An expedient synthesis of flexible nucleosides via a regiocontrolled enzymatic glycosylation of functionalized imidazoles.

A versatile two-step synthesis of C4- and C5-arylated 2'-deoxyribosylimidazoles was elaborated using enzymatic N-transglycosylation followed by microwave-assisted Pd-catalysed arylation reactions. We report herein the reaction conditions that permit managing regioselectivity (N3 versus N1-isomers) in the enzymatic glycosylation of 4-iodoimidazole using the nucleoside N-deoxyribosyltransferase from L. leichmannii. Regiocontrolled glycosylation was also observed among several other imidazole derivatives studied, providing simple access to isomers not readily accessible by chemical routes. Finally, a series of flexible nucleosides was obtained in one step from 4- or 5-iodo-imidazole nucleosides by the Suzuki-Miyaura cross-coupling reaction with (hetero)aryl-boronic acids in aqueous media. Moreover, this chemoenzymatic approach is compatible with a one-pot two-step process affording a straightforward access to a broad array of potential anticancer and antiviral drugs as well as new DNA building blocks.

Parallel genetic divergence among coastal-marine ecotype pairs of European anchovy explained by differential introgression after secondary contact.

Ecophenotypic differentiation among replicate ecotype pairs within a species complex is often attributed to independent outcomes of parallel divergence driven by adaptation to similar environmental contrasts. However, the extent to which parallel phenotypic and genetic divergence patterns have emerged independently is increasingly questioned by population genomic studies. Here, we document the extent of genetic differentiation within and among two geographic replicates of the coastal and marine ecotypes of the European anchovy (Engraulis encrasicolus) gathered from Atlantic and Mediterranean locations. Using a genome-wide data set of RAD-derived SNPs, we show that habitat type (marine vs. coastal) is the most important component of genetic differentiation among populations of anchovy. By analysing the joint allele frequency spectrum of each coastal-marine ecotype pair, we show that genomic divergence patterns between ecotypes can be explained by a postglacial secondary contact following a long period of allopatric isolation (c. 300 kyrs). We found strong support for a model including heterogeneous migration among loci, suggesting that secondary gene flow has eroded past differentiation at different rates across the genome. Markers experiencing reduced introgression exhibited strongly correlated differentiation levels among Atlantic and Mediterranean regions. These results support that partial reproductive isolation and parallel genetic differentiation among replicate pairs of anchovy ecotypes are largely due to a common divergence history prior to secondary contact. They moreover provide comprehensive insights into the origin of a surprisingly strong fine-scale genetic structuring in a high gene flow marine fish, which should improve stock management and conservation actions.

Expedient and generic synthesis of imidazole nucleosides by enzymatic transglycosylation.

A straightforward route to original imidazole-based nucleosides that makes use of an enzymatic N-transglycosylation step is reported in both the ribo- and deoxyribo-series. To illustrate the scope of this approach, a diverse set of 4-aryl and 4-heteroaryl-1H-imidazoles featuring variable sizes and hydrogen-bonding patterns was prepared using a microwave-assisted Suzuki-Miyaura cross-coupling reaction. These imidazole derivatives were examined as possible substrates for the nucleoside 2'-deoxyribosyltransferase from L. leichmannii and the purine nucleoside phosphorylase from E. coli. The optimum transglycosylation conditions, including the use of co-adjuvants to address solubility issues, were defined. Enzymatic conversion of 4-(hetero)arylimidazoles to 2'-deoxyribo- or ribo-nucleosides proceeded in good to high conversion yields, except bulky hydrophobic imidazole derivatives. Nucleoside deoxyribosyltransferase of class II was found to convert the widest range of functionalized imidazoles into 2'-deoxyribonucleosides and was even capable of bis-glycosylating certain heterocyclic substrates. Our findings should enable chemoenzymatic access to a large diversity of flexible nucleoside analogues as molecular probes, drug candidates and original building blocks for synthetic biology.

An X-chromosome linked locus contributes to abnormal placental development in mouse interspecific hybrid.

Interspecific hybridization between closely related species is commonly associated with decreased fertility or viability of F1 hybrids. Thus, in mouse interspecific hybrids, several different hybrid sterility genes that impair gametogenesis of the male hybrids have been described. We describe a novel effect in hybrids between different mouse species that manifests itself in abnormal growth of the placenta. Opposite phenotypes, that is, placental hypotrophy versus hypertrophy, are observed in reciprocal crosses and backcrosses. The severity of the phenotype, which is mainly caused by abnormal development of the spongiotrophoblast, is influenced by the sex of the conceptus. In general, placental hypertrophy is associated with increased fetal growth. Hypotrophy of the placenta frequently leads to growth impairment or death of the fetus. One of the major genetic determinants of placental growth maps to the proximal part of the mouse X chromosome.

[Perioperative management of new antiplatelet drugs]. / Gestion péri-interventionnelle des nouveaux agents antiplaquettaires.

The management of antiplatelet drugs in the perioperative setting is based on an individual evaluation of the thrombotic and bleeding risks. When the bleeding risk is deemed low, continuation of the dual antiplatelet therapy is usually recommended, especially in high thrombotic risk settings. When the bleeding risk is deemed moderate, at least one antiplatelet agent should be continued, usually aspirin, and clopidogrel and ticagrelor should be discontinued 5 days and prasugrel 7 days before surgery. In some rare instances of high bleeding risk, discontinuation of aspirin 3 days before surgery is usually acceptable. In high thrombotic settings, bridging with an intravenous antiplatelet drug with a short half-life may be considered.

Expanding hybrid zone between Solea aegyptiaca and Solea senegalensis: genetic evidence over two decades.

Several marine hybrid zones have been described and studied during the last years. Assessing the movements of extending hybrid zones is central to improve our understanding of evolutionary processes. We have re-examined the hybrid zone between Solea aegyptiaca and Solea senegalensis that was first described 22 years ago in northern Tunisia when introgressed S. senegalensis individuals were found in the Gulf of Tunis, whereas locally caught S. aegyptiaca were genetically pure. Six population samples harvested both inside and outside the area where the two fish species coexist were genotyped for allozymes and exon-primed intron length polymorphism. Both types of markers were congruent and revealed that introgression takes place indeed in both directions. A high introgression level (36.4%) in the Bizerta lagoon and much less outside indicate that this is the main area where hybridization occurs while introgression clines towards the south in S. aegyptiaca and towards the north in S. senegalensis plead in favour of the existence of a unimodal hybrid zone. The higher introgression level calculated in the current study (when compared to 16% reported formerly) and the newly found introgressed S. aegyptiaca in Bizerta lagoon seem to indicate that the genetic exchanges occurring between the two taxa are evolving and not stabilized yet.

Variance in the reproductive success of flat oyster Ostrea edulis L. assessed by parentage analyses in natural and experimental conditions.

In order to document further the phenomena of variance in reproductive success in natural populations of the European flat oyster Ostrea edulis, two complementary studies based on natural and experimental populations were conducted. The first part of this work was focused on paternity analyses using a set of four microsatellite markers for larvae collected from 13 brooding females sampled in Quiberon Bay (Brittany, France). The number of individuals contributing as the male parent to each progeny assay was highly variable, ranging from 2 to more than 40. Moreover, paternal contributions showed a much skewed distribution, with some males contributing to 50-100% of the progeny assay. The second part of this work consisted of the analysis of six successive cohorts experimentally produced from an acclimated broodstock (62 wild oysters sampled in the Quiberon Bay). Allelic richness was significantly higher in the adult population than in the temporal cohorts collected. Genetic differentiation (F(st) estimates) was computed for each pair of samples and all significant values ranged from 0.7 to 11.9%. A limited effective number of breeders (generally below 25) was estimated in the six temporal cohorts. The study gives first indications of the high variance in reproductive success as well as a reduced effective size, not only under experimental conditions but also in the wild. Surprisingly, the pool of the successive cohorts, based on the low number of loci used, appeared to depict a random and representative set of alleles of the progenitor population, indicating that the detection of patterns of temporal genetic differentiation at a local scale most likely depends on the sampling window.

Adaptive evolution of interferon-gamma in Glire lineage and evidence for a recent selective sweep in Mus. m. domesticus.

Interferon-gamma plays a key role in the immune response against intracellular pathogens. Its gene is located inside a cluster of cytokines from the interleukin-10 family. A comparison of the coding sequences in the mammalian Glire lineage indicates a possible action of positive Darwinian selection promoting rapid amino-acid changes in the branch leading to murine rodents represented by Mus and Rattus. Looking at genomic diversity of this gene inside the genus Mus, we could propose that a recent selective sweep has affected M. m. domesticus, this subspecies harbouring predominantly a single Ifng haplotype that differs from that of the other subspecies by a unique amino-acid difference in a key position of the molecule. The sweep seems to have affected a region of at most 50 kb as recombinants could be found at flanking conserved non-coding sequences. Functional differences were clearly apparent in cis-regulation of Ifng transcription between the domesticus and the musculus-type haplotypes. As the presence of the musculus haplotype in a predominantly domesticus background seems to promote susceptibility to chronic infection by Theiler's virus, these findings open interesting avenues for documenting immune system gene co-evolution.

Management of antiplatelet therapy for non elective invasive procedures of bleeding complications: proposals from the French working group on perioperative haemostasis (GIHP), in collaboration with the French Society of Anaesthesia and Intensive Care Medicine (SFAR).

The French Working Group on Perioperative Haemostasis (GIHP) and the French Study Group on Haemostasis and Thrombosis (GFHT) in collaboration with the French Society of Anaesthesia and Intensive Care Medicine (SFAR) drafted up-to-date proposals on the management of antiplatelet therapy for non-elective invasive procedures or bleeding complications. The proposals were discussed and validated by a vote; all proposals could be assigned with a high strength. Emergency management of oral antiplatelet agents (APA) requires knowledge on their pharmacokinetic/pharmacodynamics parameters, evaluation of the degree of the alteration of haemostatic competence and the associated bleeding risk. Platelet function testing may be considered. When APA-induced bleeding risk may worsen the prognosis, measures should be taken to neutralise antiplatelet therapy by considering not only the efficacy of available means (which can be limited for prasugrel and even more for ticagrelor) but also the risks that these means expose the patient to. The measures include platelet transfusion at the appropriate dose and haemostatic agents (tranexamic acid; rFVIIa for ticagrelor). When possible, postponing non-elective invasive procedures at least for a few hours until the elimination of the active compound (which could compromise the effect of transfused platelets) or if possible a few days (reduction of the effect of APA) should be considered.

Prevalence and evolutionary origins of autoimmune susceptibility alleles in natural mouse populations.

The evolutionary origin of genetic diversity in the SLAM/CD2 gene cluster, implicated in autoimmune lupus susceptibility in mice, was investigated by sequence analysis of exons from six members of the cluster in 48 wild mouse samples derived from the global mouse population. A total of 80 coding region SNPs were identified among the six genes analyzed, indicating that this gene cluster is highly polymorphic in natural mouse populations. Phylogenetic analyses of these allelic sequences revealed clustering of alleles derived from multiple Mus species and subspecies, indicating alleles at several SLAM/CD2 loci were present in ancestral Mus populations prior to speciation and have persisted as polymorphisms for more than 1 million years. Analyses of nonsynonymous/synonymous ratios using likelihood codon substitution models identified several segments in Cd229, Cd48 and Cd84 that were impacted by positive diversifying selective pressures. These findings support the interpretation that selection favoring the generation and retention of functional polymorphisms has played a role in the evolutionary origin of genetic polymorphisms that are predisposing to autoimmunity.

Evidence for a slightly deleterious effect of intron polymorphisms at the EF1alpha gene in the deep-sea hydrothermal vent bivalve Bathymodiolus.

A multilocus analysis was initiated in order to infer the general effect of demography and the indirect effect of positive selection on some chromosome segments in Bathymodiolus. Mussels of the genus Bathymodiolus inhabit the very hostile, fragmented and variable environment of deep-sea hydrothermal vents which is thought to cause recurrent population bottlenecks via extinction/colonisation processes and adaptation to new environmental conditions. In the course of this work we discovered that the assumption of neutrality of non-coding polymorphisms usually made in genome scan experiments was likely to be violated at one of the loci we analysed. The direct effect of slight purifying selection on non-coding polymorphisms shares many resemblances with the indirect effect of positive selection through genetic hitchhiking. Combining polymorphism with divergence data for several closely related species allowed us to obtain different expectations for the direct effect of negative selection and the indirect effect of positive selection. We observed a strong excess of rare non-coding polymorphisms at the second intron of the EF1alpha gene in the two species Bathymodiolus azoricus and Bathymodiolus thermophilus, while two other loci, the mitochondrial COI gene and an intron of the Lysozyme gene, did not exhibit such a deviation. In addition, the divergence rate of the EF1alpha intron was estimated to be unexpectedly low when calibrated using the closure of the Panama Isthmus that interrupted gene flow between the two species. The polymorphism to divergence ratio was similar to the one observed for the other two loci, in accordance to the hypothesis of purifying selection. We conclude that slight purifying selection is likely to act on polymorphic intronic mutations of the EF1alpha second intron and discuss the possible relationship with the specific biology of Bathymodiolus mussels.

Crystal and electronic structures of magnesium(II), copper(II), and mixed magnesium(II)-copper(II) complexes of the quinoline half of styrylquinoline-type HIV-1 integrase inhibitors.

A new target in AIDS therapy development is HIV-1 integrase (IN). It was proven that HIV-1 IN required divalent metal cations to achieve phosphodiester bond cleavage of DNA. Accordingly, all newly investigated potent IN inhibitors contain chemical fragments possessing a high ability to chelate metal cations. One of the promising leads in the polyhydroxylated styrylquinolines (SQLs) series is (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinoline carboxylic acid (1). The present study focuses on the quinoline-based progenitor (2), which is actually the most probable chelating part of SQLs. Conventional and synchrotron low-temperature X-ray crystallographic studies were used to investigate the chelating power of progenitor 2. Mg2+ and Cu2+ cations were selected for this purpose, and three types of metal complexes of 2 were obtained: Mg(II) complex (4), Cu(II) complex (5) and mixed Mg(II)-Cu(II) complexes (6 and 7). The analysis of the crystal structure of complex 4 indicates that two tridentate ligands coordinate two Mg2+ cations, both in octahedral geometry. The Mg-Mg distance was found equal to 3.221(1) A, in agreement with the metal-metal distance of 3.9 A encountered in the crystal structure of Escherichia coli DNA polymerase I. In 5, the complex is formed by two bidentate ligands coordinating one copper ion in tetrahedral geometry. Both mixed Mg(II)-Cu(II) complexes, 6 and 7 exhibit an original arrangement of four ligands linked to a central heterometallic cluster consisting of three octahedrally coordinated magnesium ions and one tetrahedrally coordinated copper ion. Quantum mechanics calculations were also carried out in order to display the electrostatic potential generated by the dianionic ligand 2 and complex 4 and to quantify the binding energy (BE) during the formation of the magnesium complex of progenitor 2. A comparison of the binding energies of two hypothetical monometallic Mg(II) complexes with that found in the bimetallic magnesium complex 4 was made.

Growth hormone and Prolactin-1 gene transcription in natural populations of the black-chinned tilapia Sarotherodon melanotheron acclimatised to different salinities.

The effects of salinity on the expression of genes coding for growth hormone (GH) and prolactin-1 (PRL1) were studied in various natural populations of the black-chinned tilapia Sarotherodon melanotheron from West Africa. Individuals were sampled in June 2005 in six locations in Senegal and the Gambia, at various salinities between 0 and 101. The poorest condition factors were recorded in the most saline sampling site and the best growth in the fish from a marine environment. The pituitary GH mRNA levels were significantly higher in fish adapted to seawater, whereas the PRL1 mRNA levels were highest in fish adapted to fresh- and brackish water. These results show that the PRL1 mRNA levels seem to reflect relatively well the differences in environmental salinity, in contrast to those of GH, which would tend instead to reflect the individual growth in each environment. However, no relation could be found between growth in the hypersaline areas and the expression profile of GH. Although the fish analysed were morphologically identical, the expression of genes coding for GH and PRL1 showed large differences between individuals. This inter-individual variation in gene expression remains poorly understood.

The effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.).

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II beta-antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.

A transcriptomic approach of salinity response in the euryhaline teleost, Dicentrarchus labrax.

Euryhaline teleosts possess the capacity to osmoregulate under various environmental conditions (freshwater to hypersaline water). This physiological capacity is generally monitored using enzyme activity assays (Na+/K+ -ATPase...), hormones quantification (prolactine, growth hormone) or their mRNAs expression. To date, few studies addressed the genetic correlates of adaptation to varying salinity at a molecular level in such fish. In the sea bass Dicentrarchus labrax, genetic differentiation was observed at specific allozyme loci between lagoon- and open-sea populations. In the present study, we investigated transcriptomic response of D. labrax to salt- and freshwater acclimation in two organs involved in osmoregulation, gill and intestine. By using suppression subtractive hybridisation, we characterised 586 partial cDNA sequences encoding proteins potentially involved in the metabolism of sea bass acclimated to salt- or freshwater under experimental conditions. Using these results, we first characterised complete genomic sequence of a carbonic anhydrase and then analysed mRNA expression of genes potentially involved in osmoregulation mechanisms (Na+/K+ -ATPase, carbonic anhydrase, angiotensin-converting enzyme and claudin-3), cell-cycle regulation (secretagogin) and immune system (nephrosin) in gill and intestine of wild fish from open sea and lagoons. Our analyses indicate a strong tissue- and environmental-dependant expression pattern for all the genes studied. A transcriptomic approach such as described in the present paper provides thus a first description of genes involved in metabolic or structural functions important for coping with environmental salinity variations in a euryhaline fish like the common sea bass D. labrax. It should be supplemented by proteomics to check the direct involvement of the gene products at the protein level, and by polymorphism analyses if one is to understand population or individual fluctuations in acclimation to salinity variation.

[The role of point-of-care platelet function testing in guiding platelet transfusion]. / Tests d'évaluation de la fonction plaquettaire délocalisés : un guide pour la transfusion de plaquettes ?

Several point-of-care platelet function devices are available and may be used to assess the effect of antiplatelet drugs. With a view to streamlining the management of bleeding and transfusion, these tests might be a support for platelet transfusion. However, their current place to guide transfusion is still unclear and their routine use cannot be recommended.

H-2 polymorphisms are more uniformly distributed than allozyme polymorphisms in natural populations of house mice.

Patterns of H-2 and allozyme polymorphism in natural populations of house mice from Europe, North Africa and South America were analyzed. The purpose of the analysis was to determine whether H-2 and allozyme polymorphisms were similarly distributed both geographically and temporally in wild mice. Two subspecies of house mice, Mus musculus domesticus and M. m. musculus were sampled and the polymorphisms of two H-2 class I genes, H-2K and H-2D, and 34 allozyme-encoding genes were surveyed. The three kinds of analyses that were conducted included a hierarchical gene diversity analysis, an analysis of the effects of barriers to gene flow, and an analysis of similarity networks. Each of the comparisons demonstrated that H-2 polymorphisms were more uniformly distributed than allozyme polymorphisms and provided additional evidence that H-2 and allozyme polymorphisms are subject to different evolutionary pressures. The analysis of similarity networks also demonstrated that H-2 genes provide little information about the phylogeny of wild mice.

Early effect of inbreeding as revealed by microsatellite analyses on Ostrea edulis larvae.

This paper reports new experimental evidence on the effect of inbreeding on growth and survival in the early developmental phase of a marine bivalve, the flat oyster Ostrea edulis. Two crosses between full sibs were analyzed using four microsatellite markers. Samples of 96 individuals were taken just after spawning (day 1), at the end of the larval stage before metamorphosis (day 10) and at the postlarval stage (day 70). Significant departure from Mendelian expectation was observed at two loci in the first cross and two loci in the second. Departure from 1:1 segregation occurred in one parent of the first cross at three loci and genotypic selection, which resulted in highly significant heterozygote excesses, was recorded at three out of four loci in cross C1 and at two out of three loci in cross C2. Across the four markers, there were similar significant excesses of multilocus heterozygosity, and significant multilocus heterozygosity-growth correlations were recorded for both crosses at all stages. These results suggest that microsatellite markers, often assumed to be neutral, cosegregated with fitness-associated genes, the number of which is estimated to be between 15 and 38 in the whole genome, and that there is a potentially high genetic load in Ostrea edulis genome. This load provides a genetic basis for heterosis in marine bivalves.

Haplotypes that are mosaic for wild-type and t complex-specific alleles in wild mice.

Two outstanding problems pertaining to the population dynamics and evolution of the t complex in mice concern the frequency of t haplotypes in the wild and the degree to which these haplotypes recombine with their wild-type homologs. To address these problems, the frequency and distribution of several t complex-associated restriction fragment variants in wild mice were estimated. Sixty-four versions of chromosome 17 from wild-derived Mus musculus musculus and Mus musculus domesticus were examined with DNA probes for six loci within the t complex that exhibit restriction fragment variation. All six probes detect variants that have heretofore been found exclusively associated with the t complex. Haplotype analysis of wild-derived chromosomes revealed a high frequency (45.3%) of "mosaic" haplotypes with a mixture of t-specific and wild-type variants and only one haplotype with t-specific variants at all six loci. When 12 well-characterized t haplotypes isolated from diverse geographic regions were analyzed, only three had a complete set of t-specific restriction fragments for the six loci examined. The preponderance of mosaic haplotypes in both groups of mice can be explained by any one of the following hypotheses: genetic recombination between t haplotypes and their wild-type homologs, the persistence in wild populations of haplotypes that have descended from ancestral partial t haplotypes, or that the restriction fragment variants fixed in the ancestral t haplotype were also fixed in some wild-type haplotypes. There is evidence to support all three of these hypotheses in our data. The allelic composition of some mosaic haplotypes indicates that they may have been formed by segmental recombination, either double crossing over or gene conversion, rather than by simple single crossovers. The occurrence of indistinguishable mosaic haplotypes in both M. m. musculus and M. m. domesticus suggests that these haplotypes are ancestral rather than recently derived.

"New" allotypes of the murine gamma 1 immunoglobulins.

SPE wild-derived inbred mice belonging to Mus spretus species can produce antibodies against gamma 1 isotypic determinants, common to European mice belonging to the Mus subgenus, with the exception of the Mus spretus species. They are also able to produce antibodies which define a "new" Igh-4 allotypic specificity (designated Igh-4.4).