Imaging malaria sporozoites in the dermis of the mammalian host

The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. the steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo.Inst Pasteur, Unite Biol & Genet Paludisme, F-75724 Paris 15, FranceWeb of Scienc

Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis

After the deciphering of the genome sequences of several Plasmodium species, efforts must turn to elucidating gene function and identifying essential gene products. However, random approaches are lacking and gene targeting is inefficient in Plasmodium. Here, we established shuttle transposon mutagenesis in Plasmodium berghei. We constructed a mini-Tn5 derivative that can transpose into parasite genes cloned in Escherichia coli, providing an efficient means of generating knockout fragments. A 10(4)-fold increase in frequencies of double-crossover homologous recombination in the parasite using a new electroporation technology permits to reproducibly generate pools of distinct mutants after transfection with mini-Tn5-interrupted sequences. The procedure opens the way to the systematic identification of essential genes in Plasmodium

Host cell traversal is important for progression of the malaria parasite through the dermis to the liver

The malaria sporozoite, the parasite stage transmitted by the mosquito, is delivered into the dermis and differentiates in the liver. Motile sporozoites can invade host cells by disrupting their plasma membrane and migrating through them (termed cell traversal), or by forming a parasite-cell junction and settling inside an intracellular vacuole (termed cell infection). Traversal of liver cells, observed for sporozoites in vivo, is thought to activate the sporozoite for infection of a final hepatocyte. Here, using Plasmodium berghei, we show that cell traversal is important in the host dermis for preventing sporozoite destruction by phagocytes and arrest by nonphagocytic cells. We also show that cell infection is a pathway that is masked, rather than activated, by cell traversal. We propose that the cell traversal activity of the sporozoite must be turned on for progression to the liver parenchyma, where it must be switched off for infection of a final hepatocyte.Inst Pasteur, Unite Biol & Genet Paludisme, F-75724 Paris 15, FranceUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Montpellier 2, CNRS, UMR 5539, F-34095 Montpellier 05, FranceMie Univ, Sch Med, Tsu, Mie 5140001, JapanUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilWeb of Scienc

Vers un outillage informatique optimisé pour corpus langagiers oraux en vue d’une exploitation textométrique : le cas des interrogatives partielles dans ESLO

Pour répondre aux problématiques engendrées par la diffusion de plus en plus massive des corpus linguistiques et à l’hétérogénéité de leurs formats, nous proposons une méthode permettant de prendre en main des corpus langagiers oraux et de les convertir dans un format permettant leur exploitation outillée. Pour cette recherche, le corpus ESLO nous sert d’exemple par sa licence de diffusion, son format, son volume et ses atouts sociolinguistiques et diachroniques. Notre travail se fonde sur la compilation de ce corpus pour le rendre compatible avec l’outil de textométrie TXM. Nous opérons un ensemble de transformations des données pour l’utiliser au mieux. Enfin, pour illustrer les apports de ces avancées méthodologiques, nous proposons une analyse fine et multidimensionnelle de l’usage des interrogatives dans le corpus ESLO.To answer the increasing trend of corpora sharing and data format heterogeneity, we present a method for converting spoken language corpora to several tool formats in order to facilitate linguistic analysis. For this research, we take as an example the ESLO corpus for several reasons: its open-source licence, its standard format used for its construction, its size, and its sociolinguistic and micro-diacronic characteristics. Our study is based on a compilation of the ESLO corpus in order to make it compatible with the textometric tool TXM. We operate a set of operations to use all the possibilities the tool offers. Finally, we present a fine-grained and multidimensional analysis of the interrogatives utterances used in the ESLO corpus

From array-based hybridization of Helicobacter pylori isolates to the complete genome sequence of an isolate associated with MALT lymphoma

<p>Abstract</p> <p>Background</p> <p><it>elicobacter pylori </it>infection is associated with several gastro-duodenal inflammatory diseases of various levels of severity. To determine whether certain combinations of genetic markers can be used to predict the clinical source of the infection, we analyzed well documented and geographically homogenous clinical isolates using a comparative genomics approach.</p> <p>Results</p> <p>A set of 254 <it>H. pylori </it>genes was used to perform array-based comparative genomic hybridization among 120 French <it>H. pylori </it>strains associated with chronic gastritis (n = 33), duodenal ulcers (n = 27), intestinal metaplasia (n = 17) or gastric extra-nodal marginal zone B-cell MALT lymphoma (n = 43). Hierarchical cluster analyses of the DNA hybridization values allowed us to identify a homogeneous subpopulation of strains that clustered exclusively with <it>cag</it>PAI minus MALT lymphoma isolates. The genome sequence of B38, a representative of this MALT lymphoma strain-cluster, was completed, fully annotated, and compared with the six previously released <it>H. pylori </it>genomes (i.e. J99, 26695, HPAG1, P12, G27 and Shi470). B38 has the smallest <it>H. pylori </it>genome described thus far (1,576,758 base pairs containing 1,528 CDSs); it contains the <it>vacA</it>s2m2 allele and lacks the genes encoding the major virulence factors (absence of <it>cag</it>PAI, <it>bab</it>B, <it>bab</it>C, <it>sab</it>B, and <it>hom</it>B). Comparative genomics led to the identification of very few sequences that are unique to the B38 strain (9 intact CDSs and 7 pseudogenes). Pair-wise genomic synteny comparisons between B38 and the 6 <it>H. pylori </it>sequenced genomes revealed an almost complete co-linearity, never seen before between the genomes of strain Shi470 (a Peruvian isolate) and B38.</p> <p>Conclusion</p> <p>These isolates are deprived of the main <it>H. pylori </it>virulence factors characterized previously, but are nonetheless associated with gastric neoplasia.</p

Secondary metabolite contents in the forests of Mayotte and Madagascar, and their incidence on two leaf-eating lemur species (Eulemur spp.)

Nous avons comparé la richesse en composés phénoliques et en alcaloïdes de six massifs forestiers tropicaux (quatre forêts denses humides plus une forêt sèche dans l'île de Mayotte, et une forêt humide anthropisée à Madagascar). Les dosages ont porté sur les phénols totaux, les tannins condensés, les alcaloïdes totaux présents dans les feuilles des lianes, des arbres et des palmiers, et ont été complétés par un test de précipitation de l'albumine de sérum bovin (BSA). Les profils chimiques ont été établis pour chaque site en pondérant les concentrations de métabolites dans les feuilles matures des arbres par l'abondance relative de ceux-ci (d'après les recensements sur transects et les mesures de diamètres). Les alcaloïdes sont généralement peu fréquents, à l'exception du site anthropisé où les manguiers et les anacardiers ont été introduits. Au contraire, la richesse en phénols totaux, en tannins condensés, ou en métabolites précipitant la BSA diffère considérablement entre les forêts en fonction de la prédominance locale de certaines espèces à fortes concentrations en composés phénoliques. Cette variabilité s'accorde difficilement avec la prévision théorique d'une même « tactique » de défense chimique des plantes contre les herbivores (tannins versus alcaloïdes, sensu Lebreton ou Feeny) dans les milieux à faible diversité spécifique. La nature insulaire de la plupart des forêts étudiées, avec des peuplements relativement récents, peut cependant déterminer les particularismes locaux des processus coévolutifs liant les populations d'herbivores et de végétaux. La relative pauvreté en phénols et en alcaloïdes des forêts humides de Mayotte pourrait aussi s'expliquer par des conditions édaphiques (sol fertile sur un socle volcanique) favorables au renouvellement à coût limité des tissus végétaux endommagés, selon la théorie de Coley et al. (1985). Les espèces de primates frugivores-folivores (Eulemur fulvus et Eulemur macaco) habitant les forêts étudiées se différencient nettement quant à leur tolérance vis-à-vis des composés phénoliques. Contrairement à E. fulvus, les choix alimentaires de feuilles par E. macaco sont négativement corrélés à la teneur en phénols totaux et à la capacité de précipiter la BSA. Les différences interspécifiques de tolérance sont discutées en relation avec la possibilité d'élargir le répertoire alimentaire, les densités de population et l'ubiquité d'une espèce de lémurien par rapport à l'autre. La relation positive entre phénols totaux et efficacité de précipitation de la BSA (alors qu'elle est faible ou nulle entre tannins condensés et BSA), suggère que la mesure des phénols totaux, de préférence aux tannins condensés, soit intégrée à l'analyse prédictive des densités de primates consommateurs de feuillage en fonction de la qualité nutritionnelle de l'environnement.We measured concentrations of total phenolics (TP), condensed tannins (CT), and total alkaloids, as well as bovine serum albumin precipitation (BSAp), in leaves of lianas, trees, and palms from six tropical forests (four rainforests plus one dry forest in Mayotte Island, and one anthropogenic rainforest in Madagascar). We established chemical profiles for each site, weighting metabolite concentrations found in mature tree leaves by relative tree species abundance (as measured by dbh along transects). Alkaloids were generally rare, except in the anthropogenic forest, in which introduced mango trees and cashew trees are abundant. In contrast, the six forest sites differed considerably with regard to TP, CT, and BSAp, due to variation in the local abundance of a few antinutrient-rich plant species. Such variation does not conform in any simple way to current theories on the evolution of plant chemical defences. Current theories predict that environments with similar plant diversity, as in all 6 forests studied here (mostly including insular forests), should exhibit the same category of anti-herbivore chemical defence (tannin versus alkaloid strategy sensu Lebreton or Feeny). It is possible that distribution patterns of secondary compounds in Mayotte forests actually result from peculiar dynamics of plant-herbivore interactions, as may be expected in insular ecosystems with low biodiversity, high rates of endemism, and eventually lack of equilibrium between populations of herbivores and plants. The relative lack of phenolics and alkaloids in the humid forests growing on Mayotte’s fertile volcanic soils may also be interpreted according to the resource availability theory of Coley et al. (1985). In environments where nutrients are widely available, costs of replacing leaves ingested by herbivores may be lower than costs of investing in chemical defences. Frugivorous/ folivorous lemurs (Eulemur fulvus and Eulemur macaco) occurring in the study forests differed markedly with regard to their ability to tolerate phenolic compounds. Leaf choice of E. macaco (but not E. fulvus) was negatively correlated to TP and BSAp. The differential deterrent effect of secondary metabolites has ramifications for lemur diet width, population densities, and ecological plasticity. The positive correlation between TP and BSAp (and lack of correlation between CT and BSAp) suggests that total phenolics should be used instead of condensed tannins in models predicting densities of leaf-eating primates based on the nutritional quality of the environment

Imaging cortical dynamics in GCaMP transgenic rats with a head-mounted widefield macroscope

Widefield imaging of calcium dynamics is an emerging method for mapping regional neural activity but is currently limited to restrained animals. Here we describe cScope, a head- mounted widefield macroscope developed to image large-scale cortical dynamics in rats during natural behavior. cScope provides a 7.8 by 4 mm field of view, dual illumination paths for both fluorescence and hemodynamic correction, and can be fabricated at low cost using readily attainable components. We also report the development of Thy-1 transgenic rat strains with widespread neuronal expression of the calcium indicator GCaMP6f. We combined these two technologies to image large-scale calcium dynamics in the dorsal neocortex during a visual evidence accumulation task. Quantitative analysis of task-related dynamics revealed multiple regions having neural signals that encode behavioral choice and sensory evidence. Our results provide a new transgenic resource for calcium imaging in rats and extend the domain of headmounted microscopes to larger-scale cortical dynamics.Accepted manuscrip