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BVES is required for maintenance of colonic epithelial integrity in experimental colitis by modifying intestinal permeability.
Choksi YA, Reddy VK, Singh K, Barrett CW, Short SP, Parang B, Keating CE, Thompson JJ, Verriere TG, Brown RE, Piazuelo MB, Bader DM, Washington MK, Mittal MK, Brand T, Gobert AP, Coburn LA, Wilson KT, Williams CS
(2018) Mucosal Immunol 11: 1363-1374
MeSH Terms: Adult, Animals, Caco-2 Cells, Cell Line, Cell Line, Tumor, Citrobacter rodentium, Coculture Techniques, Colitis, Ulcerative, Colon, Dextran Sulfate, Epithelial Cells, Escherichia coli, Female, HEK293 Cells, Humans, Intestinal Absorption, Intestinal Mucosa, Male, Membrane Proteins, Mice, Mice, Inbred C57BL, Middle Aged, Permeability, RNA, Messenger, Signal Transduction, Tight Junctions
Show Abstract · Added June 23, 2018
Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.
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3 Members
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26 MeSH Terms
Mechanistic insight into the interaction of gastrointestinal mucus with oral diblock copolymers synthesized via ATRP method.
Liu J, Cao J, Cao J, Han S, Liang Y, Bai M, Sun Y
(2018) Int J Nanomedicine 13: 2839-2856
MeSH Terms: Administration, Oral, Animals, Caco-2 Cells, Drug Carriers, Humans, Hydrophobic and Hydrophilic Interactions, Indoles, Intestinal Absorption, Intestinal Mucosa, Male, Methacrylates, Methylmethacrylates, Mice, Nanoparticles, Nylons, Particle Size, Polymers, Propionates, Tissue Distribution
Show Abstract · Added April 2, 2019
Introduction - Nanoparticles are increasingly used as drug carriers for oral administration. The delivery of drug molecules is largely dependent on the interaction of nanocarriers and gastrointestinal (GI) mucus, a critical barrier that regulates drug absorption. It is therefore important to understand the effects of physical and chemical properties of nanocarriers on the interaction with GI mucus. Unfortunately, most of the nanoparticles are unable to be prepared with satisfactory structural monodispersity to comprehensively investigate the interaction. With controlled size, shape, and surface chemistry, copolymers are ideal candidates for such purpose.
Materials and methods - We synthesized a series of diblock copolymers via the atom transfer radical polymerization method and investigated the GI mucus permeability in vitro and in vivo.
Results - Our results indicated that uncharged and hydrophobic copolymers exhibited enhanced GI absorption.
Conclusion - These results provide insights into developing optimal nanocarriers for oral administration.
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1 Members
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MeSH Terms
A neutralizing antibody that blocks delivery of the enzymatic cargo of toxin TcdB into host cells.
Kroh HK, Chandrasekaran R, Zhang Z, Rosenthal K, Woods R, Jin X, Nyborg AC, Rainey GJ, Warrener P, Melnyk RA, Spiller BW, Lacy DB
(2018) J Biol Chem 293: 941-952
MeSH Terms: Antibodies, Monoclonal, Antibodies, Neutralizing, Bacterial Toxins, Caco-2 Cells, Clostridium difficile, Crystallography, X-Ray, Cytosol, Enterotoxins, Humans, Hydrogen-Ion Concentration, Microscopy, Electron, Rubidium, rac1 GTP-Binding Protein
Show Abstract · Added March 15, 2018
infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
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2 Members
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13 MeSH Terms
Shear stress induces noncanonical autophagy in intestinal epithelial monolayers.
Kim SW, Ehrman J, Ahn MR, Kondo J, Lopez AAM, Oh YS, Kim XH, Crawley SW, Goldenring JR, Tyska MJ, Rericha EC, Lau KS
(2017) Mol Biol Cell 28: 3043-3056
MeSH Terms: Actins, Autophagy, Caco-2 Cells, Cell Culture Techniques, Epithelium, Humans, Intestinal Mucosa, Intestines, Microvilli, Stress, Physiological, Vacuoles
Show Abstract · Added April 3, 2018
Flow of fluids through the gut, such as milk from a neonatal diet, generates a shear stress on the unilaminar epithelium lining the lumen. We report that exposure to physiological levels of fluid shear stress leads to the formation of large vacuoles, containing extracellular contents within polarizing intestinal epithelial cell monolayers. These observations lead to two questions: how can cells lacking primary cilia transduce shear stress, and what molecular pathways support the formation of vacuoles that can exceed 80% of the cell volume? We find that shear forces are sensed by actin-rich microvilli that eventually generate the apical brush border, providing evidence that these structures possess mechanosensing ability. Importantly, we identified the molecular pathway that regulates large vacuole formation downstream from mechanostimulation to involve central components of the autophagy pathway, including ATG5 and LC3, but not Beclin. Together our results establish a novel link between the actin-rich microvilli, the macroscopic transport of fluids across cells, and the noncanonical autophagy pathway in organized epithelial monolayers.
© 2017 Kim et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
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3 Members
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MeSH Terms
Functional defects in TcdB toxin uptake identify CSPG4 receptor-binding determinants.
Gupta P, Zhang Z, Sugiman-Marangos SN, Tam J, Raman S, Julien JP, Kroh HK, Lacy DB, Murgolo N, Bekkari K, Therien AG, Hernandez LD, Melnyk RA
(2017) J Biol Chem 292: 17290-17301
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Bacterial Proteins, Bacterial Toxins, CHO Cells, Caco-2 Cells, Cercopithecus aethiops, Chondroitin Sulfate Proteoglycans, Clostridium difficile, Cricetinae, Cricetulus, Glucosyltransferases, HEK293 Cells, Humans, Membrane Proteins, Protein Binding, Protein Domains
Show Abstract · Added April 3, 2018
is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Use of a neutralizing antibody helps identify structural features critical for binding of toxin TcdA to the host cell surface.
Kroh HK, Chandrasekaran R, Rosenthal K, Woods R, Jin X, Ohi MD, Nyborg AC, Rainey GJ, Warrener P, Spiller BW, Lacy DB
(2017) J Biol Chem 292: 14401-14412
MeSH Terms: Amino Acid Sequence, Anti-Bacterial Agents, Antibodies, Monoclonal, Humanized, Antibodies, Neutralizing, Bacterial Proteins, Bacterial Toxins, Binding Sites, Antibody, Caco-2 Cells, Clostridium difficile, Conserved Sequence, Crystallography, X-Ray, Enterocytes, Enterotoxins, Epitope Mapping, Glucosyltransferases, Humans, Immunoglobulin Fab Fragments, Models, Molecular, Peptide Fragments, Protein Conformation, Protein Interaction Domains and Motifs, Recombinant Proteins, Repetitive Sequences, Amino Acid
Show Abstract · Added March 15, 2018
is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.
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2 Members
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23 MeSH Terms
Structure of Myo7b/USH1C complex suggests a general PDZ domain binding mode by MyTH4-FERM myosins.
Li J, He Y, Weck ML, Lu Q, Tyska MJ, Zhang M
(2017) Proc Natl Acad Sci U S A 114: E3776-E3785
MeSH Terms: Adaptor Proteins, Signal Transducing, Caco-2 Cells, Humans, Multiprotein Complexes, Myosins, PDZ Domains, Protein Structure, Quaternary
Show Abstract · Added April 10, 2018
Unconventional myosin 7a (Myo7a), myosin 7b (Myo7b), and myosin 15a (Myo15a) all contain MyTH4-FERM domains (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in their cargo binding tails and are essential for the growth and function of microvilli and stereocilia. Numerous mutations have been identified in the MyTH4-FERM tandems of these myosins in patients suffering visual and hearing impairment. Although a number of MF domain binding partners have been identified, the molecular basis of interactions with the C-terminal MF domain (CMF) of these myosins remains poorly understood. Here we report the high-resolution crystal structure of Myo7b CMF in complex with the extended PDZ3 domain of USH1C (a.k.a., Harmonin), revealing a previously uncharacterized interaction mode both for MyTH4-FERM tandems and for PDZ domains. We predicted, based on the structure of the Myo7b CMF/USH1C PDZ3 complex, and verified that Myo7a CMF also binds to USH1C PDZ3 using a similar mode. The structure of the Myo7b CMF/USH1C PDZ complex provides mechanistic explanations for >20 deafness-causing mutations in Myo7a CMF. Taken together, these findings suggest that binding to PDZ domains, such as those from USH1C, PDZD7, and Whirlin, is a common property of CMFs of Myo7a, Myo7b, and Myo15a.
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7 MeSH Terms
Abnormal Rab11-Rab8-vesicles cluster in enterocytes of patients with microvillus inclusion disease.
Vogel GF, Janecke AR, Krainer IM, Gutleben K, Witting B, Mitton SG, Mansour S, Ballauff A, Roland JT, Engevik AC, Cutz E, Müller T, Goldenring JR, Huber LA, Hess MW
(2017) Traffic 18: 453-464
MeSH Terms: Caco-2 Cells, Cell Membrane, Enterocytes, Humans, Malabsorption Syndromes, Male, Microvilli, Mucolipidoses, Mutation, Myosin Type V, Protein Transport, Qa-SNARE Proteins, Secretory Vesicles, rab GTP-Binding Proteins
Show Abstract · Added April 18, 2017
Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed "secretory granules." However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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2 Members
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14 MeSH Terms
BVES regulates c-Myc stability via PP2A and suppresses colitis-induced tumourigenesis.
Parang B, Kaz AM, Barrett CW, Short SP, Ning W, Keating CE, Mittal MK, Naik RD, Washington MK, Revetta FL, Smith JJ, Chen X, Wilson KT, Brand T, Bader DM, Tansey WP, Chen R, Brentnall TA, Grady WM, Williams CS
(2017) Gut 66: 852-862
MeSH Terms: Animals, Biomarkers, Tumor, Caco-2 Cells, Carcinogenesis, Cell Adhesion Molecules, Colitis, Colitis, Ulcerative, Colon, Colonic Neoplasms, DNA Methylation, Dextran Sulfate, Down-Regulation, Female, Gene Expression Profiling, HEK293 Cells, Humans, Male, Membrane Proteins, Mice, Mice, Knockout, Muscle Proteins, Promoter Regions, Genetic, Protein Phosphatase 2, Proto-Oncogene Proteins c-myc, RNA, Messenger, Wnt Signaling Pathway
Show Abstract · Added April 15, 2017
OBJECTIVE - Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD.
DESIGN - We determined promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured mRNA levels in a tissue microarray consisting of normal colons and CAC samples. and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels.
RESULTS - mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, was underexpressed in experimental inflammatory carcinogenesis, and mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction.
CONCLUSION - Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. promoter methylation status may serve as a CAC biomarker.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
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4 Members
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26 MeSH Terms
Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.
Chandrasekaran R, Kenworthy AK, Lacy DB
(2016) PLoS Pathog 12: e1006070
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Bacterial Toxins, Blotting, Western, Caco-2 Cells, Clathrin, Clostridium Infections, Clostridium difficile, Endocytosis, Enterotoxins, Fluorescent Antibody Technique, Gene Knockdown Techniques, HEK293 Cells, Humans, Image Processing, Computer-Assisted, Mice, Microscopy, Confocal, Protein Transport, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Virulence Factors
Show Abstract · Added April 26, 2017
Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.
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21 MeSH Terms