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  • 1
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    Online Resource
    EpCAM Mutant Enteroids Show Alterations in Differentiation and Permeability
    Wiley ; 2019
    In:  The FASEB Journal Vol. 33, No. S1 ( 2019-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 33, No. S1 ( 2019-04)
    Abstract: Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is expressed in the basolateral cell surface of most of the epithelial tissues with the highest expression in the gut. Apart from its role in homotypic cellular adhesion and tight junctions, EpCAM is known to be involved in cellular signaling that promotes proliferation, migration, and maintenance of the undifferentiated state of pluripotent stem cells. Several tumors and carcinomas have been linked with EpCAM overexpression, while the mutations in EpCAM is directly associated with a severe diarrheal disease of infancy known as Congenital Tufting Enteropathy (CTE). Alterations in intestinal cell types and barrier permeability have been observed in the patient and the murine model with CTE. We hypothesize that EpCAM plays a pivotal role in intestinal cell type differentiation and intestinal barrier function. In the current study, we evaluate whether the mutation of EpCAM has an effect on the differentiation and permeability of the intestinal epithelial cells (IECs). We have developed an inducible EpCAM mutant enteroid model based on mutations found in CTE patients (deletion of EpCAM exon 4) to allow for ex vivo study of the consequences of EpCAM mutation on all types of IECs. We first established the mutant enteroid model by confirming the deletion of EpCAM exon 4. Immuno‐fluorescence study demonstrates the mislocalization of mutant EpCAM expression away from the cell surface. The different IECs markers were studied by RNA and protein. The gene expression of different transcription factors that are involved in the secretory pathway of intestinal cell differentiation were analyzed. We observe that mutant enteroids have significantly decreased (n=3; p 〈 0.0001) gene expression of the differentiated goblet and paneth cells markers ( Muc2, Lysozyme respectively) than the control enteroids. The expression of other cell types and differentiation markers (enteroendocrine cell marker ChgA , intestinal stem cell marker Lgr5 , intestinal differentiation marker Krt20 ) are also decreased in the mutant model (n=3) as shown by qPCR. Moreover, the gene expression of Hnf1b and ATOH1 , two early transcription factors for secretory pathway differentiation, are significantly decreased (n=3; p 〈 0.05) in mutant enteroids compared with controls. The enteroids were polarized onto monolayers to check the trans‐epithelial resistance and barrier integrity with FITC‐dextran flux assay. Trans‐epithelial resistance values remained intact between the control and mutant enteroid derived monolayer, while the epithelial permeability was significantly impaired in the EpCAM mutant enteroid model compared to the control (n=3; p 〈 0.0001). In this study, we develop the first EpCAM mutant enteroid model. With this model, we note the role of EpCAM in the differentiation of IECs from intestinal crypt progenitor/stem cells. The involvement of EpCAM in barrier permeability is also confirmed. This study further establishes a new facet of EpCAM biology which will help in understanding the pathophysiology CTE and other EpCAM associated diseases. Support or Funding Information NIH R01 DK107764 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v33.S1
    DOI: 10.1096/fasebj.2019.33.1_supplement.869.23
    Language: English
    Publisher: Wiley
    Publication Date: 2019
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  • 2
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    Involvement of in vivo induced cheY‐4 gene of Vibrio cholerae in motility, early adherence to intestinal epithelial cells and regulation of virulence factors
    Wiley ; 2002
    In:  FEBS Letters Vol. 532, No. 1-2 ( 2002-12-04), p. 221-226
    Details
    In: FEBS Letters, Wiley, Vol. 532, No. 1-2 ( 2002-12-04), p. 221-226
    Abstract: Using a global transcription profile approach cheY‐4 of Vibrio cholerae was identified as an in vivo induced gene. In the present study, duplication of the gene in the chromosome resulted in increased motility, increased chemotactic response towards isolated intestinal mucus layer and stronger adhesion to human intestinal epithelial cell line at an early phase of infection compared to wild type and a null mutant strain. In contrast to the cheY‐4 null mutant, duplication of cheY‐4 gene resulted in increased expression of ctxAB and tcpA , the two major virulence genes of V. cholerae .
    Type of Medium: Online Resource
    ISSN: 0014-5793 , 1873-3468
    URL: Article
    DOI: 10.1016/S0014-5793(02)03678-5
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2002
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  • 3
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    Recurrent diarrhoea in a renal transplant recipient: Early immunoproliferative small intestinal disease may be missed if not considered
    Wiley ; 2017
    In:  Nephrology Vol. 22, No. 3 ( 2017-03), p. 265-267
    Details
    In: Nephrology, Wiley, Vol. 22, No. 3 ( 2017-03), p. 265-267
    Type of Medium: Online Resource
    ISSN: 1320-5358 , 1440-1797
    URL: Issue
    URL: Article
    DOI: 10.1111/nep.2017.22.issue-3
    DOI: 10.1111/nep.12779
    Language: English
    Publisher: Wiley
    Publication Date: 2017
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    detail.hit.zdb_id: 2008235-6
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  • 4
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    Protective Effects of Human Milk Oligosaccharides on Intestinal Epithelial Function Assessed in Enteroid‐Derived Monolayers
    Wiley ; 2018
    In:  The FASEB Journal Vol. 32, No. S1 ( 2018-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 32, No. S1 ( 2018-04)
    Abstract: Human milk oligosaccharides (HMOs) are structurally diverse carbohydrates that are unique to human milk and have beneficial effects on the growth and development of the intestine and immune system in breastfed infants. Infectious diarrheal diseases account for significant morbidity and mortality in young children and breastfeeding is protective against non‐typhoidal Salmonella infections and associated diarrhea in infants. Several studies suggest that HMOs may prevent enteric infections in breastfed infants and/or improve barrier function in transformed cell lines. Some of the beneficial effects of HMOs may be attributable to changes in the microbiota, but they likely have direct effects on the epithelium as well. The purpose of this study was therefore to evaluate mechanisms whereby HMOs may improve intestinal integrity and any protective effects of HMOs on Salmonella infection in a more clinically‐relevant model. We previously showed that Salmonella infection decreases epithelial barrier function in murine colon and in murine enteroid‐derived monolayers (EDM). In the present study, EDM were developed on permeable supports and, after differentiation, were exposed to pooled HMOs and then subsequently infected with Salmonella typhimurium at a multiplicity of infection of 10:1. Barrier integrity was determined by measuring transepithelial electrical resistance (TEER). We also measured bacterial internalization using a gentamicin protection assay followed by colony counts. Exposure of EDM to 10 mg/mL pooled HMOs for 2–8 h progressively increased TEER. Prior exposure to pooled HMOs prevented the decrease in TEER that was otherwise produced by Salmonella infection at 1 h after infection, but this effect was lost at later time‐points. Pooled HMOs had no effect on numbers of intracellular bacteria and did not alter bacterial growth when added to bacteria directly. Our results suggest that HMOs act directly on intestinal epithelial cells to strengthen intestinal barrier function both at baseline and when compromised by a pathogen. These effects may contribute to protection against the adverse outcomes of neonatal enteric infections. Support or Funding Information Larsson Rosenquist Foundation Mother Milk Infant Center of Research Excellence This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v32.S1
    DOI: 10.1096/fasebj.2018.32.1_supplement.873.22
    Language: English
    Publisher: Wiley
    Publication Date: 2018
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  • 5
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    Reduced Expression of Down‐Regulated in Adenoma (DRA) in Murine Enteroid‐Derived Monolayers Following Infection with Salmonella
    Wiley ; 2017
    In:  The FASEB Journal Vol. 31, No. S1 ( 2017-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 31, No. S1 ( 2017-04)
    Abstract: Non‐typhoidal Salmonella spp. are the second leading cause of foodborne illness worldwide. Despite its prevalence, the mechanism through which Salmonella infection causes diarrhea is not fully understood. The intestinal epithelium plays a major role in both interacting with bacteria and fluid accumulation. To understand the basic mechanism of Salmonella infection in intestinal epithelial cells, we developed a murine enteroid model that mimics the crypt‐villus axis of the intestines, and contains the four different epithelial subtypes found in the intestine (enterocytes, enteroendocrine, goblet, and Paneth cells). We have previously shown that infection with Salmonella causes decreased expression of the chloride/bicarbonate exchanger SLC26A3 (DRA; Downregulated in Adenoma) in the murine colon. We developed enteroid‐derived monolayers (EDM) from mouse ileum and colon to test whether DRA was similarly down‐regulated following infection with Salmonella . Monolayers were generated from enteroids on either permeable transepithelial supports or cell culture plates. Monolayers were also grown in 8‐well chamber slides to visualize DRA and bacterial internalization. After differentiation, EDM were infected with Salmonella enterica serovar Typhimurium. Expression of DRA and markers for different cell subtypes were studied using RT‐PCR, western blotting, and confocal microscopy. We also measured bacterial internalization using a gentamicin protection assay followed by colony counts. We found bacterial internalization after 1 h followed by decreased expression of DRA after 6 h of Salmonella infection. Interestingly, following infection the proliferation marker Ki67 was upregulated. The intestines contain both secretory (enteroendocrine, goblet, and paneth cells) and absorptive (enterocytes) cell types and their expression was evaluated following infection. The Wnt/NOTCH signaling pathways control cell fate decisions in the intestines. We found that Hes1, a downstream signaling molecule in the NOTCH signaling pathway, was up regulated following infection. Our work shows the potential for EDM to identify molecular mechanisms that may underlie intestinal dysfunction during infection. Ultimately, this model may identify signals that can be targeted for therapeutic development.
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v31.S1
    DOI: 10.1096/fasebj.31.1_supplement.1007.15
    Language: English
    Publisher: Wiley
    Publication Date: 2017
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  • 6
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    The host engulfment pathway in enteroids is linked to intestinal inflammation following bacterial infection
    Wiley ; 2017
    In:  The FASEB Journal Vol. 31, No. S1 ( 2017-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 31, No. S1 ( 2017-04)
    Abstract: Intestinal homeostasis is maintained through regulated interactions between epithelial cells, innate immune cells and the microbiota. The immune homeostasis is lost following interaction of the host with enteric pathogens that generate inflammation. Microbes are recognized by specific receptors such as Pattern Recognition Receptors (PRRs). PRRs can bind pathogens as well as commensals, but in most cases inflammation is generated only from pathogenic encounters. Previously, we identified Brain Angiogenesis Inhibitor 1 (BAI1) as a PRR that recognizes bacterial lipopolysaccharide (LPS). BAI1 binds Engulfment and Cell Motility Protein‐1 (ELMO1), which facilitates the engulfment of bacteria by host cells. We have shown that ELMO1 in phagocytes regulates the entry of bacteria and subsequent intestinal inflammation. Cytokine arrays and ELISA showed reduced release of pro‐inflammatory cytokines, including TNF‐α, monocyte chemoattractant protein (MCP‐1) and IL‐1β, from ELMO1‐depleted macrophages. Interestingly, higher expression of ELMO1 in biopsy specimens from inflammatory bowel disease patients correlates with the expression of MCP‐1 and TNF‐a. We developed enteroid‐derived monolayers (EDMs) that mimic the architecture and cell populations of the in‐vivo intestinal epithelium. Here we show that human as well as murine enteroids express ELMO1. We used Salmonella as well as adherent‐invasive E. coli as enteric pathogens. We found a significant reduction of bacterial internalization and pro‐inflammatory cytokines in EDMs generated from ELMO1 knockout mice compared to WT mice. EDMs infected with enteric bacteria generate MCP‐1 in an ELMO1‐dependent manner that attracts monocyte at the site of inflammation. The expression of MCP‐1 is higher in several inflammatory diseases including the atherosclerosis and inflammatory bowel diseases. Therefore, elevated expression of MCP‐1 via ELMO1 may be an early biomarker in inflammatory diseases. Our work also uncovers a central role of ELMO1 in the induction of inflammatory cytokines in non‐phagocytic cells. Support or Funding Information UCSD, Academic Senate (Das), NIH‐NIDDK R01 (Das)Clinical and Transitional Research Institute/NIH‐NCATS (Das)
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v31.S1
    DOI: 10.1096/fasebj.31.1_supplement.695.2
    Language: English
    Publisher: Wiley
    Publication Date: 2017
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  • 7
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    Salmonella biases epithelial differentiation in murine enteroids through Wnt and Notch signaling, which may contribute to diarrheal pathogenesis
    Wiley ; 2020
    In:  The FASEB Journal Vol. 34, No. S1 ( 2020-04), p. 1-1
    Details
    In: The FASEB Journal, Wiley, Vol. 34, No. S1 ( 2020-04), p. 1-1
    Abstract: Salmonella enterica serovar Typhimurium is one of the most burdensome foodborne diarrheal pathogens worldwide. However, despite its prevalence, the mechanism by which Salmonella causes diarrhea is not entirely known. We have shown in mice that Salmonella infection decreases expression of SLC26A3 (Down‐Regulated in Adenoma; DRA), a chloride/bicarbonate exchanger. In order to understand the mechanism of this effect, we employed an enteroid model from murine colon. Enteroid‐derived monolayers (EDM) that recapitulate both the absorptive and secretory cell lineages of the native intestinal epithelium (enterocytes vs. enteroendocrine, goblet, and Paneth cells, respectively) were infected apically with Salmonella and relevant protein expression was studied using qRT‐PCR and western blot. Infection significantly reduced expression of DRA (40%, p = 0.0124) and Hes1 (45%, p = 0.0004) while upregulating expression of ATOH1 (3‐fold, p = 0.0292) and mucin 2 (Muc2) (2‐fold, p = 0.0134). Hes1 is a Notch pathway downstream signaling molecule and thus a precursor to the absorptive epithelial lineage that expresses DRA. ATOH1, on the other hand, is a Wnt pathway downstream signaling molecule and a precursor to secretory lineages, including goblet cells that express Muc2. Infection of EDMs also reduced levels of the Notch intracellular domain. The involvement of Notch was further investigated by inhibiting Notch signaling using a γ‐secretase inhibitor, which reproduced the downregulation in Hes1 and DRA and upregulation in ATOH1 and Muc2 as seen with infection. Because the balance of Wnt and Notch signaling determines relative intestinal epithelial differentiation, our findings suggest that the diarrheal pathogenesis of Salmonella may reflect Notch inhibition and an accompanying shift in epithelial differentiation from absorptive to secretory cell types, a decreased capacity for absorption, and thus the accumulation of diarrheal fluid.
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v34.S1
    DOI: 10.1096/fasebj.2020.34.s1.02677
    Language: English
    Publisher: Wiley
    Publication Date: 2020
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  • 8
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    Host engulfment pathway controls inflammation in inflammatory bowel disease
    Wiley ; 2020
    In:  The FEBS Journal Vol. 287, No. 18 ( 2020-09), p. 3967-3988
    Details
    In: The FEBS Journal, Wiley, Vol. 287, No. 18 ( 2020-09), p. 3967-3988
    Abstract: Chronic diseases, including inflammatory bowel disease (IBD) urgently need new biomarkers as a significant proportion of patients, do not respond to current medications. Inflammation is a common factor in these diseases, and microbial sensing in the intestinal tract is critical to initiate the inflammation. We have identified ELMO1 (engulfment and cell motility protein 1) as a microbial sensor in epithelial and phagocytic cells that turns on inflammatory signals. Using a stem cell‐based ‘gut‐in‐a‐dish’ coculture model, we studied the interactions between microbes, epithelium, and monocytes in the context of IBD. To mimic the in vivo cell physiology, enteroid‐derived monolayers (EDMs) were generated from the organoids isolated from WT and ELMO1−/− mice and colonic biopsies of IBD patients. The EDMs were infected with the IBD‐associated microbes to monitor the inflammatory responses. ELMO1‐depleted EDMs displayed a significant reduction in bacterial internalization, a decrease in pro‐inflammatory cytokine productions and monocyte recruitment. The expression of ELMO1 is elevated in the colonic epithelium and in the inflammatory infiltrates within the lamina propria of IBD patients where the higher expression is positively correlated with the elevated expression of pro‐inflammatory cytokines, MCP‐1 and TNF‐α. MCP‐1 is released from the epithelium and recruits monocytes to the site of inflammation. Once recruited, monocytes require ELMO1 to engulf the bacteria and propagate a robust TNF‐α storm. These findings highlight that the dysregulated epithelial ELMO1 → MCP‐1 axis can serve as an early biomarker in the diagnostics of IBD and other inflammatory disorders.
    Type of Medium: Online Resource
    ISSN: 1742-464X , 1742-4658
    URL: Issue
    URL: Article
    DOI: 10.1111/febs.v287.18
    DOI: 10.1111/febs.15236
    Language: English
    Publisher: Wiley
    Publication Date: 2020
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  • 9
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    Deregulation of ceRNA Networks in Frontal Cortex and Choroid Plexus of Brain during SARS‐CoV‐2 Infection Aggravates Neurological Manifestations: An Insight from Bulk and Single‐Cell Transcriptomic Analyses
    Wiley ; 2022
    In:  Advanced Biology Vol. 6, No. 8 ( 2022-08)
    Details
    In: Advanced Biology, Wiley, Vol. 6, No. 8 ( 2022-08)
    Abstract: Although transcriptomic studies of SARS‐CoV‐2‐infected brains have depicted variability in gene expression, the landscape of deregulated cell‐specific regulatory circuits has not been elucidated yet. Hence, bulk and single‐cell RNA‐seq data are analyzed to gain detailed insights. Initially, two ceRNA networks with 19 and 3 differentially expressed (DE) hub lncRNAs are reconstructed in SARS‐CoV‐2 infected Frontal Cortex (FC) and Choroid Plexus (CP), respectively. Functional and pathway enrichment analyses of downstream mRNAs of deregulated ceRNA axes demonstrate impairment of neurological processes. Mapping of hub lncRNA‐mRNA pairs from bulk RNA‐seq with snRNA‐seq data has indicated that NORAD, NEAT1, and STXBP5‐AS1 are downregulated across 4, 4, and 2 FC cell types, respectively. At the same time, MIRLET7BHG and MALAT1 are upregulated in excitatory neurons of FC and neurons of CP, respectively. Here, it is hypothesized that downregulation of NORAD, NEAT1, and STXBP5‐AS1, and upregulation of MIRLET7BHG and MALAT1 might deregulate respectively 51, 6, and 37, and 31 and 19 mRNAs in cell types of FC and CP. Afterward, 13 therapeutic miRNAs are traced that might safeguard against deregulated lncRNA‐mRNA pairs of NORAD, NEAT1, and MIRLET7BHG in FC. This study helps to explain the plausible mechanism of post‐COVID neurological manifestation and also to devise therapeutics against it.
    Type of Medium: Online Resource
    ISSN: 2701-0198 , 2701-0198
    URL: Issue
    URL: Article
    DOI: 10.1002/adbi.v6.8
    DOI: 10.1002/adbi.202101310
    Language: English
    Publisher: Wiley
    Publication Date: 2022
    detail.hit.zdb_id: 3027224-5
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  • 10
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    Complex‐forming proteins escape the robust regulations of miRNA in human
    Wiley ; 2013
    In:  FEBS Letters Vol. 587, No. 14 ( 2013-07-11), p. 2284-2287
    Details
    In: FEBS Letters, Wiley, Vol. 587, No. 14 ( 2013-07-11), p. 2284-2287
    Abstract: Most proteins carry out their functions by participating in protein complexes. Recently, miRNAs were identified as promising post‐transcriptional regulators that influence a large proportion of genes in higher eukaryotes. We aim to understand the role of miRNAs in the regulation of human proteins that are present in protein complexes. Here, we show that robust regulation by miRNA is absent in human complex‐forming proteins. Moreover, the numbers of miRNA hits cannot direct the evolutionary fate of complex‐forming proteins independently. However, the duplicated complex‐forming proteins having a severe effect on organismal fitness are profoundly targeted by miRNA, probably to reduce the chances of dosage imbalance.
    Type of Medium: Online Resource
    ISSN: 0014-5793 , 1873-3468
    URL: Article
    DOI: 10.1016/j.febslet.2013.05.062
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2013
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