Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii

International audienceToxoplasma gondii virulence depends on the expression of factors packed into specific organelles such as rhoptry and microneme. Although virulence factor expression is tightly regulated, the molecular mechanisms controlling their regulation remain poorly understood. ApiAP2 are a family of conserved transcription factors (TFs) that play an important role in regulating gene expression in apicomplexan parasites. TgAP2XI-5 is able to bind to transcription-ally active promoters of genes expressed during the S/M phase of the cell cycle, such as virulence genes (rhoptries and micronemes genes). We identified proteins interacting with TgAP2XI-5 including a cell cycle-regulated ApiAP2 TF, TgAP2X-5. Using an inducible knock-down strategy and RNA-seq, we demonstrated that the level of expression of number of virulence factors transcripts is affected by the disruption of TgAP2X-5 expression. While TgAP2X-5 disruption has mild effect on parasite invasion, it leads to the strain avirulence in mice. To better understand the molecular mechanisms at stake, we investigated the binding of TgAP2XI-5 at promoters in the TgAP2X-5 mutant strain in a genome-wide assay. We show that disruption of TgAP2X-5 expression leads to defects in TgAP2XI-5 binding to multiple rhoptry gene promoters. Taken together, these data suggest a cooperative contribution of two ApiAP2 TF in the regulation of virulence genes in T. gondii

Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex

International audienceThe nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes

Dietary Intakes and Nutritional Status of Mother-Child (6-23 Months Old) Pair Targeted through the "Organic Residual Products for Biofortified Foods for Africa Project" in Rural Area in Senegal

Background: Despite micronutrient supplementation and food fortification strategies carried out for decades, micronutrient deficiencies remain prevalent among children under 5 years old in rural area in Senegal. The OR4FOOD project was implemented as a preventive and long-term approach to reduce malnutrition through biofortification. Objective: We aimed to assess the baseline dietary intakes and nutritional status of the mother-child (6-23 months old) pair in a rural community in Senegal. Methods: Dietary intakes were assessed using dietary recall questionnaires and weight food records. All foods and beverages consumed from waking to bedtime were quantified, and nutrient intakes were calculated. The nutritional status was measured by anthropometry. Results: Results showed that 77.2% of children had low dietary diversity score. Only 18% of them received an appropriate complementary feeding according to the minimum acceptable diet. Cereals and legumes were among the most consumed food groups, whereas orange-fleshed sweet potato (OFSP) and animal food products were rarely consumed. Median dietary intakes of iron, zinc, and vitamin A were lower than the recommended dietary allowances. Acute malnutrition and stunting affected 14.6% and 16.9% of children, respectively. Overall, 20.8% of mothers were underweighted, and overweight/obesity affected 23.1% of them. Conclusion: Malnutrition remains prevalent in rural areas of Senegal and affects both mothers and children. Furthermore, their nutrient requirements were not covered by the diet. Millet and cowpea being widely consumed, optimizing their iron and zinc content through biofortification and the introduction of OFSP might improve micronutrient intakes and would be promising strategies to prevent child malnutrition

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson's disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2's phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation.</p

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson's disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2's phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation.</p

Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle

Utilización de herramientas de simulación en la robótica industrial

Análisis de herramienta de simulación utilizada en el entorno industrial y estudio de las ventajas de la programación "offline" en un entorno productivo. Programación de una célula de trabajo industrial y ampliación posterior de la misma utilizando la herramienta de simulación industrial

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson’s disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2’s phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation

Time to treatment with bridging intravenous alteplase before endovascular treatment:subanalysis of the randomized controlled SWIFT-DIRECT trial.

BACKGROUND We hypothesized that treatment delays might be an effect modifier regarding risks and benefits of intravenous thrombolysis (IVT) before mechanical thrombectomy (MT). METHODS We used the dataset of the SWIFT-DIRECT trial, which randomized 408 patients to IVT+MT or MT alone. Potential interactions between assignment to IVT+MT and expected time from onset-to-needle (OTN) as well as expected time from door-to-needle (DTN) were included in regression models. The primary outcome was functional independence (modified Rankin Scale (mRS) 0-2) at 3 months. Secondary outcomes included mRS shift, mortality, recanalization rates, and (symptomatic) intracranial hemorrhage at 24 hours. RESULTS We included 408 patients (IVT+MT 207, MT 201, median age 72 years (IQR 64-81), 209 (51.2%) female). The expected median OTN and DTN were 142 min and 54 min in the IVT+MT group and 129 min and 51 min in the MT alone group. Overall, there was no significant interaction between OTN and bridging IVT assignment regarding either the functional (adjusted OR (aOR) 0.76, 95% CI 0.45 to 1.30) and safety outcomes or the recanalization rates. Analysis of in-hospital delays showed no significant interaction between DTN and bridging IVT assignment regarding the dichotomized functional outcome (aOR 0.48, 95% CI 0.14 to 1.62), but the shift and mortality analyses suggested a greater benefit of IVT when in-hospital delays were short. CONCLUSIONS We found no evidence that the effect of bridging IVT on functional independence is modified by overall or in-hospital treatment delays. Considering its low power, this subgroup analysis could have missed a clinically important effect, and exploratory analysis of secondary clinical outcomes indicated a potentially favorable effect of IVT with shorter in-hospital delays. Heterogeneity of the IVT effect size before MT should be further analyzed in individual patient meta-analysis of comparable trials. TRIAL REGISTRATION NUMBER URL: https://www. CLINICALTRIALS gov ; Unique identifier: NCT03192332