A novel mode of translocation for cytolethal distending toxin

Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB. (c) 2008 Elsevier B.V. All rights reserved

Murine Model for Measuring Effects of Humanized-Dosing of Antibiotics on the Gut Microbiome.

There is a current need for enhancing our insight in the effects of antimicrobial treatment on the composition of human microbiota. Also, the spontaneous restoration of the microbiota after antimicrobial treatment requires better understanding. This is best addressed in well-defined animal models. We here present a model in which immune-competent or neutropenic mice were administered piperacillin-tazobactam (TZP) according to human treatment schedules. Before, during and after the TZP treatment, fecal specimens were longitudinally collected at established intervals over several weeks. Gut microbial taxonomic distribution and abundance were assessed through culture and molecular means during all periods. Non-targeted metabolomics analyses of stool samples using Quadrupole Time of Flight mass spectrometry (QTOF MS) were also applied to determine if a metabolic fingerprint correlated with antibiotic use, immune status, and microbial abundance. TZP treatment led to a 5-10-fold decrease in bacterial fecal viability counts which were not fully restored during post-antibiotic follow up. Two distinct, relatively uniform and reproducible restoration scenarios of microbiota changes were seen in post TZP-treatment mice. Post-antibiotic flora could consist of predominantly Firmicutes or, alternatively, a more diverse mix of taxa. In general, the pre-treatment microbial communities were not fully restored within the screening periods applied. A new species, closely related t

A Host-Specific Factor Is Necessary For Efficient Folding Of The Autotransporter Plasmid-Encoded Toxin

Autotransporters are the most common virulence factors secreted from Gram-negative pathogens. Until recently, autotransporter folding and outer membrane translocation were thought to be self-mediated events that did not require accessory factors. Here, we report that two variants of the autotransporter plasmid-encoded toxin are secreted by a lab strain of Escherichia coli. Biophysical analysis and cell-based toxicity assays demonstrated that only one of the two variants was in a folded, active conformation. The misfolded variant was not produced by a pathogenic strain of enteroaggregative E. coli and did not result from protein overproduction in the lab strain of E. coli. Our data suggest a host-specific factor is required for efficient folding of plasmid-encoded toxin. © 2009 Elsevier Masson SAS. All rights reserved

Structural and Functional Interactions between the Cholera Toxin A1 Subunit and ERdj3/HEDJ, a Chaperone of the Endoplasmic Reticulum▿

Cholera toxin (CT) is endocytosed and transported by vesicle carriers to the endoplasmic reticulum (ER). The catalytic CTA1 subunit then crosses the ER membrane and enters the cytosol, where it interacts with its Gsα target. The CTA1 membrane transversal involves the ER chaperone BiP, but few other host proteins involved with CTA1 translocation are known. BiP function is regulated by ERdj3, an ER-localized Hsp40 chaperone also known as HEDJ. ERdj3 can also influence protein folding and translocation by direct substrate binding. In this work, structural and functional assays were used to examine the putative interaction between ERdj3 and CTA1. Cell-based assays demonstrated that expression of a dominant negative ERdj3 blocks CTA1 translocation into the cytosol and CT intoxication. Binding assays with surface plasmon resonance demonstrated that monomeric ERdj3 interacts directly with CTA1. This interaction involved the A12 subdomain of CTA1 and was further dependent upon the overall structure of CTA1: ERdj3 bound to unfolded but not folded conformations of the isolated CTA1 subunit. This was consistent with the chaperone function of ERdj3, as was the ability of ERdj3 to mask the solvent-exposed hydrophobic residues of CTA1. Our data identify ERdj3 as a host protein involved with the CT intoxication process and provide new molecular details regarding CTA1-chaperone interactions

Cognitive Enhancement Drug Use Among Future Physicians: Findings from a Multi-Institutional Census of Medical Students

BACKGROUND: Nonmedical use of prescription psychostimulants such as methylphenidate and amphetamine salts for the purpose of cognitive enhancement is a growing trend, particularly in educational environments. To our knowledge, no recent studies have evaluated the use of these psychostimulants in a medical academic setting. OBJECTIVE: To conduct an online census of psychostimulant use among medical students. DESIGN: In 2011, we conducted a multi-institutional census using a 31–48 item online survey regarding use of prescription psychostimulants. PARTICIPANTS: 2,732 actively enrolled medical students at four private and public medical schools in the greater Chicago area. MAIN MEASURES: Prevalence and correlates of psychostimulant use KEY RESULTS: 1,115 (41 %) of students responded to the web-based questionnaire (range 26–47 % among schools). On average, students were 25.1 years of age (SD = 2.7, range 20–49), and single (70 %). Overall, 18 % (198/1,115) of this medical student sample had used prescription psychostimulants at least once in their lifetime, with first use most often in college. Of these, 11 % (117/1,115) of students reported use during medical school (range 7–16 % among schools). Psychostimulant use was significantly correlated with use of barbiturates, ecstasy, and tranquilizers (Pearson’s correlation r > 0.5, Student’s t-test p < 0.01); male gender (21 % male versus 15 % female, Chi squared p = 0.007); and training at a medical school which by student self-report determined class rank (68 % versus 51 %, Chi-squared p = 0.018). Non-users were more likely to be first year students (Chi-squared p = 0.048) or to have grown up outside of the United States (Chi-squared p = 0.013). CONCLUSIONS: Use of psychostimulants, including use without a prescription, is common among medical students. Further study of the side effects, medical implications, and use during post-graduate medical training and medical practice is needed to inform evidence-based policy