Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing, and incorporation into mature Sindbis virions

We have obtained a vaccinia virus recombinant which contains a complete cDNA copy of the 26S RNA of Sindbis virus within the thymidine kinase gene of the vaccinia virus genome. This recombinant constitutively transcribed the Sindbis sequences throughout the infectious cycle, reflecting the dual early-late vaccinia promoter used in this construction. The Sindbis-derived transcripts were translationally active, giving rise to both precursor and mature structural proteins of Sindbis virus, including the capsid protein (C), the precursor of glycoprotein E2 (PE2), and the two mature envelope glycoproteins (E1 and E2). These are the same products translated from the 26S mRNA during Sindbis infection, and thus these proteins were apparently cleaved, glycosylated, and transported in a manner analogous to that seen during authentic Sindbis infections. By using epitope-specific antibodies, it was possible to demonstrate that recombinant-derived proteins were incorporated into Sindbis virions during coinfections with monoclonal antibody-resistant Sindbis variants. These results suggest that all the information necessary to specify the proper biogenesis of Sindbis virus structural proteins resides within the 26S sequences and that vaccinia may provide an appropriate system for using DNA molecular genetic manipulations to unravel a variety of questions pertinent to RNA virus replication

Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest

Cryocalcites de nappe : proxys de l’extension des environnements périglaciaires et glaciaires.

National audienceLes cristallisation de calcite des sables du Bassin de paris ont été datées par les méthodes 14C et U-Th. Les datations couvrent les dernières glaciations (Thiry et al., 2013). Le positionnement de ces datations sur les courbes du d18O des carottes de glace montrent qu’elles se forment lors des périodes de refroidissement. Ce sont des cryocalcites de nappe qui se forment apparemment par réchauffement des eaux froides d’infiltration au contact des eaux plus chaudes de la nappe. Par leur large distribution et leurs signatures isotopiques elles apparaissent comme des proxys particulièrement prometteurs pour tracer et suivre l’extension des pergélisols et des permafrosts, au moins sur l’Europe du Nord-Ouest, et des cristallisations de ce type sont également connues en Amérique du Nord

Isotope signature of sand calcites in the Paris Basin: a proxy for permafrost/groundwater paleoconditions

International audienceSand calcites occurring as rhombohedral crystals and spherolitic concretions in several Tertiary sandstone formations in the Paris basin have been dated to the cooling periods of the four last glacial stages, using 14C and U-Th methods. Sand calcites constitute a novel proxy to estimate paleo-groundwater levels and the extension of permafrost conditions. The δ13C and δ18O compositions of sand calcites are a function of precipitation depth in relation with the warming of surface water during infiltration. Sand calcites appear as precious markers for Pleistocene paleo-landscape reconstructions

Development of microstructured and protein patterned hydrogels to investigate the influence of the microenvironment on cancer cells.

Ovarian cancer is one of the most lethal gynaecological diseases. One of the reasons for its poor survival rates is its typically late diagnosis at an advanced stage of tumour progression. A better understanding of why and how cancer initiates and develops is crucial for early diagnosis and the development of improved treatment methods. To this point, investigations of mutations on genes of cells to reduce mortality of ovarian cancer have not lead to the desired outcome. Recently, increased attention has been paid to the influencing factors in cancer cell progression such as the cellular microenvironment. It is known that the natural microenvironment of tumours differs greatly from that of healthy tissues and that modification of the cell’s environment causes cellular responses such as changes in morphology, protein expressions, cell division and migration behaviour. In this work, an experimental set-up was developed to trap cells in defined 3D wells to investigate how physical properties of the microenvironment influence ovarian can- cer cells. Polyacrlyamide gels were simultaneously structured and protein patterned to create a platform for cell experiments, which allows the tuning of individual physical properties of the microenvironment of cells (such as stiffness, available volume and protein compositions for cell attachment) independently of each other. The designed and optimised fabrication process begins with optical lithography to transfer a pattern onto a Silicon (Si) substrate and a dry etching step to obtain an array of pillars while transforming the Si-substrate into a Si-mould. The Si-mould serves as a stamp during a µ-contact printing approach to transfer defined patterns of protein, and simultaneously as a mould during polyacrylamide polymerisation. The resulting microstructured and protein patterned polyacrylamide gels can then be used as cell culture substrates for cell experiments. Si-moulds and polyacrylamide gels were characterised with scanning electron microscopy, atomic force microscopy and confocal laser scanning microscopy. By investigating four differently sized circular patterns (with diameters of 20 µm, 30 µm, 40µm and 60 µm) and four different stiffnesses of the polyacrylamide gels (1 kPa, 8 kPa, 30 kPa and 100 kPa), it was shown that the fabrication process is robust and easy to adjust. Computational analysis protocols were developed and established for traction force microscopy and brightness fluctuation analysis of cells, including the corresponding bright- ness autocorrelation functions. It was shown that microstructured and protein patterned polyacrylamide gels can be used for investigations of protein expressions, cellular traction forces and brightness fluctuations of cells. While the analyses of protein expressions and autocorrelation functions of the brightness fluctuations need further improvement, the results of the traction force experiments allow a first hypothesis to be formulated: cellular traction forces increase with decreasing volume available to the cell, as indicated by analysis of cell experiments on ovarian cancer cells of the cell line SKOV3. The developed microstructured and protein patterned polyacrylamide gels are an important step to gain a better understanding on how mechanical properties of the microenvironment influence cellular responses. This experimental setup can be easily adapted and optimised for further investigations of cancer cells and can thus help in the development of new treatment approaches

The Fontainebleau Sandstone: bleaching, silicification and calcite precipitation under periglacial conditions

It will be show that the typical Fontainebleau Sandstone facies are in fact secondary facies, developed post‐deposition, and related to surface exposure of the formation. Quartzite pans, white sands, and even the mineralogical composition are related to subsurface (groundwater) alterationsconnected to geomorphology and result from exposure during the last glacial periods.The Fontainebleau Sandstone example is extremely spectacular. Yet, such alteration features also exist in many other formations and may extent to 50‐100 m depth. One has to be attentive to recognize them. The goal of the field trip is to bring insight into what is related to primary and to secondary processes in the outcropping formation. This is of prime/particular importance for better understanding of the geology of the Fontainebleau Sandstone … and the geochemical processes that led to make up theses exceptionally pure sandstones mined to be used in all high quality and luxury glass manufactories throughout Europe. and supply guides for prospecting such sand ores.But in a general way, when such alteration happen, it is essential to recognize them before any geochemical study because interpretations, in particular isotopic interpretations may completely diverge as a primary or a secondary hypothesis is envisaged for some facies … like marine deposits vs groundwater precipitation, dry and warm vs glacial climates, sedimentary deposition vs acidic leaching, etc

Technique de préparation des minéraux argileux en vue de l'analyse par diffraction des Rayons X et introduction à l'interprétation des diagrammes

Les techniques de préparation des échantillons pour l'analyse minéralogique des minéraux argileux sont présentées. Ces techniques consistent essentiellement à extraire une fraction fine (enrichie en argiles) à partir d'échantillons géologiques. Puis sont présentés les traitements de cette fraction enrichie en argiles en vue de leur analyse par diffraction des Rayons X. Enfin sont présentées les bases de la détermination des minéraux argileux par diffraction RX et l'interprétation des paramètres de cristallinité qui peuvent être extraits de ces diagrammes

Caractérisation des cycles oxydo-reducteurs du fer en Seine

Des études physico-chimiques ont été effectuées sur les matières en suspension (MES) de la Seine, et plus particulièrement sur les phases sulfurées et oxydées porteuses de polluants métalliques. Les résultats montrent qu'il existe différents types de micropolluants métalliques liés aux particules de la colonne d'eau. Le fer est à la base de nombreuses phases porteuses de métaux et à ce titre son devenir et ses transformations dans le milieu méritent d'être étudiées pour mieux comprendre et quantifier les mécanismes de mobilisation, altération et re-fixation des polluants métalliques. Connaître en détail les (oxyhydr)oxydes et sulfures de fer présents, leur minéralogie, taille et morphologies spécifiques, voire leur composition en impuretés est un atout majeur pour comprendre le devenir des métaux dans un système fluviatile. Par ailleurs, le fer est également un excellent indicateur des processus diagénétiques. Par exemple, on peut s'attendre à ce qu'une certaine fraction des nanoparticules de magnétite, porteuses d'un signal magnétique intense et très distinctif, soit fabriquée en Seine par les bactéries magnéto-tactiques qui vivent dans les environnements à la limite de l'oxique à l'interface sédiment/eau. Au contraire, une série de sulfures de fer est formée par des dynamiques post-dépôts pour des durées ou intensités de diagénèse plus grandes. En connaissant le cycle de formation de ces phases dans des milieux de degrés d'anoxie différents et en connaissant la dynamique de leur dissolution dans la colonne d'eau, on peut développer des indicateurs très pertinents sur l'intensité des échanges entre les particules en suspension et le fond sédimentaire. Un échantillonnage mensuel des MES a été mis en place entre l'été 2011 et l'hiver 2012 Une vingtaine des sites d'échantillonnage ont été choisis le long du cours de la Seine entre Saron (proche confluence Aube/Seine) et Amfreville (~ Rouen) et sur les principaux affluents. Les sites ont été sélectionnés en fonction des résultats d'études antérieures et en fonction de la disponibilité d'autres informations complémentaires (débit, teneur en oxygène de l'eau, accessibilité du site, échantillonnage en commun, etc.). Les premières analyses d'hystèresis magnétiques montrent qu'il y a une tendance générale dans la concentration de la faction métallique qui monte 'en palier' de l'amont vers l'aval en fonction de la contribution des différents affluents mais surtout en se rapprochant de l'agglomération parisienne. En regardant les différentes saisons échantillonnées, on remarque des 'groupements' entre les mois de sept-oct-nov 2011, à concentration élevée, et fev-mars-mai 2012 de concentration plus faible, les autres mois montrent des niveaux de concentration 'intermédiaires'. Le paramètre de taille de grain magnétique est un bon indicateur pour l'apport anthropique car les particules magnétiques de cette origine sont d'une manière générale de plus grand taille que les particules d'origine détritique ou biogénique. Les résultats préliminaires montrent que ce paramètre ne semble pas être influencé par les différentes saisons; par contre il augmente clairement en se rapprochant de Paris ce qui se corrèle bien avec l'augmentation d'une potentielle pollution métallique. Il diminue ensuite de nouveau vers Amfreville Les premières analyses au MEB étaient d'abord ciblées sur deux échantillons prélevés en août 2011 et mettent en comparaison le site de Bougival à l'aval de Paris et le site de Marnay très à l'amont de la Seine. Ces résultats confirment bien la présence des particules métalliques d'origine anthropique pour le site de Bougival ainsi qu'un signal de " fond détritique " pour le site rural de Marnay

High mobility group proteins of amphibian oocytes: a large storage pool of a soluble high mobility group-1-like protein and involvement in transcriptional events

No abstract availabl

Predicting instrumental mass fractionation (IMF) of stable isotope SIMS analyses by response surface methodology (RSM) [Dataset]

The dataset contains four files. File S1 corresponds to cathodoluminescence and BSE images of standard minerals. File S2 contains geochemical data of mineral standards ans samples obtained by SIMS and EPMA. File S3 contains the parameters used to obtain response surface models of IMF in mineral standards. File S4 is a response surface methodology tutorial.Instrumental mass fractionation (IMF) of isotopic SIMS analyses (Cameca 1280HR, CRPG Nancy) was predicted by response surface methodology (RSM) for 18O/16O determinations of plagioclase, K-feldspar and quartz. The three predictive response surface models combined instrumental and compositional inputs. The instrumental parameters were: (i) X and Y position, (ii) LT1DefX and LT1DefY electrostatic deflectors, (iii) chamber pressure and, (iv) primary-ion beam intensity. The compositional inputs included: (i) anorthite content (An%) for the plagioclase model and, (ii) orthoclase (Or%) and barium (BaO%) contents for the K-feldspar model. The three models reached high predictive powers. The coefficients R2 and prediction-R2 were, respectively, 90.47% and 86.74% for plagioclase, 87.56% and 83.17% for K-feldspar and 94.29% and 91.59% for quartz. The results show that RSM can be confidently applied to IMF prediction in stable isotope SIMS analyses by the use of instrumental and compositional variables.