Alteration of gut microbial colonization process may influence susceptibility of the newborn/infant to infectious and chronic disease. Infectious disease risk leads to widespread use of non-prescription antimicrobials in household products such as Triclocarban (TCC), an antimicrobial compound in personal care products. TCC concentrates in and is transferred through the milk to suckling offspring. TCC exposure during gestation and lactation significantly reduced phylogenetic diversity (PD) among exposed dams and neonates. Among dams using weighted UniFrac distances, TCC induced significant dysbiosis of gut microbiota by gestational day (GD) 18, a trend that continued after delivery. Similarly, an overall restructuring of gut microbiota occurred in neonates. By postnatal day (PND) 12, communities separated based on exposure status and became significantly different at PND 16. The ability of TCC to drive microbial dysbiosis warrants future investigation to evaluate the safety of non-prescription antimicrobial use, including TCC, during critical exposure windows. Though it has commonly been estimated that bacteria cells outnumber human cells 10 to 1, more recent estimates suggest that a 1 to 1 ratio may be a more appropriate calculation2At birth, microbial colonization is largely a product of the host environment and is tied to a variety of factors including delivery  and infant feeding mode (breast/formula feeding)68The composition of the gut microflora can have a broad impact on the health of the host; it is well established that prescription antibiotic exposure can disrupt the balance of the intestinal microbiota potentially leading to unintentional side-effects; alteration of the colonization process may influence susceptibility of newborn/infant to infectious disease in the short-term and lead to immune mediated and metabolic disorders later in life3111112Triclocarban  is a high production volume antimicrobial, used in personal care products, at a mass of up to 1.5% in certain brands of bar soaps111516Previously our group reported that TCC concentrates in the breast milk after dietary exposure in a rodent model indicating potential neonatal TCC exposure via lactationAfter quality filtering and removal of any OTU present at less than 0.005% of the total read count, 54 samples comprised of 4,931,803 sequences remained with an average of 91,330 sequences per sample. Neither Shannon’s index nor phylogenetic diversity differed between control and exposed dams prior to TCC exposure at baseline (GD 4). TCC exposure significantly reduced the diversity of microbiota in feces of treated animals compared to controls at 7 days after treatment  and remained different until AD 16 . Repeated measures analysis revealed a significant interaction between time and treatment . Post-hoc analysis demonstrated that a significant time-treatment interaction occurred beginning at GD 11 after which microbial communities in control and treatment dams behaved differently.Increasing microbial community dissimilarity of TCC exposed vs control dams was pronounced at GD11 and maintained over time . MicrobiS24-7 dominated fecal microbiota. While the Bacteroideaceae family was overrepresented in the microbiota of dams exposed to 0.1% w/w TCC. Significant differences at the phylum and family level across time and exposure groups are shown in 2 = 0.87, Adonis p < 0.05). Repeated measures analysis revealed an effect of collection date (R2 = 0.25).Among neonates, Shannon’s index did not differ between the two groups across the study period Given the pattern of diversity shown in Proteobacteria, Firmicutes, and Bacteroidetes regardless of collection date or treatment status  and the fact that the neonatal samples were pooled is noted. Pooling samples reduces variability, but could also reduce the overall sample number and statistical power. However, because TCC was administered through the chow to dams and neonatal exposure occurred through the milk to the whole litter, statistical analysis based on the whole litter instead of using individual neonates within each litter has been proposedEnterobacteriaceae that lower the redox potential allowing for growth of strictly anaerobic bacteriaEnterobacteriaceae dominated in both control and treated groups  in the short-term and chronic disease (i.e. asthma) throughout life. Like prescription antibiotics, we demonstrated the ability of a non-prescription antimicrobial TCC, to induced gut microbial dysbiosis during sensitive exposure windows in a rat model. Collectively, our results add to the growing public concern related to the potential human health impact of non-prescription antimicrobial exposure and should guide regulatory agencies in policy decisions regarding the use of non-prescription antimicrobials in personal care products during critical physiological stages.Oscillations in the gut microbial community structure can occur with exposure to prescription antibiotics leading to dysbiosis of the gut ecosystemTimed-pregnant Sprague Dawley (SD) rats were purchased from Harlan Laboratory . The day after mating was designated as gestational day (GD) 1. Upon arrival, animals were weight ranked and randomly assigned to control or treatment groups (n = 4/group). Animals were housed individually under specified conditions  with ad libitum access to water and commercial Harlan ground 2020X chow or 2020X supplemented with 0.1% w/w TCC  daily from GD 4 until 16 days after delivery. This period was chosen as we demonstrated that TCC could cross the placental barrier and accumulate in the milk of lactating rats21Fecal samples from dams (n = 4/group) and cecum content (n = 3/group) from neonates were collected at designated dates throughout gestation and lactation. Briefly, for fecal collection, an individual dam was removed from the home cage and placed in a clean cage free of bedding. The tail of the rat was lifted to facilitate the discharge of feces. Stainless steel forceps were used to collect fecal pellets immediately after the samples were produced. All tools were autoclaved prior to use and changed between cages. Fecal samples were collected at baseline (GD 4), 7 days post-treatment (GD 11), 14 days post-treatment (GD 18), and 16 days after delivery (AD) corresponding to 34 days after the initiation of exposure regimen. No fecal samples were collected from dams between GD 18 and any days prior to the final day of the study to reduce disturbance prior to the delivery and during lactation.Collection of cecum content from neonates was a terminal procedure. Samples were collected between 8:00 AM and 12:00 PM on the day of sacrifice. At postnatal day (PNDs) 3, 6, 12 and 16, within each group, two female neonates were randomly selected from each dam. Cecum content from each neonate was removed and combined into three pools so that no individual pool contained two neonates born to the same dam. The maternal origin of the composition of each pool was made consistent at each subsequent collection date. In other words, if cecum content from a neonate born to a specific dam was added to a designated pool on PND 3, cecum content from an additional neonate born to the same dam was used to create the same pool on PND 6. Fecal/cecum samples were snap frozen immediately following collection and stored at −80 ° C until analysis.At PND 12, neonatal gastrointestinal tracts (jejunum and cecum) were collected from male neonates with or without TCC exposure during lactation and fixed in 10% formalin. Tissue sections were examined with hematoxylin-eosin (H&E) staining. A board-certified pathologist, blinded to treatment status, evaluated histological changes.et al.DNA was extracted from frozen fecal/cecum samples with the Power fecal DNA isolation kit  following manufacturer’s instructions. Extracted DNA samples were quantified with Nanodrop 1000™ and stored at −80 °C until PCR amplification. DNA was amplified by targeting the V4 region of the bacterial 16S rRNA gene as described by Caporaso The initial PCR product was purified with DNA gel electrophoresis to remove DNA impurities and primer dimers. The concentration of purified amplicon product was measured with Qubit dsDNA HS Assay Kit  and normalized to an equal concentration to create a single amplicon pool.Pooled amplicons were purified with SPRIselect  following the manufacturer’s protocol . The products were analyzed with Agilent High Sensitivity DNA Analysis (CHIP) Kit for quality assurance on a 2100 Bioanalyzer .The pooled amplicon library concentration was quantified with the Illumina Library Quantification kit  prior to sequencing. Quantitative® FAST qPCR Master Mix (2X). The amplicon library was diluted to a starting concentration of 10 nM and sequenced on the Illumina MiSeq sequencer .PCR was performed with the KAPA SYBRThe resulting raw sequencing data was analyzed using the QIIME (v1.8.0) pipeline404346Statistical analysis was conducted in R (version 3.1.2) using Phyloseq unless otherwise noted50How to cite this article: Kennedy, R. C. et al. Temporal Development of Gut Microbiota in Triclocarban Exposed Pregnant and Neonatal Rats. Sci. Rep.6, 33430; doi: 10.1038/srep33430 (2016).


Donor organ quality affects long term outcome after renal transplantation. A variety of prognostic molecular markers is available, yet their validity often remains undetermined. A network-based molecular model reflecting donor kidney status based on transcriptomics data and molecular features reported in scientific literature to be associated with chronic allograft nephropathy was created. Significantly enriched biological processes were identified and representative markers were selected. An independent kidney pre-implantation transcriptomics dataset of 76 organs was used to predict estimated glomerular filtration rate (eGFR) values twelve months after transplantation using available clinical data and marker expression values. The best-performing regression model solely based on the clinical parameters donor age, donor gender, and recipient gender explained 17% of variance in post-transplant eGFR values. The five molecular markers EGF, CD2BP2, RALBP1, SF3B1, and DDX19B representing key molecular processes of the constructed renal donor organ status molecular model in addition to the clinical parameters significantly improved model performance  explaining around 33% of the variability of eGFR values twelve months after transplantation. Collectively, molecular markers reflecting donor organ status significantly add to prediction of post-transplant renal function when added to the clinical parameters donor age and gender. As the pathophysiology of this process is probably complex and hitherto only incompletely understood, prognostic markers available lack sensitivity and/or specificity, and treatments applied are often not successful2. Next to postoperative and procedure related complications like cold ischemia time, infections, rejection episodes, or the toxicity of immunosuppressive therapy, the “quality” of the donor organ has often also been associated with mid- to long term transplant survival. A proof of concept being the fact that living donation provides superior results when compared to deceased donor transplantation1. Unfortunately especially for deceased donors the exact procedure how to optimally describe “organ quality” is still under discussion and this uncertainty has important clinical consequences. Rejection of allocated organs by transplant centres based on opinion, rather than evidence leads to an unjustified discard of a scarce resource3. Kidney transplant recipients, who suffer from allograft failure after transplantation, on the other hand show even greater morbidity and mortality than patients on dialysis4. These caveats have fostered efforts for matching donor organ quality to recipient characteristics, but this approach needs high quality data for modelling. Donor organ quality description currently follows two concepts, one using clinical information available at the time of the offer. As an example the kidney donor profile index (KDPI)5, which is used for organ allocation in the US, includes among other variables donor age and the last serum creatinine. Recently van Balkom et al. studied the proteomic signature of the preservation fluid to derive biomarkers to predict immediate postoperative transplant function6. Another intuitively superior way is to use information obtained from pre-implantation biopsies. Next to unresolved technical issues  it has recently been shown that conventional histological workup does provide reliable information in some7 but not all situations8. Researchers have started to look into molecular signatures in the biopsy tissue that predict immediate, but also mid- to long term post-transplant renal function16. Some of these studies are hypothesis driven. Donor age for example is an often used  surrogate for organ quality. Koppelstätter and colleagues extended this concept by showing that markers of biological age in the biopsy (telomere length and expression of cell cycle inhibitors) better than chronological donor age predict post-transplant renal function as assessed by serum creatinine one year after transplantation17. Günther and colleagues very recently could show that the activating cytotoxicity receptor NKG2D was associated with chronological age and was indicative for post-transplant outcome18. However age is only one factor in a likely complex molecular interaction phenotype that determines organ quality in a way that it affects transplant outcome. Hypothesis driven approaches looking into single markers or pathways therefore are most likely inappropriate, a problem that could be solved by studying marker combinations or panels. However, as long as the molecular pathophysiology of chronic allograft failure is not completely understood unbiased approaches like whole-genome expression data sets obtained from pre-implantation biopsies are an important data source. Strategies on the identification of biomarkers making use of computational modelling and data integration approaches seem reasonable in this context and were elegantly summarized by Wang and Sarwal19. Recently O’Connel and colleagues published a study on the use of transcriptomic profiling using tissue obtained from protocol biopsies taken three months after transplantation to predict long term outcome20.Short-term renal allograft survival increased continuously during the last decades but the rate, at which transplants are lost long term remained disappointingly stable at a high levelIn this study we used a data integration approach to build a molecular model reflecting pre-implantation donor organ status. This model was based on features associated with mid- to long term allograft function from published hypothesis driven research articles and from transcriptomics data sets. Bioinformatics network analysis reduced the number of genes by focusing on those, which are more likely important features in interaction networks. A major limitation of published data on biomarkers in this context is that they are usually not validated in external cohorts. We therefore finally tested our in-silico derived markers in an independent group of renal allograft recipients, in whom gene profiling data were available from pre-implantation biopsies.Figure A set of 548 molecular features associated with renal function 12 months after transplantation could be derived based on molecular feature sets from LIT-CAN, TX-PERCO, TX-KAINZ, and TX-SCIAN. A brief description of datasets used in the analysis is listed in Table LIT-CAN consisted of eight genes, the transcriptomics datasets TX-PERCO, TX-KAINZ, and TX-SCIAN held 260, 46, and 259 molecular features respectively. 391 of the molecular features shared at least one protein interaction to another member of the combined set thus forming the induced subgraph with omicsNET as the underlying biological network. Eleven clusters of highly interconnected proteins were identified by the MCODE algorithm with 89 proteins assigned to these eleven clusters. A set of 34 GO biological processes could be identified as being enriched based on the set of 89 proteins being part of the donor organ status molecular model. The top-ranked GO biological processes were the neurotrophin TRK receptor signaling pathway (FDR < 0.001), the Fc-epsilon receptor signaling pathway (FDR < 0.001), the epidermal growth factor receptor signaling pathway (FDR < 0.001), as well as the fibroblast growth factor receptor signaling pathway (FDR = 0.0027).2 ranging from 6.38 ml/min/1.73 m2 to 100.74 ml/min/1.73 m2.Average donor age in our cohort was 54 years ranging from 18 to 86. Donor age was significantly correlated to recipient age  see Fig. . The ratThe best performing linear model solely holding clinical parameters consisted of donor age, donor gender, and recipient gender and explained about 17% of variance of recipient eGFR values twelve months after transplantation .The best performing linear model holding both clinical parameters and molecular markers consisted of donor age and gender along with the expression levels of the markers EGF , CD2BP2 (CD2 cytoplasmic tail binding protein 2), RALBP1 , SF3B1 (splicing factor 3b subunit 1), and DDX19B (DEAD-box helicase 19B). This set of parameters explained around 33% of post-transplant eGFR values  thus significantly outperforming the statistical model only holding clinical parameters following analysis of variance (ANOVA) . Detailed model characteristics of the statistical models are given in Table Significant correlations between predictor variables were identified for the molecular markers CD2BP2 and DDX19B , SF3B1 and RALBP1 , as well as RALBP1 and DDX19B . As the maximum absolute Pearson correlation was not above 0.5, inclusion of selected molecular markers into one regression model was justifiable. The full correlation matrix of continuous variables is given in Fig. 2 value of the model only holding clinical parameters increased from 0.17 to 0.28  and the adjusted R2 value of the best performing model based on molecular markers further increased from 0.33 to 0.42 . No significant association of the five markers with either delayed graft function nor rejection episodes could be found in our dataset.Whereas the add-on information on rejection episodes did not improve model performance, knowledge on the occurrence of delayed graft function significantly improved both models. The adjusted RIn this study we used a systematic data integration approach to develop a molecular model of highly interconnected proteins being associated with renal function as assessed by eGFR 12 months after transplantation. Our in-silico approach reduced the number of genes of interest and in addition allowed to focus on features with a high interaction potential thus more likely reflecting core pathophysiological processes. As the genes reported in the literature and used in our modelling approach had not been validated in independent datasets we performed an additional study with this focus. Our final prediction model, holding the five selected molecular markers EGF, CD2BP2, RALBP1, SF3B1, and DDX19B on top of two clinical parameters significantly outperformed a model only based on clinical parameters. No significant correlations between molecular markers and donor age could be detected indicating that molecular markers hold complementary information to donor age. Information on delayed graft function significantly added to predictive power whereas information on rejection episodes did not further add in explaining variability of eGFR values one year after transplantation. Rejection episodes have been reported to be associated with renal outcome in the past. This is however mostly confined to rejection episodes not responsive to steroid bolus therapy, which were not observed in our cohort. The fact that rejections were rare (n = 9) and usually mild may very well be due to the fact, that no highly immunized subjects were included and subjects suffering irreversible rejection leading to graft loss within the follow up period were excluded.21. The authors could further demonstrate that intrarenal EGF values are highly correlated to urinary EGF concentrations. A positive regulatory effect of EGF in progressive kidney disease has also been previously discussed by Rudnicki and colleagues22. This finding could be confirmed by a study from Betz and colleagues investigating EGF levels in a diabetic nephropathy animal model as well as in a cohort of normoalbuminuric type 2 diabetes patients23. EGF values were also found to be downregulated in donor kidney biopsies showing reduced eGFR values after transplantation in the TX-SCIAN dataset that entered our molecular model generation workflow16. Dosanjh and colleagues showed that members of both the hepatocyte growth factor (HGF) as well as the EGF signaling pathway are differentially expressed in renal allografts with chronic allograft nephropathy and interstitial fibrosis as well as tubular atrophy24.Higher EGF levels were associated with higher post-transplant eGFR levels and thus with better renal function in our dataset. This is in-line with recent findings that EGF was downregulated in progressive DNet al. collected biopsies from allograft recipients with stable renal function three months after transplantation and identified a set of 13 genes that were predictive of the development of fibrosis at one year. Although this final set did not contain EGF the latter was found to be negatively associated with the chronic allograft damage index three months after transplantation, which is in line with our findings20.O’Connel 25.Surprisingly and contradictory to all other studies Rintala and colleagues demonstrated that inhibition of the EGF signalling cascade with the EGFR inhibitor erlotinib prevented chronic rejection and led to improved allograft function as their reasoning had been that EGF in part mediated chronic allograft nephropathy26. CD2BP2 was however also very recently shown to be an important factor for podocyte function. Depletion of CD2BP2 in podocytes led to proteinuria and ultimately kidney function decline in a mouse model as reported by Albert and colleagues27.As with EGF, also higher levels of CD2BP2 were associated with higher post-transplant eGFR levels indicating that reduced expression of CD2BP2 is detrimental to renal function. CD2BP2 was initially described as factor involved in T cell activation and enhancer of interleukin 2 expression28. Enhanced expression of RALBP1 was also found to enhance cisplatin-induced nephrotoxic acute kidney injury in an in-vitro model by inducing necroptosis of cultured tubular cells29. Higher RALBP1 expression levels were also detected in an animal model of cisplatin-induced nephrotoxicity with inhibitors of necroptosis providing protection from kidney injury in this mouse model30.Higher expression of RALBP1, also known as RIP1 or receptor-interacting protein 1, in our dataset as indicated by the negative parameter estimate in the statistical model were associated with lower eGFR values post-transplant and thus reduced kidney function. RALBP1 was reported as inducer of necroptosis, a novel form of cell death, which was also linked to myocardial, renal, and cerebral ischemia-reperfusion injury31. SF3B1 was also reported as a direct target of the hypoxia inducible factor 1 alpha (HIF1A) containing a hypoxia response element in the promoter region when investigated in an animal model of cardiac hypertrophy32. No information on involvement in pathophysiological renal processes could be identified in scientific literature for DDX19B being involved in mRNA transport from the nucleus. This molecule might serve as sensor of deregulated transcriptional activity in deceased donor organs after brain death. Of note that DDX19B was the least significant parameter in the constructed regression model with a p-value of 0.0449. Excluding DDX19B from the model led to a drop of the adjusted R2 from 0.33 to 0.30 for overall model performance. Removal of either one of the other molecular markers on the contrary led to much steeper drops of adjusted R2 values to 0.26 down to 0.22 for overall model performance.SF3B1 encodes for a subunit of the splicing factor 3b,being involved in RNA splicing and gene expression, which was previously reported to be detectable in exosomes of transformed Madin-Darby canine kidney cells with the potential of inducing epithelial to mesenchymal transition14, while others dealt with expression patterns in the recipient after transplantation34. We identified three studies reporting on transcriptional changes in the donor organ being associated with long term transplant outcome16. Based on these transcripts complemented by genes reported in scientific literature we constructed a network-based molecular model or organ damage and validated a set of five markers in an independent dataset in the present study. Especially the mechanistic roles of EGF, CD2BP2, and RALBP1 in kidney tissue might hold the potential to also serve as targets for therapeutic intervention.Most transcriptomics studies dealing with expression profiles and outcome in the renal donor setting focused on short term outcome such as delayed graft function35. Delayed graft function indeed improved model performance significantly indicating that delayed graft function holds information being complementary to the information of our selected markers. Rejection episodes interestingly did not add to prediction in our dataset with nine patients having biopsy-proven rejections in our cohort.As we were primarily interested in prediction models holding parameters known at the time of transplantation, we deliberately did not include post-operative parameters in the first place. We however evaluated the impact of the two post-operative parameters rejection episodes as well as delayed graft function on performance of our generated prediction models. Delayed graft function is a known predictor of long term graft function which we also observed in our cohortA limitation of our study is that no functional experiments were conducted thus further exploring the mechanistic role of the validated markers in renal tissue damage which however was not scope of this work.In summary renal transplant donor organ expression of the five molecular markers EGF, CD2BP2, RALBP1, SF3B1, and DDX19B significantly adds in predicting post-transplant renal function when added to the clinical parameters donor age and gender.kidney transplantation [mh] AND  NOT review [publication type]. Titles and abstracts of 242 publications were screened for gene expression studies reporting on human deceased donor kidney biopsy workup with a focus on investigating correlations of gene expression to renal function 12 months after transplantation (either based on serum creatinine or eGFR). Three studies were identified as relevant and molecular features were extracted from these publications. These three studies were the only ones using an unbiased gene expression profiling approach in donor kidney biopsies and were thus considered for further analysis in the current study. From the studies by Kainz et al.15 [TX-KAINZ] as well as Scian et al.16 [TX-SCIAN] the reported sets of transcripts were used as input for molecular model generation. The expression dataset published by Perco and colleagues [TX-PERCO] was re-analysed with respect to correlation of transcripts to twelve months post-transplant eGFR values9. P-values of the correlation analysis were corrected with the R package fdrtool, setting the false discovery rate to <5%36. All transcripts were mapped to their respective genes using the Ensembl Gene ID as common denominator. All datasets used in the integrative analysis of this work are listed in Table Transcriptomics studies were identified in Pubmed using the following query: “chronic allograft nephropathy” OR “chronic allograft dysfunction” AND humans NOT review [publication type] resulted in 1075 publications. Publications mentioning at least one gene  in title or abstract were manually reviewed in order to extract deregulated genes in the donor organ having impact on post-transplant renal function. Next, all molecular features of donor organ status affecting outcome identified above were mapped on a hybrid interaction network including protein-protein interaction data from IntAct, BioGrid, and Reactome together with computationally inferred relations37. The organ status-transplant outcome specific molecular interaction subgraph was extracted from the network discarding molecular features not being connected to at least one other member from the signature. This subgraph was forwarded to the Molecular Complex Detection algorithm for identifying clusters of nodes, in the following denoted as molecular processes39. These processes are characterised by an accumulation of genes with a high level of interaction and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 was used to identify significantly enriched Gene Ontology (GO) biological process terms in these processes reflecting donor organ status40. This in silico workflow, which was developed within the European Framework 7 project SYSKID43, reduced the number of genes derived from the literature from 548 to 89. Additionally the genes remaining for validation are part of molecular processes characterized by a high level of interaction and thus most likely hold information on relevant pathophysiology.The set of transcripts from the omics studies was complemented by a set of molecular features reported in scientific literature described in the donor organ and having impact on medium-term graft function [LIT-CAN]. The Pubmed query 44. The study was approved by the Ethics Board of the Medical University Innsbruck. Details on the population are provided in Table Wedge biopsies from 78 deceased donor kidneys were obtained prior to reperfusion. Biopsy workup, RNA extraction and hybridization to Agilent Human Gene Expression 4x44 oligonucleotide microarrays was performed as described in a previous publicationThe Agi4x44PreProcess R module was used for gene expression analysis. For 27 of the 78 samples, the Agilent chip version 2 was used. Raw gene expression values obtained from the two chip versions were combined into a joined dataset using shared probe identifiers. Quantile-quantile normalization was used to normalize raw mean intensity values. Summarization of probes was successively performed on the level of the human Gene Symbol. Two samples had to be excluded from further analysis based on hierarchical cluster analysis resulting in an analysis dataset of 76 samples. Gene expression data were available for 74 of the 89 molecular features in the donor organ status molecular model described above thus qualifying for inclusion in successive multiple regression analysis.2 values, leave-one-out cross validated (LOOCV) R2 values and LOOCV root-mean-square-error values.Linear regression models were derived using all clinical parameters listed in Table The clinical parameters together with all molecular features of the donor organ status molecular model were used as input for delineating improved multiple regression models for predicting eGFR values twelve months after transplantation. Simulated annealing was used to identify models holding between six and eight parameters minimizing LOOCV root-mean-square error. Individual non-significant parameters were removed in a post-processing step in order to achieve statistical significance for all single parameters in the final model. The best performing regression model was compared against the regression model only holding clinical parameters using ANOVA.Pairwise Pearson correlation coefficients were determined between molecular markers of the best performing regression model and all continuous clinical parameters listed in Table The primary aim of our study was to derive prediction models based solely on data available at the time of transplantation. In addition we were interested to decipher if postoperative complications like delayed graft function or acute rejection episodes have an impact on identified associations. We therefore analysed the clinical course of the study population with regard to the need for postoperative dialysis as an indication for delayed graft function  or the incidence of biopsy-proven acute rejections (regardless of Banff categorization). We first tested whether the molecular marker panel was capable of predicting these postoperative events and successively also determined the add-on value of postoperative parameters on predicting eGFR values one year after transplantation.The study protocol and the use of surplus zero hour biopsy tissue after routine workup was approved by the local IRB. As no living donors were included no informed consent from the organ donors could be obtained.Raw gene expression data used in this study are available at NCBI Gene Expression Omnibus with the accession number GSE95026.


Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system. No single technology can adequately capture all forms of structural variations to provide accurate and contiguous genome assembly3. Despite the enormous progress in NGS techniques, SVs remain difficult to detect reliably due to the short read lengths3. In addition, NGS sequencing platforms rely on the alignment of millions of short sequence reads4, thus NGS results require careful examination, particularly when investigating long-range genetic information5. Single-molecule DNA optical mapping, pioneered in mid-1990s by the Schwartz laboratory7, was first proposed as a powerful tool for genome sequence assembly in 19998. Since then optical mapping has developed rapidly. It is nowadays used to improve the assembly of fragmented genome sequences that cannot be resolved by NGS methods9, as optical maps provide long-range scaffolds onto which short NGS reads can be assembled10. Thus, the long-range genome sequence information is preserved, enabling direct investigation of structural variants in sub- to 2 megabasepair-long single DNA molecules with a resolution of 1–5 kilobasepairs12. One important parameter in optical mapping is the degree of stretching of the DNA molecule. While high throughput methods exist, they either suffer from uneven or incomplete stretching (<85% of the contour length of the DNA molecule), compromising the optical mapping sensitivity. Stretching above 90% provides a sufficient resolution for single molecule sensitivity of the optical mapping. Such stretching can be achieved by manipulation of individual molecules16 but this lowers throughput. Methods that can enrich the regions of interest might circumvent the need for high throughput.Structural variants (SVs) are genomic rearrangements that may lead to phenotypic diversity and human diseases17. Target enrichment is a more cost-effective method compared to whole genome sequencing, as many genes can be sequenced simultaneously with higher coverage and deeper sequencing across many samples20. Customized sequencing of targeted genes opens new perspectives for implementing NGS as a routine tool in medical diagnostics laboratories, as it requires significantly less input material and offers straightforward data analysis21.Methods of target enrichment, also termed reduced representation, are commonly used in NGS workflows to enrich for subsets of the genome before sequencing23 could be combined with subsequent optical mapping, since fragmentation of the target sequence to less than 5 kilobasepairs (kb) pieces is a key step for preparing DNA for any NGS analysis24.None of the existing target enrichment methods, commonly used in NGS workflows25. In order to isolate intact, very long chromosomal DNA molecules, cell nuclei are embedded in agarose plugs, and digested with the rarely cutting restriction endonuclease NotI, followed by size selection of the DNA fragments by pulsed-field gel electrophoresis (PFGE), which enables separation of large DNA molecules up to 10 Mb in size26. Molecules, recovered from the PFGE gel, were analysed by NGS. Size selected DNA from the same PFGE gels were analysed by single-molecule optical mapping to demonstrate that the method indeed produced long, intact DNA molecules from regions specified by NotI-cutting. Figure We hereby enrich for megabase-sized single DNA molecules from specified human genomic regions for optical mapping in nanodevices and NGS. Genomic DNA molecules longer than 0.5 megabase (Mb) are fragile and shear easily when stirred or pipetted(i) there is no need for site-specific labelling, and (ii) multifold coverage of the genome and construction of consensus maps are not required because the probability of aligning a DR map from a single 100 kbp molecule to an incorrect position in the human reference genome is negligible11. Single DNA molecules with DR patterns of bright (GC-rich) and dark (AT-rich) regions are loaded into the nanofluidic chip, stretched up to 98% of their contour length by a cross-flow, and imaged , which linearize nicked and fluorescently labelled genomic DNA molecules in nanochannel arrays30. Our enrichment method increases the efficiency of DR mapping and thus lowers the need for high throughput.Here we applied a DNA optical mapping technique called denaturation-renaturation (DR) mapping (Supplementary Information) that has some unique advantages compared to other optical mapping techniques, as ged Fig. . The blaged Fig.  resembleNocardia otitidis-caviarum with recognition sequence 5′-GC^GGCCGC-3′ has been reported to cut only upon recognition of two unmethylated CpG sites within the sequence (5′-GC^GGCCGC-3′)31. From the human reference genome and the methylation map, we define a NotI fragment as a genomic region between two NotI recognition sites with two unmethylated CpG sites. IMR-90 female human fetal lung fibroblasts was used in this study, since the single-base methylation data is available for this cell line in the UCSC Genome Browser. Thus the theoretical prediction of NotI cutting was performed taking the impact of methylation into consideration32. Theoretical NotI fragments longer than 1 Mb is 13.5% of the total number of fragments , where l is the DNA length, l0 = 3 Mb is a characteristic length scale, and the prefactor is d0 = 44 mm. Inverting this expression, we find that DNA molecules that have travelled a distance d have a length l = −l0ln(d/d0) for d < 44 mm. A plot of this expression is shown in Fig. Genomic DNA extracted from IMR-90 cells was digested with NotI within agarose gel plugs and separated according to size by pulsed-field gel electrophoresis Fig. . DNA fro33, as molecules do not remain fully stretched after the electrical field is switched off34. In total, 606 DNA molecules were extracted from the PFGE samples, captured, and imaged in the nanofluidic chip. All of the DR maps were then compared with an in silico theoretical DR map extrapolated from the sequence of the hg19 human reference genome, and 566 DR maps could be aligned unambiguously to specific positions in the human genome. The numbers of molecules both analysed and aligned are an order of magnitude higher than in previous studies . Moreover, 190 genomic regions were covered by more than one DR map due to the enrichment protocol described in this study . We conclude that the enrichment procedure gives an enhanced fraction of DR maps of NotI fragments of the specified sizes, and that the enrichment method indeed produces megabase-long DNA molecules after recovery from the gel.Of the 566 DR maps from individual molecules that were successfully aligned to the hg19 human genome assembly, 468 molecules were extracted from the double-fractions 4–5, 51 molecules were from fractions 6–7, and 47 molecules were from fractions 8–9. The locations of these molecules were then compared with the positions of the different groups of NotI fragments. As DR maps are on average only 0.7 Mb long, a DR map is defined as overlapping with a NotI fragment if the center of the DR map is on the NotI fragment. So here a DR map can only overlap with a single NotI fragment. Results are illustrated in Fig. 38. This figure agrees well with our detection of 40 out of 606 (6.6%) unalignable molecules. Especially interesting molecules would be those that border an annotated with an unannotated region, since it would provide a specific localization to the unannotated part. Indeed, we encountered one molecule, M1, which overlapped partly with one of the few annotated centromeric regions, on chromosome 8 39 we calculated the fraction of closed basepairs (helicity) versus temperature inside the centromeric region and in two neighbouring regions of the same size  preparation and handling of megabase-long DNA molecules, (ii) size-selection of megabase DNA molecules from NotI digested human genomes by PFGE, and (iii) subsequent recovery of the molecules from the gel without breakage. This enrichment method is the first attempt to analyse non-randomly selected single molecules from human cells by denaturation mapping in nanodevices. We did a comparison of the theoretical predictions for NotI cut DNA from the PFGE experiment, the NGS sequencing results, and the positions of the localized DR maps to validate the enrichment procedure. The analysis showed that we can indeed enrich for megabase-long molecules from specified regions in the genome. A key point in the method is the two-step preparation of the gel used for PFGE, where we include a low melting point agarose that enables further recovery of long DNA molecules from the gel. The study presents detailed NGS data of a global PFGE separation map of NotI digested human genomic DNA from IMR-90 cells. As we observed that some genomic regions were not separated according to the theoretical prediction, NGS can help to specify where the region of interest is located on a PFGE lane.43. For example, Teague et al.43 described the analysis of SVs in four human genomes by using shotgun single-molecule restriction optical mapping and presented an impressive number of 4,205 of previously unreported SVs. However, as their analysis was based on 10 kb long fragments, the assembly of many single-molecule maps at each locus of a consensus map was required. In our method, multifold coverage of the genome and construction of consensus maps are not required because the probability of aligning a DR map from a single 100 kbp molecule to an incorrect position in the human reference genome is negligible11.Single-molecule DNA optical mapping that enables assembly of genomes and detection of SVs was presented previously by other groups28. This number can be further improved, as automation of single DNA molecules capturing and visualization in a nanofluidic device has been demonstrated44. The throughput of the flow-stretch device is still limited compared to the commercially available Irys System (BioNano Genomics), which is based on stretching the DNA through confinement in nanochannels, and allows imaging of several gigabases of DNA per hour, but at a somewhat lower stretching. The enrichment method presented here could, however, potentially be improved by applying various restriction enzymes or other genomic editing tools such as the CRISPR/Cas9 system45. Moreover, we have demonstrated recently that cheap, disposable polymer devices (3 USD per device) can be used for single-molecule optical mapping. The high-yield production potential allowing several thousands of chips to be produced within a few days46 will greatly facilitate future optical mapping studies of megabase-size DNA molecules. Additionally, in contrast to many other optical mapping methods, there is a possibility to recover DNA molecules for further analysis11. The enrichment method presented here can also be used for sample preparation for commercial optical mapping platforms. Limiting the input DNA to specific genomic regions decreases the time and effort needed for mapping and data analysis.The new design of our previous nanofluidic device allowed capturing of DNA molecules as short as 0.7 Mb, which significantly increased the number of imaged molecules compared with previously published reports47, many of which are evolutionarily conserved since they contain arrays of long-range cis-regulatory sequences that regulate key developmental genes which can be located more than 1 Mb away51. The disruption of such cis-regulatory domains by structural chromosomal rearrangements have been associated with long-range position effects in a number of human disorders and mouse models53. 112 out of 168 evolutionarily stable gene deserts50 overlap more than 80% with NotI fragments longer than 1.5 Mb. This is exemplified by a stable gene desert on chromosome 12 covered by four DR-maps from the combined fractions 4 and 5, and by NGS reads from fraction 4  in agarose plugs was based on a previously published method55 with some modifications described in the Supplementary Information.Preparation of cell nuclei from IMR-90 female human fetal lung fibroblasts (ATCCThe two-step gel preparation was developed for pulsed-field gel electrophoresis (Supplementary Information). The DNA in plugs was digested with NotI enzyme (NEB) for 4 hours at 37 °C (50 units of enzyme per plug). Pulsed-field gel electrophoresis was performed on 1% UltraPure™ Low Melting Point Agarose (Invitrogen) at 11 °C for 120 hours in 0.5xTBE buffer with following parameters: voltage, 50 V linear to 45 V; angle, from 110° linear to 100°; interval, from 5000 sec logarithmic to 1000 sec. The gel was stained with SYBR® Safe DNA Gel Stain (Invitrogen) and visualized using a Syngene transilluminator. PFGE lanes were sliced into 2 mm- or 4 mm wide agarose fractions for NGS or single-molecule denaturation mapping, respectively.56 and aligned to the human reference genome hg19 with bwa57. Alignments were generated in BAM format and uploaded to the SeqMonk software (http://www.bioinformatics.bbsrc.ac.uk/projects/seqmonk/). The DataStore summary report from SeqMonk provides information about the analysed data stores  and sequenced on the Ion Proton™ semiconductor sequencer (Life Technologies). Each chip was run with six samples. Reads from the Ion Proton sequencing were trimmed for adapter-sequences using cutadapt28. Compared to the previous device design28, the present device has been modified so that the slit length is shorter (200 µm) and the cross-shaped slit is narrower at its centre  and partially denaturated to produce the DR maps (Supplementary Information). Basic design and working principle of the device used in the experiment is described elsewhere60. Theoretical prediction of NotI digestion was performed in silico by use of Matlab scripts developed in-house.Single-base resolution DNA methylation data for IMR90 was downloaded from the UCSC Genome Browser (track name: Human_IMR90_Meth) by use of the UCSC Table Browser data retrieval toolSupplementary Information


NOD2 and TNF-α, dysregulated expression in let-7 and miR-124 were associated with paediatric IBD, but not as yet replicated. Highly credible SNPs associated with paediatric IBD have also been implicated in adult IBD, with similar magnitudes of associations. Early onset and distinct phenotypic features of paediatric IBD might be due to distinct epigenetic changes, but these findings need to be replicated. Further progress identifying genetic and epigenetic susceptibility of paediatric IBD will require international collaboration, population diversity and harmonization of protocols.We provide a comprehensive field synopsis of genetic and epigenetic associations for paediatric Inflammatory Bowel Disease (IBD). A systematic review was performed and included 84 genetic association studies reporting data for 183 polymorphisms in 71 genes. Meta-analyses were conducted for 20 SNPs in 10 genes of paediatric Crohn’s disease (CD) and for 8 SNPs in 5 genes of paediatric ulcerative colitis (UC). Five epigenetic studies were also included, but formal meta-analysis was not possible. Venice criteria and Bayesian false discovery probability test were applied to assess the credibility of associations. Nine SNPs in 4 genes were considered to have highly credible associations with paediatric CD, of which four variants  were not previously identified in paediatric GWAS. Differential DNA methylation in  Crohn’s disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are chronic inflammatory gastrointestinal disorders that most commonly arise in adolescents and young adultsThe increasing incidence and prevalence with decreased quality of life in child IBD patients has spurred ongoing search for risk factors of paediatric IBD. Familial, twin and phenotypic concordance studies suggest that IBD is highly heritable458Although genetic studies have identified a large number of IBD risk loci, the majority of the identified variants are located in noncoding regions and explain a small proportion of the variance in disease risk (13.6% for CD and 7.5% for UC)We report a comprehensive review of genetic and epigenetic studies in paediatric IBD, using the guidelines established by Human Genome Epidemiology Network (HuGENet)We searched three databases  . StudiesFor each study, the following data were extracted by two authors (XL and PS) independently: (1) identification details: the authors, publication year, study location, study design, sample size, age cut-off of paediatric IBD, diagnosis criteria, and sample source; (2) participant characteristics: age of disease onset, age structure of study population, and ethnicity; (3) genetic information: genes, polymorphisms, reference allele, allele frequency, allelic and genotypic counts; (4) epigenetic changes: loci with differential DNA methylation and miRNAs with dysregulated expression. Double extracted data were checked by a third author (XM) and any discrepancies were resolved by discussion.p-value <0.05) and I2 metric were calculated to quantify heterogeneity. Funnel plot analysis with an Egger test was conducted to test for small study effect. Statistical power was estimated by the Power and Sample Size Program. Hardy–Weinberg equilibrium (HWE) in controls was analysed by chi-square or Fisher’s exact test for each individual study. Sensitivity analysis was performed by excluding studies violating HWE or studies about non-white populations. Linkage disequilibrium (LD) between SNPs in the same genes was checked using the databases of 1000 Genomes (http://www.1000genomes.org/).Statistical analysis was conducted using the R software (R x64 3.0.1). Meta-analyses were performed only for the variants with data available from at least three independent studies. Summary crude odds ratios (ORs) and 95% confidence intervals (95% CI) for four genetic models were calculated by using either the fixed-effect or random-effect modelWe applied the Venice criteria2 value): A, 25% or less; B, 25–49%; C, 50% or more. For protection from bias, complete assessment is difficult. We assigned “B” to associations for which no small study effect was detected; otherwise, “C” was assigned.With Venice criteria, the credibility of associations was assessed for three criteria: the amount of evidence, the extent of replication and protection from biashttp://faculty.washington.edu/jonno/cv.html) on a prior level of 0.05BFDP was estimated to assess the noteworthiness of observed associations by using the Excel Calculation Spreadsheet  in at least two genetic models; (2) the BFDP value was less than 0.20 ; (3) the statistical power was greater than 80%; and (4) I2 was less than 50%. “Less credible” associations were defined as associations that were statistically significant  in at least one genetic model, with BFDP value greater than 0.20, and statistical power between 50% and 79%. All other associations were considered to be “not credible”.Genetic associations were classified into three categories based on the above assessment. Associations were classified as “highly credible” when (1) the association was statistically significant . Thirty five variants were found to be positively associated with paediatric CD in genetic association studies and twenty of them (57.1%) were confirmed in GWAS. Fifteen variants were identified to have positive associations with paediatric UC and ten of them (66.7%) were confirmed in GWAS .p < 0.05). For two variants  in paediatric CD, one studies was conducted in African Americans. Four pairs of SNPs were found in LD with r2 threshold ≥0.60  were considered as the most highly-credible, due to the high statistical power (>0.80), small heterogeneity (I2 < 50%) and low BFDP (<0.20); the other 2 variants  were considered as less-credible SNPs. ORs and 95% CI of individual studies for each variant and each model are shown in forest plots in Meta-analyses of 20 variants in four genetic models for paediatric CD are presented in Among the highly credible variants, rs2066844, rs2066845, rs11209026, rs7517847 and rs2241880 achieve genome-wide significance in both paediatric and adult-onset IBD GWAS . Rs20668p-values <0.05 in at least two genetic models, but none of them were classified as highly-credible associations either because of the high BFDP (>0.20) or low statistical power (<0.80). ORs and 95% confidence intervals of individual studies for each variant and each model are shown in forest plots in p < 0.05) with small changes in summary ORs and credibility assessment showed no inconsistencies with the previous analysis.Sensitivity analysis was conducted in three variants  for paediatric CD and one variant (rs2066844) for paediatric UC by excluding a study of non-white population and a study with variants violating HWE . SensitiAfter screening 408 titles and abstracts, 62 papers were reviewed in full text. Five studies met the inclusion and exclusion criteria, with three of them focusing on changes in DNA methylation and two investigating miRNA expression changes , Table 7NOD2, two variants  in IL23R, one variant (rs2241880) in ATG16L1, two variants  in the IBD5 region and one variant (rs1800629) in TNF-α represented the most credible findings and were referred as highly-credibly associated with paediatric IBD. Four  of these variants have not been identified in the GWAS of paediatric IBD. In addition, all of the identified variants have been found also to be associated with adult IBD either by candidate gene studies or GWAS.This study offers a comprehensive and up-to-date assessment of genetic and epigenetic literature in paediatric IBD. We systematically evaluated data from 84 studies for 183 variants in 71 candidate genes. It differs from other meta-analyses of genetic effect on IBD1516NOD2 gene to CD risk has been confirmed in multiple GWAS of European ancestry adultsNOD2 were each highly associated with increased paediatric CD risk. Compared with a meta-analysis of adult-onset CD, the effect sizes of these three SNPs in children were similar to those in adultsNOD2 gene and UC remains somewhat controversial. Most of the available data suggest no association between NOD2 mutations and UC susceptibility. The findings of this study indicated that the frequency of rs2066844 and rs2066847 variants was significantly higher in paediatric UC patients than in healthy children suggesting a possible involvement of NOD2 in paediatric UC. However, due to the limited statistical power and high probability of false discovery, this association between the NOD2 gene variant and paediatric UC susceptibility is considered as “less credible” and needs further study and elucidation.The contribution of IL23R with adult IBD risk was established in a GWASet al. replicated this association in childrenIL23R association in both paediatric CD and UC. It was estimated that the rs11209026 variant conferred approximately a 3-fold reduction in the risk of paediatric CD. For paediatric UC, the protective effect was significant but less-credible  due to the high probability of false discovery. In addition, homozygosity for the rs7517847 variant was also credibly associated with paediatric CD . A recent meta-analysis of rs7517847 estimated an odds ratio of 0.49 (95% CI: 0.38–0.64) for adult-onset CD riskThe association of IBD5 region encompasses several candidate genes which were initially suggested to be associated with early-onset IBDIBD5 region had strong association with early-onset CD, but only exerted a moderate effect on adult-onset CD. Subsequently, Mirza et al. reported a correlation between genes in IBD5 region and age of CD onsetIBD5 region with paediatric CD, but no stronger effects than those observed in adult population. Variant homozygotes of rs26313667 had an estimated OR of 1.68 (95% CI: 1.26–2.23), which was not significantly greater than that in adult-onset CD et al., which reported an effect size of 1.68 (95% CI: 1.21–2.33) for childrenIBD5 region increase CD susceptibility with no distinct specification to paediatric CD. Of particular note, identification of the precise causal gene in IBD5 region has proved to be challenging, due to the extensive degree of linkage disequilibrium between the examined polymorphisms.ATG16L1 gene, the rs2241880 polymorphism was identified to be associated with paediatric CD. Association of rs2241880 with adult-onset CD has been confirmed in multiple GWAS8ATG16L1 is involved in autophagy and plays an important role in the degradation of intracellular pathogens and antigen processingIn the DLG5 is the gene coding for an important scaffolding protein in maintaining epithelial structure and colonic barrier function. Since the original report identified DLG5 as a candidate gene for IBD risk25DLG5 did not achieve genome-wide significanceDLG5 variant (rs1248696) was not correlated with paediatric IBD. Despite the inability to replicate this association, we cannot rule out the possibility that DLG5 plays a role in IBD pathogenesis in specific populations. The varied association of DLG5 gene may be explained by genetic heterogeneity and a moderate effect. It is important to emphasize that, as a weak factor, a disease-causative effect could be easily modified in either a positive or negative way by gene-environment interactions. Therefore, even in the same population with the application of identical diagnostic criteria, variation in environmental exposures could lead to differences in genetic association.PSMG1 gene. This variant was identified as linked to early onset IBD in both paediatric IBD GWAS8PSMG1 is a paediatric-specific IBD related gene. However, association with PSMG1 was later detected in a large scale meta-analysis of adult-onset GWASPSMG1 gene was confirmed in both paediatric and adult-onset GWAS, the candidate gene studies presented inconsistent results. Four candidate gene studies were conducted to verify the role of PSMG1 gene in paediatric IBD27282929PSMG1 is required to elucidate the role of this gene in the pathogenesis of IBD.The polymorphic rs2836878 variant is located on chromosome 21q22, a region of linkage disequilibrium that harbours no genes but is close to the TNF-α gene has been identified as an IBD-susceptibility locus in genome wide scansTNF-α gene, have been studied extensively. Conflicting findings were observed in the associations between these polymorphisms and IBD development. A meta-analysis of genetic association studies of rs1800629 polymorphism in adulthood CD suggested no associationThe PTPN2 gene and IBD has been confirmed in GWAS among adultsPTPN2 gene and paediatric IBD, and found no association, which is consistent with the findings of paediatric GWAS8PTPN2 gene had a role in patients with older age-at-diagnosis or who were smokersAn association between the NOD2, which in the present study is classified as highly-credibly associated with paediatric IBD, have been reported in whole blood samples of IBD patientsNOD2 has been reported to regulate the expression of miRNA-29 to reduce the release of IL-23, which indirectly influences the Th17 pathway in human dendritic cellsNOD2 activationTNF-α, identified as associated with paediatric CD with high credibility, is located in a transcription factor AP2 binding site, which is sensitive to methylationThe identified epigenetic studies found differential DNA methylation in IBD susceptibility loci involved in the immune responseCFI, TNF-α and STAT3 have been replicated in adults, but not in the same tissues1041Two systematic reviews have been conducted to summarize observed associations between differential methylated DNA and dysregulated miRNA expression and adult IBD10In spite of the systematic internationally recognised approaches applied in this study, there is still room for future improvement in assessing the credibility of association. Venice criteria and BFDP improve the consistency and objectiveness of the interpretation and reporting of genetic associations, but this evidence does not prove causality. Additional evidence, such as gene knock-out experiments, gene expression microarray experiments or other mechanistic data are necessary to understand the specific functions of variants or genes. Associations with high credibility deserve in-depth evaluation including biological investigations; associations with moderate credibility indicate more genetic and biological studies are needed; weakly genetic associations may be not worthy of further investigation unless strong mechanistic evidence has been demonstrated.One of the potential obstacles to distinguish the genetic susceptibility of paediatric IBD from adult-onset disease is the difficulty of recruiting affected children in sufficient numbers to have adequate statistical power to detect and characterize association. GWAS have shown that odds ratios of genome wide significant genetic variants in IBD are small and thus sample sizes need to be very large to identify variants specific to paediatric IBD with moderate or small effect sizes78For epigenetic studies, there is widespread concern about the gene-set analysis approach generally applied in genome-wide methylation assays, which is severely biased as a result of differences in the numbers of CpG sites associated with different classes of genes and gene promotersNOD2 and TNF-α, dysregulated expression in let-7 and miR-124 were putatively associated with paediatric IBD, and worthy of further investigation. There is the possibility that genetic variations outside the investigated genes and unexplored genetic-environment interactions may modulate the early age of disease onset. Further progress identifying genetic susceptibility loci and epigenetic changes specific to paediatric IBD will require international collaboration to increase statistical power, population diversity, and for epigenetic changes, harmonization of protocols for investigation and an open-ness to share data.In summary, our study supports the highly credible associations of 9 genetic variants with paediatric IBD, of which 4 variants had not been previously identified in paediatric GWAS. SNPs identified for paediatric IBD have been found also to be implicated in adult IBD and there is no evidence that genetic effects are of greater magnitude in children. Differential DNA methylation in How to cite this article: Li, X. et al. Systematic meta-analyses and field synopsis of genetic and epigenetic studies in paediatric inflammatory bowel disease. Sci. Rep. 6, 34076; doi: 10.1038/srep34076 (2016).


BRCA1 gene impaired the homologous recombination pathway in a subset of TNBCs, which exhibited similar phenotypes to tumors with BRCA1 mutations; they harbored many structural variations (SVs) with relative enrichment for tandem duplication. Clonal analysis suggested that TP53 mutations and methylation of CpG dinucleotides in the BRCA1 promoter were early events of carcinogenesis. SVs were associated with driver oncogenic events such as amplification of MYC, NOTCH2, or NOTCH3 and affected tumor suppressor genes including RB1, PTEN, and KMT2C. Furthermore, we identified putative TGFA enhancer regions. Recurrent SVs that affected the TGFA enhancer region led to enhanced expression of the TGFA oncogene that encodes one of the high affinity ligands for epidermal growth factor receptor. We also identified a variety of oncogenes that could transform 3T3 mouse fibroblasts, suggesting that individual TNBC tumors may undergo a unique driver event that can be targetable. Thus, we revealed several features of TNBC with clinically important implications.Triple-negative breast cancer (TNBC) cells do not express estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2. Currently, apart from poly ADP-ribose polymerase inhibitors, there are few effective therapeutic options for this type of cancer. Here, we present comprehensive characterization of the genetic alterations in TNBC performed by high coverage whole genome sequencing together with transcriptome and whole exome sequencing. Silencing of the  TGFA gene that encodes one of the high affinity ligands for epidermal growth factor receptor (EGFR). Although TGFA expression is known to be elevated in a subset of TNBC tumors, this is the first report of the mechanistic basis of this phenomenon. It is of particular importance considering that anti-EGFR agents are possible therapeutic options for TNBC patients. Our study also revealed several features associated with “BRCAness”, which is critical for identification of patients who may be responsive to platinum agents and/or poly ADP-ribose polymerase inhibitors. Thus, the data presented in this report may advance our understanding of the pathogenesis of TNBC.Cancer can result from genetic alterations, some of which can be good drug targets. To reveal genetic alterations that provide important information for the development of ideal therapeutic strategies for triple-negative breast cancer (TNBC), TNBC tumor samples were subjected to comprehensive genomic analyses. We identified novel recurrent structural variations associated with enhanced expression of the  ERBB2 gene amplification, which encodes human epidermal growth factor receptor 2 (HER2)  at the indicated times. Data are the means ± standard deviation.3T3 cells expressing wild-type or mutant forms of the indicated proteins were injected subcutaneously into the shoulders of nude mice. ((TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Oscillatory activity in the gamma frequency range is a critical mechanism, which integrates neural networks within and across brain structures during cognitive processes. In schizophrenia, abnormalities in high gamma oscillations are ubiquitous and most likely reflect dysfunction in neuronal networks. In conscious rats, disturbed network oscillations associated with positive symptoms and cognitive deficits were modeled in different cortical areas by the dopaminergic agonist (amphetamine) and the N-methyl-D-aspartate (NMDA) receptor antagonists (PCP and MK801). Subsequently, the efficacies of marketed atypical antipsychotics  to normalize dysfunctional oscillations and network connectivity were examined. Acute NMDA antagonists elicited aberrant synchrony in the gamma frequency oscillations. In addition, coherent slow alpha network activity was observed with MK801 and amphetamine, both of whose oscillatory rhythms were correlated with pronounced locomotor activity. All antipsychotics commonly decreased slow alpha and high gamma network oscillations in different cortical regions as well as motion behavior. In the combined treatments, antipsychotics attenuated NMDA antagonist-induced abnormalities in functional network oscillations and connectivity, whose effects on motor behavior is mechanistically related. These results suggest that pharmacologically induced disruption of cortical gamma oscillations and network connectivity in rats is a candidate model to study dysfunctional oscillatory patterns described in positive and negative symptoms of schizophrenia. The efficacy of antipsychotics to rescue cortical network oscillatory patterns is in line with the idea that glutamatergic and dopaminergic systems play a role in maintaining the integrity of cortical circuits. Thus, gamma oscillations could provide a powerful translational index to assess the integrity of neural networks and to evaluate the efficacy of drugs with potential antipsychotic properties. Temporal oscillation on alpha rhythm reflects an active inhibitory mechanism of task-irrelevant information, whereas gamma rhythm is critical for the maintenance of working memory13.Ongoing brain oscillations determine the dynamic changes in brain states, and influence alertness such as cortical computations, cognitive perceptual grouping, attention-dependent stimulus selection, subsystem integration, working memory, and consciousness15. Alteration in GABA-mediated neurotransmission has been proposed as a candidate mechanism that impairs gamma oscillations21. Postmortem studies in schizophrenics confirmed deficits in GABA-mediated synaptic transmission and reduced GABA synthesis in the parvalbumin (PV) containing subpopulation of inhibitory neurons22. PV interneurons are crucial in the genesis of gamma oscillations in cortical circuits, as they exert powerful, precisely timed recurrent inhibition onto their target pyramidal cells and inhibitory interneurons24. These GABAergic interneurons appear to be under the control of glutamatergic system, which is also known to be abnormal in schizophrenia29.Network oscillations have received much interest in contemporary schizophrenia research as the same cognitive processes driven by gamma rhythm are disrupted in this disorder30. Gamma oscillatory rhythm can be assessed across species and at various spatial levels, from single unit to large-scale networks' electroencephalographic (EEG) recordings, which offers the possibility of applying findings from basic neuroscience models to clinical studies. Activation of dopaminergic receptors by amphetamine or blockade of N-methyl-d-aspartatic acid (NMDA) receptor by ketamine, PCP, and MK801 has widely been used in humans, primate, and rodents to recreate core symptoms of schizophrenia, such as hallucinations, thought disorder, negative symptoms, and cognitive deficits36. Previous reports showed aberrant increases of gamma frequency oscillations in acute psychotic unmedicated schizophrenic patients, and activation of dopaminergic receptors or blockade of NMDA receptors resulted in a short-term pathological increases of gamma oscillations in local cortical circuits in humans and animals42. Thus, network gamma oscillatory rhythm may provide a valuable window for investigating the contribution of dopaminergic and glutamatergic transmission in the disturbance of integrative circuitry of cognitive processing and could possibly help to improve detection of more effective and targeted novel pharmacological therapies45.Electrophysiological findings have firmly established the role of abnormal oscillatory processes in schizophreniaThe present studies aimed to evaluate whether EEG network oscillations in conscious rats are candidate quantitative markers to pharmacologically recreate cardinal features of dysfunctional cortical networks described in schizophrenia, and to subsequently assess the efficacy of antipsychotic drugs to normalize aberrant functional network activities and associated disorganized motion behavior.All procedures were performed in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, and of the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the Janssen Pharmaceutica ethical committee. The experiments were carried out in male adult Sprague–Dawley rats, supplied by Harlan (the Netherlands), and weighing ~250 g at the time of surgery. Animals were housed in full-view Plexiglas cages  that belong to IVC racks  located in a sound-attenuated chamber. Rats received a chip for identification purpose by Animal Inventory and Weighing system, and were maintained under controlled environmental conditions throughout the study: 22 ± 2 °C ambient temperature, the relative humidity at 60%, 12:12 light–dark cycle  and food and water available ad libitum.46 In brief, animals were equipped with six stainless steel-fixing screws (diameter 1 mm) for the recording of EEG activities inserted bilaterally in the left and right hemispheres along the anteroposterior axes at the locations . All electrodes were placed stereotaxically . In addition, stainless steel wires  were placed in the muscle of the neck to record the electromyogram activity (EMG). Electrodes were connected to a pin (Future Electronics: 0672-2-15-15-30-27-10-0) with a small insert  and were fit into a 10-hole connector, after which the whole assembly was fixed with dental cement to the cranium.Surgery was performed under Isoflurane anesthesia as described earlier  filter at 50 Hz was applied to avoid voltage related to power line interferences.Two weeks after recovery, all EEG recordings from six brain regions were performed under vigilance-waking condition during the dark circadian phase as described earliern = 8 for each condition). For reversal challenge experiments, antipsychotics or vehicle were administered, followed 30 min later by a challenge drug and EEGs were further recorded for 2 h (n = 8 for each condition). Continuous EEG and EMG field potentials were acquired at 2 kHz sample rate with an input range of ±500 mV through a Biosemi ActiveTwo system , which replaces the conventional ground electrodes by two separate electrodes: the common mode sense active electrode and the driven right leg passive electrode. This common mode reference for online data acquisition and impedance measures is a feedback loop driving the average potential across the montage close to the amplifier zero. In addition to motion levels, the EMG signals were used to differentiate active vs. passive behavior. The signals were amplified and analog band-pass-filtered between 1 and 100 Hz and was digitized with 24-bit resolution.All experimental baseline EEG recording sessions of 30 min started 2 h after the light offset to avoid confounding circadian effect on EEG. In case of dose–response experiments, signals were recorded for 2 h after vehicle or drug administration  constructed from overlapping windows of 4 s data and plotted for post-drug periods windowing over 1–100 Hz. Each frequency band was averaged for each 15-min interval in the following frequency bands: Delta band (1–4 Hz), Theta band , Alpha band , Beta band , and Gamma band .Analysis was performed using a method described earlierIn each experiment, values of baseline 4 s waking epochs were averaged for 30 min followed by 15 min average for the remaining time after the pharmacological treatments. Drug-induced changes in different cortical networks and EEG power were calculated for each 15 min time block over 2 h as the ratio of mean spectral power obtained following the administration of test drug vs. the mean spectral power obtained during 30 min baseline period. This procedure allows for assessment of drug-induced changes in each frequency band as a percentage of the original power, compared to the vehicle condition.47. The magnitude of EEG coherence ranges between 0 and 1: a low value indicates no similarity between the two signals, whereas values close to 1 indicate a high similarity between two-time series. Functional connectivity between the time series in different electrodes was quantified by coherence at different time points before and after administration of drugs using a generalized additive mixed model where the coherence over the frequencies was fitted using a cubic spline for the different treatment groups and the heterogeneity between the animals49. Subsequently, coherence between each pair of electrodes was concatenated every 4 s and aggregated over 15 min in each frequency bands for each animal, time point, and treatment group. Frequency bands were selected for comparison between groups based on the most prominent oscillatory and coherent activities across different brain regions. To reduce the impact of volume conduction of signals propagated from common generators, computation of all edges in which the maximum absolute value of coherence occurred at zero time lags between time series was removed.To assess effects of antipsychotics on arousal level, the time spent in active waking was quantified and expressed in % of total duration of the recording session during baseline condition and within discrete time points 45–60 and 105–120 min post dosing. Network connectivity between cortical regions was computed by phase coherence, which is a measure commonly used to probe the integrity of cortical neural pathways and index the functional coupling between different cortical structures at various frequencies50. The doses of risperidone and olanzapine were selected based on occupancy of dopamine D2 receptor and their potency to attenuate the challenge-induced hyperlocomotor behavior. The dose of clozapine was selected based on its ability to antagonize apomorphine-induced stereotypy behavior51.All drugs were synthesized in Janssen Research and Development laboratories. For acute experiments, olanzapine , clozapine , and risperidone  were formulated in H2O + 2H2T (tartaric acid) + NaCl. PCP , MK801 , and amphetamine  were dissolved in saline solution. Solutions were subcutaneously (s.c.) administered in a volume of 5 ml/kg body weight. In the case of combined pharmacological studies, antipsychotics were administered 30 min before the drug challenge. The doses selected for PCP, MK801, and amphetamine were repeatedly found to elicit marked alterations in motor behavior and EEG patterns52. p values from the associated F-tests were reported as well as Least squares means from the corresponding ANOVA models. Associations between EEG parameters and activity levels were evaluated using Spearman rank-correlation analyses.The values of consecutive 4 s epochs were averaged and drug-induced changes in EEG oscillations were calculated in blocks of 15 min for 2 h as the ratio of mean spectral power obtained following the administration of test drug vs. the mean spectral power obtained during 30 min baseline period. This procedure allows for assessment of drug-induced changes in EEG power expressed at each frequency band as a percentage of the original power, compared to the vehicle condition. Time course EEG spectral changes were submitted to a two-way multivariate ANOVA for repeated measures with two main factors (treatment and period of recordings), followed by pairwise comparisons between treatment levels for each of the 15 min periods. A one-way ANOVA was applied to compare effects of different antipsychotics on average percent time spent in active waking within discrete time point 45–60 and 105–120 min. Sample sizes including animal numbers were chosen to ensure adequate statistical power comparable to previously published papers. Data distribution of experiments was assumed to be normal and was presented as the means ± SEM. The aggregated coherences as well as the changes from baseline were analyzed per frequency band using an ANOVA with time, group, and its interaction as covariates while considering the heterogeneity between the different animalsThe NMDA antagonists and the dopaminergic agonist were used to model the aberrant cortical network oscillations in rats that resemble the dysfunctional network oscillations widely described in schizophrenia. Acute administration of PCP dose-dependently enhanced aberrant network oscillatory synchrony in higher gamma frequencies, which was apparent in different brain areas Fig. . As chanIn addition to their effects on high oscillatory rhythm, both MK801 and amphetamine increased the overall slow alpha oscillatory activity in different brain regions ; Olanzapine  = 0.48, p = 0.7); and risperidone  = 0.27, p = 0.84). Approximately 80% of time during baseline (Condition 0) was spent in active waking  = 0.23, The potency of antipsychotics to reverse the challenge-induced aberrant network synchrony in slow alpha and higher gamma frequencies is shown in Figs. Risperidone normalized the pathological synchronous high gamma and slow alpha network oscillations induced in the MK801-treated rats Fig. . SimilarQualitative evaluation of the efficacy of clozapine to reverse abnormalities in EEG network oscillations and associated disorganized behavior in the amphetamine-treated rats are shown in Fig. Analysis of motion activity at different time points showed that drug challenges enhanced, whereas different antipsychotics consistently decreased locomotor activity levels . The scatter plot in Fig. p values < 0.05, for MK801: rS ranges between 0.53 and 0.81, all P values < 0.001, and for amphetamine: rS ranges between 0.48 and 0.77, all p values < 0.05).The results of a Spearman rank-correlation analysis between the higher gamma activity and motor activity at 30 min after the administration of challenge drugs are shown in Fig. r = 0.41, p < 0.02, MK801 r = 0.81, p < 0.0001 and amphetamine r = 0.82, p < 0.001), which was maintained across all time intervals of the recording session  and motor activity levels  all NMDA receptor antagonists elicited aberrant network oscillations in the higher gamma frequency range associated with pronounced hyperactivity, while a consistent abnormal slow alpha coherent oscillatory activity was observed with both amphetamine and MK801, (2) all antipsychotics decreased EEG slow alpha and higher network gamma oscillations as well as decreased motion levels, (3) pre-treatment with antipsychotics attenuated drug challenge-induced abnormal slow alpha and higher gamma network synchrony, which was mechanism-dependent.55. Schizophrenia is considered as a disconnection syndrome associated with failure in early automatic neural connectivity of important sensory inputs leading to cognitive impairments56. These widespread structural abnormalities are accompanied by alterations in glutamatergic and GABAergic neurotransmission and decreased expression of NMDA receptors on inhibitory interneurons, which lead to an imbalance between excitation and inhibition, hyperexcitability, and unstable coordination of cortical networks59. Aberrant synchronization in the gamma oscillatory range has been repeatedly shown to accompany many disturbed neurocognitive functions in schizophrenic patients61. Positive symptoms of schizophrenia are correlated with enhanced gamma oscillatory synchrony and had been associated with an attempt to adapt to a progressive loss of cortical gray matter and associated changes in cognitive and emotional function, whereas negative symptoms have been related to both enhanced and reduced high-frequency oscillations64. Therefore, network gamma oscillations may provide a plausible animal–clinical interface on studying dysfunction in the integrity of glutamatergic, dopaminergic, and GABAergic neuronal circuits in schizophrenia.Network gamma synchrony selectively facilitates the communication among synchronized assemblies to ensure large numbers of cortical computationsAdministration of NMDA receptor antagonists enhanced aberrant cortical network gamma oscillations.65. The NMDA receptors have been proposed as a candidate for the synchronization of local circuits, which are prominent in the superficial cortical layers that are the main recipients of long corticocortical connections. Disinhibition of principal cells that may result from reduced interneuron excitation would facilitate the transient pathological generation of gamma oscillations. Our present results further extend previous observations showing abnormal cortical gamma oscillations associated with hyperlocomotion following systemic injection of PCP, MK801, and ketamine41. The potential mechanism underlying aberrant gamma oscillations might involve the release of monoamines such as dopamine and serotonin, which in turn increases glutamate transmission in the prefrontal cortex to elicit transient aberrant neuronal network activation67. PCP strongly binds in the hippocampus leading to increased metabolic activity and schizophrenic-like symptoms as well as disorganized behavior by activating dopaminergic neurotransmission in the nucleus accumbens, which may in turn drive the nucleus at gamma oscillatory rhythm68. Accordingly, muscimol-induced inactivation of the hippocampal medial septum concomitantly suppressed the occurrence of gamma oscillations and associated disorganized behavior40. Thus, disruption of NMDA receptor signaling may release local gamma frequency oscillations from the inhibitory control of extended networks leading to pathological increase of this rhythm.In human and animal studies, acute and chronic administration of PCP, ketamine, and MK801 produced schizophrenia-like behavior with positive and negative symptoms associated with cognitive impairments69. In general, theta rhythmicity has been shown to organize gamma frequency dynamics of neuronal populations during cortical computing, both of which rhythms are accompanied with reduced synchrony of alpha oscillations. Here, the enhanced alpha network activity by MK801 may silence theta and gamma activities to disengage and/or inhibit network computations by decreasing the demand of those cortical regions.The thalamocortical coupling generates a large network oscillatory activity in the alpha frequency range (8–11 Hz) during cortical operations. However, this rhythm may also emerge during loss of attention in the immobile state or during failure to process sensory input, and may serve to functionally disengage and reduce the processing capabilities of a given brain region. The alpha activity is decreased in engaged brain regions, whereas it increased in disengaged regions and human74, motor activity is accompanied with changes in alpha power. The strong correlation between the magnitude of the alpha band oscillation at the onset of motion and the duration of spontaneous movement epoch is consistent with a mechanical connection to motor planning networks in parietal cortex76. Pharmacological evidence has shown a functional interaction between glutamatergic–serotoninergic–dopaminergic systems and behaviors related to NMDA receptor. On the one hand, inhibition of NMDA receptors has been shown to reduce the inhibitory GABAergic signaling from PV-containing interneurons and to consequently increase the rate of pyramidal neurons and glutamate release78 as well as psychotomimetic activity79. On the other hand, a variety of antagonists having selectivity at 5-HT2a and D2 receptors attenuated the disruptive effect of NMDA blockade on locomotor activity84.The mechanism by which MK801 elicited aberrant slow alpha oscillation associated with hyperactivity is not clear. In non-pharmacologically treated animals.In the present study, MK801-induced aberrant slow network alpha frequency oscillation associated with hyperactivity was attenuated by risperidone, possibly due to its atypical character ascribed to combined antagonism at 5-HT2a/D2 receptors. Thus, these results further extend the evidence for functional interactions between NMDA and 5-HT2a/D2 receptor systems and suggest that abnormal EEG power and behavioral disinhibition actions of MK801 may largely be attributed to pronounced enhancement of glutamate neurotransmission following the activation of presynaptic and postsynaptic serotonin and/or dopaminergic receptorsAdministration of amphetamine elicited robust coherent EEG slow alpha network activity at the dose that induced stereotypic behavior.35. Amphetamine is known to produce active arousal stereotypic behavior, i.e., sniffing and licking, which could be suppressed by intra-accumbens injection of either dopamine antagonist85, or glutamate receptor blockade86. There is scarce evidence for a direct influence of the dopaminergic transmission on neuronal oscillations in schizophrenia. Administration of psychomotor stimulant amphetamine or the dopamine agonist apomorphine causes a prolonged pathological increase of cortical gamma oscillations likely through increased excitability of fast-spiking GABAergic interneurons88.Amphetamine is widely used to model positive symptoms of schizophrenia such as delusions, hallucinations, and disorganized behavior by increasing dopaminergic function in the brain, particularly in the nucleus accumbens89. In a similar vein, amphetamine may enhance 5-HT neurotransmission in a similar way as hallucinogenic LSD or DOM, which have been shown to attenuate 5-HT release through activation of presynaptic 5-HT2a/2c leading to increased glutamate release in the prefrontal cortex, stereotypic behavior, and increased the power of the alpha1 rhythm90.In the present work, amphetamine consistently induced coherent alpha and gamma activities associated with signs of disorganized behavior. The enhancement of higher gamma oscillations may result from its influence on fast-spiking PV-containing interneurons, while the particular enhancement of slow alpha oscillations might derive from the stimulation of presynaptic dopaminergic D1 receptors and postsynaptic dopaminergic D2 receptorsClozapine, olanzapine, and risperidone had highly distinctive EEG fingerprints and caused similar attenuation of the NMDA antagonists and amphetamine-induced aberrant network oscillations in different cortical regions.92. Interestingly, the effectiveness of antipsychotic drugs in relieving schizophrenic symptoms was correlated with the decrease in fast cortical activity94. In addition, both typical and atypical antipsychotics demonstrated a potency to decrease basal gamma and theta power; however, it is not clear whether this medication provides benefit for cognitive function in schizophrenia96. Moreover, the typical antipsychotic haloperidol at a therapeutic dose that occupies >70% of D2 receptors significantly reduced PCP, MK801, and ketamine-induced higher gamma oscillatory rhythm and hyperlocomotion98. Likewise, all effective antipsychotics inhibit dopaminergic neurotransmission and exhibit different affinities for 5-HT receptors , and serotonin transporter98. The increased coherent slow alpha rhythm elicited by amphetamine was likely mediated through presynaptic D1 and/or postsynaptic D2 dopaminergic.Increased gamma oscillations have consistently been observed in schizophrenic patients99. The current study extends on a previous report showing a late decrease of striatal alpha1 spectral power by atypical antipsychotic drugs in rats100. The fact that antipsychotics attenuated the NMDA receptor antagonist-induced aberrant oscillations associated with hyperactivity may suggest an indirect interaction with the glutamatergic system rather than the dopaminergic system. Assessment of specific pharmacological ligands into D1/2 receptor function would be an obvious way to examine this hypothesis and possible relationship to sedative effects of antipsychotics.As the atypical antipsychotics display combined antagonist activity at D2 and 5-HT2a/c receptors, their efficacy to increase 5-HT/DA release would decrease the slow alpha network synchrony, an effect that may contribute to antisychotic therapeutic actions101. Alpha power has been associated with stereotypic behavior elicited by different hallucinogenic and psychostimulant drugs90. This raises the question as to whether the effects of antipsychotics on EEG power are a result of the reduced locomotion102, rather than a direct effect on cortical processing.Gamma power in the rodent prefrontal cortex increases with running speed, and gamma power has been shown to be higher in fast-moving rats under saline and in rats treated with an NMDA antagonist90. Both risperidone and olanzapine attenuated concomitantly evoked gamma network oscillations and hyperlocomotor behavior, as well as additional alpha rhythm in case of MK801. However, clozapine decreased amphetamine-induced gamma oscillatory rhythm appeared to last longer and dissociated from behavioral hyperactivity (Figure In the present work, PCP had no consistent effect on theta frequency rhythm critically involved in the moment-to-moment voluntary movement indicating that this rhythm is not a key activity underlying the hyperactive behavior in the PCP model. However, both MK801 and amphetamine increased the power in the 8–10 Hz known to be linked to abnormal behavior elicited by hallucinogenic and psychostimulant drugs40. Accordingly, the administration of the GABAA agonist muscimol in the medial septum suppressed completely locomotor activity in animals treated with PCP and only partially in amphetamine-treated animals103. Consistent with this view, antipsychotics completely suppressed the hyperactivity that accompanied the increase in the gamma rhythm elicited by NMDA receptor blockade but not in dopaminergic receptor activation. These findings undermine the argument that attenuation of aberrant network oscillations may underlie the antipsychotic activity, which may be mechanistically associated or dissociated from having an active role in the hyperactive behavior in the NMDA receptor model or the dopaminergic receptor model, respectively.Gamma oscillatory rhythm has been suggested to influence behavioral response via the nucleus accumbens by interfering with glutamate or dopamine release104. Novel therapeutic strategies that could normalize aberrant high gamma network oscillations and/or elevate gamma power during cognitive paradigms may be promising candidates for restoring gamma abnormalities in schizophrenia and associated cognitive impairments. Therefore, the ability to induce and maintain network oscillations within the gamma range in the rat offers a promising translational tool in preclinical research that may enhance the chance of screening and identifying novel drugs with antipsychotic potential with cognitive enhancing properties.Convergent clinical literature indicates abnormalities in synchronization of EEG gamma oscillation as an endophenotype in schizophrenic patients. In rodents, acute NMDA receptor blockade produced schizophrenia-like behavior and cognitive deficits associated with increased cortical network excitability and impaired gamma oscillation synchrony. Synchronization of gamma oscillations has a wider relevance in cortical networks for behavioral and cognitive phenomena, and emerging views suggest that development of medications that could improve cognition is a major step forward in achieving better functional outcome in schizophrenic patients. Targeting gamma-frequency deficits and the circuit insults, the index could have implications to improve perceptual abnormalities in a broad group of schizophrenia patients