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We aimed to determine microbial signature linked with lung cancer (LC) diagnosis and to define taxa linked with durable clinical benefit (DCB) of advanced LC patients. Stool samples for microbial 16S amplicon sequencing and clinical data were collected from 75 LC patients (50 of which were treated with checkpoint inhibitors) and 31 matched healthy volunteers. We compared LC to healthy controls and patients with DCB to those without. LC patients had lower α-diversity and higher between-subject diversity. Random Forests model to differentiate LC cases from controls ROC-AUC was 0.74. Clostridiales, Lachnospiraceae, and Faecalibacterium prausnitzii taxa abundance was decreased in LC compared to controls. High Akkermansia muciniphila correlated with DCB (HR 4.26, 95% CI 1.98–9.16), not only for the immunotherapy-treated patients. In addition, high Alistipes onderdonkii (HR 3.08, 95% CI 1.34–7.06) and high Ruminococcus (HR 7.76, 95% CI 3.23–18.65) correlated with DCB.Our results support the importance of gut microbiome in LC. We have validated the apparent predictive value of Akkermansia muciniphila, and highlighted Alistipes onderdonkii and Ruminococcus taxa correlation with DCB. Upon additional validations those can be used as biomarkers or as targets for future therapeutic interventions.

Background and Aims Widespread dysregulation of long non-coding RNAs (lncRNAs) including a reduction in GATA6-AS1 was noted in inflammatory bowel disease (IBD). We previously reported a prominent inhibition of epithelial mitochondrial functions in UC. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not yet defined. Methods Mucosal transcriptomics was used to conform GATA6-AS1 reduction in several treatment naïve independent human cohorts (n=673). RNA pull-down followed by mass-spectrometry was used to determine GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2, were used to elaborate on GATA6-AS1 functions. Results GATA6-AS1 showed predominant expression in gut epithelia using single cell datasets. GATA6-AS1 levels were reduced in Crohn disease (CD) ileum and in ulcerative colitis (UC) rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to more severe UC form, and to less favorable UC course. GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in celiac disease that is induced in UC, CD, and celiac, in contrast GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in reduction of metabolites linked with aerobic respiration relevant to mucosal inflammation. TGM2 knockdown in GATA6-AS1 deficient cells rescued mitochondrial respiration. Conclusions GATA6-AS1 levels are reduced in UC, CD, and celiac, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 levels.

The coronavirus disease 2019 (COVID-19) has rapidly spread around the world, impacting the lives of many individuals. Growing evidence suggests that the nasopharyngeal and respiratory tract microbiome are influenced by various health and disease conditions, including the presence and the severity of different viral disease. To evaluate the potential interactions between Severe Acute Respiratory Syndrome Corona 2 (SARS-CoV-2) and the nasopharyngeal microbiome. Microbial composition of nasopharyngeal swab samples submitted to the clinical microbiology lab for suspected SARS-CoV-2 infections was assessed using 16S amplicon sequencing. The study included a total of 55 nasopharyngeal samples from 33 subjects, with longitudinal sampling available for 12 out of the 33 subjects. 21 of the 33 subjects had at least one positive COVID-19 PCR results as determined by the clinical microbiology lab. Inter-personal variation was the strongest factor explaining > 75% of the microbial variation, irrespective of the SARS-CoV-2 status. No significant effect of SARS-CoV-2 on the nasopharyngeal microbial community was observed using multiple analysis methods. These results indicate that unlike some other viruses, for which an effect on the microbial composition was noted, SARS-CoV-2 does not have a strong effect on the nasopharynx microbial habitants.

Background Gut microbial alteration is implicated in inflammatory bowel disease but is noted in other diseases. Systematic comparison to define similarities and specificities is hampered since most studies focus on a single disease. Results We develop a pipeline to compare between disease cohorts starting from the raw V4 16S amplicon sequence variants. Including 12,838 subjects, from 59 disease cohorts, we demonstrate a predominant shared signature across diseases, indicating a common bacterial response to different diseases. We show that classifiers trained on one disease cohort predict relatively well other diseases due to this shared signal, and hence, caution should be taken when using such classifiers in real-world scenarios, where diseases are intermixed. Based on this common signature across a large array of diseases, we develop a universal dysbiosis index that successfully differentiates between cases and controls across various diseases and can be used for prioritizing fecal donors and samples with lower disease probability. Finally, we identify a set of IBD-specific bacteria, which can direct mechanistic studies and design of IBD-specific microbial interventions. Conclusions A robust non-specific general response of the gut microbiome is detected in a large array of diseases. Disease classifiers may confuse between different diseases due to this shared microbial response. Our universal dysbiosis index can be used as a tool to prioritize fecal samples and donors. Finally, the IBD-specific taxa may indicate a more direct association to gut inflammation and disease pathogenesis, and those can be further used as biomarkers and as future targets for interventions.

Background Crohn disease (CD), environmental enteric dysfunction (EED) and celiac disease are prevalent enteropathies linked to chronic gut inflammation. However, unlike celiac and Crohn that are prevalent in westernized countries, EED is seen in only in low- and middle-income countries of the developing world. Recent work has defined specific EED duodenal microbiota associated with stunting, and transmission of EED histologic changes and weight loss by microbial transfer to germ free mice. Methods We performed high throughput transcriptomics of 94 duodenal samples from the SEEM cohort:52 undernourished EED participants, 17 celiac and 25 healthy North American controls. We compared those to our previously reported transcriptomics of 248 newly diagnosed ileal CD from RISK. Comparison were performed between cases and controls within each cohort. Results Above 90% of the expressed protein coding genes are shared between duodenum samples within SEEM, and Crohn ileal within RISK enabling the anticipated comparison. A substantial number of the down-regulated genes in undernourished EED participants overlapped with celiac (212/481, 44%) and with Crohn Disease (249/481, 52%). Shared reduced signatures and functions included decrease in epithelial lipid metabolism, vitamins, and brush border transport signatures. Relative to celiac disease, suppression of antioxidant and detoxification genes were EED- and Crohn-specific, which include the reduction of metallothioneins (MT family) that buffer against toxic metals, and aldo-keto NADPH- genes (AKR1C family) involved in detoxification of environmental compounds. A smaller proportion of the EED up-regulated signal overlapped with celiac (85/781), and we noted a more significant overlap with Crohn disease (248/781, Chi squares p Conclusion We highlight important biologic signals widely overlapping between Crohn Disease (CD) and EED transcriptome in the small intestine, suggesting mechanisms involving altered gut microbiota. Inhibiting innate immune activation or reverting Crohn dysbiosis may be beneficial future therapy for Crohn Disease and EED.

Background and Aims Ileal strictures are the major indication for resective surgery in Crohn’s disease [CD]. We aimed to define ileal gene programmes present at diagnosis and linked with future stricturing behaviour during 5-year follow-up, and to identify potential small molecules to reverse these gene signatures. Methods Antimicrobial serologies and pre-treatment ileal gene expression were assessed in a representative subset of 249 CD patients within the RISK multicentre paediatric CD inception cohort study, including 113 that are unique to this report. These data were used to define genes associated with stricturing behaviour and for model testing to predict stricturing behaviour. A bioinformatics approach to define small molecules which may reverse the stricturing gene signature was applied. Results A total of 19 of the 249 patients developed isolated B2 stricturing behaviour during follow-up, while 218 remained B1 inflammatory. Using deeper RNA sequencing than in our previous report, we have now defined an inflammatory gene signature including an oncostatin M co-expression signature, tightly associated with extra-cellular matrix [ECM] gene expression, in those who developed stricturing complications. We further computationally prioritise small molecules targeting macrophage and fibroblast activation and angiogenesis which may reverse the stricturing gene signature. A model containing ASCA and CBir1 serologies and a refined eight ECM gene set was significantly associated with stricturing development by Year 5 after diagnosis {AUC (area under the curve) (95th CI [confidence interval]) = 0.82 [0.7–0.94)}. Conclusions An ileal gene programme for macrophage and fibroblast activation is linked to stricturing complications in treatment of naïve pediatric CD, and may inform novel small molecule therapeutic approaches.

High fructose consumption is one of the hallmarks of Western diets and has been found to induce MeS symptoms in parallel to gut microbial dysbiosis. However, the causality between those two is still elusive. Here, we studied whether a significant modification of gut microbial composition by antibiotics can influence the fructose-induced metabolic changes. Male Sprague-Dawley (SD) rats were divided into four groups including controls, controls + antibiotics, high fructose diet (HFrD, 60% fructose), HFrD + antibiotics (n = 7&ndash, 8 in each group) for a period of 8-weeks. The high fructose diet increased blood pressure (BP), triglyceride (TG), fatty liver and the expression of hepatic genes related to lipogenesis, and fructose transport and metabolism. In addition, fructose changed the microbial composition and increased acetic and butyric acids in fecal samples but not in the blood. Antibiotic treatment significantly reduced microbial diversity and modified the microbial composition in the samples. However, minimal or no effect was seen in the metabolic phenotypes. In conclusion, high fructose consumption (60%) induced metabolic changes and dysbiosis in rats. However, antibiotic treatment did not reverse the metabolic phenotype. Therefore, the metabolic changes are probably independent of a specific microbiome profile.

Motivation RNA viruses generate a cloud of genetic variants within each host. This cloud contains high-frequency genotypes, and many rare variants. The dynamics of these variants is crucial to understand viral evolution and their effect on their host. Results We use an experimental evolution system to show that the genetic cloud surrounding the Coxsackie virus master sequence slowly, but steadily, evolves over hundreds of generations. This movement is determined by strong context-dependent mutations, where the frequency and type of mutations are affected by neighboring positions, even in silent mutations. This context-dependent mutation pattern serves as a spearhead for the viral population’s movement within the adaptive landscape and affects which new dominant variants will emerge. The non-local mutation patterns affect the mutated dinucleotide distribution, and eventually lead to a non-uniform dinucleotide distribution in the main viral sequence. We tested these results on other RNA viruses with similar conclusions. Supplementary information Supplementary data are available at Bioinformatics online.

Background Long non-coding RNAs (lncRNAs) have attenuated expression in several immune-mediated disorders. Using mRNAseq of intestinal biopsies, we identified a widespread dysregulation of 459 lncRNAs in the ileum of treatment-naïve pediatric Crohn Disease (CD) patients. We noted that large fraction of downregulated lncRNAs were correlated with epithelial functions. CDKN2B-AS1 (ANRIL) is one of the top most down-regulated lncRNA in CD, and showed decreased expression in Ulcerative Colitis (UC) colon tissues in recent studies. Methods Transcriptomics data is used to evaluate CDKN2B-AS1 expression in mucosal biopsies datasets and in gut epithelia. siRNA oligonucleotides targeting most CDKN2B-AS1 transcripts using Lipofectamine RNAiMAX is used to modulate CDKN2B-AS1 function, and RT-PCR to evaluate mRNA expression. xCELLigence system with gold microelectrodes in plate base is used to measure the impedance as an indicator for cell index. Results CDKN2B-AS1 is down-regulated in bulk mucosal biopsies obtained from CD ileum [fold change (FC) -8, corrected p=1.6E-5], in UC rectum (FC -9.4, corrected p=3.9E-18), and in celiac duodenum (FC -2.3, corrected p=0.03) in comparison to controls. CDKN2B-AS1 is detected under basal conditions in HT-29 cells. Two sets of CDKN2B-AS1 siRNA oligonucleotides (targeting most transcripts) achieved up to 60% reduction in CDKN2B-AS1 expression in HT-29 cells (p=1E-04) as confirmed by RT-PCR using two sets of primers. CDKN2B-AS1 reduction significantly increased cell index as measured by xCELLigence (doubled the index, p=1.7E-03). mRNAseq of the CDKN2B-AS1 reduced HT-29, showed reduction of the tumor suppressor gene APC in CDKN2B-AS1 siRNA treated cells (FC=-1.75, p=0.04) and of TGFBR2 (FC=-1.75, p=0.04). In contrast, reduction of CDKN2B-AS1 resulted in increase in WNT11 (FC=2.1, p=0.02) and TGFB1 (FC=1.9, p=0.04), and induction of the replication associated topoisomerase TOP1MT (FC=2.7, p=0.001). We confirmed these mRNAseq results using RT-PCR. Finally, mimicking inflammation by treating HT-29 with IL1β and LPS, reduced CDKN2B-AS1 expression (by 70%, p=6.3E-04) and doubled the cell index (p=1.8E-03). Conclusion We detected reduced CDKN2B-AS1 expression in three inflammatory disease affecting the gut in different location along the GI tract. Using HT-29 model system we were able to show, as was previously shown, an increase in cell index in CDKN2B-AS1 siRNA treated cells. We supplement those showing effect on down-stream genes that may be relevant in controlling cell proliferation. We further show that upon inflammatory triggering of HT-29 cells, CDKN2B-AS1 expression is reduced and cell index is increased, which may suggest a potential role in epithelial renewal in inflammation.

Background The serum metabolome contains potential disease biomarkers, some of which are produced endogenously, and some are generated by the gut microbiome. We recently found that Crohn’s Disease (CD) patients in remission still show significantly reduced gut microbial richness and increased dysbiosis compared to healthy controls (PMID: 30741738). Methods Serum untargeted metabolomics, fecal V4 16S microbial characterization, and fecal calprotectin were longitudinally tested in CD patients enrolled in remission. Metabolomics was performed on 139 samples (25 CD and 12 controls) by liquid chromatography coupled with Q-Exactive Orbitrap Mass Spectrometer (LC/MS), and Thermo Xcalibur was used for data acquisition. PERMANOVA on beta diversity was used to define factors affecting serum metabolome. Multivariate analyses in MaAsLin were applied for specific metabolites differential abundance (FDR Results During 24-months of prospective follow-up, 11/25 of the CD patients, who started in remission, flared. PERMANOVA on the serum metabolomic data indicated subject-specific signals. Despite this, using multivariate analyses and controlling for age, gender, and subject, we identified specific metabolites that were significantly different between CD and controls. CD samples were significantly enriched for purine derivatives, simple sugars, histidine and oxalate, compared to healthy controls. In contrast, amino-acids, TCA cycle-related metabolites, saturated fatty acids and acylcarnitines were consistently downregulated in CD. Interestingly, most of those CD-associated metabolic signatures were similar between CD patients with mucosal healing (calprotectin 100, Median 197; IQR 143,300). Interestingly, α-ketoglutarate (aKG) and decanoic-acid were further downregulated in active samples compared to remission (q= 0.2 and 0.16, respectively). Looking into those metabolites in samples prior to the flare, flared patients showed a negative trend in aKG, and a significant reduction in decanoic acid pre-flare (p Conclusion CD patients in remission exhibit a distinct serum metabolic signature, which may be linked in part with the gut microbial diversity and composition. Several metabolite signals may precede disease flare.

Background Microbial dysbiosis is widely described in inflammatory bowel disease (IBD), and has been shown to predict IBD state. However, many other diseases including neuro-psychiatric, metabolic, and malignancies, most of which do not result in gut inflammation, are also linked with gut microbial alteration. Since most studies focus on a single disease, the extent of similarity between different diseases is usually not examined. Methods We reanalyzed raw sequencing data from 12,838 human gut V4 16Sseq samples, spanning 59 case-controls comparisons and 28 unique diseases. Novel statistical approach was applied to reduce the effect of the different cohorts; all samples were processed uniformly, and differentially expressed amplicon sequence variants (ASVs) were identified within each cohort. The resulting behavior (direction of change and effect size) of each ASV were then combined across all studies. We used random forest as our classifier and generated non-specific dysbiosis index (NSDI). Results For the disease prediction, each cohort was randomly subsampled to 23 healthy and 23 disease samples. Random forest classifier was trained on one disease/control cohort, and the trained classifier was then used to predict the status of a different disease/control cohort. Disease classifiers performed well in identifying many sick vs. healthy states but failed to differentiate between different diseases. For example, a classifier trained on IBD cohort classified relatively good also disease/control in lupus, schizophrenia, or Parkinson’s from different cohorts. We show this cross-identification is due to a large number of shared disease-associated bacteria and utilize these bacteria to define a novel non-specific dysbiosis index (NSDI). After, we identified 114 non-disease specific ASVs (86 up and 28 down regulated ASVs across diseases in comparison to controls), we calculate the per-sample NSDI by rank-transforming the bacteria within the sample and computing the normalized log ratio of the sum of the ranks of the 86 down and 28 up regulated ASVs. The resulting NSDI is shown to perform better than the previously published CD dysbiosis index (Gevers et al, 2014; PMID: 24629344) indicating that NSDI can successfully differentiate between most cases and controls across a wide variety of diseases. Conclusion A robust non-specific general response of the gut microbiome is detected across different diseases, some of which is shared with IBD. Classifiers trained on a single disease may identify the general non-specific signal and therefore care should be taken when interpreting the classifier predictions. Finally, our NSDI can be used to prioritize the per-sample degree of dysbiosis.

Background & Aims Environmental enteric dysfunction (EED) limits the Sustainable Development Goals of improved childhood growth and survival. We applied mucosal genomics to advance our understanding of EED. Methods The Study of Environmental Enteropathy and Malnutrition (SEEM) followed 416 children from birth to 24 months in a rural district in Pakistan. Biomarkers were measured at 9 months and tested for association with growth at 24 months. The duodenal methylome and transcriptome were determined in 52 undernourished SEEM participants and 42 North American controls and patients with celiac disease. Results After accounting for growth at study entry, circulating insulin-like growth factor-1 (IGF-1) and ferritin predicted linear growth, whereas leptin correlated with future weight gain. The EED transcriptome exhibited suppression of antioxidant, detoxification, and lipid metabolism genes, and induction of anti-microbial response, interferon, and lymphocyte activation genes. Relative to celiac disease, suppression of antioxidant and detoxification genes and induction of antimicrobial response genes were EED-specific. At the epigenetic level, EED showed hyper-methylation of epithelial metabolism and barrier function genes, and hypo-methylation of immune response and cell proliferation genes. Duodenal coexpression modules showed association between lymphocyte proliferation and epithelial metabolic genes and histologic severity, fecal energy loss, and wasting (weight-for-length/height Z < -2.0). Leptin was associated with expression of epithelial carbohydrate metabolism and stem cell renewal genes. Immune response genes were attenuated by giardia colonization. Conclusions Children with reduced circulating IGF-1 are more likely to experience stunting. Leptin and a gene signature for lymphocyte activation and dysregulated lipid metabolism are implicated in wasting, suggesting new approaches for EED refractory to nutritional intervention. ClinicalTrials.gov, Number: NCT03588013. (https://clinicaltrials.gov/ct2/show/NCT03588013), Graphical abstract

Age-of-diagnosis associated variation in disease location and antimicrobial sero-reactivity has suggested fundamental differences in pediatric Crohn Disease (CD) pathogenesis. This variation may be related to pubertal peak incidence of ileal involvement and Peyer’s patches maturation, represented by IFNγ-expressing Th1 cells. However, direct mucosal evidence is lacking. We characterize the global pattern of ileal gene expression and microbial communities in 525 treatment-naive pediatric CD patients and controls (Ctl), stratifying samples by their age-of-diagnosis. We show a robust ileal gene signature notable for higher expression of specific immune genes including GM-CSF and INFγ, and reduced expression of antimicrobial Paneth cell α-defensins, in older compared to younger patients. Reduced α-defensin expression in older patients was associated with higher IFNγ expression. By comparison, the CD-associated ileal dysbiosis, characterized by expansion of Enterobacteriaceae and contraction of Lachnospiraceae and Ruminococcaceae, was already established within the younger group and did not vary systematically with increasing age-of-diagnosis. Multivariate analysis considering individual taxa, however did demonstrate negative associations between Lachnospiraceae and IFNγ, and positive associations between Bacteroides and α-defensin expression. These data provide evidence for maturation of mucosal Th1 immune responses and loss of epithelial antimicrobial α-defensins which are associated with specific taxa with increasing age-of-diagnosis in pediatric CD.

Molecular mechanisms driving disease course and response to therapy in ulcerative colitis (UC) are not well understood. Here, we use RNAseq to define pre-treatment rectal gene expression, and fecal microbiota profiles, in 206 pediatric UC patients receiving standardised therapy. We validate our key findings in adult and paediatric UC cohorts of 408 participants. We observe a marked suppression of mitochondrial genes and function across cohorts in active UC, and that increasing disease severity is notable for enrichment of adenoma/adenocarcinoma and innate immune genes. A subset of severity genes improves prediction of corticosteroid-induced remission in the discovery cohort; this gene signature is also associated with response to anti-TNFα and anti-α4β7 integrin in adults. The severity and therapeutic response gene signatures were in turn associated with shifts in microbes previously implicated in mucosal homeostasis. Our data provide insights into UC pathogenesis, and may prioritise future therapies for nonresponders to current approaches., The severity of ulcerative colitis, and response to treatment, is highly variable. Here, the authors examine rectal gene expression signatures and faecal microbiomes of children and adults with the disease and provide new insights in to pathogenesis.

The optimal monitoring strategy for predicting disease course in Crohn's disease remains undefined. We aimed to evaluate the accuracy, safety, and tolerability of an intensive monitoring strategy designed to predict the future course of Crohn's disease in patients with quiescent disease.In a prospective observational cohort study, we recruited patients older than 18 years with quiescent (for 3-24 months) Crohn's disease involving the small bowel with confirmed small bowel patency from three tertiary medical centres in Israel. Enrolled patients underwent baseline magnetic resonance enterography (MRE) and patency capsule, clinical or biomarker assessment every 3 months, and video capsule endoscopy (VCE) at baseline and every 6 months for 2 years or until a clinical flare (the primary outcome, defined as an increase in the Crohn's disease activity index score by 70 points or more) or disease worsening necessitating treatment intensification. We assessed the ability of the different Crohn's disease monitoring methods used to predict the occurrence of a flare during the 24-month follow-up period.Of 90 screened patients, 29 were excluded (17 because of non-patent small bowel). Of the 61 patients enrolled between July 3, 2013, and Feb 1, 2015, 17 (28%) had a flare during the 24-month follow-up. No clinicodemographic parameter predicted future flare. A baseline VCE Lewis score of 350 or more identified patients with future flare (area under the curve [AUC] 0·79, 95% CI 0·66-0·88; p0·0001; hazard ratio 10·7, 3·8-30·3). C-reactive protein at baseline had an AUC of 0·73 (0·6-0·84; p=0·0013) for predicting flare. The AUC of baseline faecal calprotectin for the prediction of flare occurring within 2 years was 0·62 (0·49-0·74; p=0·17), but progressively improved for shorter timespans and reached an AUC of 0·81 (0·76-0·85) for the prediction of flare occurring within 3 months. Of four MRE-based indices, only MRE global score correlated with 2-year flare risk (AUC 0·71, 0·58-0·82; p=0·024). During follow-up, a Lewis score increase of 383 points or more from baseline predicted imminent disease exacerbation within 6 months (AUC 0·79, 0·65-0·89; p=0·011). The safety and tolerability of the 231 VCEs ingested was excellent, with none being retained.In patients with quiescent Crohn's disease involving the small bowel, faecal calprotectin predicts short-term flare risk, whereas VCE predicts both short-term and long-term risk of disease exacerbation. If corroborated by additional studies, protocols incorporating VCE could expand the scope of available methods for monitoring disease activity and predicting outcomes in small bowel Crohn's disease.The Leona MHarry B Helmsley Charitable Trust.

Celiac disease is provoked by gluten exposure, but the complete pathogenic process in the duodenum and the loss of tolerance to gluten is not well understood. We aimed to define the core celiac transcriptomic signature and pathologic pathways in pre-treatment formalin-fixed paraffin-embedded (FFPE) duodenum biopsies used for clinical diagnosis. We use mRNAseq to define pre-treatment diagnostic duodenum gene expression in 54 pediatric celiac patients and non-celiac controls, and we validate our key findings in two independent cohorts of 67 adults and pediatric participants that used fresh frozen biopsies. We further define similar and divergent genes and pathways in 177 small bowel Crohn disease patients and controls. We observe a marked suppression of mature epithelial metabolic functions in celiac patients, overlapping substantially with the Crohn disease signature. A marked adaptive immune response was noted for the up-regulated signature including interferon response, alpha-beta, and gamma-delta T-cells that overlapped to some extent with the Crohn disease signature. However, we also identified a celiac disease specific signature linked to increased cell proliferation, nuclear division, and cell cycle activity that was localized primarily to the epithelia as noted by CCNB1 and Ki67 staining. Lastly, we demonstrate the utility of the transcriptomic date to correctly classify disease or healthy states in the discovery and validation cohorts. Our data supplement recently published datasets providing insights into celiac pathogenesis using clinical pathology FFPE samples, and can stimulate new approaches to address this highly prevalent condition.

OBJECTIVES Crohn's disease (CD) is a chronic relapsing-remitting gut inflammatory disorder with a heterogeneous unpredictable course. Dysbiosis occurs in CD; however, whether microbial dynamics in quiescent CD are instrumental in increasing the risk of a subsequent flare remains undefined. METHODS We analyzed the long-term dynamics of microbial composition in a prospective observational cohort of patients with quiescent CD (45 cases, 217 samples) over 2 years or until clinical flare occurred, aiming to identify whether changes in the microbiome precede and predict clinical relapse. Machine learning was used to prioritize microbial and clinical factors that discriminate between relapsers and nonrelapsers in the quiescent phase. RESULTS Patients with CD in clinical, biomarker, and mucosal remission showed significantly reduced microbial richness and increased dysbiosis index compared with healthy controls. Of the 45 patients with quiescent CD, 12 (27%) flared during follow-up. Samples in quiescent patients preceding flare showed significantly reduced abundance of Christensenellaceae and S24.7, and increased abundance of Gemellaceae compared with those in remission throughout. A composite flare index was associated with a subsequent flare. Notably, higher individualized microbial instability in the quiescent phase was associated with a higher risk of a subsequent flare (hazard ratio 11.32, 95% confidence interval 3-42, P = 0.0035) using two preflare samples. Importantly, machine learning prioritized the flare index and the intrapersonal instability over clinical factors to best discriminate between relapsers and nonrelapsers. DISCUSSION Individualized microbial variations in quiescent CD significantly increase the risk of future exacerbation and may provide a model to guide personalized preemptive therapy intensification.

Background Stricturing complications account for substantial morbidity in Crohn’s disease (CD). We aimed to define ileal gene programmes present at diagnosis in paediatric CD associated with future stricturing behaviour (B2), and to identify potential small molecules to reverse these gene signatures. Methods Antimicrobial serologies and ileal gene expression (RNASeq) were assessed at diagnosis in 249 CD patients enrolled in a 5-year inception cohort study. These data were used to define genes associated with stricturing behaviour and for model testing to predict stricturing. Sirius Red immuno-histochemistry was utilised to determine the extent of collagen infiltration into the sub-cryptal space. A bioinformatics approach defined small molecules which may reverse the stricturing gene signature. Results Of 249 (8%) patients, 19 developed B2 behaviour during the 5-year follow-up, while 218 remained B1 inflammatory. We defined 518 genes that were differentially expressed in the ileum at diagnosis (FC≥1.5, FDR Conclusion An ileal gene program for macrophage and fibroblast activation is linked to future stricturing complications in treatment naïve paediatric CD, and may inform small-molecule therapeutic approaches.

Background Crohn disease and coeliac disease are two inflammatory conditions known to cause small intestine inflammation. Using high throughput transcriptomics, microbial, and bioinformatics approaches we aimed to capture differences and similarities linked to the pathogenesis and to future potential interventions for those chronic manifestations. Methods We performed high throughput transcriptomics and 16S microbial characterisation of 55 paediatric new-onset coeliac patients and controls using clinical pathology specimens, and compared those signatures to our previously reported 248 RISK Crohn’s disease newly diagnosed cohort. ToppGene/ToppCluster and ClueGO platforms were used for functional annotation enrichment analyses, and MaAsLin for microbial differential abundance. Results A substantial number (>90%) of genes passed the expression filtering criteria in both studies enabling the comparison. Of the 354 coeliac down-regulated genes, 59% (209/354) overlapped with the reduced Crohn signature. Shared reduced signatures and functions included a decrease in epithelial lipid metabolism, oxidoreductase activity, and brush border transport signatures. In contrast, a significantly smaller proportion [19% (97/427, Chi-squares p < 0.001] of the coeliac disease 524 up-regulated genes overlapped with the induced Crohn disease signature. We noted shared enriched signatures for adaptive immune-related pathways and interferon-γ in both coeliac and Crohn diseases. However, the Crohn disease signature exhibited more specific enrichments for signatures associated with innate immune pathways and with a strong signal for granulocytes, an extracellular matrix signature, and CXCR chemokines signalling, while the coeliac up-regulated signature showed unique enrichment for cell cycle and mitosis. As opposed to the robust dysbiosis previously characterised in Crohn disease, we were only able to identify significant enrichment for Bacteroidetes taxa in coeliac patients in comparison to controls. Conclusion We highlight important biologic differences between Crohn and coeliac diseases emphasising an intensified innate granulocytes activation signature in Crohn disease and a specific epithelial proliferative signal in coeliac disease. Unlike the robust dysbiosis linked to Crohn disease, the coeliac patient showed only modest enrichment for several Bacteroidetes taxa in comparison to controls. It is possible that microbial alteration in Crohn disease triggers granulocytes activation, and that this signal inhibits epithelial proliferation/renewal, eventually leading the epithelial damage seen in Crohn but not coeliac disease. Inhibiting innate immune activation or reverting Crohn dysbiosis may be a beneficial future therapy for Crohn disease.

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease. Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample. Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

Background Long noncoding RNAs (lncRNA) are key regulators of gene transcription and many show tissue-specific expression. We previously defined a novel inflammatory and metabolic ileal gene signature in treatment-naive pediatric Crohn disease (CD). We now extend our analyses to include potential regulatory lncRNA. Methods Using RNAseq, we systematically profiled lncRNAs and protein-coding gene expression in 177 ileal biopsies. Co-expression analysis was used to identify functions and tissue-specific expression. RNA in situ hybridization was used to validate expression. Real-time polymerase chain reaction was used to test lncRNA regulation by IL-1β in Caco-2 enterocytes. Results We characterize widespread dysregulation of 459 lncRNAs in the ileum of CD patients. Using only the lncRNA in discovery and independent validation cohorts showed patient classification as accurate as the protein-coding genes, linking lncRNA to CD pathogenesis. Co-expression and functional annotation enrichment analyses across several tissues and cell types 1showed that the upregulated LINC01272 is associated with a myeloid pro-inflammatory signature, whereas the downregulated HNF4A-AS1 exhibits association with an epithelial metabolic signature. We confirmed tissue-specific expression in biopsies using in situ hybridization, and validated regulation of prioritized lncRNA upon IL-1β exposure in differentiated Caco-2 cells. Finally, we identified significant correlations between LINC01272 and HNF4A-AS1 expression and more severe mucosal injury. Conclusions We systematically define differentially expressed lncRNA in the ileum of newly diagnosed pediatric CD. We show lncRNA utility to correctly classify disease or healthy states and demonstrate their regulation in response to an inflammatory signal. These lncRNAs, after mechanistic exploration, may serve as potential new tissue-specific targets for RNA-based interventions.

Hospitalized patients are at increased risk for acquiring healthcare-associated infections (HAIs) and inadequate nutrition. The human intestinal microbiota plays vital functions in nutrient supply and protection from pathogens, yet characterization of the microbiota of hospitalized patients is lacking. We used 16S rRNA amplicon sequencing to characterize the global pattern of microbial composition of fecal samples from 196 hospitalized patients with suspected infectious diarrhea in comparison to healthy, non-hospitalized subjects (n = 881), and to traditional culture results. We show that hospitalized patients have a significant rise in α-diversity (richness within sample) from birth to Campylobacter, Salmonella, or Shigella entero-pathogens in traditional culturing methods. As hospitalized patients have increased risk for HAIs and inadequate nutrition, our data support the consideration of nutritional and/or microbial modification in this population.

Background Avian avulavirus-1 (AAvV-1, previously Newcastle Disease Virus) is responsible for poultry and wild birds' disease outbreaks. Numerous whole genome sequencing methods were reported for this virus. These methods included cloning, specific primers amplification, shotgun PCR approaches, Sequence Independent Single Primer Amplification and next generation sequencing platform kits. Methods Three methods were used to sequence 173 Israeli Avian avulavirus-1 field isolates and one vaccine strain (VH). The sequencing was performed on Proton and Ion Torrent Personal Genome Machine and to a lesser extent, Illumina MiSeq and NextSeq sequencers. Target specific primers (SP) and Sequence Independent Single Primer Amplification (SISPA) products sequenced via the Ion torrent sequencer had a high error rate and truncated genomes. All the next generation sequencing platform sequencing kits generated high sequence accuracy and near-complete genomic size. Results A high level of mutations was observed in the intergenic regions between the avian avulavirus-1 genes. Within genes, multiple regions are more mutated than the Fusion region currently used for typing. Conclusions Our findings suggest that the whole genome sequencing by the Ion torrent sequencing kit is sufficient. However, when higher fidelity is desired, the Illumina NextSeq and Proton torrent sequencing kits were found to be preferable., Highlights • PCR amplification of the viral genome, results in partial genome sequences (72% and 98% respectively). • Primer based sequencing methods have 5 to 5.6 times more N reads compared to NGS platform sequencing kits, indicating poor sequence quality. • The Ion Torrent PGM sequencing results in sequencing 99% of the viral genome. • ProtonTorrent and Illumina Nextseq sequenced 99-100% of the viral genome, leading a to full genome sequence at high coverage. • The virus is mostly mutated within a host at intergenic regions.

Emerging data indicate a correlation between gut microbial composition and cardiovascular disease including hypertension. The host&rsquo, s diet greatly affects microbial composition and metabolite production. Short chain fatty acids (SCFAs) are products of microbial fermentation, which can be utilized by the host. It has been suggested that SCFAs play a pivotal role as mediators in a microbiome host: microbial interactions occur in health and disease. The aim of this study was to evaluate the effect of a high salt diet (HSD) on microbial variation and to determine whether this effect is accompanied by an alteration in fecal SCFAs. To this end, Dahl salt-sensitive rats were divided into two groups (n = 10 each): (A) Control: fed regular chow, and (B) Fed HSD. High-throughput pyrosequencing of the 16S rRNA amplicon sequencing was used for microbiome characterizing. Chromatography-mass spectrometry was used to measure the levels of SCFAs: acetic acid, propionic acid, butyric acid, and isobutyric acid in fecal samples. Differences in microbial composition were noted between groups. Principal Coordinate Analysis (PCoA) principal coordinate 1 (PC1) primarily separated controls from the HSD. Four taxa displayed significant differences between HSD and controls. Taxa from the Erwinia genus, the Christensenellaceae and Corynebacteriaceae families, displayed an increased abundance in HSD versus control. In contrast, taxa from the Anaerostipes genus displayed a decreased abundance in HSD. We were able to identify seven unique taxa that were significantly associated with blood pressure. There was a significant difference in fecal acetic acid, as well as propionic and isobutyric acid, but not in the butyric acid composition between groups. Adding salt to a diet impacts the gut&rsquo, s microbial composition, which may alter fecal SCFA production.

Objective:Emerging data demonstrate correlation between microbiota and cardiovascular disease including hypertension. The microbiota is exposed to the host diet, thus different diet components may change microbiota composition and metabolites production. Short chain fatty acids (SCFAs) are major pro