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ObjectiveLoss-of-function mutations in genes generating reactive oxygen species (ROS), such asNOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined.DesignROS measurements and single-cell transcriptomics were performed on colonoids stratified byNOX1genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury.ResultsTNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta),DNMT1(DNA methyltransferase) andHIF1A(hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstratedGP2(glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation.ConclusionsNOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.

Summary An important goal of clinical genomics is to be able to estimate the risk of adverse disease outcomes. Between 5% and 10% of individuals with ulcerative colitis (UC) require colectomy within 5 years of diagnosis, but polygenic risk scores (PRSs) utilizing findings from genome-wide association studies (GWASs) are unable to provide meaningful prediction of this adverse status. By contrast, in Crohn disease, gene expression profiling of GWAS-significant genes does provide some stratification of risk of progression to complicated disease in the form of a transcriptional risk score (TRS). Here, we demonstrate that a measured TRS based on bulk rectal gene expression in the PROTECT inception cohort study has a positive predictive value approaching 50% for colectomy. Single-cell profiling demonstrates that the genes are active in multiple diverse cell types from both the epithelial and immune compartments. Expression quantitative trait locus (QTL) analysis identifies genes with differential effects at baseline and week 52 follow-up, but for the most part, differential expression associated with colectomy risk is independent of local genetic regulation. Nevertheless, a predicted polygenic transcriptional risk score (PPTRS) derived by summation of transcriptome-wide association study (TWAS) effects identifies UC-affected individuals at 5-fold elevated risk of colectomy with data from the UK Biobank population cohort studies, independently replicated in an NIDDK-IBDGC dataset. Prediction of gene expression from relatively small transcriptome datasets can thus be used in conjunction with TWASs for stratification of risk of disease complications.

Background & Aims Recent literature has implicated a key role for mast cells in murine models of colonic inflammation, but their role in human ulcerative colitis (UC) is not well established. A major advance has been the identification of mrgprb2 (human orthologue, MRGPX2) as mediating IgE-independent mast cell activation. We sought to define mechanisms of mast cell activation and MRGPRX2 in human UC. Methods Colon tissues were collected from patients with UC for bulk RNA sequencing and lamina propria cells were isolated for MRGPRX2 activation studies and single-cell RNA sequencing. Genetic association of all protein-altering G-protein coupled receptor single-nucleotide polymorphism was performed in an Ashkenazi Jewish UC case-control cohort. Variants of MRGPRX2 were transfected into Chinese hamster ovary (CHO) and human mast cell (HMC) 1.1 cells to detect genotype-dependent effects on β-arrestin recruitment, IP-1 accumulation, and phosphorylated extracellular signal–regulated kinase. Results Mast cell-specific mediators and adrenomedullin (proteolytic precursor of PAMP-12, an MRGPRX2 agonist) are up-regulated in inflamed compared to uninflamed UC. MRGPRX2 stimulation induces carboxypeptidase secretion from inflamed UC. Of all protein-altering GPCR alleles, a unique variant of MRGPRX2, Asn62Ser, was most associated with and was bioinformatically predicted to alter arrestin recruitment. We validated that the UC protective serine allele enhances β-arrestin recruitment, decreases IP-1, and increases phosphorylated extracellular signal–regulated kinase with MRGPRX2 agonists. Single-cell RNA sequencing defines that adrenomedullin is expressed by activated fibroblasts and epithelial cells and that interferon gamma is a key upstream regulator of mast cell gene expression. Conclusion Inflamed UC regions are distinguished by MRGPRX2-mediated activation of mast cells, with decreased activation observed with a UC-protective genetic variant. These results define cell modules of UC activation and a new therapeutic target.

Background and Aims Crohn’s disease [CD] recurrence following ileocolic resection [ICR] is common. We sought to identify blood-based biomarkers associated with CD recurrence. Methods CD patients undergoing ICR were recruited across six centres. Serum samples were obtained at post-operative colonoscopy. A multiplex immunoassay was used to analyse 92 inflammation-related proteins [Olink Proteomics]. Bayesian analysis was used to identify proteins associated with increasing Rutgeerts score. Identified proteins were used in receiver operating characteristic [ROC] analysis to examine the ability to identify CD recurrence [Rutgeerts score ≥i2]. Existing single cell data were interrogated to further elucidate the role of the identified proteins. Results Data from 276 colonoscopies in 213 patients were available. Median time from surgery to first and second colonoscopy was 7 (interquartile range [IQR] 6–9) and 19 [IQR 16–23] months, respectively. Disease recurrence was evident at 60 [30%] first and 36 [49%] second colonoscopies. Of 14 proteins significantly associated with Rutgeerts score, the strongest signal was seen for CXCL9 and MMP1. Among patients on anti-tumour necrosis factor drugs, CXCL9 and CXCL11 were most strongly associated with Rutgeerts score. Both are CXCR3 ligands. Incorporation of identified proteins into ROC analysis improved the ability to identify disease recurrence as compared to C-reactive protein alone: area under the curve [AUC] 0.75 (95% confidence interval [CI]: 0.66–0.82] vs 0.64 [95% CI 0.56–0.72], p = 0.012. Single cell transcriptomic data provide evidence that innate immune cells are the primary source of the identified proteins. Conclusions CXCR3 ligands are associated with CD recurrence following ICR. Incorporation of novel blood-based candidate biomarkers may aid in identification of CD recurrence.

Liver fibrosis is the excessive accumulation of extracellular matrix that can progress to cirrhosis and failure if untreated (1). The mechanisms of fibrogenesis are multi-faceted and remain elusive with no approved antifibrotic treatments available (2). Here we use single-cell RNA sequencing (scRNA-seq) of the adult zebrafish liver to study the molecular and cellular dynamics of the liver at a single-cell level and demonstrate the value of the adult zebrafish as a model for studying liver fibrosis. scRNA-seq reveals transcriptionally unique populations of hepatic cell types that comprise the zebrafish liver. Joint clustering with human liver scRNA-seq data demonstrates high conservation of transcriptional profiles and human marker genes in zebrafish cell types. Human and zebrafish hepatic stellate cells (HSCs), the driver cell in liver fibrosis (3), specifically show conservation of transcriptional profiles and we uncover Colec11 as a novel, conserved marker for zebrafish HSCs. To demonstrate the power of scRNA-seq to study liver fibrosis, we performed scRNA-seq on our zebrafish model of a pediatric liver disease with characteristic early, progressive liver fibrosis caused by mutation in mannose phosphate isomerase (MPI) (4–6). Comparison of differentially expressed genes from human and zebrafish MPI mutant HSC datasets demonstrated similar activation of fibrosis signaling pathways and upstream regulators. CellPhoneDB analysis revealed important receptor-ligand interactions within normal and fibrotic states. This study establishes the first scRNA-seq atlas of the adult zebrafish liver, highlights the high degree of similarity to the human liver, and strengthens its value as a model to study liver fibrosis.Significance StatementTo our knowledge, this is the first single-cell characterization of the adult zebrafish liver, both in a normal physiologic state and in the setting of liver fibrosis. We identify transcriptionally distinct zebrafish liver cell populations and a high degree of transcriptional conservation between human and zebrafish cells across the majority of hepatic cell types. Furthermore, using this scRNA transcriptome, we identify key signaling pathways in zebrafish HSCs that are replicated in human HSCs and implicated in the regulation of liver fibrosis. Our work provides a useful resource that can be used to aid research using the zebrafish liver and asserts the usefulness of the adult zebrafish to study liver fibrosis.

Genome-wide association studies (GWAS) have identified hundreds of loci associated with Crohns disease (CD), however, as with all complex diseases, deriving pathogenic mechanisms from these non-coding GWAS discoveries has been challenging. To complement GWAS and better define actionable biological targets, we analysed sequenced data from more than 30,000 CD patients and 80,000 population controls. We observe rare coding variants in established CD susceptibility genes as well as ten genes where coding variation directly implicates the gene in disease risk for the first time.

SUMMARYAn important goal of clinical genomics is to be able to estimate the risk of adverse disease outcomes. Between 5% and 10% of ulcerative colitis (UC) patients require colectomy within five years of diagnosis, but polygenic risk scores (PRS) utilizing findings from GWAS are unable to provide meaningful prediction of this adverse status. By contrast, in Crohn’s disease, gene expression profiling of GWAS-significant genes does provide some stratification of risk of progression to complicated disease in the form of a Transcriptional Risk Score (TRS). Here we demonstrate that both measured (TRS) and polygenic predicted gene expression (PPTRS) identify UC patients at 5-fold elevated risk of colectomy with data from the PROTECT clinical trial and UK Biobank population cohort studies, independently replicated in an NIDDK-IBDGC dataset. Prediction of gene expression from relatively small transcriptome datasets can thus be used in conjunction with transcriptome-wide association studies to stratify risk of disease complications.

Crohn's disease is a chronic inflammatory intestinal disease that is frequently accompanied by aberrant healing and stricturing complications. Crosstalk between activated myeloid and stromal cells is critical in the pathogenicity of Crohn's disease1,2, and increases in intravasating monocytes are correlated with a lack of response to anti-TNF treatment3. The risk alleles with the highest effect on Crohn's disease are loss-of-function mutations in NOD24,5, which increase the risk of stricturing6. However, the mechanisms that underlie pathogenicity driven by NOD2 mutations and the pathways that might rescue a lack of response to anti-TNF treatment remain largely uncharacterized. Here we use direct ex vivo analyses of patients who carry risk alleles of NOD2 to show that loss of NOD2 leads to dysregulated homeostasis of activated fibroblasts and macrophages. CD14+ peripheral blood mononuclear cells from carriers of NOD2 risk alleles produce cells that express high levels of collagen, and elevation of conserved signatures is observed in nod2-deficient zebrafish models of intestinal injury. The enrichment of STAT3 regulation and gp130 ligands in activated fibroblasts and macrophages suggested that gp130 blockade might rescue the activated program in NOD2-deficient cells. We show that post-treatment induction of the STAT3 pathway is correlated with a lack of response to anti-TNF treatment in patients, and demonstrate in vivo in zebrafish the amelioration of the activated myeloid-stromal niche using the specific gp130 inhibitor bazedoxifene. Our results provide insights into NOD2-driven fibrosis in Crohn's disease, and suggest that gp130 blockade may benefit some patients with Crohn's disease-potentially as a complement to anti-TNF therapy.

BACKGROUND AND AIMS: Polygenic risk scores (PRS) may soon be used to predict inflammatory bowel disease (IBD) risk in prevention efforts. We leveraged exome-sequence and single nucleotide polymorphism (SNP) array data from 29,358 individuals in the multiethnic, randomly ascertained health system-based BioMe biobank to define effects of common and rare IBD variants on disease prediction and pathophysiology. METHODS: PRS were calculated from European, African American, and Ashkenazi Jewish (AJ) reference case-control studies, and a meta-GWAS run using all three association datasets. PRS were then combined using regression to assess which combination of scores best predicted IBD status in European, AJ, Hispanic, and African American cohorts in BioMe. Additionally, rare variants were assessed in genes associated with very early-onset IBD (VEO-IBD), by estimating genetic penetrance in each BioMe population. RESULTS: Combining risk scores based on association data from distinct ancestral populations improved IBD prediction for every population in BioMe and significantly improved prediction among European ancestry UK Biobank individuals. Lower predictive power for non-Europeans was observed, reflecting in part substantially lower African IBD case-control reference sizes. We replicated associations for two VEO-IBD genes, ADAM17 and LRBA, with high dominant model penetrance in BioMe. Autosomal recessive LRBA risk alleles are associated with severe, early-onset autoimmunity; we show that heterozygous carriage of an African-predominant LRBA protein-altering allele is associated with significantly decreased LRBA and CTLA-4 expression with T-cell activation. CONCLUSIONS: Greater genetic diversity in African populations improves prediction across populations, and generalizes some VEO-IBD genes. Increasing African American IBD case-collections should be prioritized to reduce health disparities and enhance pathophysiological insight.

Duchenne muscular dystrophy is initiated by dystrophin deficiency, but downstream pathophysiological pathways such as membrane instability, NFĸB activation, mitochondrial dysfunction, and induction of TGFβ fibrosis pathways are thought to drive the disability. Dystrophin replacement strategies are hopeful for addressing upstream dystrophin deficiency; however, all methods to date use semi-functional dystrophin proteins that are likely to trigger downstream pathways. Thus, combination therapies that can target multiple downstream pathways are important in treating DMD, even for dystrophin-replacement strategies. We sought to define blood pharmacodynamic biomarkers of drug response in the mdx mouse model of Duchenne muscular dystrophy using a series of repurposed drugs. Four-week-old mdx mice were treated for four weeks with four different drugs singly and in combination: vehicle, prednisolone, vamorolone, rituximab, β-aminoisobutyric acid (BAIBA) (11 treatment groups; n = 6/group). Blood was collected via cardiac puncture at study termination, and proteomic profiling was carried out using SOMAscan aptamer panels (1,310 proteins assayed). Prednisolone was tested alone and in combination with other drugs. It was found to have a good concordance of prednisolone-responsive biomarkers (56 increased by prednisolone, 39 decreased) focused on NFκB and TGFβ cascades. Vamorolone shared 45 (80%) of increased biomarkers and 13 (33%) of decreased biomarkers with prednisolone. Comparison of published human corticosteroid-responsive biomarkers to our mdx data showed 14% (3/22) concordance between mouse and human. Rituximab showed fewer drug-associated biomarkers, with the most significant being human IgG. On the other hand, BAIBA treatment (high and low dose) showed a drug-associated increase in 40 serum proteins and decreased 5 serum proteins. Our results suggest that a biomarker approach could be employed for assessing drug combinations in both mouse and human studies.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6‐months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease., Heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu induce the integrated stress response and metabolic rearrangements, reducing penetrance and promoting spontaneous recovery in a rare mitochondrial myopathy.

Inflammatory bowel disease (IBD) is a group of chronic diseases, affecting different parts of the gastrointestinal tract, that mainly comprises Crohn’s Disease (CD) and Ulcerative Colitis (UC). Most IBD genomic research to date has involved genome-wide association studies (GWAS) of common genetic variants, mostly in Europeans, resulting in the identification of over 200 risk loci. The incidence of IBD in Ashkenazi Jews (AJ) is particularly high compared to other population groups and rare protein-coding variants are significantly enriched in AJ. These variants are expected to have a larger phenotypic effect and are hypothesized to complement the missing heritability that cannot be fully addressed by GWAS in IBD. Therefore, we genetically identified 4,974 AJs IBD cases and controls from whole exome sequencing (WES) data from the NIDDK IBD Genetics Consortium (IBDGC). We selected credible rare variants with high predicted impact, aggregated them into genes, and performed gene burden and pathway enrichment analyses to identify 7 novel plausible IBD-causing genes:NCF1, CES1, ICAM1, INPP5D, ABCB1, IL33andTLR4. We further perform bulk and single-cell RNA sequencing, demonstrating the likely relatedness of the novel genes to IBD. Importantly, we demonstrate that the rare and high impact genetic architecture of AJ adult IBD displays a significant overlap with very early onset IBD (VEOIBD) genetics. At the variant level, we performed Phenome-wide association studies (PheWAS) in the UK Biobank to replicate risk sites in IBD and reveal shared risk sites with other diseases. Finally, we showed that a polygenic risk score (PRS) has high power to differentiate AJ IBD cases from controls when using rare and high impact variants.

Genome-wide association studies have identified ~170 loci associated with Crohn's disease (CD) and defining which genes drive these association signals is a major challenge. The primary aim of this study was to define which CD locus genes are most likely to be disease related. We developed a gene prioritization regression model (GPRM) by integrating complementary mRNA expression datasets, including bulk RNA-Seq from the terminal ileum of 302 newly diagnosed, untreated CD patients and controls, and in stimulated monocytes. Transcriptome-wide association and co-expression network analyses were performed on the ileal RNA-Seq datasets, identifying 40 genome-wide significant genes. Co-expression network analysis identified a single gene module, which was substantially enriched for CD locus genes and most highly expressed in monocytes. By including expression-based and epigenetic information, we refined likely CD genes to 2.5 prioritized genes per locus from an average of 7.8 total genes. We validated our model structure using cross-validation and our prioritization results by protein-association network analyses, which demonstrated significantly higher CD gene interactions for prioritized compared with non-prioritized genes. Although individual datasets cannot convey all of the information relevant to a disease, combining data from multiple relevant expression-based datasets improves prediction of disease genes and helps to further understanding of disease pathogenesis.

PurposeWhole body insulin sensitivity (Si) typically improves after aerobic exercise training; however, individual responses can be highly variable. The purpose of this study was to use global gene expression to identify skeletal muscle genes that correlate with exercise-induced Si changes.M

We recently evaluated two of the original three patients (siblings) diagnosed with Proximal Myopathy with Focal Depletion of Mitochondria. The condition was named for the distinctive pattern of enlarged mitochondria around the periphery of muscle fibres with a complete absence in the middle. These siblings, aged 37 and 40, are cognitively normal with mild non-progressive muscle weakness and a susceptibility to rhabdomyolysis. Both were shown to be compound heterozygotes for novel mutations (c.263C>T + c.950T>A) in CHKB , the gene currently associated with Megaconial Congenital Muscular Dystrophy. Individuals with this condition have early-onset muscle weakness and profound intellectual disability but share the same unique pattern on muscle biopsy as was noted in Proximal Myopathy with Focal Depletion of Mitochondria; focal depletion of mitochondria was surrounded by abnormally large "megaconial" mitochondria. Thus the phenotypic spectrum of CHKB mutations ranges from a congenital muscular dystrophy with intellectual disability to a later-onset non-progressive muscular weakness with normal cognition.

SummaryClinical benefits to cytokine blockade in ileal Crohn’s disease (iCD) have been limited to a subset of patients. Whether cellular and molecular heterogeneity contributes to variability in treatment responses has been unclear. Using single cell technologies combining scRNAseq, CyTOF and multiplex tissue imaging, we mapped the cellular landscape of inflamed ileum lesions, adjacent non-inflamed ileum and matched circulating blood cells of iCD patients. In inflamed tissues, we identified a pathogenic module characterized by an inflammatory mononuclear phagocyte (Inf.MNP)-associated cellular response organized around inflammatory macrophages and mature dendritic cells in a subset of iCD patients. We confirmed the Inf.MNP-associated cellular response in 4 independent iCD cohorts (n=441) and showed that presence of this pathogenic module at diagnosis correlated with primary resistance to anti-TNF therapy. Single cell mapping of iCD tissues identifies a complex cellular signature of anti-TNF resistance thereby revealing novel biomarkers of treatment response and tailored therapeutic opportunities.

Genome-wide association studies have identified over 200 genomic loci associated with inflammatory bowel disease (IBD). High-effect risk alleles define key roles for genes involved in bacterial response and innate defense. More high-throughput in vivo systems are required to rapidly evaluate therapeutic agents. We visualize, in zebrafish, the effects on epithelial barrier function and intestinal autophagy of one-course and repetitive injury. Repetitive injury induces increased mortality, impaired recovery of intestinal barrier function, failure to contain bacteria within the intestine and impaired autophagy. Prostaglandin E2 (PGE2) administration protected against injury by enhancing epithelial barrier function and limiting systemic infection. Effects of IBD therapeutic agents were defined: mesalamine showed protective features during injury, whereas 6-mercaptopurine displayed marked induction of autophagy during recovery. Given the highly conserved nature of innate defense in zebrafish, it represents an ideal model system with which to test established and new IBD therapies targeted to the epithelial barrier. This article has an associated First Person interview with the first author of the paper., Summary: As IBD attempts to enter the era of precision medicine, our models provide an opportunity for the rapid and sophisticated determination of time course, cellular contributions and mechanisms of new drugs.

In this paper, we present miRTarVis+, a Web-based interactive visual analytics tool for miRNA target predictions and integrative analyses of multiple prediction results. Various microRNA (miRNA) target prediction algorithms have been developed to improve sequence-based miRNA target prediction by exploiting miRNA-mRNA expression profile data. There are also a few analytics tools to help researchers predict targets of miRNAs. However, there still is a need for improving the performance for miRNA prediction algorithms and more importantly for interactive visualization tools for an integrative analysis of multiple prediction results. miRTarVis+ has an intuitive interface to support the analysis pipeline of load, filter, predict, and visualize. It can predict targets of miRNA by adopting Bayesian inference and maximal information-based nonparametric exploration (MINE) analyses as well as conventional correlation and mutual information analyses. miRTarVis+ supports an integrative analysis of multiple prediction results by providing an overview of multiple prediction results and then allowing users to examine a selected miRNA-mRNA network in an interactive treemap and node-link diagram. To evaluate the effectiveness of miRTarVis+, we conducted two case studies using miRNA-mRNA expression profile data of asthma and breast cancer patients and demonstrated that miRTarVis+ helps users more comprehensively analyze targets of miRNA from miRNA-mRNA expression profile data. miRTarVis+ is available at http://hcil.snu.ac.kr/research/mirtarvisplus.

Summary Clinical benefits of cytokine blockade in ileal Crohn’s disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.

Background Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs) that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV) to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome) and examine the changes during rhinovirus (RV) infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood. Methods Nasal airway secretions were obtained from children (≤3 yrs. old) during PCR-confirmed RV infections (n = 10) and age-matched controls (n = 10). Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC) differentiated at air-liquid interface (ALI). Bioinformatics tools were used to determine the unified (nasal and bronchial) signature airway secretory miRNAome and changes during RV infection in children. Results Multiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV. Conclusions Comparative analysis of the airway secretory microRNAome in children indicates that RV infection is associated with airway secretion of EVs containing miR-155, which is predicted in silico to regulate antiviral immunity. Further characterization of the airway secretory microRNAome during health and disease may lead to completely new strategies to treat and monitor respiratory conditions in all ages.

Background. BP1 (Beta Protein 1) belongs to the Distal-less family of homeobox genes. We have demonstrated that BP1 is activated in over 80% of invasive ductal breast tumors, where it is associated with breast cancer progression. The mechanism(s) of BP1 involvement in breast cancer progression, invasion and metastasis are still not known. Homeobox genes contribute to the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells acquire mesenchymal features which lead to motility, invasiveness and resistance to apoptosis. EMT is also characterized by changes in apico-basal polarity and a dramatic remodeling of the cytoskeleton. During progression toward metastasis, cancer cells acquire a mesenchymal gene expression phenotype and increased motility. This transition allows the tumor cells to metastasize and establish secondary tumors at distant sites. One of the drivers of the EMT is the transcription factor Twist. Twist is a member of highly conserved family of basic helix-loop-helix transcription factors and is involved in the specification and differentiation of mesenchymal tissue in embryos. Twist overexpression can induce EMT, generate cancer stem cells, and promote metastasis in vivo. We hypothesized that BP1 might promote cancer metastasis and invasiveness mediated though Twist and the EMT. Methods. We generated BP1 overexpressing cell lines, MCF-7/BP1 and HS578T/BP1, along with the associated vector controls. Expression of BP1 was tested by both protein and transcript levels by Western blotting and qRT-PCR, respectively. BP1 binding to Twist was assayed by chromatin immunoprecipitation. Confocal microscopy was used to localize BP1 expression and Boyden chamber assays were employed to assess the migratory and invasive ability of these cells. ELISA assays were employed to study IL6 expression in BP1 expressing cells. Results. BP1 expression led to an increase in Twist expression. Mechanistically, we demonstrated that BP1 bound proximally to the Twist promoter and regulated its expression. BP1 induced an EMT in both cell lines as seen by (a) lack of expression of E-cadherin, and an increase of vimentin and fibronectin expression, as well as (b) morphological changes, including a spindle-like, and more migratory phenotype. We also observed that BP1 induced expression of the cytokine IL6 in both cell lines. We conclude that BP1 regulates the EMT in breast cancer cells via the Twist and IL6 pathways. Citation Format: Farhad Vesuna, Bin-Jin Hwang, Jinguen Rheey, Mamta Giri, Mandeep Gill, Sidney W. Fu, Ashley Irving, Ala Lisok, Yehudit Bergman, Venu Raman, Patricia E. Berg. BP1 induces an epithelial to mesenchymal transition in breast cancer cells by modulating the Twist/IL6 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3338.

Two patients with exercise-induced myalgias and rhabdomyolysis with myoglobinuria were evaluated with muscle biopsy and comprehensive myopathy next generation sequencing (NGS) gene panels. Genetic analysis revealed homozygosity for two known pathogenic SGCA mutations (R284C in Patient 1 and V247M in Patient 2). Muscle biopsy showed minimal changes with normal immunohistochemistry for α-sarcoglycan. Western blotting showed 27% and 35% of normal α-sarcoglycan immunoreactivity when compared to age matched controls, confirming the diagnosis of α-sarcoglycanopathy in both patients. The sarcoglycan genes should be added to the differential diagnosis for cases that present with rhabdomyolysis, exercise intolerance, and hyperCKemia, even in the absence of muscle weakness or normal α-sarcoglycan immunohistochemistry. Work-up of patients with these types of non-specific presentation may be best facilitated through the use of non-specific NGS myopathy panels.

Epigenomic regulation of the transcriptome by DNA methylation and posttranscriptional gene silencing by miRNAs are potential environmental modulators of skeletal muscle plasticity to chronic exercise in healthy and diseased populations. We utilized transcriptome networks to connect exercise-induced differential methylation and miRNA with functional skeletal muscle plasticity. Biopsies of the vastus lateralis were collected from middle-aged Polynesian men and women with morbid obesity (44 kg/m2 ± 10) and Type 2 diabetes before and following 16 wk of resistance ( n = 9) or endurance training ( n = 8). Longitudinal transcriptome, methylome, and microRNA (miRNA) responses were obtained via microarray, filtered by novel effect-size based false discovery rate probe selection preceding bioinformatic interrogation. Metabolic and microvascular transcriptome topology dominated the network landscape following endurance exercise. Lipid and glucose metabolism modules were connected to: microRNA (miR)-29a; promoter region hypomethylation of nuclear receptor factor ( NRF1) and fatty acid transporter ( SLC27A4), and hypermethylation of fatty acid synthase, and to exon hypomethylation of 6-phosphofructo-2-kinase and Ser/Thr protein kinase. Directional change in the endurance networks was validated by lower intramyocellular lipid, increased capillarity, GLUT4, hexokinase, and mitochondrial enzyme activity and proteome. Resistance training also lowered lipid and increased enzyme activity and caused GLUT4 promoter hypomethylation; however, training was inconsequential to GLUT4, capillarity, and metabolic transcriptome. miR-195 connected to negative regulation of vascular development. To conclude, integrated molecular network modelling revealed differential DNA methylation and miRNA expression changes occur in skeletal muscle in response to chronic exercise training that are most pronounced with endurance training and topographically associated with functional metabolic and microvascular plasticity relevant to diabetes rehabilitation.

Although it is well known and identified that drug-drug interactions exist, the recognition of importance of food and drug interactions to practice has been growing much slower. On the other hand, drug-food/nutrient interactions continue to grow with the common use of medications. Beside the awareness of this type of interactions, food-drug interaction studies are critical to evaluate appropriate dosing, timing, and formulation of new drug candidates. Drug-food interactions take place mechanistically due to altered intestinal transport and metabolism, or systemic distribution, metabolism and excretion. In addition, some people have greater risk of food and drug interactions that have a poor diet, have serious health problems, children's and pregnant women. In this article, basic information's about importance, classifications, transporters and enzymes of drug and nutrient interaction are given and some specific examples of both drug and nutrients and influences on each other are included.

BACKGROUND Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. OBJECTIVE To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. METHODS We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. RESULTS We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. CONCLUSIONS Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.