Gut-associated cGMP mediates colitis and dysbiosis in a mouse model of an activating mutation in GUCY2C

Activating mutations in receptor guanylyl cyclase C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial heat-stable enterotoxins cause early-onset diarrhea and chronic inflammatory bowel disease (IBD). GC-C regulates ion and fluid secretion in the gut via cGMP production and activation of cGMP-dependent protein kinase II. We characterize a novel mouse model harboring an activating mutation in Gucy2c equivalent to that seen in an affected Norwegian family. Mutant mice demonstrated elevated intestinal cGMP levels and enhanced fecal water and sodium content. Basal and linaclotide-mediated small intestinal transit was higher in mutant mice, and they were more susceptible to DSS-induced colitis. Fecal microbiome and gene expression analyses of colonic tissue revealed dysbiosis, up-regulation of IFN-stimulated genes, and misregulation of genes associated with human IBD and animal models of colitis. This novel mouse model thus provides molecular insights into the multiple roles of intestinal epithelial cell cGMP, which culminate in dysbiosis and the induction of inflammation in the gut.

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Role of Various Enterotoxins in Aeromonas hydrophila-Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity

Infection and Immunity ◽

10.1128/iai.70.4.1924-1935.2002 ◽

2002 ◽

Vol 70 (4) ◽

pp. 1924-1935 ◽

Cited By ~ 137

Author(s):

Jian Sha ◽

E. V. Kozlova ◽

A. K. Chopra

Keyword(s):

Biological Activity ◽

Mouse Model ◽

Aeromonas Hydrophila ◽

Fluid Secretion ◽

The Other ◽

Enterotoxin Gene ◽

Knockout Mutant ◽

Heat Stable ◽

Isogenic Mutants

ABSTRACT Three enterotoxins from the Aeromonas hydrophila diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (act)-positive or -negative background strain of A. hydrophila. We developed two single knockouts with truncation in either the heat-labile (alt) or the heat-stable (ast) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i) alt and ast, (ii) act and alt, and (iii) act and ast genes; and a triple-knockout mutant with truncation in all three genes, act, alt, and ast. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type A. hydrophila; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected A. hydrophila mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in A. hydrophila-induced gastroenteritis in a mouse model with the greatest contribution from the cytotoxic enterotoxin Act, followed by the Alt and Ast cytotonic enterotoxins.

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A Rare p.T599dup BRAF Mutant NSCLC in a Non-Smoker

Current Oncology ◽

10.3390/curroncol28010021 ◽

2020 ◽

Vol 28 (1) ◽

pp. 196-202

Author(s):

Alla Turshudzhyan ◽

James Vredenburgh

Keyword(s):

Lung Cancer ◽

Targeted Therapies ◽

Kinase Activity ◽

Mitogen Activated Protein Kinase ◽

Braf Mutations ◽

Activating Mutations ◽

Extracellular Signal ◽

Activating Mutation ◽

Mitogen Activated Protein ◽

Murine Sarcoma

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) mutated non-small-cell lung cancer (NSCLC) is an exceptionally rare form of lung cancer, found only in one to two percent of patients with an NSCLC diagnosis. BRAF NSCLC traditionally affects former or active smokers. BRAF mutations have always been of special interest to the oncological community, as they offer potential for targeted therapies. BRAF mutation spectrum includes mutations that are of both V600 and non-V600 types. BRAF V600 is an activating mutation, which results in high kinase activity and overproduction of active oncoproteins such as rapidly accelerated fibrosarcoma (RAF). This makes them susceptible to targeted therapies with RAF inhibitors. There has been little evidence, however, regarding efficacy of RAF inhibitors towards non-activating mutations that have intermediate to low kinase activity, such as non-V600 BRAF mutations. While several approaches have been investigated to overcome the limitations of RAF inhibitors, such as use of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) inhibitors or combination of MEK and RAF inhibitors, none of them have been proven to have a superior efficacy for low kinase activity non-V600 BRAF tumors. We present a case of an extremely rare variant of NSCLC BRAF p.T599dup mutation in a non-smoker that responded to a targeted combination therapy with RAF and MEK inhibitors. The patient responded well to therapy that usually targets high kinase activity V600 mutations. Our hope is to bring more attention to non-V600 mutations and document their responses to existing and new therapies.

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The Capsule Encoding the viaB Locus Reduces Interleukin-17 Expression and Mucosal Innate Responses in the Bovine Intestinal Mucosa during Infection with Salmonella enterica Serotype Typhi

Infection and Immunity ◽

10.1128/iai.01571-06 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4342-4350 ◽

Cited By ~ 69

Author(s):

Manuela Raffatellu ◽

Renato L. Santos ◽

Daniela Chessa ◽

R. Paul Wilson ◽

Sebastian E. Winter ◽

...

Keyword(s):

Mouse Model ◽

Salmonella Enterica ◽

Intestinal Inflammation ◽

Fluid Secretion ◽

Interleukin 17 ◽

Loop Model ◽

Wild Type ◽

Ileal Loop ◽

Chemokine Production

ABSTRACT The viaB locus contains genes for the biosynthesis and export of the Vi capsular antigen of Salmonella enterica serotype Typhi. Wild-type serotype Typhi induces less CXC chemokine production in tissue culture models than does an isogenic viaB mutant. Here we investigated the in vivo relevance of these observations by determining whether the presence of the viaB region prevents inflammation in two animal models of gastroenteritis. Unlike S. enterica serotype Typhimurium, serotype Typhi or a serotype Typhi viaB mutant did not elicit marked inflammatory changes in the streptomycin-pretreated mouse model. In contrast, infection of bovine ligated ileal loops with a serotype Typhi viaB mutant resulted in more fluid accumulation and higher expression of the chemokine growth-related oncogene alpha (GROα) and interleukin-17 (IL-17) than did infection with the serotype Typhi wild type. There was a marked upregulation of IL-17 expression in both the bovine ligated ileal loop model and the streptomycin-pretreated mouse model, suggesting that this cytokine is an important component of the inflammatory response to infection with Salmonella serotypes. Introduction of the cloned viaB region into serotype Typhimurium resulted in a significant reduction of GROα and IL-17 expression and in reduced fluid secretion. Our data support the idea that the viaB region plays a role in reducing intestinal inflammation in vivo.

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Activated CARD11 accelerates germinal center kinetics, promoting mTORC1 and terminal differentiation

Journal of Experimental Medicine ◽

10.1084/jem.20180230 ◽

2018 ◽

Vol 215 (9) ◽

pp. 2445-2461 ◽

Cited By ~ 6

Author(s):

Michelle N. Wray-Dutra ◽

Raghav Chawla ◽

Kerri R. Thomas ◽

Brenda J. Seymour ◽

Tanvi Arkatkar ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Terminal Differentiation ◽

B Cell Differentiation ◽

Activating Mutations ◽

Class Switch ◽

Mtorc1 Signaling ◽

Activating Mutation ◽

Novel Strategy

Activating mutations in the adapter protein CARD11 associated with diffuse large B cell lymphomas (DLBCLs) are predicted to arise during germinal center (GC) responses, leading to inappropriate activation of NF-κB signaling. Here, we modeled the B cell–intrinsic impact of the L251P activating mutation in CARD11 (aCARD11) on the GC response. Global B cell aCARD11 expression led to a modest increase in splenic B cells and a severe reduction in B1 B cell numbers, respectively. Following T cell–dependent immunization, aCARD11 cells exhibited increased rates of GC formation, resolution, and differentiation. Restriction of aCARD11 to GC B cells similarly altered the GC response and B cell differentiation. In this model, aCARD11 promoted dark zone skewing along with increased cycling, AID levels, and class switch recombination. Furthermore, aCard11 GC B cells displayed increased biomass and mTORC1 signaling, suggesting a novel strategy for targeting aCARD11-driven DLBCL. While aCARD11 potently impacts GC responses, the rapid GC contraction suggests it requires collaboration with events that limit terminal differentiation to promote lymphoma.

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Abstract 1079: Prevention of Fatal Arrhythmia in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin-2 Mutation

Circulation ◽

10.1161/circ.116.suppl_16.ii_216-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Hiroko Wakimoto ◽

Ronny Alcalai ◽

Lei Song ◽

Michael Arad ◽

Christine E Seidman ◽

...

Keyword(s):

Ventricular Tachycardia ◽

Mouse Model ◽

Peak Height ◽

Mutant Mice ◽

Catecholaminergic Polymorphic Ventricular Tachycardia ◽

Polymorphic Ventricular Tachycardia ◽

Study Drug Administration ◽

Intracellular Ca

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmia syndrome caused by mutations in the ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2) genes and characterized by exercise or emotional stress-induced sudden death. Beta-adrenergic blockers are only partially effective and other agents have not been widely tested. Recent studies have shown that CPVT is mediated by increased Ca 2+ leak through the RyR2 channel. Our aim was to determine whether agents that inhibit intracellular Ca 2+ leak can effectively prevent CPVT. Methods: The efficacy of intraperitoneal (IP) propranolol (1mcg/g), Mg 2+ (0.002mEq/g), verapamil (8 mcg/g) and diltiazem (8 mcg/g) were tested both in vivo and in vitro using CASQ2 mutant mouse CPVT model. In vivo studies included ambulatory ECG recordings at rest and following epinephrine stress (0.4 mcg/g IP) at baseline and after study drug administration. Experiments for each drug were performed on separate days to avoid confounding effects. In vitro studies included intracellular Ca 2+ transient analysis on isolated cardiomyocytes from mutant mice with and without epinephrine (5.5 μM). Results: All 4 drugs restored sinus rhythm and reduced the frequency of VT episodes induced by epinephrine in CASQ2 mutant mice. Only verapamil completely prevented epinephrine-induced VT in 87% of the mice (p<0.01). Cardiomyocyte studies in basal conditions revealed that Mg 2+ and verapamil inhibited sarcomere contraction and normalized the prolonged Ca 2+ reuptake period in CASQ2 mutants, but did not decrease baseline Ca 2+ peak height. Epinephrine-stressed mutant myocytes had increased diastolic Ca 2+ levels, lower Ca 2+ peak height and spontaneous SR Ca 2+ release events that were partially prevented by verapamil and Mg 2+ . Verapamil was more effective than Mg 2+ in reducing the frequency of spontaneous Ca 2+ releases induced by epinephrine. Conclusions: All 4 agents can inhibit ventricular arrhythmia in CPVT mouse model; however verapamil appears most effective in preventing arrhythmia in vivo and in modifying intracellular abnormal calcium handling. Calcium antagonists might have therapeutic value in CPVT and other RyR2-mediated arrhythmias and should be considered for human clinical studies.

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Inhibition of Escherichia coli heat-stable enterotoxin by indomethacin and chlorpromazine

Infection and Immunity ◽

10.1128/iai.29.3.908-913.1980 ◽

1980 ◽

Vol 29 (3) ◽

pp. 908-913

Author(s):

R N Greenberg ◽

F Murad ◽

B Chang ◽

D C Robertson ◽

R L Guerrant

Keyword(s):

Escherichia Coli ◽

Guanylate Cyclase ◽

Fluid Secretion ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Enterotoxigenic Escherichia Coli ◽

Fluid Accumulation ◽

Intestinal Tissue ◽

Suckling Mice ◽

Heat Stable

Purified heat-stable enterotoxin (ST) from a procine strain of enterotoxigenic Escherichia coli activates quanylate cyclase in particulate fractions of rat intestinal tissue and induces fluid accumulation in suckling mice. These effects of ST were examined in the presence of either indomethacin or chlorpromazine. We also examined the effects of these two drugs on fluid accumulation in suckling mice induced by the 8-bromo analog of cyclic guanosine monophosphate. Either indomethacin or chlorpromazine reduced ST activation of guanylate cyclase. Both drugs also reduced intestinal fluid accumulation in suckling mice that resulted from submaximal doses of ST (both P < 0.001). However, there was no reduction in fluid secretion by either drug when a maximally effective dose of ST was used, suggesting that inhibition of fluid secretion by both drugs can be overcome by increasing the ST dose and that a threshold level of guanylate cyclase activity results in maximal secretory response. Both drugs also reduced basal guanylate cylase activity in rat intestinal tissue and fluid secreton in suckling mice. Chlorpromazine also reduced intestinal secretion mediated by 8-bromo cyclic guanosine monophosphate (P < 0.001). These findings indicate that chlorpromazine interferes with the effects of ST both before and after its activation of guanylate cyclase, whereas indomethacin interfers with ST only before its activation of guanylate cyclase.

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The Craniofacial Phenotype of the Crouzon Mouse: Analysis of a Model for Syndromic Craniosynostosis Using Three-Dimensional MicroCT

The Cleft Palate-Craniofacial Journal ◽

10.1597/05-212 ◽

2006 ◽

Vol 43 (6) ◽

pp. 740-747 ◽

Cited By ~ 55

Author(s):

Chad A. Perlyn ◽

Valerie B. DeLeon ◽

Christian Babbs ◽

Daniel Govier ◽

Lance Burell ◽

...

Keyword(s):

Mouse Model ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Distance Matrix ◽

Mutant Mice ◽

Matrix Analysis ◽

Crouzon Syndrome ◽

Wild Type ◽

Skull Morphology ◽

Euclidean Distance Matrix

Objective: To characterize the craniofacial phenotype of a mouse model for Crouzon syndrome by a quantitative analysis of skull morphology in mutant and wild-type mice and to compare the findings with skull features observed in humans with Crouzon syndrome. Methods: MicroCT scans and skeletal preparations were obtained on previously described Fgfr2C342Y/+ Crouzon mutant mice and wild-type mice at 6 weeks of age. Three-dimensional coordinate data from biologically relevant landmarks on the skulls were collected. Euclidean Distance Matrix Analysis was used to quantify and compare skull shapes using these landmark data. Results: Obliteration of bilateral coronal sutures was observed in 80% of skulls, and complete synostosis of the sagittal suture was observed in 70%. In contrast, fewer than 40% of lambdoid sutures were found to be fully fused. In each of the 10 Fgfr2C342Y/+ mutant mice analyzed, the presphenoid-basisphenoid synchondrosis was fused. Skull height and width were increased in mutant mice, whereas skull length was decreased. Interorbital distance was also increased in Fgfr2C342Y/+ mice as compared with wild-type littermates. Upper-jaw length was shorter in the Fgfr2C342Y/+ mutant skulls, as was mandibular length. Conclusion: Skulls of Fgfr2C342Y/+ mice differ from normal littermates in a comparable manner with differences between the skulls of humans with Crouzon syndrome and those of unaffected individuals. These findings were consistent across several regions of anatomic interest. Further investigation into the molecular mechanisms underlying the anomalies seen in the Crouzon mouse model is currently under way.

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Organic acid proton donors decrease intestinal secretion caused by enterotoxins

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1981.241.3.g227 ◽

1981 ◽

Vol 241 (3) ◽

pp. G227-G234

Author(s):

G. W. Forsyth ◽

R. A. Kapitany ◽

D. L. Hamilton

Keyword(s):

Cholera Toxin ◽

Intestinal Secretion ◽

Fluid Secretion ◽

Weak Acid ◽

Aminobenzoic Acid ◽

Control Loops ◽

Heat Stable ◽

Acid Proton ◽

Proton Donors ◽

Sodium Flux

The effects of several weak acids on the secretory actions of cholera toxin and the heat-stable enterotoxin of Escherichia coli (ST) have been examined in ligated jejunal loops in weanling pigs. Ascorbic and acetic acids had no effect, but L-lactic acid reduced the net fluid secretion caused by cholera toxin. Glutaric acid and p-aminobenzoic acid blocked net fluid secretion caused by cholera toxin or by ST. Antisecretory effects were pH dependent for p-aminobenzoic acid in this study and for nicotinic acid in a previous report (6). At a pH of 5.0, p-aminobenzoic acid treatment increased lumen-to-blood sodium flux and decreased the blood-to-lumen sodium flux caused by cholera toxin. These weak acid effects were more marked on fluid fluxes in enterotoxin-treated loops than in control loops and persisted for 20–30 min after acid removal from loops. These findings are discussed in terms of requirements for antisecretory activity and possible modes of action of antisecretory compounds.

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GSK3β inhibition restores cortical gamma oscillation and cognitive behavior in a mouse model of NMDA receptor hypofunction relevant to schizophrenia

Neuropsychopharmacology ◽

10.1038/s41386-020-00819-0 ◽

2020 ◽

Vol 45 (13) ◽

pp. 2207-2218

Author(s):

Kazuhito Nakao ◽

Mahendra Singh ◽

Kiran Sapkota ◽

Bailey C. Hagler ◽

Robert N. Hunter ◽

...

Keyword(s):

Working Memory ◽

Mouse Model ◽

Spatial Working Memory ◽

Gamma Oscillations ◽

Gamma Oscillation ◽

Mutant Mice ◽

Patch Clamp Recording ◽

Behavioral Deficits ◽

Gamma Power ◽

Sensory Evoked

Abstract Cortical gamma oscillations are believed to be involved in mental processes which are disturbed in schizophrenia. For example, the magnitudes of sensory-evoked oscillations, as measured by auditory steady-state responses (ASSRs) at 40 Hz, are robustly diminished, whereas the baseline gamma power is enhanced in schizophrenia. Such dual gamma oscillation abnormalities are also present in a mouse model of N-methyl-D-aspartate receptor hypofunction (Ppp1r2cre/Grin1 knockout mice). However, it is unclear whether the abnormal gamma oscillations are associated with dysfunction in schizophrenia. We found that glycogen synthase kinase-3 (GSK3) is overactivated in corticolimbic parvalbumin-positive GABAergic interneurons in Grin1 mutant mice. Here we addressed whether GSK3β inhibition reverses both abnormal gamma oscillations and behavioral deficits with high correlation by pharmacological and genetic approach. We demonstrated that the paralog selective-GSK3β inhibitor, but not GSK3α inhibitor, normalizes the diminished ASSRs, excessive baseline gamma power, and deficits in spatial working memory and prepulse inhibition (PPI) of acoustic startle in Grin1 mutant mice. Cell-type specific GSK3B knockdown, but not GSK3A knockdown, also reversed abnormal gamma oscillations and behavioral deficits. Moreover, GSK3B knockdown, but not GSK3A knockdown, reverses the mutants’ in vivo spike synchrony deficits. Finally, ex vivo patch-clamp recording from pairs of neighboring cortical pyramidal neurons showed a reduction of synchronous spontaneous inhibitory-postsynaptic-current events in mutants, which was reversed by GSK3β inhibition genetically and pharmacologically. Together, GSK3β inhibition in corticolimbic interneurons ameliorates the deficits in spatial working memory and PPI, presumably by restoration of synchronous GABA release, synchronous spike firing, and evoked-gamma power increase with lowered baseline power.

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