Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.     

Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV-1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV-1 infected monocyte-derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV-infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV-1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.     

Opposing actions of endocannabinoids on cholangiocarcinoma growth: recruitment of Fas and Fas ligand to lipid rafts

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a G(i/o) protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other G(i/o) protein-coupled receptor. Using the lipid raft disruptors methyl-beta-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.     

Heritability and genetic correlations of metabolic disease-related phenotypes in Mexico: preliminary report from the GEMM Family Study

Cardiovascular disease (CVD) is a major cause of mortality in the Republic of Mexico, and metabolic syndrome, a complex of CVD risk factors, is increasingly prevalent. To date, however, there have been few studies of the genetic epidemiology of metabolic syndrome in Mexico. As a first step in implementing the GEMM Family Study, a large, multicenter collaborative study, we recruited 375 individuals in 21 extended families, without ascertainment on disease, at 9 medical institutions across Mexico. Participants were measured for anthropometric (stature, weight, waist circumference) and hemodynamic (blood pressure, heart rate) phenotypes; glucose, cholesterol, and triglyceride levels were measured in fasting blood. Variance components-based quantitative genetic analyses were performed using SOLAR. All phenotypes except diastolic blood pressure were significantly heritable. Consistent with the definition of metabolic syndrome, many phenotypes exhibited significant environmental correlation, and significant genetic correlations were found between measures of adiposity and fasting glucose and fasting triglyceride levels. These preliminary data represent the first heritability estimates for many of these phenotypes in the Republic of Mexico and indicate that this study design offers excellent power for future gene discovery relative to metabolic disease.     

Small molecule ago-allosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor

Following our previous publication describing the biological profiles, we herein describe the structure-activity relationships of a core set of quinoxalines as the hGLP-1 receptor agonists. The most potent and efficacious compounds are 6,7-dichloroquinoxalines bearing an alkyl sulfonyl group at the C-2 position and a secondary alkyl amino group at the C-3 position. These findings serve as a valuable starting point for the discovery of more drug-like small molecule agonists for the hGLP-1 receptor.     

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition

Background  

Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition.     

Effects of an invasive consumer on zooplankton communities are unaltered by nutrient inputs

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Secreted Bacillus anthracis proteases target the host fibrinolytic system

The fibrinolytic system is often the target for pathogenic bacteria, resulting in increased fibrinolysis, bacterial dissemination, and inflammation. The purpose of this study was to explore whether proteases NprB and InhA secreted by Bacillus anthracis could activate the host's fibrinolytic system. NprB efficiently activated human pro-urokinase plasminogen activator (pro-uPA), a key protein in the fibrinolytic cascade. Conversely, InhA had little effect on pro-uPA. Plasminogen activator inhibitors (PAI)-1, 2 and the uPA receptor were also targets for NprB in vitro. InhA efficiently degraded the thrombin-activatable fibrinolysis inhibitor (TAFI) in vitro. Mice infected with B. anthracis showed a significant decrease in blood TAFI levels. In another mouse experiment, animals infected with isogenic inhA deletion mutants restored TAFI levels, while the levels in the parent strain decreased. We propose that NprB and InhA may contribute to the activation of the fibrinolytic system in anthrax infection.