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      Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

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          Summary

          Clostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infection (CDI). Here we carried out CRISPR/Cas9-mediated genome-wide screens and identified the members of the Wnt receptor Frizzled (FZDs) family as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity toward FZD1, 2, and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signaling. FZD1/2/7 triple-knockout (KO) cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7 KO mice, combined with knock-down of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7 KO mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

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          Most cited references45

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          In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.

          It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell-enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell-enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.
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            Organizing cell renewal in the intestine: stem cells, signals and combinatorial control.

            The lining of the intestine is renewed at an extraordinary rate, outpacing all other tissues in the vertebrate body. The renewal process is neatly organized in space, so that the whole production line, from the ever-youthful stem cells to their dying, terminally differentiated progeny, is laid out to view in histological sections. A flurry of recent papers has clarified the key regulatory signals and brought us to the point where we can begin to give a coherent account, for at least one tissue, of how these signals collaborate to organize the architecture and behaviour of a stem-cell system.
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              Defining human ERAD networks through an integrative mapping strategy

              SUMMARY Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin and proteasome dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organised into a functional network that coordinates recognition, ubiquitination, and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organisation of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics, and the transcriptional response to ER stress. This analysis supports an adaptive organisation for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                24 September 2016
                28 September 2016
                20 October 2016
                20 July 2017
                : 538
                : 7625
                : 350-355
                Affiliations
                [1 ]Department of Urology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
                [2 ]Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA
                [3 ]Department of Microbiology and Physiological Systems (MaPS), University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
                [4 ]Division of Endocrinology, Boston Children’s Hospital, Boston, Massachusetts 02115, USA
                [5 ]Center for Infectious and Inflammatory Diseases, Texas A & M Health Science Center, Houston, Texas 77030, USA
                [6 ]Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany
                [7 ]The F. M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
                [8 ]Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA
                [9 ]Tumor Microenvironment and Cancer Immunology Program, Sanford-Burnham Prebys Medical Discovery Institute, Cancer Center, La Jolla, California 92037, USA
                [10 ]Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA
                [11 ]Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA
                [12 ]Gastroenterology Division, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
                Author notes
                Correspondence to: Min Dong, Ph.D. Telephone: 857-218-4232; min.dong@ 123456childrens.harvard.edu
                [*]

                These authors contribute equally to the study.

                Article
                NIHMS813402
                10.1038/nature19799
                5519134
                27680706
                19a95cb1-4033-40d8-8052-9892005338e0

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