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Abstract: Background: Alterations of site‐specific gene expression profiles in disease‐relevant networks within the different layers of the intestinal wall may contribute to the onset and clinical course of gastrointestinal disorders. To date, no systematic analysis has assessed and compared sub‐regional gene expression patterns in all distinct layers of the gut using fresh frozen human samples. Our aim was to establish an optimized protocol for site‐specific RNA isolation in order to achieve maximum RNA quality and amount for subsequent gene expression analysis combining laser‐capture microdissection (LCM) with a probe‐based technology, the NanoString nCounter Analysis system. Methods: Four full‐thickness colon samples from patients who underwent surgery due to pathological conditions were processed and separated into epithelium, lamina propria, myenteric plexus, submucosa, and tunica muscularis by LCM. Site‐specific marker expression by nCounter technology was performed on total RNA from each sub‐region, respectively. Key Results: Collecting ~10 mm² (~100 000‐250 000 cells) of tissue from the epithelial layer, lamina propria, and myenteric plexus provided sufficient amounts of RNA of appropriate quality for subsequent analyses. In contrast, ~40 mm² (~250 000‐650 000 cells) of tissue were dissected from the less cell‐rich submucosal and tunica muscularis layer. nCounter analysis revealed a site‐specific expression pattern of marker genes in the different layers of the colonic wall which were highly correlating (r > .9). Conclusions and Inferences: LCM in combination with nCounter expression analysis enables site‐specific, sensitive, reliable detection, and quantification of mRNA from histologically heterogeneous tissues. [ABSTRACT FROM AUTHOR]

Purpose: Until recently, tissue fibrosis-ultimately leading to permanent scaring-has been considered an irreversible process. However, recent findings indicate that it may be reversible after all. Vesicourethral anastomotic stenosis (VUAS) as fibrous narrowing is a frequent complication after radical prostatectomy with high recurrence rates and requires invasive treatment. The pathophysiology is poorly understood. Therefore, a combined mRNA and miRNA transcription profiling in tissue from VUAS was performed using nCounter technology. Methods: To assess tissue morphology and fiber composition, histochemical staining was performed. RNA expression of healthy and fibrotic tissue of twelve patients was analyzed using the human miRNA panel v3 and mRNA PanCancer pathway panel on the nCounter gene1 system and qRT-PCR. Differential expression data analysis was performed using the nSolver software implementing the R-based advanced pathway analysis tool. miRWalk2.0 was used for miRNA target prediction. Results: More linearized tissue architecture, increased collagens, and decreased elastic fibers were observed in VUAS samples. 23 miRNAs and 118 protein coding genes were differentially expressed ( p < 0.01) in fibrotic tissue. miRNA target prediction and overlap analysis indicated an interaction of the strongest deregulated miRNAs with 29 deregulated mRNAs. Pathway analysis revealed alterations in DNA repair, cell cycle regulation, and TGF-beta signaling. qRT-PCR confirmed differential expression of top deregulated miRNAs and mRNAs. Conclusions: In VUAS tissue, severe alterations on mRNA and miRNA level are found. These consistent changes give insights into the pathogenesis of VUAS after radical prostatectomy and point to future options for transcriptomics-based risk stratification and targeted therapies. [ABSTRACT FROM AUTHOR]

Background Impaired 5- HT3 receptor function is likely involved in the pathogenesis of functional gastrointestinal disorders ( FGID) and 5- HT3 receptor antagonists are effective treatments for chemotherapy-induced nausea and vomiting ( CINV) and irritable bowel syndrome ( IBS). The monoterpene alcohol menthol and the aporphine alkaloid boldine combat symptoms of gastrointestinal diseases; both interact with other members of the Cys-loop ligand-gated ion channel family and may therefore also act on 5- HT3 receptors. Methods The impact of boldine and menthol on human recombinant homomeric 5- HT3A- and heteromeric 5- HT3 AB receptors in HEK293 cells was determined by radioligand binding, a luminescence-based Ca2+ assay, and a membrane potential assay. 5- HT3 protein and m RNA expression was assessed in human colon tissue. Key Results Boldine and menthol inhibited the 5- HT-induced activation of 5- HT3 receptors in the low and middle micromolar range, respectively. Boldine was a competitive antagonist of both receptors being 6.5- to 10-fold more potent at 5- HT3A- vs 5- HT3 AB receptors. Menthol non-competitively and stereoselectively inhibited both receptors: In contrast to (+)-menthol, (−)-menthol was significantly more potent toward 5- HT3A- vs 5- HT3 AB receptors. We show co-expression of 5- HT3A and 5- HT3B subunits in the human gut epithelium, the lamina propria, the myenteric plexus, and the muscular cell layer. Conclusions & Inferences The demonstrated 5- HT3 inhibitory effects may be relevant for boldine's and menthol's alleviating properties on FGID and may encourage clinical studies with the compounds or the plant extracts for CINV and IBS treatment. The found receptor-discriminative properties make boldine and (−)-menthol to potentially useful tools for analyzing structural differences between these receptor subtypes. [ABSTRACT FROM AUTHOR]