Genomic and Phenotypic Characterization of Vibrio cholerae Non-O1 Isolates from a US Gulf Coast Cholera Outbreak

Between November 2010, and May 2011, eleven cases of cholera, unrelated to a concurrent outbreak on the island of Hispaniola, were recorded, and the causative agent, Vibrio cholerae serogroup O75, was traced to oysters harvested from Apalachicola Bay, Florida. From the 11 diagnosed cases, eight isolates of V. cholerae were isolated and their genomes were sequenced. Genomic analysis demonstrated the presence of a suite of mobile elements previously shown to be involved in the disease process of cholera (ctxAB, VPI-1 and -2, and a VSP-II like variant) and a phylogenomic analysis showed the isolates to be sister taxa to toxigenic V. cholerae V51 serogroup O141, a clinical strain isolated 23 years earlier. Toxigenic V. cholerae O75 has been repeatedly isolated from clinical cases in the southeastern United States and toxigenic V. cholerae O141 isolates have been isolated globally from clinical cases over several decades. Comparative genomics, phenotypic analyses, and a Caenorhabditis elegans model of infection for the isolates were conducted. This analysis coupled with isolation data of V. cholerae O75 and O141 suggests these strains may represent an underappreciated clade of cholera-causing strains responsible for significant disease burden globally.     

1H NMR spectroscopic studies on the characterization of renal cell lines and identification of novel potential markers of in vitro nephrotoxicity

Cell cultures are increasingly used in the evaluation of chemically-induced nephrotoxicity. The utili of renal cell culture systems in toxicology would be improved, however, if better characterized and more specific markers of toxicity were available. High resolution proton nuclear magnetic resonance (¹H NMR) spectroscopy is well suited to the study of toxicological events and has identified many novel markers of nephrotoxicity in vivo. In this study, ¹H NMR spectroscopy has been used to characterize the biochemical composition of two renal cell lines of different nephronal origin, LLC-PK₁ (pig proximal tubule) and Madin-Darby canine kidney (MDCK, distal tubule). The early biochemical responses of these cell lines to the model proximal tubular toxin S-(1,2dichlorovinyl)i-L-cysteine (DCVC) and the renal medullary toxin 2-chloroethanamine (CEA) have also been investigated. For each line, 500 MHz ¹H NMR spectra of protein-free acetone extracts of cells and culture medium gave characteristic and reproducible profiles of low MW constituents, including amino and organic acids, glucose and soluble membrane precursors, such as choline and myo-inositol. Treatment-related changes in several low MW compounds not routinely measured in toxicological studies were revealed by NMR specboscopy before marked cytotoxicity was observed by phase contrast microscopy. For example, LLC-PK₁ cells treated with 60 μM DCVC showed a marked decrease in intracellular choline levels within 3 h which suggests an effect on the balance of choline synthesis and utilization. Wrthin 9 h of treatment with DCVC there were decreases in intracellular acetate and alanine concentrations which may be indicative of a decrease in fatty acid oxidation and biglyceride metabolism accompanied by an increase in gluconeogenesis. In MDCK cells, 1 h post treatment with 5 mM CEA, intracellular glycine was decreased. This study indicates the potential power and applicability of ¹H NMR spectroscopy for evaluating the biochemical and metabolic effects of toxins in cell culture systems and provides a novel approach to identifying new markers of tissue damage.     

-Subunits of Ns are released from the plasma membrane following cholera toxin activation

Cholera toxin (CT) and islet-activating protein (IAP, a Bordetella pertussis toxin) were employed to test the hypothesis that GTP-binding regulatory proteins are released from plasma membranes to a greater extent when ‘activated’ than when ‘inactivated’. CT, which activates N_s (the stimulatory GTP-binding regulatory protein of the adenylate cyclase system), catalyzed the incorporation of radioactivity from [³²P]NAD into 45 and 47.5 kDa peptides associated with rat liver plasma membranes. Following ADP-ribosylation and centrifugation at 100000 × g for 1 h, approx. 30–35% of these CT-labelled peptides were no longer associated with the plasma membranes, but were recovered from the supernatant fraction. IAP, which inactivates N_i (the inhibitory GTP-binding regulatory protein of the adenylate cyclase system) catalyzed the incorporation of radioactivity from [³²P]NAD into a 41 kDa peptide associated with the membranes. However, in contrast to the CT-labelled peptides, typically less than 5% of the lAP-labelled peptide was found in the 100000 × g supernatant fraction, but rather was almost exclusively associated with the membrane pellet. The data indicate that the -subunits of N_s are released from the plasma membrane following activation, and support the hypothesis that the βγ-subunits act to anchor the -subunits to the plasma membrane. Cholera toxin Islet-activating protein GTP-binding protein     

In vitro fertilisation with recombinant follicle stimulating hormone requires less IU usage compared with highly purified human menopausal gonadotrophin: results from a European retrospective observational chart review

Background  

Previous studies have reported conflicting results for the comparative doses of recombinant follicle stimulating hormone (rFSH) and highly purified human menopausal gonadotrophin (hMG-HP) required per cycle of in vitro fertilisation (IVF); the aim of this study was to determine the average total usage of rFSH versus hMG-HP in a 'real-world' setting using routine clinical practice.     

Pollen morphology and infra-generic evolutionary relationships in some Chinese species of Pedicularis (Scrophulariaceae)

 The pollen grains of 32 Chinese species of Pedicularis representing 13 groups within the genus were investigated using light microscopy and scanning electron microscopy. The genus is eurypalynous, the apertures may be tricolpate, trisyncolpate, or bisyncolpate and five types of surface ornamentation (microscabrate, microrugulate, retipilate, microreticulate and microfoveolate) were observed. Comparison of the different aperture types using light microscopy allows three pollen types to be distinguished. Examination of exine ornamentation with scanning electron microscopy enabled each pollen type to be divided into two or three sub-types (giving a total of seven sub-types). Pollen morphology within the genus is discussed in relation to infrageneric relationships, evolutionary and pollination biology. There is little correlation with the existing infrageneric taxonomy (some taxonomic groups have more than one pollen type, while the same pollen type may be found in several different species-groups), but greater correlation with the corolla morphology. Received January 10, 2002; accepted March 5, 2002 Published online: March 10, 2003