A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

International audienceIn Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii

A Temporal Role Of Type I Interferon Signaling in CD8+ T Cell Maturation during Acute West Nile Virus Infection

A genetic absence of the common IFN- α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR-/- mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8+ T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8+ T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8+ T cell development requires type I IFN signaling. WNV infection experiments in BATF3-/- mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8+ T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8+ T cell response at a stage distinct from the initial priming event

COMPARISON OF OPTIMIZATION FORMULATIONS TO DESIGN AN HYBRID RAILWAY POWER SUBSTATION

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Toward a local drift flux model for high-pressure, subcooled, convective boiling flows

International audienceForced convective boiling is of great interest for several applications in the power and process industry,particularly in nuclear plants. Under certain nominal, incidental or accidental conditions, a boiling crisismay occur resulting in the meltdown of the heating surface. It is then essential to predict as accuratelyas possible the thermal-hydraulic conditions leading to the occurrence of this boiling crisis. Such an ob-jective cannot reasonably be achieved without a good description of the associated two-phase flow. Theobjective of the present study is twofold: (1) to identify the necessary key parameters for correctly de-scribing boiling flows, and (2) to present in a didactic way an original stationary and local model involv-ing these parameters. This new model is primarily based on four mixture balance equations, a submodelfor the local vapor generation rate, and a turbulence submodel inspired by the pioneering work of [25].The results obtained with this original boiling flow model are then compared to an extensive experimental data set obtained on a R12/R134a experimental facility. The comparison clearly demonstrates that this new model contains the fewer necessary submodels to describe the structure of a boiling two-phase flow under pressurized water reactor conditions. Subcooled boiling is acceptably described by the model. However, for higher values of void fraction, the model always predicts a nonexistent void fraction peak near the heating wall and over predicts the wall and liquid temperatures. This behavior may be explained by: (i) the inadequacy of the radial turbulence modeling, (ii) the use of Prandtl’s analogy whose validity under boiling conditions is questionable, and (iii) too simplistic a model for the vapor generation rate

Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery

BACKGROUND: Apicomplexan parasites are responsible for some of the most deadly parasitic diseases afflicting humans, including malaria and toxoplasmosis. These obligate intracellular parasites exhibit a complex life cycle and a coordinated cell cycle-dependant expression program. Their cell division is a coordinated multistep process. How this complex mechanism is organised remains poorly understood.\ud \ud METHODS AND FINDINGS: In this study, we provide evidence for a link between heterochromatin, cell division and the compartmentalisation of the nucleus in Toxoplasma gondii. We characterised a T. gondii chromodomain containing protein (named TgChromo1) that specifically binds to heterochromatin. Using ChIP-on-chip on a genome-wide scale, we report TgChromo1 enrichment at the peri-centromeric chromatin. In addition, we demonstrate that TgChromo1 is cell-cycle regulated and co-localised with markers of the centrocone. Through the loci-specific FISH technique for T. gondii, we confirmed that TgChromo1 occupies the same nuclear localisation as the peri-centromeric sequences.\ud \ud CONCLUSION: We propose that TgChromo1 may play a role in the sequestration of chromosomes at the nuclear periphery and in the process of T. gondii cell division

TgChromo1 participates to the nuclear organisation of the nucleus.

<p>TgChromo1 participates in the functional organisation of the nucleus. The schematic represents the chromosomes in the nucleus with the centromere and telomere clusters occupied by TgChromo1 and their position at the periphery of the nucleus.</p

TgChromo1 binds to peri-centromeric heterochromatin.

<p>ChIP on chip was performed with the TgChromo1 antibody (anti-CHD1, red) or the anti-HA antibody (HA, black) and hybridized on a genome-wide tiling microarray. The regions of enrichment for H3K9me3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032671#pone.0032671-Brooks1" target="_blank">[12]</a> are represented in blue. A snapshot of the 12 chromosomes where a centromere was identified <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032671#pone.0032671-Brooks1" target="_blank">[12]</a> is presented. ChIP on chip signals are represented as a log2 ratio of the signal given by the immunoprecipitated DNA over the input and plotted according to the genomic position of the oligonucleotide.</p

Subtelomeric repeats occupy the same nucleus territory as TgChromo1.

<p>FISH/IFA of TgChromo1-HA (green) and chromosome IX telomeric repeats (red). Parasite nuclei are labelled with DAPI (blue). Colocalising signals from FISH and IFA are arrowed.</p

TgChromo1 expression is cell cycle regulated.

<p><b>A:</b> IFA of TgChromo1-HA (green) and IMC1 (red), a marker of the inner membrane complex, throughout the cell cycle. Parasites representative of interphase (G1) and mitosis are presented. Parasite nuclei are labelled with DAPI. <b>B:</b> Comparison of the intensity of the signal produced by IFA of TgChromo1-HA in interphase (G1) and during budding (Budding, B). IMC1, a marker of the inner membrane complex, is used to identify emerging daughter cells during the budding. Parasites during budding (B) are arrowed. Parasite nuclei are labelled with DAPI. <b>C:</b> TgChromo1 is concentrated in foci of different intensity. IFA was performed using an anti-HA antibody and the IMC1 antibody. Parasites nuclei are labelled with DAPI. Foci of lesser intensity are arrowed.</p