bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the Antioxidant Glutathione by Enteric Glia During Inflammation
Authors: Brown Isola A.M. , Gulbransen Brian D. 
Published: November 2023

Enteric glia are a unique type of peripheral neuroglia that surround and support neurons in the enteric nervous system. Enteric glia serve a dichotomous protective/pathogenic role where activated glia contribute to neurodegeration via mechanisms involving oxidative stress (Brown et. al, 2015). Conversely, the ablation of glia leads to neurodegeneration and altered gastrointestinal (GI) motility patterns (Aubé et. al, 2006; Abdo et. al, 2010). This suggests that a loss of glial neuroprotective factors such as reduced glutathione (GSH) may also contribute to neurodegeneration in disease. Here, we investigate the role of glial GSH in GI pathology. Specifically, we tested the hypotheses that glial GSH content is dysregulated during GI inflammation and that inhibition of glial GSH production can contribute to neurodegeneration.We measured neuronal and glial content of reduced/oxidized glutathione (GSH/GSSG) using antibodies against glutaradehyde‐linked and free GSH/GSSG. We measured neuronal survival by immunohistochemistry and inhibited GSH synthesis using Buthionine Sulfoximine (BSO), an inhibitor of the GSH synthesis enzyme glutamyl‐cysteine ligase. We induced inflammation in vivo using 2,4‐dinitrobenzene sulfonic acid (DNBS) and in situ using the P2X7 receptor (P2X7R) agonist BzATP. All experiments used male C57Bl/6 mice, aged 8–10 weeks. Data was analyzed using a Student's t‐test or one‐way ANOVA as appropriate.Total glial glutaradehyde‐linked GSH/GSSG content was not altered following in vivo DNBS colitis (p= 0.81). Free neuronal oxidized glutathione (GSSG) was increased during DNBS colitis (p<0.001) with no corresponding change in neuronal reduced glutathione (GSH), thus increasing the ratio of oxidized/reduced glutathione (GSSG/GSH) (p<0.05). Surprisingly, the ratio of neuronal oxidized/reduced glutathione did not increase during an in situ model of neuroinflammation with the P2X7R agonist BzATP. In situ inhibition of endogenous GSH synthesis decreased myenteric plexus neuronal density (p<0.01) but did not potentiate P2X7R mediated neuron loss.Our data suggests a role for glial GSH where glia produce neuroprotective GSH in response to increased neuronal oxidative stress during GI pathophysiology. Inhibition of this protective capacity of glia with BSO makes enteric neurons more susceptible to neurodegeneration and may be a potential mechanism explaining the susceptibility of specific neuronal populations to oxidative stress and neurodegeneration during inflammation.Support or Funding InformationNIEHS Training Grant in Environmental Toxicology to I.B. and Crohn's and Colitis Foundation of America Senior Research Award to B.G.
Bile acids regulate intestinal epithelial restitution: implications for pathogenesis and therapy of IBD
Authors: Lajczak Natalia Katarzyna , Mroz Magdalena Slawa , Saint‐Criq Vinciane , Keely Stephen 
Published: November 2023

Epithelial restitution is an essential process for maintenance of intestinal barrier function. Increased levels of colonic bile acids have been proposed to be involved in the pathogenesis of inflammatory bowel disease (IBD) but their roles in regulating restitution are not yet known. Here, we investigated the effects of bile acids on epithelial restitution and molecular pathways involved in colonic epithelial healing.T84 colonic epithelial cells, grown as monolayers on transparent permeable supports, were wounded by scratching with a pipette tip at T=0. Cells were treated with either the most abundant colonic bile acid, deoxycholic acid (DCA; 150 μM), the “therapeutic” bile acid, ursodeoxycholic acid (UDCA; 100 μM), a Farnesoid X Receptor agonist, GW4064 (5 μM), or a cystic fibrosis transmembrane conductance regulator (CFTR) channel blocker, CFTR(inh)‐172 (10 μM). Restitution was measured as wound area after 48 h expressed as % T=0 wound area. HEK‐293 cells were transfected with vector expressing luciferase gene under control of the CFTR promoter and vectors expressing FXR. Protein expression was assessed by western blotting and cell migration by Boyden chamber assay.After 48 h post‐wounding, wound closure in untreated cells was 63.3 ± 13.5% of that at T=0, while in cells treated with DCA (150 μM) it was reduced to 24.5 ± 13.1 % (n = 5; p <0.001), whereas UDCA enhanced healing to 88 ± 4 (n = 5; p < 0.001). Furthermore, UDCA prevented inhibition of wound closure by DCA. The effects of DCA are mediated via a decrease in cell migration to 0.7 ± 0.1 fold (n = 5, p < 0.05) of that in untreated controls, rather than inhibition of cell growth. Furthermore, DCA decreased cell surface CFTR expression to 23 ± 5% of controls (n = 3, p < 0.001), while a CFTR inhibitor, CFTR(inh)‐172 (10 μM), attenuated wound closure to 37 ± 2 % (n = 5; p < 0.01), compared to. Moreover, DCA decreased CFTR promoter activity, in a concentration‐dependent manner that was also dependent on co‐expression of FXR. Finally, GW4064 (5 μM), an agonist of FXR, mimicked DCA effects on wound healing and CFTR expression.Our data suggest that colonic bile acids differentially regulate intestinal epithelial restitution and that UDCA promotes healing and protects against the detrimental effects of DCA. Thus, manipulation of the colonic bile acid pool may prove to be a useful approach for promoting intestinal barrier function in IBD.
IBS‐induced constipation alters gut microbiota stability leading to intestinal transit time changes in the host
Authors: Touw Ketrija , Nadimpalli Anuradha , Hubert Nathaniel , Leone Vanessa , Kashyap Purna , Chang Eugene 
Published: November 2023

Pathogenesis of Irritable Bowel Syndrome (IBS) is multifactorial, and gut microbes are sought to play a role in development and progression of disease. In this study we examined how constipation‐predominant Irritable Bowel Syndrome (IBS‐C) microbiota affect host intestinal transit and signaling pathways. To determine IBS‐C dysbiosis effects on the host, we introduced fecal microbiota from constipated mice to germ‐free recipients. Recipient mice of IBS‐C microbiota developed delayed GI transit time when compared to controls. Bacterial DNA analysis by HiSeq sequencing showed significant increase in Bacteroides uniformis, Bacteroides ovatus and Parabacteroides distasonis species from Bacteroidetes phylum in constipated mice. Fecal microbiota metabolic potential was measured with tetrazolium dye assay and showed significant differences between recipients of control and IBS‐C microbiota. Microbial metabolites such as short chain fatty acids (SCFA) are known to alter intestinal transit. SCFA levels in cecal contents of recipeints were measured by GC‐MS, and showed lower butyrate, propionate and acetate concentration in IBS‐C mice when compared to controls. To determine contractile properties of intestine after introduction of control and IBS‐C microbiota, we stimulated colonic rings with carbachol and KCl, and observed hypercontractility in IBS‐C microbiota recipients. To determine mechanism for delayed GI transit we analyzed colon tissues in fecal microbiota recipients by qRT‐PCR, and determined dysregulated neuromediator pathways. Overall, these results suggest that IBS‐C dysbiosis contributes to development of delayed transit time through microbial metabolite production and changes in colonic contractile properties.Support or Funding InformationR01 DK097268 and T32 DK007074
The Baylor KOMP2 Program and BaSH Consortium: A Resource for Producing and Phenotyping Knockout Mice
Authors: Reynolds Corey L , Beaudet Arthur , Dickinson Mary , Seavitt John , Heaney Jason 
Published: November 2023

The Knockout Mouse Phenotyping Program (KOMP2) was launched in the Fall of 2011 with the goal of having three mouse production and cryopreservation centers generate a knockout mouse strain for every protein coding gene by using embryonic stem cell resources generated by the International Knockout Mouse Consortium (IKMC). In addition to the generation of these knockout lines of mice, each center is responsible for the systematic, broad‐based, phenotyping of each line using standardized procedures. Phenotyping procedures start when the animal is nine weeks of age and conclude at sixteen weeks of age. The tests include behavioral, metabolic, cardiovascular, auditory, ocular, and histopathological assays. Gene‐to‐phenotype associations are made by a versioned statistical analysis with all data (quantitative and images) freely available on a web portal which offers several data download features. New technology development has been supplemented throughout the projects lifespan to include the standardized phenotyping of embryos from embryonic lethal mouse lines, usage of CRISPR/Cas9 technology to increase productivity and a subset of lines will be examined for small molecule metabolites to detect metabolic abnormalities. At present, the KOMP2 project has produced over 2,500 lines of mice and phenotyping of these lines is on track to be completed by Fall 2016 with all data currently being freely available to the scientific community.Support or Funding InformationNIH U54‐HG006348
The Micro‐RNA let‐7f Regulates Expression of the Voltage‐gated Mechanosensitive Ion Channel NaV1.5 in Human Gastrointestinal Smooth Muscle
Authors: Mazzone Amelia , Strege Peter R , Bernard Cheryl E , Cima Robert R , Larson David W , Dozois Eric J , Hayashi Yujiro , Ordog Tamas , Gibbons Simon J , Beyder Arthur , Farrugia Gianrico 
Published: November 2023

BackgroundSCN5A‐encoded NaV1.5 is a mechanosensitive voltage‐gated sodium channel expressed in human gastrointestinal smooth muscle cells. In the gastrointestinal tract, alterations in NaV1.5 current density by naturally occurring mutations or by pharmaceutical block of channel function are associated with constipation. In the heart loss of NaV1.5 current is associated with cardiac contractility disorders such as dilated cardiomyopathy. Ion channel expression level is regulated by miRNAs, and NaV1.5 is known to be regulated by miRNA in heart. However, it is not known if miRNAs regulate NaV1.5 expression in GI smooth muscle.AimTo identify miRNAs that regulate NaV1.5 expression in gastrointestinal smooth muscle in health and disease.MethodsA qPCR array was used to screen for differential expression of 372 of the most common miRNAs in the miRBase database to test the differential expression of miRNA between control tissue and tissues from patients with slow transit constipation. qRT‐PCR was done to determine expression levels of SCN5A mRNA. A cell line (HuJej) was derived from human jejunum smooth muscle cells by immortalization using the SV40 large T antigen. Whole cell voltage clamp experiments were done by standard patch clamp techniques.ResultsSCN5A mRNA expression was reduced by 66±0.08% in patients with slow transit constipation (57.5±1.8 years) compared to age matched controls (53±1.7 years). qPCR array screening showed that 12 miRNAs were significantly upregulated in colonic muscularis externa from patients with STC compared to controls. Let‐7f miRNA was increased 6.46‐fold in slow transit constipation compared to controls and was the only miRNA with strong negative correlation to SCN5A mRNA expression in the same tissues. HuJej cells express NaV1.5; therefore, we investigated the effect of let‐7f on NaV1.5 currents by transfection with a stable miRNA mimic. 48 hours after transfection with let‐7f mimic, NaV1.5 currents were significantly decreased compared to untransfected cells or to cells transfected with non‐targeting miRNA (let‐7f, −0.6±0.1 pA/pF versus untransfected, −3.9±0.6 pA/pF or non‐targeting, −2.3±0.6 pA/pF).Summary and ConclusionsSCN5A mRNA is decreased in slow transit constipation. Expression levels of let‐7f and NaV1.5 RNA are negatively correlated in human colonic smooth muscle. miRNA let‐7f regulates NaV1.5 current density. Regulation of NaV1.5 current density may have relevance in health and disease.Support or Funding InformationThis work was supported by grant DK52766.
miR429 is Upregulated in Inflammatory Monocytes and Regulates Monocyte Migration
Authors: Barry Zachary R , Kynast Ross W , Stinson W Alexander , Fox David A , Amin M Asif , Rabquer Bradley James 
Published: November 2023

PurposeRheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Inflammation is characteristic of an immune response, brought about by increased migration of leukocytes to the damaged area. Monocytes migrate from the blood into the tissue to exert their effects. A multitude of genes involved in inflammatory pathways regulate monocyte migration. miRNAs are post transcriptional regulators that effect gene expression in a variety of cell types and in response to various stimuli. We hypothesized that inflammation regulates miRNA expression, including miR429, which then plays a specific role in regulating the expression of monocyte genes involved in monocyte migration.Methods and ResultsMonocytes were isolated from the blood of normal subjects (n=5) and patients with RA (n=8). miRNA was isolated and cDNA prepared. qPCR was performed using primers specific for miR429. There was a 2.6 fold increase in the expression of miR429 in the RA patients compared to the normal subjects. The miRNA databases, miRanda and MiRwalk, were used to identify gene targets for miR429 that are involved in monocyte and inflammation pathways. This analysis identified 38 gene targets with possible relevance to monocyte inflammatory processes, including MAP4K4, PIKfyve, and PAK2. miR429 mimics and inhibitors were then used to assess the role of miR429 in regulating gene expression and migration. After confirming mimic and inhibitor specificity and efficiency in U937 monocytes, we found miR429 had an effect on PAK2 expression. The mimic reduced the expression of PAK2 by a fold change of 0.57, and the inhibitor increased the expression of PAK2 by a fold change of 28.6. Chemotaxis was performed to test the effect of miR429 on monocyte migration in response to monocyte chemoattractant protein (MCP‐1). The miR429 mimic treated U937 cells (n=11 replicates) had similar migration to MCP‐1 when compared to control cells treated with only transfection reagent (n=10). However, migration was decreased by 47% in U937 cells treated with the miR429 inhibitor (n=10 replicates) compared to the control.ConclusionOur results suggest miR429 is upregulated in RA monocytes, and that miR429 plays a role in monocyte migration by regulating genes such as the cytoskeletal regulator, PAK2.Support or Funding InformationHewlett‐Mellon Fund for Faculty Development at Albion College
Comparative analysis of methods to profile mRNA expression of G‐protein coupled receptors
Authors: Sriram Krishna , Zhou Shu , Lowy Andrew M. , Insel Paul A. 
Published: November 2023

G‐Protein coupled receptors (GPCRs) comprise the largest family of cell surface receptors and proteins used as targets of approved drugs. Due to their low abundance, paucity of specific and sensitive antibodies and the challenge of identifying receptors in individual cell types, efforts to profile GPCR expression typically involve mRNA‐based methods and the relative quantification of gene expression. In the current study we compared in parallel the use of Affymetrix microarrays, RNAseq and qPCR‐based arrays for the measurement of mRNA expression of non‐chemosensory endogenously expressed (endo)GPCRs. We analyzed samples of patient‐derived pancreatic cancer‐associated fibroblasts (CAFs). RNAseq was done with a read depth of ~25 million reads/sample with single‐end 75bp reads. Affymetrix samples were run on the GeneChip Human Genome U133 Plus 2.0 Array chip and for qPCR, we used Taqman GPCR arrays from Life Technologies.Our results demonstrate a high degree of correlation between results obtained from the GPCR arrays and RNAseq. In general, the number of GPCRs detected (~100 per sample), their identity and magnitude of expression were closely correlated with those 2 methods. The validity of the results was verified by independent qPCR. By comparison, the Affymetrix arrays failed to identify a large number of GPCRs (~30) detected by both RNAseq and Taqman arrays. In addition, different probe sets for the same GPCR on Affymetrix arrays sometimes resulted in drastically divergent mRNA intensities, even though both values were reported as being “statistically significant” (P<0.05). We conclude that RNAseq and qPCR‐based arrays both provide appropriate methods to assess GPCR expression (although each method has advantages and disadvantages) and are preferable to Affymetrix arrays for the detection of GPCR mRNAs, especially low abundance GPCRs. Importantly, our mRNA analyses of GPCR expression by both RNA seq and GPCR arrays indicate that CAFs and other individual cell types express >100 different GPCRS, including a large number of orphan (without known physiologic agonists) receptors, certain of which we find can regulate cell function and may be novel drug targets.Support or Funding InformationSupported by NIH R21CA189477 and T32CA121938
The Effect of Phenylalanine Exposure on Cell Migration
Authors: Vaughan Melville B , Alkadhem Niyaf J , Seagraves Nikki J 
Published: November 2023

Phenylketonuria (PKU) is an inborn error of metabolism that results when the amino acid phenylalanine (Phe) cannot be catabolized to tyrosine due to a mutation in phenylalanine hydroxylase enzyme. The inability to regulate Phe levels in individuals with PKU leads to mental retardation, seizures and other symptoms that can be avoided with early detection and a Phe‐restricted diet. When PKU mothers do not adhere to a Phe‐restricted diet during pregnancy, Phe can become teratogenic to the fetus and result in a condition known as Maternal PKU. Maternal PKU is characterized by multiple congenital defects, including cardiovascular malformations (CVM) such as coarctation of the aorta, tetralogy of Fallot, ventricular septal defects and hypoplastic left heart syndrome. However, the mechanisms responsible for Phe‐induced CVMs are poorly understood. Previous studies have shown that cardiac neural crest cells are important in formation of the outflow tract (OFT) and aortic arch arteries (AAA). Cell migration of the neural crest cells is a central process in the development of the heart.Study ObjectiveSince congenital CVM of the OFT and AAA are often observed in maternal PKU, in this study we aimed to determine if exposure to high Phe levels perturbs cell migration and proliferation.MethodsWe conducted migration assays on human fibroblasts cells and chick neural crest cells tubes to determine if migration was affected by Phe. Migration was assessed by classic scratch wound assay and by Silicon elastomeric masks migration study. Additionally, in the human fibroblast cells we stained for markers of proliferation to determine if it was affected by the Phe exposure. The chick neural tubes were dissected and plated on a 3‐D collagen matrix. Migration of neural crest cells was observed over 12–24 hours and the area of migration was quantified.ResultsPhe doses tested were 0.1mM, 1mM, and 10mM. We observed migration in human fibroblast was decreased in the higher doses (1mM and 10mM) but was increased in the 0.1mM dose in both methods observed. Proliferation was decreased in the cells exposed to 0.1mM Phe but was not changed in the higher doses (1mM and 10mM). Chick neural tubes were exposed to 1.5mM Phe and we observed increase in the area of migration.ConclusionPhenylalanine has a dose‐dependent effect on cell migration in vitro. Additionally, it has an effect on proliferation rates. While these results are preliminary they provide a foundation for more mechanistic studies to understand the effects of MPKU on development.Support or Funding Information2015 Fulbright Visiting Scholar Program for Iraq, UCO CMS CURE STEM program, and UCO office of Research and Grants
Role of Skeletal Muscle in Inner Ear Development
Authors: Rot Irena , Kablar Boris 
Published: November 2023

We are interested in developmental morphodynamics and the ability of muscle to provide mechanical cues for organogenesis during development. Our study of compound mouse knockout (Myf5−/−:MyoD−/) embryos that entirely lack skeletal musculature has revealed that these embryos develop abnormal phenotypes in many organs, such as skeleton (palate, mandible, clavicle), lungs, retina and inner ear. In this study we analyzed the altered development of the mouse inner ear and its sensory fields. Amyogenic fetuses lack skeletal muscles that move the chain of three middle ear ossicles responsible for the transfer of sound vibrations. They are also unable to tilt their head which prevents the perception of angular acceleration. The inner ear areas that were most affected in amyogenic embryos were vestibular sensory fields, i.e., cristae ampullaris, sensitive to angular acceleration. Our finding that a specific vestibular hair cell type 1 was absent in mutants was further used to identify a profile of genes specific for the lacking cell type. We performed a DNA microarray analysis to compare gene expression between inner ear sensory fields of amyogenic and wild type mouse embryos. This analysis generated a list of 10 up‐ and 87 down‐regulated genes in mutant inner ear tissues. We subsequently performed a detailed consultation of web‐accessible mouse databases regarding the genes with altered expression in mutants. This allowed us to identify 6 candidate genes with a possible role in the development of the inner ear sensory organs: Actc1, Pgam2, Ldb3, Eno3, Hspb7 and Smpx. They were established as candidate genes based on their altered expression in our mutant embryos compared to the wild type, as well as their previously documented expression in the sensory organs of the mouse inner ear. Further analysis of inner ear tissues of knockout embryos for these specific molecules is necessary to confirm, or rule out, the role of candidate genes in the inner ear development. Additionally, we searched for human homologues of the candidate genes since a number of syndromes in humans have inner ear abnormalities associated with them. Mutations in one of our candidate genes, Smpx, have been reported as the cause of X‐linked deafness in humans. Our current study suggests an epigenetic role that skeletal muscle might play in organogenesis, and offers an approach to finding novel genes responsible for altered morphology in the inner ear.Support or Funding InformationNatural Sciences and Engineering Research Council of CanadaCanada Foundation for Innovation