Colon Epithelial MicroRNA Network in Fatty Liver.

Background & Aims: Intestinal barrier alterations are associated with fatty liver (FL) and metabolic syndrome (MetS), but microRNA (miR) signaling pathways in MetS-FL pathogenesis remain unclear. This study investigates an epithelial-focused miR network in colorectal cell models based on the previously reported MetS-FL miR trio of hsa-miR-142-3p, hsa-miR-18b, and hsa-miR-890. Methods: Each miR mimic construct of MetS-FL miR trio was transfected into human colorectal cells, CRL-1790 or Caco-2. Global miRNome changes posttransfection were profiled (nCounter® Human v3 miRNA, NanoString Technologies). Changes in barrier (transepithelial electrical resistance, TEER) and epithelial cell junction structure (Occludin and Zona Occludens-1/ZO-1 immunofluorescence staining-confocal microscopy) were examined pre- and posttransfection in Caco-2 cell monolayers. A signaling network was constructed from the MetS-FL miR trio, MetS-FL miR-induced colorectal miRNome changes, ZO-1, and Occludin. Results: Transfection of CRL-1790 cells with each MetS-FL miR mimic led to global changes in the cellular miRNome profile, with 288 miRs being altered in expression by more than twofold. Eleven miRs with known cytoskeletal and metabolic roles were commonly altered in expression by all three miR mimics. Transfection of Caco-2 cell monolayers with each MetS-FL miR mimic induced barrier-associated TEER variations and led to structural modifications of ZO-1 and Occludin within epithelial cell junctions. Pathway analysis incorporating the MetS-FL miR trio, eleven common target miRs, ZO-1, and Occludin revealed a signaling network centered on TNF and AKT2, which highlights injury, inflammation, and hyperplasia. Conclusions: Colon-specific changes in epithelial barriers, cell junction structure, and a miRNome signaling network are described from functional studies of a MetS-FL miR trio signature.

Eating Behavior, Stress, and Adiposity: Discordance Between Perception and Physiology.

The purpose of the study was to examine the interrelationships among stress, eating behavior, and adiposity in a cohort of normal- and overweight individuals. Clinical markers of physiological stress (fasting serum cortisol) and adiposity (body mass index [BMI] and percent body fat) were obtained from participants selected for a natural history protocol ( n = 107). Self-reported data on eating behavior (using the Three-Factor Eating Questionnaire subscales such as Cognitive Restraint, Disinhibition, and Hunger) and psychological stress (via the Perceived Stress Scale) were evaluated. Demographic information was incorporated using principal component analysis, which revealed sex- and weight-based differences in stress, adiposity, and eating behavior measures. Following a cross-sectional and descriptive analysis, significant correlations were found between the Disinhibition and Hunger eating behavior subscales and measures of adiposity including BMI ( r = .30, p = .002 and r = .20, p = .036, respectively) and percent body fat ( r = .43, p = .000 and r = .22, p = .022, respectively). Relationships between stress measures and eating behavior were also evident in the analysis. Disinhibition and Hunger correlated positively with perceived stress ( r = .32, p .001 and r = .26, p = .008, respectively). However, Disinhibition varied inversely with serum cortisol levels ( r = -.25, p = .009). Future studies are warranted to better understand this paradox underlying the effects of perceived and physiological stress on eating behavior.

Simple lateral flow assays for microbial detection in stool.

Diarrheal diseases claim the lives of 1300 children daily, mostly in the developing world. We have developed a simple lateral flow assay capable of detecting E. coli and EPEC DNA and RNA rapidly (<15 minutes) at the point-of-need, directly from stool without nucleic acid extraction or molecular amplification. The limit of detection of the method is 1 nM using synthetic DNA target substrates spiked into stool. However, due to the endogenous amplification of the 23S rRNA targets, we were able to detect the endogenous EPEC in pea-sized (5 mg) stool without labor-intensive and time-consuming nucleic acid purification or target amplification using enzymes. The significance of this method is that it is rapid (<15 minutes) and simple (without nucleic acid purification or molecular amplification) and does not require instrumentation, or access to a laboratory, cold chain or electric power. Thus, it is well-suited for point-of-need use in remote and/or resource-limited settings in the developing world where the mortality due to diarrheal diseases is especially high. The rapid testing of stool pathogens in real time at the point-of-need will decrease the loss of patients to follow-up, and enable patients to be treated earlier with the appropriate therapeutics in both the developed and developing world settings.

Gender and Weight Influence Quality of Life in Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain and bowel dysfunction in the absence of structural abnormality. Diagnosis can be challenging and often leads to extensive medical tests, non-effective therapeutic modalities, and reduced quality of life (QOL). Identifying factors associated with dysfunction have the potential to enhance outcomes. Participants with IBS (n = 41) and healthy volunteers (n = 74) were recruited into this cross-sectional, descriptive, natural history protocol at the National Institute of Health, Clinical Center. Demographic characteristics were self-reported. QOL was assessed with the Irritable Bowel Syndrome Quality of Life (IBS-QOL) questionnaire. Statistical analysis included descriptive statistics, factorial ANOVA, and multiple regression. Individuals with IBS reported lower QOL scores across all QOL-subscales compared to healthy controls. Normal-weight women and overweight men with IBS reported the greatest QOL impairment. Body fat percent had confounding effects on the relationship between IBS and QOL. The disparity between QOL scores in participants with IBS by both gender and weight groups may reflect different social pressures perceived by normal and overweight women and men. These findings enhance the recognition of the disparities in patients with chronic symptoms and thereby lead to personalized assessment and interventions to improve their QOL.

Data supporting the effects of lysozyme on mRNA and protein expression in a colonic epithelial scratch wound model.

Colonic epithelial health is implicated in a host of gastrointestinal (GI) diseases and disorders. Lysozyme is suspected to play a role in the ability of the epithelium to recover from injury (Abey et al., in press; Gallo, 2012; Rubio, 2014) [1], [2], [3]. Disrupted repair mechanisms may lead to delayed or ineffective recovery and disruptions to epithelial biology resulting in GI symptoms and altered barrier function (Peterson and Artis, 2014) [4]. The effect of lysozyme on the transcriptomic and proteomic profile of healthy colonic epithelial cells was investigated. Epithelial cells in culture were scratch wounded and treated with lysozyme. mRNA and protein profiles were simultaneously quantified in the same sample using a digital counting technology. Gene and protein expressions altered by the presence or absence of lysozyme are described in this article. Extensive statistical and bioinformatic analysis, and interpretation of the results can be found in "Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress" (Abey et al., in press) [1].

Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress.

BACKGROUND: Stress has demonstrated effects on inflammation though underlying cell-cell communication mechanisms remain unclear. We hypothesize that circulating RNAs and extracellular vesicles (EVs) in patients with chronic stress contain signals with functional roles in cell repair. METHODS: Blood transcriptome from patients with Irritable Bowel Syndrome versus controls were compared to identify signaling pathways and effectors. Plasma EVs were isolated (size-exclusion chromatography) and characterized for effectors' presence (immunogold labelling-electron microscopy). Based on transcriptome pathways and EV-labelling, lysozyme's effects on cell migration were tested in human colon epithelial CRL-1790 cells and compared to the effects of CXCL12, a migration inducer (wound assay). The effect of lysozyme on immune-linked mRNA and protein levels in cells which survived following serum starvation and scratch wound were investigated (NanoString). RESULTS: Blood transcriptomes revealed pyridoxal 5'phosphate salvage, pyrimidine ribonucleotides salvage pathways, atherosclerosis, and cell movement signaling with membrane CD9 and extracellular lysozyme as effectors. Plasma EVs showed labelling with CD9, mucins, and lysozyme. This is the first identification of lysozyme on plasma EVs. In CRL-1790 cells, lysozyme induced migration and repaired scratch wound as well as CXCL12. Immune mRNA and protein expressions were altered in cells which survived following serum starvation and scratch wound, with or without lysozyme in serum-free media post-wounding: CD9, IL8, IL6 mRNAs and CD9, NT5E, PD-L1 proteins. CONCLUSIONS: Repair and inflammatory signals are identified in plasma EVs and circulating RNAs in chronic stress. Registered clinicaltrials.gov #NCT00824941. GENERAL SIGNIFICANCE: This study highlights the role of circulating RNAs and EVs in stress.

Structural and functional alterations in the colonic microbiome of the rat in a model of stress induced irritable bowel syndrome.

Stress is known to perturb the microbiome and exacerbate irritable bowel syndrome (IBS) associated symptoms. Characterizing structural and functional changes in the microbiome is necessary to understand how alterations affect the biomolecular environment of the gut in IBS. Repeated water avoidance (WA) stress was used to induce IBS-like symptoms in rats. The colon-mucosa associated microbiome was characterized in 13 stressed and control animals by 16S sequencing. In silico analysis of the functional domains of microbial communities was done by inferring metagenomic profiles from 16S data. Microbial communities and functional profiles were compared between conditions. WA animals exhibited higher α-diversity and moderate divergence in community structure (ß-diversity) compared with controls. Specific clades and taxa were consistently and significantly modified in the WA animals. The WA microbiome was particularly enriched in Proteobacteria and depleted in several beneficial taxa. A decreased capacity in metabolic domains, including energy- and lipid-metabolism, and an increased capacity for fatty acid and sulfur metabolism was inferred for the WA microbiome. The stressed condition favored the proliferation of a greater diversity of microbes that appear to be functionally similar, resulting in a functionally poorer microbiome with implications for epithelial health. Taxa, with known beneficial effects, were found to be depleted, which supports their relevance as therapeutic agents to restore microbial health. Microbial sulfur metabolism may form a key component of visceral nerve sensitization pathways and is therefore of interest as a target metabolic domain in microbial ecological restoration.

The Gastrointestinal Pain Pointer: A Valid and Innovative Method to Assess Gastrointestinal Symptoms.

Abdominal pain is a chronic condition experienced by approximately 20% of individuals in the United States. The purpose of the study was to assess the validity of the Gastrointestinal Pain Pointer as a measure of abdominal pain intensity. A prospective longitudinal time-series study design was utilized. The sample included 93 outpatients (58.1% female). Participants met Rome III criteria for irritable bowel syndrome (n = 32) or were healthy controls (n = 61). The Gastrointestinal Pain Pointer, a new electronic pain assessment tool, was used to assess self-reported abdominal pain intensity among participants before and after ingestion of an intestinal permeability test solution across 11 time points over a 5-hour time period. The results were compared with the Short-Form McGill Pain Questionnaire. The Gastrointestinal Pain Pointer was found to be valid in the assessment of abdominal pain intensity. The tool is a novel and valid measure of abdominal pain intensity that enhances the ability for clinicians to better quantify, in real time, patient-related pain outcomes for both clinical care and research.

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3' end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

Emerging Role of Nutri-Epigenetics in Inflammation and Cancer.

Nutrition is a factor involved in inflammation and a modulator of risk toward some cancers, and the complexity of linkages between dietary components and epigenetics mechanisms (e.g., DNA methylation, histone modification, chromatin remodeling) may affect the inflammation phenotype and the development of cancer. An increasing number of studies support the role of diet in cancer development, prevention, and treatment. Although current knowledge regarding nutri-epigenetics is expanding, more work is needed, and nurse scientists have the potential to significantly contribute to the expansion of this knowledge.

The microbiome of the oral mucosa in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a poorly understood disorder characterized by persistent symptoms, including visceral pain. Studies have demonstrated oral microbiome differences in inflammatory bowel diseases suggesting the potential of the oral microbiome in the study of non-oral conditions. In this exploratory study we examine whether differences exist in the oral microbiome of IBS participants and healthy controls, and whether the oral microbiome relates to symptom severity. The oral buccal mucosal microbiome of 38 participants was characterized using PhyloChip microarrays. The severity of visceral pain was assessed by orally administering a gastrointestinal test solution. Participants self-reported their induced visceral pain. Pain severity was highest in IBS participants (P = 0.0002), particularly IBS-overweight participants (P = 0.02), and was robustly correlated to the abundance of 60 OTUs, 4 genera, 5 families and 4 orders of bacteria (r2 > 0.4, P < 0.001). IBS-overweight participants showed decreased richness in the phylum Bacteroidetes (P = 0.007) and the genus Bacillus (P = 0.008). Analysis of ß-diversity found significant separation of the IBS-overweight group (P < 0.05). Our oral microbial results are concordant with described fecal and colonic microbiome-IBS and -weight associations. Having IBS and being overweight, rather than IBS-subtypes, was the most important factor in describing the severity of visceral pain and variation in the microbiome. Pain severity was strongly correlated to the abundance of many taxa, suggesting the potential of the oral microbiome in diagnosis and patient phenotyping. The oral microbiome has potential as a source of microbial information in IBS.

Global analysis of posttranscriptional gene expression in response to sodium arsenite.

BACKGROUND: Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription. OBJECTIVES: We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts. METHODS: We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: In arsenite-exposed cells, 186 probe set-identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set-identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA (HMOX1; 68-fold increase), the rate-limiting enzyme in heme catabolism. CONCLUSIONS: We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.

Elevated circulating miR-150 and miR-342-3p in patients with irritable bowel syndrome.

BACKGROUND AND AIMS: MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression and are thus of interest as diagnostic markers, and as clues to etiology and targets of intervention. This pilot study examined whether circulating miRNAs are differentially expressed in patients with IBS. METHODS: miRNA microarrays (NanoString) were run on the whole blood of 43 participants. RESULTS: hsa-miR-150 and hsa-miR-342-3p were found to be significantly elevated (FDR adjusted p≤0.05, ≥1.6 fold change) in IBS patients compared to healthy controls. Neither of these miRNAs showed any relationship to race or sex. hsa-miR-150 is associated with inflammatory bowel disorders and pain, and interacts with a protein kinase (AKT2) through which it may affect inflammatory pathways. hsa-miR-342-3p is predicted to interact with mRNAs involved in pain signaling, colonic motility, and smooth muscle function. CONCLUSIONS: This preliminary study reports the association of two miRNAs, detected in whole blood, with IBS. These miRNAs link to pain and inflammatory pathways both of which are thought to be dysregulated in IBS. Larger sample sizes are needed to confirm their importance and potential as biomarkers.