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      The Water Channel Aquaporin 8 is a Critical Regulator of Intestinal Fluid Homeostasis in Collagenous Colitis

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          Abstract

          Background and Aims

          Diarrhoea is a common, debilitating symptom of gastrointestinal disorders. Pathomechanisms probably involve defects in trans-epithelial water transport, but the role of aquaporin [AQP] family water channels in diarrhoea-predominant diseases is unknown. We investigated the involvement of AQPs in the pathobiology of collagenous colitis [CC], which features chronic, watery diarrhoea despite overtly normal intestinal epithelial cells [IECs].

          Methods

          We assessed the expression of all AQP family members in mucosal samples of CC patients before and during treatment with the corticosteroid drug budesonide, steroid-refractory CC patients and healthy controls. Samples were analysed by genome-wide mRNA sequencing [RNA-seq] and quantitative real-time PCR [qPCR]. In some patients, we performed tissue microdissection followed by RNA-seq to explore the IEC-specific CC transcriptome. We determined changes in the protein levels of the lead candidates in IEC by confocal microscopy. Finally, we investigated the regulation of AQP expression by corticosteroids in model cell lines.

          Results

          Using qPCR and RNA-seq, we identified loss of AQP8 expression as a hallmark of active CC, which was reverted by budesonide treatment in steroid-responsive but not refractory patients. Consistently, decreased AQP8 mRNA and protein levels were observed in IECs of patients with active CC, and steroid drugs increased AQP8 expression in model IECs. Moreover, low APQ8 expression was strongly associated with higher stool frequency in CC patients.

          Conclusion

          Down-regulation of epithelial AQP8 may impair water resorption in active CC, resulting in watery diarrhoea. Our results suggest that AQP8 is a potential drug target for the treatment of diarrhoeal disorders.

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          Most cited references 38

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          Primer3Plus, an enhanced web interface to Primer3

          Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.
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            SARTools: A DESeq2- and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data

            Background Several R packages exist for the detection of differentially expressed genes from RNA-Seq data. The analysis process includes three main steps, namely normalization, dispersion estimation and test for differential expression. Quality control steps along this process are recommended but not mandatory, and failing to check the characteristics of the dataset may lead to spurious results. In addition, normalization methods and statistical models are not exchangeable across the packages without adequate transformations the users are often not aware of. Thus, dedicated analysis pipelines are needed to include systematic quality control steps and prevent errors from misusing the proposed methods. Results SARTools is an R pipeline for differential analysis of RNA-Seq count data. It can handle designs involving two or more conditions of a single biological factor with or without a blocking factor (such as a batch effect or a sample pairing). It is based on DESeq2 and edgeR and is composed of an R package and two R script templates (for DESeq2 and edgeR respectively). Tuning a small number of parameters and executing one of the R scripts, users have access to the full results of the analysis, including lists of differentially expressed genes and a HTML report that (i) displays diagnostic plots for quality control and model hypotheses checking and (ii) keeps track of the whole analysis process, parameter values and versions of the R packages used. Conclusions SARTools provides systematic quality controls of the dataset as well as diagnostic plots that help to tune the model parameters. It gives access to the main parameters of DESeq2 and edgeR and prevents untrained users from misusing some functionalities of both packages. By keeping track of all the parameters of the analysis process it fits the requirements of reproducible research.
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              Reproducible RNA-seq analysis using recount2

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                Author and article information

                Journal
                J Crohns Colitis
                J Crohns Colitis
                eccojc
                Journal of Crohn's & Colitis
                Oxford University Press (UK )
                1873-9946
                1876-4479
                July 2020
                04 February 2020
                04 February 2020
                : 14
                : 7
                : 962-973
                Affiliations
                [1 ] Department of Biomedical and Clinical Sciences [BKV), Linköping University , Linköping, Sweden
                [2 ] Division of Gastroenterology and Hepatology, Department of Biomedical and Clinical Sciences [BKV), Faculty of Health Science, Linköpings University , Linköping, Sweden
                [3 ] Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology (NTNU) , Trondheim, Norway
                [4 ] Department of Gastroenterology and Hepatology, St Olav’s University Hospital , Trondheim, Norway
                [5 ] Clinic of Medicine, St Olav’s University Hospital , Trondheim, Norway
                [6 ] Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology (NTNU) , Trondheim, Norway
                [7 ] Wallenberg Centre for Molecular Medicine (WCMM), Linköping University , Linköping, Sweden
                Author notes
                Corresponding authors: Andreas Münch, MD PhD, Division of Gastroenterology and Hepatology, Department of Biomedical and Clinical Sciences [BKV), Faculty of Health Sciences, Linköping University, Linköping, 58185, Sweden. Tel: +46 100130000; Email: andreas.munch@ 123456regionostergotland.se ; Stefan Koch, PhD, BKV/MII—Plan 13, s-581 83 Linköping, Sweden. Tel: +46 13 282969; Email: stefan.koch@ 123456liu.se .
                Article
                jjaa020
                10.1093/ecco-jcc/jjaa020
                7393183
                32016376
                © European Crohn’s and Colitis Organisation (ECCO) 2020.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                Page count
                Pages: 12
                Product
                Funding
                Funded by: Ferring Pharmaceuticals, DOI 10.13039/501100004914;
                Funded by: Magtarmfonden;
                Funded by: Mucosal Infection and Inflammation Centre;
                Funded by: Norwegian Research Council, DOI 10.13039/501100005416;
                Funded by: Knut and Alice Wallenberg Foundation, DOI 10.13039/501100004063;
                Funded by: Norges Teknisk-Naturvitenskapelige Universitet, DOI 10.13039/100009123;
                Categories
                Original Articles
                Eccojc/1000
                AcademicSubjects/MED00260

                microscopic colitis, permeability, rna sequencing

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