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Search Results: 1 - 10 of 9638 matches for " Nicolas Tromas equal contributor "
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Within-Host Spatiotemporal Dynamics of Plant Virus Infection at the Cellular Level
Nicolas Tromas equal contributor,Mark P. Zwart equal contributor ,Guillaume Lafforgue,Santiago F. Elena
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004186
Abstract: A multicellular organism is not a monolayer of cells in a flask; it is a complex, spatially structured environment, offering both challenges and opportunities for viruses to thrive. Whereas virus infection dynamics at the host and within-cell levels have been documented, the intermediate between-cell level remains poorly understood. Here, we used flow cytometry to measure the infection status of thousands of individual cells in virus-infected plants. This approach allowed us to determine accurately the number of cells infected by two virus variants in the same host, over space and time as the virus colonizes the host. We found a low overall frequency of cellular infection (<0.3), and few cells were coinfected by both virus variants (<0.1). We then estimated the cellular contagion rate (R), the number of secondary infections per infected cell per day. R ranged from 2.43 to values not significantly different from zero, and generally decreased over time. Estimates of the cellular multiplicity of infection (MOI), the number of virions infecting a cell, were low (<1.5). Variance of virus-genotype frequencies increased strongly from leaf to cell levels, in agreement with a low MOI. Finally, there were leaf-dependent differences in the ease with which a leaf could be colonized, and the number of virions effectively colonizing a leaf. The modeling of infection patterns suggests that the aggregation of virus-infected cells plays a key role in limiting spread; matching the observation that cell-to-cell movement of plant viruses can result in patches of infection. Our results show that virus expansion at the between-cell level is restricted, probably due to the host environment and virus infection itself.
Electrochemical Regulation of Budding Yeast Polarity
Armin Haupt equal contributor,Alexis Campetelli equal contributor,Daria Bonazzi equal contributor,Matthieu Piel,Fred Chang ,Nicolas Minc
PLOS Biology , 2014, DOI: 10.1371/journal.pbio.1002029
Abstract: Cells are naturally surrounded by organized electrical signals in the form of local ion fluxes, membrane potential, and electric fields (EFs) at their surface. Although the contribution of electrochemical elements to cell polarity and migration is beginning to be appreciated, underlying mechanisms are not known. Here we show that an exogenous EF can orient cell polarization in budding yeast (Saccharomyces cerevisiae) cells, directing the growth of mating projections towards sites of hyperpolarized membrane potential, while directing bud emergence in the opposite direction, towards sites of depolarized potential. Using an optogenetic approach, we demonstrate that a local change in membrane potential triggered by light is sufficient to direct cell polarization. Screens for mutants with altered EF responses identify genes involved in transducing electrochemical signals to the polarity machinery. Membrane potential, which is regulated by the potassium transporter Trk1p, is required for polarity orientation during mating and EF response. Membrane potential may regulate membrane charges through negatively charged phosphatidylserines (PSs), which act to position the Cdc42p-based polarity machinery. These studies thus define an electrochemical pathway that directs the orientation of cell polarization.
Parasitic Loads in Tissues of Mice Infected with Trypanosoma cruzi and Treated with AmBisome
Sabrina Cencig equal contributor,Nicolas Coltel equal contributor,Carine Truyens,Yves Carlier
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001216
Abstract: Background Chagas disease is one of the most important public health problems and a leading cause of cardiac failure in Latin America. The currently available drugs to treat T. cruzi infection (benznidazole and nifurtimox) are effective in humans when administered during months. AmBisome (liposomal amphotericin B), already shown efficient after administration for some days in human and experimental infection with Leishmania, has been scarcely studied in T. cruzi infection. Aims This work investigates the effect of AmBisome treatment, administered in 6 intraperitoneal injections at various times during acute and/or chronic phases of mouse T. cruzi infection, comparing survival rates and parasitic loads in several tissues. Methodology Quantitative PCR was used to determine parasitic DNA amounts in tissues. Immunosuppressive treatment with cyclophosphamide was used to investigate residual infection in tissues. Findings Administration of AmBisome during the acute phase of infection prevented mice from fatal issue. Parasitaemias (microscopic examination) were reduced in acute phase and undetectable in chronic infection. Quantitative PCR analyses showed significant parasite load reductions in heart, liver, spleen, skeletal muscle and adipose tissues in acute as well as in chronic infection. An earlier administration of AmBisome (one day after parasite inoculation) had a better effect in reducing parasite loads in spleen and liver, whereas repetition of treatment in chronic phase enhanced the parasite load reduction in heart and liver. However, whatever the treatment schedule, cyclophosphamide injections boosted infection to parasite amounts comparable to those observed in acutely infected and untreated mice. Conclusions Though AmBisome treatment fails to completely cure mice from T. cruzi infection, it impedes mortality and reduces significantly the parasitic loads in most tissues. Such a beneficial effect, obtained by administrating it over a short time, should stimulate studies on using AmBisome in association with other drugs in order to shorten recovery from T. cruzi infection.
Complete In Vitro Life Cycle of Trypanosoma congolense: Development of Genetic Tools
Virginie Coustou equal contributor ,Fabien Guegan equal contributor,Nicolas Plazolles,Théo Baltz
PLOS Neglected Tropical Diseases , 2010, DOI: 10.1371/journal.pntd.0000618
Abstract: Background Animal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses. Methodology/Principal Findings We optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement. Conclusions/Significance We devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model.
Targeted Deficiency of the Transcriptional Activator Hnf1α Alters Subnuclear Positioning of Its Genomic Targets
Reini F. Luco equal contributor,Miguel A. Maestro equal contributor,Nicolas Sadoni,Daniele Zink,Jorge Ferrer
PLOS Genetics , 2008, DOI: 10.1371/journal.pgen.1000079
Abstract: DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary β-cells and hepatocytes freshly isolated from mice lacking Hnf1α, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a?/? cells inactive endogenous Hnf1α-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1α-targets in Hnf1a?/? cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.
Emergence of Young Human Genes after a Burst of Retroposition in Primates
Ana Claudia Marques equal contributor,Isabelle Dupanloup equal contributor,Nicolas Vinckenbosch,Alexandre Reymond,Henrik Kaessmann
PLOS Biology , 2005, DOI: 10.1371/journal.pbio.0030357
Abstract: The origin of new genes through gene duplication is fundamental to the evolution of lineage- or species-specific phenotypic traits. In this report, we estimate the number of functional retrogenes on the lineage leading to humans generated by the high rate of retroposition (retroduplication) in primates. Extensive comparative sequencing and expression studies coupled with evolutionary analyses and simulations suggest that a significant proportion of recent retrocopies represent bona fide human genes. We estimate that at least one new retrogene per million years emerged on the human lineage during the past ~63 million years of primate evolution. Detailed analysis of a subset of the data shows that the majority of retrogenes are specifically expressed in testis, whereas their parental genes show broad expression patterns. Consistently, most retrogenes evolved functional roles in spermatogenesis. Proteins encoded by X chromosome?derived retrogenes were strongly preserved by purifying selection following the duplication event, supporting the view that they may act as functional autosomal substitutes during X-inactivation of late spermatogenesis genes. Also, some retrogenes acquired a new or more adapted function driven by positive selection. We conclude that retroduplication significantly contributed to the formation of recent human genes and that most new retrogenes were progressively recruited during primate evolution by natural and/or sexual selection to enhance male germline function.
Cyclin A Degradation by Primate Cytomegalovirus Protein pUL21a Counters Its Innate Restriction of Virus Replication
Nicolas Caffarelli equal contributor,Anthony R. Fehr equal contributor,Dong Yu
PLOS Pathogens , 2013, DOI: 10.1371/journal.ppat.1003825
Abstract: Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV) can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC), but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL) cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.
Synaptonemal Complex Components Promote Centromere Pairing in Pre-meiotic Germ Cells
Nicolas Christophorou equal contributor,Thomas Rubin equal contributor,Jean-René Huynh
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1004012
Abstract: Mitosis and meiosis are two distinct cell division programs. During mitosis, sister chromatids separate, whereas during the first meiotic division, homologous chromosomes pair and then segregate from each other. In most organisms, germ cells do both programs sequentially, as they first amplify through mitosis, before switching to meiosis to produce haploid gametes. Here, we show that autosomal chromosomes are unpaired at their centromeres in Drosophila germline stem cells, and become paired during the following four mitosis of the differentiating daughter cell. Surprisingly, we further demonstrate that components of the central region of the synaptonemal complex are already expressed in the mitotic region of the ovaries, localize close to centromeres, and promote de novo association of centromeres. Our results thus show that meiotic proteins and meiotic organization of centromeres, which are key features to ensure reductional segregation, are laid out in amplifying germ cells, before meiosis has started.
Fertility, Gestation Outcome and Parasite Congenital Transmissibility in Mice Infected with TcI, TcII and TcVI Genotypes of Trypanosoma cruzi
Sabrina Cencig equal contributor,Nicolas Coltel equal contributor,Carine Truyens,Yves Carlier
PLOS Neglected Tropical Diseases , 2013, DOI: 10.1371/journal.pntd.0002271
Abstract: This work aims to compare the effects of acute or chronic infections with the T. cruzi genotypes TcI (X10 strain), TcII (Y strain) and TcVI (Tulahuen strain) on fertility, gestation, pup growth and the possible vertical transmission of parasites in BALB/c mice. The occurrence of congenital infection was evaluated by microscopic examination of blood and/or qPCR on blood and heart in newborn pups and/or older offspring submitted to cyclophosphamide-induced immunosuppression in order to detect possible cryptic congenital infection. Altogether, the results show that: i) for the three strains tested, acute infection occurring after the embryo implantation in the uterus (parasite inoculation 4 days before mating), or close to delivery (parasite inoculation on day 13 of gestation), prevents or severely jeopardizes gestation outcome (inducing pup mortality and intra-uterine growth retardation); ii) for the three strains tested, gestation during chronic infection results in intra-uterine growth retardation, whereas re-inoculation of TcVI parasites during gestation in such chronically infected mice, in addition, strongly increases pup mortality; iii) congenital infection remains a rare consequence of infection (occurring in approximately 4% of living pups born to acutely infected dams); iv) PCR, detecting parasitic DNA and not living parasites, is not convenient to detect congenial infection close to delivery; v) transmission of parasites by breast milk is unlikely. This study should encourage further investigations using other parasite strains and genotypes to explore the role of virulence and other factors, as well as the mechanisms of such effects on gestation and on the establishment of congenital infection.
The Master Activator of IncA/C Conjugative Plasmids Stimulates Genomic Islands and Multidrug Resistance Dissemination
Nicolas Carraro equal contributor,Dominick Matteau equal contributor,Peng Luo,Sébastien Rodrigue ,Vincent Burrus
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004714
Abstract: Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.
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