bluuebunny/crossref_metadata_2025_split huggingface dataset:
Local Colonic Delivery of Vasoactive Intestinal Peptide (VIP) Nanomedicine Alleviates Colitis in Mice
Authors: Jayawardena Dulari , Anbazhagan Arivarasu Natarajan , Onyuksel Hayat , Dudeja Pradeep K. 
Published: June 2021

Inflammatory Bowel Disease (IBD) is an unmet medical need encompassing two entities; Ulcerative Colitis (UC) and Crohn's Disease (CD). We have previously demonstrated the superior therapeutic effects of systemically administered VIP nanomedicine (VIP in sterically stabilized micelles; VIP‐SSM), in alleviating colitis in mice. In addition, we have also characterized a luminally expressed VPAC1 receptor in human and mouse colonic mucosa. Thus, the current studies were undertaken to determine if delivering the nanomedicine directly to the colonic lumen could have similar therapeutic benefits.MethodsVIP‐SSM was prepared by mixing aqueous solutions of lipid and peptide in a predetermined molar ratio and was characterized for its size by dynamic light scattering. Colitis was induced in C57Bl/6 mice with 3.5% w/v dextran sulfate sodium (DSS) in drinking water for 7 days. Animals were randomly divided into 5 groups; (Control, DSS, VIP‐SSM, DSS+VIP‐SSM and DSS+ VIP). On the 5th day mice were anesthetized with ketamine/xylazine and catheterized to intra rectally instill 100 μls of, VIP‐SSM (0.25 nmol), free VIP (0.25 nmol) or SSM (100 nmol). At day 7, mice were switched to tap water and sacrificed at day 10. Body weights of mice were recorded daily. Colon appearance was observed for diarrheal phenotype, distal colonic mucosa was used for RNA and protein isolation for qPCR and western blot analysis, respectively. Frozen sections of colon were used for H & E and immunostaining.ResultsThe prepared nanoparticle hydrodynamic particle sizes were approximately 15 nm. At day 10, VIP‐SSM treated mice showed significant improvement in body weight compared to DSS and free VIP treated DSS mice. The diarrheal phenotype seen with DSS indicated by loose stool in colon and reduced length was alleviated with VIP‐SSM (showing presence of solid fecal pellets) and improved colon length. In parallel, reduced expression of ion transporters such as SLC26a3 or down regulated in adenoma (DRA) seen in colitis was abrogated with VIP‐SSM intra rectal treatment but not with the free VIP peptide. Furthermore, increased pro‐inflammatory cytokine mRNA expression and damaged distal colonic histology and goblet cell loss seen in DSS was also significantly alleviated with VIP‐SSM treatment. The VPAC1 receptor expression was similar across all groups.ConclusionsThese results demonstrate that local administration of VIP nanomedicine is effective in alleviating severe inflammation and associated diarrhea in colitis. Thus, delivering VIP‐SSM nanoparticle orally with site‐specific release to the colon could be a potential option in managing UC in future.Support or Funding InformationNIH/NIDDK, Department of Veteran AffairsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Sprouty2 enhances IL‐33 expression and protects against experimental colitis.
Authors: Schumacher Michael A. , Hsieh Jonathan J. , Liu Cambrian Y. , Almohazey Dana , Warburton David , Washington M. Kay , Frey Mark R. 
Published: June 2021

OBJECTIVESprouty2 (Spry2) is an intracellular modulator of MAPK signaling. It is present in the intestinal tract, but its function there is unknown. Aberrant MAPK signaling is associated with altered cytokine production in disorders such as inflammatory bowel disease, raising the possibility that Spry2 may affect the response to colitis. In particular, MAPK regulates IL‐33 expression in response to bacterial infection, and IL‐33 has been shown to be protective in some forms of colitis. Furthermore, both MAPK and IL‐33 regulate secretory cell differentiation. Here we tested the hypothesis that Spry2 control of MAPK signaling alters cytokine production and/or secretory cell development, thus altering the intestinal response to injury.METHODSMice that lack Spry2 in the intestinal epithelium (VillinCre;Spry2flox/flox, hereafter Spry2KOIE) and littermate controls were given 3% dextran sodium sulfate (DSS) in drinking water for 4 days followed by 3 days without DSS, and colitis severity was assessed. Cytokine levels and epithelial marker expression were determined by qPCR in challenged and unchallenged mice. GSK3beta phosphorylation was determined by western blot. In vitro, IEC6 and YAMC intestinal epithelial cells were treated with GSK3beta and MAPK inhibitors and cytokine expression was assessed.RESULTSColonic Spry2 levels were reduced in DSS colitis by 59% (p<0.05). Spry2KOIE mice subjected to DSS had lower levels of pro‐inflammatory cytokines versus wildtype littermates, and were protected from histological damage (injury score reduced by 30%, p=0.037), weight loss, and colon shortening. Unchallenged Spry2KOIE mice displayed elevated GSK3beta phosphorylation on the inactivating Ser9 site in epithelial cells (88% increase, p<0.01). These mice also had increased IL‐33 expression (80% increase, p<0.05) and more DCLK1+ tuft cells in the colon (2.2‐fold increase, p<0.01). In vitro, inactivation of GSK3beta in both IEC6 and YAMC cells mimicked the increased IL‐33 observed in vivo; this effect was attenuated by ERK or p38 MAPK inhibitors. Expression of the epithelial‐derived cytokine CXCL2 was unaffected by GSK3beta inhibition, suggesting an IL‐33 specific response.CONCLUSIONColonic epithelial Spry2 is downregulated in response to injury and inflammation. This may represent an integration point of environmentally‐driven MAPK signals that leads to altered Wnt signaling, elevated IL‐33 expression, and promotion of intestinal tuft cell development as a protective or regenerative mechanism.Support or Funding InformationNIH R01DK095004This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Early weaning in pigs induces long‐term alterations in intestinal nutrient transporter function and expression
Authors: Li Yihang , Rajput Mrigendra , Fernandez Karina Matos , Moeser Adam J. 
Published: June 2021

Early life adversity (ELA) is associated with increased disease risk throughout the lifespan but the developmental mechanisms underlying the association remain elusive. Altered intestinal nutrient transporter function has been implicated as a mechanism contributing to a number of diseases associated with ELA such as Type 2 diabetes and inflammatory and functional GI disorders in humans and impaired health and growth performance in agricultural animals. Using an early weaning stress model of ELA in pigs, we investigated the link between ELA and long‐term intestinal nutrient transporter function and expression. Yorkshire breed female piglets were weaned from their sows at 15 d (Early Weaning; EW) or 28 d of age (Late Weaning; LW). Early and LW piglets were co‐housed and received the same diets. At 70 d of age, mid‐jejunum and distal ileum were harvested from EW and LW pigs and mounted on Ussing chambers to evaluate Na+‐dependent electrogenic nutrient transporter function, determined as the change in short‐circuit current (ΔIsc) in response to select luminal nutrients. Villus morphology and epithelial gene expression of nutrient transporters were assessed. Compared with age‐matched LW pigs, EW pigs exhibited reduced jejunal and ileal Na+ glucose linked transporter (SGLT1) function (by ~30%; P < 0.05) and amino transporter function for glutamate, glutamine, and alanine by (~30–35%; P < 0.05). In contrast, jejunal and ileal lysine transporter function was upregulated (by 110% and 25%, respectively; P < 0.05) in EW pigs. Gene expression for nutrient transporters (SGLT1, EAAT3, and B0AT1) were reduced (P < 0.05) while the lysine transporter CAT1 was enhanced in the EW jejunum. There were no differences observed in villus morphology between EW and LW pigs. Enteric neuronal inhibitors tetrodotoxin, atropine and propranolol, enhanced ΔIsc responses to glucose, lysine and alanine transporter function in EW but not in LW pig ileum. Together, these data show that EW in pigs induces lasting and divergent alterations in small intestinal nutrient transporter function and expression which is mediated in part by the enteric nervous system. These findings could provide new insight into the mechanisms of increased disease risk linked with ELA and thus lead to novel dietary or therapeutic strategies.Support or Funding InformationNIH R01 HD072968USDA‐NIFA #2017‐67015‐26673This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Helicobacter pylori Alters the Expression of Circadian Clock Components PER2 and BMAL1 During Infection
Authors: Teal Emma L. , Li Jing , Chakrabarti Jayati L. , Steele Nina , Rosselot Drew , Sundaram Nambirajan , Hawkins Jennifer , Hong Christian , Helmrath Michael A. , Diwan Tayyab L. , Zavros Yana L. 
Published: June 2021

BackgroundIndividuals with persistent Helicobacter pylori (H. pylori) infection are known to have an increased risk of gastric adenocarcinoma development. The circadian clock has been shown to be intimately involved in various gastrointestinal functions and clock components BMAL1 and PER2 have been found to be disrupted in colorectal cancer tissues. Aged individuals have a dampened expression of such components which may further increase their susceptibility to gastric cancer development.HypothesisPersistent infection of H. pylori induces an unregulated proliferation of gastric epithelial cells via activation of BMAL1 leading to a predisposition to gastric cancer development.MethodsGastric organoids (hFGOs) were generated from either primary gastric tissue derived from aged (> 55 years of age) and young (14–20 years) individuals. Gastric organoids were then infected with H. pylori. BMAL1 and PER2 expression was then quantified over a period of 24 hours. Proliferation and expression of gastric cancer markers were quantified by qRT‐PCR.ResultsH. pylori infection correlated with a significant upregulation of cytoplasmic BMAL1 expression within the gastric fundus of infected individuals. Robust circadian rhythmicity of clock genes Bmal1 and Per2 were observed in human hFGOs. Phosphorylation (inactivation) of GSK‐3b showed circadian changes in hFGOs over 52 hours. H. pylori infection of hFGOs resulted in sustained GSK‐3b phosphorylation, increased BMAL1 expression and epithelial cell hyperproliferation when compared to uninfected controls. Organoids derived from elderly patients exhibited a dampened rhythmicity of of clock genes Bmal1 and Per2. Dampened rhythmicity correlated with increased gastric cancer biomarkers of intestinal metaplasia including Cdx1, Cdx2 and MUC2.ConclusionsChronic H. pylori infection induces a hyperproliferative response in gastric epithelial cells by inactivating GSK‐3β leading to the activation of BMAL1:CLOCK. This may be a plausible mechanism that predisposes individuals with persistent H. pylori infection to gastric cancer development and also heightens this risk in aged individuals.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nucleophosmin Is Essential for Stimulation of Rac1 Nuclear Accumulation by RNA‐binding Protein HuR in the Intestinal Epithelium
Authors: Liu Lan , Chung Hee Kyoung , Kalakonda Sudhakar , Yu Tingxi , Xiao Lan , Rao Jaladanki N. , Wang Jian‐Ying 
Published: June 2021

The mammalian intestinal epithelium is a rapidly self‐renewing tissue in the body and its homeostasis is tightly regulated by numerous factors including the RNA‐binding protein HuR. HuR is highly expressed in the intestinal mucosa and acts as a key regulator of the epithelial homeostasis by regulating the stability and translation of target mRNAs. We have recently demonstrated that targeted deletion of HuR in intestinal epithelial cells (IECs) inhibits growth of the small intestinal mucosa and delays healing after injury in mice partially by altering subcellular distribution of a small GTP‐binding protein Rac1. HuR knockout in IECs increases the levels of cytoplasmic Rac1 in vivo as well as in vitro, but the exact mechanism underlying this process remains unknown. Nucleophosmin (NPM) is a phosphoprotein that binds to proteins containing nuclear localization signals for their import and functions as a molecular chaperone. In this study, we investigated the role of NPM in HuR‐dependent nuclear accumulation of Rac1 in the intestinal epithelium.MethodsStudies in vivo were conducted in intestinal epithelial tissue‐specific HuR knockout (IE‐HuR−/−) mice, and experiments in vitro were carried out in cultured IEC‐6 cells that were derived from rat small intestinal crypts. Functions of NPM and HuR in vitro were investigated via their gene silencing by using siRNAs specifically targeting NPM (siNPM) or HuR (siHuR) and their overexpression. Levels of NPM, Rac1, and HuR proteins were measured by Western blotting analysis, and the cellular distributions of Rac1 and NPM were examined by immunofluorescent staining.ResultsThe levels of NPM protein in the intestinal mucosa decreased significantly in IE‐HuR−/− mice compared with control littermates. Decreased NPM was associated with elevated cytoplasmic Rac1 in the HuR‐deficient epithelium, along with a decrease in nuclear Rac1 abundance. In cultured IEC‐6 cells, HuR silencing by transient transfection with siHuR also decreased cellular NPM, but it increased the levels of cytoplasmic Rac1. NPM was found to physically interact with Rac1 and formed NPM/Rac1 complex in normal IEC‐6 cells. NPM silencing by transfection with siNPM increased cytoplasmic Rac1 levels and decreased nuclear Rac1 abundance. Furthermore, ectopically expressed NPM abolished the increased levels of cytoplasmic Rac1 in HuR‐silenced cells and restored the nuclear Rac1 levels to near normal.ConclusionsThese results indicate that 1) HuR regulates NPM expression in the intestinal epithelium; and 2) HuR regulates Rac1 subcellular trafficking by altering NPM.Support or Funding InformationNational Institutes of Health (JYW) and US Department of Veterans Affairs (JYW and JNR)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An iron supplement containing L. plantarum increases ferric iron and up‐regulates the ferric reductase Dcytb in human Caco‐2/HT29 MTX co‐cultures
Authors: Scheers Nathalie , Önning Gunilla , Björklund Malin , Sandberg Ann‐Sofie 
Published: June 2021

Iron absorption from a bread meal, added an encapsulated iron‐containing supplement, may be increased if the lactic acid bacteria L. plantarum (DSM 9843) is present. The aim of this study was to investigate the mechanism by which L. plantarum increases iron absorption. We have previously shown that lactic fermentation of mixed vegetables increased iron absorption in humans and voltammetric studies revealed that the level of ferric iron [Fe3+ (H2O)6] was increased in the fermented vegetables, compared to unfermented control. In this study, we detected a similar increase in ferric iron in the in vitro digested bread meal containing the supplement with L. plantarum. In a co‐culture of intestinal Caco‐2 cells and mucus‐producing goblet cells (HT29 MTX E12), we observed that the ferric reductase Dcytb increased in the presence of L. plantarum‐containing iron supplements while the production of mucin (muc5a1c) decreased independent of L. plantarum. This suggests that the L. plantarum‐effect on iron absorption is mediated through enhancing the Fe3+/Dcytb system.Support or Funding InformationThe work was financially supported by Probi ABThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unlocking gut health with the prebiotic galactooligosaccharide (GOS) and probiotic L. reuteri to keep pathogenic S. typhimurium at bay
Authors: Forshee Micah D. , Sharda Daniel R. 
Published: June 2021

The microbiome is a dynamic community that can positively and negatively influence host health. Lactobacillus reuteri is a probiotic that has received much attention for its ability to inhibit pathogens such as Salmonella typhimurium, Escherichia coli, and Clostridium difficile. It does so by its unique ability to metabolize glycerol into the antimicrobial compound 3‐HPA, which is commonly referred to as reuterin. The ability to secrete reuterin is dependent not only on glycerol availability but also the concentration of glucose. In fact, there appears to be a “goldilocks” ratio between glucose and glycerol as either too much or too little glucose significantly diminishes reuterin production. Since L. reuteri primarily resides in distal regions of the intestine and colon where most of the glucose has already been absorbed, it seems unlikely that reuterin production would be promoted at the physiological level via this mechanism.Prebiotics are carbohydrates that are indigestible by the host and remain for enzymatic digestion by intestinal probiotics. Inulin and galactooligosaccharides (GOS) are two widely studied prebiotics that are known for their ability to promote the growth of a wide range of lactobacilli, and have been shown to promote L. reuteri growth to varying degrees. Here, we asked if prebiotics such as inulin and GOS promote the production of reuterin in the absence of glucose. L. reuteri were cultured in TSB with or without glycerol in the presence of either glucose, inulin, or GOS, and assessed for their ability to produce reuterin. While inulin did not enhance the production of reuterin, GOS induced reuterin production, although 45% less than that of glucose. Moreover, unlike the dose‐dependence observed with glucose, incubation with GOS induced similar reuterin production regardless of concentration. This suggests that an enzymatic equilibrium may exist where glucose/galactose is cleaved from GOS only as needed by L. reuteri. Finally, to confirm the biopotency of reuterin production, we cultured S. typhimurium with supernatants from L. reuteri that were grown with various carbohydrates. Supernatant dilutions as low as 1:15 were able to significantly retard growth of S. typhimurium with ratios of 1:1 completely inhibiting growth. Together, these results suggest that prebiotics such as GOS may be able to elicit physiologically relevant production of reuterin, which may shape the flora of the microbiome and reduce incidence and severity of pathological infections. Further, as GOS are particularly abundant in breast milk, it suggests a possible link for early immunoprotection from intestinal pathogens while the infant is still immunologically naïve.Support or Funding InformationOlivet Nazarene University Honors Program and Department of Biological SciencesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepcidin interaction with ferroportin in the Xenopus oocyte expression system
Authors: Ruwe T Alex , Vieth Kyle R. , Aschemeyer Sharraya , Qiao Bo , Ganz Tomas , Nemeth Elizabeta , Mackenzie Bryan 
Published: June 2021

The cellular iron‐export protein ferroportin (Fpn), expressed in enterocytes and macrophages, is essential for iron homeostasis. Fpn operates under the control of the iron‐regulatory hormone hepcidin. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and ubiquitin‐dependent degradation of the transport protein [Drakesmith H et al (2015) Cell Metab 22, 777–787]. Here we explored hepcidin inhibition of 55Fe efflux from RNA‐injected Xenopus oocytes expressing human Fpn. Hepcidin treatment induced a rapid inhibition (t½ = 8.2 ± [SEM] 0.5 min; r2 > 0.99, P < 0.001) of Fpn‐mediated 55Fe efflux that was not dependent on endocytosis, a conclusion based on the following four observations: (i) Inhibition of Fpn by hepcidin was only modestly temperature‐dependent (Q10 = 2.5 ± [SEM] 1.4 over the range 17–27 °C) whereas endocytosis is expected to be substantially more temperature‐dependent, Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem 256, 2615–2617]. (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin‐mediated (Dynasore) and clathrin‐independent (filipin, genistein) endocytosis. (iii) Hepcidin treatment inhibited 55Fe efflux in oocytes expressing the K8R mutant, in which 8 Lys residues required for ubiquination and endocytosis of Fpn were mutated to Arg, to the same degree as in wildtype Fpn. (iv) Confocal LSM imaging of wildtype Fpn‐GFP and K8R‐Fpn‐GFP expression in oocytes revealed no detectable endocytosis of either protein after 30‐min treatment with hepcidin. Our data reveal that hepcidin can directly block Fpn activity independent of Fpn internalization. We speculate that the Xenopus oocyte lacks the specific signal by which hepcidin triggers Fpn internalization. The Xenopus oocyte is therefore an ideal model system in which to study the mechanism by which hepcidin directly blocks Fpn activity.Support or Funding InformationNIH–NIDDK grant R01 DK107309This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.