However, the subjacent molecular mechanisms are not yet understood. Our previous work suggests that early neuronal progenitors were not differentiating correctly in the developing spinal cord. As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis. Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down. The strength of this approach is that it focused on the NSC population while tackling the biological issue in an in vivo context in whole zebrafish embryos. After sequencing the transcriptome by RNA-sequencing, we analyzed the genes whose expression was changed upon disruption of glycine signaling and we confirmed the differential expression by independent RTqPCR assay. While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signaling pathways  that are likely to mediate the promotion of neurogenesis by glycine.How is the initial set of neurons correctly established during the development of the vertebrate central nervous system? In the embryo, glycine and GABA are depolarizing due the immature chloride gradient, which is only reversed to become hyperpolarizing later in post-natal development. We previously showed that glycine regulates neurogenesis  Neurogenesis is a crucial step in vertebrate development and it requires multiple and integrated signals throughout development to be achieved correctly . Many faDuring development, prior to glutamatergic innervation, neural activity mainly relies on glycinergic and GABAergic neurotransmission . InteresPreviously, our group discovered an early role of glycine during neurogenesis in the spinal cord, which starts during the first day post-fertilization (dpf), prior to hatching after 2 dpf. Glycine acts to promote the differentiation of neural progenitors specifically into interneurons  in a proglra4a morpholino-injected versus control morpholino-injected and non-injected zebrafish embryos and we confirmed their differential expression by independent RTqPCR assay. Using pathway analysis and clustering tools, we identified several canonical signaling pathways involved in glycine-dependent neurogenesis.Although the requirement for glycine signaling for interneuron neurogenesis is now well established, the subjacent molecular mechanisms are not yet understood. In this work, we aimed at shedding light on the molecular action of glycine signaling during neural development and to identify the downstream molecular pathways involved. We took advantage of a transgenic line in which NSCs are fluorescently labeled in order to develop a protocol to specifically purify the NSC population from whole embryos by fluorescence-activated cell sorting (FACS). After mRNA purification, we performed next generation sequencing to access the transcriptome of NSCs purified from embryos in which glycine signaling had been disrupted by morpholino knockdown. This allowed us to compare the set of genes differentially expressed in NSCs purified from Danio rerio) were reared at 28.5°C, kept under a 12-h dark, 12-h light cycle, and staged as described previously . Primers were designed to flank the part of the 5′UTR/5′CDS of zebrafish glra4a gene containing the morpholino binding site targeted in this study. The resulting 0.1 kb segment was sub-cloned in-frame into pEGFP-N1 to generate a fusion protein (primer sequences are available upon request). Thirty nanogram of glra4a-EGFP plasmid was microinjected with 0.6 ng of glra4a or control morpholino, respectively. The fluorescence was analyzed by confocal microscopy  at 18 hpf.Total RNA was isolated from 24 hpf zebrafish embryos using TRIZOLg centrifugation for 5 min. They were briefly washed with FACSmax cell dissociation solution (Genlantis) and transferred in a 60 mm petri dish with fresh FACSmax solution, then incubated at 28.5°C. Single-cell dissociation was carefully monitored every 5 min and was generally achieved within 30 min of incubation. Efficient dissociation was helped by firmly tapping the petri dish and by gentle pipetting. Single cells were exhaustively washed twice in cold PBS, pelleted and resuspended in 500 μL of cold PBS. Single cells were filtered in a Falcon tube with a cell strainer cap (Fisher Scientific) and placed on ice until cell sorting. Sorting was performed using a BDARIA IIIu FACS with DIVA 8 sofware . GFP expressing cells were identified using a 488 nm laser and a 530/30 BP filter. Cells were collected in cold PBS on ice.Embryos at 20 hpf were briefly washed in calcium-free Ringer’s solution and deyolked by up and down pipetting. Deyolked embryos were pelleted by 500 ×5 cells by RNAsolv reagent manufacturer’s protocol (Omega Biotek). Absence of contamination with chemicals was assessed by nanodrop using 260/280 and 260/230 ratios. Quantification of total RNA was made by nanodrop and 0.1 to 1.44 g of total RNA was used for sequencing. Quality of total RNA was assessed with the BioAnalyzer Nano (Agilent) and all samples had a RNA Integrity Number (RIN) above 8.Two independent clutches were injected, by each of two experimenters, corresponding to experimental duplicates. Total RNA from RNAlater-fixed embryos (Ambion) was extracted using RNAsolv reagent (Omega Biotek) following the manufacturer’s standard protocol. RNA extraction was made using between 5.19–9.75 × 102. Around 65 to 89 million reads were generated for each sample. Library preparation and sequencing was done at the Institute for Research in Immunology and Cancer’s Platform . About 86% of high quality reads were mapped onto the zv9 version of the zebrafish genome (ensemble release 77) using TopHat version 2.0.10.Library preparation was done with the Truseq RNA (Illumina). 18 PCR cycles was required to amplify cDNA libraries. Libraries were quantified by nanodrop and BioAnalyzer. All librairies were diluted to 10 nM and normalized with the Miseq SR50 v2. Libraries were pooled to equimolar concentration and multiplexed by six samples per lane. Sequencing was performed with the Illumina Hiseq2000 using the SBS Reagent Kit v3  with 1.6 nM of the pooled library. Cluster density was targeted at around 800 k clusters/mmp-value) < 0.05. Pathway analysis was performed using DAVID bioinformatics resources (www.genome.jp/kegg/pathway).Differential gene expression analysis was assessed by DeSeq2 package using R software. Differential gene expression was filtered on an absolute value of LogFC > 1 and a False Discovery Rate (or adjusted esources . The lispolr2d gene (ENSDART00000108718) was used as a reference gene for ddCt quantificationQuantitative PCR (qPCR) primers for each tested gene were designed using the Universal Probe Library tool from Roche. Total RNA was extracted from FACS-purified NSCs using RNAsolv reagent (Omega Biotek) and chloroform followed by isopropanol precipitation. Reverse transcription was performed from 1 μg of total RNA using the superscript VILO reverse transcription mix (Invitrogen). Quantitative PCR was performed on 2 μL of 1:10-diluted cDNA using SYBR Green I master (Roche) on a LightCycler 80 thermocycler. gfap:GFP transgenic line that has been reported to express GFP in NSCs , we successfully dissociated 20 hpf embryos into single cells and we were able to perform a FACS purification of GFP+ cells  . As shown in Figure 1A, the quality of the single-cell dissociation was suitable for subsequent FACS with more than 50% healthy single cells. After doublet elimination and using a 530/30 BP filter, the GFP+ population  was easily distinguishable from the negative batch as separate peaks in the cell count . In order to prevent any contamination with GFP- cell types, we increased the stringency of the sorting by only selecting cells with a high GFP expression . Finally, to ensure the specificity of the sorting and the integrity of the purified cells, we performed a quality control by further analyzing the GFP+ sorted cells . We found that about 81% of cells survived and more than 97% of them were GFP+. Together these results indicate that live NSCs can indeed be purified by FACS and so used to explore the neurogenic role of glycine.In order to identify the signaling pathways involved in glycine-mediated NSC differentiation, we took advantage of the  in NSCs . Using t1 allele , we wereglra4a morpholino . To validate the in vivo efficacy and selectivity of our morpholino, we cloned a 0.1 kb fragment of the glra4a region containing the morpholino target site  in frame with the coding sequence of GFP. After co-injection of this construct and our control morpholino, 92% of the embryos (n = 49/53) depicted broad GFP fluorescence, thus validating the expression of the fusion protein . However, when co-injected with glra4a morpholino, GFP was undetectable in 36 out of 38 of the injected embryos (95%). These results validate the selectivity of our glra4a morpholino compared to the 5 base-mismatch control morpholino. For each condition , we dissociated between 60 and 80 gfap:GFP+ embryos from which we successfully FACS-purified between 5.19 and 9.75 × 105 GFP+ cells . Each treatment condition was processed in duplicate from independent batches of eggs and total RNA was extracted reaching a RIN higher than 8.50.We used the same procedures to purify NSCs from (i) uninjected 20 hpf embryos, (ii) embryos injected with a 5 base-mismatch control morpholino, and (iii) embryos knocked-down for GlyR using a specific Figure 3A) and found 6,971 differentially expressed genes .Between 65 and 89 million high quality reads per condition were sequenced and we were able to map about 86% of these reads onto the zv9 version of the zebrafish genome (ensemble release 77). Using DESeq2 algorithm, we assessed the differentially expressed genes comparing the dataset from GlyR knockdown NSCs with uninjected WT NSCs . Most likely, we assume that these genes (6%) are morpholino off-targets and were removed for further analysis. To this aim, we also ran a third differential analysis test (#3) comparing the GlyR knockdown dataset with control-morpholino knockdown. This analysis revealed that the expression of 215 genes from this overlapping list were not statistically different between GlyR and control knockdown. Thus, we considered these genes as off-targets and we removed them as well from the final list. However, we kept those genes whose differential expression between GlyR and control knockdown was significant  since although being found in the control condition, the difference of expression for these genes seemed to be specific to GlyR knockdown .We assumed that some of these genes could be misregulated because of the injection itself rather than being specific to the GlyR knockdown. To circumscribe this issue, we therefore performed a differential gene expression analysis in NSCs from control-morpholino injected embryos compared to uninjected (test#2). We found that 635 genes were misregulated in NSCs upon injection of the control morpholino (the full list of these genes is available upon request). Moreover, 402 of them were overlapping with the list of genes found to be differentially expressed upon GlyR knockdown  and this number decreases to 1,162 after filtering with a |logFC| greater than one . As visualized in the volcano plot , 77% of these genes were upregulated upon GlyR knockdown  and 23% were downregulated . By filtering more stringently with a |logFC| > 2 (at least 4 fold up- or down-regulation), we could narrow down this number to 141 genes .We concluded that a total of 6,756 genes were specifically and differentially expressed in NSCs upon GlyR knockdown : signaling by (i) calcium, (ii) TGF-beta, (iii) sonic hedgehog, (iv) Wnt, and (v) p53-related apoptosis. Regarding calcium signaling, we found CACNA2D4B and other calcium channels  to be upregulated. Consistently, upon GlyR knockdown, the ITPR1 intracellular calcium channel expressed at the endoplasmic reticulum was also upregulated. Besides, the expression of genes encoding the Gαq GNA14 and the downstream phospholipases C (PLCD1A/4A), which activate ITRP1, were also upregulated in our dataset. Interestingly, we found that some calcium-dependent proteins such as calmodulin kinases CAMK2D1 and CAMK4, the proline-rich tyrosine kinase PTK2BB and the nitric oxide synthase 1 NOS1 were upregulated in NSCs upon GlyR knockdown. Finally, some transcription factors regulated by calmodulin kinases and belonging to the CREB complex were downregulated in our dataset  .Using the freely available DAVID bioinformatics resources  and throFigure 4B). In the same pathway, the transcription factor SMAD3A was found to be downregulated in response to GlyR knockdown in NSCs.Furthermore, we found some genes involved in the Transcription Growth Factor TGF-beta signaling pathway such as Bone Morphogenetic Proteins (BMP) 2B, 4 and 6, TGF-beta receptor 2 (TGFBR2) and the TGF inhibitor Lefty1 (LFT1) as upregulated in our knockdown dataset  in which they are activated by GLI2B zinc-finger transcription factor (GLI-Kruppel family member 2B) and the expression of this gene was downregulated in our GlyR knockdown dataset. We also observed the upregulation of SHH1B gene itself and of the SHH interacting protein HHIP.According to DAVID ressources, BMP2B and BMP4 are also involved in Sonic HedgeHog signaling (SHH) . Moreover, we found that the secreted frizzled-related protein 1a (SFRP1A) and the target transcription factor TCF7L1A were downregulated after GlyR knockdown. On the contrary, the expression of the leucine zipper FOS-like antigen 1A (FOSL1A) was upregulated in our dataset.The fourth pathway identified is the WNT signaling pathway in which two inhibitor proteins WIF1 (Wnt inhibitory factor 1) and DKK1B (Dickkopf wnt inhibitor 1B) were upregulated upon GlyR knockdown .Lastly, we found p53 itself and numerous downstream targets of the p53-related apoptosis pathway  to be activated upon GlyR knockdown, while SESN2 was downregulated  involved in each of these five pathways by RTqPCR . For all of these genes we found a positive correlation between the RNAseq data and the trend of expression observed by RTqPCR upon GlyR knockdown in NSCs. More importantly, for all these genes, we observed no difference in the expression between uninjected and control knockdown NSCs, thus confirming the specificity of our transcriptomic observations.In order to validate these transcriptomic results, we repeated the FACS purification on new independent clutches of embryos that we injected with control or In this work, we aimed at identifying the molecular mechanisms involved in glycine-dependent neurogenesis during embryonic development, precisely in NSCs. By specifically looking into these cells after GlyR gene knockdown, we identified five signaling pathways that are very likely to play a role downstream to glycine. One of the most interesting pathways that we found is calcium signaling. Its involvement is consistent with our previous work in which we found that glycine-induced calcium transients in neuronal progenitors are required for neurogenesis . Our preThree other signaling pathways identified  are also very good candidate since their implication in neurogenesis has been exhaustively reviewed . Indeed,Figures 5G,H). According to the literature, this finding would be consistent with an inhibition of neurogenesis. Moreover, SMAD3 transcription factor, a target of TGFs that is also a crucial player promoting neurogenesis in adult mice . Altogether, these results suggest that the abnormal neurogenesis induced by GlyR knockdown could be, at least in part, due to misregulation of TGF-related proteins.Bone Morphogenetic Proteins are the largest subgroup of the TGFs and they have been involved in numerous biological processes from cell survival and proliferation to differentiation in particular during the development of the central nervous system . They hault mice , is downSonic HedgeHog signaling is important for the patterning of the dorso-ventral neuron types within the developing neural tube where it promotes ventral identities . TherefoFigures 5K,L), thus allowing us to assume that Wnt-dependent neurogenic processes could be silenced in NSCs when glycine signaling is impaired. Consistently, it has been reported that WNT inhibition through DKK1 overexpression prevents neuronal production during mouse embryonic neurogenesis  is a transcription factor regulating the expression of a broad panel of genes, some of them being necessary for cell proliferation although in endothelial cells . Interestingly, P53 activation has been recently shown as regulating neurogenesis by increasing proliferation and differentiation of hippocampal NSCs in vitro . As a result, more discrete cell death could arise in a subpopulation of neural progenitors in the developing spinal cord.Lastly, our gene expression analysis identified P53 as being upregulated specifically upon GlyR knockdown (in vitro . Howeverin vitro , althougin vivo context in which cells develop. As a result, zebrafish embryos can be considered as in vivo test tubes in which it becomes more and more easy to dig for molecular characterization of phenotypes.The next step in deciphering the molecular aspects of glycine-dependent neurogenesis will be to perform functional genomics on the candidate genes we identified. Such a characterisation is beyond the scope of the present work as a large effort needs now to be deployed to understand the function of these molecular actors. Zebrafish would be a suitable model for such an analysis since the embryo is easily genetically accessible. Lastly, our present work widens the use of zebrafish as a model of developmental biology. Indeed, we took advantage of advanced cell purification techniques to precisely look within our cells of interest but without dismantling the in vivo morpholino knockdown. AB and ES designed FACS experiments. ES performed and interpreted the transcriptomic and pathway analyses and did RTqPCR validations. ES and PD wrote the manuscript.AB validated the 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.


Drinking water distribution systems, including premise plumbing, contain a diverse microbiological community that may include opportunistic pathogens. On-site supplemental disinfection systems have been proposed as a control method for opportunistic pathogens in premise plumbing. The majority of on-site disinfection systems to date have been installed in hospitals due to the high concentration of opportunistic pathogen susceptible occupants. The installation of on-site supplemental disinfection systems in hospitals allows for evaluation of the impact of on-site disinfection systems on drinking water system microbial ecology prior to widespread application. This study evaluated the impact of supplemental monochloramine on the microbial ecology of a hospital’s hot water system. Samples were taken three months and immediately prior to monochloramine treatment and monthly for the first six months of treatment, and all samples were subjected to high throughput Illumina 16S rRNA region sequencing. The microbial community composition of monochloramine treated samples was dramatically different than the baseline months. There was an immediate shift towards decreased relative abundance of Betaproteobacteria, and increased relative abundance of Firmicutes, Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria and Actinobacteria. Following treatment, microbial populations grouped by sampling location rather than sampling time. Over the course of treatment the relative abundance of certain genera containing opportunistic pathogens and genera containing denitrifying bacteria increased. The results demonstrate the driving influence of supplemental disinfection on premise plumbing microbial ecology and suggest the value of further investigation into the overall effects of premise plumbing disinfection strategies on microbial ecology and not solely specific target microorganisms. Drinking water distribution systems, including premise plumbing, contain a diverse microbiological population Legionella spp., non-tuberculous Mycobacteria, Pseudomonas spp., Acinetobacter spp., Stenotrophomonas spp., Brevundimonas spp., Sphingomonas spp., and Chryseobacterium spp. The complex microbial ecology of premise plumbing systems can serve as a reservoir for opportunistic pathogens, such as Premise plumbing systems have an approximately ten-times greater microbial load than full-scale drinking water distribution systems, due to many factors including greater water stagnation and surface area to volume ratio Nitrospira spp. and Nitrosomonas spp. In addition to use in on-site systems, monochloramine as a secondary disinfectant has been advocated in the US as an effective method to reduce the production of disinfection-by-products LegionellaLegionella control demonstrated a significant reduction in L. pneumophila and no change in nitrate or nitrite levels The effectiveness of chloramination in removing opportunistic pathogens in premise plumbing remains unclear Despite the obvious importance of the microbial ecology of drinking water systems in modulating disinfectant effectiveness and as a reservoir for opportunistic pathogens, there is a notable lack of studies detailing the shift in microbial diversity and composition in response to on-site disinfection. The objective of this study was to determine the effects of on-site monochloramine disinfection on the microbial ecology of a hospital hot water system. Both the microbial ecology of hot water systems and the response of premise plumbing microbial ecology to on-site disinfection are not currently well described in the literature. This study utilizes 216 samples taken from 27 sites and pooled into five composites for two time points prior to and six time points following the addition of on-site monochloramine addition. Samples were analyzed utilizing Illumina DNA sequencing of the microbial community 16S rRNA region and results demonstrate a dynamic shift of the microbial ecology of a hospital’s hot water system in response to monochloramine addition.2. Details regarding monochloramine dosing and water chemistry are included in For these activities no specific permissions were required for these locations. This study took place in a 495-bed tertiary care hospital complex in Pittsburgh, PA. The building has 12 floors and receives chlorinated, municipal cold water. The hospital’s hot water system was treated with the Sanikill monochloramine injection system . Monochloramine was dosed to a target concentration between 1.5 and 3.0 ppm as ClLegionella isolation. Five mL of each concentrated sample was frozen at −80°C until DNA extraction.Hot water was collected from 27 sites throughout the hospital at two time points before monochloramine injection (three months and immediately prior) and monthly for the first six months of monochloramine application. Water samples were collected from a variety of locations throughout the hospital . SamplesFrozen water samples were thawed and pooled as described in http://www.wernerlab.org/software/macqiime) implementation of QIIME 1.7.0 Stenotrophomonas spp. following monochloramine addition; closed-reference OTU picking is presented for higher-quality taxonomic assignment. Morisita-Horn indices were calculated as previously described Data was analyzed within the MacQIIME  and open reference  in Alpha diversity (number of observed OTUs) of samples treated with monochloramine was significantly higher than samples from the baseline months . Prior tBeta diversity (sample interrelatedness) was analyzed using weighted UNIFRAC The samples from the hot water tank (HWT) from pre-treatment months (B3 and B0) were approximately 60% Betaproteobacteria with approximately 35% Firmicutes, Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria in aggregate  Panel A.The microbial community profile of samples from the lower floors of the hospital  was slightly different than those of the hot water tank samples but a similar trend was observed  Panel B.In spite of being from the same rooms, the taxonomic composition of samples from F6A and F6S differed after treatment  Panel C.Prior to treatment, the microbial community in samples from the upper floors of the hospital  resembled most of the other baseline samples with over 70% Betaproteobacteria and approximately 20% of Firmicutes, Bacteroidetes, Alphaproteobacteria, Gammaproteobacteria, Acidobacteria, and Cyanobacteria  Panel E.Separately amplified and barcoded technical replicates of sample pool F8 for 7 of the 8 sample pools were also sequenced to verify technical reproducibility. There is no replicate for month B0. UNIFRAC analysis demonstrated that the replicates from each month cluster very closely . All of Legionella spp., Pseudomonas spp., Acinetobacter spp., and Stenotrophomonas spp. (Gammaproteobacteria group); Brevundimonas spp. and Sphingomonas spp. (Alphaproteobacteria group); Chryseobacterium spp. (Bacteroidetes group); and Mycobacterium spp. (Actinobacteria group). These genera are of special interest as some to all of the species contained within them are pathogens; however, the nature of short-read 16S rRNA region sequence analysis is such that species-level pathogens cannot be definitively identified. Trends demonstrated by this analysis could be used to direct future analyses targeting opportunistically pathogenic organisms more specifically. Analysis of the relative abundance of each of these organism groups over time shows a statistically significant increase in relative abundance for Acinetobacter (p = 0.0054), Mycobacterium (p = 0.0017), Pseudomonas (p = 0.031) and Sphingomonas (p = 0.034) as treatment progressed , Mycobacterium (p = 0.002), Pseudomonas (p = 0.015), and Sphingomonas (p = 0.025), but also showed a significant increase in the genera Stenotrophomonas (p = 0.03)  .Nitrospira and Nitrosomonadaceae, before (mean = 0.0015±0.0018) and after treatment (mean = 0.0005±0.0011) (p = 0.175). Other nitrifier-containing genera such as Nitrosococcus, Nitrobacter, Nitrospina, or Nitrococcus, were not identified in any samples. The total relative abundance of genera containing denitrifiers  underwent a statistically significant increase before (mean = 0.00005±0.000074) and after treatment with monochloramine (mean = 0.0029±0.0029) (p = 0.026). The denitrifier-containing genera Rhizobiales and Rhodanobacter were not identified in any samples. The same trends were observed in open-reference picked data . This study took place during the first U.S. trial of the Sanikill on-site monochloramine generation system  The microbial population shift in response to monochloramine addition was immediate. The number of OTUs observed  significantly increased following monochloramine treatment . It is pLegionellaPseudomonas aeruginosaWe investigated the possible differences in microbial ecology between automatic and standard faucets as it has been previously demonstrated that opportunistic pathogens, including There was an overall shift towards less relative abundance of Betaproteobacteria, and more relative abundance of Firmicutes, Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria and Actinobacteria after monochloramine treatment. A previous microbial ecology study of a simulated drinking water distribution system treated with monochloramine demonstrated a different trend, with an increase in specific genera within the Actinobacteria, Betaproteobacteria, and Gammaproteobacteria phyla Acinetobacter, Mycobacterium, Pseudomonas, and Sphingomonas, showed an increase after monochloramine treatment. Other studies have described an increase in some of these organisms including Legionella, Mycobacterium, and Pseudomonas in chloraminated water Mycobacterium spp. can be enriched in showerhead biofilms compared to the source water Mycobacterium spp. due to monochloramine treatment is of concern, specifically if this increase in relative abundance is due to the presence of more viable mycobacterial cells. These microorganisms may pose a specific threat of aerosol exposure to immunocompromised patients who reside in buildings with an increased abundance of these organisms in hot water Legionella species following monochloramine treatment  was observed following monochloramine treatment, from an average of 4.1±4.5% of the microbial population prior to treatment to an average of 40.9±27.1% following treatment (p<0.001). This genera is comprised primarily of spore-formers that are of concern in food spoilage Alicyclobacillus spp. suggests a potentially dominant role in chloraminated hot water system microbial ecology worthy of future investigation.A notable increase in the relative abundance of the genus Leginonella and other opportunistic waterborne pathogens. Additionally, the American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASHRAE) has recently proposed Standard 188P for the prevention of legionellosis associated with premise plumbing systems Legionella infections through a risk management approach The incidence of reported Legionnaires’ disease cases increased threefold from 2000 to 2009 Legionella such as the potential enrichment of other waterborne pathogens, including Acinetobacter, Mycobacterium, Pseudomonas, and Sphingomonas. Understanding the impact of supplemental disinfection on water system microbial ecology, through a holistic approach, is necessary to maximize disinfectant effectiveness and to ensure that supplemental disinfectant does not select for alternative opportunistic pathogens. A recent review emphasizes not only the role of disinfectants but also other system factors that may impact microbial ecology such as temperature, pipe material, organic carbon, presence of automatic faucets, and point-of-use filtration This study demonstrates that there exists the potential for unwanted consequences of supplemental disinfectant addition for the removal of Acinetobacter, Mycobacterium, Pseudomonas, and Sphingomonas and genera associated with denitrification. The benefits and risks of each supplemental disinfection strategy should be evaluated before implementation in any building, especially in hospitals, long term care facilities, and other buildings housing immunocompromised patients. This work demonstrates the effects of a supplemental monochloramine disinfection system on the microbial ecology of premise plumbing biofilms. Given the importance of premise plumbing microbial ecology on opportunistic pathogen presence and persistence, understanding the driving influence of supplemental disinfectants on microbial ecology is a crucial component of any effort to rid premise plumbing systems of opportunistic pathogens. As additional facilities turn to on-site water disinfection strategies, more long-term studies on the effects of disinfectants on microbial ecology in premise plumbing are needed as well as those evaluating a probiotic approach to opportunistic pathogen eradication.In conclusion, we observed a shift in the microbial ecology of a hospital’s hot water system treated with on-site chloramination. This shift occurred immediately following monochloramine treatment. Prior to treatment, the bacterial ecology of all samples was dominated by Betaproteobacteria; following treatment, members of Firmicutes and Alphaproteobacteria dominated. Differences in community composition were seen in different locations within the hospital as well as between automatic and standard faucets. This suggests that water from different locations and outlet types should be sampled to get a more thorough picture of the microbiota of a system. There was an increase in the relative abundance of several genera containing opportunistic waterborne pathogens following the onset of monochloramine treatment, including Figure S1Sample evenness for closed-reference OTU picking. No statistically significant different was observed for samples taken prior to or following monochloramine addition.(TIF)Click here for additional data file.Figure S2Alpha diversity for open-reference OTU picking. A statistically significant difference was observed for samples taken prior to or following monochloramine addition (p = 0.046).(TIF)Click here for additional data file.Figure S3Beta diversity for open-reference OTU picking. Samples from before monochloramine treatment clustered together whereas following treatment samples clustered by location more so than month of treatment.(TIF)Click here for additional data file.Figure S4Taxonomic assignment of sequences from HWT (hot water tank samples) (Panel A), F3 (floors 3–5) (Panel B), F6A (floors 6 and 7 automatic faucets) (Panel C), F6S (floors 6 and 7 standard faucets) (Panel D), F8 (floors 8–12) and F8rep (replicate barcoded PCRs of samples from floors 8–12) (Panel E) for open-reference OTU picking.(TIF)Click here for additional data file.Figure S5Relative abundance of waterborne pathogen containing genera for open-reference OTU picking. A statistically significant increase in Acinetobacter spp., Mycobacterium spp., Pseudomonas spp., Sphingomonas spp., and Stenotrophomonas spp. was observed following treatment.(TIF)Click here for additional data file.Figure S6Relative abundance genera containing nitrifying (Nitrospira and Nitrosomonadacea) and denitrifying bacteria  for open-reference OTU picking. No other genera containing nitrifying bacteria  or denitrifying bacteria  were found in our samples.(TIF)Click here for additional data file.Table S1Physicochemical data obtained during the study.(DOCX)Click here for additional data file.Text S1Supplementary Information. Water chemistry and monochloramine dosing methods, description of minor phyla observed, and open-reference OTU picking results.(DOCX)Click here for additional data file.


Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas.Global gene transcription was measured in pancreatic islets isolated from young and old rats by Ilumina BeadChip arrays. Promoter DNA methylation was measured by Sequenom MassArray in 46 genes that showed differential expression with age, and correlations with expression were established. Alterations in morphological and cellular processes with age were determined by immunohistochemical methods.Enpp1, Abcc8), type 2 diabetes , inflammatory processes  and extracellular matrix organisation . Age-associated transcriptional differences negatively correlated with promoter DNA methylation at several loci related to inflammation, glucose homeostasis, cell proliferation and cell–matrix interactions .Age-related changes in gene expression were found at 623 loci , with a significant (FDR < 0.05) enrichment in genes previously implicated in islet-cell function genetic signatures of the ageing process in islets.The online version of this article (doi:10.1007/s00125-015-3837-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users. The normal process of ageing is a complex phenomenon associated with a variety of physiological alterations in the function of cells and organs. These alterations are thought to be major determining risk factors for many traditionally adult-onset diseases, including type 2 diabetes . The proMore recently, epigenetic processes have been suggested to underpin the loss of plasticity of cells that occurs during ageing . DNA metn = 4–10 samples per measurement).All procedures were conducted in accordance with the 1986 British Home Office Animals Act. Each sample was composed of islets isolated from two to four male Wistar rats , which were killed at 3 months or 15 months (±3 days) of age after overnight fasting . Analysis of microarray data was performed using GeneSpring GX 12.1 . Signal intensity levels across arrays were normalised to the 75th percentile, with baseline transformation to the median intensity of all samples. Genes with signal intensities <50 after normalisation were classified as non-expressed. Hierarchical clustering was performed using a Euclidean distance metric and a centroid distance rule. Criteria used for filtering ‘ageing genes’ were: false discovery rate (FDR) <0.05 from an unpaired n = 10 samples per group) was used to synthesise cDNA with the RevertAid H Minus Reverse Transcriptase kit . Quantification of gene expression was performed using the ABI Prism 7900 system  and primers for SYBR Green or TaqMan probes (Applied Biosystems)  to assess whether there was enrichment for genes implicated in particular biological processes (background used: RatRef-12_V1_0_R4_11222119_A). Enriched gene ontology (GO) terms with an FDR < 0.05 were considered significant. These terms were then clustered semantically using the REViGO  server ) was measured by qRT-PCR. We found a strong positive correlation between microarray and qRT-PCR measurements  and 15-month-old (15M) male rats, using Illumina’s Expression BeadChips, which interrogate ~21,900 genes. Information on the preparation and purity of the rat islet samples is summarised in the Using DAVID functional annotation, followed by REViGO filtering see , we founTo test the hypothesis that the mRNA expression changes with age were associated with alterations in DNA methylation levels at promoter regions we used bisulfite MassArray assays  to measuIl33, Cxcl9], glucose homeostasis  and extracellular matrix organisation ) (ESM Table The methylation status of 26 of the loci studied 56.5%) changed with age , consistent with demethylation leading to increased transcription. In contrast, zebularine treatment had no effect on the expression of the remaining four genes  /histochemical staining in sections of young and old islets and performed a semiquantitative analysis Fig. . Strong It is well established that ageing is associated with the functional decline of endocrine organs and therefore a risk factor for metabolic conditions, such as type 2 diabetes, in later life. Little work has been done to define the effects of ageing on the transcriptome of the endocrine pancreas and how epigenetic mechanisms could contribute to these effects. In this study we analysed the dynamics of gene transcription during ageing of the endocrine pancreas and measured promoter DNA methylation at a selected number of loci with age-associated transcriptional changes. We found age-related molecular signatures of ‘inflammageing’ and ‘type 2 diabetes’, with parallel changes in DNA methylation of genes contributing to this effect. We propose that these (epi)genetically regulated ‘signature’ genes represent novel mediators of the age-associated decline in islet-cell function.Ccl2 (encoding chemokine [C-C motif] ligand 2) in their beta cells developed diabetes [Ccl2 being detrimental for endocrine pancreatic function. Our findings are also consistent with studies showing that cells undergoing senescence start secreting a large number of pro-inflammatory cytokines and chemokines, as part of the so-called ‘senescence-associated secretory phenotype’ (SASP) [Igfbp6, Mmp12, Mmp14, Timp2 and Fn1.We identified 3.7% genes within the islet transcriptome that were differentially expressed between young and old islets, by 1.5-fold or more, with the majority (84.3%) showing increases in expression with age. Pathway analysis revealed an enrichment of upregulated genes implicated in inflammation and immune responses , suggestive of age-associated increases in endocrine pancreas inflammation (or inflammageing). Inflammageing, a term used to describe an imbalance between inflammatory and anti-inflammatory networks that causes lifelong antigenic burden and exposure to damaging agents, is thought to be a major driving force for frailty and common age-related pathologies . Our findiabetes ; this is’ (SASP) . We idenENPP1, TSPAN4, TSPAN8, CSRP1, REG3A and REG3G [TSPAN8, KCNQ1 and ITGB6 loci (upregulated with age in rat islets) and some of these polymorphisms have been predicted to affect beta cell function. Other genes expressed differentially with age have been previously implicated in endocrine pancreas function  had similar age-related gene expression changes to the ones reported in a recent meta-analysis of mouse, rat and human tissues (over ten tissues but not including pancreatic islets) [As expected from the nature of this study, we found a number of gene expression ‘signatures’ of the ageing process. Fifteen of the genes upregulated with age ( islets) , 33. The islets) , 33.n = 46), based on the microarray differential expression analysis, with the aim of establishing correlations between DNA promoter methylation and transcriptional activity. Globally, 19 of the 46 loci analysed (41%) showed absolute differences in methylation of between 5% and 28%. Fourteen of the 19 differentially methylated genes (74%) demonstrated a direction of change consistent with differences in expression, suggesting that DNA methylation may contribute to the transcriptional changes. Notably, 13 of these 14 loci reflected hypomethylation with age and were associated with increased expression. These changes were observed at loci related to ‘inflammageing’ and endocrine function. Global analysis of DNA methylation, as measured by methylation levels of LINE-1 repeats, further suggested that hypomethylation might be an epigenetic hallmark of ageing in rat islets. Importantly, a major preponderance for loss of DNA methylation has been observed recently in pancreatic islets of type 2 diabetes patients [Age-related changes in DNA methylation in the endocrine pancreas have not been reported prior to this study. We focused our analysis on a restricted number of loci . Cxcl9 is of particular interest as it is upregulated at both the mRNA  has been shown to increase with age in a number of tissues [Our current study has one important limitation, which is the use of islet cells instead of purified cell types. A clear advantage of using islet cells is that this reflects the physiology of the endocrine pancreas and takes into account the milieu of insulin-secreting cells. However, the transcriptomic and epigenetic data analyses are made more complex given potential confounding effects brought about by morphology and cell-type composition changes with age. In this study, we found clear evidence for cellular senescence, collagen deposition and macrophage infiltration in a significant proportion of aged islets . Associated with these morphological changes, 11 collagen genes were upregulated in our dataset   did not Future studies aimed at assessing the biological roles of the genes highlighted in this study, using in vitro assays and/or in vivo animal models, have the potential to expand our knowledge on type 2 diabetes pathogenesis. It will be important to investigate whether the same mechanisms reported in this study are also observed in aged human islets.ESM Table 1(PDF 22 kb)ESM Table 2(XLS 52 kb)ESM Table 3(PDF 10 kb)ESM Table 4(XLS 146 kb)ESM Table 5(XLS 305 kb)ESM Fig. 1(PDF 100 kb)ESM Fig. 2(PDF 1212 kb)ESM Fig. 3(PDF 280 kb)ESM Fig. 4(PDF 631 kb)


To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer  and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish.Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out  Salmo salar L.) are a major problem of fish farming as cultured fish are kept in open sea cages during ongrowthdead end (dnd), as shown in zebrafish, medaka, loach, goldfish and sturgeon3456dnd gene leads to germ cell-free sterile gonadspiwil1, piwil2 and vasa, which are sufficient to initiate germ cell formation in homozygous mutant fish for these genes, although germ cell loss then occurs at later stages1112dnd when both maternal and zygotic dnd mRNA is lost during early development34dnd mRNA can rescue the survival of germ cells in a dnd KO fish. Loss of germ cells in fish also affects the somatic sex of the gonad differently since loss of PGCs leads to an all-male phenotype in medaka, zebrafish and tilapia while both males and females develop in germ cell-free loach and goldfish34514dnd gene leads to an all-male offspringEscaped Atlantic salmon  pigmentation gene resulted in a range of mosaic phenotypes from the lack of pigmentation in a few cells to completely albino animals. This graded mosaicism is a general problem in F0 KO animals. However, working with F0 animals is often the only feasible approach for functional studies in non model species with a long generation time like the Atlantic salmon.Recently, the CRISPR-Cas9 system for targeted mutagenesis has been implemented in zebrafish using a codon-optimized Cas915171820dnd or both dnd and slc45a2 . Since CRISPR-Cas9 RNAs targeting alb produce a complete albino phenotype when the mutation is double allelic, we hypothesized that this feature could indicate double allelic gene loss following the injection of a second, unrelated CRISPR-RNA into the same embryo. Hence loss of pigmentation in double injected embryos could also indicate a high mutation degree of the second targeted gene. Here we show that albino dnd/alb KO fish were germ cell free in F0. Hence, we could describe for the first time in fish that CRISPR-Cas9-mediated KO of dnd leads to complete loss of germ cells in the F0 generation, also demonstrating that maternal RNA cannot compensate for the zygotic loss of the gene. Finally, we found that germ cells are not required for female sex differentiation in Atlantic salmon but possibly for establishing a normal structure of the somatic compartment in the female gonad.In the present study we carried out double and single KO experiments in Atlantic salmon using CRISPR/Cas9, targeting Xenopus, zebrafish, tilapia and Atlantic salmon, thereby allowing functional characterization after a comparatively short period151718alb and tyralb or tyr produced fish with different degrees of mosaicisms. This feature is a general problem when studying gene function in F0: It is not known to what extent wildtype sequences may rescue the phenotype produced by (incomplete) mutagenesis. In CRISPR-alb individuals many fish completely lacked pigmentation and fin clip analyses confirmed the absence of wild type variants in those, analyzing about 100 clones from PCR productstyr KO also causes loss of pigmentation in F0 but unfortunately, this phenotype is not maintained in juvenile and adult fish (data not shown), making tyr an unsuitable tracer for phenotypic identification of mutants. Based on our previous studies, we hypothesized that CRISPR-Cas9-induced double KO of alb and dnd would exhibit similar efficiencies for both genes. Especially since both genes induced mutations (in whole embryo assays) in 40% of the injected embryos (4 of 10 embryos for each targeted gene). Double and multiple gene knockouts using CRISPR-Cas9 have previously been reported for zebrafish and mouse models23alb and dnd (dnd of 271 fin clips from juvenile fish resulted in 17% (n = 45) fish that carried indel mutations in dnd (dnd gene (dnd KO fish (44/45) also carried mutations in the albino gene. This shows that CRISPR-Cas9-mediated alb KO can be used as a visual tracer for gene editing in salmon, facilitating the phenotypic analysis of mutations in genes with unknown functions. We also attempted single gene mutations for dnd  where 28% (63/222) of injected fish carried indels.Targeted mutagenesis in species with long generation times  is challenging because it takes a long time to produce F2 individuals of a single double allelic mutation. The recently developed CRISPR-Cas9 system efficiently generates double allelic mutations in the F0 generation in many species, such as  and dnd . Furthers in dnd , althougdnd gene  and 98% dnd mutations in finclips. In the 30 fish sampled, 10 were dnd/alb KO with complete albino phenotype, 14 were gene-edited for dnd only, and 6 control fish . These fish could have a mosaic mutation pattern and only some tissues would be depleted of functional dnd. Among those, one of the females had gonads containing germ cells, indicating the absence of dnd mutations in cells responsible for normal germ cell development . For the dnd/alb KO analysis we selected only individuals completely lacking pigmentation since these were more likely to have a double allelic mutation for the alb and thereby also for the dnd. This demonstrates the benefit of using alb KO as a tracer for double allelic mutants in functional studies of candidate genes in Atlantic salmon.From the ten male vasa to confirm absence of these cells in gonads of dnd KO fish by qPCR analyses of whole gonad preparations26vasa, six other tissues in proximity of the gonads were assayed for vasa expression. Results confirmed tissue specificity of vasa in gonads  catalyzes the conversion of testosterone into estrogen and is highly expressed in granulosa cells of the ovary and is thereby a reliable marker of somatic sex of the ovarycyp19a1a in 7 other tissues close to the gonads nor in testis tissue . This gene is highly expressed in the immature testis prior to the onset of gonad maturationamh is highly expressed in immature wild type testes and in the dnd KO male gonad, confirming the male phenotype of the germ cell-free gonad , and one that does not . There is no obvious phylogenetic aspect in this grouping, considering that goldfish and zebrafish are closely related species. Recent work showed that zebrafish have lost a sex-determining region on chromosome 4 during domesticationalb KO phenotype is a reliable tracer for the concomitant KO of genes involved in unrelated processes (100% accuracy in our study using dnd). Importantly, the biallelic KO allows functional studies in F0 of Atlantic salmon, and with high probability also in other species with a long life cycle which usually prohibits generating an F2 generation. We reveal that dnd/alb F0 mutants completely lacking pigmentation are also devoid of germ cells, suggesting that maternally transferred dnd mRNA cannot compensate for the zygotic loss of this gene. In addition, our experiments show that germ cells are not essential for gonadal sex differentiation in Atlantic salmon since the somatic sex was maintained in male and female germ cell free fish. Finally, we saw disorganized, very thin gonads only in germ cell free females while the somatic compartment in sterile males appeared normal, supporting the notion that germ cells exert an organizing effect on the somatic compartment in the salmon ovary but not in the testis.Our study shows that the complete Salmo salar dnd mRNA sequence ). Dnd target site sequences and CRISPR oligos are listed in Table S1. Oligonucleotides for slc45a2  are described in Edvardsen et al.dnd target site was selected in the exon encoding the DNA-binding domain. To avoid off-target affects we searched the whole salmon genome (No. AGKD00000000.4) for binding sites for the oligos selected for the CRISPR target for both dnd and alb, no specific binding was identified for the targets used. Cloning of CRISPR target sequences and preparation of cas9 mRNA and in-vitro transcription was conducted as previously describedin-vitro transcription of gRNA we used the HighScribe T7 High Yield RNA Synthesis Kit (NEB) according to the protocol for short transcripts and purified gRNA via an RNeasy column (Qiagen) using 3.5 vol. of 100% EtOH.The cas9 mRNA. At 8–10 months after fertilization, when fish were around 10–50 g they were tagged intraperitoneally with 8 mm passive inductive transponder-tags  and the adipose fin was clipped as a source for DNA extraction. Visual inspection revealed the number of alb KO fish due to their albino phenotypednd mutations in the fin-clip sample. Fish bearing dnd mutations but no visual albino phenotype were screened for alb mutations as previously publisheddnd KO fish were screened for their sex using double exon sdY PCRdnd KO fish  were sedated and euthanized using 10 mg/l metomidate . Fish were dissected and the whole length of one gonad was immediately frozen in liquid nitrogen for RNA extraction and qPCR analyses. The whole length of the other gonad was fixed in 4% glutaraldehyde for embedding in plastic (Technovit 7100).Injection procedures were carried out as described previouslyet al.dnd indel variations because we expected a high degree of mosaicism in the F0 generation and no wildtype variants in full albino fish due to our previous analyses of alb (slc45a2) and tyr indel variationsTo screen for mutations, 10 embryos from each injection batch were dechorionated and frozen for DNA extraction at the 17 somite stage at approx. 2 weeks of age. DNA extraction from embryos and screening for indel mutations was done as described in Edvardsen For morphological analyses, the whole length of one gonad was fixed in 4% phosphate-buffered glutaraldehyde at 4 °C overnight. Subsequently, the tissue was dehydrated, embedded in Technovit 7100 (Electron Microscopy Sciences), sectioned at 4 μm thickness, and stained with toluidine blue, according to conventional histological procedures.vasa, cyp19a1a and amh Fast Sybr green was used (Applied Biosystems). Primers and probes for the amplicons are listed in elongation factor1α . qPCR was performed on a SDS 7900HT Fast Real-Time PCR system (Applied Biosystems),with the following thermal cycling conditions: 95 °C for 20 sec, and 40 cycles of 95 °C for 1 sec followed by 60 °C for 20 sec. For screening of tissue specific gene expression we used cDNA prepared from tissue samples presented in Kleppe et al. 2015RNA was extracted from the whole length of one gonad using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. Purified RNA was then treated with the turbo DNAfree kit (Ambion) to remove trace amounts of contaminating DNA. The treated RNA served as input for reverse transcription reaction using the VILO cDNA synthesis kit (Invitrogen). 2 μl of a 1/20 dilution of the final cDNA was used in a 6 μl Fast Taqman qPCR reaction (Applied Biosystems) for The statistical tests on qPCR results were performed using GraphPad Prism 5.04 (GraphPad Software Inc). A p-value of ≤0.05 indicates significant difference. Due to the low number of replicate qPCR samples (n = 3–4) none of the samples showed a normal distribution. Therefore, the non parametric Kolmogorov-Smirnov test was applied to calculate significant differences between samples.th of June 2009, in force from 1st of January 2010.All experiments herein have been approved by the Norwegian Animal Research Authority . Welfare and use of these experimental animals was performed in strict accordance with the Norwegian Animal Welfare Act of 19How to cite this article: Wargelius, A. et al.Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon. Sci. Rep.6, 21284; doi: 10.1038/srep21284 (2016).


The molecular classification of human breast tumors has afforded insights into subtype specific biological processes, patient prognosis and response to therapies. However, using current methods roughly one quarter of breast tumors cannot be classified into one or another molecular subtype. To explore the possibility that the unclassifiable samples might comprise one or more novel subtypes we employed a collection of publically available breast tumor datasets with accompanying clinical information to assemble 1,593 transcript profiles: 25% of these samples could not be assigned to one of the current molecular subtypes of breast cancer. All of the unclassifiable samples could be grouped into a new molecular subtype, which we termed “luminal-like”. We also identified the luminal-like subtype in an independent collection of tumor samples (NKI295). We found that patients harboring tumors of the luminal-like subtype have a better prognosis than those with basal-like breast cancer, a similar prognosis to those with ERBB2+, luminal B or claudin-low tumors, but a worse prognosis than patients with luminal A or normal-like breast tumors. Our findings suggest the occurrence of another molecular subtype of breast cancer that accounts for the vast majority of previously unclassifiable breast tumors. The cellular heterogeneity and genetic complexity of breast carcinomas cannot be completely captured using only clinico-pathological tumor characteristics Studies performed using global gene expression profiling introduced molecular classification of breast tumors based on ∼500 ‘intrinsic’ genes known to exhibit high variability of expression between different groups of tumors, but to have a stable level of expression in the same tumor over time and/or after one or another therapy GRB7 located on the 17q ERBB2 amplicon Tumors of the luminal A and luminal B subtypes are distinguished based on their relative expression of the ER, ER-regulated genes, the progesterone receptor (PR) and other genes such as GATA-binding protein 3, X-binding protein and hepatocyte nuclear factor 3 alpha, which are also expressed by normal breast luminal cells All breast tumor samples cannot be assigned to one or another molecular subtype. For example an early study examining 3 independent collections of gene expression profiles of human breast tumors comprising 115, 117 and 49 samples revealed that 35.2%, 25.8% and 6.1% respectively of these specimens could not be assigned to one of the known molecular subtypes Because the unclassifiable breast tumor samples comprise a high fraction of all tumors and could potentially comprise novel molecular subtypes, we investigated means of classifying these samples. To this end we used a collection of publically available gene expression profiles obtained from 1,593 human breast tumor samples. We could not assign 24.9% of these samples to any of the 7 identified molecular subtypes. We subsequently discovered that all of these unclassified samples comprised a new molecular subtype, which we termed “luminal-like”. Luminal-like subtype tumors are characterized by increased interferon alpha/beta signaling, and a decrease in metabolic processes, ERBB2 signaling and integrin cell-surface interactions. Patients with tumors of the luminal-like subtype are predicted to have a better prognosis than those who experienced basal-like breast cancer, a similar prognosis to those with ERBB2+, luminal B, or claudin-low tumors, but a worse prognosis than patients with luminal A and normal-like breast tumors.in silico of 7 publically available datasets, obtained using Affymetrix HG-U133A GeneChip arrays. These profiles were deposited in the Gene Expression Omnibus (GEO)  and comprise a total of 2,027 samples. All the samples are accompanied with clinical follow-up data. However, because some of the datasets were contributed by the same source, there were redundant samples in several datasets. Hence these redundant sample profiles were removed thus reducing the number of unique samples to 1,695. All samples used for our study were normalized with frozen Robust Multi-array Analysis (fRMA), a procedure that allows one to pre-process microarrays individually or in small batches and to then combine the data into a single dataset for further analysis Patient gene expression profiles and accompanying clinical data was publically available and as such their use did not require local ethics board approval. In the course of our study we analyzed the gene expression profiles Samples were classified as basal-like, ERBB2+, luminal A, luminal B, claudin-low, normal-like or apocrine by assigning them to a cluster representing that subtype to a standardized centroid of which they had the highest Spearman rank correlation https://genome.unc.edu/pubsup/clow/UNC337arraydata_imputedCollapsedannotation.txt). Expression profiles obtained from the Farmer study The standardized centroid was computed for each subtype as follows: the average expression of each gene across the subtype was divided by the standard deviation of expression of that gene across that subtype. Reference samples used to calculate standardized centroids for the apocrine subtype were taken from Farmer et al PAM50 http://www.rii.com/publications/2002/default.html. This dataset was combined with the reference samples from Prat et al The reproducibility of clusters 1 and 2 (described in Results), and that of the luminal-like cluster (Results) was examined by using the IGP (In-Group Proportion) metric Because the majority of the samples did not have information about their ER and PR protein status, we established the ER and PR status of samples by using our previously published gene signatures predictive of ER and PR status TFRC (N21329), MYBL2 (AA456878), KIF23 (AA452513), LAPTM4B , GGH (AA455800), FJL10511 (AA115275), YBX1 (AA599175), EBNA1BP2 (T74979), YWHAZ (AA609598), and SQLE (R01118)). Two genes  were not represented on the arrays used in our study; therefore, 9 of 11 genes were used for our analysis. Cluster G comprises 13 genes/hypothetical proteins including GPR160 (H50224), ACADSB (H95792), ESR1 (AA291749), TFF3 (N74131), GATA3 , XBP1 (W90128), FOXA1 (T74639), AFF3 (H99588), LIV1 (H29315), NPNT (AA029948), TUBA1C (N54508), NAT1 (R91802), MYO6 . Two of these genes (GPR160 and NPNT) were not represented by probe sets on the arrays used in our study; therefore, 11 genes were used for further analysis. We used these 20 genes to determine whether the transcript profiles of the unclassified samples could be assigned to the luminal C subtype by performing hierarchical clustering  and examining whether these genes were up-regulated in samples of the luminal-like cluster.Because the raw data used to build a standardized centroid for the luminal C subtype was not available, we used information reported in the study that defined this subtype http://cran.r-project.org/web/packages/survival/index.html). The analysis included the “disease-free” and “overall” survival parameters. Because clinical information supplied with the breast tumor samples did not always include both clinical parameters, 928 and 963 among the 1,593 samples were available for disease-free and overall survival analyses respectively.To perform survival analysis we used the “survival” package  genes that were expressed at their highest level in the luminal-like and another subtype(s) compared to the remaining subtypes; (2) genes that were expressed at their lowest level in the luminal-like and another subtype(s) compared to the remaining subtypes; and (3) genes that were expressed at an intermediate level in the luminal-like and another subtype(s) compared to the remaining subtypes.We selected genes for pathway analysis representative of patterns 1 and 2 described above. A complete list of these genes is shown in As reported previously, subtype assignment is performed by identifying a centroid representing a subtype to which a sample has the highest correlation When attempting to assign molecular subtypes to 1,593 publically available human breast tumor global transcript profiles we found that 397 samples could not be assigned to one or another molecular subtype because they had a low correlation (Spearman rank correlation coefficient <0.3) to the standardized centroids previously established for each of the 7 molecular subtypes  with 710 genes used for the classification of the samples . As can To examine the relatedness of the candidate luminal-like molecular subtype to those identified previously, we performed hierarchical clustering of the standardized centroids for all of the subtypes. Centroids for the established subtypes  and the mApo subtype were computed based on reference samples from Prat We wondered whether the luminal-like subtype was an artifact, resulting from the fact that the luminal A and luminal B standardized centroids do not reflect the variability of all samples that comprise these two subtypes. Hence we tested whether the 397 samples could be classified as either luminal A or luminal B if a correlation coefficient of less than 0.3 was used. As shown in We subsequently compared the maximal correlation coefficients used for the subtype assignment of the 397 luminal-like samples across all subtypes. Only the basal and claudin-low subtypes displayed lower correlation coefficients than did the luminal subtypes to the luminal-like subtype . Hence tA luminal C molecular subtype has previously been reported As shown in Patients who harbored tumors of one or another molecular subtype have different prognoses As shown in The finding that the survival rate of patients with luminal-like tumors is different than that of patients with tumors of some of the other subtypes could represent the fact that had the tumors from patients with a good prognosis not been classified into the luminal-like subtype, then they would have been assigned to either the luminal A or normal-like subtype, whereas tumors from the patients with poor prognosis would have been assigned to either the basal, luminal B or ERBB2+ subtypes. To explore this potential, we examined the prognosis of patients with luminal-like tumors across the subtypes the tumors would have been assigned had they not been assigned to the luminal-like subtype Click here for additional data file.Table S1710 intrinsic genes used for assigning molecular subtypes to 1,593 tumor samples.(XLS)Click here for additional data file.Table S2518 intrinsic genes used for assigning molecular subtypes to tumor samples from NKI cohort.(XLS)Click here for additional data file.Table S3Genes used for pathway analysis. Event identifiers were obtained from Reactome analysis; p-values were adjusted by using Benjamini&Yekutieli method for FDR. Hierarchy of events, when present, was marked by “•”, their number corresponding to the level within the hierarchical tree of events (Pathway Browser at http://www.reactome.org/ReactomeGWT/entrypoint.html).(XLS)Click here for additional data file.Table S4Molecular subtypes of the samples following classification with and without maximal correlation coefficient threshold of 0.3.(XLS)Click here for additional data file.Table S5Genes differentially expressed between Luminal-like and the other subtypes. Differentially expressed genes were found from pair-wise comparisons between Luminal-like and the rest of the molecular subtypes. Total of 11,982 were used for the analyses.(XLS)Click here for additional data file.Table S6Genes differentially expressed between Luminal-like and the other subtypes. Differentially expressed genes were found from pair-wise comparisons between Luminal-like and the rest of the molecular subtypes. Total of 710 genes were used for the analyses.(XLS)Click here for additional data file.Table S7Processes up- and down-regulated in samples belonging to Luminal-like subtype. Event identifiers were obtained from Reactome analysis; p-values were adjusted by using Benjamini&Yekutieli method for FDR (see Methods). Hierarchy of events, when present, was marked by “•”, their number corresponding to the level within the hierarchical tree of events (Pathway Browser at http://www.reactome.org/ReactomeGWT/entrypoint.html).(DOC)Click here for additional data file.


Summary: Seq2pathway is an R/Python wrapper for pathway  analysis of genomic loci, adapted for advances in genome research. Seq2pathway associates the biological significance of genomic loci with their target transcripts and then summarizes the quantified values on the gene-level into pathway scores. It is designed to isolate systematic disturbances and common biological underpinnings from next-generation sequencing (NGS) data. Seq2pathway offers Bioconductor users enhanced capability in discovering collective pathway effects caused by both coding genes and cis-regulation of non-coding elements.Availability and implementation: The package is freely available at http://www.bioconductor.org/packages/release/bioc/html/seq2pathway.html.Contact: xyang2@uchicago.eduSupplementary information:Supplementary data are available at Bioinformatics online. Functional gene-set analysis (fGSA) improves biological interpretability of genomic features with respect to prior knowledge and has led to important biological discoveries , a popular tool for discovering cis-regulatory regions before any subsequent fGSA, links genomic loci to only the nearest genes   to compare the cumulative quantitative effects of genes inside an ontology  with those outside, thus overcoming a number of difficulties in prior fGSA methods .Seq2pathway is designed for knowledge discovery using a variety of NGS data  by taking the contribution of non‐coding loci and their experimental significance scores into consideration. Users can apply the ‘seq2pathway’ function jointly, or in a two-step algorithm consisting of ‘seq2gene’ and ‘gene2pathway’ components separately  . We also provide other three alternative methods: Fisher’s exact test, the Kolmogorov–Smirnov test, and the cumulative rank test .The gene2pathway step integrates several cutting-edge fGSA algorithms, characterized by the improved FAIME method .Significance of biologically-defined loci of genomic aberrations or functional elements has been lacking of attention than their recurrence. Confidence of threshold-based significance is likely to depend on the choice of genomic background and the test statistic; thus, empirical approaches are recommended .Additionally, we collapsed significant scores per gene when a gene was mapped to multiple neighboring genomic loci, removing the bias of linkage disequilibrium for the downstream pathway analysis . This feature essentially allows unbiased comparisons of methods and results.Supplementary Document).To highlight the earlier features, we demonstrate the application of seq2pathway on a variety of different NGS datasets, point out the novel insights obtained only by seq2pathway, and summarize the take home message  < 0.01, count ≥5 and search radius = 5 kb). Both seq2pathway and GREAT identified the biological process GO:0008285 (negative regulation of cell proliferation), while seq2pathway concentrates better with less identified GO terms than the other two methods. Interestingly, the significance of GO:0003705 , a molecular function significantly enriched by bivalent genes marked with both H3K4me3 and H3K27me3 .We first compared seq2pathway with ChIP-Enrich and GREAT to test GO enrichment in a set of H3K27me3 peaks using the same criteria (in utero embryonic development) that was predicted recently (We next tested 460 human height-associated SNP loci and compared the output with the seq2pathway (setting the parameter SNP = T) to the results of GREAT . We applied a search radius = 5 kb for both methods. The valuable biological processes obtained only with seq2pathway include GO:0001701 (recently  in a comWhen we applied seq2pathway to RNA-seq data, individualized gene-set profiles allow the derivation of statistics at the pathway level directly rather than at the gene level. The advantages of seq2pathway lie in noise and dimension reduction, its desired biological interpretability, as well as a platform for novel integrative systems biology analysis (It is a breakthrough in genome analytics coordinates to assign region significance scores such as binding affinity estimates from ChIP-Seq onto a uniform gene-set scale. This feature facilitates the integrative analysis of multiple datasets (


Bovine viral diarrhea virus (BVDV) is desirable in dairy and beef operations to reduce clinical disease and prevent spread of the virus among cattle. Although protection from clinical disease by multivalent, modified-live virus (MLV) vaccines has been demonstrated, the ability of MLV vaccines to prevent viremia and viral shedding in young calves possessing passive immunity is not known. The purpose of this study was to compare the ability of three different MLV vaccines to prevent clinical disease, viremia, and virus shedding in early weaned beef calves possessing maternal immunity that were vaccinated once at 45 days prior to challenge with virulent BVDV 2.Vaccination of young calves against At 45 days following vaccination, calves that received vaccines B and C had significantly higher BVDV 1 and BVDV 2 serum antibody titers compared with control calves. Serum antibody titers for BVDV 1 and BVDV 2 were not significantly different between control calves and calves that received vaccine D. Following BVDV 2 challenge, a higher proportion of control calves and calves that received vaccine D presented viremia and shed virus compared with calves that received vaccines B and C. Rectal temperatures and clinical scores were not significantly different between groups at any time period. Calves that received vaccines B and C had significantly higher mean body weights at BVDV 2 challenge and at the end of the study compared with control calves.Moderate to low maternally-derived BVDV antibody levels protected all calves against severe clinical disease after challenge with virulent BVDV 2. Vaccines B and C induced a greater antibody response to BVDV 1 and BVDV 2, and resulted in reduced viremia and virus shedding in vaccinated calves after challenge indicating a greater efficacy in preventing virus transmission and reducing negative effects of viremia. Bovine viral diarrhea virus (BVDV) is an important cause of respiratory, enteric, and reproductive disease in cattle and has been associated with major economic losses in cattle operations worldwide  was performed using an appropriate distribution function. The repeated nature of this experiment, viz. multiple observations on the same experimental unit = animal over time, implies non-independence of residuals. Hence, the residual variance was modelled to arrive at a reasonable residual covariance structure using Akaike’s Information criterion corrected for small sample sizes (AICc) to determine the best structure. A first-order autoregressive structure with heterogeneous variances (ARH 1) was most commonly fitted. This structure allows for a separate residual variance at each time point and a correlation among time points that diminishes with the lag. Because clinical scores for respiratory distress and diarrhea were binary in nature (only scores 1 and 2 were given) the binary distribution function was used in the abovementioned procedure; this analysis approach is commonly referred to as logistic regression. No analysis was performed for clinical depression score as all scores equalled zero. Dunnett’s test for multiple comparisons was used to detect differences between vaccinated groups and the control group; a probability of P ≤ 0.05 was considered statistically significant for all tests. The FREQ procedure in the abovementioned software package h was used to analyze the proportion of calves with viremia and virus shedding with a χ2 test.All statistical analyses were performed using the GLIMMIX procedure in the SAS 9.3 software package aBovi-Shield Gold 5, Zoetis Animal Health, Florham Park, NJbMicrosoft Excel 2010, Redmond, WAcBRD-Shield, Novartis Animal Health, Larchwood, IAdExpress 5, Boehringer Ingelheim Vetmedica, Ridgefield, Conn.ePowder River Cattle & Livestock Equipment, Provo, UTfTrue-test Inc. Mineral Wells, TXgSAS Institute Inc., Cary, NChPROC FREQ SAS 9.2, SAS Institute Inc., Cary, NC


Arsenic is one of the most commonly encountered environmental toxicants, and research from model systems has suggested that one mode of its toxic activity may be through alterations in DNA methylation. In utero exposure to arsenic can affect fetal, newborn, and infant health, resulting in a range of phenotypic outcomes.This study examined variation in placental DNA methylation and its relationship to arsenic exposure in 343 individuals enrolled in the New Hampshire Birth Cohort Study.Linear regression models using a reference-free correction to account for cellular composition were employed to determine CpG loci affected by arsenic levels.Total arsenic measured in maternal urine during the second trimester was not associated with methylation in the placenta, whereas arsenic levels quantified through maternal toenail collected at birth were associated with methylation at a single CpG locus (p = 4.1 × 10–8). Placenta arsenic levels were associated with 163 differentially methylated loci , with 11 probes within the LYRM2 gene reaching genome-wide significance (p < 10–8). Measurement of LYRM2 mRNA levels indicated that methylation was weakly to moderately correlated with expression . In addition, we identified pathways suggesting changes in placental cell subpopulation proportions associated with arsenic exposure.These data demonstrate the potential for arsenic, even at levels commonly experienced in a U.S. population, to have effects on the DNA methylation status of specific genes in the placenta and thus supports a potentially novel mechanism for arsenic to affect long-term children’s health.Green BB, Karagas MR, Punshon T, Jackson BP, Robbins DJ, Houseman EA, Marsit CJ. 2016. Epigenome-wide assessment of DNA methylation in the placenta and arsenic exposure in the New Hampshire Birth Cohort Study (USA). Environ Health Perspect 124:1253–1260; http://dx.doi.org/10.1289/ehp.1510437 One hundred million individuals worldwide, 13 million of whom reside within the United States, are exposed to arsenic in drinking water at levels above the World Health Organization and U.S. Environmental Protection Agency recommended limit of 10 μg/L . Arsenic+ T cells was described as a function of arsenic exposure , maternal postpartum toenail samples and placenta from 342 individuals with downstream analysis conducted on samples with values above the detectable limit. Arsenic in all three tissues was quantified using inductively coupled plasma dynamic reaction cell–mass spectrometry. The protocol has been described previously . A totalp-values > 0.01 in at least one sample were removed. This resulted in 344,348 autosomal probes from 343 unique samples being included within our analysis.Isolated DNA underwent bisulfite modification using the EZ Methylation kit . Samples of bisulfite-modified DNA were then randomized across several plates and assessed for epigenome-wide DNA methylation levels using the Infinium HumanMethylation450 (450K) Bead Chip  profiling methylation status for approximately 486,000 CpG loci. The microarrays were processed at the Biomedical Genomics Center at the University of Minnesota . Data was assembled using the BeadStudio methylation software package (Illumina) and then processed using the minfi package in R . For ourhttp://bioconductor.org/packages/release/bioc/html/minfi.html). A principal-component analysis (PCA) was then used to determine whether technical aspects of our design (bisulfite conversion plate or microarray slide) might be influencing underlying variation within the data set. We examined the two principal components accounting for the largest degree of variation and found that plate was associated with a significant portion of the variation. To correct this, we adjusted our data for this effect using the “ComBat” method  protocol within the minfi package as specified by the software authors  correction of q < 0.05. All p-values reported are the product of the multiple comparisons correction.Using normalized and ComBat adjusted methylation data, we proceeded to investigate the relationship between placental methylation and measured levels of arsenic exposure. Of the 343 samples within our study, 285, 257, and 271 had available placenta, toenail, and total urinary arsenic measurements, respectively. These subsets were used to determine the relationship between arsenic and methylation. The relationships between methylation and each measure of arsenic were evaluated via epigenome-wide association studies (EWAS) using the R package RefFreeEWAS, which fits a series of linear models and subsequently accounts for cell proportion variability within tissue samples without a reference set . Althougne cells . Using Dp-value for δ, with a null hypothesis of β* = β. We considered all loci with p < 0.10 to have undergone significant cell mixture corrections while including placental arsenic levels within the model. A Cochran–Mantel–Haenszel test was then employed to determine associations between these loci and GO term pathways while accounting for the locus’s relation to a CpG island as well as position relative to a gene. This would then allow us to indirectly determine how exposure to arsenic in utero may be altering cellular composition within the placenta based upon pathways overrepresented among these loci.The RefFreeEWAS method produces an effect estimate for each of the CpG loci investigated and returns results for both a covariate-only–adjusted (β) and a covariate- and cell-mixture–adjusted (β*) model. Those loci exhibiting the large differences between the β and β* estimates would represent those most confounded by differences in cell subpopulation proportions. By linking the genes associated with these highly confounded CpG sites to gene ontology (GO) terms, we can make an indirect assessment of the representation of these genes within specific functions or pathways, and then infer the types of cellular functions and thus types of cells, most likely demonstrating altered subpopulations by arsenic exposure. To decide which loci to include, we calculated the difference (δ) between β* and β for each locus, and used the resulting value and standard error to derive a Several recent papers have investigated the role of arsenic exposure on an epigenome-wide scale. Although none of these reports have examined placenta-specific DNA methylation, the use of an identical or similar platform does allow for some comparisons to be made in the findings. These studies investigated the effects of arsenic on DNA methylation as measured in both maternal  and cordLYRM2, were used for gene expression analysis. Due to sample failure, we were able to collect expression data from 93 individuals. For each sample, cDNA as well as a non-template control were produced using the iScript cDNA master mix  as specified within manufacturer’s instructions and stored at –4°C. mRNA levels were quantified using the iQ SYBER Green Supermix (Bio-Rad) on the CFX Connect Real-Time PCR Detection System (Bio-Rad) per manufacturer’s instructions. Primers specific to LYRM2 were used from the TaqMan Gene Expression Assays kit  following recommended PCR (polymerase chain reaction) conditions. Expression levels were normalized against the housekeeping gene succinate dehydrogenase (SDHA) using TaqMan Gene Expression Assay primers (Life Technologies). We selected SDHA for our housekeeping gene because it has previously been shown to be the most effective for use in placental gene expression studies due to stable expression across samples , or between placenta and urine (r = 0.03), and only a weak correlation between maternal toenail samples and urine (r = 0.19).The characteristics of the 343 mother–infant pairs examined in this analysis are presented in q < 0.05) with placental arsenic concentration (see Excel File Table S1). Of those, 13 attained genome-wide significance following multiple comparisons correction with the more stringent Bonferroni correction method (cg08640609) tracked to calmodulin-binding transcription activator-1 (CAMTA1), 1 (cg01777586) to coiled-coil domain containing-57 (CCDC57), and 11 to LYR-motif containing 2 (LYRM2) (cg01021483), within the 3´UTR of mini-chromosome maintenance complex component 5 (MCM5), was genome-wide significantly  associated with toenail arsenic  . Only 1  arsenic . No loci arsenic . Estimatexposure , and theLYRM2 were in the promoter region within approximately 600 bp of one another. These probes all showed a negative relationship with arsenic, demonstrating a decreased level of methylation as arsenic exposure increased. The methylation β-values displayed a strong positive correlation between each of the eleven sites, allowing downstream analysis to focus on mean methylation levels for these sites in order to reduce the number of comparisons being made  between LYRM2 expression within the placenta and mean promoter methylation that approached (p = 0.058) statistical significance (The 11 probes associated with ing made . In a suificance .p < 0.10) correction based upon the δ estimate, representing the difference in effect estimates of placental arsenic on methylation with and without cell mixture control. A total of 319 GO terms demonstrated significant overrepresentation  among loci that were most affected by cellular subpopulation correction. The top 20 GO terms overrepresented amongst the genes associated with these loci are provided in Due to the use of the RefFreeEWAS methodology, in addition to examining CpG loci with variable methylation most associated with placental arsenic exposure independent of cellular mixture, we can also identify those loci whose effect estimates are changed the most when cell mixture is controlled. Of the 344,348 loci analyzed from the methylation array, 10,596 sites showed substantial  within our data set. Again, the majority of these were from the single In the present study, we evaluated previously reported loci from other arsenic based epigenome-wide studies  using a n = 766) observed a significant relationship between placental arsenic levels and maternal urine or toenail   were comparable with prior subsets examined from within this cohort  , though ctively) . In the LYRM2 were linked to placental arsenic exposure burden, with 11 of these sites maintaining significance after the more conservative Bonferroni multiple tests correction. Further, we provide evidence that the loci identified within the LYRM2 promoter region may influence gene transcription, consistent with the canonical role of promoter DNA methylation on gene transcription potential. Samples with the highest levels of methylation showed a trend toward muted expression levels, whereas samples with low methylation displayed a range of expression, though contained a high proportion of individuals with the highest levels of expression. The variability in the relationship between methylation and expression observed in this data, particularly at the lowest extents of DNA methylation, is expected, because the absence of methylation does not guarantee high expression levels, but instead endows a gene with the potential for expression. In addition, although DNA methylation is a major contributor to regulation of gene expression, there is not always a guaranteed relationship between these two biological measurements. Because of this, it is important to examine the relationship between identified differentially methylated regions and the expression of the associated gene, and to highlight the strength of the nearly significant relationship identified within this study.Using placental tissue from 343 mother–infant pairs, we identified that variation in DNA methylation of 163 CpG loci was statistically associated with arsenic levels at a 5% FDR. Among these 163 sites, 15 within the promoter region of LYRM2 is a member of the LYR-motif–containing family of genes. Although LYRM2 currently lacks a known function within the placenta or other tissues, several members of this gene family have been ascribed an important role in iron–sulfur cluster (ISC) protein production  ZIPClick here for additional data file.