Surveillance for norovirus and enteric bacterial pathogens as etiologies of acute gastroenteritis at U.S. military recruit training centers, 2011-2016.

An estimated 179 million cases of acute gastroenteritis (AGE) occur each year in the U.S. and AGE is commonly reported within both training and deployed U.S. military populations. Beginning in 2011, the Operational Infectious Diseases laboratory at Naval Health Research Center (NHRC) has undertaken routine surveillance of four U.S. military training facilities to systematically track the prevalence of AGE and to establish its etiologies among U.S. military recruits. Employing both molecular and standard microbiological techniques, NHRC routinely assays for pathogens of direct military relevance, including norovirus genogroups I and II, Salmonella, Shigella, and Campylobacter. During its initial surveillance efforts (2011-2016), NHRC identified norovirus as the primary etiology of both sporadic cases and outbreaks of AGE among trainees.

Rapid steps in the glmS ribozyme catalytic pathway: cation and ligand requirements.

The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5' UTR of the glmS mRNA, resulting in downregulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured. This does not provide data regarding cation and ligand specificities of the glmS ribozyme during the rapid ligand binding chemical catalysis events. Prefolding of the ribozyme in Mg(2+)-containing buffers effectively isolates the rapid ligand binding and catalytic events (k(obs) > 60 min(-1)) from rate-limiting folding (k(obs) < 4 min(-1)). Here we employ this experimental design to assay the cations and ligand requirements for rapid ligand binding and catalysis. We show that molar concentrations of monovalent cations are also capable of inducing the formation of the native GlcN6P binding structure but are unable to promote ligand binding and catalysis rates of >4 min(-1). Our data show that the sole obligatory role for divalent cations, for which there is crystallographic evidence, is coordination of the phosphate moiety of GlcN6P in the ligand-binding pocket. In further support of this hypothesis, our data show that a nonphosphorylated analogue of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations. Folding of the ribozyme is, therefore, relatively independent of cation identity, but the rapid initiation of catalysis upon the addition of ligand is stricter.

Inhibitors of cellular signalling are cytotoxic or block the budded-to-hyphal transition in the pathogenic yeast Candida albicans.

The pathogenic yeast Candida albicans can grow in multiple morphological states including budded, pseudohyphal and true hyphal forms. The ability to interconvert between budded and hyphal forms, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans virulence, and is regulated by multiple environmental and cellular signals. To identify small-molecule inhibitors of known cellular processes that can also block the BHT, a microplate-based morphological assay was used to screen the BIOMOL-Institute of Chemistry and Cell Biology (ICCB) Known Bioactives collection from the ICCB-Longwood Screening Facility (Harvard Medical School, Boston, MA, USA). Of 480 molecules tested, 53 were cytotoxic to C. albicans and 16 were able to block the BHT without inhibiting budded growth. These 16 BHT inhibitors affected protein kinases, protein phosphatases, Ras signalling pathways, G protein-coupled receptors, calcium homeostasis, nitric oxide and guanylate cyclase signalling, and apoptosis in mammalian cells. Several of these molecules were also able to inhibit filamentous growth in other Candida species, as well as the pathogenic filamentous fungus Aspergillus fumigatus, suggesting a broad fungal host range for these inhibitory molecules. Results from secondary assays, including hyphal-specific transcription and septin localization analysis, were consistent with the inhibitors affecting known BHT signalling pathways in C. albicans. Therefore, these molecules will not only be invaluable in deciphering the signalling pathways regulating the BHT, but also may serve as starting points for potential new antifungal therapeutics.

A rate-limiting conformational step in the catalytic pathway of the glmS ribozyme.

The glmS ribozyme is a conserved riboswitch in numerous Gram-positive bacteria and is located upstream of the glucosamine-6-phosphate (GlcN6P) synthetase reading frame. Binding of GlcN6P activates site-specific self-cleavage of the glmS mRNA, resulting in the downregulation of glmS gene expression. Unlike other riboswitches, the glmS ribozyme does not undergo structural rearrangement upon metabolite binding, indicating that the metabolite binding pocket is preformed in the absence of ligand. This observation led us to test if individual steps in the reaction pathway could be dissected by initiating the cleavage reaction before or after Mg(2+)-dependent folding. Here we show that self-cleavage reactions initiated with simultaneous addition of Mg(2+) and GlcN6P are slow (3 min(-1)) compared to reactions initiated by addition of GlcN6P to glmS RNA that has been prefolded in Mg(2+)-containing buffer (72 min(-1)). These data indicate that some level of Mg(2+)-dependent folding is rate-limiting for catalysis. Reactions initiated by addition of GlcN6P to the prefolded ribozyme also resulted in a 30-fold increase in the apparent ligand K(d) compared to those of reactions initiated by a global folding step. Time-resolved hydroxyl-radical footprinting was employed to determine if global tertiary structure formation is the rate-limiting step. The results of these experiments provided evidence for fast and largely concerted folding of the global tertiary structure (>13 min(-1)). This indicates that the rate-limiting step that we have identified either is a slow folding step between the fast initial folding and ligand binding events or represents the rate of escape from a nativelike folding trap.

Increased male-male courtship in ecdysone receptor deficient adult flies.

Male-male courtship is infrequent among mature adult Drosophila melanogaster. After pairs of mature adult males expressing a temperature-sensitive allele of the ecdysone receptor (EcR) gene were treated at a restrictive temperature, however, they engaged in elevated levels of male-male courtship. EcR-deficient males courted wildtype males and females, but were not courted by wildtype males. These results suggest that the ecdysone steroid hormone system may have a role in courtship initiation by adult male fruit flies.