A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors

We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5′ end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.     

A Nonintegrative Lentiviral Vector-Based Vaccine Provides Long-Term Sterile Protection against Malaria

Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS) have shown that protective immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV) hold promise as mass pediatric vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP) and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5–62.5) of the animals were fully protected (p = 0.0072 and p = 0.0008 respectively when compared to naive mice). The remaining mice with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection was long-lasting with 42.8% sterile protection six months after the last immunization (p = 0.0042). Post-challenge CD8+ T cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r = −0.6615 and p = 0.0004 between the frequency of IFN-g secreting specific T cells in spleen and parasitemia). However, while NILV and RAS immunizations elicited comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine.     

Constitutive autophagy contributes to resistance to TP53-mediated apoptosis in Epstein-Barr virus-positive latency III B-cell lymphoproliferations

The Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and lymphomas. We have previously demonstrated that treating wild-type TP53-expressing B cell lines with the TP53 pathway activator nutlin-3 induced apoptosis in EBV-negative and EBV-positive latency I cells whereas EBV-positive latency III cells remained much more apoptosis-resistant. Here, we report a constitutively high level of autophagy in these resistant cells which express high levels of the proautophagic protein BECN1/Beclin 1 based, at least in part, on the activation of the NFKB signaling pathway by the viral protein LMP1. Following treatment with nutlin-3, several autophagy-stimulating genes were upregulated both in EBV-negative and EBV-positive latency III cells. However the process of autophagy was only triggered in the latter and was associated with an upregulation of SESN1/sestrin 1 and inhibition of MTOR more rapid than in EBV-negative cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy.     

Comparison of the lipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure

A new two-step gradient technique has been used in the separation of the different classes of lipoproteins from the serum of cows, horses, dogs, pigs, rabbits and rats. Total lipoproteins were first isolated at d 1.21 then floated through a d 1.006 to d 1.21 gradient. Collection by mean of a gradient fractionator provided directly comparable lipoprotein profiles, allowed the determination of the exact density range of each lipoprotein class and the fraction by fraction analysis of composition. Cholesterol and apo AI recoveries were high. Horse, dog, rabbit and pig exhibited three distinct lipoprotein classes: VLDL, LDL and HDL. LDL were polydisperse in the pig (three components), light in the rabbit and scarce in the horse. In the Sprague-Dawley rat, LDL could not be individualized from HDL. In the bovine, LDL overlapped with a light form of HDL. Although AI was the main apoprotein in the HDL of all species, it ranged in proportion from 35% in the rat to 75% in the bovine. Apo AII was dimeric in the dog, as already known, but also in the horse, rabbit and bovine (MW:17,000) and in the pig MW:13,000). Apo AIV was present in the heavier HDL of all species. Rabbit, horse and pig HDL contained only one species of apo C which, in the pig was identified as apo CII.     

Radiotherapy setup displacements in breast cancer patients: 3D surface imaging experience

Aim

In this study, we intend to compare two different setup procedures for female breast cancer patients.

Characterization of DCL4 missense alleles provides insights into its ability to process distinct classes of dsRNA substrates

In the model plant Arabidopsis thaliana, four Dicer‐like proteins (DCL1–4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII‐like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4‐dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4‐dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold‐back double‐stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR‐dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.     

Adjacent woodlands rather than habitat connectivity influence grassland plant,carabid and bird assemblages in farmland landscapes

One response to biodiversity decline is the definition of ecological networks that extend beyond protected areas and promote connectivity in human-dominated landscapes. In farmland, landscape ecological research has focused more on wooded than open habitat networks. In our study, we assessed the influence of permanent grassland connectivity, described by grassland amount and spatial configuration, on grassland biodiversity. We selected permanent grasslands in livestock farming areas of north-western France, which were sampled for plants, carabids and birds. At two spatial scales we tested the effects of amount and configuration of grasslands, wooded habitats and crops on richness and abundance of total assemblages and species ecological groups. Grassland connectivity had no significant effects on total richness or abundance of any taxonomic group, regardless of habitat affinity or dispersal ability. The amount of wooded habitat and length of wooded edges at the 200 m scale positively influenced forest and generalist animal groups as well as grassland plant species, in particular animal-dispersed species. However, for animal groups such as open habitat carabids or farmland bird specialists, the same wooded habitats negatively influenced richness and abundance at the 500 m scale. The scale and direction of biodiversity responses to landscape context were therefore similar among taxonomic groups, but opposite for habitat affinity groups. We conclude that while grassland connectivity is unlikely to contribute positively to biodiversity, increasing or maintaining wooded elements near grasslands would be a worthwhile conservation goal. However, the requirements of open farmland animal species groups must be considered, for which such action may be deleterious.     

Étude craniométrique du complexe d’espèces Meriones shawii–grandis (Mammalia : Rodentia) au Maroc,en Algérie et en Tunisie

In North Africa, the rodents of the species complex Meriones shawii–grandis have a considerable ecological, economic and epidemiological importance. Until now, the systematics of these species was subject to discussion due to the presence of populations displaying high morphological variability. By means of an approach of traditional morphometrics based on cranial distances and by using the method of the log shape-ratio, we attempt to characterize morphologically these two taxa. The results show significant differences in size and shape between the specimens of Morocco, on the one hand, and those of Algeria and Tunisia, on the other hand. The samples of Morocco that have been molecularly typed and attributed to M. grandis have larger tooth rows and narrower skulls, as well as relatively small tympanic bullae. On the other hand, those of Algeria and Tunisia assigned to M. shawii are characterized by small tooth rows and wide skulls with well-developed tympanic bullae. The morphological distance is relatively strong between both clades (79.5%), which corresponds to the molecular distance. However, the discriminant analysis performed after molecularly-typed specimens allows the correct classification of only 91.8% of the individuals.