Rapid Proliferation of Vibrio parahaemolyticus,Vibrio vulnificus,and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae of the non-O1/non-O139 serotype are present in coastal lagoons of southern France. In these Mediterranean regions, the rivers have long low-flow periods followed by short-duration or flash floods during and after heavy intense rainstorms, particularly at the end of the summer and in autumn. These floods bring large volumes of freshwater into the lagoons, reducing their salinity. Water temperatures recorded during sampling (15 to 24°C) were favorable for the presence and multiplication of vibrios. In autumn 2011, before heavy rainfalls and flash floods, salinities ranged from 31.4 to 36.1‰ and concentrations of V. parahaemolyticus, V. vulnificus, and V. cholerae varied from 0 to 1.5 × 10³ most probable number (MPN)/liter, 0.7 to 2.1 × 10³ MPN/liter, and 0 to 93 MPN/liter, respectively. Following heavy rainstorms that generated severe flash flooding and heavy discharge of freshwater, salinity decreased, reaching 2.2 to 16.4‰ within 15 days, depending on the site, with a concomitant increase in Vibrio concentration to ca. 10⁴ MPN/liter. The highest concentrations were reached with salinities between 10 and 20‰ for V. parahaemolyticus, 10 and 15‰ for V. vulnificus, and 5 and 12‰ for V. cholerae. Thus, an abrupt decrease in salinity caused by heavy rainfall and major flooding favored growth of human-pathogenic Vibrio spp. and their proliferation in the Languedocian lagoons. Based on these results, it is recommended that temperature and salinity monitoring be done to predict the presence of these Vibrio spp. in shellfish-harvesting areas of the lagoons.     

Hair to document exposure to glibenclamide

Among the drugs that are used to incapacitate victims such as kids or elderly for sedation or for criminal gain such as sexual offences or robberies, glibenclamide, an antidiabetic was never mentioned. To document the interest of hair testing in such forensic situations, we have developed an original method to test for glibenclamide. A 30-year-old man was admitted to the Emergency Unit for coma and seizures after a party with some members of his family. Blood glucose was 0.40 g/l. A hair specimen was collected several weeks after the event and divided into two segments of 2 cm. Twenty milligrams of each segment cut into small pieces were incubated overnight in a phosphate buffer (pH 5.5), in presence of gliclazide used as internal standard (IS). A liquid/liquid extraction was realized with a mixture of diethyl ether/methylene chloride, and hair extract was separated on a XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection of glibenclamide was achieved using two transitions: m/z 493.9 to 168.9 and 493.9 to 368.8. Linearity was observed from 5 to 1000 pg/mg (r2 = 0.956) with a limit of quantification at 5 pg/mg and a clean-up recovery of about 61%. Within-batch precision and bias were 9.0 and 9.5%, respectively. Ion suppression tested on drug-free hair was about 50%. Glibenclamide tested positive in the two consecutive segments (root to 2 cm: 23 pg/mg and 2-4 cm: 31 pg/mg). These findings were in accordance with a repetitive exposure to the drug. The concentrations were compared with those obtained after a single and a daily dose administration. In the hair of a subject receiving a single 5mg dose and collected 4 weeks later, glibenclamide was detected in the proximal segment at 5 pg/mg. After a 20 mg/day dose, the hair concentration of a subject under glibenclamide therapy was 650 pg/mg.     

Dynamics of microbial planktonic food web components during a river flash flood in a Mediterranean coastal lagoon

Episodic river flash floods, characteristic of Mediterranean climates, are suspected to greatly affect the functioning of microbial food webs. For the first time, the abundance, biomass and diversities of microbial food web components were studied before and during 4 consecutive days after a flash flood that occurred in November 2008, in the surface waters of five stations along a salinity gradient from 20 to 36 in the Thau lagoon. Eukaryotic pico- and nanophytoplankton were discharged from the river into the lagoon and increased by 30- and 70-fold, respectively. Bacteria increased by only 2-fold in the lagoon, from around 4–8 × 10⁶ cells ml⁻¹, probably benefiting from river nutrient input. Chlorophyll a increased 4-fold, and pigment biomarkers showed that the dinophyceae, prasinophyceae and prymnesiophyceae were sensitive to the flood perturbation, whereas the bacillariophyceae, cryptophyceae and chlorophyceae were resistant and/or transported to the lagoon from the river. Predator responses were more complex as total heterotrophic flagellate abundance decreased slightly, whereas those of specific naked ciliates increased, particularly for Uronema sp. The flood also induced a specific change in diversity, from a community dominated by Strobilidium spiralis to a community dominated by Uronema sp. The tintinnid community was particularly sensitive to the flood event as the abundance of all species decreased greatly. The high increases in biomass, mainly brought by the river during the flood, could have eventually sedimented to the benthic layer and/or been transported further into the lagoon, supporting the pelagic food web, or have even been exported to the Mediterranean Sea.     

Intracellular Routing and Release of Caseins and Growth Hormone Produced into Milk from Transgenic Mice

Growth hormone (GH) secretion, in mammary tissue from transgenic mice, containing a chimeric gene composed of the regulatory region of whey acidic protein gene and the structural region of GH gene, was compared to casein secretion. GH was expressed in milk and for a small percentage (1:1000) in blood as revealed by SDS-polyacrylamide gel electrophoresis and radio-immunoassay. As attested by immunofluorescence and immunogold electron microscopy, caseins and GH followed the same secretory pathway. However, contrary to caseins, which are essentially in micellar form, GH was detected in a nonaggregated form in secretory vesicles and in the lumen of the acini. Newly synthesized caseins and GH were carried simultaneously, mainly to the lumen of the acini, but also to the base of the cell. Secretion of newly synthesized proteins was increased by prolactin (PRL). As shown by immunoblotting, the proportion of GH versus other proteins, secreted in the presence of PRL was not modified, suggesting that GH secretion is subjected to the same hormonal regulation by PRL as other milk proteins. These results show that, in lactating mammary epithelial cells from transgenic mice, a recombinant GH and the caseins are carried simultaneously to the lumen and suggest that secretion of both proteins is increased by PRL during the same time course. Transport of these newly synthesized proteins occurs also to the base of the cell.     

Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis

A stable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase II alpha (topoII alpha) protein was downregulated and replaced by the expression of topoII alpha fused with enhanced green fluorescent protein (EGFP-topoII alpha). The EGFP-topoII alpha faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP-topoII alpha accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP-topoII alpha diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP-topoII alpha associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topoII alpha was essential for rapid dynamics, as ICRF-187, an inhibitor of topoII alpha, blocked recovery after photobleaching. Although some topoII alpha may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topoII alpha in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation.     

Targeted Expression of the only Zinc Finger Gene in Transgenic Mice is Associated with Impaired Mammary Development

The only zinc finger (OZF) gene encodes a protein consisting mainly of 10 zinc finger motifs of the Krüppel type of yet unknown function. To potentially assess its in vivo role, mammary targeted deregulation of the expression of the murine gene was performed in transgenic mice using a goat -casein-based transgene. Mammary expression of the transgene was observed in the 11 lines obtained. In three expressing lines, this expression was tissue-specific and developmentally regulated. Further analysis of mice from two expressing lines revealed that transgene-homozygous females could not sustain full growth of their pups. This phenotype was associated with an impaired mammary gland development noticeable only after mid-gestation. It was characterised by an increase of the adipocyte to acini ratio and low or absence of fat globules within these acini compared to non-transgenic control animals. These transgenic observations strongly suggest that OZF is active in the mammary gland, interfering with the lactation process and thus that the described transgenic mice could be useful models to search for the cellular partner(s) of this protein.     

Promotion time models with time-changing exposure and heterogeneity: application to infectious diseases

Promotion time models have been recently adapted to the context of infectious diseases to take into account discrete and multiple exposures. However, Poisson distribution of the number of pathogens transmitted at each exposure was a very strong assumption and did not allow for inter-individual heterogeneity. Bernoulli, the negative binomial, and the compound Poisson distributions were proposed as alternatives to Poisson distribution for the promotion time model with time-changing exposure. All were derived within the frailty model framework. All these distributions have a point mass at zero to take into account non-infected people. Bernoulli distribution, the two-component cure rate model, was extended to multiple exposures. Contrary to the negative binomial and the compound Poisson distributions, Bernoulli distribution did not enable to connect the number of pathogens transmitted to the delay between transmission and infection detection. Moreover, the two former distributions enable to account for inter-individual heterogeneity. The delay to surgical site infection was an example of single exposure. The probability of infection was very low; thus, estimation of the effect of selected risk factors on that probability obtained with Bernoulli and Poisson distributions were very close. The delay to nosocomial urinary tract infection was a multiple exposure example. The probabilities of pathogen transmission during catheter placement and catheter presence were estimated. Inter-individual heterogeneity was very high, and the fit was better with the compound Poisson and the negative binomial distributions. The proposed models proved to be also mechanistic. The negative binomial and the compound Poisson distributions were useful alternatives to account for inter-individual heterogeneity.