Discovery of parvovirus-related sequences in an unexpected broad range of animals.

Our knowledge of the genetic diversity and host ranges of viruses is fragmentary. This is particularly true for the Parvoviridae family. Genetic diversity studies of single stranded DNA viruses within this family have been largely focused on arthropod- and vertebrate-infecting species that cause diseases of humans and our domesticated animals: a focus that has biased our perception of parvovirus diversity. While metagenomics approaches could help rectify this bias, so too could transcriptomics studies. Large amounts of transcriptomic data are available for a diverse array of animal species and whenever this data has inadvertently been gathered from virus-infected individuals, it could contain detectable viral transcripts. We therefore performed a systematic search for parvovirus-related sequences (PRSs) within publicly available transcript, genome and protein databases and eleven new transcriptome datasets. This revealed 463 PRSs in the transcript databases of 118 animals. At least 41 of these PRSs are likely integrated within animal genomes in that they were also found within genomic sequence databases. Besides illuminating the ubiquity of parvoviruses, the number of parvoviral sequences discovered within public databases revealed numerous previously unknown parvovirus-host combinations; particularly in invertebrates. Our findings suggest that the host-ranges of extant parvoviruses might span the entire animal kingdom.

Comparative population genomics in animals uncovers the determinants of genetic diversity.

Genetic diversity is the amount of variation observed between DNA sequences from distinct individuals of a given species. This pivotal concept of population genetics has implications for species health, domestication, management and conservation. Levels of genetic diversity seem to vary greatly in natural populations and species, but the determinants of this variation, and particularly the relative influences of species biology and ecology versus population history, are still largely mysterious. Here we show that the diversity of a species is predictable, and is determined in the first place by its ecological strategy. We investigated the genome-wide diversity of 76 non-model animal species by sequencing the transcriptome of two to ten individuals in each species. The distribution of genetic diversity between species revealed no detectable influence of geographic range or invasive status but was accurately predicted by key species traits related to parental investment: long-lived or low-fecundity species with brooding ability were genetically less diverse than short-lived or highly fecund ones. Our analysis demonstrates the influence of long-term life-history strategies on species response to short-term environmental perturbations, a result with immediate implications for conservation policies.

Development of SNP-genotyping arrays in two shellfish species.

Use of SNPs has been favoured due to their abundance in plant and animal genomes, accompanied by the falling cost and rising throughput capacity for detection and genotyping. Here, we present in vitro (obtained from targeted sequencing) and in silico discovery of SNPs, and the design of medium-throughput genotyping arrays for two oyster species, the Pacific oyster, Crassostrea gigas, and European flat oyster, Ostrea edulis. Two sets of 384 SNP markers were designed for two Illumina GoldenGate arrays and genotyped on more than 1000 samples for each species. In each case, oyster samples were obtained from wild and selected populations and from three-generation families segregating for traits of interest in aquaculture. The rate of successfully genotyped polymorphic SNPs was about 60% for each species. Effects of SNP origin and quality on genotyping success (Illumina functionality Score) were analysed and compared with other model and nonmodel species. Furthermore, a simulation was made based on a subset of the C. gigas SNP array with a minor allele frequency of 0.3 and typical crosses used in shellfish hatcheries. This simulation indicated that at least 150 markers were needed to perform an accurate parental assignment. Such panels might provide valuable tools to improve our understanding of the connectivity between wild (and selected) populations and could contribute to future selective breeding programmes.

Population genomics of eusocial insects: the costs of a vertebrate-like effective population size.

The evolution of reproductive division of labour and social life in social insects has lead to the emergence of several life-history traits and adaptations typical of larger organisms: social insect colonies can reach masses of several kilograms, they start reproducing only when they are several years old, and can live for decades. These features and the monopolization of reproduction by only one or few individuals in a colony should affect molecular evolution by reducing the effective population size. We tested this prediction by analysing genome-wide patterns of coding sequence polymorphism and divergence in eusocial vs. noneusocial insects based on newly generated RNA-seq data. We report very low amounts of genetic polymorphism and an elevated ratio of nonsynonymous to synonymous changes ­ a marker of the effective population size ­ in four distinct species of eusocial insects, which were more similar to vertebrates than to solitary insects regarding molecular evolutionary processes. Moreover, the ratio of nonsynonymous to synonymous substitutions was positively correlated with the level of social complexity across ant species. These results are fully consistent with the hypothesis of a reduced effective population size and an increased genetic load in eusocial insects, indicating that the evolution of social life has important consequences at both the genomic and population levels.

Reference-free transcriptome assembly in non-model animals from next-generation sequencing data.

Next-generation sequencing (NGS) technologies offer the opportunity for population genomic study of non-model organisms sampled in the wild. The transcriptome is a convenient and popular target for such purposes. However, designing genetic markers from NGS transcriptome data requires assembling gene-coding sequences out of short reads. This is a complex task owing to gene duplications, genetic polymorphism, alternative splicing and transcription noise. Typical assembling programmes return thousands of predicted contigs, whose connection to the species true gene content is unclear, and from which SNP definition is uneasy. Here, the transcriptomes of five diverse non-model animal species (hare, turtle, ant, oyster and tunicate) were assembled from newly generated 454 and Illumina sequence reads. In two species for which a reference genome is available, a new procedure was introduced to annotate each predicted contig as either a full-length cDNA, fragment, chimera, allele, paralogue, genomic sequence or other, based on the number of, and overlap between, blast hits to the appropriate reference. Analyses showed that (i) the highest quality assemblies are obtained when 454 and Illumina data are combined, (ii) typical de novo assemblies include a majority of irrelevant cDNA predictions and (iii) assemblies can be appropriately cleaned by filtering contigs based on length and coverage. We conclude that robust, reference-free assembly of thousands of genes from transcriptomic NGS data is possible, opening promising perspectives for transcriptome-based population genomics in animals. A Galaxy pipeline implementing our best-performing assembling strategy is provided.

The evolutionary fate of recently duplicated retrogenes in mice.

Inferences about the evolutionary impact of gene duplications often rely on the analysis of their long-term outcome. The fate of the majority of them must, however, be decided shortly after duplication. Here we analysed the evolutionary pattern of 10 mouse genes very recently duplicated by retrotransposition, by sequencing the retroposed copy in five to 10 closely related mouse species. In all cases the retroposed copy experienced accelerated nonsynonymous evolution whereas the divergence pattern of the source copy appeared unaffected by the duplication, consistent with the neofunctionalization model. The analysis further revealed that most retrogenes, including pseudogenes, did not experience a period of relaxed neutral evolution, but have been submitted to purifying selection ever since their retroposition. We propose that these duplicates play a biochemical role but are not indispensable. Purifying selection prevents them from acquiring a negative role until they are lost or silenced. This period of unnecessary redundancy could in rare cases give the time for new functions to evolve.

[Fluctuations of serotonin and its metabolites in the lung and intestinal mucosa in the rat infected with Nippostrongylus brasiliensis]. / Fluctuations de la sérotonine et de ses métabolites au niveau du poumon et du mucus intestinal chez le rat infesté par Nippostrongylus brasiliensis.

The influence of parasitism on host biogenic amine levels was investigated in Nippostrongylus brasiliensis infected rats. Amine levels were estimated in tissues surrounding Nematods in their biological environment: the lung and intestinal mucus. D0 being the day of infestation, tissues were obtained at 24, 30 and 45 hrs, and every day between D4 and D14 (when the rat was completely deparasited by the self-cure phenomenon). Biogenic amines belonging to the serotoninergic pathway were quantified by HPLC with electrochemical detection. In the lungs and mucus, parasitism resulted in an important decrease in serotonine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels, as opposed to the immediate 5-HT precursor, the 5-hydroxy-tryptophane (5-HTP). Host response to parasitism is translated by serotoninergic pathway levels. This leads to two hypotheses: 5-HT turn-over may be accelerated, but the inhibition of 5-HT synthetic enzyme, 5-hydroxytryptophane hydroxylase, by the parasite present in the host seems more probable.

Epoxyethane-/ethynesulfonamides with antifilarial activities. Degradation kinetics and inhibitory effect on filarial malate dehydrogenase and lactate dehydrogenase.

Some epoxyethane-/ethynesulfonamides had shown strong filaricidal activity with inconstant reproducibility as a result of a lack of stability in aqueous solution. The degradation in hydroxylic and aprotic solutions of two epoxyethanesulfonamides and one ethynesulfonamide was investigated using TLC, HPLC, GC and mass spectrometry. For both epoxydes, the degradation rate followed first-order kinetics and was more rapid in hydroxylic than in aprotic solutions. The degradation increased with the temperature whereas it was not modified with and without light exposure. Four kinds of degradation products were found: the first one involved the oxidation of the epoxyde bond, the second the breaking of the N-S bond, the third a desulfonation product and the fourth was not identified. In contrast, the stability of ethynesulfonamide was better than those of epoxyethanesulfonamide. These results suggest that epoxyethanesulfonamides should be kept at +4 degrees C before being injected to animals during the study of biological activity. Since epoxyde compounds are known to have inhibitory effects on parasite energy metabolism enzymes, the compunds were evaluated on two major filarial enzymes: lactate dehydrogenase (LDH) and cytoplasmic malate dehydrogenase (MDH). Both epoxyethanesulfonamides showed only a slight inhibitory effect on filarial LDH and MDH confirming the evidence that the main mode of action of these compounds remains to discover. Moreover, ethynesulfonamide and the degradation products of both epoxyethane-sulfonamides had no effect on LDH and MDH.

Levels of biogenic amines in larvae and adults of the rat hookworm, Nippostrongylus brasiliensis (Nematoda).

Norepinephrine, 5-hydroxytryptophan, octopamine, dihydroxyphenylacetic acid, N-acetyldopamine, dopamine, 5-hydroxyindoleacetic acid, N-acetylserotonin, tyramine, tryptophan and serotonin in larvae (third free stage and parasitic stages) and adult males and females (at defined ages during the intestinal phase) of the parasitic nematode Nippostrongylus brasiliensis were quantified simultaneously by high-performance liquid chromatography with electrochemical detection. Biogenic amine levels depended on the stage, the age and the sex of parasites and on environmental conditions. Their physiological roles in reproductively competent adults of this nematode are discussed in relation to exuviation and egg laying. Parallel fluctuations in free ecdysteroids and norepinephrine were observed in females from the same worm populations.

Tissue distribution and metabolism in rat of an ethynesulphonamide with filaricidal activity.

1. The tissue distribution and metabolism of a new filaricidal agent P903 (N-[(2-phenylethynyl)sulfonyl]morpholine) were studied in rat. 2. After s.c. administration of 14C and 13C P903, the Tmax in the blood was observed on day 2. Elimination was slow and > 95% was bound to protein. Radioactivity was distributed in the whole organism but particularly in erythrocytes and the lymphatic channel. Four days later, > 60% of the radioactivity was excreted in urine and faeces at equal amounts and 15% remained at the injection point. 3. In all biological fluids tested no P903 was found but only its metabolites. 4. One principal metabolite, the N-[(2-phenyloxo-2-ethane) sulphonyl] morpholine or oxosulphonamide was identified in blood, urine and faeces as compared with the reference compound by GC/MS and NMR. This latter molecule was detected following hydrolysis by hydrochloric acid but not with beta glucuronidase/sulphatase. 5. Unconjugated and conjugated oxosulphonamide represented > 85% of the radioactivity at all times tested in blood but only 38 and 35% respectively of urinary and faecal radioactivity on day 1 after the administration of the labelled drug. 6. Thus, P903 is rapidly converted to a reactive metabolite, probably an oxirene, which is then conjugated with endogenous components to form conjugated oxosulphonamide and an unknown metabolite. The role of this reactive metabolite in antifilarial activity seems to be very important in understanding the mechanism of action of P903.

[Experimental studies of filaricides]. / Etudes expérimentales de filaricides.

Several chemical compounds have shown filarical activities, which are presented and compared in this presentation: diethylcarbamazin, ivermectin, benzimidazoles and original compounds as epoxy and ethynesulphonamide, or carboxamides. Their metabolism and tissue distribution are depending of the host, and whether hosts are parasitized or not. Reference compounds have filaricidal activities depending of the stage of Filaria, and of the experimental model. The necessity of a good model is essential to extrapolate the results. Biochemical targets which can be identified from these animals models may be inappropriate for use in human filariasis; that is why fundamental research is still necessary in this area.

[Leponex: experience of the hospital pharmacist]. / Leponex: l'expérience du pharmacien hospitalier.

Clozapine's monitoring, with a co-responsibility between psychiatrists and pharmacists, is very efficient for the prevention of neutropenia's side effects. This intensive drug safety has lowered the cases of agranulocytosis in France to a 0.5% prevalence. However the cost of clozapine led to a strict estimation for health expenditures. Our study, performed in a university department of psychiatry in Sainte-Anne Hospital (Paris), has included 14 patients treated with clozapine for at least 12 months and has displayed a decrease of 10% in their annual global cost, comparing with the same group of patients treated by classical neuroleptics during the preceding year. This global cost includes the treatment, the blood monitoring and the cost of different hospital or community cares. Quality of life, in the clozapine group, was much improved, as illustrated by the shortened full time hospitalization which was followed at an earlier stage by community care and earlier social readaptation.

Presence of norepinephrine and other biogenic amines in stonefish venom.

Although fish venoms exert a cardiovascular effect, the presence of adrenergic substances was not previously demonstrated. Chromatographic analysis with electrochemical detection showed the presence of substances co-migrating with norepinephrine, dopamine and tryptophan. Serotonin, which was thought to be implicated in the intense pain following fish envenomation, was not detected. Norepinephrine was identified as a component of the stonefish Synanceia verrucosa venom by gas chromatography-mass spectrometry.

In vitro and in vivo antifilarial activity of ethynesulfonamides, epoxyethanesulfonamides and carboxamide analogues.

A series of 2-substituted trans-1,2-epoxyethanesulfonamides (1T), ethynesulfonamides (2), and their carboxamide analogues 3 (cis and trans) and 4 were evaluated for their antifilarial activity, first in vitro against the infective larvae of the filaria Molinema dessetae, then in vivo against the same filaria in Proechimys oris, its natural host. On the whole, compounds 2 displayed a high level of activity in vitro, while 4 showed a wider range and 3 were virtually inactive. The modest activity found within the series 1T was assumed to be due, in part, to the instability of the products under the conditions of the biological tests. Five new compounds within the series 1T, 2 and 4 showed a macrofilaricidal activity in vivo. There is no clear correlation between in vivo and in vitro data. It was observed, however, that within the series 2 and 4 all the compounds active in vivo were among the most potent compounds in vitro. Nevertheless, the in vitro model, although of limited value, could help in selecting compounds for further evaluation within a given series.

[Leponex: experience of the hospital pharmacist]. / Leponex: l'expérience du pharmacien hospitalier.

Clozapine's monitoring, with a co-responsibility between psychiatrists and pharmacists, was very efficient for the prevention of neutropenia's side effects. This intensive drug safety has lowered the agranulocytosis' cases in France to a 0.5% prevalence. However the cost of clozapine led to a strict estimation for Health expenditures. Our study, trained in an university department of psychiatry in Sainte-Anne Hospital (Paris), has included 14 patients treated with clozapine during at least 12 months and has displayed a decrease of 10% in their annual global cost, comparing to the same group of patients treated by classical neuroleptics during the preceding year. This global cost includes the treatment, the blood monitoring and the cost of different hospital or community cares. Quality of life, in clozapine group, was much improved as illustrated by lowing full time hospitalization relayed earlier by community care and precocious social readaptation.

Synthesis and orally macrofilaricidal evaluation of niclosamide lymphotropic prodrugs.

The development of new macrofilaricidal drugs is described following a strategy for the promotion of the lymphatic transport of anthelminthic drugs by-passing the liver. The selected compound was niclosamide (CAS 50-65-7) which is very effective in vitro against infective larvae but has no significant antifilarial activity when orally administered at 200 mumol/kg. To estimate the interest of such an approach, the synthesis of 5 prodrugs was achieved in a first stage. The intrinsic antifilarial activity and the delayed effect of these compounds were evaluated in vitro. Then, in vivo tests were performed with Molinema dessetae infective larvae to select the best ligands. The prodrug V 1,3-dihexadecanamido-2-[4-chloro(2- chloro-4-nitroanilinocarbonyl)phenyloxy-carbonylpropanoyl oxy]propane (having a diamide function) was responsible for an in vitro delayed effect and an orally in vivo activity (200 mumol/kg when administered in a single dose). The biological improvement of this easily micellizable prodrug which is stable to intestinal enzymes in respect to Niclosamide confirms such a strategy.