Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes.

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This paper presents a highly reproducible method for the precise measurement of pH within the lumina of 3D human gastric organoids via micromanipulator-controlled microelectrodes. The pH microelectrodes are commercially available and consist of beveled glass tips of 25 µm in diameter. For measurements, the pH microelectrode is advanced into the lumen of an organoid (>200 µm) that is suspended in Matrigel, while a reference electrode rests submerged in the surrounding medium in the culture plate. Using such microelectrodes to profile organoids derived from the human gastric body, we demonstrate that luminal pH is relatively consistent within each culture well at ~7.7 ± 0.037 and that continuous measurements can be obtained for a minimum of 15 min. In some larger organoids, the measurements revealed a pH gradient between the epithelial surface and the lumen, suggesting that pH measurements in organoids can be achieved with high spatial resolution. In a previous study, microelectrodes were successfully used to measure luminal oxygen concentrations in organoids, demonstrating the versatility of this method for organoid analyses. In summary, this protocol describes an important tool for the functional characterization of the complex luminal space within 3D organoids.

YAP1 regulates the self-organized fate patterning of hESC-derived gastruloids.

The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.

Cellular Plasticity, Reprogramming, and Regeneration: Metaplasia in the Stomach and Beyond.

The mucosa of the body of the stomach (ie, the gastric corpus) uses 2 overlapping, depth-dependent mechanisms to respond to injury. Superficial injury heals via surface cells with histopathologic changes like foveolar hyperplasia. Deeper, usually chronic, injury/inflammation, most frequently induced by the carcinogenic bacteria Helicobacter pylori, elicits glandular histopathologic alterations, initially manifesting as pyloric (also known as pseudopyloric) metaplasia. In this pyloric metaplasia, corpus glands become antrum (pylorus)-like with loss of acid-secreting parietal cells (atrophic gastritis), expansion of foveolar cells, and reprogramming of digestive enzyme-secreting chief cells into deep antral gland-like mucous cells. After acute parietal cell loss, chief cells can reprogram through an orderly stepwise progression (paligenosis) initiated by interleukin-13-secreting innate lymphoid cells (ILC2s). First, massive lysosomal activation helps mitigate reactive oxygen species and remove damaged organelles. Second, mucus and wound-healing proteins (eg, TFF2) and other transcriptional alterations are induced, at which point the reprogrammed chief cells are recognized as mucus-secreting spasmolytic polypeptide-expressing metaplasia cells. In chronic severe injury, glands with pyloric metaplasia can harbor both actively proliferating spasmolytic polypeptide-expressing metaplasia cells and eventually intestine-like cells. Gastric glands with such lineage confusion (mixed incomplete intestinal metaplasia and proliferative spasmolytic polypeptide-expressing metaplasia) may be at particular risk for progression to dysplasia and cancer. A pyloric-like pattern of metaplasia after injury also occurs in other gastrointestinal organs including esophagus, pancreas, and intestines, and the paligenosis program itself seems broadly conserved across tissues and species. Here we discuss aspects of metaplasia in stomach, incorporating data derived from animal models and work on human cells and tissues in correlation with diagnostic and clinical implications.

Use of human tissue stem cell-derived organoid cultures to model enterohepatic circulation.

The use of human tissue stem cell-derived organoids has advanced our knowledge of human physiological and pathophysiological processes that are unable to be studied using other model systems. Increased understanding of human epithelial tissues including intestine, stomach, liver, pancreas, lung, and brain have been achieved using organoids. However, it is not yet clear whether these cultures recapitulate in vivo organ-to-organ signaling or communication. In this work, we demonstrate that mature stem cell-derived intestinal and liver organoid cultures each express functional molecules that modulate bile acid uptake and recycling. These organoid cultures can be physically coupled in a Transwell system and display increased secretion of fibroblast growth factor 19 (FGF19) (intestine) and downregulation of P450 enzyme cholesterol 7 α-hydroxylase (CYP7A) (liver) in response to apical exposure of the intestine to bile acids. This work establishes that organoid cultures can be used to study and therapeutically modulate interorgan interactions and advance the development of personalized approaches to medical care.NEW & NOTEWORTHY Interorgan signaling is a critical feature of human biology and physiology, yet has remained difficult to study due to the lack of in vitro models. Here, we demonstrate that physical coupling of ex vivo human intestine and liver epithelial organoid cultures recapitulates in vivo interorgan bile acid signaling. These results suggest that coupling of multiple organoid systems provides new models to investigate interorgan communication and advances our knowledge of human physiological and pathophysiological processes.

Methods to improve efficacy of orally administered bioactive peptides using bovine colostrum as an exemplar.

BACKGROUND: Oral administration of bioactive peptides has potential clinical advantages, but its applicability is limited due to gastric and pancreatic enzyme proteolysis. OBJECTIVE: To examine whether the co-packaging of bovine colostrum (BC), a rich source of IgG, immune and growth factors, with the food additives trehalose (carbohydrate), stearine (fat), casein (protein present in BC) or soy flour (plant based with high protease inhibitory activity) enhances the stability of BC against digestion. DESIGN: Samples alone and in combination (BC+ 10% wt/wt trehalose, stearine, casein or soy) were exposed to HCl/pepsin, followed by trypsin and chymotrypsin ("CT"). Assessment of proliferation used gastric AGS cells (Alamar blue), IgG function measured bovine IgG anti-E.coli binding and ELISAs quantified growth factor constituents. In vivo bioassay assessed ability of BC alone or with soy to reduce injury caused by dextran sodium sulphate (DSS, 4% in drinking water, 7 days, test products started 2 days prior to DSS). RESULTS: Proliferative activity of BC reduced 61% following HCl/pepsin and CT exposure. This was truncated 50% if soy was co-present, and also protected against loss of total IgG, IgG E.coli binding, TGFß, lactoferrin and EGF (all P<0.01 vs BC alone). Co-packaging with trehalose was ineffective in preventing digestion whereas casein or stearine provided some intermediate protective effects. Rats given BC alone showed beneficial effects on weight gain, disease activity index, tissue histology and colonic MPO. Soy alone was ineffective. BC+ soy combination showed the greatest benefit with a dose of 7 mg/kg (6.4 BC + 0.6 soy flour) having the same degree of benefit as using 20 mg/kg BC alone. CONCLUSION: Soy, and to a lesser extent casein, enhanced the biostability of BC against digestive enzymes. Co-packaging of BC with other food products such as soy flour could result in a decreased dose being required, improving cost-effectiveness and patient compliance.

Gastric Serotonin Biosynthesis and Its Functional Role in L-Arginine-Induced Gastric Proton Secretion.

Among mammals, serotonin is predominantly found in the gastrointestinal tract, where it has been shown to participate in pathway-regulating satiation. For the stomach, vascular serotonin release induced by gastric distension is thought to chiefly contribute to satiation after food intake. However, little information is available on the capability of gastric cells to synthesize, release and respond to serotonin by functional changes of mechanisms regulating gastric acid secretion. We investigated whether human gastric cells are capable of serotonin synthesis and release. First, HGT-1 cells, derived from a human adenocarcinoma of the stomach, and human stomach specimens were immunostained positive for serotonin. In HGT-1 cells, incubation with the tryptophan hydroxylase inhibitor p-chlorophenylalanine reduced the mean serotonin-induced fluorescence signal intensity by 27%. Serotonin release of 147 ± 18%, compared to control HGT-1 cells (set to 100%) was demonstrated after treatment with 30 mM of the satiating amino acid L-Arg. Granisetron, a 5-HT3 receptor antagonist, reduced this L-Arg-induced serotonin release, as well as L-Arg-induced proton secretion. Similarly to the in vitro experiment, human antrum samples released serotonin upon incubation with 10 mM L-Arg. Overall, our data suggest that human parietal cells in culture, as well as from the gastric antrum, synthesize serotonin and release it after treatment with L-Arg via an HTR3-related mechanism. Moreover, we suggest not only gastric distension but also gastric acid secretion to result in peripheral serotonin release.

Sodium sulfite causes gastric mucosal cell death by inducing oxidative stress.

Sulfites are commonly used as a preservative and antioxidant additives in the food industry. Sulfites are absorbed by the gastrointestinal tract and distributed essentially to all body tissues. Although sulfites have been believed to be safe food additives, some studies have shown that they exhibit adverse effects in various tissues. In this study, we examined the cytotoxic effect of sodium sulfite (Na2SO3) against rat gastric mucosal cells (RGM1) and further investigated its underlying molecular mechanism. We demonstrated that exposure to Na2SO3 exerts significant cytotoxicity in RGM1 cells through induction of oxidative stress. Exposure of RGM1 cells to Na2SO3 caused a significant formation of protein carbonyls and 8-hydroxy-2'-deoxyguanosine, major oxidative stress markers, with a concomitant accumulation of carbonylated protein-related aggregates. Furthermore, we found that incubation of lysozyme with Na2SO3 evokes protein carbonylation and aggregation via the metal ion-catalyzed free radical formation derived from Na2SO3. Our results suggest that Na2SO3 might lead to gastric tissue injury via induction of oxidative stress by the formation of Na2SO3-related free radicals.

Mouse Spexin: (III) Differential Regulation by Glucose and Insulin in Glandular Stomach and Functional Implication in Feeding Control.

Spexin (SPX), a neuropeptide with diverse functions, is a novel satiety factor in fish models and its role in feeding control has been recently confirmed in mammals. In mouse, food intake was shown to trigger SPX expression in glandular stomach with parallel rise in serum SPX and these SPX signals could inhibit feeding via central actions within the hypothalamus. However, the mechanisms for SPX regulation by food intake are still unclear. To examine the role of insulin signal caused by glucose uptake in SPX regulation, the mice were IP injected with glucose and insulin, respectively. In this case, serum SPX was elevated by glucose but not altered by insulin. Meanwhile, SPX transcript expression in the glandular stomach was up-regulated by glucose but the opposite was true for insulin treatment. Using in situ hybridization, the differential effects on SPX gene expression were located in the gastric mucosa of glandular stomach. Co-injection experiments also revealed that glucose stimulation on serum SPX and SPX mRNA expressed in glandular stomach could be blocked by insulin. In gastric mucosal cells prepared from glandular stomach, the opposite effects on SPX transcript expression by glucose and insulin could still be noted with similar blockade of the stimulatory effects of glucose by insulin. In this cell model, SPX gene expression induced by glucose was mediated by glucose uptake via GLUT, ATP synthesis by glycolysis/respiratory chain, and subsequent modulation of KATP channel activity, but the voltage-sensitive Ca2+ channels were not involved. The corresponding inhibition by insulin, however, was mediated by PI3K/Akt, MEK1/2/ERK1/2, and P38MAPK cascades coupled to insulin receptor but not IGF-1 receptor. Apparently, glucose uptake in mice can induce SPX expression in the glandular stomach through ATP synthesis via glucose metabolism and subsequent modification of KATP channel activity, which may contribute to SPX release into circulation to act as the satiety signal after food intake. The insulin rise caused by glucose uptake, presumably originated from the pancreas, may serve as a negative feedback to inhibit the SPX response by activating MAPK and PI3K/Akt pathways in the stomach.

Age-related changes in NG2-expressing telocytes of rat stomach.

NG2 immunoreactive cells (NG2 cells) are found in the brain and peripheral tissues including the skin, intestinal tracts, and bladder. In a previous study, we observed the presence of NG2 cells in the stomach using bioluminescence imaging techniques in NG2-firefly luciferase (fLuc) transgenic (Tg) rats. Here, we aimed to identify and characterize NG2 cells in the adult rat stomach. Immunohistochemical studies showed that NG2 cells were mainly present in the lamina propria and most of the cells were gastric telocytes, co-expressing CD34, and platelet-derived growth factor receptor alpha (PDGFRα), with a small oval-shaped cell body and extremely long and thin cellular prolongations. In the rat stomach, NG2-expressing telocytes comprised two subpopulations: NG2+/CD34+/PDGFRα+ and NG2+/CD34+/PDGFRα-. Furthermore, we showed that the expression of NG2 gene in the aged rat stomach decreased relative to that of the young rat stomach and the decline of NG2 expression in aged rats was mainly observed in NG2+/CD34+/PDGFRα+ telocytes. These findings suggested age-related alterations in NG2+/CD34+/PDGFRα+ telocytes of rat stomach.

Drug-exposed cancer-associated fibroblasts facilitate gastric cancer cell progression following chemotherapy.

BACKGROUND: Cancer progression following chemotherapy is a significant barrier to effective cancer treatment. We aimed to evaluate the role of drug-exposed cancer-associated fibroblasts (CAFs) in the growth and progression of drug-exposed gastric cancer (GC) cells and to explore the underlying molecular mechanism. METHODS: The human GC cell line 44As3 and CAFs were treated with 5-fluorouracil and oxaliplatin (5FU + OX). 5FU + OX-pretreated 44As3 cells were then cultured in a conditioned medium (CM) from 5FU + OX-pretreated CAFs, and the growth and migration/invasion ability of the cells were evaluated. We also compared the clinicopathological characteristics of the GC patients treated with S1 + OX in accordance with the properties of their resected specimens, focusing on the number of CAFs. Changes in gene expression in CAFs and 44As3 cells were comprehensively analyzed using RNA-seq analysis. RESULTS: The CM from 5FU + OX-pretreated CAFs promoted the migration and invasion of 5FU + OX-pretreated 44As3 cells. Although the number of cases was relatively small (n = 21), the frequency of positive cases of lymphovascular invasion and the recurrence rate were significantly higher in those with more residual CAF. RNA-seq analysis revealed 5FU + OX-pretreated CAF-derived glycoprotein 130 (gp130) as a candidate factor contributing to the increased migration of 5FU + OX-pretreated 44As3 cells. Administration of the gp130 inhibitor SC144 prevented the increased migration ability of 5FU + OX-pretreated 44As3 cells owing to drug-treated CAFs. CONCLUSIONS: Our findings provide evidence regarding the interactions between GC cells and CAFs in the tumor microenvironment following chemotherapy, suggesting that ligands for gp130 may be novel therapeutic targets for suppressing or preventing metastasis in GC.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Organ-specific, multimodal, wireless optoelectronics for high-throughput phenotyping of peripheral neural pathways.

The vagus nerve supports diverse autonomic functions and behaviors important for health and survival. To understand how specific components of the vagus contribute to behaviors and long-term physiological effects, it is critical to modulate their activity with anatomical specificity in awake, freely behaving conditions using reliable methods. Here, we introduce an organ-specific scalable, multimodal, wireless optoelectronic device for precise and chronic optogenetic manipulations in vivo. When combined with an advanced, coil-antenna system and a multiplexing strategy for powering 8 individual homecages using a single RF transmitter, the proposed wireless telemetry enables low cost, high-throughput, and precise functional mapping of peripheral neural circuits, including long-term behavioral and physiological measurements. Deployment of these technologies reveals an unexpected role for stomach, non-stretch vagal sensory fibers in suppressing appetite and demonstrates the durability of the miniature wireless device inside harsh gastric conditions.

A novel method for isolation and culture of primary swine gastric epithelial cells.

BACKGROUND: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach. RESULTS: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William's E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively. CONCLUSIONS: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William's E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells.

An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3ß, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.

Autophagy induced by Helicobacter pylori infection is necessary for gastric cancer stem cell emergence.

BACKGROUND: The main cause of gastric cancer is the infection by the bacterium Helicobacter pylori which induces a chronic inflammation and an epithelial-to-mesenchymal transition (EMT) leading to the emergence of cells with cancer stem cell (CSC) properties. However, the underlying mechanisms have not been fully characterized. Moreover, H. pylori modulates the host cell autophagic process, but a few studies have investigated the role of this process in tumoral transformation. The aim of this study was to determine whether H. pylori-induced autophagy has a role in CSC emergence. METHODS: Autophagic flux in response to H. pylori infection was characterized in AGS cell line expressing the tandem-tagged mCherry-GFP-LC3 protein and using a ratiometric flow cytometry analysis. Then, AGS and MKN45 cell lines were treated with bafilomycin or chloroquine, two pharmaceutical well-known inhibitors of autophagy, and different EMT and CSC characteristics were analyzed. RESULTS: First, a co-expression of the gastric CSC marker CD44 and the autophagic marker LC3 in mice and human stomach tissues infected with H. pylori was observed. Then, we demonstrated in vitro that H. pylori was able to activate the autophagy process with a reduced autophagic flux. Finally, infected cells were treated with autophagy inhibitors, which reduced (i) appearance of mesenchymal phenotypes and migration ability related to EMT and (ii) CD44 expression as well as tumorsphere formation capacities reflecting CSC properties. CONCLUSION: In conclusion, all these data show that H. pylori-induced autophagy is implicated in gastric CSC emergence and could represent an interesting therapeutic target.

Gastric ghrelin cells in obese patients are hyperactive.

BACKGROUND/OBJECTIVES: Distribution and activity of ghrelin cells in the stomach of obese subjects are controversial. SUBJECTS/METHODS: We examined samples from stomachs removed by sleeve gastrectomy in 49 obese subjects (normoglycemic, hyperglycemic and diabetic) and quantified the density of ghrelin/chromogranin endocrine cells by immunohistochemistry. Data were compared with those from 13 lean subjects evaluated by gastroscopy. In 44 cases (11 controls and 33 obese patients) a gene expression analysis of ghrelin and its activating enzyme ghrelin O-acyl transferase (GOAT) was performed. In 21 cases (4 controls and 17 obese patients) the protein levels of unacylated and acylated-ghrelin were measured by ELISA tests. In 18 cases (4 controls and 14 obese patients) the morphology of ghrelin-producing cells was evaluated by electron microscopy. RESULTS: The obese group, either considered as total population or divided into subgroups, did not show any significant difference in ghrelin cell density when compared with control subjects. Inter-glandular smooth muscle fibres were increased in obese patients. In line with a positive trend of the desacylated form found by ELISA, Ghrelin and GOAT mRNA expression in obese patients was significantly increased. The unique ghrelin cell ultrastructure was maintained in all obese groups. In the hyperglycemic obese patients, the higher ghrelin expression matched with ultrastructural signs of endocrine hyperactivity, including expanded rough endoplasmic reticulum and reduced density, size and electron-density of endocrine granules. A positive correlation between ghrelin gene expression and glycemic values, body mass index and GOAT was also found. All obese patients with type 2 diabetes recovered from diabetes at follow-up after 5 months with a 16.5% of weight loss. CONCLUSIONS: Given the known inhibitory role on insulin secretion of ghrelin, these results suggest a possible role for gastric ghrelin overproduction in the complex architecture that takes part in the pathogenesis of type 2 diabetes.

N-Terminally Added Tag Selectively Enhances Heterologous Expression of VacA Cytotoxin Variants from Helicobacter pylori.

BACKGROUND: Gastric pathogen Helicobacter pylori secretes VacA cytotoxin displaying a high degree of polymorphic variations of which the highest VacA pathogenicity correlates with m1-type variant followed by VacA-m2. OBJECTIVE: To comparatively evaluate expression in Escherichia coli of the mature VacA variants (m1- and m2-types) and their 33- and 55/59-kDa domains fused with His(6) tag at N- or C-terminus. METHODS: All VacA clones expressed in E. coli TOP10™ were analyzed by SDS-PAGE and Western blotting. VacA inclusions were solubilized under native conditions (~150-rpm shaking at 37°C for 2 h in 20 mM HEPES (pH7.4) and 150 mM NaCl). Membrane-perturbing and cytotoxic activities of solubilized VacA proteins were assessed via liposome-entrapped dye leakage and resazurin- based cell viability assays, respectively. VacA binding to human gastric adenocarcinoma cells was assessed by immunofluorescence microscopy. Side-chain hydrophobicity of VacA was analyzed through modeled structures constructed by homology- and ab initio-based modeling. RESULTS: Both full-length VacA-m1 and 33-kDa domain were efficiently expressed only in the presence of N-terminal extension while its 55-kDa domain was capably expressed with either N- or Cterminal extension. Selectively enhanced expression was also observed for VacA-m2. Protein expression profiles revealed a critical period in IPTG-induced production of the 55-kDa domain with N-terminal extension unlike its C-terminal extension showing relatively stable expression. Both VacA- m1 isolated domains were able to independently bind to cultured gastric cells similar to the full- length toxin, albeit the 33-kDa domain exhibited significantly higher activity of membrane perturbation than others. Membrane-perturbing and cytotoxic activities observed for VacA-m1 appeared to be higher than those of VacA-m2. Homology-based modeling and sequence analysis suggested a potential structural impact of non-polar residues located at the N-terminus of the mature VacA toxin and its 33-kDa domain. CONCLUSION: Our data provide molecular insights into selective influence of the N-terminally added tag on efficient expression of recombinant VacA variants, signifying biochemical and biological implications of the hydrophobic stretch within the N-terminal domain.

Wnt-induced, TRP53-mediated Cell Cycle Arrest of Precursors Underlies Interstitial Cell of Cajal Depletion During Aging.

BACKGROUND & AIMS: Gastric dysfunction in the elderly may cause reduced food intake, frailty, and increased mortality. The pacemaker and neuromodulator cells interstitial cells of Cajal (ICC) decline with age in humans, and their loss contributes to gastric dysfunction in progeric klotho mice hypomorphic for the anti-aging Klotho protein. The mechanisms of ICC depletion remain unclear. Klotho attenuates Wnt (wingless-type MMTV integration site) signaling. Here, we examined whether unopposed Wnt signaling could underlie aging-associated ICC loss by up-regulating transformation related protein TRP53 in ICC stem cells (ICC-SC). METHODS: Mice aged 1-107 weeks, klotho mice, APCΔ468 mice with overactive Wnt signaling, mouse ICC-SC, and human gastric smooth muscles were studied by RNA sequencing, reverse transcription-polymerase chain reaction, immunoblots, immunofluorescence, histochemistry, flow cytometry, and methyltetrazolium, ethynyl/bromodeoxyuridine incorporation, and ex-vivo gastric compliance assays. Cells were manipulated pharmacologically and by gene overexpression and RNA interference. RESULTS: The klotho and aged mice showed similar ICC loss and impaired gastric compliance. ICC-SC decline preceded ICC depletion. Canonical Wnt signaling and TRP53 increased in gastric muscles of klotho and aged mice and middle-aged humans. Overstimulated canonical Wnt signaling increased DNA damage response and TRP53 and reduced ICC-SC self-renewal and gastric ICC. TRP53 induction persistently inhibited G1/S and G2/M cell cycle phase transitions without activating apoptosis, autophagy, cellular quiescence, or canonical markers/mediators of senescence. G1/S block reflected increased cyclin-dependent kinase inhibitor 1B and reduced cyclin D1 from reduced extracellular signal-regulated kinase activity. CONCLUSIONS: Increased Wnt signaling causes age-related ICC loss by up-regulating TRP53, which induces persistent ICC-SC cell cycle arrest without up-regulating canonical senescence markers.

5HT expression in the stomach and duodenum of the Rhesus Monkey (Macaca mulatta).

This study aims to provide a histological description of different regions of the gastric and duodenal mucosa in Rhesus monkey, as well as to analyze the distribution and the relative frequency of 5-HT. The cardia region mucosa consists of simple columnar epithelium PAS + and AB + and the 5-HT cells were observed at the base of the gland (QA [5-HT cells]/mm²) = 8.72 ±â€¯4.98). The body region, has a smaller number of glands. The 5-HT cells were found predominant in the base of the gastric glands. QA= 6.96 ±â€¯3.81. When compared to body region, the stomach fundus has smaller gastric pits. The 5-HT cells are found at the base of the glands near the main cells. QA = 5.29 ±â€¯2.09. The pylorus region was found to have deep pits and well-developed gastric glands. The 5-HT cells are scarce, at the base of the pyloric gland. QA = 1.18 ±â€¯1.36. The duodenum presented goblet cells strong PAS + and AB +. 5-HT cells were found both in the lining epithelium and in the intestinal glands. QA = 8.16 ±â€¯2.59.

Nutrient-sensing components of the mouse stomach and the gastric ghrelin cell.

BACKGROUND: The ability of the gut to detect nutrients is critical to the regulation of gut hormone secretion, food intake, and postprandial blood glucose control. Ingested nutrients are detected by specific gut chemosensors. However, knowledge of these chemosensors has primarily been derived from the intestine, while available information on gastric chemosensors is limited. This study aimed to investigate the nutrient-sensing repertoire of the mouse stomach with particular emphasis on ghrelin cells. METHODS: Quantitative RT-PCR was used to determine mRNA levels of nutrient chemosensors (protein: G protein-coupled receptor 93 [GPR93], calcium-sensing receptor [CaSR], metabotropic glutamate receptor type 4 [mGluR4]; fatty acids: CD36, FFAR2&4; sweet/umami taste: T1R3), taste transduction components (TRPM5, GNAT2&3), and ghrelin and ghrelin-processing enzymes (PC1/3, ghrelin O-acyltransferase [GOAT]) in the gastric corpus and antrum of adult male C57BL/6 mice. Immunohistochemistry was performed to assess protein expression of chemosensors (GPR93, T1R3, CD36, and FFAR4) and their co-localization with ghrelin. KEY RESULTS: Most nutrient chemosensors had higher mRNA levels in the antrum compared to the corpus, except for CD36, GNAT2, ghrelin, and GOAT. Similar regional distribution was observed at the protein level. At least 60% of ghrelin-positive cells expressed T1R3 and FFAR4, and over 80% expressed GPR93 and CD36. CONCLUSIONS AND INFERENCES: The cellular mechanisms for the detection of nutrients are expressed in a region-specific manner in the mouse stomach and gastric ghrelin cells. These gastric nutrient chemosensors may play a role modulating gastrointestinal responses, such as the inhibition of ghrelin secretion following food intake.