Live cell immunogold labelling of RNA polymerase II

Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range

Distant visual acuity loss among Japanese grammar school children: The roles of heredity and the environment

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32243/1/0000305.pd

Les vaccins antivarioliques (pathogénicité-innocuité, immunogénicité humorale et cellulaire, protection)

La variole, éradiquée en 1980, a représenté l un des plus grands fléaux. Le risque de réémergence du virus de la variole rend nécessaire l évaluation de nouveaux vaccins. Le but de notre étude était d étudier les facteurs immunitaires essentiels à la résistance naturelle contre les orthopoxvirus et d évaluer de nouveaux vaccins antivarioliques. La caractérisation du modèle d infection souris/cowpox virus nous a permis de constater que la protection de la souris était due à l ensemble des réponses immunitaires spécifiques. Un vaccin de 2ème génération (2G) et trois souches de vaccines non réplicatives (3G) ont été évaluées dans notre modèle d infection. Le vaccin de 2G a montré la même efficacité vaccinale que le vaccin traditionnel de référence. Parmi les trois vaccins de 3G, seule la souche MVA a induit chez la souris une protection similaire au vaccin traditionnel à long terme après un rappel. Cependant, son immunogénicité induite à long terme reste inférieure au vaccin traditionnel.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Construction et évaluation de différents mutants du virus de la vaccine exprimant un gène suicide dans la cadre d'une thérapie anti-cancéreuse

Les virus de la vaccine délétés du gène de la thymidine kinase ont montré, après une injection par voie systémique, une certaine efficacité sur des tumeurs implantées dans la souris. Ce virus utilisé comme vecteur pour exprimer le gène suicide FCU1, capable de métaboliser la prodrogue 5-FC non toxique pour l homme en métabolites cytotoxiques, montre une augmentation de la réponse anti-tumorale. De plus, afin d améliorer la spécificité tumorale et diminuer la toxicité virale, nous avons construit plusieurs virus délétés de différents gènes combinés à la délétion du gène TK. Les premiers résultats in vitro montre une réplication virale modifiée suivant l état des cellules, réplicatives ou non. Des études thérapeutiques in vivo mettent en évidence une activité oncolytique équivalente avec cependant une diminution des signes secondaires de toxicité. Ces travaux illustrent l'efficacité des virus de la vaccine recombinants dans le cadre d'une thérapie gène suicide anti-cancéreuse.Recombinant thymidine kinase-deleted vaccinia virus vector targets tumor tissue after systemic delivery, making it ideal for a tumor-directed enzyme/prodrug approach. We have generated a tk-deficient recombinant expressing the suicide gene FCU1 which catalyses the direct conversion of the nontoxic 5-fluorocytosine into toxic metabolites. This vaccinia virus demonstrated a significant tumor regression after systemic injection in combination with administration of 5-FC. Moreover, to enhance the safety and efficacy of this vector, deletion of other genes was combined with thymidine kinase gene deletion. We evaluated in vivo the therapeutic efficacy after intravenous injection in nude mice bearing subcuteanous human tumors and we compared the toxicity between single and double-deleted viruses. The results of this study demonstrate that double-deleted viruses are capable of selective tumor replication and are significantly less pathogenic than the single-deleted virus.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus Ankara.

International audienceVaccinia virus is a structurally complex virus that multiplies in the cell cytoplasm. The assembly of Vaccinia virus particles and their egress from infected cells exploit cellular pathways. Most notably, intracellular mature viral particles are enwrapped by Golgi-derived or endosomal vesicles. These enveloped particles, enriched in virus-encoded proteins, migrate to the cell surface where they are released into the extracellular space through fusion of their outer envelope with the cell membrane. We report that baby hamster kidney cells productively infected with the modified vaccinia virus Ankara strain (MVA) also release extracellular vesicles containing virus-encoded envelope proteins but devoid of any virus cargo. Such vesicles were visualized on the cell surface by electron microscopy and immunogold labelling of the B5 envelope protein. A portion of the B5 protein was found to be associated with non-viral material in high speed ultracentrifugation pellets and displayed a buoyant density characteristic of exosomes released by some cell types. An unrelated transmembrane protein (CD40 ligand) encoded by the MVA genome was also incorporated into extracellular vesicles but not into the envelopes that surround extracellular enveloped virus. High speed pellets obtained by centrifugation of culture medium from cells infected with MVA encoding CD40 ligand displayed the ability to induce dendritic cell maturation suggesting that the ligand is on the outer surface of the extracellular vesicles. We propose that the formation of extracellular vesicles after vaccinia virus infection is a byproduct of the pathway leading to the formation of extracellular enveloped virus

The cowpox virus host range gene, CP77, affects phosphorylation of eIF2 α and vaccinia viral translation in apoptotic HeLa cells

AbstractHost restriction of vaccinia virus has been previously described in CHO and RK13 cells in which a cowpox virus CP77 gene rescues vaccinia virus growth at the viral protein translation level. Here we investigate the restrictive stage of vaccinia virus in HeLa cells using a vaccinia mutant virus (VV-hr) that contains a deletion of 18-kb genome sequences resulting in no growth in HeLa cells. Insertion of CP77 gene into VV-hr generated a recombinant virus (VV-36hr) that multiplied well in HeLa cells. Both viruses could enter cells, initiate viral DNA replication and intermediate gene transcription. However, translation of viral intermediate gene was only detected in cells infected with VV-36hr, indicating that CP77 relieves host restriction at the intermediate gene translation stage in HeLa cells.Caspase-2 and -3 activation was observed in HeLa cells infected with VV-hr coupled with dramatic morphological alterations and cleavage of the translation initiation factor eIF4G. Caspase activation was reduced in HeLa cells infected with VV-36hr, indicating that CP77 acts upstream of caspase activation. Enhanced phosphorylation of PKR and eIF2α was also observed in cells infected with VV-hr and was suppressed by CP77. Suppression of eIF4G cleavage with the caspase inhibitor ZVAD did not rescue virus translation, whereas expression of a mutant eIF2α protein with an alanine substitution of serine at amino acid position 51 (eIF2αS51A) partially restored viral translation and moderately increased virus growth in HeLa cells

Inefficient Measles Virus Budding in Murine L.CD46 Fibroblasts

International audienc

Structural basis for allosteric regulation of Human Topoisomerase IIα

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6–7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme’s activities and opens perspective for the analysis of post-translational modifications