subcellular-fractionation-from-fresh-frozen-gastrointestinal

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

UPV-EHU, CIBERDEM

Type 1 diabetes (T1D) is a complex autoimmune disease in which pancreatic beta cells are specifically destroyed by the immune system. The disease has an important genetic component and more than 50 loci across the genome have been associated with risk of developing T1D. The molecular mechanisms by which these putative T1D candidate genes modulate disease risk, however, remain poorly characterized and little is known about their effects in pancreatic beta cells. Functional studies in in vitro models of pancreatic beta cells, based on techniques to inhibit or overexpress T1D candidate genes, allow the functional characterization of several T1D candidate genes. This requires a multistage procedure comprising two major steps, namely accurate selection of genes of potential interest and then in vitro and/or in vivo mechanistic approaches to characterize their role in pancreatic beta cell dysfunction and death in T1D. This chapter details the methods and settings used by our groups to characterize the role of T1D candidate genes on pancreatic beta cell survival and signaling pathways, with particular focus on potentially relevant pathways in the pathogenesis of T1D, i.e., inflammation and innate immune responses, apoptosis, beta cell metabolism and function.

Pancreatic Beta Cell Survival and Signaling Pathways: Effects of Type 1 Diabetes-Associated Genetic Variants.

Neonatal diabetes mellitus (NDM) is a rare form of diabetes diagnosed within the first 6 months of life. Genetic studies have allowed the identification of several genes linked to the development of NDM; however, genetic causes for ∼20% of the cases remain to be clarified. Most cases of NDM involve isolated diabetes, but sometimes NDM appears in association with other pathological conditions, including autoimmune diseases. Recent reports have linked activating mutations in  with early-onset autoimmune disorders that include diabetes of autoimmune origin, but the functional impact of -activating mutations have not been characterized at the pancreatic β-cell level. By using whole-exome sequencing, we identified a novel missense mutation in the binding domain of the STAT3 protein in a patient with NDM. The functional analyses showed that the mutation results in an aberrant activation of STAT3, leading to deleterious downstream effects in pancreatic β-cells. The identified mutation leads to hyperinhibition of the transcription factor Isl-1 and, consequently, to a decrease in insulin expression. These findings represent the first functional indication of a direct link between an NDM-linked activating mutation in  and pancreatic β-cell dysfunction.

An Activating Mutation in  Results in Neonatal Diabetes Through Reduced Insulin Synthesis.

To identify additional celiac disease associated loci in the Major Histocompatibility Complex independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level.

A Celiac Diasease Associated lncRNA Named HCG14 Regulates NOD1 Expression in Intestinal Cells.

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

Article	Year
Pancreatic Beta Cell Survival and Signaling Pathways: Effects of Type 1 Diabetes-Associated Genetic Variants. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26936771	2016
An Activating Mutation in Results in Neonatal Diabetes Through Reduced Insulin Synthesis. Diabetes\| PubMed ID: 28073828	2017
A Celiac Diasease Associated lncRNA Named HCG14 Regulates NOD1 Expression in Intestinal Cells. Journal of pediatric gastroenterology and nutrition\| PubMed ID: 29601440	2018
Transcription Factor Binding Site Enrichment Analysis in Co-Expression Modules in Celiac Disease. Genes\| PubMed ID: 29748492	2018

Izortze Santin

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