MOLECULAR CHARACTERIZATION OF THE DIVERSITY AND POTENTIAL TOXICITY OF CYANOBACTERIAL MATS IN TWO TROPICAL LAGOONS IN THE SOUTH PACIFIC OCEAN1

Marine benthic cyanobacteria in tropical areas have recently been associated with several human poisoning events. To enhance the characterization of these microorganisms and their potential toxicity, benthic cyanobacterial communities were sampled in the lagoons of two islands (Raivavae and Rurutu) located in French Polynesia where human poisoning events by seafood had been reported. The morphological appearance of the mats was used to identify four types of cyanobacterial mat. By a 16S rRNA sequencing approach, it appeared that these mats were usually dominated by a restricted number of operational taxonomic units (OTUs), which were closely related to Leptolyngbya, Oscillatoria, Hydrocoleum, and Anabaena sequences, as previously reported in other tropical lagoons. Interestingly, we determined that these dominant filamentous OTUs were associated in the mats with other cyanobacteria, including unicellular species. By using a population genetic approach based on the sequencing of the internally transcribed spacer (ITS) of the rRNA operon, we found a very restricted genetic diversity in the most common OTU, which displayed a high sequence similarity with Leptolyngbya sp. In addition, there was no geographic differentiation at various spatial scales in the distribution of the different genotypes, suggesting that this species is able to spread over large distances. Finally, PCR screening of genes involved in the biosynthesis of known cyanotoxins revealed the presence of the saxitoxin gene (stxG) in two mats containing a mix of filamentous and unicellular cyanobacterial species.     

Uncoupled IP3 receptor can function as a Ca2+-leak channel: cell biological and pathological consequences

Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways.     

A thermodynamic approach to PCR primer design

We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.     

Metagenomic approach studying the taxonomic and functional diversity of the bacterial community in a mesotrophic lake (Lac du Bourget – France)

The main goals of this work were to identify the metabolic pathways of the bacterial community in a lacustrine ecosystem and to establish links between taxonomic composition and the relative abundances of these metabolic pathways. For this purpose, we analysed a 16S rRNA gene library obtained by gene amplification together with a sequence library of both insert ends on c . 7700 fosmids. Whatever the library used, Actinobacteria was the most abundant bacterial group, followed by Proteobacteria and Bacteroidetes . Specific aquatic clades such as acI and acIV ( Actinobacteria ) or LD12 and GOBB-C201 ( Alphaproteobacteria ) were found in both libraries. From comparative analysis of metagenomic libraries, the metagenome of this lake was characterized by overrepresentation of genes involved in the degradation of xenobiotics mainly associated with Alphaproteobacteria . Actinobacteria were mainly related to metabolic pathways involved in nucleotide metabolism, cofactors, vitamins, energy, replication and repair. Betaproteobacteria appeared to be characterized by the presence of numerous genes implicated in environmental information processing (membrane transport and signal transduction) whereas glycan and carbohydrate metabolism pathways were overrepresented in Bacteroidetes . These results prompted us to propose hypotheses on the ecological role of these bacterial classes in lacustrine ecosystems.     

Spatiotemporal Variations in Microcystin Concentrations and in the Proportions of Microcystin-Producing Cells in Several Microcystis aeruginosa Populations

With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (mcyB⁺) were predominant (70 to 100%) in all of these M. aeruginosa populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of mcyB⁺ cells. Finally, the variations in total MC concentrations (0.3 to 15 μg liter⁻¹) and in the MC cellular quotas (0.01 to 3.4 pg cell⁻¹) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of mcyB⁺ cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in M. aeruginosa populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of Microcystis blooms.Microcystins (MCs) are the most common cyanotoxins and have been involved in several animal and human poisoning episodes (8). These hepatotoxic cyclic heptapeptides are synthesized by a multifunctional enzyme complex (10, 40), and the discovery of the gene cluster encoding this complex has made it possible in recent years to develop molecular tools for studying the relative proportions of MC-producing and non-MC-producing cells in natural cyanobacterial populations. Potentially MC-producing and non-MC-producing cells can coexist in these populations, but the factors and processes governing the dynamics of these subpopulations remain unclear.Some recent papers on the Microcystis genus have shown that the proportions of potentially MC-producing cells can differ considerably from lake to lake. For example, in Lake Wannsee, Germany, this proportion was between 0 and 40% (28), as it was in Lake Oneida, United States (18), and in Lake Mikata, Japan (48). In contrast, large variations over time (6 to 93%) of potentially MC-producing cells were found in the Grangent Reservoir, France (4). Major variations (30 to 80%) were also found in a natural French population of Planktothrix agardhii (3), and the variations in the proportions of potentially MC-producing cells reflected those of the MC concentrations. However, only 54% of the variation in MC concentrations could be explained by changes in the proportion of MC-producing cells, suggesting that a considerable part of the MC concentrations was also due to variations in MC cell quotas. These findings suggest that the toxic risks during cyanobacterial proliferations are due to variations in both the proportion of MC-producing cells and the production of MC by the toxic cells.Numerous papers have already investigated the impact of various biotic and abiotic environmental factors on microcystin production by toxic cells. These studies demonstrate that MC production can be influenced by temperature (35), light (46), nutrients such as nitrogen and phosphorus (12, 32), pH (39), iron (42), xenobiotics (17, 34, 45), and predators (22, 23, 47). Despite inconsistent results, the production of microcystins by the cells does seems to be linked to their growth rate (11, 31, 33), which is itself affected by environmental conditions. On the other hand, several studies of variations in the proportions of MC-producing cells have demonstrated the potential influence of nutrient concentrations (9, 48) and light and temperature (5), and two papers (3, 5) have suggested that there is a negative correlation between the proportions of MC-producing cells and the abundance of cyanobacterial cells. These findings are consistent with the data of Kardinaal and Visser (26), showing that in Dutch lakes there is a negative relationship between the densities of cyanobacterial cells and the mean MC concentration in the cells.In an overall attempt to explain the variations of toxicity during cyanobacterial blooms, we studied the spatiotemporal variations in MC concentrations and in the proportions of MC-producing and non-MC-producing cells in several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. The point of this study was to analyze these variations in terms of the characteristics of these ecosystems and the population dynamics of the M. aeruginosa populations. In addition, these data were compared to the variations in the intergenic transcribed spacer (ITS) composition of the same populations recently reported by Sabart et al. (38). The proportion of potentially MC-producing cells was estimated by a real-time quantitative PCR approach, the change in threshold cycle (ΔC_T) method recently developed by Briand et al. for Planktothrix (3) and Microcystis (4) and targeting the mcyB (mcyA for Planktothrix) and phycocyanin (PC) genes.     

Discovery of a novel series of CXCR3 antagonists

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC₅₀ 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

Benfluorex and Unexplained Valvular Heart Disease: A Case-Control Study

Background

Recent case reports suggest that benfluorex, a fenfluramine derivative used in the management of overweight diabetic patients and dyslipidemia, is associated with cardiac valve regurgitation.

Methods

We conducted a case-control study. Eligible patients were those admitted in the cardiology or the cardiac surgery units of our hospital between January, 1^st 2003 and June 30^th 2009, with mitral insufficiency diagnostic codes (ICD-10 I340 and I051). Patients with either a primary cause (degenerative, known rheumatic heart disease, infectious endocarditis, congenital, radiation-induced valvular disease, associated connective and/or vasculitis disease, trauma, tumor) or a secondary (functional) cause were considered as having an “explained” mitral regurgitation. Other patients were considered as having an “unexplained” mitral regurgitation and were included as cases. For each case, two controls were matched for gender and for the closest date of birth, among a list of patients with an “explained” mitral regurgitation. Drug exposures were assessed blindly regarding the case or control status, through contacts with patients, their family and/or their physicians.

Results

Out of the 682 eligible patients, 27 cases and 54 matched controls were identified. The use of benfluorex was reported in 22 patients: 19 of the 27 cases, versus 3 of the 54 controls, odds-ratio 17.1 (3.5 to 83), adjusted for body mass index, diabetes and dexfenfluramine use.

Conclusion

The use of benfluorex is associated with unexplained mitral regurgitation.