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.

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.

[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.

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.

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.

[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.

[Rare bleeding disorders and invasive procedures]. / Déficits rares de la coagulation et gestes invasifs.

Rare inherited bleeding disorders include fibrinogen disorders, and deficiencies of factors II (prothrombin), V, VII, X, XI, XIII, and combined V+VIII, and combined vitamin K-dependent factors, with general population prevalence rates between 1/500,000 and 1/2,000,000. These inherited disorders, transmitted as autosomal recessive traits, are characterized by a heterogeneous clinical presentation (asymptomatic, mild, moderate or severe bleeding tendency); this variability is more important for deficiencies with factor levels ranging from 5 to 50%. Individual bleeding risk assessment before an invasive procedure or during peri-partum period remains difficult, although an essential step to decide whether a substitution with clotting factor is necessary or not. Because there is a poor correlation between factor activity levels and the severity of bleeding symptoms, factor correction before an invasive procedure should not be based on factor level only, but physicians must also take into account the patient phenotype as well as the haemorrhagic risk of the procedure.

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.

Induction of a melanoma-specific antibody response by a monovalent, but not a divalent, synthetic GM2 neoglycopeptide.

The GM2 ganglioside represents an important target for specific anticancer immunotherapy. We designed and synthesized a neoglycopeptide immunogen displaying one or two copies of the GM2 tetrasaccharidic moiety. These glycopeptides were prepared using the Huisgen cycloaddition, which enables the efficient ligation of the alkyne-functionalized biosynthesized GM2 with an azido CD4(+) T cell epitope peptide. It is worth noting that the GM2 can be produced on a gram scale in bacteria, which can be advantageous for a scale-up of the process. We show here for the first time that a fully synthetic glycopeptide, which is based on a ganglioside carbohydrate moiety, can induce human tumor cell-specific antibodies after immunization in mice. Interestingly, the monovalent, but not the divalent, form of GM2 peptide construct induced antimelanoma antibodies. Unlike traditional vaccines, this vaccine is a pure chemically-defined entity, a key quality for consistent studies and safe clinical evaluation. Therefore, such carbohydrate-peptide conjugate represents a promising cancer vaccine strategy for active immunotherapy targeting gangliosides.

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.

Genetic variation and phylogeography of free-living mouse species (genus Mus) in the Balkans and the Middle East.

This work presents a study of the distribution and pattern of variation throughout the ranges of three free-living mouse species of the genus Mus-M. macedonicus, M. spicilegus, and a M. cypriacus - based on sequencing of two segments of the mitochondrial DNA (mtDNA) control region. The study shows a similar level of variability in the three species and suggests their recent population expansion. The highest proportion of variation is found within populations indicating low genetic structuring. Phylogenetic analysis confirms the significant divergence of a mitochondrial lineage of M. macedonicus from Israel, recently described as a new subspecies, M. macedonicus spretoides. Conversely, no genetic hiatus is revealed between European and Asian populations of M. macedonicus macedonicus. Although phylogenetic relationships among M. spicilegus populations could not be unravelled precisely, the results suggest a recent westward expansion of the species. The mtDNA divergence between M. macedonicus and M. spicilegus is 7.3%, suggesting their split between c. 700,000 and 1 million years ago. These dates correspond with a coalescent estimate about 720,000 years ago. On the other hand, M. cypriacus appeared almost twice as divergent from the former species (4.5%) as from the latter (8.8%) suggesting a divergence of c. 430,000-610,000 years ago (coalescent approximately 490,000 years ago) and 830,000-1.2 million years ago (coalescent approximately 780,000 years ago), respectively. Approximate times of population expansion have also been estimated for all taxa and groups of populations. Existence of several glacial refuges and various colonization scenarios are discussed; since all estimated divergence times fall within interglacial periods it seems that climatic oscillations did not play a crucial role in the evolution of the three species.

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.

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.

Inferring gene flow in coral reef fishes from different molecular markers: which loci to trust?

Contrasting results are usually reported in the literature regarding the factors influencing observed structuring of genetic variability. The goals of this study were, for five coral reef fishes in French Polynesia, (1) to infer the theoretical variance of single locus F(ST) estimates expected under neutrality in order to exclude outlier loci before inferring gene flow and (2) to test thereafter whether species laying pelagic eggs effectively disperse more than species laying benthic eggs in this system. For this purpose, a total of 952 individuals from five species belonging to two families (Chaetodontidae and Pomacentridae) were screened among populations sampled within a 60-600 km spatial range for intron length polymorphism at 11 loci in order to illuminate contrasting results previously published on allozymes and mitochondrial DNA (mtDNA) control region polymorphisms. Statistically speaking, among the five species, four loci (three allozymes and one intron) were identified as outliers and discarded before interpretation of genetic differentiation in terms of effective dispersal. Biologically speaking, our results suggest that the observed genetic structure is not significantly related to the reproductive strategy of coral reef fish in the island system we analysed and that observed random genetic differentiation accommodates Wright's island model in all five species surveyed. Overall, our study emphasizes how cautious one has to be when trying to interpret present-day genetic structure in terms of gene flow while using a limited number of loci and/or different sets of loci.

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.

A change of expression in the conserved signaling gene MKK7 is associated with a selective sweep in the western house mouse Mus musculus domesticus.

Changes in gene expression are known to occur between closely related species, but it is not yet clear how many of these are due to random fixation of allelic variants or due to adaptive events. In a microarray survey between subspecies of the Mus musculus complex, we identified the mitogen-activated protein-kinase-kinase MKK7 as a candidate for change in gene expression. Quantitative PCR experiments with multiple individuals from each subspecies confirmed a specific and significant up-regulation in the testis of M. m. domesticus. Northern blot analysis shows that this is due to a new transcript that is not found in other tissues, nor in M. m. musculus. A cis-trans test via allele specific expression analysis of the MKK7 gene in F1 hybrids between domesticus and musculus shows that the expression change is mainly caused by a mutation located in cis. Nucleotide diversity was found to be significantly reduced in a window of at least 20 kb around the MKK7 locus in domesticus, indicative of a selective sweep. Because the MKK7 gene is involved in modulating a kinase signalling cascade in a stress response pathway, it seems a plausible target for adaptive differences between subspecies, although the functional role of the new testis-specific transcripts will need to be further studied.