes in non-serous ovarian samples compared to serous ovarian and serous endometrial: PTEN, PIK3R1, CTNNB1, ARID1A, and KRAS. Many of these genes are frequently mutated in non-ultramutated endometrioid uterine cancer and another p.V411L (TG10).One endometrioid sample (OV4) was observed to have very high mutation count Fig.\u00a0 Supplem. Furtheri.e. the mutational signatures, in a tumor can suggest mechanisms that may have contributed to tumorigenesis. All three endometrioid ovarian tumors containing POLE proofreading domain mutations consisted almost entirely of the previously reported POLE mutation signature (http://cancer.sanger.ac.uk/cosmic/signatures)38 . In ES samples, we observed signature 1b \u201cAge\u201d (likely age-related cytosine-deamination) in all samples and signature 18, which has unknown etiology, as the predominant signature in two samples of different histologies (OV10: mucinous and OV6: endometrioid) 38 Fig.\u00a0. This siid) Fig.\u00a0.Figure 339 were identified in the CNA regions.Tumor specific copy number alterations (CNA) were examined in the ES dataset. A median of 25 events was observed in each sample (range 5\u2013192) covering a median of 76,303,334 bases had the most CNAs. This tumor had the third lowest number of mutations with no identified common cancer mutations had a TP53 mutation and no samples had deletions in the region. We examined the fraction of bases with depth of coverage\u2009\u2265\u200910 across the TP53 gene, and found an average of 73.7% per sample, suggesting our coverage was sufficient to have observed mutations. In the TGS cohort, mutations were prevalent in mucinous , endometrioid and clear cell subtypes but still markedly lower than high grade serous. This is line with previous studies of clear cell carcinomas reporting 10\u201315% TP53 mutation frequency40. Interestingly, comparative studies of TP53 mutations in uterine cancer also reports widely different mutation frequencies between the endometrioid (11.4\u201317%) and serous carcinomas (>90%)41.First, non-serous ovarian tumors seem to be distinct from serous subtypes in their prevalence of POLE, and mutations are not found in other non-serous subtypes. One ES sample harbored a POLE mutation in the proofreading domain, and showed a mutational signature matching a previously reported signature of POLE deficiency38. In the TGS set we observed recurrent POLE mutations in 14% (2/14) of endometrioid ovarian cancers. A screen for POLE mutations in a study of 251 Chinese samples of different subtypes of ovarian carcinomas, identified mutations in 3 out of 37 (8.1%) patients with ovarian endometrioid carcinoma, but not observed in the other subtypes42. Interestingly, POLE hotspot mutations were found in 7% (17/248) of uterine carcinomas, all of endometrioid histology36.Second, a small but significant fraction of endometrioid tumors display mutations in the proofreading domain of FOXL2 in adult granulosa cell tumors. This is consistent with existing reports showing a prevalence ranging from 70.4% to 100%50.Third, we confirmed the high prevalence of pathognomonic mutations in KRAS mutations are observed in significant fraction of mucinous and, to a lesser extent, in endometrioid but much less frequently in high grade serous ovarian tumors8. A total of 71% (5/7) of mucinous samples carried mutations in KRAS, confirming its high frequency in this subtype53. We also observed 20% (4/20) of endometrioid tumors with mutations in KRAS. Mutations in PIK3CA and ARID1A are the most frequent alteration in clear cell carcinomas found in combination in 2/5 (40%) of all CCC tumors in the present study. Sequencing of 97 OCCC tumors identified a high frequency of mutations in PIK3CA (33%) and in TP53 (15%)9.Fourth, different non-serous subtypes have distinct mutation profiles. 31. Our findings also offer molecular evidence to support continued distinction of non-serous ovarian cancers by histology, and may offer the opportunity for future work to investigate subtype-specific interventions targeting unique molecular alterations.Finally, the finding that deletion in genes implicated in the DNA damage response are relatively common suggest that therapeutic approaches targeting these pathways, such as the use of PARP inhibitors may be provide effective options in treatment. In summary, our results show that non-serous ovarian tumors are mutationally distinct from serous ovarian tumors. Interestingly, a similar but not identical mutational profile can be found in endometrioid ovarian and endometrioid uterine tumors. These similarities support the link between endometrioid ovarian cancers and endometriosisFor the Exome Sequencing (ES) cohort all subjects were women between the ages of 28 and 63 median 51) from Moffitt Cancer Center. Tumor and matched normal samples were retrieved from Moffitt\u2019s Total Cancer Care tissue bank. Tumor samples for sequencing were selected to have >80% tumor cellularity. Six endometrioid, three mucinous, and one granulosa cell ovarian tumors were sequenced (Table\u00a0 from MofFor the Target Gene Sequencing (TGS) cohort all samples were from the Total Cancer Care tissue bank. Patients were women between the ages of 30 and 79 (median 60) diagnosed in 1993\u20132012. Tumors underwent macrodissection to achieve >80% tumor cellularity in subsequent molecular studies. Samples were grouped by subtype: fourteen endometrioid, four mucinous, five clear cell, and five granulosa cell tumors were target sequenced . DNA from blood was isolated using Qiagen Gentra Puregene Blood Kit or Qiagen PAXgene blood DNA kit.54. The Genome Analysis ToolKit (GATK)55 was used for insertion/deletion realignment, and quality score recalibration.Paired-end libraries were constructed and whole exome capture was performed using Roche NimbleGen SeqCap EZ v3 (Roche). Captured libraries were sequenced using 100 base-pair reads on an Illumina HiScanSQ platform following the manufacturer\u2019s protocols. Reads were aligned to the human genome reference hs37d5 using the Burrows-Wheeler Aligner (BWA)56 or MuTect57. Strelka 1.0.13 was run with default settings except the following: ssnvNoise\u2009=\u20090.00000005, sindelNoise\u2009=\u20090.0000001. MuTect 1.1.4 was run with default settings except: max_alt_alleles_in_normal_count\u2009=\u20093, max_alt_allele_in_normal_fraction\u2009=\u20090.05. ANNOVAR58 was used to annotate variants. Final read depth averaged 39.2\u00d7, and a median of ~87% of bases across samples had \u226510 reads. Coverage in each sample across genes of interest can be seen in Supplementary Figure\u00a059. Circos was used for visualization of somatic alterations60. Blood was unavailable for case OV7, and although plasma was subjected to exome capture, sequencing coverage was low (0.2% target bases having \u226510 reads) and duplicate rates were high (87%) preventing sensitive tumor/normal mutation detection. Case OV7 was analyzed with GATK, and mutations were enriched by filtering out known variants.Somatic mutations were detected with either Strelka63: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS1, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, and TP53.We also examined normal DNA to identify germline mutations implicated in ovarian cancer in breast/ovarian cancer families or in Lynch syndromeTarget gene capture was performed using SureSelect custom designs targeting 1,321 genes 55 was used for insertion/deletion realignment, quality score recalibration, and variant identification. ANNOVAR58 was used to annotate mutations. Matched normal samples were not available for comparison so somatic mutations were enriched via population filtering, including 1000 Genomes and 238 unmatched normal samples. Final read depth averaged ~140x across the targeted regions and a median of ~94% of bases across samples had \u226510 reads 65 using the \u201cOvarian Serous Cystadenocarcinoma8 and \u201cUterine Corpus Endometrial Carcinoma36.Mutation frequencies were downloaded from cBioportal38. Curated and updated ICGC signatures can be found at http://cancer.sanger.ac.uk/cosmic/signatures.Mutational signatures were derived from the ES samples using previously described methodsSupplementary DataSupplementary Tables 2\u201311"} +{"text": "Leishmania infantum might be affected by other vector-borne organisms that mimic its clinical signs and clinicopathological abnormalities. The aim of this study was to determine co-infections with other vector-borne pathogens based on serological and molecular techniques in dogs with clinical leishmaniosis living in Spain and to associate them with clinical signs and clinicopathological abnormalities as well as disease severity.The severity of canine leishmaniosis (CanL) due to Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum and Bartonella henselae antigens by the immunofluorescence antibody test (IFAT) and for E. canis, Anaplasma spp., Hepatozoon spp., Babesia spp. and filarioid DNA by polymerase chain reaction (PCR).Sixty-one dogs with clinical leishmaniosis and 16 apparently healthy dogs were tested for R. conorii, 57% for E. canis, 44% for A. phagocytophilum and 37% for B. henselae; while the prevalences found by PCR were: 8% for Ehrlichia/Anaplasma, 3% for Anaplasma platys and 1% for H. canis. No other pathogen DNA was detected. Statistical association was found between dogs with clinical leishmaniosis and seroreactivity to R. conorii antigen and A. phagocytophilum antigen and being positive to more than one serological or molecular tests (co-infections) when compared with healthy dogs. Interestingly, a statistical association was found between the presence of R. conorii, E. canis, A. phagocytophilum and B. henselae antibodies in sick dogs and some clinicopathological abnormalities such as albumin and albumin/globulin ratio decrease and increase in serum globulins. Furthermore, seroreactivity with A. phagocytophilum antigens was statistically associated with CanL clinical stages III and IV.Among the dogs examined by IFAT, the seroprevalences were: 69% for This study demonstrates that dogs with clinical leishmaniosis from Catalonia (Spain) have a higher rate of co-infections with other vector-borne pathogens when compared with healthy controls. Furthermore, positivity to some vector-borne pathogens was associated with more marked clinicopathological abnormalities as well as disease severity with CanL. Leishmania infantum endemic in the Mediterranean basin. Phlebotomus spp. sand flies are the only vector adapted for the biological transmission of L. infantum in Europe . When comparing our results to those of these studies, we found a high increase of seropositivity rates to other pathogens when studying dogs with clinical leishmaniosis. For example, the seroprevalence found for , 10, 12.A. platys and H. canis were only confirmed by PCR in dogs from the Tarragona area. In the Mediterranean basin, where mosquitoes and R. sanguineus (s.l.) ticks are common, it would be expected that the pathogens related to this tick species would be also prevalent [E. canis, Hepatozoon spp. Babesia spp. and Dirofilaria spp. are circulating abundantly in those countries while, the results suggest that they are less common in Catalonia.Combining the serological and molecular results of the present study with the findings from previous literature, it is noteworthy to remark that co-infections patterns are different in several geographical regions where the dogs with leishmaniosis live and there is variability in their life style, exposure to ticks and fleas, the species of ectoparasites present in the area, and also on the preventative measures applied against ticks and fleas. For example, in the present study, revalent , 24, 60.revalent , Greece revalent , 63, Correvalent , Cyprus revalent , Tunisiarevalent and Israrevalent , it is eRickettsia spp. such as R. conorii due to the low rickettsiaemia usually found in dogs [Bartonella was also not performed. These bacteria are often cultured with an enrichment medium for insect cell culture growth (BAPGM) prior to PCR testing to increase the likelihood of detecting this species [PCR is a technique that detects pathogen DNA and can, therefore, confirm infection although a negative result does not completely exclude it. Serological techniques, such as ELISA and IFAT, on the other hand, detect antibodies formed due to current infection or past exposure to the pathogen studied. Quantitative serology may be used to detect seroconversion, but seropositivity may also result from cross-reaction with antibodies formed against other organisms with similar antigens. PCR also allows identification of the pathogen. Due to the aforementioned characteristics, it is recommended to use both techniques for diagnosis of some infectious diseases , 68, 69. in dogs , 19, 71. species .R. conorii in dogs might be due to infection with other spotted fever group (SFG) Rickettsia spp. such as R. massiliae, R. slovaca or R. aeschlimannii, which are common in ticks in the Mediterranean basin countries [A. phagocytophilum and A. platys is common, due to their antigenic similarity [A. phagocytophilum is usually transmitted by I. ricinus ticks while A. platys is suspected to be transmitted by R. sanguineus (s.l.) [R. sanguineus (s.l.) [A. platys and not A. phagocytophilum. Similarly, E. canis can also have some degree of cross-reactivity with Anaplasma spp. [E. canis and A. phagocytophilum, without positive PCR and sequencing. It could be suggested that these dogs were exposed only to one of the two vector-borne pathogens detected and they could have been infected by A. platys, the only Anaplasmataceae species detected by PCR. In addition, other species of Bartonella apart from B. henseale such as Bartonella vinsonii berkhoffii are associated with clinical illness in dogs. Therefore, the present study might have detected Bartonella seroreactivity related to infection with these other Bartonella species [The different cross-reactions that could have occurred in this study should also be considered. It has been reported that positive reaction found in serological tests for ountries , 73, 74.milarity , 75, 76.s (s.l.) \u201317. Takis (s.l.) , it can sma spp. , 79. In species .R. conorii infection was significantly associated with older age. However, a recent study [Leishmania and Babesia spp. and Mir\u00f3 et al. [E. canis and Borrelia burgdorferi compared to dogs older than one year. Further studies are needed to understand the relationships between age and different vector-borne diseases, taking into account other factors such as lifestyle and location.Another finding of the present study was the detection of a higher number of pathogens by IFAT and PCR in older dogs compared to young dogs. It is reasonable that older dogs would have more time and opportunity to be exposed to the different pathogens studied, although young dogs could be more susceptible to infections due to the immaturity of the immune system \u201384. In ant study found th\u00f3 et al. found thA. phagocytophilum antigen showed an association between seropositivity and season, in this case spring or winter. The vector for A. phagocytophilum present in Spain is the I. ricinus tick [A. phagocytophilum were infested with these ticks and a subsequent infection occurred. However, I. ricinus is usually not found in the Mediterranean area [A. phagocytophilum are likely to have been formed against A. platys, for which the tick R. sanguineus (s.l) is suspected as its main vector. Different studies [R. sanguineus (s.l.) is in summer, this tick can infest dogs during all seasons [A. platys is known to cause subclinical infections [E. canis [When studying vector-borne pathogens, it is also expected to find a relationship between the time of infection detection and the season when the vector is more active. In this study, only the results of IFAT for nus tick \u201317, whicnus tick . When evean area , 77, 86 ean area , 77. Con studies , 87, 88 seasons , 87. Furfections , 55, 89 E. canis with theE. canis , 4, 6.R. conorii, E. canis, A. phagocytophilum and B. henselae antigens was associated with more pronounced clinicopathological abnormalities when compared with sick dogs that were seronegative to the same individual antigen. Interestingly, only seroreactivity of leishmaniotic dogs to A. phagocytophilum was associated with increased disease severity of clinical leishmaniosis.This study demonstrates that dogs with clinical leishmaniosis from the Barcelona and Tarragona areas have a higher rate of co-infections with other vector-borne pathogens when compared with healthy controls. Furthermore, individual seroreactivity to"} +{"text": "Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls.In the Mediterranean basin, L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls.We conducted a prospective case-control study of dogs with ClinL for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or \u201cCandidatus Mycoplasma haematoparvum\u201d DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis.From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR.Dogs with ClinL are at a higher risk of co-infection with Leishmania infantum, is transmitted by a phlebotomine sand fly vector compared to control dogs was found. We did not identify any association for A. platys, Hepatozoon spp. and M. haemocanis between the two groups. There were no statistically significant differences between the ClinL cases and controls in terms of age, sex, breed, lifestyle, and use of ectoparasitic prevention.Using multivariable logistic regression analysis, a significant association between ClinL and E. canis, younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, although only a trend was identified for the latter, and a trend existed for co-infections between Hepatozoon spp. and M. haemocanis to occur. The SEM showed that there was otherwise negligible evidence of determinants of, or correlations among, VBPs.The SEM supported four main associations among variables Fig.\u00a0, Table\u00a02P\u2009=\u20090.022) more likely to be co-infected with E. canis compared to healthy controls. This further supports the concept of synergism between L. infantum and E. canis during co-infection in dogs in which, as previous studies have suggested, there are more commonly clinical signs [In this first comprehensive case-control study assessing the risk of VBP co-infection in dogs with leishmaniosis, our key finding shows that dogs with ClinL are 12 times , more seht loss) , 27\u201329 aht loss) comparedL. infantum and E. canis in dogs has not been investigated. Due to the zoonotic nature of canine leishmaniosis there have been extensive studies on the immunopathology of this disease, and it is the best understood canine VBP [L. infantum infection promotes a mixed Type 1\u00a0T helper (Th1) and Th2 response that will determine the clinical outcome [E. canis infection or facilitate the establishment of a new E. canis infection in dogs. While little is known regarding the immunopathology of canine monocytic ehrlichiosis, there is evidence of downregulation of major histocompatibility complex (MHC) class II molecules in a macrophage cell line infected with E. canis compared with uninfected macrophages [Leishmania infection outcome as MHC class II antigen presentation is likely to be an important mechanism in generating an effective cell mediated response to L. infantum. Furthermore, MHC Class II genotype has been associated with Leishmania specific antibody level and parasite load but not with clinical outcome [The pathogenesis behind the speculated synergetic action of nine VBP . It is w outcome , with in outcome \u201335. The rophages . This do outcome .Leishmania causing a more rapid progression to AIDS [L. infantum and E. canis in dogs, since both microorganisms infect monocytes and macrophages. This hypothetical mechanism is supported by the findings of our clinical case-control study in which an association with ClinL was only found with E. canis co-infection, but not with A. platys, Hepatozoon spp. or M. haemocanis that infect predominantly platelets, neutrophils and erythrocytes, respectively [L. infantum and E. canis in dogs. Therefore, further studies are needed to investigate how the co-infection of these two pathogens potentially affect the dog\u2019s immune response.In humans there is a well-established synergism between leishmaniasis and human immunodeficiency virus (HIV) , with Le to AIDS and HIV to AIDS . The imm to AIDS , 41, 42.ectively \u201345. EquaHepatozoon spp. (46%), with H. canis being the only species identified by sequencing, as well as a reasonably high prevalence for M. haemocanis (23%). Similar prevalences have been reported for Hepatozoon spp. and haemoplasmas in the cat population of this island [E. canis of 5% (7/142), and for A. platys of 4% (5/142) in this canine population, are similar to those reported in dogs from other Mediterranean countries [Although, our study is not a cross-sectional epidemiological research project, and the dog population recruited is heavily biased by the inclusion and exclusion criteria, it does provide information for the prevalence of the various VBP tested in the area of Paphos in Cyprus, especially since 65% (92/142) of the samples we collected were from apparently healthy dogs. In the studied population of 142 dogs there is a noticeably high prevalence of s island , suggestoon spp. %, with HE. canis and allowed us to simultaneously investigate the effects of demographic, lifestyle and breed on VBP infection, and the associations between the different VBP. Two additional findings were made. The first one was that dogs infected with Hepatozoon spp. were more likely to be infected with M. haemocanis and, to the authors\u2019 knowledge, this is the first time such an association has been reported. This is probably due to the fact that both VBP are suspected to have the same vector R. sanguineus, despite their different routes of transmission: host ingestion of the tick for Hepatozoon spp. transmission and a tick bite for M. haemocanis transmission [Hepatozoon spp. and M. haemocanis, which is in agreement with a previous study on dogs infected with canine haemoplasmas from other Mediterranean countries [The use of SEM strengthens the findings of our study by confirming the association found between ClinL and smission , 47. Secountries and coulE. canis and other VBPs.Limitations of our study include selection and observer bias as this is a case-control study, and the geographical restriction of only including one district of Cyprus. Furthermore, the control dogs were recruited on the basis of being clinically healthy, thus they might not be representative of the general canine population. A multicentre prospective longitudinal study design with follow-up monitoring from birth until death would be ideal, but difficult to implement. Even so, the adequate sample size and conclusions which were based on statistical analysis employing different methodologies should allow some generalisation of our findings to other countries with similar environmental conditions and canine VBP prevalence as Paphos, Cyprus. Studies in the future over longer time periods would be beneficial to investigate the possibility of seasonal effects and to determine if the prognosis of leishmaniosis is different when dogs are also co-infected with E. canis infection compared to healthy dogs, could impact upon the diagnostic and monitoring management of canine leishmaniosis. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR on EDTA peripheral blood [E. canis infection but should be interpreted appropriately [E. canis PCR testing on EDTA peripheral blood if there is clinical or haematological deterioration, such as thrombocytopenia, despite the dog receiving the appropriate anti-Leishmania treatment.Our finding, that dogs with ClinL are at increased risk of al blood . Quantitpriately . Whilst E. canis infection, we recommend simultaneous treatment of both infections. For E. canis, the treatment of choice is oral doxycycline at 5\u00a0mg/kg twice daily or 10\u00a0mg/kg once daily for 4\u00a0weeks [E. canis by R. sanguineus and avoid transmission of L. infantum to sand flies.If a dog with ClinL is diagnosed with concurrent 4\u00a0weeks and for 4\u00a0weeks . FurtherE. canis than clinically healthy dogs in Cyprus. These findings are of a value in the diagnosis and management of leishmaniosis in dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR. Further studies should be targeted in investigating the underlying pathology of this association.We showed that dogs with ClinL are 12 times more likely to be co-infected with"} +{"text": "Tongue cancer is still one of the leading causes of mortality around the world. Recently, the ubiquitin system has been established as a critical modulator of tumors. In order to find the oral cancer related E3 ubiquitin ligases, we screened the human E3 ubiquitin ligase library and found that RING finger protein 139 (RNF139) regulated the biological behavior of tongue cancer cells.MTT assay was used to analyze the cell viability changes of tongue cancer SCC9 and SCC25 cells caused by RNF139. The invasion ability of SCC9 and SCC25 cells with or without the knockdown of RNF139 was evaluated through transwell assay. The immunoblotting was recruited to determine the expression level of RNF139 in human tongue cancer tissues and para-carcinoma tissues. The effect of RNF139 on tumorigenicity of tongue cancer cells was analyzed by xenograft model on immunodeficient Balb/c nude mice.Overexpression of RNF139 inhibits the viability of tongue cancer cells since day 2. The colony formation ability of SCC9 and SCC25 cells was also decreased with the overexpression of RNF139. Knockdown of RNF139 significantly promoted the invasion ability of SCC9 and SCC25 cells. Furthermore, knockdown of RNF139 also induced the activation of AKT signaling pathway. While human tongue cancer tissues had low expression of RNF139. In nude mice, knockdown of RNF139 promoted the tumorigenicity of the SCC25 cells.Our data establish a role for RNF139 in regulating the progression of tongue cancer. Tongue cancer is one of the leading cancers in prevalence and around 16,400 new American cases are estimated in 2017 [Ubiquitination is one of the post-translational modification which involves in several cellular activity, including gene transcription, cell-cycle control, DNA repair and protein degradation \u20135. It isIn order to find the oral cancer related E3 ubiquitin ligases, we screened the human E3 ubiquitin ligase library and founLipofectamine 2000 (Life Technologies), 3--2,5-diphenyltetrazolium bromide (MTT) (Antgene), mouse antibodies against \u03b2-actin (Sigma) and RNF139 (Santa Cruz); rabbit polyclonal antibodies against AKT (CST), phospho-AKT (308 and 473) (CST), phospho-FoxO1 (CST), phospho-GSK3\u03b2 (CST), and phospho-mTOR (CST) were purchased from the indicated manufacturers. SCC9 (CRL 1629\u2122), SCC25 (CRL 1628\u2122) and HEK293 (CRL 1573\u2122) cells were obtained from ATCC.Human tongue cancer SCC9 and SCC25 cells were maintained in DMEM medium with the supplement of 10% FBS and 1% Penicillin-Streptomycin. Mammalian expression plasmids for the RNF139 and RNF139-RNAi were constructed according to the instructions of molecular cloning: a laboratory manual.The protocol was the same with our previous report . In brie3) were seeded on the 96 well plates. MTT (5\u00a0\u03bcg/ml) was used and incubated at 37\u00a0\u00b0C for 4\u00a0h. Then the cells were incubated with Dimethyl sulfoxide (DMSO) for 30\u00a0min. The cell viability was determined by microplate reader at day 2, 4 and 6.The stable RNF139 overexpression/knockdown SCC9 and SCC25 cells . Protein were seperated with the polyacrylamide gel electrophoresis and transferred into polyvinylidene membrane. The membrane was immunoblotted with the correspondent antibodies and developed with the ECL reagent.7) cells were injected subcutaneously into the flank. Tumor diameters were recorded every 4\u00a0days. Specimens were harvested at 40\u00a0days.Eight-week-old male athymic immunodeficient Balb/c nude mice were purchased from Shanghai Laboratory Animal Center. The stable RNF139-knockdown SCC25 .P value <0.05.The tumor volume of mice xenograft was analyzed by two-way ANOVA. One-way ANOVA was used to analyze the results of cell viability assay, cell invasion assays and colony formation assays. All data were analyzed by the SPSS package for Windows . The statistically significant refers to the In order to elaborate the role of RNF139 in regulating the biological behavior of tongue cancer, we analyzed the viability changes of tongue cancer cells, SCC9 and SCC25 cells, induced by RNF139. The results suggested that overexpression of RNF139 significantly inhibited the viability of SCC9 and SCC25 cells Fig. . While kWe next investigated the functions of RNF139 on cell invasion. The transwell assay was used to analyze the invasion changes of tongue cancer cells. Knockdown of RNF139 significantly promoted the invasion ability of SCC9 and SCC25 cells Fig. . Taken tTo analyze the role of RNF139 in human lung cancers, we detected the protein level of RNF139 in 23 tongue cancer patients\u2019 tumor tissues through western blotting. Most of these patients were 51\u201370\u00a0years old. Squamous cell carcinoma accounted for 91.30% (21 out of 23). The protein level of RNF139 was significantly decreased in the tongue cancer tissues in comparison to para-carcinoma tissues Fig. &b.Fig. 2After determining the correlation between RNF139 and tongue cancer, we further analyze the regulating mechanism of RNF139 on tongue cancer. We screened the expression changes of critical components in PI3K-AKT, JAK-STAT, p53 and MAPK signaling pathway. The AKT signaling pathway had evident changes with the knockdown of RNF139. As shown in Fig. Next, we performed in vivo xenograft analysis by subcutaneously injecting stable RNF139 knockdown SCC25 cells into nude mice to further analyze the function of RNF139. As shown in Fig. Despite advances made in detection and diagnosis as well as treatments in the past ten years, tongue cancer is still one of the leading causes of mortality around the world . The bodUbiquitin is a widely existed protein which can be found in all eukaryotic cells. It consists of 76 amino acids with an exposed C-terminal. The isopeptide bond connection between the carboxyl-terminal glycine residue of ubiquitin and an internal K residue or the amino-terminal methionine (M1) of another ubiquitin forms the polyubiquitin chains. Ubiquitination is indispensable for several biological processes. Several E3 ubiquitin ligases have been confirmed to be related with initiation and progression of tumor , 13.E3 ubiquitin ligases mainly include RING and HECT type E3 ubiquitin ligases. The multimembrane-spanning protein RNF139 belongs to the RING type E3 ubiquitin ligases and has a RING-H2 domain with E3 ubiquitin ligase activity at the COOH-terminal. It locates in the endoplasmic reticulum (ER) and transfers the ubiquitin from the E2 ubiquitin conjugating enzymes to substrate. Previous study suggested that RNF139 could utilize several E2 ubiquitin conjugating enzymes for ubiquitylation. RNF139 ubiquitinated and degraded the antioxidant enzyme heme oxygenase-1 (HO-1) and suppressed HO-1-induced cancer cell growth, migration and invasion . In thisOur data clearly establish a correlation between RNF139 and tongue cancer. Of clinical relevance is the fact that our results contribute to the new molecule treatment targets for tongue cancer."} +{"text": "Tight regulation of inflammation is very important to guarantee a balanced immune response without developing chronic inflammation. One of the major mediators of the resolution of inflammation is the transcription factor: the nuclear factor erythroid 2-like 2 (Nrf2). Stabilized following oxidative stress, Nrf2 induces the expression of antioxidants as well as cytoprotective genes, which provoke an anti-inflammatory expression profile, and is crucial for the initiation of healing. In view of this fundamental modulatory role, it is clear that both hyper- or hypoactivation of Nrf2 contribute to the onset of chronic diseases. Understanding the tight regulation of Nrf2 expression/activation and its interaction with signaling pathways, known to affect inflammatory processes, will facilitate development of therapeutic approaches to prevent Nrf2 dysregulation and ameliorate chronic inflammatory diseases. We discuss in this review the principle mechanisms of Nrf2 regulation with a focus on inflammation and autophagy, extending the role of dysregulated Nrf2 to chronic diseases and tumor development. NFE2L2) gene pyrene, Nrf2 knockout (KO) mice exhibited a significantly higher gastric tumor burden compared to wild-type mice . SimilarKeap1 and Nrf2 genes have been identified in several cancers, as elegantly reviewed by Taguchi, et al. [Nrf2 are located either within the DLGex or the ETGE motifs, preventing regular Keap1 binding and subsequent proteasomal degradation. Besides the obstruction of the Keap1-Nrf2 pathway by mutations, transcriptional alterations have also been observed. Hypermethylation of the Keap1 promoter is observed in lung, prostate and kidney cancer [Keap1 transcription and thereby reduced expression, leads to elevated Nrf2 synthesis and activity.However, there is a dark side to Nrf2. Many types of cancer exhibit aberrant levels of Nrf2, as a result of the dysregulation of the Keap1-Nrf2 pathway. Correspondingly, many mutations within , et al. . All muty cancer ,104,105.The binding capacity of Keap1 to Nrf2 can also be decreased despite its unrestricted expression. Recently, Ge, et al. showed tSimilarly to iASPP, p62/SQSTM1 can interfere with Keap1-Nrf2 binding. Impaired autophagy and the resulting accumulation of p62/SQSTM1 leads to an increase in Nrf2 activity. p62/SQSTM1 competes with Nrf2 for binding to Keap1, thereby leading to decreased degradation of Nrf2 and increased activity ,107,108.Kras, Braf, and Myc in primary murine cells and human pancreatic cancer [Increased Nrf2 activity was also observed upon expression of the oncogenes c cancer . Kras-dec cancer , furtherThis increased Nrf2 activity in tumors leads to an increase in target genes, which mainly account for the pro-tumorigenic properties of Nrf2. The ARE-regulated antioxidant target genes of Nrf2 allow cancer cells to maintain elevated ROS levels for pro-tumorigenic cell signaling and proliferation, without leading to ROS-mediated cell death . In addiNrf2-dependent chemoresistance is reflected by enhanced resistance of cancer cells to chemotherapeutics when Nrf2 is stably overexpressed . In contThese contrasting roles of Nrf2 are strikingly demonstrated by data from the group of Donna D. Zhang in a recent study and summAccordingly, Nrf2 activation and antioxidants are able to reduce the risk of environmentally-induced cancer, but as cancer treatment, the inhibition of Nrf2 in combination with chemotherapeutics seems to be a promising approach. Therefore, there is a need to identify therapeutics and mechanisms to inhibit Nrf2 activity.Peng, et al. identifiA positive effect was also obtained by combined treatment with brusatol and cisplatin of the lung cancer cell line A549, which exhibits high levels of Nrf2 due to a mutation in Keap1 . The quaA more radical chemotherapeutic strategy to disturb tumor capacity by dealing with elevated ROS levels, was shown in head and neck cancer (HNC). By simultaneously targeting the glutathione-(GSH), thioredoxin-(Trx) and Nrf2-system, Roh et al. achievedKras-mutant lung adenocarcinoma, with a loss-of-function in the Keap1 gene, shows enhanced dependency on glutaminolysis. Consequently, growth of Keap1-mutant lung tumors was markedly reduced upon inhibition of glutaminase [Instead of targeting Nrf2 directly, therapeutic approaches are also exploiting the resulting phenotype of a dysregulated Keap1-Nrf2 pathway. Recently, it was reported that taminase .Additionally, Nrf2 has been shown to modulate anabolic metabolism in cancer to allow tumor cell proliferation. This is achieved by redirecting glucose and glutamine into anabolic pathways . In geneTaken together, targeting the Nrf2-redox-system in combination with chemotherapeutics could be a promising treatment for several cancers. Nevertheless, the success of these therapies depends on the identification of specific inhibitors. Moreover, it will be important to specifically target tumor cells to maintain the Nrf2-ARE pathway intact in healthy tissue, thereby protecting it from damage by environmental stressors, xenobiotics and ROS and preventing further malignant degeneration.In addition to the use of CDDO-Me for APAP intoxication and the mechanistic basis for Nrf2 activation in COPD, there is an extensive literature on the potential for therapeutic modulation of Nrf2 in inflammatory and immunological disorders . Many ofNrf2 gene itself or its regulatory partners such as Keap1, allowing constitutive activation of Nrf2. This will facilitate the more selective use of current therapeutic approaches and point towards potentially new ones.Taken together, Nrf2 is an important mediator of antioxidant signaling during inflammation. Its function is based mainly on induction of the expression of target genes responsible for detoxifying and anti-oxidant effects, although a role of Nrf2 as a transcriptional repressor is also well established. Dysregulation of Nrf2, leading to hyper- or hypo-activation, frequently due to excessive environmental stress, is often accompanied by the development of chronic inflammatory diseases. New insights into the mechanisms responsible for aberrant Nrf2 function are needed. These include, on the one hand, those caused by overloading of Nrf2-dependent signaling and on the other hand, the processes initiated by mutations in the"} +{"text": "Identification and characterization of somatic mutations in cancer have important prognostication and treatment implications. Genes encoding the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), are frequently mutated in cancer. These mutations drive constitutive NRF2 activation and correlate with poor prognosis. Despite its apparent significance, a comprehensive catalogue of somatic NRF2 mutations across different tumor types is still lacking. Here, we catalogue NRF2 mutations in The Cancer Genome Atlas (TCGA) database. 226 unique NRF2-mutant tumors were identified from 10,364 cases. NRF2 mutations were found in 21 out of the 33 tumor types. A total of 11 hotspots were identified. Of these, mutation to the R34 position was most frequent. Notably, R34 and D29 mutations were overrepresented in bladder, lung, and uterine cancers. Analyses of corresponding RNA sequencing data using a de novo derived gene expression classifier showed that the R34 mutations drive constitutive NRF2 activation with a selection pressure biased against the formation of R34L. Of all R34 mutants, R34L conferred the least degree of protein stabilization, suggesting a pro-tumor NRF2 half-life threshold. Our findings offer a comprehensive catalogue of NRF2 mutations in cancer that can help prognostication and NRF2 research. When activated, NRF2 promotes the transcription of its target genes, many of which are involved in the augmentation of cellular reducing capacity and in the detoxification and efflux of xenobiotics4. Thus, NRF2 activation protects cells against chemical and oxidative insults.The NRF2 (Nuclear factor (erythroid-derived 2)-like 2) transcription factor is the master regulator of cellular antioxidant responsesNRF2, together with its negative regulator KEAP1 (Kelch-like ECH-associated protein 1), are frequently mutated in cancer11. These mutations phenotypically converged at the constitutive activation of NRF213. Since NRF2 activation protects cells against xenobiotics and oxidative insults, these mutations correlate with chemo- and radio-resistance, and result in poor clinical outcomes13. Indeed, patient survival has been shown to be significantly poorer in tumors harboring NRF2 activation, including lung15, gallbladder16, esophageal17, ovarian18, head and neck19, and gastric cancers20. As such, identification of cancer cases with constitutive NRF2 activation has important prognostic implication.Several studies have shown that 21. These interactions enable KEAP1 to function as a substrate adaptor protein for a Cullin-3 (CUL3) containing E3 ubiquitin ligase complex that mediates NRF2 ubiquitylation22. In this system, KEAP1 functions as a cellular redox sensor, whereby critical cysteine residues on its surface are amenable to covalent modification by electrophiles and reactive oxygen species25. These modifications render KEAP1 unable to mediate NRF2 ubiquitylation, enabling NRF2 to accumulate and perform its transcriptional function. Accordingly, somatic NRF2 mutations in cancer mainly occurred within the ETGE and DLG motifs8. The focal nature of somatic NRF2 mutations presents an attractive genetic screening modality that could be used to identify cancers with constitutive NRF2 activation. Moreover, the mutant forms of NRF2 are unique to cancer cells and therefore may be targeted as a treatment strategy. However, despite the focal nature of NRF2 mutation, a thorough cataloging of NRF2 mutation in cancer has yet to be reported, hampering the development of an easy NRF2 mutation screening method.Mechanistic studies have revealed that KEAP1 interacts with NRF2 through the conserved ETGE and DLG motifs residing in the N-terminal tail of NRF2NRF2 mutations in cancer cases reported in The Cancer Genome Atlas (TCGA). By cross analyzing somatic mutations with gene expression analyses, we report NRF2 mutations that result in constitutive NRF2 activation.Here we catalogue somatic 27. For the purpose of cataloging somatic NRF2 mutations, we used mutation sites that were called by at least two of the 4 mutation calling algorithms to achieve a compromise between mutation calling accuracy and sensitivity. This restricted the total mutation calls from 2,851,982 down to 2,143,125 unique mutation sites , followed by esophageal carcinoma (ESCA) and uterine corpus endometrial carcinoma (UCEC) cancers , followed by LUSC and liver hepatocellular carcinoma (LIHC) show increased frequency of mutations , but not to transition mutation events , was also reported as a reliable biomarker for smoking in lung cancers, and its expression is a good surrogate for NRF2 activation in lung cancer30. More recently, Orr\u00f9 and co-workers demonstrated that diethylnitrosamine, an alkylating agent, induced a high frequency of NRF2 gain-of-function mutations in a rat liver carcinogenesis model. The mutations were found in the preneoplastic lesions. Moreover, they showed that NRF2 gain-of-function mutations were critical for the onset of hepatocellular carcinoma in the model31.There were a total of 226 cases of tumors with somatic one Fig.\u00a0. Consisters Fig.\u00a0. SomaticHC) Fig.\u00a0. There ions Fig.\u00a0. Moreove23) Fig.\u00a0. These cNRF2 with KEAP1 mutations. The 6 NRF2 mutant cases were chosen based on mutation at the DLG or ETGE motifs, which are known to activate NRF2, while the 6 KEAP1 mutant cases were chosen based on low KEAP1 expression levels. We performed differential gene expression analysis to identify genes that are differentially regulated between tumor and normal tissues. This analysis filtered the number of transcripts from 24,507 to 2,112. Since the training set consisted of two tumor types (LUSC and LUAD), we devised a scoring algorithm to remove the tumor type biased (described in Methods section). Using this scoring system, genes were ranked and the 28 top scoring genes were chosen as the NRF2 activation signature enhancer. NRF2 binds to ARE sequence to drive expression of a luciferase reporter that serves as an indicator for NRF2-mediated transcriptional activation. We developed NRF2 expression constructs for the non-DLG and non-ETGE NRF2 mutants found in the activated signature, as well as the R34L NRF2 mutant. All mutants enhanced luciferase activity to levels similar as wildtype NRF2, indicating none of the mutants were deleterious to NRF2 activity of each mutant. The analysis showed that R34L is the least stable mutant and vemurafenib (against BRAF V600E) have a good degree of success in cancer management41. The development of such mutant specific compounds requires that cancer specific mutations happen in a predictable manner. For example, oncogenic BRAF mutations regularly occur at the P-loop and the activation domain (V600 is located within the activation domain)42. Thus, drug development efforts can concentrate on developing compounds that target the cancer specific variant, while leaving wild-type protein in normal cells untouched. This study demonstrates that somatic NRF2 mutations in cancer fulfill this criterion. Specifically, the distribution of somatic NRF2 mutations are very similar to those sustained by oncogenes whereby the mutation are focal at certain locations. As such, it opens up opportunities to develop mutant specific NRF2 inhibitors, allowing tumor specific NRF2 inhibition while leaving wild type NRF2 in normal tissues to carry out its protective functions. Additionally, the nature of the focal mutations also allow for individualized mutation screening, whereby the mutational hotspots can be amplified with appropriate PCR primers followed by Sanger sequencing. Such screening strategy may be developed into a diagnostic method when mutant specific NRF2 inhibitors become available.Activating the NRF2 transcription factor has long been recognized as a means to protect cells against chemical carcinogenesis and environmental insults8, our results also showed that somatic NRF2 mutations primarily occurred at the ETGE and the DLG motifs, which interfere with KEAP1 binding. Several mutations outside the DLG/ETGE motifs to NRF2 were also frequently mutated (p\u2009<\u20090.05), including W24, Q25, D77, and R34. While a few isolated mutations to these locations have been reported and shown to mitigate NRF2-KEAP1 interactions32, we focused on the R34 mutation and found it to be the most frequently mutated amino acid position and the only position significantly enriched across many tumor types. We identified 34 cases harboring R34 mutations; these are high confidence mutations, as they were called by at least two of the mutation-calling algorithms used, and 30/34 mutations were called by all four algorithms. Upon evaluation of possible R34 mutations, there is a selection bias against R34L mutation. Using ARE-Luciferase reporter assay, we found that R34L mutant could still evade KEAP1-mediated decrease in NRF2 transcriptional activity. Half-life analysis showed that R34L mutation still stabilizes NRF2. However, it is the least stable among the NRF2 R34 mutants, indicating a potential minimal stability threshold for NRF2 to benefit cancer growth. Although mechanistic underpinning behind the overrepresentation of longer half-life NRF2 mutants in cancer is still lacking, constitutive/sustained versus intermittent NRF2 activation has been proposed as the distinguishing feature that separate cancer prevention and cancer promotion properties of NRF2 activation43. Given that NRF2 R34L has the shortest half-life and is significantly negatively selected for in cancer, future studies into the potential periodicity of NRF2-R34L activation may offer clues into the hypothesis behind constitutive versus intermittent NRF2 activation in cancer prevention and promotion.Consistent with previous studiesNRF2 mutation, somatic mutations that activate NRF2 can range from direct loss-of-function KEAP1/CUL3 mutations10, to KEAP1 gene silencing44, to somatic mutations that lead to accumulation of oncometabolites47, or ETGE/ETGE-like motifs containing proteins48. Intriguingly, our analysis revealed several KEAP1 mutations that were significantly enriched; some of these locations have been characterized, including KEAP1-G333C which has been shown to not bind NRF2 and subsequently not suppress NRF2-mediated transcription. In contrast, KEAP1-R470C mutants have exhibited enhanced NRF2 binding: these \u201csuperbinder\u201d mutants were shown to not suppress NRF2-mediated transcription, albeit through an unknown mechanism49. Identifying which biochemical class of KEAP1 mutant those identified in this manuscript fall under could lead to novel insights into NRF2-KEAP1 relationships.Apart from somatic NRF2 or KEAP1 mutation. Thus, mutations to other genes may also contribute to the observed NRF2 activation phenotype. Given the prognostic and potential treatment implications of identifying cases with NRF2 activation, there is a need to identify all NRF2 regulatory genes, which when mutated drive constitutive NRF2 activity. To date, much of what is known is based on piece-meal efforts of identifying one regulatory gene at a time; genome wide systematic identification of the NRF2 regulatome may offer a more powerful way of reanalyzing TCGA and other legacy data to identify the mechanisms by which NRF2 becomes activated in tumors.Stratification of the lung cancer cases based on our NRF2 activation identifier revealed a large number of cases appeared to have NRF2 activation yet did not harbor an AKR1B10 was the only bona fide NRF2 target gene. AKR1B10 can be found in other NRF2-activation gene expression signatures50, and its overexpression has been associated with lung cancers in particular51. Besides AKR1B10, one gene in our signature, NR0B1, has been utilized in other NRF2-activation signatures53; how NRF2 regulates NR0B1 expression has not been identified. The other genes identified in our signature have not been found in several other signature we looked at for NRF2 activation53. It remains unclear if and how these genes are regulated by NRF2.Of the genes identified in our NRF2 activation signature, NRF2 and KEAP1 mutations in tumor types with known association with carcinogen exposure provides a glimpse into the roles of NRF2 in cancer development and progression. We identified that tumors with NRF2 or KEAP1 mutations show higher frequencies of transversion mutations. Many carcinogen-associated Michael acceptors, such as acrolein in cigarette smoke or quinone metabolites of polyaromatic hydrocarbons found in exhaust fumes, are known to cause both transversion mutations and NRF2 activation60. Given the role of NRF2 in protecting cells against environmental insults, exposure to environmental carcinogens like cigarette smoke may exert a selection pressure for cells with constitutive NRF2 activation. Thus, NRF2 activation may allow cells to endure the insults from mutagens and carcinogens, and survive to undergo malignant transformation. Indeed, recent work by Orr\u00f9 and co-workers showed that NRF2 activating mutations were acquired during early carcinogenesis in a rat carcinogenesis model using diethylnitrosamine as the carcinogen. They also showed that NRF2 activation was necessary for expansion of initiated cells31. However, we cannot rule out the possibility that NRF2-activating gene mutations contribute toward cancer progression rather than development.Apart from mutation sites and gene expression changes, the overrepresentation of NRF2 mutation hotspots that fall outside the well-established DLG and ETGE motifs, and five KEAP1 mutation hotspots. Of all NRF2 mutant hotspots, R34 was the most frequently mutated. Functional and transcript analyses revealed R34 mutation prevents KEAP1-mediated NRF2 degradation, stabilizes the protein, and leads to NRF2 activation. Future computational approaches may focus on the development of a web application that integrates existing knowledge on NRF2 signaling, allowing easy indexing and consolidation of knowledge that may guide prediction of cellular changes following specific alterations to the NRF2 signaling pathway.This study provides an overview of NRF2 mutations in cancer in one of the largest curated datasets presented. We identified four http://cancergenome.nih.gov/) on March 2, 2017. Somatic mutation data from 33 tumor types identified using 4 different somatic mutation-calling algorithms were utilized in the analyses.Somatic mutations and RNASeq data were downloaded from The Cancer Genome Atlas (TCGA) consortium \u2009=\u2009(b\u2009+\u20091)/(m\u2009+\u20091), where b is the number of permutations with enrichment frequency greater than or equal to that observed in the TCGA data and m is the number of random permutations, which is 5\u2009\u00d7\u2009106 for each estimated p-value. For efficiency, permutations were performed using an in-house C++ implemented R function utilizing the Rcpp package62. All p-values were adjusted for multiple testing according to the method proposed by Benjamini and Hotchberg. An estimated p-value of <0.05 is deemed significant.All statistical analyses were performed in R statistical environment63. Genes with an adjusted p-value of <0.05 and at least 2.5 log2 fold change were deemed significant and utilized for downstream analysis.Raw RNA sequencing count data for non-small cell lung cancers (LUAD and LUSC) were used in RNAseq analyses. Differential gene expression between tumor and normal lung tissues was assessed using the DESeq 2 packageNRF2-DLG or -ETGE motif mutations (3 LUAD and 3 LUSC cases) and 6 cases with low relative KEAP1 expression amongst KEAP1 mutant cases (3 LUAD and 3 LUSC cases), while the 12 normal tissues were consisted of 6 cases of LUSC and 6 cases of LUAD normal tissues. To minimize tumor type effects (LUSC vs LUAD), the absolute difference in relative gene expression level between LUAD and LUSC, |dr| were calculated and ranked in increasing order so that genes with the lowest |dr| (genes with the least inter tumor type difference will be on the top of the list). Next, sequential hierarchical clustering that progressively adds one gene from the list (starting with the top two genes) into a developing signature per iteration was performed. With each iteration, a machine learning score (MLS) was calculated whereby MLS\u2009=\u2009LInter/LIntra where LInter is the intergroup Euclidean distance , and LIntra represents the intragroup Euclidean distance . From this sequential clustering analysis, we found that the top 28 genes from the list gave the best distance between the NRF2 activated tumor cases from the NRF2 low normal cases. This 28 genes signature was then used in the testing set, which consisted of all lung cancer cases to test its ability to stratify samples based on NRF2 activation.Variance stabilized transformed relative expression levels were used to develop an NRF2 activation signature. Twelve tumor and 12 normal tissues were set aside as a training set. The 12 tumors in the training set consisted of 6 cases with either 2. For both sustained culture and experiments, HEK293 cells were cultured on flasks and dishes coated with poly-D-lysine.HEK293 cells were obtained from American Type Culture Collection (ATCC) . HEK293 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium with high glucose (4.5\u2009g/L) (DMEM) and 10% fetal bovine serum (FBS). All FBS was heat inactivated at 56\u2009\u00b0C for 30\u2009minutes. Cells were cultured at 37\u2009\u00b0C in atmospheric air enriched with 5% CO64. HA-Ubiquitin was a gift from Edward Yeh (Addgene #18712)65. Site-directed mutagenesis was carried out on MYC3-NRF2 to generate NRF2 mutations. The KEAP1 open reading frame was isolated from HA2-KEAP1 and cloned into pCDNA3.1(+) to generate PCDNA3-KEAP1. Site-directed mutagenesis was carried out on PCDNA3-KEAP1 to generate KEAP1 mutations.MYC3-NRF2 (Addgene #21555) and HA2-KEAP1 (Addgene #21556) were obtained from Addgene following their characterizationTo evaluate the role of KEAP1 in mediating the degradation of different NRF2 mutants, HEK293 cells were transfected with the different NRF2 mutant plasmids and either PCDNA3-KEAP1 or empty PCDNA3.1(+) using Attractene transfection reagent . 24\u2009hours post-transfection, cells were prepared for immunoblot analysis. Three biological replicates were used to quantify relative band intensities with ImageJ software.luc2P/ARE/Hygro] , pRL Renilla Luciferase (Promega), and indicated empty vectors, NRF2 expression vectors, and KEAP1 expression vectors. 48\u2009hours after transfection with indicated plasmids, cells were assayed for luciferase activity using dual-luciferase reporter assay system (Promega).Cells were transfected with pGL4.37[Primary antibodies used in this study were raised against \u03b2-actin (ACTB) , HA MYC , and KEAP1 .Cells were transfected with indicated plasmids for 48\u2009hours. For ubiquitylation analyses, cells were treated with 10\u2009\u03bcM MG132 for four hours prior to harvesting. Cells were harvested in radioimmunoprecipitation buffer and immunoprecipitated overnight at 4\u2009\u00b0C.HEK293 cells were transfected with indicated plasmids. 48\u2009hours post-transfection, cells were washed with phosphate-buffered saline (PBS) and 100\u2009\u03bcg/mL cycloheximide in serum-free DMEM was added to all plates; plates were placed back in 37\u2009\u00b0C incubator. After five minutes, cells were lysed in 1X Laemmli sample buffer as a zero timepoint. Lysates were then collected, boiled, and frozen for later immunoblot analysis. Subsequent plates were similarly harvested every 30\u2009minutes for the duration of the chase assay.N(t)\u2009=\u2009N0e\u03bbt\u2212 where N(t) is the quantity at time t with N0\u2009=\u2009N(0) and \u03bb is the rate constant such that t-test was used to evaluate significance between WT and mutant NRF2 half-lives, with p\u2009<\u20090.05 deemed significant.Following immunoblotting, three biological replicates of blots were quantified using ImageJ software with ACTB as a loading control. Half-life was calculated according to the exponential decay equation Supplementary FiguresSupplemental Table S1Supplemental Table S2"} +{"text": "Oxidative stress (OS) is associated with many diseases ranging from cancer to neurodegenerative disorders. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is one of the most effective cytoprotective controller against OS. Modulation of Nrf2 pathway constitutes a remarkable strategy in the antineoplastic treatments. A big number of Nrf2-antioxidant response element activators have been screened for use as chemo-preventive drugs in OS associated diseases like cancer even though activation of Nrf2 happens in a variety of cancers. Research proved that hyperactivation of the Nrf2 pathway produces a situation that helps the survival of normal as well as malignant cells, protecting them against OS, anticancer drugs, and radiotherapy. In this review, the modulation of the Nrf2 pathway, anticancer activity and challenges associated with the development of an Nrf2-based anti-cancer treatment approaches are discussed. Cancer is the second leading cause of death both for men and women, behind cardiovascular diseases . AccordiThere are many types of cancer treatment. The types of treatment that that patient will receive will depend on the type of cancer and how advanced it is. Today, we can talk about surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy and stem cell transplants processes that are there to treat cancer. In addition, precision medicine helps doctors select treatments that are most likely to help patients, based on a genetic understanding of their disease.Types of immunotherapy that help the immune system act directly against the cancer include: Checkpoint inhibitors, adoptive cell transfer, monoclonal antibodies, treatment vaccines, cytokines, BCG . Although there are good advantages, immunotherapy is not yet as widely used as surgery, chemotherapy, and radiation therapy. Many new immunotherapies are being studied in clinical trials ,5.Targeted therapy is the foundation of precision medicine. Most targeted therapies are either small-molecule drugs or monoclonal antibodies. Generally, targeted therapies help the immune system destroy cancer cells, stop cancer cells from growing, stop signals that help form blood vessels, deliver cell-killing substances to cancer cells, cause cancer cell death, starve cancer of the hormones it needs to grow. The important drawbacks of targeted therapy include resistance of cancer cells to the therapy and difficulties of developing drugs to some targets ,7.Stem cell transplants are most often used to help people with leukemia and lymphoma. They may also be used for neuroblastoma and multiple myeloma. Stem cell transplants for other types of cancer are being studied in clinical trials ,9.Precision medicine may be called personalized medicine. The idea of this treatment is to develop a treatment that will be tailored to the genetic changes in each person\u2019s cancer. However, the precision medicine approach to cancer treatment is not yet part of routine care for most patients ,11.OS plays a crucial role in determining cell fate. As a reaction to the excessive reactive oxygen species (ROS) load, apoptotic-signaling pathway is stimulated to promote normal cell death. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) looks as if to be as a chief regulator, which defends cells . Nrf2 isNrf2 belongs to the cap \u2019n\u2019 collar type of basic region leucine zipper factor family (CNC-bZip) that is a group of transcription factors that are activated in response to cellular stress . Nrf2 is\u03b1) and retinoic acid receptor, (RAR-\u03b1)) regulate Nrf2 transcription negatively, other transcriptional factors can induce Nrf2 activation [The Nrf2 signaling pathway contributes to the maintenance of cellular and tissue homeostasis and protects cells against OS. The repressor protein Keap1, an adaptor component of Cullin 3-based ubiquitin E3 ligase complex, tightly regulates the activities and the protein level of Nrf2 . Under btivation ,37,38,39tivation . ROS and reactive nitrogen species (RNS) are continuously produced in humans from internal metabolism and external exposure . OxidantNrf2 acts as a mediator to induce drug-metabolizing enzymes (DMEs) with the help of species like antioxidants and electrophiles . ResearcNrf2, a transcription factor with a great affection to OS, binds to AREs in the nucleus and stimulates the transcription of many antioxidant genes. OS makes Nrf2 separate from Keap1 and to transfer into the nucleus, which results in its binding to AREs ,48. ExceROS and other endogenous reactive molecules may promote the release of Nrf2 that binds to the ARE in the nucleus. This binding motivates gene transcription and stimulates the antioxidant defenses ,55. The Nrf2 is a main controller of the antioxidant reaction and xenobiotic metabolism via several of antioxidant and Phase II detoxification genes . Nrf2 deIt is widely accepted that Nrf2 is an important player in the cellular defense mechanism that protects cells from cancer progression and promotes cell survival under stress conditions in normal cells A. HoweveSomatic mutations in NRF2 (gain of function mutations) and KEAP1 (loss of function mutations) result in constitutive activation of Nrf2 and its target genes, and these mutations have been identified in many different cancers ,75,81,82Besides somatic mutations of KEAP1, epigenetic alterations in promoter regions of the KEAP1 gene can lead to aberrant activation and nuclear accumulation of Nrf2 protein in cancer cells. Hypermethylation of the promoter region of KEAP1 was reported in several cancers, including lung and prostate cancer, causing down-regulation of Keap1 expression and accumulation of Nrf2 ,87. On tMicro RNAs (miRNAs) are small non-coding RNA molecules containing about 18\u201325 nucleotides that regulate the post-transcriptional activity of many genes by sequence-specific binding to mRNA . InteresStudies have demonstrated that different proteins in cancer progression can deregulate Nrf2 signaling by altering the Nrf2\u2013Keap1 binding . Nrf2 siIt has been reported that Nrf2 level is also modulated through protein-protein interactions with Nrf2 or Keap1. Wilms tumor gene on the X chromosome (WTX) prevents Nrf2 degradation by binding to Keap1 . SimilarSome Nrf2 inhibitors have been stated for the treatment of Nrf2-addicted cancers. One of them is brusatol, which is a natural quassinoid. It was found that brusatol stimulates poly-ubiquitination of Nrf2, which decreases the Nrf2 protein level. The inhibitory effect of brusatol to Nrf2 is revealed to not be dependent of its repressor Keap1. Brusatol was found beneficial for the inhibition of Nrf2 signaling .It has been established that stimulation of Nrf2 helps to lower chemo-resistance in NSCLC cells . Many flAnother Nrf2 inhibitor, halofuginone, was established to develop a chemosensitizing influence on Nrf2-addicted cancer cells . In thisThe effective protection activity of Nrf2 has been stated generally during tumor initiation. However, it is now well recognized that Nrf2 shows a dual effect in carcinogenesis. Growing number of research showed the oncogenic properties of Nrf2 in lung cancer, esophagus and skin and renal cell cancer ,113,114.Nrf2 is found in practically all cell types and tissues, but it is prominent in tissues where main detoxification reactions take place, such as intestine, lung, and kidney. It has a protective role against oxidative damage and carcinogenesis through binding to AREs .Recent studies specify that targeting Nrf2 may be a new therapy to diminish tumor and develop a defense. It has been established that Nrf2 is defending normal cells under OS. The excess Nrf2 in tumor cells carry on its defense toward cytoprotection. This might help to defend tumor cells against OS. This process by which cancer cells adjust themselves to survive in augmented OS and resist treatment is called \u201credox adaptation\u201d. Therefore, it is debatable whether the activation, or the inhibition, of Nrf2 is beneficial for the prevention or treatment of cancer .Numerous compounds modulate the Nrf2 pathway to perform anticancer activity. It is obvious that both Nrf2 inducers and inhibitors could be valuable as anticancer policy. Nevertheless, due to modulating effects of Nrf2, it is active in the detoxification procedure of anticancer drugs, and its activation in cancer cells possibly will lead to chemo-resistance. A beneficial or unfavorable process of Nrf2 in cancer cells basically depends on the close control of its action, surroundings of tumor and cell type . Over thNrf2 is crucial to providing defense against OS and also preventing tumor promotion and progression ,126. StuN-acetyl-4-aminophenol (APAP)) [The role of Nrf2 in defense against toxic effects of chemicals was proven on Nrf2 knockout mice using acetaminophen ( (APAP)) . The micNrf2 stabilizes oxidative tissue damage and inflammation through transcriptional activation via the ARE. Protecting activity of Nrf2 in the development of emphysema was observed by testiCitrus coumarin auraptene was studied in order to identify the possible effect on premalignant mammary lesions via activation of Nrf2/ARE . While tROS and RNS are believed to be a main cause underlying the contribution of chronic inflammation to cancerogenic alteration . Nrf2 shThe pharmacological significance of Nrf2 has been revealed by many studies mainly using Nrf2-deficient mice and research of single nucleotide polymorphism in the NRF2 gene . Nrf2 acSince Nrf2 shows boundless benefits on cancer cells, including therapeutic resistance, improved antioxidant capability and aggressive tumorigenic ability, cancer cells with Nrf2 activation often develop \u201cNrf2 addiction\u201d. Even though constant stimulation of Nrf2 helps growth and survival benefits on cancer cells, high stimulation of Nrf2 in normal cells is quite toxic. These findings suggest that definite requirements allow for the formation of Nrf2-addicted cancers .Some Nrf2 inhibitors have been stated for the treatment of Nrf2-addicted cancers. Brusatol, a natural quassinoid, stimulates poly-ubiquitination of Nrf2 and decreases the Nrf2 protein level . HalofugInhibition of Nrf2 is an encouraging approach for the treatment of Nrf2-addicted cancers. Nevertheless, using systemic Nrf2 inhibitors may have unwanted effects on cancer-bearing hosts, seeing the essential roles of Nrf2 in cytoprotection. Finding novel therapeutic targets besides Nrf2 for Nrf2-addicted cancers have been still under investigation.It is known that one of the chief pathways in charge for cell protection against OS is the Nrf2/Keap1-signaling pathway . Nrf2 haThe characterization of the crystal structure of Keap1 in complex with the Neh2 domain of Nrf2 may deliver chances to design molecules that specially and selectively interfere with the binding of Keap1 and Nrf2 . Nrf2 haNrf2 has a significant part in cellular defense to OS and exogenous toxic materials, and it is strictly associated to inflammatory reactions, respiratory system diseases, cardiovascular diseases, and malignant tumors . ROS eliOS is associated with cancer initiation and progression. The Nrf2 transcription factor is the main regulator of antioxidant genes and has a critical role in regulating the metabolic pathways important in cancer cells. Undeniably dissimilar results show that there are beneficial and harmful effects of targeting Nrf2 in some cancer cells. Antioxidant agents may help explain the activity of Nrf2 in cancer, as well as its power as a biomarker of cancer progression and therapy.Both Nrf2 inducers and inhibitors could be beneficial in cancer treatment approaches. Nonetheless, Nrf2 is able to modulate many systems possibly involved in the detoxification procedure of anticancer drugs; its activation in cancer cells may cause chemo-resistance. The beneficial or disadvantageous role of Nrf2 in cancer cells basically depends on the constricted control of its action . Discove"} +{"text": "MMP-9), B-cell lymphoma 2 (BCL-2), B-cell lymphoma-extra large (BCL-xL), Tumour Necrosis Factor \u03b1 (TNF-\u03b1), and Vascular endothelial growth factor A (VEGF-A). We also did a brief analysis of The Cancer Genome Atlas (TCGA) data of lung adenocarcinoma concerning the effects of radiation therapy and found that the therapy-induced Nrf2 activation is not universal. For instance, in the case of recurrent disease and radiotherapy, we observed that, for the majority of Nrf2-targeted genes, there is no change in expression level. This proves that the universal, axiomatic rationale that Nrf2 is activated as a response to chemo- and radiation therapy is wrong, and that each scenario should be carefully evaluated with the help of Nrf2-targeted genes. Moreover, there were nine genes involved in lipid peroxidation, which showed underexpression in the case of new radiation therapy: ADH1A, ALDH3A1, ALDH3A2, ADH1B, GPX2, ADH1C, ALDH6A1, AKR1C3, and NQO1. This may relate to the fact that, while some studies reported the co-activation of Nrf2 and other oncogenic signaling pathways such as Phosphoinositide 3-kinases (PI3K), mitogen-activated protein kinase (MAPK), and Notch1, other reported the inverse correlation between Nrf2 and the tumor-promoter Transcription Factor (TF), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-\u03baB). Lastly, Nrf2 establishes its activity through interactions at multiple levels with various microRNAs. MiR-155, miR-144, miR-28, miR-365-1, miR-93, miR-153, miR-27a, miR-142, miR-29-b1, miR-340, and miR-34a, either through direct repression of Nrf2 messenger RNA (mRNA) in a Kelch-like ECH-associated protein 1 (Keap1)-independent manner or by enhancing the Keap1 cellular level, inhibit the Nrf2 activity. Keap1\u2013Nrf2 interaction leads to the repression of miR-181c, which is involved in the Nuclear factor kappa light chain enhancer of activated B cells (NF-\u03baB) signaling pathway. Nrf2\u2019s role in cancer prevention, diagnosis, prognosis, and therapy is still in its infancy, and the future strategic planning of Nrf2-based oncological approaches should also consider the complex interaction between Nrf2 and its various activators and inhibitors.Nrf2 is a transcription factor that stimulates the expression of genes which have antioxidant response element-like sequences in their promoter. Nrf2 is a cellular protector, and this principle applies to both normal cells and malignant cells. While healthy cells are protected from DNA damage induced by reactive oxygen species, malignant cells are defended against chemo- or radiotherapy. Through our literature search, we found that Nrf2 activates several oncogenes unrelated to the antioxidant activity, such as Matrix metallopeptidase 9 ( NFE2L2 gene encodes the Nrf2 protein [The process of tumor formation and dissemination of malignant cells is strictly regulated by the external exposure of cells to harmful or protective elements. The interplay between these factors is seen at the cellular level through the strict modulation of signaling pathways ,2,3 for protein , which i protein .\u03b1 (TNF-\u03b1) and Interleukin 1 beta (IL-1\u03b2) in adipocyte-associated macrophage, but also raises the level of p62, a negative regulator of pro-inflammatory cytokines [When reactive oxygen species (ROS) level rises, Nrf2 disassociates from its inhibitor, Keap1, and enters into the nucleus. Keap1 belongs to the Kelch family of proteins (KLHL), and it binds to the Nrf2-ECH homology (Neh2) domain of Nrf2 in a region containing 69\u201384 amino acids flanked by the ETGE motif . Translaytokines . P62 binytokines ,15.NQO1), Glutathione S-transferase 1 (GST) [HMOX1), glutamate\u2013cysteine ligase (GCL), peroxiredoxins, and Glutathione (GSH) synthesis enzyme [Inside the nucleus, NRF2 forms a heterodimer with small V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene MAF proteins (sMAF) and regulates the expression of genes that contain the antioxidant response elements (AREs) or the MAF recognition elements (MARE) in their promoter region ,17. Nrf2 1 (GST) , heme oxs enzyme . Mutatios enzyme .Several authors already showed the defensive role of Nrf2 in various malignancies, neurodegenerative diseases, cardiovascular disorders, aging, inflammation, or photo-oxidative stress . MoreoveThere are several types of cancers in which this mechanism was demonstrated, and tumors were proven to use Nrf2 as a self-protective mechanism .In this review, we analyze the involvement of Nrf2 in cancer installment and progression focusing on the interactions this signaling pathway establishes with various kinds of regulators: proteins, microRNAs, and interactions with other signaling pathways and transcription factors. We also take a look at the The Cancer Genome Atlas (TCGA) data regarding the activity of Nrf2 in lung cancer and how it affects the cellular response to radiation therapy.Nrf2 is a cytoprotective transcription factor which has both a positive effect and a negative effect on cancer ,27. Nrf2The different roles of Nrf2 in cancer progression were analyzed in a study from 2016, with Keap1 knockdown mice. The mice grew smaller urethane-induced tumors and had the following antioxidant genes upregulated: Catalase (Cat), Glutathione peroxidase 2 (Gpx2), Glutathione-S-transferase a4 (Gsta4), Ppargc1A and Glutathione reductase (Gsr). However, when these tumors were transplanted into nude mice, the tumors were more aggressive .The reasons behind the overactivation of Nrf2 in cancer are DNA mutations, epigenetic changes, and modifications in the protein structure. In some cancer types, one or both Keap1 and Nrf2 are mutated, which does not allow a proper chemical interaction between the two ,31,32,33In many cancers, the general methylation pattern of the DNA is changed, and the promoter region of Keap1 becomes hypermethylated, resulting in the decreased transcription of the Keap1 protein and the release of Nrf2 ,35,36,37In classical Hodgkin lymphomas, it was found that the increased expression of Nrf2 is associated with a limited form of the disease as opposed to the advanced forms . In glioMMP9 and Bcl-xL (B-cell lymphoma extra large). MMP-9 is a metalloproteinase involved in cancer invasion through degradation of the basal membrane, and Bcl-xL is an anti-apoptotic factor [MMP9 [Bcl-2. There is an ARE region in the \u22123148 and \u22123140 reverse strands of the promoter region of Bcl-2 [Nrf2 is involved in maintaining cancer cell proliferation and invasion. In hepatocellular carcinoma, it was proven that the upregulation of the Nrf2 signaling pathway was correlated with the increased level of c factor . In glioor [MMP9 . The Nrfof Bcl-2 .FAK , MLC (modulator of volume-regulated anion channel current 1), and ROCK (Rho-associated coiled-coil-containing protein kinase 1), while inhibiting the expression of estrogen-related receptor \u03b1 (ERR1). Nrf2 also directly interacts with Breast cancer type 1 susceptibility protein (BRCA1), leading to increased stability of the BRCA protein. In the absence of BRCA expression, estrogen restores Nrf2 activation, causing decreased production of ROS in vitro and the protection of mammary gland cells [PHGPx), or pro-oxidant, 15-lipoxygenase (15-LOX), inhibited the expression of the vascular cell adhesion molecule (VCAM) via Nrf2 interaction in the promoter region of this gene [In cervical cancer, the Nrf2 signaling pathway leads to increased proliferation and inhibits apoptosis . In breand cells . The exohis gene .Snail genes [There is a negative regulation between E-cadherin and the Nrf2 protein. E-cadherin impairs the nuclear localization of Nrf2, with the help of \u03b2-catenin. In hepatocellular carcinoma, however, E-cadherin is inhibited due to the action of Transforming Growth Factor Beta 1 (TGF\u03b2) . Howeveril genes .HO-1 was correlated with the increased expression of Nrf2, HIF-1\u03b1, HIF-2\u03b1, and VEGFA. In sera of bladder cancer patients, the pro-inflammatory cytokines IL-6 and IL-8 were also elevated, along with the pro-angiogenic factor VEGFA [In bladder cancer tissue, the overexpression of or VEGFA .The TNF-\u03b1 cytokine functions both as a promoter and as an inhibitor of the Nrf2 pathway. At average concentrations (2\u20135 ng/mL), TNF-\u03b1 mediated the nuclear import of Nrf2 and the transcription initiation of its target genes, while, at high concentrations (> 10\u201350 ng/mL), it inhibited the Nrf2 pathway .HMOX1, than in the case of acute hypoxia [Hypoxic conditions are also regulators of the Nrf2 stimulation of transcription. In acute hypoxia, this TF binds less commonly in the enhancer or promoter region of hypoxia .GCLc). The stimulated expression of GCLc leads to the synthesis of glutathione, an important antioxidant, thus protecting the cancerous cells from the damage caused by high oxidative stress [The exposure of HepG2 cells to homocysteine results in the stabilization of Nrf2, and the transcription activation of glutamate cysteine ligase , pro-inflammatory cytokines , and the pro-angiogenic factor VEGFA [In mucoepidermoid carcinoma of the lung, HMOX1 overexpression was also associated with the inhibition of cell-cycle progression proteins Cyclin D1 (CCND1) and CCND2, and the stimulated transcription of the cell-cycle arrest proteins Gastrin (GAS) and Cyclin-dependent kinase inhibitor 1C (CDKN1C). The small interfering RNA (siRNA)-mediated silencing of HO-1 decreased the expression level of invasion promoters in its promoter, which can explain the synchronous expression of Nrf2 and MRP1 [ABCG2) [GSTA2), Glutathione S-Transferase Pi 1 (GSTP1), Cytochrome P450 Family 3 Subfamily A Member 4 (CYP3A4), Heme Oxygenase 1 (HO-1), MRP5 [ABCF2) [GCLC), and Glutamate-Cysteine Ligase Modifier Subunit (GCLM) [G6PD), Phosphogluconate Dehydrogenase (PGD), Transaldolase 1 (TALDO1), and Transketolase (TKT), and the synthesis of purines. Its activity involves the inhibition of Keap1 and the activation of the signaling pathway hosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (Akt). Moreover, for metabolism reprogramming, Nrf2 also interacts and modulates the activity of other TFs such as p53, c-Myc, and HIF1\u03b1 [PHGDH), Phosphoserine Aminotransferase 1(PSAT1), and Serine Hydroxymethyltransferase 2 (SHMT2) [ABHD14B), Acyl-CoA Thioesterase 13 (ACOT13), Aldo-Keto Reductase Family 1 Member C1 (AKR1C1), Aldehyde Dehydrogenase 3 Family Member B1 (ALDH3B1), Glutamate-Cysteine Ligase Catalytic Subunit(GCLC), Kynureninase (KYNU), LDL Receptor Related Protein 8 (LRP8), Nicotinamide Phosphoribosyltransferase (NAMPT), Prostaglandin E Synthase (PTGES), Prostaglandin Reductase 1 (PTGR1), Solute Carrier Family 27 Member 5 (SLC27A5), and Thioredoxin Reductase 1 (TXNRD1) [TKT) gene, which encodes for an enzyme involved in the pentose phosphate pathway (PPP) and protects cancerous cells from treatment-induced oxidative stress [In cancer cells, it was proven that Nrf2 stimulates the multidrug-resistance-associated protein-1 (MRP1). Moreover, the and MRP1 . Nrf2 al [ABCG2) , Glutath1), MRP5 , ATP Bin [ABCF2) , Glutamat (GCLM) . In colot (GCLM) . Nrf2 ist (GCLM) , or it it (GCLM) . Nrf2 rend HIF1\u03b1 . Nrf2 re (SHMT2) , or the (TXNRD1) . Nrf2 tae stress .The Nrf2 signaling pathway is, in turn, repressed by multiple nuclear receptors. Retinoid X receptor alpha (RXR\u03b1) and Peroxisome proliferator-activated receptor gamma (PPAR\u03b3) bind to the retinoic acid response element in DNA or the peroxisome proliferation-activated receptors. Estrogen receptor alpha (ER\u03b1), Estrogen-related receptor \u03b2 (ERR\u03b2), and Glucocorticoid receptor (GR) bind in the promoter region of their targeted genes .Nrf2 stability is increased through the binding of Inhibitor of apoptosis-stimulating protein of p53 (iASPP), a negative regulator of the p53 signaling pathway, which cooperates with Keap1 for Nrf2 binding . Nrf2-meP53 is a gene with essential functions in the progression of various cancer types, such as breast cancer [The wild-type p53 interacts with the promoter of Nrf2, and it induces its suppression; however, in the case of cancer, the mutated p53 can no longer bind to the promoter region of Nrf2, and it leads to the stimulated transcription of this gene and cisplatin resistance . P53 is t cancer , colorect cancer , and lunt cancer .On the other hand, lung cancer cells which have overexpression of the Nrf2/HO-1 axis have downregulated expression of IL-1\u03b2 and metallo-proteinases . The actThe Keap1/Nrf2 pathway interacts with other signaling pathways, such as NF-\u03baB, PI3K/Akt, Notch, MAPK, and Wnt Family Member 3AWNT-3A. These switch the tumor survival signal on and off as a result of Nrf2 activation or inhibition see .The mitogen-activated protein kinase kinase 1 /extracellular signal\u2013regulated kinase (ERK) signaling pathway induces the nuclear accumulation of Nrf2. In human embryonic kidney cells, HEK-293, it was proven that the MEK/ERK signaling pathway interacts with Nrf2 through IQ Motif Containing GTPase Activating Protein 1 (IQGAP1) . IQGAP1 The Nuclear factor kappa light chain enhancer of activated B cells (NF-\u03baB) signaling pathway promotes the expression of the pro-inflammatory cytokines TNF\u03b1, IL-1, IL-6, and IL-8 , and itsInhibitor of nuclear factor kappa-B kinase subunit beta (IKK\u03b2) is a member of the NF-\u03baB signaling pathway that interacts with Keap1 and induces IKK\u03b2 ubiquitination. The Keap1 genomic locus is lost or mutated in cancer . The p65The PI3K/Akt/mechanistic target of rapamycin kinase (mTOR) signaling pathway promotes cancer cell proliferation and survival , especiaPCNA) gene and the activation of the Notch signaling pathways, which augments the cellular level of NOTCH1 intracellular domain (NICD1) and Hes Family BHLH Transcription Factor 1 (HES1) genes, leading to enhanced proliferation of oral squamous cell carcinoma cells [Nrf2 also leads to the increased transcription of the proliferating cell nuclear antigen (ma cells .P38 MAP kinase (MAPK) activation leads to the turning on of Nrf2, which resulted in acquired resistance to temozolomide in glioma cells .MicroRNAs (miRNAs) are a type of non-coding RNA with a length of 19\u201325 nt which caMiR-432 is an oncomiR involved in the cisplatin resistance of cancerous cells, and it binds to the coding region of the Keap1 transcript. This is followed by the inhibition of Keap1\u2013Nrf2 interaction and the activation of Nrf2 .ERMPI expression [In bronchial epithelial cells exposed to arsenic, the level of miR-155 increased, and Nrf2 translation was inhibited . Nrf2 trpression .In breast cancer, the silencing of Nrf2 leads to the activation of the NF-\u03baB signaling pathway and the overexpression of miR-181c. This miRNA inhibits the mitochondrial production of cytochrome c oxidase, the mitochondrial potential maintenance, and oxygen consumption .Because Nrf2 is a transcription factor, it can also induce the transcription of several microRNAs by binding to the promoter region of the encoding DNA sequence. These targeted miRNAs include miR-193b, miR-365, miR-617, miR-592, miR-1207, miR-32, miR-200c, and miR-550 . Nrf2 inIn mucoepidermoid lung carcinoma (MEC), Nrf2 overexpression was correlated with HO overexpression, and it led to MMP12 and MMP9 downregulation. Nrf2 production was positively correlated with miR-181a, miR-193b, and miR-424 and negatively associated with miR-378 .During the process of carcinogenesis, miR-365-1, miR-193b, miR-28, miR-93, miR-153, miR-27a, miR-142-5p, and miR-144 are downregulated. This underexpression leads to the upregulation of their target, Nrf2 mRNA, and the stimulated phosphorylation and activity of this transcription factor. Nrf2 overstimulation results in increased cell survival, sustained tumorigenesis, and enhanced tumor growth. This is followed by an increased miR-125b1 and decreased miR-29-b1 expression level, which offers chemoresistance. During chemotherapy, ROS levels are also increased, as well as miR-141 and miR-340 expression level, which suppress the phosphorylation of Nrf2. MiR-200a is decreased during carcinogenesis, which inhibits the formation of the Nrf2\u2013Keap1\u2013Cul3 complex . MiR-200In lung fibroblasts exposed to radiation, the BRCA1 level is increased, which elevates Nrf2 nuclear import, as well as decreasing the Keap1 level. Nrf2, once in the nucleus, promotes the transcription of miR-140, which results in impaired self-renewal ability, increased cell migration, and higher contractile capabilities .HO-1 gene by increasing this gene product and triggers an elevated 5-fluorouracil resistance [An in vitro experiment proved that, in hepatocellular carcinoma, miR-141 is overexpressed, leading to Keap1 underexpression and, consequently, Nrf2 nuclear accumulation. This TF binds to the promoter region of the sistance . In the sistance .The feedback regulation between Nrf2 and its miRNA targets was also revealed in AML cells. Through Keap1 silencing, miR-125b was upregulated, and miR-29b was downregulated by Nrf2 in AML. It was further proven that the Nrf2 binds to the 5\u2019 UTR DNA region of miR-125b and downstream of miR-29b. The altered expression of these two microRNAs results in leukemic cell survival after chemotherapy treatment .In mouse keratinocytes, Nrf2 activation leads to the overstimulated transcription of miR-29a/b by Nrf2 bonding to the promoter region of the MiR-29ab1 gene. Nrf2 also leads to changes in epidermal desmosomes .SIRT) downregulation only in p53 wild-type cancer cells. This results in the upregulation of p53 and miR-34a, followed by a decreased level of Nrf2 and PPAR\u03b3 transcriptional activity [Metformin, an antidiabetic drug repositioned for cancer , inducesactivity .In human leukemic cells, Mitogen-activated protein kinase 7 and Myocyte Enhancer Factor 2 (MEF2) are phosphorylated in response to oxidative stress. MEF2 enters the nucleus, where it stimulates the transcription of miR-23a. This microRNA represses the translation of Keap1 in the cytoplasm .The multi level regulation of Keap1-Nrf2 signaling pathway by microRNAs is illustrated in Throughout our review, we analyzed the protective role of Nrf2 in the case of cancer, which leads to a weaker treatment response. In lung cancer, it was proven in vitro that A549 cells transfected with Nrf2siRNA were more prone to cell death as a response to radiotherapy than the control A549 cells . At the Our analysis of TCGA data is meant to be proof of concept, which we used to prove that gene expression analysis in regard to Nrf2signaling pathways should not be so heavily focused on the expression of Keap1\u2013Nrf2 mRNA expression, but also on the expression of their targeted genes.In order to observe the consequences following Nrf2 activation in lung cancer tissue exposed to radiation therapy, we extracted clinical data and RNA-sequencing (RNA-Seq) data of lung adenocarcinoma (LUAD) from TCGA, which included 173 samples for which there was information regarding radiation therapy status. The clinical data selected for our analysis included qualitative values describing whether the patients went through radiation therapy or not. From the RNA-Seq dataset, we looked only at the genes whose transcription is induced by Nrf2, resulting in a total of 117 genes (and gene variants) 2, which was upregulated in the case of additional radiotherapy versus non-recipients of this kind of additional therapy. At the same time, nine genes were downregulated in the case of radiotherapy versus non-recipients of this kind of therapy (ADH1A), Aldehyde Dehydrogenase 3 Family Member A1 (ALDH3A1), Aldehyde Dehydrogenase 3 Family Member A2 (ALDH3A2), Alcohol Dehydrogenase 1B (ADH1B), Glutathione Peroxidase 2 (GPX2), Alcohol Dehydrogenase 1C (ADH1C), Aldehyde Dehydrogenase 6 Family Member A1 (ALDH6A1), Aldo-Keto Reductase Family 1 Member C3 (AKR1C3), and NAD(P)H Quinone Dehydrogenase 1 (NQO1) All of these genes are involved in redox reactions.In LUAD tissue samples from patients who initially received radiation therapy, we found only two genes, Glutathione S-Transferase Omega 1 and Sulfotransferase Family 2B Member 1 , which were upregulated in the case of radiotherapy versus non-recipients of this kind of therapy . In LUAD therapy . These ghttps://gtexportal.org) on 31 October 2019. The survival analysis was done with the help of the RTCGA package and the maxstat package to determine the cutoff for each gene in R (p < 0.05). These were Glutathione S-Transferase Omega 1 (GSTO1), Sulfotransferase Family 2B Member 1 (SULT2B1), ADH1A, ALDH3A2, ADH1B, ADH1C, and Methylenetetrahydrofolate Dehydrogenase (NADP+ Dependent) 2, Methenyltetrahydrofolate Cyclohydrolase (MTHFD2) (NFE2L2 gene) has no statistically significant influence in overall survival in lung cancer; however, the lung is the tissue with top NFE2L2 expression (http://gepia.cancer-pku.cn/index.html) [GSTO1), Sulfotransferase Family 2B Member 1 (SULT2B1), ADH1A, ADH1B, and ADH1C were downregulated in lung cancer versus normal tissue, while ALDH3A2 and MTHFD2 were upregulated in lung cancer tissue versus normal adjacent tissue.The data used to look at the gene expression of each targeted gene affected by radiation therapy described in this manuscript were obtained from the GTEx Portal (ene in R . From th(MTHFD2) . Nrf2 , with a https://string-db.org/), where the outliers were excluded from the central network (The genes were then inserted in the Gene String online tool ( network ; the fun network .Nrf2 was suggested as a possible therapeutic target. Nrf2 implication in cancer remains controversial due to its protection of normal and cancerous cells. Nrf2 can be regarded as a simple response element to the signals initiated by an external factor or via its intersection with different regulatory mechanisms inside the cell. Nrf2 activation protects normal cells from malignant transformation, especially in the case of bronchial epithelial cells. At the DNA level, methylation in the Keap1 promoter, epigenetic silencing of its targeted genes, or mutations present in the binding sites of Keap1\u2013Nrf2 take place during cancer development, but also under selective pressure of different therapies.MicroRNAs can control the Nrf2 signaling pathway at different levels: repression of Keap1 by miR-27a, miR-141, miR-144, miR-153, miR-200a, miR-432, and miR-23a; repression of Nrf2 by miR-155, miR-144, miR-28, miR-365-1, miR-93, miR-153, miR-27a, miR-142, miR-29-b1, miR-340, and miR-34a; and the indirect activation of Nrf2 by miR-181a, miR-193b, miR-424, and miR-125b. Also, it is worth mentioning that the TCGA data revealed the fact that Nrf2 activation is not universal.For instance, in the case of recurrent disease and radiotherapy, we observed that, for the majority of Nrf2-targeted genes, there is no change in expression level. This proves that the universal, axiomatic rationale that Nrf2 is activated as a response to chemo- and radiation therapy is wrong, and that each scenario should be carefully evaluated with the help of Nrf2-targeted genes. There were nine genes involved in lipid peroxidation, which showed underexpression in the case of additional radiation therapy. In addition to this, we consider that it makes more sense and it will have greater scientific value if future research on Nrf2 activation/inhibition in different scenarios is evaluated not through the evaluation of Keap1\u2013Nrf2 mRNA expression, but through the mRNA expression of their targeted genes. It is also important to analyze the targeted genes at the post-transcriptional level, not the post-translational or protein levels, since there can be other inhibitory molecular mechanisms interfering in gene expression."} +{"text": "Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a major regulator of antioxidant response element- (ARE-) driven cytoprotective protein expression. The activation of Nrf2 signaling plays an essential role in preventing cells and tissues from injury induced by oxidative stress. Under the unstressed conditions, natural inhibitor of Nrf2, Kelch-like ECH-associated protein 1 (Keap1), traps Nrf2 in the cytoplasm and promotes the degradation of Nrf2 by the 26S proteasome. Nevertheless, stresses including highly oxidative microenvironments, impair the ability of Keap1 to target Nrf2 for ubiquitination and degradation, and induce newly synthesized Nrf2 to translocate to the nucleus to bind with ARE. Due to constant exposure to external environments, including diverse pollutants and other oxidants, the redox balance maintained by Nrf2 is fairly important to the airways. To date, researchers have discovered that Nrf2 deletion results in high susceptibility and severity of insults in various models of respiratory diseases, including bronchopulmonary dysplasia (BPD), respiratory infections, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), asthma, idiopathic pulmonary fibrosis (IPF), and lung cancer. Conversely, Nrf2 activation confers protective effects on these lung disorders. In the present review, we summarize Nrf2 involvement in the pathogenesis of the above respiratory diseases that have been identified by experimental models and human studies and describe the protective effects of Nrf2 inducers on these diseases. In the past few decades, environmental issues due to manmade and natural factors have sharply increased the incidence of malignant and nonmalignant respiratory diseases. Therefore, the reason underlying why the respiratory system is so easily affected by environmental problems and the pathogenesis of respiratory diseases has attracted increasing attention.As the location of gas exchange, the airways with large surface area constantly interface with the external environment and are exposed to various airborne toxicants especially inhaled oxidants . Due to \u03b3-glutamyl cysteine ligase , and NADP(H):quinone oxidoreductase (NQO1). Moreover, the stress response protein heme oxygenase (HO-1) is reported to be a particularly potent antioxidant protein [Nevertheless, during the long journey of life evolution, the organism has developed a series of antioxidant responses to counteract the toxicity of oxidative stress. The antioxidant system in cellular response includes either proteins or small molecules . As enzymes have been proven to play a significant role in life cycles, their effects on antioxidant defense have been investigated extensively. Direct antioxidant enzymes refer to classical enzymes including superoxide dismutases (SODs), catalase, and glutathione peroxidase (GPx), while indirect antioxidant enzymes mainly refer to phase 2 detoxifying enzymes such as glutathione-S-transferase (GST) isozymes, catalytic and modifier subunits of protein \u20139. AntioAlthough the functional mechanisms are diverse in the antioxidant system, a large number of typical phase 2 detoxifying enzymes and the stress response protein HO-1 are regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which indicates that this transcription factor is a possible and imperative upstream regulator of antioxidative responses that maintains cellular redox homeostasis and reduces severe oxidative damage \u201313.Nrf2, which belongs to the cap \u201cn\u201d collar (CNC) family of transcription factors, is a major transcription factor that counteracts oxidative stress and inflammation through the coordinated induction of antioxidant response element- (ARE-) driven cytoprotective gene transcription , 15. The\u03b2-TrCP (\u03b2-transducin repeat-containing protein) and proteasome degradation by the Cullin1/Rbx1 complex [Moreover, recent studies have shown that in addition to Keap1, other pathways involving cullin adaptor proteins also direct the ubiquitination of Nrf2. For instance, the phosphorylation of Nrf2 at specific serine residues in the Neh6 domain by GSK-3 forms a degradation part for recognition by the ubiquitin ligase adapter Skp1-Cul1-F-box protein (SCF)/ complex , 32. Sim complex .\u03b2 and NF-\u03baB), xenobiotic metabolism and excretion , apoptosis (Bcl-2 and BclxL), and autophagy (p62) [The functional process occurs when de novo synthesized Nrf2 translocates to the nucleus, after which Nrf2 heterodimerizes with small Maf or Jun proteins and then binds to ARE in the regulatory regions of Nrf2 target genes and either upregulates or inhibits target genes. Previous studies have shown that in the protection of the respiratory system by Nrf2, Nrf2-targeted genes are essential effectors that are identified by microarray analyses and bioinformatic studies. The Nrf2/ARE pathway regulates more than 500 genes, including genes that regulate oxidative stress , inflammation (TGF-gy (p62) , 23. Thegy (p62) , 34, 35.gy (p62) .Accordingly, previous studies have shown that in tissues where routine antioxidation and detoxification processes occur, such as the lungs, liver, and kidneys, Nrf2 is relatively abundant. The expression level of Nrf2 is highly correlated with the susceptibility, severity, and recovery of airway disorders. Data from Nrf2-knockout mice identify the protective effects of Nrf2 on airway disorders: compared to wild-type mice, Nrf2-knockout mice have enhanced lung inflammation, epithelial cell injury, and increased sensitivity to cigarette smoke, elastase, ovalbumin, bleomycin, and other stimuli \u201341. ThesAs described above, the respiratory system is directly exposed to inhaled oxidants, and this oxidative burden makes this system more vulnerable to oxidant stress, which has proven to be associated with the pathogenesis of diverse respiratory diseases. Currently, the protective roles of Nrf2 signaling in respiratory disorders have been identified with the application of Nrf2 knockout mice, including three different genetic backgrounds and lung-specific conditional knockout mice . For insBronchopulmonary dysplasia (BPD) is a chronic respiratory disease that usually occurs in premature infants with very low birth weight. BPD is mainly characterized by a failure in alveolarization, which results in impaired alveolar growth and vascular development, pulmonary inflammation, and abnormal lung function , 44. Fur2 toxicity of Nrf2 may be mediated by its regulation of the cell cycle, metabolism processes, cell\u2013cell interactions, and redox homeostasis [Although the pathogenesis of BPD has not been fully understood, the lung injury in BPD can be divided into two groups described as \u201cnew\u201d BPD and \u201cold\u201d BPD. The former refers to early developmental arrest by prenatal exposure or genetic factors, while the latter develops into surfactant-deficient premature infants who receive respiratory support. Further studies elucidate that important trigger events for BPD may include oxidative damage and inflammation. Particularly, as human infants undergo critical postnatal alveolar growth, therapeutically administered oxygen to improve the oxygenation of premature infants is a major risk factor, which may be associated with the induction of p21 and p53 cell cycle regulatory genes . For oxieostasis , 48.These findings may elucidate a possible beneficial role for Nrf2 activators in the BPD of preterm infants with respect to both lung development and hyperoxia-mediated lung injury, and several studies have attempted to unveil such potential. For instance, aurothioglucose (ATG), a TrxR1 inhibitor, inhibits thioredoxin reductase-1 (TrxR1) activity and activates the Nrf2 pathway in the lungs of newborn C3H/HeN mice exposed to hyperoxia, attenuating the decrease in body weight and alterations in alveolar development. However, the effects on alveolarization and sustained Nrf2 activation are not observed in the lungs of newborn C57BL/6 mice under the same conditions , 50. In As the respiratory tract directly contacts various pathogens from internal and external environments, the associated infections are highly prevalent and variable. Thus, there is still an unmet need for new therapeutic methods for these diseases, especially for infections with viruses. Recent studies indicate that during the process of respiratory infections, the excessive ROS produced by phagocytic cells usually causes an imbalance between oxidants and antioxidants, which may contribute to the pathogenesis of infection-related respiratory diseases. As an essential fighter against oxidative stress, Nrf2 is also considered to play a protective role in antiviral and antibacterial processes in several common infections of the respiratory tract.\u2212/\u2212 mice induced by RSV is likely due to the enhanced concurrent activation of AP-1 and NF-\u03baB. Additionally, Nrf2 deficiency makes mice more \u201cTh1-like\u201d with lower GSH and IFN-\u03b3 levels, which may impair virus clearance. Therefore, the Nrf2 pathway may be targeted for protection against RSV-induced lung injury.Respiratory syncytial virus (RSV) is not only a leading cause of acute respiratory tract infections in infants and children but also an essential factor for substantial respiratory morbidity and mortality in the elderly , 55. HowIn recent years, several Nrf2 inducers have been found to display beneficial effects on RSV infection. The potent Nrf2 inducer sulforaphane pretreatment significantly limits lung RSV replication and acute inflammation in wild-type but not Nrf2-deficient mice . ButylatInfluenza A virus (IAV) infection ranging from upper respiratory infection to pneumonia has troubled individuals for a long time , 66. AltTo date, several Nrf2 inducers have been examined for their protective role in IAV infection. It has been recently reported that the Nrf2 inducer epigallocatechin gallate decreases influenza A/Bangkok/1/79 virus entry and replication in nasal epithelial cells; however, the inhibitory effect on replication is blocked when Nrf2 is knocked down , while cRecently, Nrf2 has been reported to have a beneficial role in infection with diverse bacteria, and the application of Nrf2 inducers may represent a novel treatment for respiratory tract bacterial infections, although related studies are limited.S. pneumoniae can release several bacterial components to cause an enhanced oxidant burden on the lung epithelial surface, while the specific Nrf2 inducer resveratrol can ameliorate pneumococcal-induced oxidative stress in the airway epithelium [Staphylococcus aureus and lung inflammation in Haemophilus influenzae infections [Pseudomonas aeruginosa (PA) infection and inhibit the internalization of the bacteria in A549 cells [Streptococcus is an important bacterium that colonizes the upper respiratory airways. This bacterium is not only the most common cause of community-acquired pneumonia but also an essential risk factor for several life-threatening diseases, including sepsis . Previouithelium . Similarfections , 82. Fur49 cells . RegrettMycobacterium tuberculosis, especially in the respiratory tract [Moreover, tuberculosis (TB) is a typical example of a specific infection and the top infectious disease killer worldwide. TB mainly refers to infections with ry tract , 85. Recry tract .Acute respiratory distress syndrome (ARDS) refers to the severe clinical condition of dyspnea, refractory hypoxemia, and noncardiogenic pulmonary edema and affects millions of people throughout the world. According to existing statistics, the etiology of ARDS is fairly complex, varying from severe pneumonia, sepsis, and major trauma to massive transfusion, and severe pneumonia or sepsis may be the major cause . NeverthAlthough the mechanisms responsible for the pathogenesis of ARDS have not been fully understood, there is already evidence indicating the involvement of two essential and interactional factors, oxidative stress and inflammation. The overproduction of ROS or RNS, infiltration of inflammatory cells, and synthesis of inflammatory mediators have been proven to inflict severe lung damage. Recently, increasing evidence has demonstrated the importance of Nrf2 activation in coping with oxidative stress and inflammation in ARDS. A previous study showed that Nrf2 may act as a candidate gene of ARDS susceptibility for humans, as over 500 single-nucleotide polymorphisms (SNPs) of Nrf2 have been identified to date, and the risk of ARDS after severe trauma has increased in people with a functional NRF2 SNP in European and African American individuals . For exphttp://clinicaltrials.gov), which brings the hope of the clinical application of CDDO-Im. Similarly, vitexin and aucubin both mitigate inflammatory and oxidative injury along with the suppression of inflammatory signaling, such as NLRP3/NF-\u03baB, and the induction of Nrf2 in the LPS-induced ARDS model in wild-type but not Nrf2 knockdown mice/macrophages [\u03baB and activating the Nrf2 pathway and is also reported to alleviate lung injury in another oleic acid-induced ARDS model [Therefore, in recent years, numerous investigations have focused on the protection against ARDS by Nrf2 activators, especially in hyperoxia- or LPS-induced ARDS models. The oleanane triterpenoid CDDO-imidazole (CDDO-Im) is reported to activate Nrf2/ARE signaling by breaking the interactions between Keap1 and Nrf2 in the cytosol and confer protective effects such as the inhibition of pulmonary hemorrhage, proteinaceous edema, and inflammatory cell infiltration on hyperoxia-induced ARDS. However, these protective effects are almost abolished in Nrf2-deficient mice . Moreoverophages , 99. TheDS model , 101. InDS model \u2013107. MorDS model . FurtherDS model .Chronic obstructive pulmonary disease (COPD), a common chronic pulmonary disease characterized by irreversible airflow limitation, is projected to emerge as the third most prevalent cause of death by 2020 . The pat\u2212/\u2212 mice display augmented lung inflammation that cannot be alleviated by steroids and revealed that deficits in Nrf2 may play an essential role in steroid resistance via HDAC2 repression: the recruitment of HDAC2 is important in mediating the anti-inflammatory activities of glucocorticoids by its interaction with promoters of proinflammatory genes, while Nrf2 deficiency may significantly reduce histone deacetylase 2 (HDAC2) level and deacetylase activity [Exposure to cigarette smoke can lead to obvious oxidative stress, inflammation, and alveolar cell apoptosis. In healthy smokers, to cope with such high oxidant burden, there is a significant increase in numerous antioxidant defenses, among which Nrf2 is largely relied on. This reliance may be evidenced by the transient Nrf2 expression induced by CS in human airway cells. However, decreased levels of Nrf2 and its stabilizer DJ1 (PARK7) in lung tissues of COPD patients have been observed, and multiple human studies have demonstrated that the NRF2\u2013KEAP1\u2013BACH1 equilibrium in lung and alveolar macrophages is lowered in the population of aged smokers and COPD patients. Moreover, a cohort study on the relationship between polymorphisms of the Nrf2 gene and limitations of airflow in smokers also indicates that impaired Nrf2 may contribute to the development of COPD owing to excessive oxidant burden and apoptosis in the lungs . The sevactivity .Andrographis paniculata. Recent studies reveal that andrographolide has the ability to protect the lungs from oxidative injury caused by cigarette smoke and suppress nontypeable Haemophilus influenza- (NTHi-) increased inflammatory and oxidative lung injury in a CS-predisposed mouse model that imitates COPD exacerbation, while the mechanism of action may be attained by the induction of Nrf2-mediated cytoprotective responses [\u2212/\u2212 mice showed increased mortality and lung damage of increased severity after FluV infection [\u03b3-tocotrienol, and aspirin-triggered resolvin D1 (AT-RvD1) [Therefore, novel therapies, such as Nrf2 activators, may show promise for therapy in COPD patients. Several Nrf2 activators have the potential to protect against exposure to cigarette smoke. For example, upon chronic CS exposure, CDDO-Im induces a more significant upregulation of Nrf2 and its target genes, mitigating CS-induced lung oxidative stress, tissue destruction, and even pulmonary hypertension in wild-type mice; however, these protective effects are not obviously observed in Nrf2-deficient mice. Additionally, it is interesting that CDDO-Im shows no inhibitory effect on CS-induced inflammation, despite its prevention of emphysema development, which may suggest that the inhibition of oxidative stress is enough to interrupt the development of emphysema . Similaresponses , 123. Innfection . TherefoAT-RvD1) \u2013127.\u2212/\u2212 mice but also causes higher levels of neutrophils, which may be responsible for airway remodeling in severe asthma. In turn, eosinophils and neutrophils generate more ROS to cause damage to the lung. In addition, the disruption of Nrf2 may also be associated with more obvious AHR, goblet cell hyperplasia, epithelial cell apoptosis, and an elevated level of Th2 cytokines in this model. In the DEP-challenged asthma model, Nrf2\u2212/\u2212 mice also display increased eosinophils, AHR, IL-12, IL-13, and thymus and activation-regulated chemokines (TARC) in BALF. Moreover, when dendritic cells (DCs) from both Nrf2-deficient and wild-type cells were exposed to particulate matter (PM), Nrf2 successfully restrains the production of a proallergic phenotype via the inhibition of oxidative stress and Th2-directed proallergic immunity regulated by DCs [Asthma is a complex respiratory disorder characterized by chronic airway inflammation, airway hyperreactivity (AHR), and extensive and polytropic reversible airway obstruction . Supportd by DCs . During 2-induced murine asthma model defined as irritant-induced asthma (IIA) and DEP-stimulated airway epithelial cells, sulforaphane administration with augmented Nrf2 activity inhibited airway inflammation [\u03b3, and the mechanism of action may derive from Nrf2-regulated Th1/Th2 balance [\u03b1-tocopherol) is also reported to protect against IgE-induced asthma by reversing the impairment of Nrf2 activity in alveolar macrophages in vivo and alleviating asthma exacerbation stimulated by ozone in the OVA-induced murine model via the Nrf2 pathway, although its effect is absent on OVA-induced asthma symptoms [In recent decades, the protective role of Nrf2 inducers in asthma has been extensively investigated, and ovalbumin is used as a classical asthma inducer. Until recently, different studies have examined the protection of sulforaphane against asthma. Sulforaphane administration can suppress ovalbumin-induced allergic airway inflammation in mice, and a recent study further investigated the protective effects of sulforaphane in asthmatics. The results reveal that Nrf2 signaling may play an essential role in individuals whose bronchoprotective responses against methacholine (MCh) are improved by sulforaphane , 136. Moammation , 138. Noammation . Therefo balance . The welsymptoms , 142. Mosymptoms \u2013145.\u03b21, which can accelerate ROS generation, existing inflammation, and lung scarring as well as suppress antioxidant gene expression by mediating the interaction of Smad3-ATF3 with Nrf2. Moreover, ROS play an essential role in myofibroblastic differentiation, which is intimately involved in the pathogenesis of IPF. Therefore, it is not surprising that the dysregulation of Nrf2, a master regulator of oxidative stress, is reported to contribute greatly to pulmonary fibrosis [Idiopathic pulmonary fibrosis (IPF), an important representative of interstitial lung disease, is described as a chronic progressive lung disease with fibroproliferation and excessive extracellular matrix (ECM) deposition, which ultimately results in irreversible lung interstitial fibrosis and respiratory failure . Althougfibrosis . In prevfibrosis . In vitr\u03b2- (TGF-\u03b2-) stimulated fibroblasts and bleomycin-challenged murine models is associated with the restoration of Nrf2/Bach1 equilibrium through Bach1 inhibition and Nrf2 activation [\u03b1-SMA, collagen I, fibroblast proliferation, migration, and contraction), even under TGF-\u03b2 stimulation, and the antifibrosis activity is dependent on the restoration of redox balance by Nrf2 activation. However, studies have reported that sulforaphane cannot protect against bleomycin-induced lung fibrosis in mice, which may be related to the fact that this model does not address the effects of Nrf2 activators in lung fibrosis due to the absence of Nrf2 activation in mouse lung fibroblasts [\u03b2 and p-Smad expression. In addition, emodin can reverse recombinant TGF-\u03b21-stimulated EMT-like shifts in alveolar epithelial-cultured cells [\u03b2-induced fibrosis in fibroblasts is also at least partly mediated by HO-1, a main downstream target of Nrf2 [Several Nrf2 activators have been studied to protect against pulmonary fibrosis. Pirfenidone (PFD) is a current approved drug for the therapy of IPF, and its antifibrosis activity in transforming growth factor-tivation . The claroblasts . Anothered cells . Additio of Nrf2 , 154. Ap of Nrf2 , 155.Lung cancer is reported to be the leading cause of cancer-related mortality worldwide . In all G12D-driven genetic lung cancer mouse model, Nrf2 is able to inhibit lung cancer initiation in a vinyl carbamate/urethane-induced model but promotes existing tumor progression in either model [To decrease the incidence of cancer, people attempt to apply chemicals to detoxify or remove carcinogens . Among ter model , 164. Aner model . The poser model . In the er model . These fer model . Accordier model . Keap1 der model .Considering the double effect of Nrf2 on lung cancer, the application of the Nrf2 activator in the early stages of carcinogenesis to prevent cancer may be more promising. Indeed, several well-known Nrf2 inducers have been studied in this respect. One of the most potent activators of Nrf2, sulforaphane, activates Nrf2 signaling via its impact on Keap1 and exerts suppressive effects on benzo(a)pyrene- (B(a)P-) initiated lung carcinogenesis in mice . IntriguOxidative stress has been reported to participate in the occurrence and development of various respiratory diseases, including BPD, respiratory infection, ARDS, COPD, asthma, IPF, and lung cancer. Thus, the master antioxidant transcription factor Nrf2, which is abundant in the lungs, drives diverse cellular defense pathways to counteract detrimental stimuli that are involved in the pathogenesis of these pulmonary disorders, including oxidative stress, inflammation, apoptosis, and carcinogenesis. Currently, Nrf2-deficient mice have provided an effective tool for investigating Nrf2 function in oxidative pulmonary disease models and have led to a better understanding of Nrf2 function in the pathogenesis of related pulmonary diseases. In fact, researchers have adopted three different genetic backgrounds and lung-specific conditional knockout mice to conduct experiments to obtain insight into the protective roles of Nrf2. Therefore, in recent decades, a large number of studies have focused on the protective effects of Nrf2 activators on the above pulmonary diseases and found that certain compounds may provide new strategies to intervene or prevent oxidative airway diseases via Nrf2 induction."} +{"text": "Nrf2 is a master transcriptional regulator of antioxidant and cytoprotective pathways. Currently in its third decade, research on Nrf2 has expanded to encompass not only basic but also clinical studies. In the present bibliometric review, we employed the VOSviewer tool to describe the existing Nrf2 literature landscape. As of July 2019, 11,931 papers on Nrf2 were listed in the \u201cWeb of Science\u201d database, with more than 1000 new papers published each year. As expected, terms related to oxidative stress and antioxidant molecules occur very often in the Nrf2 literature throughout the years. Interestingly, there is also a gradual increase in the occurrence of terms related to diseases or to natural compounds, the most prominent being sulforaphane, curcumin, and resveratrol that modulate the Nrf2 pathway. Going beyond molecular biology/biochemistry and related fields, Nrf2 research has begun to spread into more clinical areas like endocrinology/metabolism, cardiology, and nephrology, likely reflecting an increased interest in clinical applications of Nrf2 pathway activators. China has become the most prolific producer of Nrf2 papers the last five years followed by the USA and Japan, a reverse pattern compared to the past. In conclusion, Nrf2 is the subject of a globally active research field that keeps growing and extends from bench to bedside. The nuclear factor, erythroid 2-like transcription factor 2 (Nrf2) pathway orchestrates the expression of antioxidant and cytoprotective genes. Its activity is induced upon exposure to oxidative or electrophilic stresses, including the so-called indirect antioxidant compounds ,2,3. TarIn the Nrf2 research community, it is well-known that historically one major research focus had been on basic studies trying to decipher the mechanism of Nrf2 regulation by its cytoplasmic inhibitor, kelch-like ECH-associated protein 1 (Keap1) . AnotherThe discovery that natural compounds, such as sulforaphane from broccoli sprouts, can activate the Nrf2 pathway has alsoDuring the last 10 years, several excellent reviews have been published about Nrf2 and have summarized the progress in Nrf2-related research through the perspective of basic ,31,32,33The Web of Science online database was accessed in July 2019 and the following search terms were employed in an \u201cadvanced search\u201d: (ALL = (Nrf2 NOT \u201cnuclear respiratory factor\u201d)), \u201clanguage: English\u201d and \u201cdocument type: \u201cArticle\u201d. The database \u201cWeb of Science Core Collection\u201d was selected. This eliminated papers related to nuclear respiratory factors 1 and 2 that are also commonly abbreviated as \u201cNrf1\u201d and \u201cNrf2\u201d, respectively, leading to confusion. Of note, inclusion or exclusion of additional search terms referring to Nrf2 synonyms such as NFE2L2, NFE2-L2, Nrf-2, etc.) had no impact on the search results. By selecting \u201ccustom year range\u201d timespan was set to between 1990 and 2019, and this was subsequently sub-divided into four periods . Web of Science citation files for selected periods were downloaded as \u201cfull record and cited references\u201d and saved in a \u201ctab-delimited\u201d file format. https://www.wordfrequency.info/free.asp?s=y), were excluded from the analysis, as previously described [The citations extracted using the aforementioned search strategy were imported into VOSviewer for biblescribed . The terThe aforementioned search for Nrf2 in \u201cWeb of Science\u201d resulted in 11,931 publications. The first paper about Nrf2 that meets the search terms was published in 1994. At that time, it was not referring to its cytoprotective roles, but it was focused on a search for regulators of the \u03b2-globin gene . Only afAs the publications per year on Nrf2 are gradually increasing, the following shorter time periods were defined: First, from 1990 to 2005, because the first publication on Nrf2 appeared in 1994, and only few publications on Nrf2 came out over the first decade that followed. Then, five-year intervals were defined, namely 2006\u20132010, 2011\u20132015, and 2016\u2013to date (July 2019). The term maps generated after analysis of each respective time period using VOSviewer are shown in The most common terms appearing in Nrf2-related publications reflect the models used for the study of the pathway. Terms related to mice and rats appear throughout the years , becauseStudies on the role of Nrf2 in the pathophysiology or prevention of diseases started to appear mainly after 2006. One exception is the potential of Nrf2 as a target for chemoprevention, which has been recognized before 2005. Starting from 2006 to 2010, besides carcinogenesis, other diseases feature prominently in Nrf2-related studies, notably Parkinson\u2019s disease, Alzheimer\u2019s disease and chronic obstructive pulmonary disease . Even moThe main research areas of Nrf2 papers are \u201cbiochemistry/molecular biology\u201d, \u201cpharmacology/pharmacy\u201d, \u201ccell biology\u201d and \u201ctoxicology\u201d as they are always in the top five research areas over the years A\u2013D. EvenInitially (between 1990 and 2005), papers about Nrf2 originated mainly in the USA (~60%) and Japan (~30%) . GradualTo obtain a better understanding of the contributions of each country to the Nrf2 research field and the collaborations between countries, data for the whole period (1990\u20132019) were analyzed with a focus on international collaborations, the times each country cites the work of another, and the number of citations these papers have received. As depicted in This bibliometric analysis of Nrf2-related literature to date revealed some interesting and useful facts. Firstly, the field of Nrf2-related research is continuously expanding, as reflected in both the accelerating pace of new Nrf2-related publications (currently close to 2000 per year) and the increasing variety of research areas involved. Specifically, Nrf2 papers are no longer limited to basic research categories , but they also encompass more clinical areas . This trFree Radical Biology and Medicine\u201d, \u201cJournal of Biological Chemistry\u201d and \u201cOxidative Medicine and Cellular Longevity\u201d that mainly publish basic studies or preclinical studies with mouse or rat models. In the top 10 are also two major open access journals that publish research from any discipline (\u201cPLoS One\u201d and \u201cScientific Reports\u201d) and \u201cBiochemical and Biophysical Research Communications\u201d that publishes short but innovative research reports. Pharmacology-toxicology journals also publish a substantial proportion of Nrf2-related research, reflecting not only the interest in Nrf2 as a drug target but also the defensive roles of Nrf2 against toxic insults. Examples of such journals in the top 10 include \u201cFood and Chemical Toxicology\u201d, \u201cToxicology and Applied Pharmacology\u201d and \u201cBiomedicine and Pharmacotherapy\u201d. The list also includes journals dedicated to specific medical fields , reflecting the attention that Nrf2 has started to attract from experimental medicine and from the various clinical specialties (The list of journals that have published at least 20 papers on Nrf2 from 1990 to 2019 indicatecialties . This isNatural compounds that activate the Nrf2 pathway were among the frequently occurring terms, including the general category of isothiocyanates, the isothiocyanate sulforaphane, curcumin, resveratrol, etc. . TherefoAnother interesting aspect of the bibliometric analysis is the visualization of global trends in the output of Nrf2-related research papers, as illustrated in One limitation of this type of bibliographic analysis is that this approach reflects only the quantity of research papers, the frequency of occurrence of specific terms, the research output of various countries and the citations their papers receive. It thus does not assess the quality of the respective publications, neither does it correct for the factors such as a country\u2019s population size, total research output, the gross domestic product, resources invested in research, etc. These considerations should be taken into account when interpreting or communicating the countries data emanating from the present bibliographic analysis. As a relative measure of quality, impact or usefulness of the published papers, the averaged citation count was calculated for each country A. This iNevertheless, the advantage of this type of analysis is that it is unbiased. It can illustrate where the Nrf2 field has been heading and it can also highlight areas where Nrf2 has not yet been thoroughly investigated.In conclusion, the present analysis revealed that Nrf2 is in the spotlight of research worldwide and it is expected that we will continue to see an increasing research output in the near future. Nrf2-focused research started from basic science but has expanded to encompass multiple clinical perspectives. A particular clinical focus is on natural compounds that modify Nrf2 activity, whose pharmacological applications in health and disease are being actively explored."} +{"text": "Remote ischemic conditioning (RIC) is a procedure applied in a limb for triggering endogenous protective pathways in distant organs, namely brain or heart. The underlying mechanisms of RIC are still not fully understood, and it is hypothesized they are mediated either by humoral factors, immune cells and/or the autonomic nervous system. Herein, heart rate variability (HRV) was used to evaluate the electrophysiological processes occurring in the heart during RIC and, in turn to assess the role of autonomic nervous system.Healthy subjects were submitted to RIC protocol and electrocardiography (ECG) was used to evaluate HRV, by assessing the variability of time intervals between two consecutive heart beats. This is a pilot study based on the analysis of 18 ECG from healthy subjects submitted to RIC. HRV was characterized in three domains that can be correlated with the autonomic nervous system function.RIC procedure increased significantly the non-linear parameter SD2, which is associated with long term HRV. This effect was observed in all subjects and in the senior (>\u200960\u2009years-old) subset analysis. SD2 increase suggests an activation of both parasympathetic and sympathetic nervous system, namely via fast vagal response (parasympathetic) and the slow sympathetic response to the baroreceptors stimulation.RIC procedure modulates both parasympathetic and sympathetic autonomic nervous system. Furthermore, this modulation is more pronounced in the senior subset of subjects. Therefore, the autonomic nervous system regulation could be one of the mechanisms for RIC therapeutic effectiveness.The online version of this article (10.1186/s12872-019-1181-5) contains supplementary material, which is available to authorized users. Organisms have developed endogenous mechanisms of defence against external aggressions. Therapeutic strategies enhancing these mechanisms can be more efficient and safer than pharmacological exogenous treatments. Hormesis or conditioning, which is a procedure by which noxious stimuli below the threshold of damage are applied to a tissue or system, promotes cellular tolerance against more severe stimuli . InteresThe involvement of the autonomic nervous system was discovered when pharmacological blockade of ganglionic neurons inhibited RIC in animal models of cerebral and heart ischemia \u20135. In exRIC clinical studies in myocardial infarction patients have reported reductions in infarcted area, as well as an improvement of left-ventricle ejection fractions, reduction of creatinine-kinase myocardial plasma release or even ST-segment elevation resolution \u201314. In aHerein, a pilot study was performed to evaluate potential alterations in autonomic nervous system due to RIC. In healthy subjects, heart rate variability (HRV) was assessed through electrocardiography (ECG) during RIC procedure. HRV studies the variation between the interval of consecutive beats and it can be described by a set of features correlating these variations with the autonomic nervous system . MeasuriA total of 20 subjects were selected according to two age subgroups: senior and young. Senior subjects were recruited in our hospital volunteers association: \u201cLiga dos Amigos do Hospital Sao Francisco Xavier\u201d, while the younger subjects were recruited among Nova Medical School. The following exclusion criteria were applied: Any previous neurological disease or neurosurgical procedure, severe heart failure (NYHA class III or higher), peripheral artery disease, skin ulcers or other severe dermatological disease. Subjects were also excluded per investigator judgment if they had any unstable/severe disease. Subjects were screened for vascular risk factors and current medication, which is summarized in Additional\u00a0file\u00a0https://www.biosignalsplux.com/datasheets/ECG_Sensor_Datasheet.pdf). The interface with the body is made through Ag/AgCl electrodes with a solid adhesive gel. PLUX\u00ae also provided two BVP sensors and the data acquisition module . The data was streamed via Bluetooth to a nearby computer at 1000\u2009Hz sampling rate and 16-bit resolution.For this study, ECG and blood volume pulse (BVP) signals were recorded during the RIC procedure. BVP signal was used to confirm blood occlusion. The ECG signal was recorded using a 1-lead local differential bipolar sensor from PLUX\u00ae , placed on the left chest, above the heart. This sensor has an input range of +\u2009\u2212\u20091.5\u2009mV, a signal band width of 0.5\u2013100\u2009Hz, an input impedance of >100GOhm and a common mode rejection ratio of 100\u2009dB to allow the occlusion of the brachial artery. Keep the cuff inflated for 5-min;Deflate the cuff. Keep the cuff deflated for another 5-min;Repeat the inflation (occlusion) and deflation (non-occlusion) 3 times more;Keep the cuff deflated for another 5-min;The protocol can be summarized as follows:All subjects were under the same conditions, namely: RIC procedure was applied between 9 to 10\u2009am, in a quiet and isolated room, free from distraction. Subjects were not fasted and have taken their usual medication.According to , the calThe features assessed from the HRV can be divided in three domains: time, frequency and non-linear domain.In the time domain, the most commonly used features are the mean R-R interval, median, root mean square of successive differences (rMSSD) and the pNN50 . Using tIn the frequency domain, the normalized low-frequency (LF) and high-frequency (HF) power spectra were calculated, as well as their ratio. The literature specifies that the LF spectrum covers frequencies from 0.04\u2009Hz to 0.15\u2009Hz, while the HF spectrum ranges from 0.15\u2009Hz to 0.4\u2009Hz . Since RHF band reflects faster changes of heart rate, which are associated with parasympathetic activity. In fact, parasympathetic activity produces responses that have a higher frequency compared to those from sympathetic branch . The LF Finally, in the non-linear domain, the Poincar\u00e9 plot is used to describe the nature of R-R interval fluctuations, by plotting a certain R-R interval (R-Rn) versus the next one R-Rn\u2009+\u20091) . The res . The reIn Eq.\u00a0In Eq.\u00a0SD1 is mostly influenced by the parasympathetic activity, while SD2 is influenced by both the sympathetic and parasympathetic activities.The analysed parameters were summarized into tables and filtered by age. In Additional\u00a0file\u00a0The non-parametric Wilcoxon signed-rank test was used to statistically analyse the RIC procedure effect on the different subjects. For each HRV parameter, it was analysed and compared: (i) sample pairs for occlusion and non-occlusion intervals and (ii) sample pairs for the first 10\u2009min before the procedure and the 10\u2009min after the last occlusion. Furthermore, same analysis was done in young and senior population subsets.p\u2009<\u20090.05). Out of the 18 test subjects, 1 of the tested subjects had to be removed from the statistical analysis due to the lack of one time interval (before procedure). The correlation studies between changes in SD2 and in pNN50 and rMSSD were also done by non-parametric Wilcoxon test.The used significance level was 0.05 and using the following libraries: NumPy (v. 1.10.4), SciPy (v. 0.16.1) and Nova instrumentation (version 1.0).n\u00a0=\u200918), 11 (61.1%) were female, and mean age was 47.0\u2009\u00b1\u200921.9\u2009years. For detailed information on vascular risk factors and current medication see Additional\u00a0file\u00a0Among the 20 subjects initially included, 10 in each age subgroup, two of the senior subjects were excluded from our analysis due to noisy segments, not allowing the R peak detection. On total are represented in Additional\u00a0file\u00a0During aging, there is a decrease of endogenous response to stress and organism defences. Thus the use of two subsets (young and senior) can eventually disclose potential different responses of autonomic nervous system to RIC and putative novel mechanisms associated with aging processes. Furthermore, co-morbidities associated with aging such as risk of cardiovascular disease or Diabetes Mellitus might also influence autonomic nervous system response.The non-linear feature SD2 is the single parameter that significantly increases after RIC procedure in both global and with rMSSD (r\u2009=\u20090.54 with p\u00a0=\u20090.01), which are both associated with parasympathetic system, being rMSSD via vagal response. Nevertheless, one cannot exclude the involvement of sympathetic activity, since SD2 is related with both branches of autonomic nervous system.Because the sample size is limited and in order to further evaluate the role of RIC on autonomic nervous system, correlations between key features were also performed for all subjects. For each subject and feature the difference between its value before and after RIC procedure was calculated. Then, the differences between features were correlated. Changes in SD2 were significantly and positively correlated with changes in pNN50 and rMSSD. These results reinforce the RIC involvement of parasympathetic system via vagal response, since SD2 positively correlates with pNN50 in senior subset . In fact, in individuals older than 60\u2009years-old, the differences in the HRV features between genders are negligible due to mIt was expected to find significant differences in HRV parameters during the alternation of occlusion and non-occlusion procedures, since there is disturbance introduced into the organism. Nevertheless, in global analysis only rMSSD parameter increased during non-occlusion period, suggesting higher parasympathetic activity.RIC-induced cytoprotection can be promoted by: (i) modulation of autonomic nervous system, (ii) production and release of bio-molecules and/or (iii) immune cells signalling. In fact, in experimental models RIC also mediates distant organ protection by the release of blood-borne factors. In mice, RIC increases nitric oxide levels, which induces vasodilation and increases cerebral blow flow, besides protecting mitochondria from oxidative stress . Other aThe first limitation concerns the sample size. In fact, the present work is a pilot study to assess the potential role of autonomic nervous system in RIC and to further explore the underlying mechanisms of RIC on distant organ protection. Further studies with increased number of subjects are crucial to deeper assess the role of autonomic nervous system. The second limitation is the time window of ECG recording and HRV analysis. RIC-induced modulation of autonomic nervous system was only assessed during RIC procedure and at short periods (10\u2009min) before and after it. Therefore, further studies must be performed to assess a potential second window of autonomic nervous system modulation, namely at 2-3\u2009h and/or 24\u2009h following RIC procedure. Finally, more frequent RIC procedures (such as daily frequency) may also amplify the effect of RIC on sympathetic and parasympathetic activities.Electrocardiography (ECG) was used to study the effects of remote ischemic conditioning (RIC) in autonomic nervous systems of healthy subjects. RIC procedure significantly increased the non-linear parameter SD2. Finally, this data suggests that autonomic nervous system involvement could be one of the mechanisms for RIC therapeutic effectiveness.Additional file 1:Figure S1. Global population boxplots for each phase of the RIC procedure. Each graph corresponds to one HRV feature: Mean R-R intervals (ms); median R-R intervals (ms); percentage of intervals falling outside a 50\u2009ms difference, pNN50 (%); root mean square of successive differences of the R-R interval values per event (rMSSD (ms); normalized low frequency power spetrum density, nuLF PSD (%); normalized high frequency power spectrum density, nuHF PSD (%); LF and HF normalized power spectrum density ratio, LF/HF; SD1 axis of the Poincar\u00e9 plot, SD1 axis (ms); SD2 axis of the Poincar\u00e9 plot, SD2 axis (ms) and SD1/SD2 per event. (TIFF 293 kb)Additional file 2:Figure S2. Global population boxplots comparing occlusion and non-occlusion intervals. Each graph corresponds to one HRV feature: Mean R-R intervals (ms); median R-R intervals (ms); percentage of intervals falling outside a 50\u2009ms difference, pNN50 (%); root mean square of successive differences of the R-R interval values per event (rMSSD (ms); normalized low frequency power spetrum density, nuLF PSD (%); normalized high frequency power spectrum density, nuHF PSD (%); LF and HF normalized power spectrum density ratio, LF/HF; SD1 axis of the Poincar\u00e9 plot, SD1 axis (ms); SD2 axis of the Poincar\u00e9 plot, SD2 axis (ms) and SD1/SD2 per event. Red lines correspond to mean value, black lines correspond to median value and * p-value <\u20090.05. (TIFF 129 kb)Additional file 3:Table S1. Subjects baseline characteristics: Demographics, relevant cardiovascular risk factors and medication. (PDF 44 kb)Additional file 4:Table S2. Global population analysis for the first and last 10\u2009min and occlusion and non-occlusion intervals. For the first and last 10\u2009min analysis, the mean values are presented as well as a comparison between them and the p-value for the Wilcoxon signed-rank test. For the occlusion and non-occlusion interval analysis, the mean values are presented as well as a comparison between them and the p-value for the Wilcoxon signed-rank test. (PDF 60 kb)Additional file 5:Table S3. Senior population analysis for the first and last 10\u2009min and occlusion and non-occlusion intervals. For the first and last 10\u2009min analysis, the mean values are presented as well as a comparison between them and the p-value for the Wilcoxon signed-rank test. For the occlusion and non-occlusion interval analysis, the mean values are presented as well as a comparison between them and the p-value for the Wilcoxon signed-rank test. (PDF 60 kb)Additional file 6:Table S4. Young population analysis for the first and last 10\u2009min and occlusion and non-occlusion intervals. For the first and last 10\u2009min analysis, the mean values are presented as well as a comparison between them and the p-value for the Wilcoxon signed-rank test. For the occlusion and non-occlusion interval analysis, the mean values are presented as well as a comparison between them and the p-value for the Wilcoxon signed-rank test. (PDF 60 kb)"} +{"text": "Type II endoleak is a common complication following endovascular aortic aneurysm repair and can lead to an increased risk of aneurysmal expansion and rupture. The most frequently employed strategies to treat Type II endoleak involves catheterization of the branch vessels responsible for the endoleak or accessing the aneurysm sac through a percutaneous approach. An endovascular transcaval approach for embolization of the aneurysmal sac provides an alternate strategy with comparable success rates. This technique is advantageous when the endoleak is predominantly on the right side of the aneurysm sac and/or when a direct access to the aneurysm sac through a percutaneous approach is not feasible. Endovascular abdominal aortic aneurysm repair (EVAR) is the standard of care for the treatment of most aneurysms greater than 5.5\u2009cm diameter and continued sac expansion (>\u20090.5\u2009cm) provides an additional strategy with comparable success rates probe is used to direct the tip of a vascular sheath against the inferior vena cava (IVC) wall near the site of the T2E identified on IVUS as patent cavities or inflow vessel tracts. The aneurysm sac is then punctured with a needle through the sheath using fluoroscopy and/or IVUS. Access of the endoleak cavity is confirmed by injection of dye. Wire and catheter are used to further select the endoleak cavity as needed. A microcatheter is eventually placed into the aneurysm sac and the endoleak cavity is embolized with coils, liquid embolics, or bothA 75-year-old man underwent EVAR for a 6.1\u2009cm abdominal aortic aneurysm. On follow-up CT angiography (CTA) imaging obtained 9\u2009months later the aneurysm measured 6.8\u2009cm and a T2E was seen arising from a lumbar artery . The right femoral vein micropuncture sheath was exchanged for a Rosch-Uchida transjugular liver access set . The left femoral micropuncture sheath was exchange for a 9F vascular sheath through which an IVUS probe was advanced into the IVC.Under fluoroscopic and IVUS guidance the aneurysm sac was accessed near the endoleak with the Rosch-Uchida liver access set Fig.\u00a0. The innNext, the microcatheter was flushed with 5% dextrose solution. Ethylene vinyl alcohol liquid embolic (Onyx\u00ae18) was then administered through the microcatheter into the aneurysm sac, in the region of the T2E, under sonographic and fluoroscopic guidance Figs.\u00a0. The embReal-time visualization of the access needle using \u201cside-firing\u201d IVUS probes is invaluable when performing TCE. First, it allows the endoleak cavity to be directly targeted. Targeted embolization of the endoleak cavity decreases the pressure within the aneurysm, increasing the likelihood of treatment success. Cessation of color Doppler flow on IVUS also determines the endpoint of embolization approach to managing T2E. We believe TCE can potentially have a higher technical success rate and a decreased risk profile when compared with other more common catheterization techniques. Operators may consider TCE as a feasible first line alternative approach in the management of T2E."} +{"text": "P. falciparum-infected erythrocytes (IEs), where they contribute to the pathogenesis of malaria and are important targets of acquired immunity. Although the PfEMP1-specific antibody response is dominated by the opsonizing and complement-fixing subclasses IgG1 and IgG3, activation of the classical complement pathway by antibody-opsonized IEs does not appear to be a major immune effector mechanism. To study the molecular background for this, we used ELISA and flow cytometry to assess activation of the classical component pathway by recombinant and native PfEMP1 antigen opsonized by polyclonal and monoclonal PfEMP1-specific human IgG. Polyclonal IgG specific for VAR2CSA-type PfEMP1 purified from a pool of human immune plasma efficiently activated the classical complement pathway when bound to recombinant PfEMP1 in ELISA. In contrast, no activation of complement could be detected by flow cytometry when the same IgG preparation was used to opsonize IEs expressing the corresponding native PfEMP1 antigen. After engineering of a VAR2CSA-specific monoclonal antibody to facilitate its on-target hexamerization, complement activation was detectable in an ELISA optimized for uniform orientation of the immobilized antigen. In contrast, the antibody remained unable to activate complement when bound to native VAR2CSA on IEs. Our data suggest that the display of PfEMP1 proteins on IEs is optimized to prevent activation of the classical complement pathway, and thus represents a hitherto unappreciated parasite strategy to evade acquired immunity to malaria.Members of the PfEMP1 protein family are expressed on the surface of Plasmodium, but P. falciparum is responsible for most severe cases and essentially all malaria mortality on the surface of the infected erythrocytes (IEs) (The particular virulence of es (IEs) . This enes (IEs) \u20137. The eP. falciparum malaria, and semi-immune individuals living in areas of stable parasite transmission possess a broad repertoire of PfEMP1-specific antibodies, dominated by the opsonizing and complement-fixing subclasses IgG1 and IgG3 was produced in ExpiCHO-S cells as a recombinant C-terminal histidine-tagged protein, as described elsewhere .1-chain and the \u03ba-chain PAM1.4 , which i \u03ba-chain . This an \u03ba-chain .1-chain, using the QuickChange site-directed mutagenesis kit (Agilent) according to manufacturer's instructions. Briefly, mutations were introduced by a PCR reaction of the entire plasmid with a high-fidelity DNA-polymerase and a complementary primer pair with the desired mutation . Plasmids were subsequently sequenced to confirm the introduction of the substitutions. All the recombinant antibodies were produced in human embryonic kidney cells according to manufacturer's instructions. Briefly, the cells were grown to ~1.5 \u00d7 106 cells/mL, and adjusted to 1 \u00d7 106 cells/mL 1 h prior to transfection, and co-transfected with heavy- and light chain plasmids (0.5 \u03bcg DNA/plasmid/mL culture) and FreeStyle MAX reagent (1 \u03bcL/\u03bcg DNA). Cultures were incubated for seven days before harvesting the supernatant. The recombinant antibodies were purified using Protein G-coupled agarose beads (Pierce).We also produced two variants of this antibody (PAM1.4-E345K and PAM1.4-E430G) by introducing single-nucleotide substitutions in the \u03b3P. falciparum parasites expressing VAR2CSA-type PfEMP1. The samples were collected in a previous study approved by the Institutional Review Board of Noguchi Memorial Institute for Medical Research, University of Ghana (study no. 038/10-11) . Samples8/10-11) .Flat-bottomed 96-well plates (Nunc) were coated with FV2 (2 \u03bcg/mL), mAbs , purified human IgG (Invitrogen), or human serum albumin . Wells were blocked with PBS supplemented with TWEEN (0.05%) and BSA (1%) and subsequently incubated with the above-mentioned VAR2CSA-specific mAbs or with control reagents and incubated in the same buffer supplemented with non-immune human serum as source of complement components. After washing as above, bound complement components were detected with polyclonal rabbit anti-human C1q , rabbit anti-human C4c , rabbit anti-human C3c , or monoclonal mouse-anti human-terminal complement complex . All the complement-specific antibody reagents were biotinylated prior to the experiments. After the last washing as above, bound antibody was detected by incubation with streptavidin-conjugated horse radish peroxidase , followed by TMB ONE (ECO-TEK). The color reaction was terminated with H2SO4 (0.2 M), and quantified at 450 nm. Arbitrary units (AU) were calculated as (ODTest - ODBlank) / (ODControl - ODBlank).After incubation with antibody reagents, the plates were washed four times in Barbital-Tween buffer [sodium barbital (4 mM), NaCl (145 mM), CaClP. falciparum laboratory isolate IT4/FCR3 was cultured in serum-free medium as described elsewhere (The lsewhere .Parasites were selected by antibody panning and density separation monthly to ensure expression of the VAR2CSA-type PfEMP1 IT4VAR04 and IE surface knobs as described previously . Briefly6 cells/mL) were incubated with mAbs (10 \u03bcg/mL) or purified pooled human IgG (1 mg/mL), followed by incubation with NHS (1%) and compstatin (to inhibit cleavage of C3). As positive and negative controls, type A and 0 erythrocytes were incubated with type 0 NHS and compstatin.Late trophozoite-stage IEs were purified by magnet-activated cell sorting (Miltenyi Biotec) in PBS supplemented with BSA (1%) . The pur++ buffer containing Ca2+ and Mg2+ (Complement Technology). Samples were run on a Cytomics FC500 flow cytometer (Beckman Coulter). Single ethidium bromide-positive cells were analyzed for complement components and antibody labeling by FlowLogic (Inivai Technologies).Complement components were detected by incubation with the same antibodies as above, but at different concentrations , followed by incubation with FITC-conjugated goat anti-rabbit IgG and ethidium bromide . Binding of human IgG to the IEs was detected in a similar way, using FITC-conjugated goat anti-human IgG . All incTo our knowledge, the ability of PfEMP1-specific human IgG to activate the classical complement pathway has not been reported previously. Binding of C1q Figure , depositWe next assessed the ability of human IgG purified from the same plasma pools to activate complement when bound to IEs expressing the native PfEMP1 (IT4VAR04) represented by FV2. IgG purified from the highly FV2-reactive pool efficiently labeled the IEs, in contrast to the low FV2-reactivity IgG Figure . HoweverIt was recently reported that complement activation by IgG requires on-target, Fc-dependent hexamerization of the antibody . We therAll three mAbs had similar ability to activate complement when coated directly to ELISA plates, as binding of C1q Figure , as wellIn the studies first identifying the complement activation-enhancing mutations, the assays involved cell lines over-expressing the targeted antigen , 29. We in vitro assays of classical complement pathway activation.Our data confirm that the E345K and E430G mutations in the Fc-region enhance the ability of IgG to activate complement, probably by facilitating on-target hexamerization. Furthermore, the results highlight antigen orientation as an important parameter in To investigate whether the lack of complement activation on IEs Figure could beP. falciparum malaria and protectin (CD59) to prevent inadvertent phagocytosis and lysis of complement-opsonized erythrocytes. Although CD59 has been reported as the factor preventing complement-mediated lysis of IEs , IE lysiP. falciparum to evade acquired protective immunity.To conclude, we report that although PfEMP1-specific IgG can activate the classical complement pathway in a system where the antigens are homogeneously distributed, this appears not to happen at the IE surface, where PfEMP1 display is restricted to well-defined knobs. The most parsimonious explanation for this discrepancy is that the focal display of native PfEMP1 interferes with the on-target hexamerization of IgG, which is a requirement for binding of C1q and activation of the classical complement cascade. The knob-restricted display may thus represent a hitherto unrealized strategy of ML, LH, and PG formulated the hypothesis and designed the experiments and analyzed the data and wrote the paper. ML, MQ, SD, and RB-O produced the recombinant proteins. ML carried out all the experiments. MO was responsible for collection of the biological samples. All authors reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The effects of pH, PAC coagulant dose alone and with polymers dose in various combinations was studied by jar tests. To compare the removal efficiencies of turbidity, total suspended solids (TSS), chemical oxygen demand (COD), and color at different levels, we run multivariate analysis of variance. Regarding the economic evaluation, we applied the incremental cost-effectiveness ratio. PAC had the best performance in pH 7 and in optimal dose of 400\u00a0mg/L; so that removal efficiency of wastewater turbidity, TSS, COD and color were 99.63%, 99.7%, 47.5% and 50.38%, respectively. The best removal efficiency for wastewater turbidity, TSS, COD and color were 99.87%, 99.89%, 87.5% and 93.02%, respectively which were obtained by combination of anionic polymer (1.5\u00a0mg/L) with PAC (300\u00a0mg/L). Furthermore, with combination of PAC\u2009+\u2009anionic\u2009+\u2009non-ionic polymers, the removal efficiency for wastewater turbidity, TSS, COD and color were 99.93%, 99.94%, 88% and 94.57%, respectively. The imposed cost for treating one cubic meter of ceramic-tile wastewater treatment by PAC\u2009+\u2009anionic and PAC\u2009+\u2009anionic and non-ionic polymers in comparison with PAC alone was reduced to 22.96% and therefore economically more affordable for the tile industry wastewater treatment.Enhanced treatment of ceramic-tile industry wastewater was investigated by modified coagulation\u2013flocculation process using combination of poly Organic materials in wastewater are mainly produced from the additives used in decorating tiles .Characteristics of the ceramic-tile industry raw wastewater are presented in Table\u00a02(OH)nCl6\u2212n. YH2O, Al2O3\u2009=\u200930%wt, basicity\u2009=\u200965% and pH\u2009=\u20093) was used as a coagulant and anionic (A300), cationic (C270) and nonionic polymers, as the coagulant aids were provided from AquaTech Company, Switzerland.Poly-aluminum chloride . In order to prepare a 0.1 percent solution as coagulant aid, 0.1\u00a0g of each polymer was provided separately. Considering the polymers, a 0.1 percent solution was prepared; 0.05\u00a0g of each polymer was separately dissolved into 100\u00a0ml of distilled water at temperature of 30\u201350\u00a0\u00b0C . All experiments were carried out at a temperature of 25\u00a0\u00b0C. In order to determine the best sedimentation time, 1\u00a0L of the wastewater samples was poured into an Erlenmeyer flask at sedimentation time of 10\u2013120\u00a0min. Later, the wastewater turbidity and TSS removal were measured to determine the best time of sedimentation before coagulation and flocculation. In order to determine the optimal pH, we used the lime solution and normal hydrochloric acid and adjusted the pH in the range of 5\u201311. Then, the constant concentration of PAC (200\u00a0mg/L) was added to them using the jar test. The optimum pH was determined for each sample by measuring the removal efficiencies of turbidity, TSS, COD, and color and color were analyzed by using standard methods , chemical oxygen demand (COD), biochemical oxygen demand (BODods APHA . ParametCH) APHA .Data normality was investigated by Kolmogorov\u2013Smirnov and the homogeneity of data was determined using Levene test. To compare the turbidity, TSS, COD, and color of wastewater with the independent variables such as PH and different concentrations, we run the multivariate analysis (MANOVA) (P\u2009=\u20090.05). The Tukey test was also used to conduct multiple average comparisons between the groups.1 and C0 are the cost and E1 and E0 are the effects in the intervention and control groups, respectively.In order to evaluate the economic efficiency, we used the ICER statistical formula Eq.\u00a0. In thisThe effects of the primary sedimentation time on the TSS and turbidity removal efficiencies before adding a coagulant are represented in Fig.\u00a0The results of determining the optimal pH using a fixed dosage of PAC (200\u00a0mg/L) are represented in the Fig.\u00a0The effects of PAC dosage on the removal efficiency of turbidity, TSS, COD and color is shown in the Fig.\u00a0300) (0.5\u00a0mg/L) was observed 99.66% for turbidity, 99.72% for TSS, 85.12% for COD and 87.2% for colors. The best removal efficiency in combination of PAC (300\u00a0mg/L)\u2009+\u2009cationic polymer C270 was observed in 99.58% for turbidity, 99.64% for TSS, 52.5% for COD and 62.4% for color respectively. The highest removal efficiency in combination of 300\u00a0mg/L of PAC with non-ionic polymer were 99.41%, 99.48%, 76.25% and 55.03% for turbidity, TSS, COD and color, respectively. As a result, the applied polymers as coagulant aids decreased the efficient dose of PAC coagulants from 400\u00a0mg/L to 300\u00a0mg/L.In this stage of experiment, a constant dose of each polymer (0.5\u00a0mg/L) was added to different doses of PAC coagulants. The results are presented in Table\u00a0300, cationic C270, and non-ionic polymers on removal efficiency of turbidity, TSS, COD, and color are represented in Fig.\u00a0300 was 1.5\u00a0mg/L with removal efficiency of turbidity, TSS, COD and color 99.87%, 99.89%, 87.5% and 93.02%, respectively. The most removal efficiency was in 2\u00a0mg/L cationic polymer for turbidity (99.85%), TSS (99.88%), COD (65%) and color (89.14%). Furthermore, the most removal efficiency of non-ionic polymer for turbidity, TSS, COD and color were 99.68%, 99.73%, 86.5% COD and 84.88%, respectively, in concentration of 2\u00a0mg/L. As it is observed in Fig.\u00a0The purpose of this stage of jar-test experiment was to identify the suitable dose of polymers, which could be used in combination with optimal dose of PAC coagulant (300\u00a0mg/L) for treatment of the ceramic wastewater. The applied polymer dose varied from 0.5 to 3\u00a0mg/L. The effect of various doses of anionic AAt this stage of the experiment, the anionic, cationic, and non-ionic polymers were combined in different doses of 0.5\u20133\u00a0mg/L. Later, we investigated the effect of this composition combined with the optimal dose of PAC. The effect of various doses of anionic-nonionic polymer combination on removal efficiencies of turbidity, TSS, COD, and color is shown in Fig.\u00a0300/nonionic) and PAC\u2009+\u2009A300 were higher than those of other options. The economic evaluation formula (ICER) was applied to compare the costs of different methods used for ceramic- tile wastewater treatment; the results of which are tabulated in Table\u00a0The comparisons of removal efficiencies attributed to different coagulant-polymers combinations are indicated in Table\u00a0Removal efficiency of heavy metals including cadmium (Cd), chromium (Cr), nickel (Ni), zinc (Zn), lead (Pb), and boron (B) in the coagulation\u2013flocculation process under the optimum condition are represented in Fig.\u00a0In this study, the TSS and turbidity removal efficiencies at the primary sedimentation time of 100\u00a0min were 39.9% and 41.97%, respectively. Fahimnia et al. studied 2+ and Al(OH)2+). So, they neutralize the charges, adsorb the organic pollutants and solids, and consequently increase the removal efficiency , in which charge neutralization and adsorption mechanisms did not happen for the pollutant removal . As a result, application of polymers as coagulant aids decreased the efficient dose of PAC coagulant. Polymers act as aids in cleansing the water and wastewater. They even can be used as primary coagulants for some purposes showed the highest removal efficiency for turbidity, TSS, COD, and color. Considering the polymers\u2019 combinations, the combination of anionic A300 and non-ionic polymers in the optimal dose had the highest removal efficiency. The treatment cost for one cubic meter of ceramic-tile wastewater using the PAC\u2009+\u2009anionic as well as PAC\u2009+\u2009anionic and non-ionic polymers was 22.96 percent less than the PAC-alone method.Finally, the results of this study show that the coagulation\u2013flocculation process as well as the PAC combination with anionic, cationic, and nonionic polymers can be used as an effective method for treatment of ceramic-tile wastewater. The PAC, as the coagulator with the optimal dose of 300\u00a0mg/L and the anionic polymer A"} +{"text": "Taken together, our data sheds light on a new mechanism whereby PGF2\u03b1 specifically recruits and signals through \u03b2-arrestin but only in the context of the AT1R/FP dimer, suggesting that this may be a new allosteric signaling entity.Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional homo- and heterodimers that act as distinct signaling hubs for cellular signal integration. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2\u03b1 (PGF2\u03b1) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells. Here, we hypothesize that both Ang II- and PGF2\u03b1-induced activation of the AT1R/FP dimer, or the parent receptors alone, differentially regulate signaling by distinct patterns of \u03b2-arrestin recruitment. Using BRET-based biosensors, we assessed the recruitment kinetics of \u03b2-arrestin1/2 to the AT1R/FP dimer, or the parent receptors alone, when stimulated by either Ang II or PGF2\u03b1. Using cell lines with CRISPR/Cas9-mediated gene deletion, we also examined the role of G proteins in such recruitment. We observed that Ang II induced a rapid, robust, and sustained recruitment of \u03b2-arrestin1/2 to AT1R and, to a lesser extent, the heterodimer, as expected, since AT1R is a strong recruiter of both \u03b2-arrestin subtypes. However, PGF2\u03b1 did not induce such recruitment to FP alone, although it did when the AT1R is present as a heterodimer. \u03b2-arrestins were likely recruited to the AT1R partner of the dimer. G\u03b1 G protein-coupled receptors (GPCRs) have historically been studied and understood as functional monomers. Although well-established for class-C GPCRs , 2, an ivice-versa. Further, GPCR dimers can also allow different intracellular signaling partners to selectively interact with only one of the two protomers or both, expanding the possibilities for the cell to adapt to different conditions. For example, a D2 dopamine receptor homodimer was found to be organized asymmetrically with respect to its G protein partners such that occupation of the first protomer facilitated downstream cellular signaling through the second protomer, while occupation of the latter (or even its constitutive activity) modulated signaling allosterically without inducing a signal on its own . \u03b2-arresr (\u03b22AR) . Some crr (\u03b22AR) , while tarrestin .A hurdle in investigating class-A GPCR dimerization has been the limited capacity to identify the respective function of the protomers within a dimer, that is which protomer binds the ligand and which protomer signals. This minimal functional unit is used to investigate the interplay between \u03b2-arrestin 1/2 and multiple G proteins at the larger intracellular interface of a GPCR dimer using a panel of BRET-based biosensors. We previously showed that AT1R and FP could be co-purified together using immunoprecipitation in HEK 293 cells and in vascular smooth muscle cells combined with photoaffinity labeling . We noteRenilla luciferase into both AT1R and FP and co-expressed them with their untagged counterparts and asymmetrically regulated in response to Ang II and PGF2\u03b1, respectively.We also engineered FlAsH tags and terparts . We agaie using) . FurtherAll cell culture media, reagents, and antibiotics were from Wisent Inc. . All DNA primers for molecular cloning were custom-made by Integrated DNA Technologies Inc. . All enzymes and other materials used for molecular cloning were from New England Biolabs Ltd. , except for the Pvu II and Taq I restriction enzymes that were both from Takara Bio Inc. . Cell transfection reagent was from Invitrogen, Thermo Fisher Scientific Inc. . All chemicals, including Ang II and the AT1R antagonists were from Sigma-Aldrich Inc. unless otherwise specified. PGF2\u03b1 and cloprostenol were from Cayman Chemical Company . Coelenterazine h was from NanoLight Technologies .q/11/12/13 cell line wherein all the genes encoding for Gq, G11, G12, and G13 proteins have been knocked out was generated by simultaneously targeting the GNA12 and the GNA13 genes of the previously established HEK 293 \u0394G\u03b1q/11 cells . The pcDNA3.1/SP-FLAG-hAT1R-KPVAT-Venus plasmid was created from pcDNA3/SP-FLAG-hAT1R-WT that was amplified by PCR using the forward primer 5\u2032-CCTAGCTAGCTCGAGGCCACCATGAACACGATCATC-3\u2032 and reverse primer 5\u2032-TACCGGTGGCGACCGGTTTCTCAACCTCAAAACATGGTGC-3\u2032 (without a stop codon). The purified PCR fragment obtained was then subcloned in-frame into the NheI and AgeI restriction sites located in 5\u2032 and 3\u2032, respectively, of pcDNA3.1/Venus, which was a kind gift from Dr. Michel Bouvier . In a same way, the pcDNA3.1/SP-FLAG-hAT1R[\u0394325]-KPVAT-Venus plasmid was generated using the forward primer 5\u2032-CCTAGCTAGCTCGAGGCCACCATGAACACGATCATC-3\u2032 and reverse primer 5\u2032-AAAGGGTGGCGACCGGTTTGGCTTTTGGGGGAATATATTTTAGAAGCTG-3\u2032 (without a stop codon) to amplified pcDNA3/SP-FLAG-hAT1R-WT, and the resulting PCR fragment was then subcloned into the same restriction sites of pcDNA3.1/Venus. The HA-hOTR-Venus plasmid was made by the PCR amplification of a previously described hOTR-YFP construct high glucose supplemented with 5% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin\u2013streptomycin (P-S) antibiotics. Cells were cultured in a controlled, humidified environment maintained at 37\u00b0C and 5% CO2 atmosphere until a confluency of 80\u2013100% was reached. For transfection, cells were trypsinized, and 125,000 cells/well in 1 mL of the same fresh media were seeded into 12-well plates. Cells were incubated overnight in at 37\u00b0C. After 24 h, media from the plate was replaced by 1 mL/well of DMEM supplemented with 5% (v/v) FBS. Cells were transfected with the appropriate plasmids encoding for AT1R (0.3 \u03bcg/well), FP (0.1 \u03bcg/well), and the sensors (\u03b2-arrestin 1/2-RlucII (0.0025\u20130.025 \u03bcg/well), along with pcDNA3.1(-) for a total of 1.0 \u03bcg per well using Lipofectamine\u00ae 2000 [2.5(Lipo2000):1(DNA) ratio] and following the manufacturer's instructions. Twenty-four hours post-transfection, the cells were trypsinized, and 40,000\u201350,000 cells/well were transferred into white 96-well plates previously coated with poly-L-ornithine hydrobromide . Cells were left in a cell culture incubator for another 24 h before performing the experiments. Mycoplasma testing was carried out periodically on all cell lines using the MycoAlertTM kit from Lonza .The HEK 293 parental and \u0394G\u03b1Renilla luciferase (Rluc) II and energy-acceptor proteins were fused to the yellow florescent protein (YFP). In all experiments, coelenterazine h was used as the Rluc/RlucII substrate to generate light with a maximal emission peak at 480 nm, allowing YFP excitation. In a typical experiment, transfected cells from a white 96-well plate were carefully washed once with 150 \u03bcL of Kreb's buffer , and then the cells were incubated in the absence or presence of pretreatments in 95 \u03bcL/well of the same fresh Kreb's buffer. The plate was protected from light, and the cells were incubated for 1 h at 37\u00b0C for BRET experiments which were conducted at 37\u00b0C. A TriStar2 LB 942 multimode microplate reader from Berthold Technologies Inc. equipped with the predetermined BRET1 filter pair F485/F530, and a VICTOR X-light multilabel plate reader from Perkin Elmer Inc. equipped with the predetermined BRET1 filter pair F460/F535 nm were used to measure BRET ratios. For enhanced bystander BRET we used 410 nm (donor) and 515/40 nm (acceptor) filters. Under these conditions, both plate readers were able to complete one cycle of BRET ratio measurement in 2 min for a 96-well plate. Experiments with 96-well plates were also carried out at once following four consecutive steps in a timely fashion so that a same time interval apply between each well throughout the process: (1) adding the coelenterazine h substrate for light generation (using a repeater pipette); (2) measuring the BRET ratios of basal, unstimulated cells; (3) stimulating the cells with either vehicle or ligand (using a multichannel pipette); (4) measuring the BRET ratios of stimulated, ligand-induced cells. Following a 1 h incubation in Krebs buffer, 25 \u03bcL/well of a 30 \u03bcM coelenterazine h solution was sequentially added to the cells so it takes 2 min to fill up the entire 96-well plate. The basal, unstimulated BRET ratios were then immediately measured over a period of 10 min (5 measuring cycles). Upon completion, the 96-well plate was rapidly removed from the plate reader, and 30 \u03bcL/well of either vehicle or 5 \u03bcM ligand was sequentially added to the cells so, again, it takes 2 min to fill up the entire 96-well plate. The ligand-induced BRET ratios were then immediately measured over a period of 90 min (45 measuring cycles).BRET experiments were performed as described elsewhere , 22, 23.530or535/\u03bb485or460, and ligand-induced BRET changes as \u0394BRET = BRETLigand \u2212 BRETVehicle. Conditions associated with ligand and vehicle were performed in triplicate for each biological replicate, and averaged data were used in all calculations. As shown in the figures, BRET data were reported as kinetic traces or bar graphs integrating the area under the curve. Statistical analysis and curve fitting were all carried out using the GraphPad PRISM software v6.0 . For U-test (p < 0.05), followed by a post-hoc Dunn's test to correct for multiple comparisons . For Figure 4 statistical analysis on \u0394BRET data was performed using an unpaired Student's t-test (p < 0.01), followed by a post-hoc Bonferroni test to correct for multiple comparisons . EC50 values from 50 \u2212 log[A])], where Top and Bottom represented maximal and minimal asymptotes of the curve, [A] is the agonist concentration expressed in (M) and EC50 is the agonist concentration (M) that generated a response half way between the top and bottom.BRET ratios were calculated as BRET = \u03bb1R/FP dimer wherein Venus was fused to either AT1R or FP. Ang II stimulation induced a rapid, robust, and sustained \u03b2-arrestin 1/2-Rluc recruitment to AT1R-Venus alone . Interestingly, in this case, re-expression of any of the four G\u03b1 subunits was able to partially rescue \u03b2-arrestin 2 recruitment mediated by PGF2\u03b1 where they are endogenously co-expressed. We analyzed several phenotypic responses, including MAPK activation and DNA and protein synthesis in both HEK 293 cells and in VSMC. We showed AT1R when treated with an antagonist strongly potentiated ERK1/2 activation by FP, which was not reciprocated by treatment of FP with its own antagonists when measuring Ang II-mediated ERK1/2 activation . We alsoporation . These rporation ], althou11, G\u03b112, or G\u03b113 but not always G\u03b1q. This is odd, as G\u03b1q is a well-known partner for both receptors. We identified G\u03b1q/11 as the conduit by which allosteric information was transferred from AT1R to FP which did not occur in the opposite direction from FP to AT1R (11 and G\u03b1q. Recent studies have suggested we need to be circumspect about cells gene-deleted for different G proteins that may get \u201crewired\u201d as a consequence (Deletion of several G\u03b1 subunits significantly abrogated both Ang II- and PGF2\u03b1-mediated \u03b2-arrestin1/2 recruitment which was in all cases restored by re-expression of G\u03b1sequence . More syWe had previously shown that the trafficking of the putative heterodimer was likely different depending on whether stimulation was via Ang II or PGF2\u03b1 . We noteSimilar asymmetric structural arrangements were also observed in other GPCR oligomers including the luteinizing hormone receptor , rhodops2CAR, and \u03b22AR receptor heterodimers (Other studies showed that AT1R may be an example of a dimer hub with manrodimers \u201342. A moThe AT1R is an important target for treatment of hypertension and heart failure and angiotensin receptor blockers remain widely prescribed . A role All datasets generated for this study are included in the manuscript and/or the supplementary files.DF and TH designed the study. DF, DD, and RS performed the experiments, analyzed the data, and generated the figures. TH, DF, DD, and AI wrote and edited the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "To optimize the focus of future public information campaigns in The Netherlands promoting the uptake of vaccines among adults and children, we quantified the contribution of several attributes to the vaccination decision.We performed a discrete choice experiment (DCE) among Dutch adults including six attributes, i.e. vaccine effectiveness, vaccine-preventable burden of disease (specified in severity and frequency), accessibility of vaccination in terms of co-payment and prescription requirements, frequency of mild side-effects, population-level vaccination coverage and local vaccination coverage among family and friends. Participants answered the DCE from their own perspective (\u2018oneself\u2019 group) or with regard to a vaccine decision for their youngest child (\u2018child\u2019 group). The data was analysed by means of panel mixed logit models.We included 1547 adult participants (825 \u2018oneself\u2019 and 722 \u2018child\u2019). Vaccine effectiveness was the most important attribute in the \u2018oneself\u2019 group, followed by burden of disease (relative importance (RI) 78%) and accessibility (RI 76%). In the \u2018child\u2019 group, burden of disease was most important, but tied closely with vaccine effectiveness (RI 97%). Of less importance was the risk of mild vaccine-related side-effects and both population and local vaccination coverage. Interestingly, participants were more willing to vaccinate when uptake among the population or family and friends was high, indicating that social influence and social norms plays a role.Vaccine effectiveness and disease severity are key attributes in vaccination decision-making for adults making a decision for themselves and for parents who decide for their children. Hence, public information campaigns for both adult and child vaccination should primarily focus on these two attributes. In addition, reinforcing social norms may be considered. Vaccination is one of the major contributors to the global improvement of health and life expectancy , 2. The Determinants of vaccine decision-making have been studied thoroughly over the past years, revealing factors like (perceived) vaccine safety, risk perception, (perceived) vaccine effectiveness, and social norms and beliefs , 8\u201311. MThe Dutch vaccination programme consists of vaccinations that target different age and risk groups, including children, adolescents, pregnant women, clinical risk groups and elderly. It is likely that these different groups have a different inclination towards vaccination, based on different perspectives on individual risk and expectations with regard to future health. For example, an adult member of a risk group may have entirely different considerations than an adolescent, or a parent who has to make the decision about vaccinating a young child. Discrete choice experiments (DCE) have been used to study vaccine decision-making, both in The Netherlands and elsewhere \u201322. ThesWe employed a recently developed DCE design that was used in a study about vaccine decision-making in Flanders and South Africa , 24. ThiWe divided study participants in two groups, referred to as the \u2018oneself\u2019 and \u2018child\u2019 group. Respondents without children under 18\u2009years of age could only be allocated to the \u2018oneself\u2019 group, remaining respondents were randomly allocated to either the \u2018oneself\u2019 or \u2018child\u2019 group. The \u2018oneself\u2019 group answered questions on vaccinating themselves, the \u2018child\u2019 group on vaccinating their youngest child. As an incentive for participation, respondents received credit rewards, transferable into coupons or air miles.The questionnaire consisted of five parts involving 1) background characteristics of the respondent; (2) vaccine-related attitudes; (3) the DCE; (4) risk perception of infectious diseases and (5) health literacy. The full questionnaire is available in the Additional Material backgrouBackground characteristics included: gender; age; 4-digit postal code; level of education; household composition; family size (number of children); age of youngest child; mother's country of birth; professional experience in the healthcare sector; previous exposure to severe diseases; eligibility for influenza vaccination ; past acceptance of influenza vaccination (if eligible); smoking status; beliefs that influence (attitude towards) vaccination decisions; religious background; and past acceptance of National Immunization Programme (NIP) vaccination for one\u2019s children (if any).The second part, surveying vaccine-related attitudes, contained 21 statements, each focussed on a particular aspect of the vaccination programme. These statements were adopted from a selection of previously conducted questionnaires on vaccine decision behaviour of parents as well as elderly , 22, 30.n\u2009=\u200916), pilot study (n\u2009=\u200941) and a soft launch with free-form feed-back (n\u2009=\u2009184) in the original Flemish target population resulted in the final selection and finetuning of the six attributes. Attributes as burden of disease, vaccine effectiveness, VRSE and accessibility are utilized frequently in DCE studies in the field of vaccination. Vaccine coverage, both at the population and the local level, is included less often in vaccine DCEs, although its importance is well described in literature about behavioural change models [The third part, the DCE, assigned each respondent to 10 choice sets of two unlabelled vaccine profiles. To capture all main and interaction effects, we created a design of 50 choice sets, divided into five subsets of 10 choice sets, using a Bayesian D-optimal design . The subtudy n\u2009=\u2009 and a soe models . The attThe fourth part of the questionnaire assessed the participants\u2019 perception of the relative severity of and susceptibility of themselves or their child for measles, influenza, a urinary tract infection and leukaemia . Also, rFinally, the respondents\u2019 health literacy was assessed using Chew\u2019s Set of Brief Screening Questions (SBSQ), which is a validated subjective measure of health literacy containing three items: \u2018How confident are you filling out forms by yourself?\u2019 (Confident with Forms), \u2018How often do you have someone help you read hospital materials?\u2019 (Help Read), and \u2018How often do you have problems learning about your medical condition because of difficulty understanding written information?\u2019 (Problems Reading) \u201345. RespNote that the entire questionnaire was identical for the \u2018oneself\u2019 and \u2018child\u2019 group except for the framing of the subject of interest with respect to the vaccination decision (oneself or one\u2019s child). As indicated, the design of our Dutch DCE was identical to the design of the South African DCE , which w\u2212log10 ). Based on the finding that both coverage attributes depict a linear trend, we used linear coding for these attributes. Therefore, the estimates for these attributes represent the marginal change in utility when vaccination coverage increases by 10%. The DCE results were analysed using the Choice Modelling platform of JMP Pro 14 [The relative importance of the attributes and the relative utility values attached to the attribute levels were obtained by a Panel Mixed Logit (PML) model using Hierarchical Bayes estimation. To accommodate for unobserved preference heterogeneity of the respondents, we assumed normally distributed preference parameters without correlation between attributes. We ran 10,000 Bayesian iterations, and used the last 5000 for estimation. The total utility of a vaccination alternative is the sum of the attributes\u2019 main and interaction effect estimates. Overall significance of the attributes was computed by likelihood ratio (LR) tests and relative importance of each attribute by the normalized logworth statistic and the \u2018child\u2019 group (n\u2009=\u2009722). The study sample was representative of the general Dutch population with respect to the background characteristics gender, age and province. There was an underrepresentation of individuals with lower educational attainment. The \u2018oneself\u2019 and the \u2018child\u2019 groups differed slightly with regard to age and gender , indicating that the vast majority of the respondents were health literate , 85.5% report that they fully vaccinated their child (ren) following the NIP guidelines, 6.5% did not vaccinate, 5.7% vaccinated their child(ren) partially and the remaining indicated \u2018n/a\u2019 (2.3%). Based on dichotomized 5-point Likert responses on three statements , at least 80% of the parents in the \u2018child\u2019 group indicated a positive attitude towards vaccination for each statement separately, whereas this held for 75% in the \u2018oneself\u2019 group. Table With regard to all respondents, both in the \u2018oneself\u2019 and the child group, with children below 18\u2009years old and susceptibility (disease frequency). Of these two elements, more weight was assigned to severity, based on the attribute level utility estimates. Irrespective of perspective, the frequency of mild VRSE, population coverage and local coverage among friends and family were relatively unimportant in the decision. When adults decided for themselves, there was no significant difference in utility between vaccinating against a common or rare mild disease. In case of the decision for their child, a vaccine against a common and mild disease was preferred over a vaccine that protects against a mild but rare disease.We found covariate interactions that differed between the two groups, whereby being among the target group for the annual influenza vaccination was the strongest in the \u2018oneself\u2019 model. Understandably, for respondents receiving this invitation, the question to vaccinate oneself was possibly related to different experiences compared to respondents not receiving the invitation, as this group is more experienced with vaccination overall compared to adults who do not qualify for influenza vaccination, and probably only have experience with travel vaccines for themselves. Following this logic, it is also not unexpected that this covariate is not explaining differences within the \u2018child\u2019 model. Notably, this also has to do with selection effects, because belonging to the risk group, thus receiving yearly influenza vaccination, is less likely in the group of parents belonging to the child group.Contrary to what we expected, we were not able to find differences in preferences between respondents belonging to the orthodox Protestant community and other respondents. It is possible that those who opt out of vaccination may have chosen not to cooperate in this survey in the first place. Furthermore, moderately negative attitudes among respondents about vaccination in general Table may haveAttitudes towards vaccination are important determinants of vaccine acceptance and vaccine hesitancy , 11, 47.Of particular interest is the finding that the marginal utility estimates of the local (friends and family) and population coverage attributes were positive. This indicates that a vaccine is more likely to be accepted by an individual if others accept it too. A possible explanation is that individuals perceive a high coverage as a social norm or as public confidence in the vaccination programme. This link is also reported in Belgium , AustralThis study has several limitations. A first limitation is that although our sample was representative with respect to gender, age and province, there could be unmeasured differences between the respondents in our sample and the general population, for example due to higher computer access in the online panel. Moreover, the sample population in this study was higher educated compared to the population\u2019s educational level.A potential weakness regarding the DCE is the sensitivity to the framing of the VRSE attribute. In the original study in Flanders, this attribute appeared most important in both the vaccination decision for oneself and one\u2019s child. In our DCE as well as in the South African DCE, this attribute was rephrased. Originally, the VRSE attribute did not provide any explanation with regard to severity of VRSE. Therefore, its description was changed such that it explicitly states that VRSE are of mild nature and rarely result in hospitalization. In our study, only the frequency of VRSE occurrence was varied. The differences in outcomes between our study and the South\u00a0African study on the one hand, and the original Flemish study on the other, highlight the influence of framing, as well as being as specific as possible with regard to VRSE.Another limitation is that due to the age structure of respondents with children under the age of 18, which are mostly between 25 and 40\u2009years old, we observed a slight underrepresentation of this age group in the \u2018oneself\u2019 group. As a result, we cannot fully explore the differences between individuals with and without young children. However, given that we found only limited differences between the two vaccination decision-making settings and the large sample size, we do not expect that additional research would lead to different conclusions.When the findings of this study are compared to both the Flemish and the South African study we find both similarities and differences. With regard to ranking of importance of the attributes the three studies all found different rank orders. As described, in the Flemish study VRSE combined with accessibility were the two most important attributes, followed by effectiveness and burden of disease. In South Africa, vaccine effectiveness was the most important attribute followed by population coverage and burden of disease. As in South Africa, here we found that vaccine effectiveness was the most important attribute, although in case of the child vaccination, this was combined with burden of disease. The importance of vaccine effectiveness was also found in other DCEs , 35, 38.We performed a discrete choice experiment in The Netherlands to gain insights into vaccination decision-making with respect to vaccinating oneself and one\u2019s child. Vaccine effectiveness, burden of disease and accessibility were the most important attributes in the decision-making process. Less influential, but still important contributors to vaccine utility were the population and local coverage. For both coverage attributes, we found positive utility estimates, indicating the effect of social influence. In the decision to vaccinate one\u2019s child, estimates were even larger in absolute magnitude due to interactions with mild VRSE and accessibility, where higher local coverage increased the utility in magnitude for the least desirable options (i.e. common VRSE and co-payment). Our findings indicate that the focus of communication about vaccination is similar for vaccinating children and oneself. The message should stress the effectiveness and low effort of vaccination and clearly explain the burden of disease against which vaccines protect. Reporting high uptake rates may help to increase uptake of vaccination in future vaccination decisions.Additional file 1. DCE Questionnaire"} +{"text": "We found that prepared TiOx thin films significantly reduce the transmittance of destructive UV radiation; a feature that can be useful for the protection of photovoltaic devices. In addition, transparent and luminescent TiOx thin films can be utilized for potential security labeling.Transparent titanium oxide thin films attract enormous attention from the scientific community because of their prominent properties, such as low-cost, chemical stability, and optical transparency in the visible region. In this study, we developed an easy and scalable solution-based process for the deposition of transparent TiOx thin films on glass substrates. We showed that the proposed method is also suitable for the fabrication of metal-doped TiOx thin films. As proof-of-the-concept, europium Eu(III) ions were introduced into TiOx film. A photoluminescence (PL) study revealed that Eu-doped TiOx thin films showed strong red luminescence associated with For example, well-known titanium dioxide TiO enamels , as a fo enamels ,4. Semit devices ,6. TiO2 devices ,8. To da devices , spray p devices , and RF devices are wideterature ,12. Howe2 nanotubes exhibited high photocatalytic activity under visible light illumination compared to a commercial P25 TiO2 powder. Another report suggested that Eu-doped TiO2 thin films can improve the performance of organic solar cells [2 [It is well-known that the doping process can significantly change the physicochemical properties of titanium oxide. For example, one can easily alter the bandgap, recombination rate of electron-hole pairs, conductivity and optical properties of prepared films . Among dar cells . It was cells [2 ,17. TherIn this study, we presented a novel and simple solution-based deposition of transparent TiOx thin films on glass slides, using spin coating at ambient conditions. Fabrication simplicity and excellent reproducibility highlight the potential applicability of the proposed method for the generation of functional coatings for security labeling, UV screening, photovoltaic devices, etc.3 \u00d7 6H2O (99.9%) were purchased from Merck & Co. and used as received. A precursor solution for TiOx film was prepared by mixing ethanol (0.5 mL), 1-butanol (1 mL), and 100 \u00b5L of TIP. For Eu-doped TiOx film, 10 mg of europium salt was firstly dissolved in 0.5 mL of ethanol and then mixed with 1 mL of 1-butanol and 100 \u00b5L of TIP. Later on, the precursor solution was spin-coated on clean glass slides (20 \u00d7 15 mm) at 500 rpm (5 s), followed by 2000 rpm (15 s). All experiments were repeated five times to ensure the reproducibility of the results. Obtained thin films were naturally dried in ambient conditions for 2 h and then annealed at 500 \u00b0C (heating rate 5 \u00b0C/min) for 1 h. Annealed thin films were used for further testing.Titanium isopropoxide TIP (>97.0%), anhydrous 1-butanol (99.8%), absolute ethanol (\u226599.8%), and EuCl\u221211 mbar and the instrumental resolution was 0.6 eV. Samples were attached to the sample holder by a copper tape. A charge neutralizer was used during the measurements. Atomic force microscope was used to obtain topographic images of film surfaces. UV-Vis light transmittance measurements were conducted using a Genesys 50 UV-Visible spectrophotometer . The optical properties of films were examined using a fluorescence spectrophotometer .X-ray diffraction (XRD) measurements were performed using a SmartLab X-ray Diffractometer with a Cu K\u03b1 radiation source. X-ray photoelectron spectroscopy (XPS) was performed in an Omicron MultiProbe XPS using a monochromized Al K\u03b1 source . The instruments\u2019 base pressure was 5 \u00d7 10It is interesting to note that this methodology can be used to fabricate metal-doped TiOx thin films. Herein, we introduced europium salt to achieve a red-emitting luminescent thin film. AFM was utilized to examine the surface topography and surface roughness of the prepared films. 3/2) and 463.46eV (Ti 2p1/2). These binding energies are consistent with the Ti3+ state [2+ oxidation state. These results suggest that the experimental conditions yielded the TiOx film with the mixed oxidation states of titanium (Ti3+/Ti2+). The incorporation of Eu ions into a TiOx matrix led to a chemical shift in the Ti 2p3/2 and Ti 2p1/2 peaks to 457.4 eV and 463.16 eV, respectively. This shift in binding energies indicates an influence of Eu ion addition on the electronic state of titanium. However, these binding energies of the shifted Ti 2p peaks in Eu-doped TiOx film could still be assigned to the Ti3+ state [3+ state . In addi3+ state . Figure 5/2) and 1165.7 eV (Eu 3d3/2). In addition, the narrow scan Eu 4d spectrum is composed of spin\u2212orbit peaks at binding energies of 142.8 eV (Eu 4d3/2) and 137.7 eV (Eu 4d5/2). These binding energies are highly consistent with Eu3+ state [Furthermore, careful examination of the wide XPS survey spectrum of the Eu-doped TiOx thin film revealed3+ state . Accordiexc. = 310 nm), measured at room temperature RT in the range of 550\u2013700 nm. A well-resolved broad emission peak, with an emission maximum at 633 nm, was detected. This emission is associated with radiative 5D0\u2192 7Fj transitions within Eu3+ ions [exc. = 302 nm). Red emission from a transparent Eu-doped TiOx film can be visually observed by a naked eye, making it suitable for the potential security labeling of valuable goods and photovoltaic applications [u3+ ions ,22. Howeications .5D0\u21927Fj radiative transitions was observed for Eu-doped TiOx film. Fabrication simplicity, chemical stability, and excellent luminescent properties make Eu-doped TiOx films promising for UV screening, security labeling, and photovoltaic applications.In summary, we introduced a novel solution-based method for the deposition of transparent TiOx thin films using a spin coating method. It was found that nearly uniform thin film with a thickness of 30\u201340 nm can be formed. We also showed that the same method can be employed for the fabrication of metal-doped TiOx films. In particular, characteristic eye-visible red emission associated with Eu(III)"} +{"text": "IntroductionPressure ulcers (PUs) are a major health problem for bedridden patients or persons with reduced mobility; individuals with spinal cord injury (SCI) are more prone to developing pressure ulcers. The purpose of this study was to determine\u00a0the efficacy of a novel negative pressure wound therapy (NPWT) system for the treatment of Grade IV PUs.MethodsA total of 34 SCI patients with Grade IV PUs\u00a0were divided into two groups: 17 cases were managed by our bellows-powered negative pressure device (NPD) and\u00a017 received wet-to-moist gauze dressing as standard wound care.ResultsWound healing outcome measures were recorded every week (at seven days) and compared at weeks 3, 6, and 9. There were no significant changes in the length and width of PUs between the groups till week 3. Significantly reduced length and width of NPD-treated PUs were found at week 6 (p=0.04) that further reduced at week 9 (p=0.001) as compared to standard wound care. Similarly, significant reduction in the depth of PUs was found in the NPD-treated group at week 9 (p<0.05). Exudate levels were significantly (p=0.001) lower in the NPD-treated group as compared to the standard wound care group from week 3 (2.96\u00b10.21 vs 2.62\u00b10.49); this difference continued through week 9 (1.35\u00b10.75 vs 0.14\u00b10.35). Disappearance of slough and formation of healthy granulation tissue was significantly higher in the NPD-treated PUs after week 6 (p=0.001).ConclusionNPWT\u00a0may be the future of wound healing. As an alternative to the existing electrically powered NPWT systems, our NPD\u00a0is safe, easy to apply, and efficacious in treating the PUs. Pressure ulcers (PUs) are wounds initiated by pressure on the skin that blocks circulation, causing the skin and underlying tissues to die ,2.\u00a0PUs aNegative pressure wound therapy (NPWT) includes a mechanical, vacuum-assisted method that exerts negative pressure of \u2212125 mm Hg on the wound bed ,10. The NPWT is evolving and is under investigation for the management of difficult, chronic, and unrelenting wounds. The available negative pressure devices (NPDs) for NPWT are expensive and hard-to-afford by patients and health systems in developing countries. As an alternative to these NPWT systems, we propose\u00a0a bellows-powered\u00a0NPD\u00a0for the management of PUs. The purpose of this study was to assess healing outcome measures such as surface area and depth, levels of exudate, and formation of granulation tissue in patients treated with our NPD compared to patients managed by wet-to-moist gauze dressings.This study was conducted at the Spinal Cord Injury Unit, Department of Orthopaedic Surgery, King George's Medical University, Lucknow, India. The study was approved by the Institutional Ethics Committee (IEC) of the University (KGMU). In this prospective non-randomized study, 34 SCI patients with PUs of Grade IV, according to the National Pressure Ulcer Advisory Panel (NPUAP), were recruited . Out of Baseline assessment of ulcersInformation regarding patient demographics, PU history, and co-morbidities was obtained from patients and/or their carers. Wound debridement for necrotic tissue and slough was done in all patients before being allocated to either group. The allocation of patients was done by the primary author throughout the study period. The PUs were measured for their length and width with a centimeter ruler. The surface area was calculated from these values. PUs in both groups were measured at each time point (weekly) using the uniform procedure. PU depth was measured with a sterilized cotton-tipped applicator, which was inserted into the ulcer and marked at the deepest level. The amount of exudate was categorized as none (0), light (1), moderate (2), or heavy (3) after the dressing was removed in both NPWT and Standard Care groups with the help of the Pressure Ulcer Scale for Healing (PUSH) tool, version 3.0 . NecrotiMethod of standard wound careThe surface of the PU was cleansed with normal saline and packed with sterilized gauze to cover the wound. Dressing changes were performed once or twice daily depending on the soakage of the dressing.Components of the novel NPDThe novel NPD\u00a0was applied exclusively as a bedside procedure. It is a low-cost device and comprises a low-power continuous suction apparatus consisting of the following: a\u00a0bellow unit of 800 mL capacity, a connecting tube with clamp, a \"Y\" connector, a curved needle with a matching catheter and a spare perforated catheter ; a sterilized piece of foam; a transparent polyurethane adhesive dressing ; and a Dynaplast elastic adhesive bandage , which sealed the adhesive dressing to the adjoining skin. We assembled these components to form a novel NPD that was applied to the PU and changed every week or earlier if there was a soakage/leakage. The cost of our NPD was obtained from the record of the hospital's central supply department. The cost of the required components of our NPD was about 40-50 USD. A single Romo Vac Set and a Dynaplast were utilized throughout the nine weeks, while one or more transparent polyurethane dressing and foam were used during the nine-week\u00a0follow-up.Application of the novel NPDApplication of the NPD and all subsequent dressing-related procedures occurred at the patient\u2019s bedside. The perforated end of the drainage tube of the Romo Vac was placed on the wound surface and its other end exited through the skin 10 cm away from the wound margin and was connected to the Romo Vac bellow. The sterilized foam was trimmed according to the size and geometry of the wound and placed on top as a cover. Opsite finally covered the wound and the adjoining healthy skin with an airtight seal. The bellow of Romo Vac is charged to attain appropriate cyclical/intermittent negative pressure , and depth of the PU, exudates \u00a0and tissue type from zero- to nine-week\u00a0follow-up. Data were recorded every seven days and analysed at weeks 3, 6, and 9 in both the groups.Data analysisThe Statistical Package for the Social Sciences, version 21 \u00a0was used for all data analyses. Descriptive findings were characterized as means \u00b1 standard deviations, and group comparisons were completed via the Student t-test. For variables that were not continuous or not normally distributed, we used a nonparametric equivalent to the Student t-test, the Mann-Whitney U test. The mixed linear model was used to find the changes from baseline to week 9. A fixed and random-effects model was used. A p-value <0.05 was considered significant.To compare the two therapies, the rate of healing was measured in terms of reduction of surface area and depth of PUs at different follow-up points. There was no significant difference in the length of PUs between the groups till week 3. A significantly reduced length of PUs in the NPWT group was observed at week 6 (p=0.04) which further reduced at week 9 p=0.001) as compared to the standard wound care group. Similarly, a significant reduction in the width and depth of PUs was observed in the NPWT group at week 9 (p<0.05) as compared to the standard wound care group lower in the NPD-treated group as compared to standard wound care group from week 3; this difference continued through week 9 (p=0.001). The disappearance of slough (dead yellowish tissue) and formation of healthy red granulation tissue was significantly higher in the NPD-treated PUs after week 6 (p=0.001) that continued at week 9 Table .PUs\u00a0are complex and chronic wounds in patients with SCI\u00a0and no gold standard has yet been established for their prevention and treatment. PUs are difficult to prevent and manage and can lead to a decline in the overall well-being of patients in SCI ,4,5,16. In this study, for removing the potential risk of bias, a standard procedure was followed for pressure offloading by application of an air tube ring\u00a0and nursing of the patients, including turning of body, positioning, and bladder and bowel management. A loss of sensation in the skin, constant pressure, moisture, and irritation to the skin in traumatic paraplegia subjects further delay\u00a0the healing process . BesidesNPWT\u00a0is a recent technical innovation in wound care with a growing number of applications. In NPWT, the application of topical negative pressure (TNP) on wound bed removes blood and serous fluid, may reduce bio-burden, and increases localized blood flow thereby supplying the wound with oxygen and nutrition to promote accelerated healing . While NA systematic report published in 2015 demonstrated the benefits of a bellows-powered NPWT device designed specifically for use in resource-constrained settings. They found that the elimination of air leaks in the simplified NPWT dressing is essential and that their system is safe and feasible for use in these environments. Application of proven therapies such as NPWT\u00a0in resource-constrained settings is limited by cost and lack of electrical supply [PU treatment with our novel device (NPD) led to accelerated healing in the majority of cases. Our study shows that PU treatment with our NPD can be used as a manageable method in primary care settings such as home care. This device provides negative pressure of \u2212125 mm Hg pressure (\u221260 to \u2212125 mm Hg) when fully charged. With time, the negative pressure is gradually lost, requiring periodic manual recharge of the device. This by itself provides an intermittent negative pressure as suggested by Morykwas et al.\u00a0.\u00a0They coMody et al. in 2008 conducted a randomized controlled trial comparing a locally constructed topical NPD with wet-to-dry gauze dressings on varied wound etiologies including diabetic foot ulcers, PUs, cellulitis/fasciitis, and other types of ulcers . Except We found that the treatment of PU with our innovative device has superior healing as compared to standard wound care with modern dressings while giving clinicians a simpler method. Two prospective non-randomized clinical trials on PUs showed positive results of NPWT on the healing process ,5. Our sSlough comprises dead white blood cells, fibrin, cellular debris, and liquefied devitalized tissue. NPWT was originally utilized to speed bedside debridement of wounds . We obseThe negative pressure speeds up the formation of granulation tissue and reduces bacterial counts in the wound ,6,9. OurWound exudate is produced as a natural and essential part of the healing process. However, the overproduction of wound exudate, in the wrong place or of the wrong composition, can adversely affect wound healing. In the normal wound healing cascade, exudate makes a moist wound environment and supports healing by facilitating the diffusion of vital healing factors (e.g.\u00a0growth and immune factors) and the migration of cells across the wound bed and prevents the wound from drying out. As healing occurs, the amount of exudate produced usually decreases . It is iNPWT\u00a0may be the future of wound healing. The commercial devices for negative pressure require rental services from the patients and the consumables are expensive. As an alternative to existing electrically powered NPWT systems, our bellows-powered novel NPD\u00a0was safe, easy to apply, and cost-effective. The patient compliance was good to excellent as the novel NPD was patient friendly and the airtight seal was very effective. Additionally, the sealed system was easy to use that made the patient's and caregiver's experience better. The procedure was also well tolerated by the patients at their home care. The procedure being safe with minimal side effects can be promoted as an OPD procedure with weekly follow-ups for change of dressings, thereby reducing the hospital stay and hence the economic burden on the patient and the hospital."} +{"text": "Malignant pleural mesothelioma (MPM) is the epitome of a recalcitrant cancer driven by pharmacologically intractable tumor suppressor proteins. A significant but largely unmet challenge in the field is the translation of genetic information on alterations in tumor suppressor genes (TSGs) into effective cancer-specific therapies. The notion that abnormal tumor genome subverts physiological cellular processes, which creates collateral vulnerabilities contextually related to specific genetic alterations, offers a promising strategy to target TSG-driven MPM. Moreover, emerging evidence has increasingly appreciated the therapeutic potential of genetic and pharmacological dependencies acquired en route to cancer development and drug resistance. Here, we review the most recent progress on vulnerabilities co-selected by functional loss of major TSGs and dependencies evolving out of cancer development and resistance to cisplatin based chemotherapy, the only first-line regimen approved by the US Food and Drug Administration (FDA). Finally, we highlight CRISPR-based functional genomics that has emerged as a powerful platform for cancer drug discovery in MPM. The repertoire of MPM-specific \u201cAchilles heel\u201d rises on the horizon, which holds the promise to elucidate therapeutic landscape and may promote precision oncology for MPM. Malignant pleural mesothelioma (MPM) is a rare but highly aggressive cancer etiologically associated with asbestos exposure and inherently resistant to treatment options . AlthougFor patients with advanced, unresectable MPM, a chemotherapy regimen that combines cisplatin and pemetrexed has for long been the only FDA (U.S. Food and Drug Administration) \u2013 approved first-line treatment, which, disappointingly, elicits only modest efficacy due to prevalence of drug resistance and no validated treatment beyond front-line therapy has emerged. However, a recent phase 3 trial has showed that overall survival of MPM patients can be further improved by cisplatin/pemetrexed plus bevacizumab, an antibody against vascular endothelial growth factor (VEGF) (CDKN2A), BRCA1 associated protein-1 (BAP1) and neurofibromatosis type 2 (NF2) in MPM, most often cyclin-dependent kinase inhibitor 2A ( 2 (NF2) , 5 , for whi 2 (NF2) . Moreove 2 (NF2) .CDKN2A is frequently inactivated in mesothelioma and p53 pathways, respectively -mediated phosphorylation of pRb, abrogates the G1/S cell-cycle arrest and promotes aberrant proliferation, functional loss of p14Arf, a central negative regulator of mouse double minute 2 homolog (MDM2), suppresses apoptosis by escape from p53-mediated anti-tumor surveillance .Recent studies showed that hibition . CDK4/6 CDKN2A alterations co-occur with biallelic deletion in type I interferon locus. The IFN-I pathway plays a key antiviral role, suggesting that CDKN2A-deficient MPM might particularly benefit from oncolytic viral therapy , contributed by failure to deubiquitinate histone H2A on chromatin, which eventually leads to accumulation of DNA mutations and chromosomal aberrations complex, rrations . BAP1 harrations . As BAP1ted ones , 18. How1 status , 20, waractivity .BAP1 mutations were reported to increase aerobic glycolysis, also known as the \u201cWarburg effect,\u201d due to impaired mitochondrial respiration and ferroptosis via modulating SLC7AL11 inhibitors (n > 600) with Vorinostat, an FDA-approved HDAC inhibitor, showed disappointing results (BAP1 loss in MPM prioritizes the enhancer of zeste homolog 2 (EZH2)-targeted therapy , and furhibitors . However results . Importa therapy , suggest therapy , might b therapy . Notablyl status , suggestBAP1 mutaions in MPM are associated with favorable prognosis in patients (BAP1-inactivated tumors might have a stronger activity of cytotoxic T cells due to increased interferon regulator factor 8 (IRF8) , 5. Fina8 (IRF8) .NF2 encodes Merlin (moesin-ezrin-radixin-like protein) that mediates tumor suppression and contact-dependent inhibition through the Hippo pathway occur in about 11% of MPM patients activity in mesothelioma . FAK is CDKN2A, BAP1, and NF2) described above, several other genes, e.g., TP53 (tumor protein p53), LATS2, SETD2 (SET domain containing 2) and oncogenic changes in the TERT (telomerase reverse transcriptase) promoter are also altered by at non-negligible frequencies in MPM.In addition to the TSGs (TP53 (encoding p53) is mutated in 6\u201316% MPM cases (CDKN2A loss that depletes p14Arf and in turn releases MDM2 (mouse double minute 2 homolog), a negative regulator of p53, also inactivates p53, a key player in G1/S cell cycle regulation and apoptosis. We and others have shown that inactivation of CDKN2A/2B and TP53 renders MPM cell particularly sensitive to G2 checkpoint inhibition, e.g., CBP501 (a peptide with G2 checkpoint-abrogating activity) and AZD1775 (selective inhibitor of the G2 checkpoint kinase WEE1) (PM cases . Moreovese WEE1) , 53.SETD2, an epigenetic tumor suppressor involved in histone methylation, are detected in more than 8% of MPM cases in PM cases . SETD2-dase EZH2 . In addiy cancer , althougTERT promoter mutations, the first recurrent oncogenic muation identified in MPM, are frequent in MPM with sarcomatoid subtype and significantly associated with worse clinical outcome , is a hallmark of cancer including MPM. Epidermal growth factor receptor (EGFR) is not mutated but overexpressed in MPM, leading to deregulation of EGFR signaling and aberrant activation of downstream pathways such as RAS/RAF/MAPK and PI3K/AKT/mTOR, which in turn promotes cell proliferation, tumor invasiveness and angiogenesis . DespiteIncreasing evidence has indicated that other RTKs, including fibroblast growth factor receptor (FGFR) , insulinGiven the crucial role of VEGF signaling in tumorigenesis, several anti-angiogenic drugs, including bevacizumab, thalidomide and nintedanib, have been investigated in MPM either alone or in combination with cisplatin plus pemetrexed over the past decade . To dateThe endoplasmic reticulum (ER) is the principal organelle monitoring proteostasis. Physiological and pathologic stimuli e.g., nutrient deprivation, aberrant glycosylation, oxidative stress and DNA damage, can disturb the ER environment, eliciting ER stress and unfolded protein response (UPR) . The UPRTumorigenesis entails aberrant proliferation, which is often confronted by limited oxygen supply and malfunctional vascularization, leading to increased demand for protein folding, assembly and transport . As suchTP53 tumor suppressor functions and increased expression of survival signals, overexpression of pro-survival B-cell lymphoma 2 (BCL-2) family proteins that dampen apoptosis by sequestering pro-apoptotic activators is another pivotal strategy to circumvent apoptosis (L/BCL-W) and a paL/BCL-W) , 92. Mord in MPM .MLH2, MSH2, and RAD51 play central roles in regulating hypoxic responses in MPM . HIF-1\u03b1/nd RAD51 . Thus, t1 is dedicated to systematic identification of cancer-specific vulnerabilities for targeted therapy and further stratification based on genomic diversity and molecular characteristics for precision oncology (The Cancer Dependency Map Project (DepMap)oncology . DepMap Despite decades of enormous efforts, cisplatin plus pemetrexed chemotherapy remains one of the few treatment options that achieve survival benefit in MPM. However, clinical evidence indicates that this combination therapy rarely achieves complete/durable clinical response in MPM patients due to drug resistance, intrinsic and/or acquired after initial treatment. Therefore, identification of therapeutic vulnerabilities to target chemoresistant MPM represents a significant yet unmet clinical challenge.Cancer cells can shift at different cell states, most prominently the transition from epithelial to mesenchymal (EMT) or vice versa (MET). The epithelial state is a differentiated cell state while the mesenchymal more undifferentiated and reminiscent of cancer stem-like cells (CSCs), so coined as they recapitulate normal stem cells characterized by the capacity of self-renewal and differentiation. Cancer cell plasticity is an important process that generates CSCs and drives therapy resistance , partly high and CD44+ in mesothelioma cell lines show increased sensitivity to cytotoxic chemotherapy due to impaired DDR (TP53 mutaions (6\u201316% in MPM) and, more often, of inactivating alterations in CDKN2A, which depletes p14Arf and promotes proteasome-mediated degradation of p53. Consequently, p53 deficient MPM cells might have greater dependence on G2/M checkpoint to protect the toxicity of chemotherapy, and abrogation of the G2/M checkpoint activity, e.g., WEE1 inhibition, sensitizes MPM cells to chemotherapy (The DNA damage response (DDR) synchronizes DNA repair and checkpoint signaling activation to arrest cell cycle progression . Compellired DDR . Moreoveired DDR , and a pired DDR . Notablyired DDR , 51, a cotherapy .Macroautophagy (hereafter autophagy) is an evolutionally conserved catabolic process, whereby long-lived proteins and damaged organelles are sequestered in a double-membraned vesicle (autophagosome) and delivered to the lysosomes for degradation and recycled to fuel cellular growth . AutophaRNAi confers transient or stable gene silencing by small interfering RNAs (siRNA) or short hairpin RNAs (shRNA). Genome-wide screens with pooled shRNA libraries are widely pursued to identify cancer drivers and context-dependent events such as synthetic lethal interactions and collateral vulnerabilities . ExperimCRISPR/Cas9 is a gene-editing technology allowing for rapid and accurate assessment of gene functions with fewer off-target effects compared to RNAi, which, due to its scalability, has emerged as an important tool for large-scale screens. Functional genomics using CRISPR have to date focused on identifying genes required by cancer cells for growth or response to a therapy, which provides a novel genetic tool to ascertain gene functions by customizing the single guide RNA (sgRNA) sequence. After transduced by pooled sgRNA library, the recipient cells become genetically heterogeneous, each one with a knockout of a different gene. Following culture and selection, e.g., drug treatment, cells expressing sgRNAs that target the genes essential for proliferation or drug resistance will die, thereby depleting them from residual tumor cells after culture or treatment .RAS and BRAF , syngeneic mouse models, and patient-derived xenografts (PDX), should be considered in systematic approaches (MPM displays high heterogeneity, characterized by inter- and intratumor variability at cellular and molecular levels , 127. Herganoids . Moreoveproaches . DespiteUnlike many other solid tumors, MPM is characterized by overwhelming prevalence of loss of function alterations in tumor suppressor genes, for which direct pharmacological targeting proves difficult. However, collateral genotoxic, proteotoxic, and metabolic stresses caused by abnormal tumor genome or anti-cancer drugs can generate context-dependent vulnerabilities and dependencies, which has profound implications for alternate treatment of TSG-driven MPM. Recent studies have identified previously unappreciated vulnerabilities contextually linked with aberrant TSGs and dependencies acquired during cancer development and drug resistance, which provides unprecedented insights into MPM pathobiology and may bring about unprecedented hopes for the development of biomarker-guided precision medicine for the disease . IntegraDX conducted literature search, drafted and revised the manuscript, and prepared the figures and table. HY and RS reviewed the manuscript. R-WP designed the study and revised and proofread the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Background: Malignant pleural mesothelioma (MPM) is driven by the inactivation of tumor suppressor genes (TSGs). An unmet need in the field is the translation of the genomic landscape into effective TSG-specific therapies. Methods: We correlated genomes against transcriptomes of patients\u2019 MPM tumors, by weighted gene co-expression network analysis (WGCNA). The identified aberrant biochemical networks and potential drug targets induced by tumor suppressor loss were validated by integrative data analysis and functional interrogation. Results: CDKN2A/2B loss activates G2/M checkpoint and PI3K/AKT, prioritizing a co-targeting strategy for CDKN2A/2B-null MPM. CDKN2A deficiency significantly co-occurs with deletions of anti-viral type I interferon (IFN-I) genes and BAP1 mutations, that enriches the IFN-I signature, stratifying a unique subset, with deficient IFN-I, but proficient BAP1 for oncolytic viral immunotherapies. Aberrant p53 attenuates differentiation and SETD2 loss acquires the dependency on EGFRs, highlighting the potential of differentiation therapy and pan-EGFR inhibitors for these subpopulations, respectively. LATS2 deficiency is linked with dysregulated immunoregulation, suggesting a rationale for immune checkpoint blockade. Finally, multiple lines of evidence support Dasatinib as a promising therapeutic for LATS2-mutant MPM. Conclusions: Systematic identification of abnormal cellular processes and potential drug vulnerabilities specified by TSG alterations provide a framework for precision oncology in MPM. Malignant pleural mesothelioma (MPM) is a deadly cancer with incidence and mortality still increasing globally . The leaCDKN2A/2B), BRCA1-associated protein-1 (BAP1), neurofibromin 2 (NF2), tumor protein p53 (TP53), SET domain containing 2 histone lysine methyltransferase (SETD2) and large tumor suppressor kinase 2 (LATS2). While the pharmacological inhibition of oncoproteins is successful, targeted therapies that exploit abnormal TSGs have proven far more difficult. Precision oncology, a burgeoning effort aimed at targeting unique molecular alterations of individual patients, has achieved great success in many cancers, but significantly lags behind in MPM. Consequently, clinical trials in MPM without biomarker-directed stratifications have generally failed [Comprehensive genomic studies in MPM have revealed a rarity of pharmacologically tractable mutations in oncogenes ,4,5, buty failed ,7,8,9.CDKN2A/2B, BAP1, NF2, TP53, SETD2, and LATS2) in MPM, and the underlying implications for precision oncology. Identification of molecular traits and the associated drug vulnerabilities co-selected by the functional loss of specific TSGs provides unprecedented insights into MPM pathobiology and may promote personalized treatment of MPM patients with molecularly guided, targeted- and immuno-therapy.Although the direct intervention of tumor suppressors is challenging, aberrant TSGs induce the reprogramming of biochemical networks, which creates cancer-specific vulnerabilities and provides an alternative venue for precision oncology in TSG-driven cancer . SystemaCDKN2A/2B (homozygous deletions (HDs)), BAP1 (HDs and point mutations), NF2 (HDs and point mutations), TP53 (point mutations), SETD2 (HDs and point mutations), and LATS2 (HDs and point mutations) or NF2 (37.8%). Importantly, analyses of RPPA data of TCGA MPM cohort (n = 61) showed that genetic alterations remarkably decreased the levels of the encoded proteins or downstream effectors occurring in TCGA MPM cohort are TSGs, including tations) A. Notabltations) B. For inffectors C.To uncover fundamental molecular features associated with the functional loss of TSGs in MPM, we performed WGCNA, based on the transcriptomic data of TCGA MPM cohort , and delCDKN2A/2B encodes three tumor suppressors, p16INK4a and p14ARF (by CDKN2A) and p15INK4b (by CDKN2B), that play critical roles in cell cycle regulation. Moreover, p16INK4a and p15INK4b are functionally redundant by inhibiting cyclin-dependent kinase (CDK) 4/6 and cyclin D, and consequently blocking cell cycle progression from G1 to S [ G1 to S .CDKN2A/2B loss in MPM was significantly positively correlated with the green module , but negatively with the red (CDKN2A/2B in cell-cycle regulation. The yellow module significantly enriched the genes of extracellular matrix (ECM)-receptor interaction, PI3K/AKT, and focal adhesion pathways , FOXM1) and PI3K ) pathways, but decreased p16INK4a and PTEN (a negative regulator of PI3K) (The correlation network showed that = 0.001) . Pathway= 0.001) , consistpathways . Interroof PI3K) E, furtheCDKN2A/2B loss enriched genes of anti-viral type I interferon signaling pathway, suggesting a link between CDKN2A/2B inactivation and impaired IFN-I pathway with PI3K/AKT, but might be associated with suppressive anticancer immunity due to high ECM. Oncolytic viral immunotherapy, a novel anticancer strategy preferentially killing proliferating cancer cells but sparing normal ones, might be particularly effective for the red module-marked subset, in which the IFN-I pathway genes are often co-deleted.Collectively, these results reveal cellular processes that may represent therapeutic vulnerabilities in BAP1 alterations in MPM are positively correlated with the red module only that enriches the IFN-I pathway ,20. Our pathway F,G, and /2B loss . This fiignature . Thus, CNF2-mutant MPM.NF2 is a plasma membrane protein binding to \u03b1-catenin and tight junctions to suppress cell growth. NF2 loss deregulates multiple signal pathways, although a prevalent notion holds that the Hippo pathway is central to the phenotype of CDKN2A loss, NF2 alterations are positively correlated with the green and the yellow modules , to a less extent with the turquoise and the green-yellow , but positively with the salmon , implying that TP53 mutations deregulate multiple biological processes in MPM limits the value of this module.The purple module enriches genes of adipocyte differentiation/lipid metabolism, suggesting that rocesses and benerocesses . Supportntiation , and thentiation E,F. HoweThe top 20 best-connected genes within the purple module are AQP7, PLIN1, ADIPOQ, TUSC5, CIDEA, THRSP, PLIN4, CIDEC, C14orf180, AQP7P1, CD300LG, C6, LIPE, LEP, NTRK2, SLC7A10, KCNIP2, GPD1, PDK4, and LPL, among which chemical agonists for PDK4, PRKAR2B and LPL are available. The top 20 best-connected genes of the green-yellow module include PDK4, TUSC5, LIPE, CIDEC, KCNIP2, CTSG, THRSP, CIDEA, AQP7P1, CD300LG, C7, C6, FREM1, THSD7B, MS4A2, TPSB2, C14orf180, FAM107A, TPSAB1, and TNMD.SETD2 is a histone-modifying enzyme responsible for trimethylation of the lysine 36 residue on Histone 3 (H3K36me3) in humans. Impaired H3K36me3 causes aberrant gene regulation and chromosomal instability [tability .SETD2 alterations is exclusively abundant in the turquoise module, consisting of 1143 genes, with functions spanning from neuronal biology and receptor tyrosine kinases (particularly EGFR family) to the potassium channel, the Hippo and Wnt and drug sensitivity data, which revealed that E-cadherin is significantly upregulated in SETD2-altered MPM (CDH1 (encoding E-cadherin) is most negatively correlated with sensitivity to various EGFR inhibitors and transcriptional co-activator with TAZ.LATS2 alterations show a negative correlation with the turquoise module , which is opposite to SETD2 alterations (positively correlated with the turquoise), but expected, in that genes involved in the Hippo and tight junction pathways are enriched in the turquoise module. Importantly, LATS2 alterations in MPM are exclusively positively correlated with the brown module is the most significantly upregulated protein in LATS2-mutant MPM , we identified Dasatinib, a potent Abl/Src inhibitor, with the efficacy negatively correlated with several immune biomarkers , that are preferentially expressed by cancer cells domains are significantly enriched in the hub proteins . By correr cells B. These LATS1/2-altered MPM cells exhibited the highest sensitivity to Dasatinib inhibits CDK4/6 [CDKN2A loss renders CDKN2A-deficient MPM particularly vulnerable to CDK4/6 inhibitors [An important finding of this study is that subsets ,36,37,38s CDK4/6 , CDK4/6 hibitors , and co-CDKN2A, suggesting a rational by oncolytic viral immunotherapy for CDKN2A-altered MPM, which is supported by a recent report [CDKN2A/2B loss is widely used in pathological diagnosis to distinguish MPM from benign pleural lesions, analyzing the mutations of IFN-I\u2013related genes will improve MPM diagnosis and patient stratification.Oncolytic viral immunotherapy shows promises in MPM , partly t report . As CDKNCDKN2A/2B and NF2 deficiency, that accounts for ~55.6% (45 of 81) of MPM cases [TP53-mutant MPM.Mutant p53 has been proposed to drive metabolic reprogramming, thereby promoting cancer progression ,46,47,48(PGC-1\u03b1) ,46,47, a(PGC-1\u03b1) . These rCDKN2A/2B and TP53 is associated with an increased dependence on the G2/M checkpoint, which represents a targetable vulnerability in MPM [Notably, synthetic lethal targets with p53 inactivation have been investigated ,50,51. Iy in MPM ,57.SETD2-altered MPM, suggesting the potential of pan-EGFR inhibitors for this MPM subset. Indeed, co-mutant EGFR and SETD2 are common in glioma and pan-cancer [SETD2-mutant cancer might have evolved a unique dependence on EGFR signaling.We showed that SETD2 might have roles beyond histone modifications. Of note, RTKs, particularly EGFR members , HER2 , HER3 (ERBB3), and HER4 (ERBB4)) were exclusively enriched in n-cancer , suggestSETD2-altered MPM and predicts the sensitivity to EGFR-targeted therapies. Our finding that E-cadherin is significantly negatively correlated with EGFR inhibitor efficacy prioritizes the need for biomarker-driven selection and pan-EGFR inhibitors that target ERBB2/3/4 as well.EGFR is not mutated, but overexpressed in MPM ,59,60. ALATS2-mutant MPM, suggesting an unanticipated role for LATS2 in immunoregulation. Supporting our finding, LATS1/2 can suppress cancer immunity, and their deletion improves tumor immunogenicity by enhancing anti-tumor immune responses [LATS2-altered MPM, although how LATS1/2 modulates the immune response awaits further studies.LATS1/2 are key players of the Hippo pathway, but only LATS2 is frequently mutated in MPM. We identified the significant enrichment of immunoregulatory pathways in esponses . These rLATS2 mutational status might be a critical factor in selecting MPM patients who can benefit from immunotherapies.Immunotherapy shows promises in MPM, but with low and heterogeneous response rates ,64, arguLATS2-altered MPM. Dasatinib shows the potential to modulate anticancer immunity . AccordiAll normal human mesothelial cells Met-5A , MPM cell lines H28 , H2452 , and H2052 were obtained from ATCC . MPM celMPM cells seeded in triplicate at 96-well plates ; for 3D: 4000\u20135000 cells/well in ultra-low attachment plate) were drugged 24 h later, over a 12-point concentration range (two-fold dilution), with DMSO as vehicle. Cell viability was determined 72 h post-treatment by the Acid Phosphatase Assay Kit . The medn = 87) were downloaded from TCGA , in which 81 samples were provided with genetic alterations data. Normalized level 4 data of reverse phase protein array (RPPA) were downloaded from The Cancer Proteome Atlas (TCPA) database [http://cicblade.dep.usal.es:8080/APID/init.action) [https://www.cbioportal.org/). Processed drug (n = 481) screening and gene expression data across solid cancer cell lines (n = 659) were downloaded and reanalyzed from a published study [RNA-sequencing data of MPM samples (g/tcpa/) , which qg/tcpa/) . Protein.action) , and co-ed study . Fisher\u2019ed study .Survival analysis was performed using \u201csurvminer\u201d and \u201csurvival\u201d R packages. Tumor samples within the TCGA MPM cohort were divided into two groups, based on each hub gene\u2019s best-separation cut-off value to plot the Kaplan\u2013Meier survival curves.VCAN, FAP, POSTN, FBLN1, COL1A1, PDPN, THY1, CSPG4, IL6, TGFB1, HGF, SERPINE1). The gene list was curated based on previous studies across different cancer lineages [The extracellular matrix (ECM)/stromal gene signature was scored as the sum of an ECM/stromal gene set representing biological replicates. Gene expression and survival data derived from the public database, as well as the correlation coefficient, were analyzed using R (version 3.6.0). p < 0.05 was considered statistically significant.Data were presented as mean \u00b1 SD, with the indicated sample size (Overall, we report the systematic identification of biochemical networks and therapeutic potential linked with aberrant TSGs, which provides a framework for biomarker-guided precision oncology for MPM subsets. Our work warrants further studies that verify the drug vulnerabilities and the stratification approaches for future clinical trials."} +{"text": "Streptococcus (GBS) infection in newborns. Antibiotic exposure unbalances women\u2019s vaginal microbiota, which is associated with the establishment of the newborn gut microbiota. However, the influence of perinatal antibiotic exposure on neonatal gut microbiota colonization and health outcomes remains unclear. In this study, we found that perinatal antibiotic exposure induced microbiota dysbiosis in a woman\u2019s vagina and the neonatal gut, and we highlight a significant decrease in the abundance of Lactobacillus spp. The influence of antibiotic use on the microbiota was greater than that from gestational age. Additionally, full-term newborns without antibiotic exposure had no evidence of early-onset sepsis, whereas in full-term or preterm newborns with antibiotic exposure before birth, at least one infant was diagnosed with early-onset sepsis. These results suggest an association between perinatal antibiotic exposure and microbial dysbiosis in maternal vaginal and neonatal gut environments, which may be related to the occurrence of early-onset sepsis.Perinatal antibiotic prophylaxis is an effective method for preventing group B Lactobacillus spp. than did the FTA and PTA groups. In addition, whether in the mother or newborn, the dissimilarity in microbiota between FT and PT was the lowest compared to that between other groups. Compared to the FT and PT groups, the dissimilarity in microbial structures between the vagina and meconium decreased in the FTA and PTA groups. The health outcome of infants reveals an association between early-onset sepsis and antibiotic-mediated microbiota dysbiosis. In conclusion, perinatal antibiotic exposure is related to the establishment of gut microbiota and health risks in newborns. Promoting the rational usage of antibiotics with pregnant women will improve neonatal health.Intrapartum antibiotic prophylaxis reduces the risk of infection to a mother and neonate, but antibiotic-mediated maternal and neonatal microbiota dysbiosis increases other health risks to newborn infants. We studied the impact of perinatal antibiotic prophylaxis on the microbiota in mothers and newborns with full-term or preterm delivery. Ninety-eight pregnant women and their neonates were divided into the following four groups: full term without antibiotic exposure (FT), full term with antibiotic exposure (FTA), preterm without antibiotic exposure (PT), and preterm with antibiotic exposure (PTA). Bacterial composition was analyzed by sequencing the 16S rRNA gene from maternal vaginal swabs (V) and neonatal meconium (F). The results showed that in maternal vaginal and neonatal meconium microbiota, FT and PT groups had a higher load of IMPORTANCE Perinatal antibiotic prophylaxis is an effective method for preventing group B Streptococcus (GBS) infection in newborns. Antibiotic exposure unbalances women\u2019s vaginal microbiota, which is associated with the establishment of the newborn gut microbiota. However, the influence of perinatal antibiotic exposure on neonatal gut microbiota colonization and health outcomes remains unclear. In this study, we found that perinatal antibiotic exposure induced microbiota dysbiosis in a woman\u2019s vagina and the neonatal gut, and we highlight a significant decrease in the abundance of Lactobacillus spp. The influence of antibiotic use on the microbiota was greater than that from gestational age. Additionally, full-term newborns without antibiotic exposure had no evidence of early-onset sepsis, whereas in full-term or preterm newborns with antibiotic exposure before birth, at least one infant was diagnosed with early-onset sepsis. These results suggest an association between perinatal antibiotic exposure and microbial dysbiosis in maternal vaginal and neonatal gut environments, which may be related to the occurrence of early-onset sepsis. Streptococcus (GBS) infection in China .On average, we generated 43,951\u2009\u00b1\u20098,572 tags and 357\u2009\u00b1\u2009228 operational taxonomic units (OTUs) for vaginal swabs, as well as 41,461\u2009\u00b1\u200911,192 tags and 319\u2009\u00b1\u2009192 OTUs for meconium samples. Additionally, no nucleic acids were amplified from the negative controls. Permutational multivariate analysis of variance (PERMANOVA) showed that the time of first maternal antibiotic exposure did not affect the vaginal and neonatal microbiota composition in each group (P = 0.001) and between PT-V and PTA-V (P = 0.000) . In addition, for vaginal swabs without antibiotic exposure, Lactobacillus spp. had about a 1.8-fold higher abundance in full-term than in preterm infants , top. Fo infants , bottom.P = 0.026). Regardless of antibiotic exposure before delivery, the Lactobacillus load in meconium microbiota with full-term delivery was significantly higher than that with preterm delivery . Nevertheless, there were no significant differences in Lactobacillus load between FT-F and FTA-F (P\u2009=\u20090.423) or between PT-F and PTA-F (P\u2009=\u20090.237).For meconium microbial samples, the dissimilarity between FTA-F and PTA-F was slightly higher than that between the FT-F and PT-F groups , top infection. For the mother of neonate P1 (P1-M), cefazolin was administered 11\u2009h before full-term delivery, and Bifidobacterium spp. (98.55%) were dominant in the meconium microbiota (Lactobacillus spp. (1.36%) was identified in neonate P2\u2019s meconium microbiota (Lactobacillus spp. (7.33%) and a high level of Staphylococcus spp. (4.27%) compared to the vaginal microbiota of mothers without antibiotic exposure in the PT-V group. In the meconium microbiota of neonate P3, Staphylococcus (20.66%) was the predominant genus (Lactobacillus (98.46%) (Sphingomonas spp. (12.48%) and by 68.19% of OTUs which could not be classified to known taxonomic units experienced antibiotic exposure because of a high risk of group B Lactobacillus spp. in vaginal microbiota are associated with a high risk of preterm delivery and impose negative effects on mother-to-infant microbiota transmission newborn infection.The pregnant women were recruited according to the following criteria: (i) 20 to 35\u2009years of age and no history of smoking, taking drugs, or alcoholic intemperance; (ii) normal rate of weight gain, body mass index, hepatorenal function, myocardial enzymes, body glucose, serum total cholesterol, and triglyceride levels during pregnancy; (iii) no family allergy history, abnormal complications, or chronic diseases in pregnancy ; (iv) absence of antibiotic exposure before our study; and (v) a vaginal delivery. The paired neonates were excluded as per the following criteria: (i) had birth asphyxia or were diagnosed with hypoxic-ischemic encephalopathy/intracranial bleeding; (ii) had a genetic metabolic disease or congenital malformation; (iii) an antibiotic or microecological preparation was used before sampling; (iv) were a twin or multiple birth; or (v) had noninfectious disease, such as neonatal cholestasis, neonatal hepatitis, and hemolytic diseases of newborns. Before birth within 48\u2009h, empirical cefazolin was used through intravenous injection with 2 g every 12\u2009h on pregnant women for the prevention of group B n\u2009=\u200923), full term with antibiotic exposure , preterm without antibiotic exposure , and preterm with antibiotic exposure . One month after birth, a follow-up investigation was conducted to assess the risk of EOS without antibiotic exposure . The collected meconium (obtained from a sterile single-use diaper) was stored at \u201380\u00b0C without buffer in 30 min . Enveloped sampling materials were also collected as negative controls for the assessment of DNA contamination. The microbial genomic DNA was extracted from vaginal and meconium samples using the DNeasy PowerSoil kit , according to the manufacturer\u2019s protocol. A Qubit fluorometer was used to qualify isolated DNA. The V4 region of the 16S rRNA gene was amplified utilizing the 515F/806R primers and sequenced on a MiSeq platform , with a 2\u2009\u00d7\u2009250-bp cartridge. Raw sequencing reads were filtered and clustered into operational taxonomic units (OTUs) with 97% similarity via USEARCH and thenPRJNA553858.The raw reads have been deposited to GenBank under BioProject number"} +{"text": "Photoredox catalysis (PRC) and synthetic organic electrochemistry (SOE) are often considered competing technologies in organic synthesis. Their fusion has been largely overlooked. We review state\u2010of\u2010the\u2010art synthetic organic photoelectrochemistry, grouping examples into three categories: 1)\u2005electrochemically mediated photoredox catalysis (e\u2010PRC), 2)\u2005decoupled photoelectrochemistry (dPEC), and 3)\u2005interfacial photoelectrochemistry (iPEC). Such synergies prove beneficial not only for synthetic \u201cgreenness\u201d and chemical selectivity, but also in the accumulation of energy for accessing super\u2010oxidizing or \u2010reducing single electron transfer (SET) agents. Opportunities and challenges in this emerging and exciting field are discussed. Photochemistry with potential: The synergy of photochemistry and electrochemistry in organic synthesis is beneficial not only for synthetic sustainability and chemical selectivity, but also in the accumulation of energy for accessing super\u2010oxidizing or \u2010reducing single electron transfer agents. Examples of synthetic organic photoelectrochemistry are dissected into three categories: electrochemically mediated photoredox catalysis, decoupled photoelectrochemistry, and interfacial photoelectrochemistry. Nature's solution is chlorophyll, a colored pigment that absorbs visible\u2010light energy to drive the process. Researchers have made efforts towards artificial photosynthesis with visible light ever since Giacomo Ciamician's vision in the turn of the 20th century (1912).II and IrIII can harvest visible\u2010light photons to become powerful excited\u2010state single electron transfer (SET) agents for redox processes, and enjoy sufficiently long lifetimes (700\u20131100\u2005ns)II and IrIII bipyridyl complexes.Chemical synthesis by visible light is the fundamental process for biological photosynthesis on Earth. However, CO century 912.1 By With sustainability and cost at the forefront of minds in academia and chemical industry,Another vehicle for SET chemistry, which has been undergoing a renaissance in recent years, is synthetic organic electrochemistry (SOE). The application of electrical current in organic synthesis dates back as far as the Faraday and Kolbe electrolysis reactions from the 1830s to 1840s;1.1II complexes.2 and H2O into glucose and water,A fundamental problem in visible\u2010light PRC is that the energy of processes is constrained by the energy of visible\u2010light photons . Inevitably, not all of this energy is accessible to the photocatalyst; losses occur due to intersystem crossing/non\u2010radiative pathways, which can account for up to approximately 0.6\u2005eV in the case of Ru3N), the formed radical anion absorbs the second quantum of visible\u2010light energy. Ultimately, a super\u2010electron donor is formed in\u2005situ by accumulation of visible photons. Although the subsequent chemistry may be redox\u2010neutral, the requirement for a sacrificial electron donor to ensure a sufficient concentration of photoexcitable radical anion is undesirable. This strategy may not be so general because it requires design of photocatalyst architectures that absorb visible light both in their ground state and in their radical ion state.Nature's solution to the \u201cenergy problem\u201d is to accumulate the energies of multiple photons.1.2n\u2010Bu4NPF6), the solution conductivity can be increased and the ohmic drop decreased;A fundamental problem in SOE is that the conductivity of organic solvents is typically low (compared to aqueous systems). A high \u201cohmic drop\u201d exists between the two separated electrodes, necessitating high cell potentials for useful reaction conversions. Such potentials may be high enough to encourage unselective, deleterious redox processes when applied to the organic substrate of interest. The cell potential is the sum of electrode potential and ohmic drop. By employing a high concentration of supporting electrolyte of DCA is first populated with an electron by cathodic current, thus becoming SOMO\u20102 (\u03c82) of DCA.\u2212. Photoexcitation promotes an electron from the MO\u20101 (\u03c81) to the SOMO\u20102 (\u03c82), thus effecting SOMO\u2013HOMO inversion.One fundamental, exciting branch of e\u2010PRC is the photoexcitation of electrochemically generated ions.E1/2\u2009(PTZ)=+0.79\u2005V vs. SCE),Ep/2ox=+1.57\u2005V vs. SCE),1 or 2 upon further oxidation/reaction with H2O. No reaction of DPE with PTZ+. occurred in the dark.The combination of photochemistry with electrochemistry within the context of organic synthesis was first disclosed by Moutet and Reverdy,N,N,N\u2032,N\u2032\u2010tetraphenyl\u2010p\u2010phenylenediamine (TPPD) radical cations and their photoexcitation with UV light (366\u2005nm) enabled oxidation of benzyl alcohol (3) to benzaldehyde developed by Fukuzumi and co\u2010workers.Epox=+2.37\u2005V vs. SCE)In terms of SET oxidation, among the most powerful photoredox catalysts are the acridinium salts that can engage unactivated or electron\u2010deficient arenes.g Figure\u200543, 44 an+ and avoids the complications associated with PRC using DDQ. In an elegant and seminal e\u2010PRC example, Lambert and co\u2010workers reported the oxidation of unactivated arenes and their coupling with heterocyclic amines was oxidized to its dication radical , which is strongly coloured. Excitation of TAC.2+ with visible light (ca. 600\u2005nm) provided the superoxidant *TAC.2+ (E1/2=+3.33\u2005V vs. SCE), which oxidized unactivated arenes to their radical cations. The remarkable potential of *TAC.2+ was rationalized by time\u2010dependent density functional theory (TD\u2010DFT) calculations, which revealed a SOMO\u2013HOMO level inversion leaving a low\u2010lying hole in the HOMO. Ethyl 1H\u2010pyrazole\u20104\u2010carboxylate (13) undergoes nucleophilic addition to the benzene radical cation, generating (upon loss of a proton) an aryl radical. Oxidation of the aryl radical, either by TAC.2+ or by the carbon (felt) anode, followed by loss of a proton, furnishes product 14. Proton reduction was proposed as the corresponding cathodic half\u2010reaction, as gas bubbles were observed. Control reactions confirmed that no reaction occurred without light, current, or TAC. For comparison, direct electrolysis was performed at fixed potential (+3.0\u2005V vs. SCE) and gave polymeric material, exemplifying the advantage of the mild conditions of e\u2010PRC. The reaction tolerated benzene and even chloroarenes to give products 15 and 16, albeit in modest yield. Substituted triazoles, benzotriazoles, and purines were successful partners, affording products such as 17 and 18. No oxidation of aldehyde\u2010, ketone\u2010, or ester\u2010bearing pyrazoles was observed. The expansion of scope to unactivated or electron\u2010deficient arenes represents a key advantage over Nicewicz's original report.Photoexcitation of electrochemically generated cations allows for redox potentials that are notably more positive than those achieved even with *Mes\u2010Acrs Figure\u2005.39 Under19 using photoexcited 9,10\u2010dicyanoanthracene radical anion (*DCA.\u2212),2pin2, Sn2Me6, or heteroarenes to give products such as 21\u201324. Oxidation of sacrificial Zn anode was proposed as the corresponding half\u2010reaction. The method provides a key advantage over palladium\u2010catalysed functionalizations used to generate similar products, which suffer when coupling partners contain Lewis basic groups (such as the precursor to 23) as they alter the course of catalysis by coordination.In a complementary fashion, cathodic current can be used to generate radical anions photoexcited to generate superreductants. Lambert, Lin, and co\u2010workers reported the reduction of chloro\u2010 and bromoarenes such as 2.1.2+) is a potent oxidant (Epred=+2.06\u2005V vs. SCE) capable of SET oxidation of isopropyl trifluoroborate 25\u2010H+ in a Minisci\u2010type manner, which, followed by loss of a proton and SET oxidation (either by ground\u2010state Mes\u2010Acr+ (Ep/2red=\u22120.57\u2005V vs. SCE) or by the anode) affords product 27.Although the former subsection likely presented a more fundamental and potentially ground\u2010breaking advantage of e\u2010PRC in organic synthesis, replacement of sacrificial redox agents is another very important aspect offered by e\u2010PRC that appeals to a sustainability and industry perspective Figure\u2005. Xu and + is regenerated by anodic oxidation of Mes\u2010Acr. at a reticulated vitreous carbon (RVC) anode. A wide substrate scope of heteroarenes were employed, including isoquinolines, phenanthridines, phthalazines, benzothiazoles, acridines, and purines, affording products such as 28\u201331. The reaction conditions tolerated secondary and tertiary amines as well as secondary alcohols and alkynes, which would all be prone to oxidation under direct electrolysis at high potentials.Mes\u2010AcrNAr reactions of unactivated aryl fluorides under e\u2010PRC to engage chlorofluoroarenes such as 32 in SET oxidation. In terms of the heteroarene partner, the substrate scope was similar to that of the previous report involving the photoexcited dication *TAC.2+.35 and 36. Alcohols such as ethanol and acetal\u2010protected galactose, as well as tert\u2010butyl carbamate, were also well\u2010tolerated as nucleophiles (products 37 and 38). Redox potentials for the oxidation of polyhalogenated benzenes are unavailable in the literature, likely because they exceed the redox potential window of the solvent. It is interesting that although *TAC.2+ (Epred= +3.33\u2005V vs. SCE) is a more potent oxidant than *DDQ, it afforded a lower yield of 34. This suggests that matching of redox potentials is not always a reliable predictor of successful SET chemistry and that other factors such as precomplexation of mediator and substrate (Section\u20053.3), might be important. Elsewhere, oxidation of unactivated alcohols was recently achieved under e\u2010PRC with riboflavin tetraacatate as the photocatalyst and thiourea as a HAT co\u2010catalyst.Lambert and Huang reported SC Figure\u2005.49 Here,2.212a (CoIII) or a CoII macrocyclic complex 42 to give CoI complex 43, which reacted with anhydride 39. Photochemical cleavage of the CoIII\u2212C bond of 44 presumably afforded an acyl radical 45, primed for 1,4\u2010addition to 40 to give 46. The authors claimed that HAT from the solvent to 46 yielded product 41. SET reductions of 45 or 46, followed by proton transfer from the solvent, could not be ruled out. The authors did not specify the anodic half\u2010reaction or the anode materials. Here, photochemistry and electrochemistry were handled as discrete processes, representing the first example of decoupled photoelectrochemistry (dPEC).Sheffold and Orlinski reported a photoelectrochemical 1,4\u2010addition of acyl groups to \u03b1,\u03b2\u2010unsaturated carbonyl compounds Figure\u2005.51 Catho3)\u2212H bonds under dPEC.3)\u2212H bonds (products 49 and 50).Stahl and Wang reported a Hofmann\u2013L\u00f6ffler\u2013Freytag\u2010type (HLF) amination of C were employed to afford oxazolines (products 51 and 52). Various heterocycle\u2010bearing substrates were tolerated despite the anodic potential and in\u2005situ generated molecular iodine. Acid hydrolysis of the oxazolines gave rise to pharmaceutically valuable (protected) 1,2\u2010amino alcohols (product 53). This work follows on from electrochemical HLF reactions reported by Mu\u00f1iz,47 into product 48, instead yielding a complex mixture of products.In addition to the HLF reactions of product . This wo product . This wo2.3EAP) is used to improve charge carrier separation upon irradiation (preventing recombination and generation of heat). For photoanodes, applied potential followed by irradiation promotes an electron from the valence band to the conductive band, generating a hole that is used for oxidation chemistry photoanode was irradiated with blue LEDs and was rendered highly oxidizing . Anisole was oxidized to its radical cation, primed to nucleophilic attack by a range of aromatic heterocycles (such as 54) in an overall C\u2212H amination of electron\u2010rich arenes to furnish products such as 55\u201359 , also known as \u201cphotoelectrocatalysis\u201d, a photoelectrode is coated in a photoresponsive material whose band gap corresponds to the energy of a visible\u2010light photon. An applied or \u201cbias\u201d potential . The markedly different ortho/para (o/p) selectivity between the two reportsortho position, and that the fundamental photoelectrochemical process proceeds through the same intermediates as the photochemical example.The fact that the arene scope was limited to electron\u2010rich arenes, mirroring the original amination report of Nicewicz and co\u2010workers,60), benzyl alcohol (3), and cyclohexene (62) in MeCN under iPEC using a BiVO4 photoanode and a 100\u2005W Xe lamp fitted with an AM1.5G filter as simulated sunlight, to give products 61, 4, and 63, respectively was employed as a soluble, transparent hole\u2010transfer mediator60 and 62, it was necessary to employ t\u2010BuOOH as the external oxygen source. The same oxidations could be achieved under electrochemical potential only (Ecell=+1.8\u2005V vs. Ag/AgCl) with a glassy carbon anode/cathode, leading the authors to assume that this potential matched the pseudo\u2010standard potential of NHS. However, the authors noted that their iPEC method, which operates at a 1.0\u2005V lower applied potential than the electrochemical cell, expects energy savings of 60\u2009%. Although product yields were modest, the authors noted that the ability to perform organic synthesis at a solar\u2010to\u2010electricity efficiency (\u03b7=1.3\u2009%) close to that of traditional photoelectrochemical water oxidation (\u03b7=1.7\u2009%) is important because of the higher value of the organic products.Several reports of oxidation of simple organic molecules by iPEC exist, for example, alcohol oxidations.y Figure\u2005.60N\u2010Hyd3 photoanode4/WO3 photoanode4/WO3 photoanode in the iPEC oxidative dimethoxylation of furan 64 mediated by bromide ions electrochemical reaction vessels. These generally fall into two categories Figure\u2005: a)\u2005an u3.2A, extinction coefficient \u03f5, path length l, and molar concentration c is given by the Beer\u2013Lambert law [Eqn.\u2005I and the absorbance A, which highlights the fundamental challenge faced in the scale\u2010up of photochemical processes. General theory predicts that for a typical 50.0\u2005mm reaction with a photocatalyst loading of 1\u2005mol\u2009% (0.5\u2005mm) and \u03f5=11280\u2009m\u22121\u2009cm\u22121 (452\u2005nm absorption band of Ru(bpy)3Cl2), 90\u2009% of the light is absorbed at l=0.2\u2005cm from the reactor surface.Both PRC and SOE suffer upon scaling up in batch mode due to the physical constraints governing transfer of photons to the reaction or electrons to/from the reaction. The relationship between absorbance law Eqn.\u2005. RearranIcell is given by a derivative of the Butler\u2013Volmer equation [Eqn.\u2005n, Faraday's constant F, electrode surface area A (cm2), mass transfer coefficient km, and reagent concentration c. Hence, the cell productivity can be increased by increasing the electrode surface area and by mixing (increasing km by decreasing the size of the diffusion layer). The time\u2010dependent fractional conversion X of a mass\u2010transfer\u2010limited reaction of volume V is given by Equation\u2005km) and largest possible electrode SAVR.While SOE in macrobatch reactors has been achieved on an industrial scale, phenomena such as interelectrode ohmic drop, mass transfer, reaction selectivity, or environmental factors have presented barriers to various synthetic processes.ion Eqn.\u2005 and is rContinuous flow (CF) is a globally recognized technology within chemical industries and academia2 by a photoanode in CF.2 photoanode under irradiation from a Hg(Xe) (200\u2005W) lamp discussed herein (Section\u20052.1.1) are rare, doublet excited states. The ultrashort lifetime of doublet excited states (fs to ps).2+ as an e\u2010PRC , sulfoxides, and sulfides may be possible. The potentials that would be required in such scenarios by SoE would no doubt lead to decomposition/poor chemoselectivity. Finally, a notable challenge is the inability to measure redox potentials of substrates that lie beyond the redox window of the solvent.The elucidation of such precomplexation mechanisms presents a challenge and demands the use of advanced spectroscopic, spectroelectrochemical, and theoretical tools.3.4One key challenge for the scientific community is the adoption of appropriate and consistent nomenclature in this rapidly developing field of synthetic photoelectrochemistry. This is important not only to avoid misunderstanding between the different strategies and concepts herein, but also to avoid confusion with other distinct research fields and phenomena such as photoelectrolysis and the photoelectric effect. In fact, reports discussed herein (that do not involve photoelectrodes) are divided in their use of the terms \u201cphotoelectrochemistry\u201d,\u201cterm applied to a hybrid field of chemistry employing techniques which combine photochemical and electrochemical methods for the study of the oxidation\u2010reduction chemistry of the ground or excited states of molecules or ions. In general, it is the chemistry resulting from the interaction of light with electrochemical systems.\u201d Therefore, we recommend \u201dsynthetic photoelectrochemistry\u201c as a blanket term that can encompass all of the examples presented herein.The IUPAC definition2),In electrochemistry, \u201celectrocatalysis\u201d refers to catalysis of electrochemical reactions by surface states of electrodes \u201d and \u201celectromediated photoredox catalysts\u201d (e\u2010PRCs).\u201cRedox catalysts\u201d and \u201cmediators\u201d are well\u2010established terms in SOE for the shuttling of electrons between electrode surfaces and substrates,4Synthetic photoelectrochemistry is a swiftly emerging research field following renaissances in its respective parent technologies, photoredox catalysis (PRC) and synthetic organic electrochemistry (SOE) that have taken place over the last decade.The authors declare no conflict of interest.Joshua P. Barham was born in Watford (UK). He received his industry\u2010based PhD in 2017 under the supervision of Prof. John A. Murphy at the University of Strathclyde and Dr. Matthew P. John at GSK (UK). His postdoctoral studies with Prof. Yasuo Norikane and Prof. Yoshitaka Hamashima at AIST and the University of Shizuoka (Japan) specialized in photoredox catalysis and microwave flow chemistry. In 2019, he was awarded a Sofja Kovalevskaja Award from the Alexander von Humboldt foundation to lead an independent research group at the University of Regensburg, investigating photo\u2010, electro\u2010, photoelectro\u2010, and flow chemistry in organic synthesis.Burkhard K\u00f6nig was born in Wiesbaden (Germany). He obtained his PhD in 1991 from the University of Hamburg and pursued postdoctoral studies with Prof. M.\u2005A. Bennett, Research School of Chemistry, Australian National University, Canberra, and Prof. B.\u2005M. Trost, Stanford University. He became full professor of organic chemistry at the University of Regensburg in 1999. His current research interests revolve around the application of visible\u2010light chemical photocatalysis towards organic synthesis."} +{"text": "Secretomes of mesenchymal stem cells (MSCs) have been successfully studied in preclinical models for several biomedical applications, including tissue engineering, drug delivery, and cancer therapy. Hydrogels are known to imitate a three-dimensional extracellular matrix to offer a friendly environment for stem cells; therefore, hydrogels can be used as scaffolds for tissue construction, to control the distribution of bioactive compounds in tissues, and as a secretome-producing MSC culture media. The administration of a polymeric hydrogel-based MSC secretome has been shown to overcome the fast clearance of the target tissue. In vitro studies confirm the bioactivity of the secretome encapsulated in the gel, allowing for a controlled and sustained release process. The findings reveal that the feasibility of polymeric hydrogels as MSC -secretome delivery systems had a positive influence on the pace of tissue and organ regeneration, as well as an enhanced secretome production. In this review, we discuss the widely used polymeric hydrogels and their advantages as MSC secretome delivery systems in biomedical applications. MSCs cater 2011 .Secretome-based therapies for tissue repair and regeneration are allogeneic and can be pre-prepared as ready-to-use therapies for a variety of conditions, similar to vaccines and monoclonal antibodies. Moreover, when compared to cell-based products, secretomes can be freeze dried or lyophilized for convenient storage and delivery ,14. SecrThe direct injection of secretomes has been shown to create secretome instability in vivo, as the secretome might be destroyed by enzymes or seep into adjacent tissues . A studyMSCs are multipotent cells that are excellent candidates for cellular therapy because of their ability to influence the tissue microenvironment via extracellular vesicles . MSCs, aMSCs have immunomodulatory abilities that regulate dendritic cells, lymphocytes, macrophages, mast cells, neutrophils, and natural killer cells, which are all involved in the immune response used to treat various inflammatory diseases . MSCs arThe bioactive factors from MSCs were harvested and cultured in vitro and called secretome or conditioned medium (CM). The MSC-derived secretome contains a wide variety of pro-regenerative factors , MSCs as a new type of regenerative medicine through non-invasive therapeutic cellular approaches so that it can overcome the poor decomposition of transplanted cells . MSCs caSecretome or CM is defined as a set of bioactive factors secreted by stem cells into the extracellular space, which includes soluble proteins , lipids, free nucleic acids, and membrane-bound extracellular vesicles containing biomolecules, such as apoptotic bodies, microparticles, and exosomes, used as a non-cellular therapeutic approach ,42. In fSecretomes represent communication pathways between cells and play important roles in several cellular mechanisms, such as the exchange of genetic material, transfer of biologically active molecules, and defense against viral attack in mammalian cells ,45. MSC-The therapeutic efficacy of secretome is related to three key mechanisms of action: (i) the homing ability, in which systemically administered cells selectively migrate to injured tissue via chemoattraction mediated by cell surface receptors of chemokines, integrins, and selectins; (ii) the ability to differentiate into new, different cell types to replace damaged cells; and (iii) the ability to secrete substances that can modulate resident cell responses via paracrine action ,47. SecrThe use of cell-free therapies in regenerative medicine, such as MSC-sourced secretions, has several advantages over stem cell-based applications: (a) the use of secretomes eliminates some of the potential safety concerns associated with the transplantation of live cell and population proliferative cells, such as immune compatibility, tumorigenicity, embolism formation, and transmission of infection; (b) secretomes sourced from MSC can be evaluated for safety, dosage, and potency in the same way as conventional pharmaceutical ingredients; (c) storage can be carried out without the use of potentially toxic cryopreservative agents for extended periods, without loss of product potency ,49,50; , vascular endothelial growth factor (VEGF), transforming growth factor beta-1 (TGF-\u03b21), basic fibroblast growth factor (bFGF), placental growth factor (PGF), nerve growth factor (NGF), interleukin-1 (IL-1), IL-6, IL-10, and Tumor Necrosis Factor alpha (TNF-\u03b1) ,38,50. TThe secretome released by MSCs can vary depending on a number of parameters, including cell species, tissue source, isolation technique, chemical and physical stimuli, and the cell microenvironment. Different secretome profiles and angiogenic potentials can be found in MSCs derived from different sources. Adipose-derived stem cell (ADSC) MSC secretome has a greater range of angiogenic factors than bone marrow stem cells (BMSCs) for angiogenesis-mediated tissue regeneration condition ,53. BecaThe reported actions of the MCS secretome products following local injection in numerous experimental in vivo models are undoubtedly the most plausible scientific evidence of their biological effects . The entIn general, secretome administration is considered safer than live cell transplantation of MSCs. In fact, this leads to a lower risk of embolic formation after intravenous infusion and a lower risk of tumorigenic or pathological transformation due to uncontrolled cell differentiation. In addition, because the secretome reflects the composition of the stem cell, it can retain the same distinctive immune properties as the MSCs, allowing the use of allogeneic preparations (even between species) without immune activation. Another important advantage to consider is that treatment with a secretome is not permanent. Regarding safety, although high doses are administered in some cases, no side effects have been reported; thus, if any side effects are present, they can be interrupted more easily than with cell administration. Doses and routes of administration vary substantially in different pre-clinical animal studies; therefore, a safe and effective dose must be determined for use in the treatment of these pathological conditions. In addition to the right dose, the appropriate time and frequency of administration also need to be determined by further research. In fact, secretome may be administered in repeated doses to maintain its therapeutic potential over time, as several authors have reported ,39,77.Secretome delivery in biomedical applications is quite difficult, mainly because the damaged tissue is not easily targeted. Various delivery systems and strategies have been developed to exploit the therapeutic potential of the stem cell secretome in organ/tissue repair and regeneration. Depending on the target site, the secretome or CM can be administered by direct injection, systemic injection, or a delivery device, such as a hydrogel .\u00ae was made to treat myocardial infarction in rats due to its strong protein adsorption capacity and contribution to the shear-thinning characteristics of hydrogels. Secretomes from spheroid cultures of human ADSCs were introduced by intramyocardial injection and administered to the peri-infarction area of the heart. Secretome delivery improved cardiac function and vascularity, while decreasing fibrosis and scar tissue development [Hydrogels are water-soluble polymer networks that can absorb more than 20% by weight in water, while maintaining their three-dimensional structure. Hydrogels have been extensively researched in therapeutic applications, such as tissue engineering and drug delivery, because of their easily modifiable chemical and physical properties, as well as their unique feature that they can be made from any hydrophilic polymer . Hydrogeelopment .Researchers in academia and business have been working on the design and development of \u201csmart polymers\u201d in general, and hydrogels in particular, in response to a wide range of therapeutic issues and improving treatment standards . HydrogeRecent research has discovered that hydrogel polymerics have chemical, physical, and biological properties that are similar to ECM in natural tissues, bringing cells together and controlling tissue structure, regulating cell function, and supporting cell attachment, proliferation, and differentiation.The outstanding properties of the hydrogel three-dimensional (3D) hierarchical structure, which allows cells to accumulate and grow inside, organizes cells into a 3D functional tissue, and enables the efficient mass transfer of nutrients, oxygen transfer, and waste exchange in cells and tissues, have resulted in their excellent performances in a variety of biomedical applications .Many different material hydrogels have been explored and used as drug delivery in tissue engineering, ranging from natural biomass to synthetic biopolymers. High elasticity and flexibility, as well as exceptional biocompatibility and biodegradability, are all characteristics of these materials. Natural biopolymers are produced by all organisms during their growth cycles and are frequently available in considerable numbers. Before tissue engineering became a research area, most natural biopolymers were well known in biomedical applications. The group includes proteins, such as collagen and fibrin, polysaccharides, such as chitosan (CS) and hyaluronic acid (HA), and some derivatives of the extracellular matrix. The inherent bioactivities of these natural biopolymers can promote cell adhesion, proliferation, and tissue recovery. The hydrogel could also be constructed from synthetic biopolymers. A commonly used synthetic biopolymer is poly(lactic acid) (PLA), an aliphatic polyester, which could degrade within the human body to form lactic acid, a naturally occurring chemical that can be easily removed from the body. Similar materials include polyglycolic acid (PGA), polycaprolactone (PCL), and poly(lactic-co glycolic acid) (PLGA). Synthetic biopolymers have a long history of use in biological applications and are generally regarded safe. However, numerous disadvantages remain, such as low hydrophilicity, bioinert characteristics, and the lack of biological signals in the ECM, which severely limit their effectiveness in tissue engineering applications. Thus, developing highly bioactive hydrogels to replace, modify, or sustain injured tissues is critical, which can be rationally accomplished by including secretomes into hydrogel systems .HA, also known as hyaluron, is a non-immune polysaccharide containing the units a-1,4-Dglucuronic acid (GlcUA) and b-1,3-acetyl-D-glucosamine (GlcNAc) . HA is aThe use of HA gel as a high-quality antiadhesive biomaterial for controlled release via intrauterine administration can restore endometrial morphology and function after electrocoagulation injury in mice. This therapeutic platform uses paracrine signaling with efficacy similar to that of cell therapy. Adhesion protection allows a fast recovery process using MSC secretomes . The conThe physical properties of the HA hydrogel can be changed to regulate the administration of MSC CM as an injection or implant material for the repair and regeneration of soft and hard tissues . In lipo2+, Sr2+, and BA2+ to improve mechanical characteristics. However, controlling the rate of degradation may be difficult; generally, cationic polymers have significant problems in this regard. Natural materials have a number of disadvantages, including poor mechanical characteristics and cell adhesion [Alginate is a naturally occurring anionic polymer electrolyte made from polysaccharides derived from brown algae. It is a tight linear copolymer consisting of the monosaccharides a-L-guluronic acid (G) and b-D-mannuronic acid (M). It has been discussed in many published studies because of its abundance, low cost, and biocompatibility, and its application has increased in recent years ,93. Algiadhesion . MSCs inadhesion .Aggregate hMSCs incorporated into an RGD-alginate hydrogel can maintain its viability and structure, as well as the formation of the ECM. The morphology of the ECM regions embedded in the alginate fragments was observed, with single cell delivery resulting in a more even distribution of pockets across the construct and aggregate delivery resulting in greater fragmentation, whereas single cells remained trapped in the alginate at 14 days after implantation . SubstraCG is a sulfated hydrophilic polysaccharide obtained from various species of red algae and is widely used by the food industry and in regenerative medicine as scaffolds and controlled release systems for the delivery of pharmaceutical drugs, growth factors, and cells. Considering its potential, we previously used CG hydrogels in conjunction with skin-derived MSCs (SD MSCs) to treat skin lesions in a murine wound healing model. The combination of CG and SD MSC is able to reduce inflammation, accelerate recovery, and increase ECM deposition in the wound area. However, the results showed that the use of CG hydrogel or polyvinyl alcohol (PVA), as well as delivery of the embedded SD MSC secretome, did not improve wound healing or lead to better outcomes of tissue vascularization in an in vivo model .A linear anionic microbial polysaccharide named gellan gum (GG) consists of repeating units of glucose, glucuronic acid, and rhamnose. The use of GG and its derivatives for hydrogels has been shown to be beneficial in disc repair/cartilage regeneration. In addition, it has previously been shown that the modification of a hydrogel with an extracellular matrix derived peptide GRGDS-fibronectin can be immobilized onto GG hydrogels. This peptide-modified gellan gel can increase the adhesion and proliferation of neural stem progenitor cells (NSPCs) more than the control GG. The results revealed that the presence of the peptide increased the proliferation of spinal cord cells (BM MSCs), and increased metabolic activity and cell morphology. It was also shown that GRGDS-GG positively modulates BM MSC secretion, which enhances the survival and differentiation of primary cultures of hippocampal neurons in vitro .Collagen is a key component of ECM proteins found in mammalian tissues. Natural collagen has been widely used as a highly adaptable biocompatible, biodegradable material and as a biomaterial in tissue engineering. Collagen has a unique helical structure in which three left-handed polypeptide helices intertwine to form a right-handed triple helix, and peptide bonds cross-link adjacent helices at the ends of each helix . As a re\u00ae via electrostatic interactions. The VEGF/phyllosilicate (LAPONITE)\u00ae complex was then encapsulated in a collagen scaffold, showing increased angiogenesis in vivo. The nanocomposite hydrogel platform can offer sustained release in the synthetic clay, allowing the full therapeutic benefits to be achieved. This method is recognized as an alternative strategy to the stem cell transplantation approach, producing more paracrine factors [Previous studies have reported the successful retention and controlled release of VEGF by surface adsorption of bioactive phyllosilicate (LAPONITE) factors . In an i factors .\u00ae nanosilicates were developed to form nanocomposite hydrogels with the aim of controlling the release of stem cell secretomes. The results showed that encapsulation of stem cell-derived secretions in nanocomposite hydrogels provides a promising alternative in cardiac tissue regeneration therapy. The cell-derived secretion-derived hydrogel nanocomposite provides a dual-action therapeutic system through its proangiogenic and cardioprotective capabilities [Gelatin is a soluble protein formed by the irreversible partial hydrolysis of collagen and can be obtained from fish, insects, and the skin of land animals. Gelatin is a high molecular weight polydisperse peptide that is widely used in food and biomedicine due to its gelling and thickening properties. Chemical and physical crosslinking methods can be used to make gelatin hydrogels. Gelatin gels have a number of disadvantages, including rapid deterioration, poor mechanical quality, and lack of heat stability . Therefobilities .In another study, MSC CM could be encapsulated into nanoparticles and then inserted into a hydrogel to form a composite without losing its functional characteristics. MSC CM nanocomposite hydrogels were prepared using gelatin, hyaluron, and Poly Latic Acid for the regulated release of MSC CM. The biocompatibility of MSC CM hydrogel is indicated by its ability to facilitate MSC adhesion and proliferation, thereby increasing the metabolic activity and potential of MSC as a regenerative drug . For thePolyethylene glycol (PEG) is also known as polyoxyethylene (POE) or polyethylene oxide (PEO), depending on the synthesis conditions and molecular weight. The Food and Drug Administration (FDA) has certified PEG as a synthetic substance with excellent biocompatibility, low cost, and water solubility. PEG is used in a variety of biomedical applications, from wound healing to drug delivery . The PEGIn recent years, polyacrylamide (PAm)-based hydrogels have been widely used in the pharmaceutical, drug delivery, and biosensor liquid sectors. One of the main characteristics that distinguishes PAm from other materials is its unique and adjustable mechanical properties: the hydrogel strength can be adjusted from less than 1000 pa to several Mpa. Nonetheless, PAm has low toxicity and weak cell adherence, both of which are problems in biomedical applications. PAm-based hydrogels require the integration of natural ingredients and peptide conjugates ,98. PolyThe encapsulation of the ECM in biodegradable and biocompatible nano or micro carriers allows it to maintain its structure and integrity, thereby maintaining biological activity. The encapsulation of BDNF in microparticles made of biocompatible poly (lactic acid-co-glycolic) (PLGA) is a strategy to protect BDNF from the environment and provide continuous delivery at the injection site, because the released BDNF undergoes increased secretion of growth factors by cells and potentially has a synergistic action. However, the efficient and sustainable release of protein from FDA-approved PLGA-based scaffolds remains a challenge due to protein instability during the formulation process. Bioactive BDNF combined with nanoprecipitation silanized-hydroxypropyl methylcellulose (Si-HPMC) hydrogels encapsulated in PAm made from polymers that are more hydrophilic than PLGA, such as the polymeric PLGA-poloxamer(P188)-PLGA triblock, are also believed to maintain the structure and biological activity of BDNF .Silanized-hydroxypropyl methylcellulose (Si-HPMC) is a non-toxic injectable hydrogel with moderate swelling qualities that forms a gel in situ at physiological pH. Si-HPMC hydrogel can be used on a continuous release basis as a therapy for cell delivery, directing neurodevelopment, and enhancing its protective/reparative properties . PolycapThe development and improvement of methodologies and technologies in MSC secretome culture, as well as a global understanding of the secretome components, will be necessary to promote secretome-based therapies and determine their safety and efficacy . RecentlIn various biomedical applications, several types of polymeric hydrogel-based systems have been shown to localize delivery and enhance the therapeutic efficacy of MSC secretomes. However, only a few studies, particularly in vivo, have fully studied the loading and release rates of MSC secretome. Hydrogels are suitable carriers for stem cells in a variety of biomedical applications due to their physical qualities similar to native ECMs. In addition, hydrogels are known to regulate the fate of stem cells to release their bioactive factors in order to produce a secretome that is more precise and efficient in its therapeutic effects, such as accelerating tissue and organ regeneration. The quality of the polymeric hydrogel material can be modified based on the characteristics of the polymer because it affects the hydrogel stiffness, biodegradability, and biocompatibility. The modification of the properties of polymeric hydrogels is very important to study and should be considered in particular because hydrogels are known to interact with the environment in which they are placed. In order to gain a better knowledge of the mechanisms underlying the action and optimize the therapeutic potential, in-depth research on the release profile of MSC secretome carrier-based with suitable controls in biomedical applications is required."} +{"text": "Cor triatriatum has been described as a heart with three atria in which the left atrium (cor triatriatum sinistrum) or right atrium (cor triatriatum dextrum) is divided into two compartments by a fold of tissue, a membrane, or a fibromuscular band. Double-chambered right ventricle, on the other hand, is identified by the presence of an anomalous muscle bundle dividing the right ventricle into two chambers.Here, we describe the case of a child who had a combination of both of these rare entities, effectively creating a heart with six chambers. The child underwent a successful intracardiac repair.The association of CTS with DCRV forming a \u201c6-chambered heart\u201d is extremely rare. Awareness of its existence and accurate preoperative diagnosis has important implications in its surgical repair with all the components of this disease spectrum, further increasing the complexity of a successful surgical repair. In cor triatriatum sinister (CTS), the proximal chamber of the left atrium (LA) receives venous blood, whereas the distal chamber is in contact with the atrioventricular valve and contains the atrial appendage and the true atrial septum bearing the fossa ovalis. The membrane that separates the atrium into two parts varies significantly in size and shape. In double-chambered right ventricle (DCRV), an anomalous muscle band divides the right ventricular cavity into a proximal and a distal chamber . In the\u00a0A 2-year-old child is presented with the complaints of shortness of breath while playing and moderate cyanosis for the past one and a half years. The symptoms had progressed over the past 3 months. On examination, the child had an oxygen saturation of 80% on room air and a systolic murmur in the pulmonary area.The child was further evaluated with basic investigations and a transthoracic echocardiography, which revealed CTS in association with DCRV, having a gradient of 70\u00a0mmHg across it, a perimembranous ventricular septal defect (VSD) in the proximal chamber of right ventricle, a small atrial septal defect (ASD) between the right atrium (RA) and distal chamber of the LA in the septum secundum and persistent left superior vena cava (PLSVC) Fig.\u00a0. The atrThe incidence of cor triatriatum has been variously reported as 0.1\u20130.4% . PatientA VSD may communicate with either the proximal or distal chamber, leading to a greater shunt in the latter situation. Development of RV outflow tract obstruction occurs in 3\u20137% of patients with membranous VSD\u2019s within the first year of life , 6.CTS is amenable to surgical correction with excellent results when diagnosed. Long-standing inflow obstruction to the left ventricle leads to pulmonary venous and arterial hypertension along with right ventricular dysfunction. Additional presence of DCRV along with CTS adds to the complexity as it leads to further RV hypertrophy, exposes the RV to pressure overload and increases the predisposition to early RV dysfunction. Hence, the need for early surgical intervention in such cases. In our case, the six chambers of the heart were correctly diagnosed by preoperative echocardiography and CT-angiogram, facilitating the surgical planning beforehand. A six-chambered heart as described here was described around 50\u00a0years back but the morphology was entirely different .The association of CTS with DCRV forming a \u201c6-chambered heart\u201d is extremely rare. Awareness of its existence and accurate preoperative diagnosis has important implications in its surgical repair with all the components of this disease spectrum further increasing the complexity of successful surgical repair."} +{"text": "Due to a production error a Data Availability statement was incorrectly added to this article. This has now been removed and the publisher apologizes for this mistake.The original article has been updated."} +{"text": "Due to a production error, The publisher apologizes for this mistake. The original article has been updated."} +{"text": "The aim of this study was to identify the perceived consequences of this on staff and their work and on students and their studies at universities.The study used a variety of methods, which involved an on-line survey on the influences of social isolation using a non-probability sampling. More specifically, two techniques were used, namely a convenience sampling , supported by a snow ball sampling (recruiting respondents among acquaintances of the participants). A total of 711 questionnaires from 41 countries were received. Descriptive statistics were deployed to analyse trends and to identify socio-demographic differences. Inferential statistics were used to assess significant differences among the geographical regions, work areas and other socio-demographic factors related to impacts of social isolation of university staff and students.The study reveals that 90% of the respondents have been affected by the shutdown and unable to perform normal work or studies at their institution for between 1\u2009week to 2\u2009months. While 70% of the respondents perceive negative impacts of COVID 19 on their work or studies, more than 60% of them value the additional time that they have had indoors with families and others. .While the majority of the respondents agree that they suffered from the lack of social interaction and communication during the social distancing/isolation, there were significant differences in the reactions to the lockdowns between academic staff and students. There are also differences in the degree of influence of some of the problems, when compared across geographical regions. In addition to policy actions that may be deployed, further research on innovative methods of teaching and communication with students is needed in order to allow staff and students to better cope with social isolation in cases of new or recurring pandemics.The online version contains supplementary material available at 10.1186/s12889-021-11040-z. During February and March 2020, following guidance from the World Health Organisation, governments around the world responded to the coronavirus pandemic by imposing restrictions on social contact. This affected almost all business sectors and public services, including the education sector , 2. OnceOne consequence is that HEIs in many countries directed their teaching staff to move teaching and learning to online platforms - where possible - without delay, and to do so as comprehensively as possible , 5. SincMore fundamentally, many HEIs, their staff and students, do not have the infrastructure to shift learning to online platforms immediately . The impApart from the hurried institutional responses to educational provision, there is emergent evidence that many individuals have struggled to cope with the multiple complications and consequences of lockdown Numerous international students were left stranded due to the travel restrictions, leaving some of them without accommodation or experiencing unexpected financial costs . SimilarSince the onset of Covid-19 and its ongoing prevalence and related lockdowns, HEIs around the world have assessed the financial impact as new - and continuing - student attendance on-campus and in residential accommodation seems increasingly unlikely. By example, Burki reports Furthermore, the looming precipitous fall in tuition fees and accommodation charges from international students has exposed the financial viability of HEIs, especially those dependent on such income, typically in native English speaking and other developed economies. Given that staff salaries constitute at least 50% of HE institutional costs, leaders of UK and Australian universities are exploring financial survival options, including voluntary and involuntary redundancies, pay cuts and freezes, and abandonment of national pay guarantees, in the face of resistance from employee unions \u201317. In asocial isolation as a primary measure to mitigate the spread of the SARS-CoV-2. People of all ages, including university students and academic staff in the majority of countries, were asked to avoid physical social contact and participation in group and community activities, family gatherings and public events. With few exceptions, self-isolation was suddenly required by nation states, particularly of individuals returning from more severely affected regions, as well as for older people and those with underlying health conditions. While self-isolation has been generally considered an act of individual responsibility, some countries introduced and enforced new specific regulation to restrict movement outside the home and to require the wearing of face masks, and established the authority to impose fines or imprisonment for non-compliance .Following the categorisation by the WHO of COVID-19 as a pandemic (11 February 2020), public health experts and authorities recommend . It is also understood as a subjective experience by individuals, such as a \u2018lack of engagement with others\u2019 [objectively real physical isolation, immediately and severely reducing direct social interaction and contact with anyone outside the household. At the same time, these conditions create circumstances in which individuals subjectively experience social isolation.Humans are fundamentally a social species: it is in their nature to interact and form various types of relationships with others. Social isolation has been understood as both an objective phenomenon experienced by individuals, such as that characterised by a \u2018lack of social interaction\u2019 , \u2018the ac others\u2019 , \u2018loneli others\u2019 or \u2018the others\u2019 . The widExtensive evidence from social science and public health studies suggest that social interaction and relationships are important for mental wellbeing throughout the lifespan. For example, Hartup and Stevens concludeSimilarly, strong correlations have been demonstrated between social relationships and physical health, such that more socially connected adults are found to be healthier and to live longer than their more isolated peers . In addiTwo decades before the COVID 19 pandemic, Killen (1998) observed an \u2018epidemic of loneliness\u2019 (according to ), partia.Research also suggests that age may be a factor influencing the level of health risks. Nonetheless, subjective perceptions of social support or isolation also play their role. Specifically, there is a tendency for young people to feel lonely even when surrounded by others or when being a member of a group of peers, while by contrast, the elderly might not feel lonely even when their social network is significantly reduced .Similar findings appear in studies on relations between age and social media use. There is some evidence that social media use may help people feel less isolated, such as through drawing support from online social networks such as Facebook, Instagram, and others; Hajek and K\u00f6nig found thThese studies show that the factors influencing or otherwise associated with social isolation as a subjective experience are interdependent and complex, but carry consequences for morbidity and mortality outcomes. The studies also highlight that the experience of social isolation is context dependent and is at least a product of psychological and social factors. Influencing factors include health, whether or not the individual is in a close social relationship and the nature of that relationship, educational level and social networks, and age, among others. From a \u2018human ecology theory\u2019 perspective (e.g. ), these It should be noted that even without the social confinement imposed as a result of the COVID-19 shutdown, an association between working conditions and wellbeing was recognised and continues to be debated . So too Changing contemporary work patterns have been shown to affect social wellbeing . Set agaAcademic staff will be familiar with these stresses and strains, balancing their high commitment to their profession and identify within a stressful working condition with the need to attend to life outside the academy , 39. In It seems likely that the overriding importance of an immediate implementation of the social lockdown will foreground its associated stressors, overshadowing the longer standing work stressors. As the lockdown persists, its stressors will compound existing work stressors and add new ones.Set against this background, the research presented here aims to identify the impact of the COVID-19 lockdown on working conditions and on the social isolation imposed on academic staff and students of HEIs around the world. This study contributes insights to the subjective experiences of staff and students working in HEIs around the world, shining a light on their subjective constructs as a response to the enforced isolation. In addition, this study contributes by highlighting far reaching policy implications for teaching and learning approaches in the emerging context of the increased reliance on social interaction in an online environment - in particular, the need to secure technological enfranchisement of all students and the wellbeing of both staff and students. This study is paralleled by a research focusing on students\u2019 mental health problems before, during, and after COVID-19 lockdown undertaken in Italy, with a sample of 358 Italian students aged 18\u201330 .Given the aim of this study, a cross-sectional survey research design was adopted to examine the experiences of academic staff and students in HEIs around the world. The design of the survey was informed by the literature on the impact of influences on work practices, and the influence of changing work practices on social isolation and wellbeing. Since the study into the links between COVID-19 lockdown and social isolation is time sensitive, a convenience sampling was appropriate, as it facilitates the timely gathering of data. It was also appropriate under the circumstances to gain responses from individuals at a time when, under the prevailing emergency circumstances, staff and students had other immediate concerns.This non-probability sampling method also involves a combination of purposive and homogeneous methods, and respondent self-selection . This strategy directly addresses respondents with experience of the questions raised in the survey and the ability to generate new insight. The online survey was conducted from 14th April to 4th May 2020 using Survey Monkey. It was initiated by the Hamburg University of Applied Sciences and disseminated via email through a web-link to the networks of the co-authors plus various mailing lists (e.g. LISTSERV). on teaching and research in higher education, thereby reaching students and academic staff across the globe. The survey link was also disseminated by email through the national and international personal and professional networks of the research team, defining this as a snowball method. Table\u00a0As a result of the promotion efforts and after two reminders, a total of 711 questionnaires from 41Figure\u00a0The instrument was designed to gather (1) socio-demographic information about the respondents, including their role in the university, area of work, age and gender, with whom they live, and where they live , and (2) perceptions of the extent to which the shutdown affected their capacity to perform their tasks as normal, in work or study (Q7-Q15). This included questions about the extent to which they felt that their institutions took adequate measures to help them perform effectively while in social isolation, namely through the (a) provision of necessary communications infrastructure and the (b) provision of other support for work/study from home. . . Descriptive statistics were analysed to establish trends and identify socio-demographic differences. Inferential statistics were used to assess significant differences among the geographical regions, work areas and other socio-demographic factors related to impacts of social isolation of university staff and students. The level of Significance was set at 0.05.: Gender, age, and role of respondents).Of the 711 responses, the larger proportion consists of students 472), either undergraduate or postgraduate (67%), and females (64%) whose ages are between 20 and 40\u2009years old (66%), and in the age band 21\u201330 .Most of the respondents worked or studied in European universities (83%). South American, North American, Central American, and African universities were represented by 1 to 14% of the respondents, and Australasian respondents by 3% Table\u00a0. More thSome 5% of the respondents had been unable to work or study at their institution for 2 to 3\u2009months, 68% for 1 to 2\u2009months, 19% for up to 1 month, and only 3% of respondents had experienced no impact at all . More than half of students (54.4%)had no particular preference , \u2009=\u20093.958, 1%, (acadp\u2009<\u20090.001). Respondents from the USA more strongly agreed with actions of their HEIs, while respondents in Serbia agreed the least.There is significant variation in how respondents of particular countries saw the need for institutional shutdown Fig.\u00a0. A one-w: Working pattern of respondents).During the 2020 period of the pandemic crisis more than 90% of academic staff and 78% of students had worked from the \u2018home office\u2019. However, a small percentage of respondents stopped working altogether, especially students (15%) compared with students: ; t(708)\u2009=\u20092.885, p\u2009<\u20090.05) regarding the performance of the communications infrastructure available at home. The mean value of both groups is above the midpoint of the scale (3) from 1 to 5 , followed by Microsoft Teams 45,76%) and Skype , independently of whether or not the University had a virtual learning environment (VLE) platform. Academic staff showed significantly higher levels of satisfaction \u2009=\u20092.183, p\u2009<\u20090.05.Mean values of both groups were above the midpoint of the scale (3) Fig.\u00a0. NeverthUsing a one-way ANOVA test we compared: a) the effect of geographical location (country) o n the respondents\u2019 evaluation of available infrastructure to work or study at home and b) the perceived support given by the institution during the shutdown. There is a statistically significant difference between the five biggest samples Fig.\u00a0. RespondThe graph in Fig. p\u2009>\u20090.05) showed either \u2018great\u2019 or \u2018moderate\u2019 agreement, with a further 20% feeling \u2018to some extent\u2019 that the shutdown affected their work or study. \u2009=\u2009\u2212\u20090.982, \u2009=\u2009\u2212\u20090.98The main functional problems that the respondents reported are: disruption of communication 51,29%) the adjustment of schedules , delays , the difficulty to combine work or studies with family , the cancellation of meetings , and the difficulty in collecting research data highlight not just functional problems but functional challenges overlaid with stress and anxiety. These include (a) feeling a lack of institutional support, (b) lacking motivation, (c) feeling the stress of living and working at home , (d) physical discomfort of working with unsuitable facilities at home and, for students e) the perception that professors were not willing or able to use online platforms.p\u2009>\u20090.05). A substantially higher proportion of students perceive a \u2018decreased\u2019 or \u2018substantially decreased\u2019 workload, which seems consistent with results reported in Table 6: Working pattern of respondents.Almost 60% of respondents considered the shutdown as having a \u2018moderate\u2019 to a \u2018greatly increased\u2019 impact on their workload, while perhaps surprisingly, 20% indicated \u2018no impact at all\u2019. Fig.\u00a0. The difAs the above results show, most respondents feel the shutdown has negatively affected their work or study. At the same time the resulting confinement with family, roommates or friends Fig.\u00a0 is consip\u00a0<\u20090.05).Religious respondents (about 25% of respondents) fe lt comparably more positive about the confinement at home, with academic staff feeling the more positive (75%) Fig.\u00a0. The difA substantial majority (70%) felt that the lockdown has adversely affected their work or study. The respondents noted that the main personal challenges due to the mandated social isolation are: a lack of personal interactions with colleagues and staff (72%), a lack of motivation (57%), anxiety, and closely followed by boredom and loneliness Fig.\u00a0.Fig. 13Respondent evaluation of communications infrastructure while home working during the shutdown, Fig. The vast majority of respondents considered that the shutdown had an adverse impact on their work, creating more of it, confirming that the shutdown disrupted their daily routines, especially with the ability to communicate with others and having to reschedule work and meetings. This is in line with results from Cao et al. with colWorking pattern of respondents mixed with new stressors . Yet another layer of anxiety among both academic staff and students is generated by the widely reported prospect of unemployment among academic staff, associated with the financial impacts on universities [Consistent with other studies noted \u201328, thisersities \u201317 and cersities a By wayersities , whose lersities survey oersities of colleersities speculatThe considerable variation between countries of respondent attitudes to HEI shutdown, and in particular to their assessment of the adequacy of ICT infrastructure at home and HEI support, warrants closer examination. There may be many reasons for such differences, some widely reported, including the distinct rates of virus infection in individual countries, and the distinct speed and effectiveness of in-country leadership responses to the threat. Informing the latter are political judgements that rely (at least in part) on track and trace strategy and ICT infrastructure. Further, the variation in respondent assessments of home-working ICT infrastructure and HEI support, reported here, highlights the uneven scope and quality of HE institutional resources and preparedness for providing support .Since COVID-19 is an ongoing, global, public health emergency and given the potential for future epidemics, attention to the concerns for psychological health and wellbeing of staff and students, will require more attention from than HE sector policy makers and HEI leaders have to date given them. h As Wigginton et al. urge, \u2018aAs pandemic continues, bodies such as the American Council on Education and Universities UK may update their guidances, to incorporate ways of embracing online support services, the use of which could improve the quality and effectiveness of not only emergency interventions, but also as part of mainstream educational provision. For example, Liu et al. reports status quo and developing new models of funding less reliant on physical consumption. This is an opportunity to cement what is being learnt about online teaching and learning . Attempts to return to this model in the medium term is fraught with risk to health, yet many HEIs will feel compelled to take that risk in order to secure financial survival. HEIs, with the support of their governments, should be reflecting on existing practices and their recent experiences of (forced) online teaching and learning and encouraging research on teaching and learning, with the aim of evolving away from the Lederman .More broadly, the pandemic has laid bare the fault lines in the existing narrow formulation of HE as an enabler of economic growth through knowledge transfer or, as is conceptualised by Etzkowitz and Zhou\u2019s , as justThe rippling impact of COVID-19 goes well beyond the internal machinations of HEIs or adjusting the ways academic staff and students interact. There is a new normal emerging, where teaching, learning and knowledge creation are unfolding in the context of social interactions (itself being reshaped) rather than in organisational contexts. As these new ways of working persist, civic society, policy makers, and HE practitioners need to reimagine how educational strategies might better support equality, the creation of knowledge, and the search for innovative ways of democratising work patterns and modes of learning, without the social cost of isolation. These seemingly divergent demands call for a broader integration of the university\u2019s role within society, in turn requiring substantial changes to the existing HE ecosystem. Such integration should redress the over-reliance on HEI competition. Significant costs accompany the disciplining benefits of operating in a competitive market. As Mintz reports,Exploring and renewing our understanding of higher education within society becomes a new research agenda. Drawing on Cai , creatinThe provision of psychological care and support to academic staff in order to better equip them to cope with the additional burdens of home schooling on the one hand, and meeting teaching schedules on the other;The provision of counselling to students, in order to reduce the anxiety caused by social isolation and foster a better work-life balance;A greater use of online activities . Many organizations offer digital gatherings of all sorts, which may be used as a means of getting in touch with more people;The set-up of informal communication channels in order to facilitate and encourage conversations in both groups, which helps people to feel less alone and more supported.Having analysed the problems, difficulties and constraints caused by and/or associated with social isolation, it is now important to look ahead. There are some measures that may be deployed in the future in order to allow staff and students to better cope with social isolation in cases of new or recurring pandemics. These are:More items may be added to the list, but the above are examples of what can be done in a rather simple way and without major costs or investments.As a complement to the above measures, a review of content delivery and the ways lectures are organized and held should also be performed. The psychological pressures that staff and students are exposed to means that traditional teaching - and evaluation \u2013 models are not suitable. Rather, academic staff needs to consider innovative ways of communicating study contents to students, in a way that takes into account the many concerns and worries they have and the pressures they are subjected to, as a result of social isolation.Despite its scope, this paper has some limitations. The first is the size of the sample, which entails 711 responses. In addition, the number of countries investigated, namely 41, cannot be regarded as representative of the world. Also, the responses varied among countries, meaning that some countries had more respondents than others. Moreover, the fact that the study looked at the attitudes of academic staff and students means that a disaggregation from their opinions is not always possible. Apart from the fact that various other studies are currently being undertaken which look at either group, the rationale behind the approach used here is that the authors wanted to offer an overall picture of the extent to which these two major groups (academic staff and students) are being affected.But despite these constraints, the study is a welcome addition to science in the sense that it offers an overview of the many aspects associated with social isolation in academic life and illustrates its impacts on academic staff and students. Also, the sampling, which involved 41 countries, allows one to build a rough international profile of the impacts of social isolation in a university context.This paper has analyzed the impacts of the lockdowns triggered by the COVID-19 pandemic on academic life, identifying the extent to which the universities\u00b4 operations were disturbed and paying special attention to aspects related to social isolation.It is evident that there were significant differences in the reactions to the lockdowns by academic staff and students. Whereas most students stopped working immediately after the lockdowns, most academic staff continued to work. In addition, academic staff showed a greater level of satisfaction with their provisions and facilities for working during the special situation caused by the pandemic, whereas students indicated they were not satisfied. In light of the growing awareness of \u2018digital poverty\u2019 and the \u2018digital divide\u2019 which define students\u2019 absolute and relative access to IT equipment and internet, an obvious reason may be that they were ill equipped to cope with the sudden change to on-line learning.There are also differences in the degree of influence of some of the problems, when compared between countries.. For instance, students in the United States considered that their institution\u2019s infrastructure was in better shape to cope with the lockdowns, when compared with those from the other sampled countries.As the world finds itself in the middle of a second wave by the time this paper has been written, it is clear that universities need to be mindful of the many impacts the pandemic will have in their operations, at present and in the future.Finally, one item that also deserves mentioning is that people have been required to remain home and avoid contact with third parties; this means that an opportunity is given for quality family time. A greater understanding of the impacts of social isolation and of some of the means by which its impacts may be mitigated, as this paper has tried to outline, may lead to a better preparedness of academic staff and students for handling such events now that pandemics are realities on our collective future horizons.Additional file 1. Questionairre."} +{"text": "V on the Fermi-surface. Our results include a rigorous confirmation for the behavior of We investigate the BCS critical temperature V, which we assume to be integrable, i.e.\u00a0T and T is below a certain critical temperature The Bardeen\u2013Cooper\u2013Schrieffer (BCS) gap equation 1\\documenV for small coupling constant BCS theory has previously been studied in the weak-coupling limit , 9 and leiringer , that \\dubiquity of superconducting domes in BCS theory, but only for pure s-wave superconductivity . If theentclass1pt{minimaS theory . At highentclass1pt{minimaV Let , the spcf.\u00a0also ). The loV depending only on V is radially symmetric, the eigenfunctions of V.In this work, we restrict ourselves to the special case of radial potentials V Assume uired in , are incentclass1pt{minimauired in , that\\do-2V from , the sec-2V from in the h. (6) in reads\\doIn order to obtain a meaningful asymptotic formula of the critical temperature at high densities in a rigorous way, the question to be addressed now is the behavior of entclass1pt{minimaaimed in , since \\\u00a0 Let V Let V. We call V an admissible potential if the following is satisfied: a around 0,There exists if if Condition (d) can be dropped, whenever we have control on the ground state space of ((i)There exists some (ii)It has a dominating attractive part (for short distances), i.e.\u00a0(iii)It is slightly less divergent at the origin than allowed by the The most relevant examples for admissible potentials are the attractive Yukawa and Gaussian potential, i.e.In a nutshell, an admissible potential is a radial potential f has some complicated (explicit) expression (see Lemma\u00a0(On condition (d) for admissible potentials) The additional mentclasspt{minimas of Eq. and compWe will show in Lemma\u00a0entclass1pt{minimamentclasspt{minimasuperconducting dome), can now be obtained by combining the decay of V, with a, and in the absence of bound states. Thus, we rigorously confirmed the ubiquity of superconducting domes in BCS theory for a general class of interaction potentials, as claimed in [The existence of a maximal critical temperature at some intermediate density . WhenevNow, we define the operatorV be an admissible potential. Then the critical temperature \u00a0 Let In other words,entclass1pt{minimaThe constant in front of the exponential is in particular relevant for obtaining the universality of the ratio of the critical temperature and the energy gap, which is achieved in , where ax and y The assumption of the interaction potential being radially symmetric enters the proofs of our main theorems in a crucial way. On the one hand, the radial symmetry allows an additional averaging over the sphere entclass1pt{minima(cf.\u00a0Eq. ) as the mentclasspt{minima The recent work by Cueniods from , where Gr by Eq. that theThe most important tool for our proofs will be the Birman\u2013Schwinger principle see , 8, 12)., 12.8, 1Let gap see , 12, 18), 18\\docuThis second term will be the dominant term, which is how the quantity entclass1pt{minimaA priori, it is not clear, how entclass1pt{minimaSince mentclass2pt{minimIn the proof of Theorem\u00a0, so Eq. gives thFix position vanish ic and C for generic positive small resp.\u00a0large constants, possibly having a different value in each appearance. If we want to emphasize the dependence on a certain parameter, we add a subscript, e.g.\u00a0by writing For the first term, note that the Fourier transform of V. In fact, since V is radially symmetric, every eigenfunction of x. Now we aim to bound V we arrive atx and y can be performed first only by the radial symmetry of V. If V were not radially symmetric, we would have had to compute the angular integral of p first and would have ended up with completely different integrals to estimate. Now, using the plane wave expansion p-integral from the second line is some function of For the third term, we will heavily use the radiality of m in Eq. as \\docuSummarizing our considerations above, we get that, since by assumption position\u00a0 can be mtain Eq. by meanstain Eq. , it remaLet We estimateWe check that entclass1pt{minima. 4) in , we find in , we entclass1pt{minimaentclass1pt{minimaThe proof of Theorem\u00a0mentclasspt{minimaLet 1} \\in 9/,3$$\\end{For admissible potentials that satisfy the V be an admissible potential in Eq. , the opementclasspt{minimaentclass1pt{minimamentclasspt{minimamentclasspt{minimaT. By continuity and monotonicity of We can thus, following the argument around Eq. (30) in , concludSince entclass1pt{minimamptotics and the entclass1pt{minimau is the normalized eigenfunction corresponding to the lowest negative eigenvalue u of Indeed, using Eqs. and 32)32), we cEquation is an ime in Eq. and sincfined in . Using tentclass1pt{minimad in Eq.\u00a0 in [\\docall Eqs. and 12)\\documentall Eqs. , 36) an an\\docum(see Eq. ), we con\\documentu resp.\u00a0u resp.\u00a0A priori, u and The proof follows a similar perturbation theoretic argument as in the proof of Lemma\u00a0\u03bc by Eq. . Using E\u03bc by Eq. and 32)\\documents of Eq. that alsith Eqs. and 42)\\documentsee Eqs. ), 43) i i\\documesee Eqs. .In case that there exists ry, Eqs. togetherFinally, we give the proofs of Lemmas\u00a0For uniform boundedness, i.e.\u00a0(ii)uniform Lipschitz continuity, i.e.\u00a0(iii)(uniform) decay, i.e.\u00a0for every (iv)uniform The first statement (i) is an elementary property of the spherical Bessel functions. The second statement (ii) follows from the uniform boundedness in (i) and the recursion relation [mentclass2pt{minimuence of , Eq.\u00a01, uence of , Eq.\u00a03, We note that (15) in , we get\\"} +{"text": "Bdellovibrio and like organisms (BALOs) can regulate prey populations, possibly in a density-dependent manner, in the naturally complex, species-rich environments of wastewater treatment plants. Abundant as well as rarer prey populations are affected, leading to an oscillating predatory landscape shifting at various temporal scales in which the total population remains stable. Shifts, along with differential prey range, explain co-existence of the numerous predators through niche partitioning. We validate these sequence-based findings using single-cell sorting combined with fluorescent hybridization and community sequencing. Our approach should be applicable for deciphering community interactions in other systems.A fundamental question in community ecology is the role of predator\u2013prey interactions in food-web stability and species coexistence. Although microbial microcosms offer powerful systems to investigate it, interrogating the environment is much more arduous. Here, we show in a 1-year survey that the obligate predators Studying the role of predator\u2013prey interactions in food-web stability and species coexistence in the environment is arduous. Here, Cohen et al. use a combination of community and single-cell analyses to show that bacterial predators can regulate prey populations in the species-rich environments of wastewater treatment plants. Inherent properties such as the prey range of a predator, i.e. being a generalist or a specialist, or spatial complexity further strongly affect the type of control, promoting top-down or bottom-up effects5.A fundamental question in community ecology is the role of predator\u2013prey interactions in trophic web stability and species coexistence. This is central to the understanding of how food webs, which are the basis of sustainable life, are maintained. Predatory interactions have profound effects on food webs, among others by promoting diversity and creating trophic cascades that may vary in length and strength, depending on various parameters, e.g. predator size, temperature and else7. However, the ecological role of other members of the predatory guild has been much less explored5. The Bdellovibrio and like organisms (BALOs) are highly motile gram-negative bacteria that obligatorily prey upon other gram-negative cells8. They are smaller than their prey, which they mainly consume after penetrating their periplasmic space, to grow as a multi-nucleoid cell which will divide into the flagellated progeny, and exit the remains of the prey8. BALOs are ubiquitous in soil and in water bodies and are considered to be \u201cintermediate\u201d versatilists, neither generalists nor specialists9. Indeed, isolated BALO strains tested with prey arrays show utilisation ranging from single10 to many prey strains12. In natural microcosms, specific BALO predators are controlled by the availability of an adequate prey9 and rapidly respond to prey abundance13; in turn, spiking microcosms with a specific BALO revealed that the predator impacted upon the bacterial community structure14. Thus, BALOs may constitute a \u201csideway control\u201d (i.e. neither top-down nor bottom-up) over food webs, affecting bacterial community structure and contributing to community succession15.In aquatic food webs, protists are essential consumers of bacteria, and along with bacteriophages they may be the largest contributors to bacterial mortality and turnover17.However, such microcosm experiments, which were also limited in time, cannot provide the accurate identity of interacting predators and prey in nature, i.e. the prey ranges of individual predators in the BALO community. This information is essential if one is to understand their impact on microbial communities, predator\u2013prey dynamics, what sustains their diversity, and to potentially manage in situ interactions for ecological and biotechnological applications. High throughput sequencing technologies may uncover the diversity of BALOs and along with quantitative PCR, network computing, and in situ detection approaches enable their identification and quantification without relying on bottlenecks created by isolating BALOs on particular prey18 and being the most microbe-diverse human-made habitat, they sustain numerous interactions, including predatory interactions20. We thus asked whether this diversity is also found in BALOs, what the predators\u2019 natural prey are and what mechanisms explain their co-existence. We hypothesized that niche differentiation sustains predator diversity by prey range partitioning, temporal differentiation22 and through fluctuating predator and prey populations. Finally, in order to validate sequence-based computing results, we developed a direct approach based on FISH tagging23 and cell sorting to identify the interacting predators and prey.Here, we specifically analysed the community dynamics of the two major BALO clades and their association with prey over a year, at three wastewater treatment plants (WWTPs), and invoke predator\u2013prey theory to explain our results. WWTPs are crucial for keeping public health and reducing environmental pollution24, a targeted 16S rRNA gene copy-based qPCR analysis of Bd and Bx absolute abundance was performed on the Shafdan (SH) samples. It showed no significant differences in absolute abundance over the time series between the two clades, averaging 0.5\u2009\u00b1\u20090.2% of the total bacterial population, for each . OTU composition of the Bx community was more diverse between samples at each site and within each fraction than that of the Bd community, as reflected by larger Bray\u2013Curtis dissimilarities and of the Bacteriovoracales (Bx) show that Bd and Bx diversity did not differ between the floc and the liquor fractions at each site but they were always higher in the former Table\u00a0. The relh Figure\u00a0. Furthers Figure\u00a0. This maBdellovibrio strain W); in Bx, only a single OTU was found in the branch constituted of known strains; all the other Bx OTUs obtained in this study formed a second, very diverse cluster which included the Bacteriovorax stolpii type strain. Thus, the BALOs populating WWTPs while diverse are almost not represented in cultures. A correlation analysis (see below) linking predator and prey OTUs revealed that prey range was not linked to predator phylogeny, i.e. predators did not \u201cspecialize\u201d in predating upon specific taxa, whether the predator had a wide (\u2009>\u200920 prey) or a narrow (1\u20135 prey) prey range were removed to restrict false-positive correlations resulting from unlinked or indirect interactions, leaving potential gram-negative prey. The analysis, based on significant negative correlations between the three annual datasets sieved >\u200999.5% of possible connections, revealing the co-occurrence of short time scale oscillations between predators and gram-negative populations, exhibiting patterns akin to predator\u2013prey cycles, defining potential predator\u2013prey interactions , creating local \u201ccommunities\u201d of potential predator\u2013prey interactions of various sizes, including large hubs . The sensitivity of a particular prey taxon to predation was also not necessarily linked to its abundance: Sphingobacteriales are the most abundant taxon were preyed upon by both Bd and Bx, the high modularity of the networks and their structures suggested differentiation in prey use by the different predators. Indeed, within each predatory family, different predators largely hunted different prey . We further examined prey resource partitioning between Bd and Bx at each site and within each fraction, observing that here also, the predators largely differed in potential prey range Fig.\u00a0.Fig. 6BAa Bdellovibrio spp. targeted BDE525-Cy5\u2019 16S rRNA nucleotide probe23 confirming single bacterial cells were measured during sorting were very largely enriched in the BALO-sorted samples . Principal coordinate analysis (PCoA) also showed that the bacterial communities in samples P1 and P14 differed significantly (p\u2009<\u20090.0005) from the P16 community and Chlorobiales. The total community was mainly composed of Rhodocyclaceae, Saprospiraceae, Comamonadaceae and Anaerolineaceae . A PCR targeting the general bacterial population yielded a positive signal in all the samples, while a PCR using y Figure\u00a0. In gates Figure\u00a0. High-thity Fig.\u00a0. This waeae Fig.\u00a0, a very Finally, prey OTUs deduced from this FISH-cell sorting experiment were compared to those of the potential prey identified in the network data derived from the floc and liquor fractions at LB. More specifically, 64 23.5%) and 46 (10.8%) of the OTUs co-sorting with BALOs, i.e. OTUs of the prey populations and identified in the P1 and P14 samples respectively, overlapped with potential prey OTUs of the LB networks were the most abundant, followed by Comamonadaceae 9, 13.8%). Chitinophagaceae , Nannocystaceae , Rhodocyclaceae , and Flavobacteriaceae diverged by no more than 3% from described strains, including 22 appeared to experience distance decay, as they were more similar at SH and AB than between these two relatively close by plants, and LB. However, for both Bd and Bx, the 20 most abundant OTUs were almost all shared between the plants, while rarer OTUs were much less common. This is suggestive of no or few constraints on dispersal as observed with other bacterial WWTP populations22, and of local selection for the less abundant OTUs. Such a pattern was observed in Chinese lakes in which the local environment selected for rare genotypes more strongly than for dominant genotypes, which in turn, were more influenced by regional conditions33.The BALO predatory communities, similar to the general microbial community12 or by spatial separation of the predators. However, flocs and liquor microhabitats did not segregate BALOs, as the Bd and the Bx communities did not differ between the floc and the liquor microhabitats. This is in contrast to the micro-eukaryote predators and the general bacterial community22. It may yet be that biofilms on constructed\u00a0surfaces or on\u00a0biological surfaces provide additional microhabitats for BALOs and prey to interact. Yet, few of the prey OTU (circa 6%) detected by network analysis were shared between flocs and liquor. BALO predation is thus not impaired by prey strain specificities promoting floc colonization35, in accordance with previous data showing that BALOs are effective predators of planktonic as well as of biofilm-associated cells38. The lack of differences in BALO distribution between flocs and liquor may be due to their ability to move between these microhabitats: BALOs infiltrate sludge bacterial biofilms20 prey upon planktonic cells39, floc surface bacteria, and enter the inner parts of a floc where oxygen can penetrate40. They also do not segregate into surface-associated and planktonic types. The propensity of floc surface-associated bacteria (including BALO-infected ones), to be washed out to the liquor fraction41 may further contribute to minimize population segregation between the microhabitats. This, in spite of flocs supporting a larger population of BALOs, a consequence of the higher bacterial density in the floc fraction, and hence of potential prey, than the liquor. Accordingly, BALOs may not impose significant differential selection between floc and liquor-associated prey, which can lead to the formation of predation-resistant floc-associated bacteria, as observed with protists42.Resource partitioning can be invoked to explain the co-existence of the numerous predators. Partitioning can be sustained through prey range differences between predators, as shown by our network-based prey range analysis and by previous culture-based studies43 or even between closely related strains44. BALO OTUs oscillated at frequencies spanning from weeks to season (winter/summer), a feature observed at the three WWTPs providing yet another mechanism to further sustain co-existence of the diverse BALO populations through temporal differentiation46. Yet, the main drivers of these two types of temporal shifts may be different: season-long oscillations in WWTPs appeared to correlate with temperature, a parameter that can directly, or indirectly through the alteration of prey abundance, affect the BALOs47. Shorter-term oscillations may be driven by other mechanisms: \u201cKill the Winner\u201d (KtW) models are based on the assumption that the fitness of an organism decreases as its frequency (relative abundance) increases, leading to predation being the major regulatory mechanism for community composition under highly productive environments48 like WWTPs. Experimental support for KtW in WWTPs was provided by Shapiro et al. 49 who showed predator (phage)-prey patterns consistent with KtW. BALO populations also fluctuate in abundance according to the presence of prey, and they can do so rapidly: Specific BALOs are indeed rapidly selected for by an increase in the abundance of specific prey13. Our network analyses based on negative Kendall correlations showed KtW-like patterns with multiple oscillations. Although oscillations in the present study were at the same time scale as those observed with phages in the Shapiro et al. study49 (tens of days to a few months), some were more rapid. Yet, not all strongly negative correlations supported clear KtW patterns, possibly because in addition to prey availability, other biotic and abiotic factors, like responses to prey depletion or the secretion of predation-inhibitory substances52, and environmental parameters (see below) may affect predatory dynamics. In both cases, no relation with prey relative abundance in the WWTP was observed as both KtW and non-KtW patterns could be seen with less and more abundant (Sphingobacteria) prey. The networks and the detailed prey-range analyses showed large numbers of connections between BALOs and orders like Rhizobiales, Xanthomonadales and Flavobacteriales, which are found at a rather low abundance, seemingly contradicting a KtW approach. Yet, KtW has been extended to include more realistic situations than when first proposed, by taking into account the selective grazing of prey by protozoa resulting in different population groups, and interactions of multiple predators with multiple prey53. BALOs similarly have differential access to prey as they have different prey ranges that potentially vary from restricted to generalist55, represented as large \u201chubs\u201d in the networks, which in turn, are thought to indicate overall community stability56.The total community size of the predators was relatively stable, fluctuating between 0.1 and 1% of the total bacterial community, suggesting that the carrying capacity of the predators is strongly linked to that of the total community. However, BALO OTUs largely fluctuated during the time series, succeeding one another, showing oscillation-like patterns. These oscillations could differ between flocs and liquor, possibly due to subtle effects such as differences in prey susceptibility between the planktonic phase and biofilms58, some studies showed the effect of particular environmental variables on BALOs. For example, selection for discrete phylogenetic Bx clusters along a salinity gradient in the Chesapeake Bay provides such an example of a strongly selective environmental parameter21. More recently, Welsh et al. 25 using network analysis showed that temperature and nutrients changed Halobacteriovorax -prey interactions in a coral microbiome. Damping of BALO-prey interaction signals resulting in their absence from the networks may, in addition to abiotic factors, be caused by direct predation upon BALOs by phages61, by generalist, top predators like protists that prey upon the bacterial predators as well as upon their prey62 or by competition between predators. Such may be the case with Myxococcales, a clade of facultative predators63 that while efficiently preying upon gram-positive prey64 can also utilize gram-negative prey and potentially compete with BALOs. We found Myxococcales to be highly connected to BALOs, representing up to 4.4% of total edges, suggesting they are largely preyed upon. This corroborates previous findings25 in which network analysis showed the potential removal of these predatory competitors by BALOs. These additional interactions between predators reflect complex intraguild predation relationships, which may have important repercussions upon the trophic network by affecting predator diversity, lowering prey suppression rates5 and indirectly constraining the evolution of predator\u2013prey interactions65.Interestingly, relatively more Bd OTUs (105 of 133 OTUs) mapped upon the predator\u2013prey networks than Bx OTUs (94 of 537 OTUs), suggesting that environmental factors may also directly affect BALO dynamics. Although unmeasured variables may contribute to about 50% of the variance in WWTP sludge communities22, including Saprospiraceae and Chitinophagaceae was largely preyed upon (10.4 %). Community analysis of BALO-spiked WW samples showed predation upon these taxa, a result further supported by predation experiments on strains isolated from WWTPs67. Sphingobacteria hydrolyse proteins and various organic compound68, likely living off dead biomass, EPS or other soluble microbial products. They are amongst the most dominant bacteria in WWTPs . They play a crucial role in sludge autolysis and reduction of sludge biomass69, which are highly important to WWTP operations, reducing sludge disposal costs. Predation by BALOs may also affect other important functional groups like the denitrifying Comamonas . Noteworthy, a previous network analysis of WWTPs bacterial communities suggested that these taxa are linked to Bdellovibrio70.Sphingobacteriales, a dominant group in WWTPs71 or directly by selectively sorting fluorescently labelled target populations using directed FISH probes74. Here, we employed this approach to sort interacting predators and prey to obtain direct identification of these populations. So far, in situ identification in environmental samples has been performed with stable isotopes labelling prey or growth substrate, leading to the identification of a succession of Halobacteriovorax 16S rRNA sequences in a tidal cycle15 and of a Micavibrio predator interacting with a sublineage type I Nitrospira in a WWTP19.Although similar linkages between BALOs and prey have been obtained by network analysis in independent studies and in different environments, providing strong support that network analysis can accurately predict predatory interactions, so far no experimental confirmation of the validity of such networks has been performed. To address this issue, FISH-labelled cells were sorted from sludge. Cell sorting can be used to separate specific populations in wastewater in a high-throughput manner, after highlighting them with fluorescent dyesBdellovibrio-targeted probe yielded no signal when hybridized to sludge samples; pulse width measurements confirmed that the gated regions were constituted of single cells; Bdellovibrio spiked into sludge samples were detectable down to low densities ; very few sequences affiliated to gram-positive taxa were detected in the sorted predator fractions, and; sequences affiliated to Bdellovibrio were only detected in the sorted predator fractions. The data revealed that Bdellovibrio contributed 12.1% of the sequences in the P1 samples, and only 3% in the P14 sample, a 12-fold and 3-fold enrichment, respectively, as compared to the control sample. The P14 sample also included events of relative lower signal intensity, contributing to the lower relative abundance of predators. It can be noted that if every sorted event originated from an AP cell or from a bdelloplast (a predator associated with a prey) one could have expected at least 50% Bdellovibrio sequences in the sorted samples. However, BALOs\u2019 genomes hold one to two rRNA gene copies75 while the largest prey populations, i.e. the Saprospiraceae, Chitinophagaceae, various Myxococales, Pseudomonadales and Comamonadaceae contain between one to six, two to four copies and up to nine rRNA gene copies per genome, respectively (https://rrndb.umms.med.umich.edu/), possibly explaining part of the discrepancy. Remarkably, the samples used in this analysis originated from another WWTP situated near the LB WWTP, which provided the samples upon which the LB network was constructed. Yet, samples originating from both were composed of the same taxa, and included a significant fraction of identical prey OTUs. Lastly, our FISH-FACS experiment employed different DNA extraction/PCR approaches than the one used to obtain sequences for community analysis. Yet, they support one another. Taken singly and together the results strongly suggest that our network approach is valid, providing a powerful tool for in situ tracking of predator and prey dynamics in complex natural, as well as in controlled, constructed environments.A number of controls were performed to assure the validity of the analysis: prey and non-prey strains were tested against a BALO predator to validate attachment to prey but not to non-prey; an anti-sense 76.This approach may be developed to precisely identify interacting individual predator and prey, for instance by using specific probes for each, based on computed interactions or by isolating single predator\u2013prey pairs (bdelloplast) and performing high-throughput single-cell sequencing or by directly linking interacting pairs using PCR technologies such as epicPCR15. So far, their relative importance in biomass control and bacterial turnover have been largely underappreciated, for instance as predators of bacteria protected from protistan grazing, which can be very abundant in WWTPs and in freshwater77. Furthermore, numerous recent studies show that BALO abundance can be greatly responsive to the type of WWT technology78, offering novel possibilities in WW treatment. Our cell sorting approach although still limited by methodological constrains like DNA extractability79 and the number of retrieved cells, fills a crucial knowledge gap and provides the mechanistic details needed to quantify BALO-imposed bacterial mortality, and their contribution to microbial turnover in trophic networks.BALOs have been proposed to form a \u201csideways\u201d control over bacterial successionSampling was performed at the WWTPs of Shafdan (Israel), Al-Bireh and Langenreichenbach (Germany). Samples were taken monthly for a year from March 2013 to February 2014 and additionally, for four consecutive weeks in August 2013 and in February 2014. Overall characteristics of the sites are described in Table\u00a022 and analysed with 16S rRNA gene amplicon sequencing targeting the Bdellovibrionales and the Bacteriovoracales. In addition, samples were taken from activated sludge basins from Eilenburg, Germany situated about 20\u2009Km from Langenreichenbach. Two hundred millilitres of samples were taken and further fixed, processed and subjected to BALO-specific FISH-labelling, flow cytometric analysis and cell sorting.One-litre samples retrieved from the activated sludge basin were transferred on ice to the lab within hours and further subjected to chemical analyses. The liquid fraction of the activated sludge was separated from its floc fraction by sedimentation on ice for 1\u2009h. Both the liquid and the sludge fractions summed up to 198 samples that were further processed as in Cohen et al. 80. DNA from samples from the Shafdan, Al-Bireh and Langenreichenbach WWTPs were amplified using the 16S rRNA gene primers of Bdellovibrionales, Bd824F (5'-ACTTGTTGTTGGAGGTAT-3')-Bd1222R (5'-TTGTAGCACGTGTGTAG-3'), and of Bacteriovoracales, Bx341F (5'-CTACGGGAGGCAGCAG-3')-Bx672RC (5'-TACCCCTACATGCGAAATTCC-3') was performed as previously described') Table\u00a0. DNA fro81. FASTA and quality data were first extracted from the raw FASTQ file. Sequences were grouped according to barcode and primer, allowing one mismatch to the barcode and two mismatches to the primer. Quality control, trimming and de-noising were performed as outlined in the standard MOTHUR MiSeq protocol (http://www.mothur.org/wiki/MiSeq_SOP). All sequences were aligned to the SILVA v. 132 reference alignment database82 and filtered, so that they all overlap perfectly (with no overhang). To further reduce sequencing errors, sequences were pre-clustered based on the algorithm of Huse et al. 83. Finally, chimeric reads were removed with MOTHUR\u2019s implementation of the UCHIME method84, and all chloroplast, mitochondria, and \u2018unknown\u2019 (i.e. unclassified at the kingdom level) reads were deleted. In total, 7.96 million reads with a uniform length of 291 nucleotides per read for the 16S rDNA amplicons, 8.1 million reads of Bdellovibrionales with a uniform length of 390\u2009bp per read and 4.1 million reads with a uniform length of 300\u2009bp per read for the Bacteriovoracales amplicons, averaging 35633\u2009\u00b1\u200910244, 39002\u2009\u00b1\u200920794, and 20207\u2009\u00b1\u200912391 reads per sample, respectively, were obtained from the Shafdan, Al-Bireh and Langenreichenbach WWTPs. In the FISH-FACS experiments, 879,042 reads using the general bacteria primers and 633,852 reads using the primers for Bdellovibrionales 16S rDNA were obtained, for 32613\u2009\u00b1\u20096142 and 24,378\u2009\u00b1\u20096224 reads per sample, respectively. Pairwise distances were calculated between all DNA reads, and reads were subsequently binned into operational taxonomic units (OTUs) at the 0.03 level (\u2009>\u200997% similarity). The taxonomic affiliation of each OTU was based on the current SILVA v.132 taxonomy85.Sequences were processed using MOTHUR v1.4http://www.mothur.org/wiki/MiSeq_SOP). Read abundance data were not rarefied86. \u03b1-diversity parameters and multivariate analysis were performed in PC-ORD v6.0 with Sorensen (Bray\u2013Curtis) distances. Ordinations were performed with non-metric multidimensional scaling (NMDS)87 at 500 iterations. Differences between sample groups were calculated with the multi-response permutation procedure (MRPP)88 a test based on the assumption that if two groups differ, the average within-group difference is smaller than the average between-group distance. The size of the difference between groups was represented by the A-statistic of the MRPP test, while its significance was identified by the MRPP P-value. Correlations between the microbial communities (as represented by the PCoA 1st axis) and the environmental parameters were calculated using Pearson\u2019s correlation coefficient.The OTUs were arranged in a data matrix where each row was a single sample and each column a specific OTU; each data point in the matrix represented the abundance of the particular OTU in a particular sample, relativized to the sampling effort (i.e. the number of MiSeq reads obtained from that sample). Rarefaction curves were calculated using MOTHUR Miseq protocol .BALOs sequences were aligned using MUSCLE (Multiple Sequence Comparison by Log-Expectation)91 between all bacteria22 and all bacterial predator OTUs which had an abundance of more than ten reads in each WWTP and in each fraction were calculated. This threshold was applied in order to filter poorly represented OTUs and reduce network complexity. Between 3374 and 3833 bacterial OTUs from our previous study22 were correlated against 99\u2013114 Bdellovibrionales OTUs and 59\u201381 Bacteriovoracales OTUs. A correlation was considered robust when the Kendall correlation coefficient (\u03c4) was both\u2009<\u2009-0.7 and statistically significant . The nodes in the constructed network represent the OTUs at 97% identity according to the taxonomy derived from the SILVA taxonomy. The topology of each network was described according to a set of measurements 27. Overlaps between the potential prey ranges were calculated using GenEvenn (http://genevenn.sourceforge.net/index.htm). Correlations and statistical analysis were carried out with SciPy package of Pandas 0.23.4 under an ipython platform and networks were calculated, explored and visualized using the interactive platform Gephi v0.9.2i92.All three annual datasets were grouped together for each WWTP and each fraction (floc/liquor). All possible Kendall rank correlationsB. bacteriovorus strain HD100 was grown in DNB medium amended with 3\u2009mM MgCl2 and 2\u2009mM CaCl2 together with Escherichia coli strain ML-35 prey cells for 24\u2009h at 28\u2009\u00b0C with vigorous aeration, yielding about 109B. bacteriovorus attack phase (AP) cells.ml\u20121. When needed, AP cells were filtered through a 0.45\u03bcm membrane to remove remaining prey cells, rinsed by centrifugation at 10,000\u2009g, 10\u2009min followed by resuspension in 5\u2009ml of amended HEPES buffer . Growth phase (GP) B. bacteriovorus cells were obtained by mixing AP cells and prey cells at a 1:10 ratio in amended HEPES, creating bdelloplasts (infected prey cells) within which the predatory cell grows. B. bacteriovorus HD100 and 109\u2009J and 109\u2009J carrying the tdtomato reporter gene93 were incubated for up to 280\u2009h in microtiter plates to test for attachment and predation of a variety of isolates from WWTPs (figures\u00a094 figures\u00a0 and S12.Bdellovibrio bacteriovorus HD100 and Bacteriovorax stolpii UKi2 strain 16S rRNA gene amplified with primers 27\u2009F and 1492\u2009R respectively into a PGEM-T easy plasmid vector system (Promega). Ten-times serial dilutions from 103 to 1010 plasmid copies per reaction were used to construct standard qPCR curves and plasmid copy numbers calculated95. For total bacteria, primer pair 1048F-1175R96 was used to quantify the 16S rDNA copy number. Each 25\u2009\u03bcl reaction consisted of 12.5\u2009\u03bcl of SYBR\u00ae Green PCR Master Mix (Applied Biosystems) 1\u2009\u03bcl of each primer, 1\u2009\u03bcl of DNA and 9.5\u2009\u03bcl of PCR grade DDW. Thermal cycling was performed as follows: 50\u2009\u00b0C (2\u2009min) and 95\u2009\u00b0C (10\u2009min), 40 cycles of 95\u2009\u00b0C (15\u2009sec), 60\u2009\u00b0C (1\u2009min), followed by dissociation; Melt curve was used for all experiments from 55\u2009\u00b0C to 95\u2009\u00b0C. For Bdellovibrio 16S rDNA quantification primers Bd347F-Bd549R97 were used in a reaction mix and thermal cycling conditions as above except for the use of 45 cycles. For Bacteriovorax 16S rDNA quantification primers BacF519 - BacR67798 were used in a reaction mix as above. Thermal cycling conditions were: 50\u2009\u00b0C (2\u2009min), 94\u2009\u00b0C (2\u2009min), 45 cycles at 94\u2009\u00b0C (30\u2009sec), 62\u2009\u00b0C (10\u2009sec) and 72\u2009\u00b0C (10\u2009sec), followed by dissociation; Melt curve was used for all experiments from 55\u2009\u00b0C to 95\u2009\u00b0C. Reactions were performed in a 96-well plate (Applied Biosystems) with MicroAmp\u00ae Optical Adhesive Film (Applied Biosystems) in a Step One plus Real-time PCR System (Applied Biosystems). Total QPCR counts were estimated based on a standard curve with 100% efficiency factoring in the cycle threshold value of the particular sample obtained, taking into account that 25\u2009mL of sample was used for DNA extraction, DNA was eluted in 50\u2009\u03bcl of DDW and 1\u2009\u03bcl of this DNA was 10 fold diluted and used in the QPCR reaction.Standards were prepared by inserting a 1467-bp fragment of the 99. In short: Samples were taken from the activated sludge basin of a wastewater treatment plant in Eilenburg, Germany, centrifuged and the supernatant discarded. The cells were washed with phosphate-buffered saline once and stabilized by adding 2\u2009ml paraformaldehyde solution to the cell pellet and incubated for 30\u2009min at room temperature (RT). After another centrifugation step , 4\u2009mL of EtOH (70%) were added for fixation and the cell solution was stored at \u201220\u2009\u00b0C. For DNA staining these samples were washed twice with PBS, and cell solutions were adjusted to an OD of 0.035 (d\u028e700nm\u2009=\u20095\u2009mm) with PBS. Two millilitres of an adjusted sample was centrifuged , and the pellet resuspended with 1\u2009mL solution A and incubated at RT for 10\u2009min in an ultrasonication bath and 10\u2009min without any further treatment. After another centrifugation step the cells were stained with solution B [0.24\u2009\u03bcM DAPI in phosphate buffer (289\u2009mM Na2HPO4 and 128\u2009mM NaH2PO4 in distil water)] overnight at RT.For flow cytometric analysis of the wastewater community, samples were treated as described in Liu Z, et al. B. bacteriovorus HD100 and E. coli ML-35 were centrifuged at 10,000\u2009g for 10\u2009min and suspended in final 1% PFA for 2\u2009h at room temperature. Fixed cells were then washed twice with 500\u2009\u03bcl PBS, pelleted as above, suspended in 50% EtOH-PBS and stored until use at \u2012\u200920\u2009\u00b0C. Samples were strongly vortexed before hybridization with FISH probes. One millilitres of fixed sample was diluted with 2\u2009ml of PBS, vortexed again, and placed in a sonication bath for 5\u2009min. Samples were then washed twice with PBS and sonicated again for 10\u2009min. When required, samples were concentrated to 100\u2009\u03bcl in PBS.For FISH experiments, AP and GP cells obtained from a dual culture of 23. Controls included target and non-target (E.coli ML-35) organisms to confirm probe specificity, and a general bacteria-directed FITC-labelled EUB338 (5'-GCTGC CTCCCGTAGGAGT-3') probe100. All experiments also included a NONBDE525 probe (5\u2019-GCGGTAAGA CGAGGGATC-3'), an antisense probe to BDE525, as a negative control for unspecific binding. Fixed samples were mixed with hybridization buffer and the appropriate probe treated for 2\u2009h at 46\u2009\u00b0C in an hybridization oven. Samples were pelleted, washed with 100\u2009\u03bcl wash buffer at 48\u2009\u00b0C for 20\u2009min. Counter-staining with DAPI was performed by adding 445\u2009\u00b5L of either PBS or DAPI-buffer (Sigma-Aldrich) and 5\u2009\u00b5L of a 50\u2009\u00b5M DAPI solution to a 50\u2009\u00b5L of BDE525-Cy5 labelled activated sludge sample. Samples were incubated at room temperature for 2.5\u2009h up to overnight, and kept in the dark at 4\u2009\u00b0C until use.Hybridization was performed with a Bdellovibrionales 16S rRNA-targeting, 5'-Cy5 labelled BDE525 (5'-GATCCCTCGTCTTACCGC-3') probeCytometric measurements were performed with a BD Influx v7 Sorter USB, equipped with a 488\u2009nm Sapphire OPS laser (400\u2009mW), a 355\u2009nm Genesis CX laser , and a red 640\u2009nm 56CRH laser . The 488\u2009nm laser light was used for the analysis of forward scatter , side scatter , and yellow\u2013green fluorescence . Cy5 fluorescence was recorded at PMT7 (670/30) after excitation with 640\u2009nm light. DAPI\u2013fluorescence was measured at PMT9 (460/50) after excitation with 355\u2009nm light.99. Briefly, cell were sorted using the \u20181.0 Drop Pure\u2019 sort mode. Either 150,000 cells per gate (P1 and P14) were sorted at a sort rate of about 80 cells.sec-1 or 500,000 cells (P16) were sorted at a sort rate of about 1,800 cells.sec-1 into tubes. Sorted cells were harvested by two successive centrifugation steps , and the cell pellets were stored at \u2013\u200920\u2009\u00b0C for subsequent downstream analysis.The fluidic system ran at 33\u2009psi using a 70\u2009\u00b5m nozzle. The sheath fluid consisted of 0.5 x FACS flow buffer (BD). For daily optical calibration of the cytometer in the linear range, 1\u2009\u00b5m blue fluorescent FluoSpheres and 2\u2009\u00b5m yellow\u2013green fluorescent FluoSpheres were used. For calibration in the log range, 0.5\u2009\u00b5m UV Fluoresbrite Microspheres , 0.5\u2009\u00b5m yellow\u2013green fluorescent Fluoresbrite - Carboxylate Microspheres , and 0.2\u2009\u00b5m crimson fluorescent Fluospheres-Carboxylate Microspheres were applied. Prior to measurement, samples were spiked with 0.5\u2009\u03bcm UV Fluoresbrite Microspheres . The microspheres served as internal standards to monitor instrument stability and to allow the correct comparison of samples. Cell data were collected in logarithmically scaled 2D-dot plots according to fluorescence and FSC for cell size-related information. Gates were set for apparent cell clusters and the cell sorting was performed according to a standardized procedure described beforeB, bacteriovorus attachment to prey and non-prey, tested strains were incubated with the predator for 24\u2009h94. Samples taken at 0, 2, 4, 6 and 24 were fixed and measured by flow cytometry using a 355\u2009nm UV-Line for excitation101. All measurements were made comparable to each other by spiking 1\u2009\u00b5m and 0.5\u2009\u00b5m calibration beads in each of the samples. All flow cytometric raw data files are deposited in the FlowRepository database (www.flowrepository.org) with the repository ID: FR-FCM-Z3WS.To compare TM according to the manufacturer\u2019s protocol. Briefly, 1\u2009\u03bcl of suspended cells was mixed with 9\u2009\u03bcl of sample buffer, denatured at 95\u2009\u00b0C for 3\u2009min, cooled on ice, added to 9\u2009\u03bcl of reaction buffer and 1\u2009\u03bcl of Phi29 DNA polymerase. The reaction was incubated at 30\u2009\u00b0C for 1.5\u2009h and then inactivated at 65\u2009\u00b0C for 10\u2009min.Sorted cells from the three gates were transferred to Eppendorf tubes (~\u2009120\u2009\u03bcl per sample), pelleted at 20,000\u2009g for 20\u2009min at 4\u2009\u00b0C, and the supernatant removed. DNA MDA was performed using the Illustra GenomiPhi V2 DNA Amplification KitThe presence of members belonging to the Bdellovibrionales was validated using the specific 16S rRNA gene primers Bd824F ('5-ACTTGTTGTTGGAGGTAT-3') and Bd1222R ('5-TTGTAGCACGTGTGTAG-3') targeting the Bdellovibrionales and polymerase chain reaction amplification as follows: 95\u2009\u00b0C \u2013 5\u2009min, 95\u2009\u00b0C -30 sec, 48\u2009\u00b0C -45 sec, 72\u2009\u00b0C -30 sec, for 28 cycles; and 72\u2009\u00b0C -7 min. The presence of general bacteria and other controls were validated using primers 515\u2009F ('5-GTGCCAGCMGCCGCGGTAA-3') and 806\u2009R ('5-GGACTACHVGGGTWTCTAAT-3') and the following PCR program: 95\u2009\u00b0C\u20145\u2009min, 95\u2009\u00b0C\u201430\u2009sec, 55\u2009\u00b0C\u201445\u2009sec, 72\u2009\u00b0C\u201430\u2009sec, for 28 cycles; and 72\u2009\u00b0C\u20147 min. PCR products were run on 1.2% agarose gels at 70\u2009V for 35\u2009min and photographed using a UV illuminator.Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary"} +{"text": "The authors regret that the name of one of the authors (Aayushi Biswas) was shown incorrectly in the original article. The corrected author\u2019s name is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that an incorrect grant number was shown in the acknowledgements section of the published article. The corrected section should read:This work was financially supported by the Chinese National Natural Science Foundation .The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "In silico and in vitro metabolism of ribociclib: a mass spectrometric approach to bioactivation pathway elucidation and metabolite profiling\u2019 by Thamer A. Alsubi et al., RSC Adv., 2020, 10, 22668\u201322683, DOI: 10.1039/D0RA01624A.Correction for \u2018 The authors regret that the email address for the corresponding author was shown incorrectly in the original article. 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The corrected author list and affiliations are as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "We regret that two of the corresponding authors, Shujun Cao and Min Shen, were not identified explicitly in the original manuscript. The corrected list of corresponding authors for this paper is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that the title shown in the original article and several sentences were incorrect due to the use of the word \u201cabsorption\u201d in place of \u201cadsorption\u201d. The correct title is as shown above and all instances of \u201cabsorption\u201d in the text should be \u201cadsorption\u201d. 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The corrected author list is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that the affiliations for the second (Rodrigo Tart\u00e9) and corresponding author (Nuria C. Acevedo) were incorrectly shown in the original article.The correct affiliations are given here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Illarionov The authors regret that one of the co-authors, Ivan Mukhin, was omitted from the original article. The correct author list is as shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret the omission of one of the authors, Michael Mak, from the original manuscript. The corrected list of authors and affiliations for this paper is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Ancistrocladus abbreviatus, with cytotoxic activity\u2019 by Shaimaa Fayez et al., RSC Adv., 2022, 12, 28916\u201328928, https://doi.org/10.1039/D2RA05758A.Correction for \u2018Naphthylisoindolinone alkaloids: the first ring-contracted naphthylisoquinolines, from the tropical liana The authors regret that the author affiliations were incorrectly shown in the original manuscript. The corrected list of affiliations is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Nguyen Minh The authors regret that in the original article the authorship was incorrect. J. Baggerman and S. P. Pujari were not included in the original author list. Additionally, J. F. van der Bent was spelled incorrectly in the original article. The correct author list is as presented above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that affiliation b was incorrect in the original version of the manuscript. The correct affiliation is presented herein.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Matson et al., RSC Adv., 2021, 11, 32269\u201332274. DOI: 10.1039/D1RA06151HCorrection for \u2018X-ray absorption spectroscopy of exemplary platinum porphyrin and corrole derivatives: metal- The authors regret that the one of the author affiliations was incorrectly shown in the original manuscript. The corrected list of affiliations is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that the primary affiliation of the corresponding author (Milica Budimir) was incorrectly designated in the original manuscript. The corrected list of affiliations is as shown herein.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Mitoraj et al., RSC Adv., 2019, 9, 23764\u201323773.Correction for \u2018Structural versatility of the quasi-aromatic M\u00f6bius type zinc( The authors regret that the affiliations of Maria G. Babashkinah and Damir A. Safin were incorrectly shown in the original manuscript. The corrected list of affiliations is as shown herein.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that In addition, a citation to Fig. 12 (inset) on page 27982 of the original article should be corrected to refer to Fig. 13 (inset).The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.RA-009-C9RA90075F-s001"} +{"text": "M. Abd-Rabboh The authors regret that the affiliations for one of the co-authors (Mohamed A. Al-Omar) were incorrectly shown in the original article. The correct affiliations are given here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that there was an error in the author list in the original article. Thomas Shean Yaw Choong should have been listed as a corresponding author. The amended author list and contact details are shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Synthesis and photophysics of sulfide, sulfoxide and sulfone based D\u2013\u03c0\u2013A compounds\u2019 by Matias Mon\u00e7alves The authors regret that The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Hussnain Ahmed Janjua's name was incorrectly spelled in the published article; the corrected author list is shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that in the published paper, the affiliations of the authors were wrongly designated. The corrected version is shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Rebekah et al., Nanoscale Adv., 2021, DOI: 10.1039/d1na00135c.Correction for \u2018NiCo The authors regret that the name of the first author (A. Rebekah) was incorrectly given in the original article. The corrected name is given here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "In the published article, Liming Bai was incorrectly not listed as the corresponding author. The correct version is shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer\u2019s disease\u2019 by Min-Che Tung et al., RSC Adv., 2021, 11, 31062\u201331072, https://doi.org/10.1039/D1RA05311F.Correction for \u2018Discovery of 8-prenylnaringenin from hop ( The authors regret that the name of one of the authors (Hsing-Mien Hsu) was shown incorrectly in the original article. The corrected author list is as shown above.The authors also regret an incorrect version of The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "DOI: 10.1039/C9RA07057ECorrection for \u2018Modelling and prediction of the thermophysical properties of aqueous mixtures of choline geranate and geranic acid (CAGE) using SAFT-\u03b3 Mie\u2019 by Silvia Di Lecce The authors regret the omission of one of the authors, David Pugh, from the original manuscript. The corrected list of authors and affiliations for this paper is as shown here.In addition, we point readers to The authors also wish to correct a number of typographical errors in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Charge partitioning theory and methodology\u2019 by Thomas A. Manz The authors regret that there was an error in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that an incorrect version of In addition, the authors regret that affiliation a was incorrectly shown in the original manuscript. The corrected list of affiliations is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The journal citations in ref. 10 and 11 were incorrect in the published article. The corrected references are shown below as The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret the omission of a funding acknowledgement in the original article. This acknowledgement is given below.The authors would like to acknowledge the financial support of the Iran National Science Foundation (INSF), grant number 98017171.In addition, the authors regret that incorrect reference numbers were given in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors wish to amend the authorship of this article by adding an additional author, Aleksandra Wiatrowska. The full author list is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that ds26, telo21 and RNA were labelled incorrectly in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Chand et al., RSC Adv., 2022, 12, 33021\u201333031, https://doi.org/10.1039/D2RA05224E.Correction for \u2018Glycal mediated synthesis of piperidine alkaloids: fagomine, 4- The authors regret that one of the authors\u2019 names, Mrityunjay K. Tiwari, was spelt incorrectly in the original manuscript. The corrected list of authors for this paper is as shown above. The authors regret that affiliation c was incorrectly shown in the original manuscript. The corrected list of affiliations is as shown below.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Acinetobacter baumannii\u2019 by Sajal Kumar Halder et al., RSC Adv., 2022, 12, 24319\u201324338, https://doi.org/10.1039/D2RA02939A.Correction for \u2018Oxa-376 and Oxa-530 variants of \u03b2-lactamase: computational study uncovers potential therapeutic targets of The authors regret that one of the authors' names, Md. Nuhu Alam, was spelt incorrectly in the original manuscript. The corrected list of authors for this paper is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "One of the affiliations of the corresponding author was omitted from the original manuscript. The correct affiliations are as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that the original author list was incorrect. The correct author list is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Aminul Islam et al., RSC Adv., 2022, 12, 7835\u20137849, DOI: 10.1039/D2RA00768A.Correction for \u2018Efficacy of surface-functionalized Mg The authors regret that the name of one of the authors (Md. Mahbubul Haque) was shown incorrectly in the original article. The corrected author list is as shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that an asterisk was not displayed next to the name of the corresponding author Chang Liu in the original manuscript. The corrected information is shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Errors were present in the published article in terms of corresponding authors and e-mail addresses; Chen Yu is the sole corresponding author, extra e-mail addresses have been deleted, and the correct version is shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Gardneria nutans Siebold & Zuccarini\u2019 by Ying-Ying Si et al., RSC Adv., 2021, 11, 27085\u201327091. DOI: 10.1039/D1RA05204G.Correction for \u2018Neuroinflammatory inhibitors from The authors regret that the first and last names for Young Jun Im were reversed in the original article. The corrected author list is shown above. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Incorrect corresponding authors were indicated in the published article while the correct version is shown above; Wei Zhang rather than Weilong Liu is the co-corresponding author.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The correct equation is shown below:The authors regret that an incorrect equation was shown in the original article. On page 98963 of the original article, in the equation for fitting power saturation data the term The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Tomane The authors regret the omission of one affiliation for one of the authors, Somia Tomane, from the original manuscript. The corrected list of authors and affiliations for this paper is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "T. H. Phong The affiliations of this author were also incomplete. The correct affiliations are as listed above.The authors regret that the email address of the author Thi Ly Mai was not listed in the original manuscript. The email address of this author is The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "H. Ji et al., RSC Adv., 2018, 8, 8302\u20138309.Correction for \u2018High-detectivity perovskite-based photodetector using a Zr-doped TiO The authors regret that the names of the authors are shown incorrectly in the original article. The corrected author list is as shown above.In addition, the authors regret that an incorrect version of The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that the funding information for the original article was incorrectly given. The corrected funding information is as follows.The authors greatly appreciate financial support from the National Natural Science Foundation of China (No. 81703664) and the China Postdoctoral Science Foundation (No. 2019M663855).The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Algethami The corrected list of affiliations is as shown in this Correction article.The authors regret that one of the affiliations (affiliation The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Singh The author regrets that the funding information was incorrectly shown in the acknowledgements section of the original manuscript. The corrected funding acknowledgement is as shown below.All authors acknowledge support for this work, in part, from the National Science Foundation under Grant No. 1609303. A. S. and S. K. S. also acknowledge support, in part, from the National Science Foundation under Grant No. 1655496 and 1661038. The electrokinetics module used in the presented NanoMagnetoElectrokinetic modeling framework was developed with support from these grants.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The e-mail contact address for the corresponding author Tao Wen, was omitted in the published article and is shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Rh-catalyzed intramolecular carbene transfer reaction of diazoacetamides' by Qingmin Song et al., RSC Adv., 2022, 12, 18197\u201318208, https://doi.org/10.1039/D2RA01298G.Correction for \u2018Theoretical study on the mechanism, chemo- and enantioselectivity of the Ag- The authors regret that the name of one of the authors (Nikolaos V. Tzouras) was shown incorrectly in the original article. The corrected author list is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "DOI: 10.1039/c9ra03560eCorrection for \u2018Role of polysilicon in poly-Si/SiO The authors regret that the list of corresponding authors was incorrect in the original article. The corrected author list and associated contact details are as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors wish to point out that in the author list of the published article, the author names were incorrectly presented, with family names incorrectly shown before given names. The corrected form of all of the author names is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Lid\u00f3n Pru\u00f1onosa Lara, who contributed significantly to the crystallography and should have been included as a co-author.The authors regret the omission of The corrected list of authors and affiliations for this paper is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret that there was an inaccuracy present in the affiliation information for one of the authors, Alexander D\u00f6mling, in the original manuscript.The work was performed at the Department of Drug Design, University of Groningen, Groningen, The Netherlands (a.s.s.domling@rug.nl) before the transition of Alexander D\u00f6mling to their current institution, CATRIN, Innovative Chemistry Group, Palack\u00fd University Olomouc, Olomouc, Czech Republic . The corrected list of author information and affiliations for this article is as shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Sci., 2022, 13, 345\u2013364, https://doi.org/10.1039/D1SC05835E.Correction for \u2018Polymers as advanced antibacterial and antibiofilm agents for direct and combination therapies\u2019 by Zhangyong Si The authors regret that the name of one of the authors (Wenbin Zhong) was shown incorrectly in the original Review Article. The corrected author list is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors regret an error in the original article whereby identical images were presented in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "In the original article in The results and conclusions of this paper are not affected by this correction.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Eurycoma spp. by micellar liquid chromatography and conventional high-performance liquid chromatography: a comparative evaluation\u2019 by Attapon Sakdamas et al., RSC Adv., 2023, 13, 6317\u20136326, https://doi.org/10.1039/D2RA07034K.Correction for \u2018Analysis of canthin-6-one alkaloids derived from The authors regret that the name of one of the authors (Fonthip Makkliang) was shown incorrectly in the original article. The corrected author list is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "A corrected y axis labelled as \u2018Weight percentage (%)\u2019 to reflect the correct TGA test curve.In the original article, the authors regret an error in An independent expert has viewed the corrected image and has concluded that it is consistent with the discussions and conclusions presented.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": \ No newline at end of file