Influence of untranslated regions on retroviral mRNA transfer and expression.

International audienceBACKGROUND: Deliberate cellular reprogramming is becoming a realistic objective in the clinic. While the origin of the target cells is critical, delivery of bioactive molecules to trigger a shift in cell-fate remains the major hurdle. To date, several strategies based either on non-integrative vectors, protein transfer or mRNA delivery have been investigated. In a recent study, a unique modification in the retroviral genome was shown to enable RNA transfer and its expression. RESULTS: Here, we used the retroviral mRNA delivery approach to study the impact of modifying gene-flanking sequences on RNA transfer. We designed modified mRNAs for retroviral packaging and used the quantitative luciferase assay to compare mRNA expression following viral transduction of cells. Cloning the untranslated regions of the vimentin or non-muscular myosin heavy chain within transcripts improved expression and stability of the reporter gene while slightly modifying reporter-RNA retroviral delivery. We also observed that while the modified retroviral platform was the most effective for retroviral mRNA packaging, the highest expression in target cells was achieved by the addition of a non-viral UTR to mRNAs containing the packaging signal. CONCLUSIONS: Through molecular engineering we have assayed a series of constructs to improve retroviral mRNA transfer. We showed that an authentic RNA retroviral genomic platform was most efficiently transferred but that adding UTR sequences from highly expressed genes could improve expression upon transfection while having only a slight effect on expression from transferred RNA. Together, these data should contribute to the optimisation of retroviral mRNA-delivery systems that test combinations of UTRs and packaging platforms

La fin d'une vicinia. Olbia à Hyères (Var) durant l'Antiquité tardive et le haut Moyen Âge (Ve-Xe s.)

International audienceL'éviction d'Olbia de l'énumération des étapes mentionnées par l’Itinéraire Maritime d’Antonin entre Marseille et Fréjus tend à confirmer que, durant la seconde moitié du IIe s., l'ancienne colonie massaliète ne jouait sans doute qu'un rôle mineur sur la carte du trafic marchand vers l'Italie. L'abandon dès le siècle précédent de certains quartiers (îlots ouest et nord-est) et la désaffectation de plusieurs entrepôts (îlots 3 et 6) incitent à placer plus haut dans le temps l'amorce de ce déclin. Aussi, l’essor non loin de là et à la même époque des ports de Telo Martius (Février, Boyer 1980, 20 ; Bérato et al. 1986, Brun 1999,777-778) et de Pomponiana (Porquerolles, Brun 1999, 483-484) s'inscrit peut-être dans un contexte de concurrence.L’importance acquise par Toulon dans les siècles qui suivent est illustrée, selon la Notitia Dignitatum, par la présence au tout début du Ve s. d'une teinturerie impériale de pourpre mais surtout, une décennie plus tard par la création de l'évêché (Février 1955, 8-9 ; Brun 1999, 778-779). Les limites du diocèse nouvellement créé absorbent alors Olbia et sa proche région. Cet événement est-il à l'origine, dans les années suivantes, de l'installation sur les îles d'Hyères des communautés de moines et d'ermites évoquées par Jean Cassien et, plus tardivement, par Ennode de Pavie ? (Palanque 1969, 85 ; Brun 1999, 485-486 ; Tréglia à paraître 2003). La faiblesse de la documentation concernant cet événement ne permet guère d'aller plus loin. Elle autorise tout au plus la formulation de quelques questions. Quels furent les effets de l'accession de Toulon au rang d'évêché sur la couronne proche des petites agglomérations ? Aggrava-t-elle, par un pouvoir d'attraction décuplé, l'hémorragie démographique qui frappait les campagnes depuis la fin du IIIe s. ou, soumise à la nécessité de conserver et d'administrer l'ancien réseau des vici, contribua-t-elle à maintenir en vie, quelques temps encore, des agglomérations depuis longtemps moribondes ? Jean-Pierre Brun note pour sa part que, durant le Ve s., un grand nombre agglomérations varoises paraît connaître une rétraction de leur surface habitée (Brun 1999, 176-178), phénomène qui s'accompagne le plus souvent d'une restructuration de l'habitat lui-même. L'abandon de certains de ces vici n'est toutefois vraiment constaté qu'à partir de la fin du Ve s. ou au début du siècle suivant (Clastre/Le Logis, Matavo, Brun 1999, 177).S'il est sans doute encore trop tôt pour inclure assurément Olbia au nombre des agglomérations qui obéissent à ce schéma d'évolution, les dernières campagnes de fouilles mettent toutefois en évidence une rupture franche de l'organisation de l'habitat dans le courant du Ve s. A un ensemble d'habitations groupées dans le quart sud-est du site succèdent semble-t-il très rapidement de vastes espaces ouverts, jardins ou terrains vagues, à vocation artisanale

Highly focused anopheline breeding sites and malaria transmission in Dakar

<p>Abstract</p> <p>Background</p> <p>Urbanization has a great impact on the composition of the vector system and malaria transmission dynamics. In Dakar, some malaria cases are autochthonous but parasite rates and incidences of clinical malaria attacks have been recorded at low levels. Ecological heterogeneity of malaria transmission was investigated in Dakar, in order to characterize the <it>Anopheles </it>breeding sites in the city and to study the dynamics of larval density and adult aggressiveness in ten characteristically different urban areas.</p> <p>Methods</p> <p>Ten study areas were sampled in Dakar and Pikine. Mosquitoes were collected by human landing collection during four nights in each area (120 person-nights). The <it>Plasmodium falciparum </it>circumsporozoite (CSP) index was measured by ELISA and the entomological inoculation rates (EIR) were calculated. Open water collections in the study areas were monitored weekly for physico-chemical characterization and the presence of anopheline larvae. Adult mosquitoes and hatched larvae were identified morphologically and by molecular methods.</p> <p>Results</p> <p>In September-October 2007, 19,451 adult mosquitoes were caught among which, 1,101 were <it>Anopheles gambiae s.l</it>. The Human Biting Rate ranged from 0.1 bites per person per night in Yoff Village to 43.7 in Almadies. Seven out of 1,101 <it>An. gambiae s.l</it>. were found to be positive for <it>P. falciparum </it>(CSP index = 0.64%). EIR ranged from 0 infected bites per person per year in Yoff Village to 16.8 in Almadies. The <it>An</it>. <it>gambiae </it>complex population was composed of <it>Anopheles arabiensis </it>(94.8%) and <it>Anopheles melas </it>(5.2%). None of the <it>An. melas </it>were infected with <it>P. falciparum</it>. Of the 54 water collection sites monitored, 33 (61.1%) served as anopheline breeding sites on at least one observation. No <it>An</it>. <it>melas </it>was identified among the larval samples. Some physico-chemical characteristics of water bodies were associated with the presence/absence of anopheline larvae and with larval density. A very close parallel between larval and adult densities was found in six of the ten study areas.</p> <p>Conclusion</p> <p>The results provide evidence of malaria transmission in downtown Dakar and its surrounding suburbs. Spatial heterogeneity of human biting rates was very marked and malaria transmission was highly focal. In Dakar, mean figures for transmission would not provide a comprehensive picture of the entomological situation; risk evaluation should therefore be undertaken on a small scale.</p

Nitrate in the coral symbiosis: from the regulation of its assimilation to its impact on the physiology of the holobiont

In oligotrophic reef systems, coral holobionts are remarkably efficient at assimilating nitrogen through heterotrophic feeding or the uptake of dissolved inorganic nitrogen. Symbiodiniaceae are vital partners of the symbiosis for nutrient assimilation. In addition to providing translocated photosynthates, they account for most of the uptake of dissolved inorganic nitrogen. Although NO3- is the most abundant source of nitrogen in the ocean, little is known about the mechanisms regulating its assimilation by the holobiont. Coral hosts are unable to reduce nitrate as they lack the necessary enzymes, whereas Symbiodiniaceae have been shown to express the enzyme nitrate reductase (NR). However, the evidence supporting the active reduction of nitrate by the symbiotic algae during symbiosis is scarce and equivocal. This research aimed at deciphering the pathways of NO3- assimilation in both free-living Symbiodiniaceae and in hospite symbionts while also investigating the relevance of inorganic nitrogen source in physiological responses to stress. We investigated the expression and regulation of NR both in free-living Symbiodiniaceae and in in hospite symbionts using a combined western blot and qRT-PCR approach. We showed that the expression and regulation of NR in free-living Symbiodiniaceae is a dynamic and reversible process impacted by NO3- and NH4+ concentrations. Symbionts from N-depleted corals incubated with NO3- enriched seawater showed an increase in NR synthesis over time. Interestingly, NR protein synthesis did not correlate with NR gene expression, hinting towards a potential post-transcriptional regulation of the enzyme. Additionally, we investigated the impacts of inorganic N source (NO3- vs NH4+ vs N depletion) in combination with stress on the physiology of Symbiodiniaceae (photosynthetic responses, ROS and NO production). The availability of inorganic nitrogen improved photosynthetic capacity while reducing ROS production. Moreover, preliminary experiments showed that NO3- and NH4+ had differential effects on the physiological responses of Symbiodiniaceae subjected to stress

Malaria transmission in Dakar: A two-year survey

<p>Abstract</p> <p>Background</p> <p>According to entomological studies conducted over the past 30 years, there was low malaria transmission in suburb of Dakar but little evidence of it in the downtown area. However; there was some evidence of local transmission based on reports of malaria among permanent residents. An entomological evaluation of malaria transmission was conducted from May 2005 to October 2006 in two areas of Dakar.</p> <p>Methods</p> <p>Mosquitoes were sampled by human landing collection during 34 nights in seven places in Bel-air area (238 person-nights) and during 24 nights in five places in Ouakam area (120 person-nights). Mosquitoes were identified morphologically and by molecular methods. The <it>Plasmodium falciparum </it>circumsporozoïte indexes were measured by ELISA, and the entomological inoculation rates (EIR) were calculated for both areas. Molecular assessments of pyrethroid knock down resistance (<it>Kdr</it>) and of insensitive acetylcholinesterase resistance were conducted.</p> <p>Results</p> <p>From May 2005 to October 2006, 4,117 and 797 <it>Anopheles gambiae s.l</it>. respectively were caught in Bel-air and Ouakam. Three members of the complex were present: <it>Anopheles arabiensis </it>(> 98%), <it>Anopheles melas </it>(< 1%) and <it>An. gambiae s.s</it>. molecular form M (< 1%). Infected mosquitoes were caught only during the wintering period between September and November in both places. In 2005 and 2006, annual EIRs were 9,5 and 4, respectively, in Bel-air and 3 and 3, respectively, in Ouakam. The proportion of host-seeking <it>An. gambiae s.l</it>. captured indoors were 17% and 51% in Bel air and Ouakam, respectively. Ace 1 mutations were not identified in both members of the <it>An. gambiae </it>complex. <it>Kdr </it>mutation frequency in <it>An. arabiensis </it>was 12% in Bel-air and 9% in Ouakam.</p> <p>Conclusion</p> <p>Malaria is transmitted in Dakar downtown area. Infected mosquitoes were caught in two subsequent years during the wintering period in two distant quarters of Dakar. These data agree with clinical data from a Senegalese military Hospital of Dakar (Hospital Principal) where most malaria cases occurred between October and December. It was the first detection of <it>An. melas </it>in Dakar.</p

Dynamics of CO2, CH4 and N2O concentrations throughout deep soil profiles in Eucalypt plantations subjected to contrasted rainfall regimes: consequences on soil effluxes

The major factors driving greenhouse gas exchanges in forest soils (substrate supply, temperature, water content) vary with soil depth. Our study aimed to assess the effects of clear-cutting and drought on the temporal variability of CO2, CH4 and N2O fluxes throughout very deep soil profiles in Brazilian eucalypt plantations conducted in coppice. Stands with 37% of through fall excluded by plastic sheets (-W) and stands without rain exclusion (+W) were compared. Every two weeks for 21 months, CO2, CH4 and N2O surface effluxes were measured using the closed-chamber method and concentrations in the soil were measured at 7 depths down to 15.5 m in -W and +W. At most measurement dates, CO2, CH4 and N2O effluxes at the soil surface were not significantly different between -W and +W. Mean CO2 and N2O concentrations in -W were 20.7% and 7.6% lower than in +W, respectively, across the sampling depths. By contrast, CH4 concentrations in -W were 44.4% higher than in +W throughout the soil profile. Across the two treatments, CO2 concentrations increased from 4446 _ 2188 ppm at 10 cm deep to 15622 _ 3523 ppm at 15.5 m, CH4 concentrations increased from 0.41 _ 0.17 ppm at 10 cm deep to 0.77 _ 0.24 ppm at 15.5 m and N2O concentrations remained roughly constant and were on average 478 _ 55 ppb from the soil surface to 15.5 m deep. A modeling approach (using the Min3P and Root Typ models) showed that the amount of water filling soil porosity accounted for a large share of the difference in gas concentration between +W and {W, and pointed out the consequences of through fall exclusion on the areas of CO2 production throughout the soil profile. Improving our understanding of the spatiotemporal dynamics of gas concentrations in deep soil layers is important to improve the current biogeochemical models predicting the effect of drought periods on greenhouse gas effluxes in eucalypt plantations established in deep tropical soils

Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates

International audienceBACKGROUND: Beta-lactamase production and porin decrease are the well-recognized mechanisms of acquired beta-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and susceptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this beta-lactam phenotype was the aim of this study. METHODOLOGY/FINDINGS: MICs of 9 beta-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other beta-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also 16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other beta-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of beta-lactams. CONCLUSION: This is the first study demonstrating that efflux mechanism plays a key role in the beta-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in beta-lactam resistance is specially underestimated in clinical isolates

TMT DMs final design and advanced prototyping results at Cilas

In order to prepare for the construction phase of the two Deformable Mirrors (DMs), which will be used in the Thirty Meter Telescope (TMT) first light Adaptive Optics (AO) system, Cilas has advanced the design of these two large size piezo DMs and has manufactured and tested a scaled demonstration prototype. The work done allowed significant reduction of the risks related to the demanding specifications of the TMT DMs; the main issues were: (i) Large pupil (up to 370 mm) and high order (up to 74x74); (ii) Relatively low operational temperature (DMs working at -30°C); (iii) New piezo material. It is important to develop such a prototype to take into account these three specifications all together (dimension, low temperature and new piezo material). The new prototype is a 6x60 actuators and has the same characteristics as the future TMT DMs. In this paper, we give the conclusions of the work through the presentation of the following items: (i) Design and finite element analysis of the two DMs and prototype; (ii) Test results obtained with the prototype with validation of the finite element analysis and compliance with the TMT AO specifications; (iii) Special focus on thermal behavior, actuator reliability and shape at rest stability

Strigolactones Stimulate Arbuscular Mycorrhizal Fungi by Activating Mitochondria

The association of arbuscular mycorrhizal (AM) fungi with plant roots is the oldest and ecologically most important symbiotic relationship between higher plants and microorganisms, yet the mechanism by which these fungi detect the presence of a plant host is poorly understood. Previous studies have shown that roots secrete a branching factor (BF) that strongly stimulates branching of hyphae during germination of the spores of AM fungi. In the BF of Lotus, a strigolactone was found to be the active molecule. Strigolactones are known as germination stimulants of the parasitic plants Striga and Orobanche. In this paper, we show that the BF of a monocotyledonous plant, Sorghum, also contains a strigolactone. Strigolactones strongly and rapidly stimulated cell proliferation of the AM fungus Gigaspora rosea at concentrations as low as 10 (−13) M. This effect was not found with other sesquiterperne lactones known as germination stimulants of parasitic weeds. Within 1 h of treatment, the density of mitochondria in the fungal cells increased, and their shape and movement changed dramatically. Strigolactones stimulated spore germination of two other phylogenetically distant AM fungi, Glomus intraradices and Gl. claroideum. This was also associated with a rapid increase of mitochondrial density and respiration as shown with Gl. intraradices. We conclude that strigolactones are important rhizospheric plant signals involved in stimulating both the pre-symbiotic growth of AM fungi and the germination of parasitic plants