The role of the vascular endothelium in arenavirus haemorrhagic fevers.

Viral haemorrhagic fevers (VHF) caused by arenaviruses are among the most devastating emerging human diseases. The most important pathogen among the arenaviruses is Lassa virus (LASV), the causative agent of Lassa fever that is endemic to West Africa. On the South American continent, the New World arenavirus Junin virus (JUNV), Machupo (MACV), Guanarito (GTOV), and Sabia virus (SABV) have emerged as causative agents of severe VHFs. Clinical and experimental studies on arenavirus VHF have revealed a crucial role of the endothelium in their pathogenesis. However, in contrast to other VHFs, haemorrhages are not a salient feature of Lassa fever and fatal cases do not show overt destruction of vascular tissue. The functional alteration of the vascular endothelium that precede shock and death in fatal Lassa fever may be due to more subtle direct or indirect effects of the virus on endothelial cells. Haemorrhagic disease manifestations and vascular involvement are more pronounced in the VHF caused by the South American haemorrhagic fever viruses. Recent studies on JUNV revealed perturbation of specific endothelial cell function, including expression of cell adhesion molecules, coagulation factors, and vasoactive mediators as a consequence of productive viral infection. These studies provided first possible links to some of the vascular abnormalities observed in patients, however, their relevance in vivo remains to be investigated.

Fine-scale genetic structure and marginal processes in an expanding population of Biscutella laevigata L. (Brassicaceae).

Evolutionary processes acting at the expanding margins of a species' range are still poorly understood. Genetic drift is considered prevalent in marginal populations, and the maintenance of genetic diversity during recolonization might seem puzzling. To investigate such processes, a fine-scale investigation of 219 individuals was performed within a population of Biscutella laevigata (Brassicaceae), located at the leading edge of its range. The survey used amplified fragment length polymorphisms (AFLPs). As commonly reported across the whole species distribution range, individual density and genetic diversity decreased along the local axis of recolonization of this expanding population, highlighting the enduring effect of the historical colonization on present-day diversity. The self-incompatibility system of the plant may have prevented local inbreeding in newly found patches and sustained genetic diversity by ensuring gene flow from established populations. Within the more continuously populated region, spatial analysis of genetic structure revealed restricted gene flow among individuals. The distribution of genotypes formed a mosaic of relatively homogenous patches within the continuous population. This pattern could be explained by a history of expansion by long-distance dispersal followed by fine-scale diffusion (that is, a stratified dispersal combination). The secondary contact among expanding patches apparently led to admixture among differentiated genotypes where they met (that is, a reshuffling effect). This type of dynamics could explain the maintenance of genetic diversity during recolonization.

Mls: a link between immunology and retrovirology.

The nature of the mysterious minor lymphocyte stimulating (Mls) antigens has recently been clarified. These molecules which were key elements for our current understanding of immune tolerance, have a strong influence on the mouse immune system and are encoded by the open reading frame (orf) of endogenous and exogenous mouse mammary tumor viruses (MMTV's). The knowledge that these antigens are encoded by cancerogenic retroviruses opens an interdisciplinary approach for understanding the mechanisms of immune responses and immune tolerance, retroviral carcinogenesis, and retroviral strategies for infection.

Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

Can we continue to neglect genomic variation in introgression rates when inferring the history of speciation? A case study in a Mytilus hybrid zone.

The use of molecular data to reconstruct the history of divergence and gene flow between populations of closely related taxa represents a challenging problem. It has been proposed that the long-standing debate about the geography of speciation can be resolved by comparing the likelihoods of a model of isolation with migration and a model of secondary contact. However, data are commonly only fit to a model of isolation with migration and rarely tested against the secondary contact alternative. Furthermore, most demographic inference methods have neglected variation in introgression rates and assume that the gene flow parameter (Nm) is similar among loci. Here, we show that neglecting this source of variation can give misleading results. We analysed DNA sequences sampled from populations of the marine mussels, Mytilus edulis and M. galloprovincialis, across a well-studied mosaic hybrid zone in Europe and evaluated various scenarios of speciation, with or without variation in introgression rates, using an Approximate Bayesian Computation (ABC) approach. Models with heterogeneous gene flow across loci always outperformed models assuming equal migration rates irrespective of the history of gene flow being considered. By incorporating this heterogeneity, the best-supported scenario was a long period of allopatric isolation during the first three-quarters of the time since divergence followed by secondary contact and introgression during the last quarter. By contrast, constraining migration to be homogeneous failed to discriminate among any of the different models of gene flow tested. Our simulations thus provide statistical support for the secondary contact scenario in the European Mytilus hybrid zone that the standard coalescent approach failed to confirm. Our results demonstrate that genomic variation in introgression rates can have profound impacts on the biological conclusions drawn from inference methods and needs to be incorporated in future studies.

Combining MAR elements and transposable vectors for more reliable transgene integration and expression

Integration without cytotoxic effects and long-term expression of a transgene constitutes a major challenge in gene therapy and biotechnology applications. In this context, transposons represent an attractive system for gene transfer because of their ability to promote efficient integration of a transgene in a variety of cell lines. However, the transgene integration can lead to insertional mutagenesis and/or unstable transgene expression by epigenetic modifications. These unwanted events may be limited by the use of chromatin control elements called MARs (matrix attachment regions). Indeed, the insertion of these DNA elements next to the transgene usually results in higher and more stable expression by maintaining transgene chromatin in an active configuration and preventing gene silencing. In this study, we tested if the inclusion of the MAR 1-68 in the piggyBac transposon system may lead to efficient and safer transgene integration and ensure reliable stable and long-term expression of a transgene. The MAR-containing transposon construct was tested in CHO cells, for biotechnology applications, and in mesoangioblast cells that can differentiate into muscle cells and are important candidates for potential stem cell therapies of myopathies. We showed that the addition of the MAR 1 -68 in the piggyBac transposon did not interfere with transposition, thereby maintaining high frequency of transgene integrations in these cells. Moreover, the MAR allowed higher transgene expression from fewer transposon integration events. We also found that enriched transgene-expressing cell populations could be obtained without the need of selection pressure. Since antibiotic-enforced selection protocols often result in a higher integrated copy number and mosaic expression patterns, this strategy could benefit many applications in which a low copy number of integrated transgenes and antibiotic-free conditions are desired. In addition, the intramuscular transplantation of mouse tibialis anterior muscles with mesoangioblasts containing the transposon led to widespread and sustained myofiber transgene expression after differentiation of these cells in vivo. These findings indicated that piggyBac vectors may provide a viable approach to achieve stable gene transfer in the context of Duchenne muscular dystrophy therapy. - L'intégration sans effets cytotoxiques et l'expression à long terme d'un transgène constituent un défi majeur en thérapie génique et en biotechnologie. Dans ce contexte, les transposons représentent un système attrayant pour le transfert de gènes en raison de leur capacité à promouvoir l'intégration efficace d'un transgène dans une variété de lignées cellulaires. Toutefois, l'intégration d'un transgène peut conduire à une mutagénèse insertionnelle et/ou à une expression instable due au silençage du transgène suite à des modifications épigénétiques. Ces événements indésirables de silençage génique peuvent être diminués par l'utilisation d'éléments de contrôle de la chromatine appelés MAR (matrix attachment region). En effet, l'insertion de ces éléments d'ADN à proximité du transgène se traduit généralement par une expression plus élevée et plus stable de celui-ci, en permettant le maintien d'une chromatine dans une configuration active autour du transgène et en empêchant l'inactivation du gène. Dans cette étude, nous avons testé si l'inclusion du MAR 1-68 dans le système transposon piggyBac peut améliorer l'efficacité d'intégration de façon sécuritaire et l'expression à long terme d'un transgène. Le transposon contenant l'élément MAR a été testé dans les cellules CHO, couramment utilisées en biotechnologie, et dans des cellules progénitrices appelées mésoangioblastes, qui peuvent se différencier en cellules musculaires, et qui constituent ainsi des candidats prometteurs pour la thérapie à partir de cellules souches de patients souffrant de myopathie. Nous avons montré que l'addition du MAR 1-68 dans le transposon piggyBac n'interfère pas avec la transposition et permet de maintenir une fréquence élevée d'intégration du transgène dans ces deux types cellulaires. De plus, il semble que cette association mène à une meilleure expression du transgène à partir de peu d'événements d'intégration du transposon. En outre, ces populations enrichies en cellules exprimant de façon stable le transgène ont pu être obtenues sans avoir recours à une pression de sélection. Etant donné que les protocoles de sélection basée sur l'utilisation d'antibiotiques conduisent souvent à un nombre plus élevé de copies intégrées et à la variégation de l'expression du transgène et qu'ils impliquent une longue culture in vitro, cette stratégie pourrait profiter à des applications pour lesquelles on souhaite un faible nombre de copies intégrées et/ou l'utilisation d'antibiotiques n'est pas souhaitable. De plus, la transplantation intramusculaire de mésoangioblastes contenant le transposon dans le muscle tibial antérieur de souris a conduit, après la différentiation de ces cellules in vivo, à une expression constante et étendue du transgène dans les myofibres. Ces résultats indiquent que les vecteurs piggyBac pourraient fournir une approche viable pour assurer un transfert de gènes stables dans le contexte d'un traitement de la dystrophic musculaire de Duchenne.