A pan-apicomplexan phosphoinositide-binding protein acts in malarial microneme exocytosis.

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is an essential step in the development of the disease. Consequently, the molecular players involved in host cell invasion represent important targets for inhibitor design and vaccine development. The process of merozoite invasion is a succession of steps underlined by the sequential secretion of the organelles of the apical complex. However, little is known with regard to how their contents are exocytosed. Here, we identify a phosphoinositide-binding protein conserved in apicomplexan parasites and show that it is important for the attachment and subsequent invasion of the erythrocyte by the merozoite. Critically, removing the protein from its site of action by knock sideways preferentially prevents the secretion of certain types of micronemes. Our results therefore provide evidence for a role of phosphoinositide lipids in the malaria invasion process and provide further insight into the secretion of microneme organelle populations, which is potentially applicable to diverse apicomplexan parasites.

Total Synthesis and Antimalarial Activity of Dominicin, a Cyclic Octapeptide from a Marine Sponge.

Dominicin, a macrocyclic peptide isolated from the marine sponge Eurypon laughlini, has been synthesized for the first time by solid-phase peptide synthesis. The strategy uses oxime resin and takes advantage of the nucleophile susceptibility of the oxime ester bond. The synthesis relies on the preparation of a linear precursor followed by on-resin head-to-tail concomitant cyclization-cleavage. This is the first report of the use of a Boc/OtBu biorthogonal protection strategy on oxime resin to facilitate concomitant N-terminal and side-chain tert-butyl ether deprotection cyclization of unprotected peptides. Also, we report the first antimalarial investigation of dominicin. Interestingly, the natural macrocyclic peptide demonstrates effective low micromolar activity (1.8 µM) against the chloroquine-mefloquine-pyrimethamine-resistant Dd2 strain of Plasmodium falciparum.

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.

Inactivation of Plasmepsins 2 and 3 Sensitizes Plasmodium falciparum to the Antimalarial Drug Piperaquine.

Dihydroartemisinin-piperaquine (DHA-PPQ), the current frontline artemisinin combination therapy used to treat Plasmodium falciparum malaria in multiple Southeast Asian countries, is now increasingly failing in Cambodia, where artemisinin resistance is nearly fixed, which suggests that PPQ resistance has emerged and is spreading rapidly in the Greater Mekong Subregion. Recent reports have shown that amplification of the genes encoding plasmepsins 2 and 3 is a molecular marker of PPQ resistance; however, whether these enzymes play a role in the mechanism of resistance is currently unknown. We show here that inactivating the genes encoding plasmepsin 2 or 3 individually in P. falciparum reference strain 3D7 results in hypersusceptibility to PPQ. Interestingly, no significant differences in the susceptibility to other antimalarials were observed, which suggests specific roles of plasmepsins 2 and 3 in PPQ susceptibility. The piperaquine hyper-sensitivity of the plasmepsin-2-and-3-inactivated lines provides direct evidence that these enzymes modulate parasite susceptibility to PPQ in the context of a single copy of PfMDR1 and independent of Kelch13 mutations conferring artemisinin resistance.

Anthropometric Profile, Overweight/Obesity Prevalence, and Socioeconomic Impact in Moroccan Children Aged 6-12 Years Old with Autism Spectrum Disorder.

BACKGROUND: In addition to the inherent challenges of their condition, children with autism spectrum disorder (ASD) are also susceptible to the global obesity epidemic. However, concerning the prevalence of obesity within the Moroccan ASD pediatric population, data remain scarce. METHODS: A total of 258 children (boys = 195) aged 6 to 12 years old (mean = 9.4 ± 1.4) diagnosed with ASD participated in this study. Besides the body mass and height, four significant anthropometric markers for assessing obesity were examined: body mass index (BMI), body surface area (BSA), waist circumference (WC), and waist-to-height ratio (WHtR). Each anthropometric marker was categorized into one of three cardiometabolic risk levels based on the Z-scores and their corresponding percentiles. The distribution was as follows: low risk (≤84th percentile), high risk (85th-94th percentile), and very high risk (≥95th percentile). Subsequently, a multiple regression analysis was employed to develop an algorithm that generates a composite risk score. This score incorporates all the anthropometric variables simultaneously, while also weighting their individual contributions to the cardiometabolic risk. RESULTS: Children with ASD exhibit an anthropometric profile that markedly increases their susceptibility to cardiometabolic issues. While roughly 11% of the general Moroccan child population is overweight or obese, this figure soars to nearly 60% among children with ASD when considering the central adiposity markers. Furthermore, children from middle-class socioeconomic backgrounds display a more than threefold greater risk of developing overweight or obesity compared to their counterparts from lower socioeconomic backgrounds. CONCLUSIONS: This study has, for the first time, provided an up-to-date overview of the cardiometabolic risk in Moroccan children with ASD using traditional anthropometric measurements. The primary risk factor is clearly linked to central (abdominal) adiposity, which is recognized as the most deleterious. This study highlights the need to include general and central obesity markers. This study underscores the importance of incorporating both general and central adiposity markers for a more comprehensive assessment, and it emphasizes the need for closer monitoring within this high-risk population.

Investigation of Underlying Association between Anthropometric and Cardiorespiratory Fitness Markers among Overweight and Obese Adolescents in Canada.

BACKGROUND: Adolescents who experience overweight or obesity commonly persist in these conditions into adulthood, thereby elevating their vulnerability to health issues. The focus of this study is on health risk markers such as body mass index (BMI), waist circumference (WC), waist-to-height ratio (WHtR), body surface area (BSA), and cardiorespiratory fitness (CRF). The objectives include updating normative values for BMI, WC, WHtR, and BSA in Canadian adolescents, establishing cardiometabolic risk zones, and developing a composite score considering both anthropometric and CRF markers. METHODS: Involving 1864 adolescents, the study used the LMS method to generate percentile norms, stratified by age and sex. Cardiometabolic risk zones were established for each marker based on Z-scores, and a composite score was created. RESULTS: An increase in WC of 5.8 and 7.4 cm for boys and girls, respectively, was observed since 1981. Forward multiple regression analyses were conducted to assess the robustness and validity of the proposed model. The results indicated that the model explained nearly 90% (R2 = 0.890) of the common variance between the composite score and the retained independent variables. Moreover, the model demonstrated a mean absolute error (MAE) of approximately 6 percentiles, confirming its high precision. Furthermore, these analyses yielded key thresholds for identifying adolescents at risk: the 70th percentile for high cardiometabolic risk and the 85th percentile for very-high risk. CONCLUSIONS: Individually, WC or WHtR seem to be better markers for evaluating cardiometabolic risk than BMI during adolescence. However, CRF showed comparable importance to anthropometric markers in determining cardiometabolic risk. The simultaneous inclusion of anthropometric and CRF markers provides a better picture of the global cardiometabolic risk in adolescents.

Convergence of TCR and cytokine signaling leads to FOXO3a phosphorylation and drives the survival of CD4+ central memory T cells.

The molecular events involved in the establishment and maintenance of CD4+ central memory and effector memory T cells (TCM and TEM, respectively) are poorly understood. In this study, we demonstrate that ex vivo isolated TCM are more resistant to both spontaneous and Fas-induced apoptosis than TEM and have an increased capacity to proliferate and persist in vitro. Using global gene expression profiling, single cell proteomics, and functional assays, we show that the survival of CD4+ TCM depends, at least in part, on the activation and phosphorylation of signal transducer and activator of transcription 5a (STAT5a) and forkhead box O3a (FOXO3a). TCM showed a significant increase in the levels of phosphorylation of STAT5a compared with TEM in response to both IL-2 (P<0.04) and IL-7 (P<0.002); the latter is well known for its capacity to enhance T cell survival. Moreover, ex vivo TCM express higher levels of the transcriptionally inactive phosphorylated forms of FOXO3a and concomitantly lower levels of the proapoptotic FOXO3a target, Bim. Experiments aimed at blocking FOXO3a phosphorylation confirmed the role of this phosphoprotein in protecting TCM from apoptosis. Our results provide, for the first time in humans, an insight into molecular mechanisms that could be responsible for the longevity and persistence of CD4+ TCM.

Squaramide Tethered Clindamycin, Chloroquine, and Mortiamide Hybrids: Design, Synthesis, and Antimalarial Activity.

Malaria remains one of the major health problems in the world. In this work, a series of squaramide tethered chloroquine, clindamycin, and mortiamide D hybrids have been synthesized to assess their in vitro antiplasmodial activity against 3D7 (chloroquine-sensitive) and Dd2 strains of Plasmodium falciparum. The most active compound, a simple chloroquine analogue, displayed low nanomolar IC50 value against both strains (3 nM for 3D7 strain and 18 nM for Dd2 strain). Moreover, all molecular hybrids incorporating the hydroxychloroquine scaffold showed the most potent activities, exemplified with a chloroquine dimer, IC50 = 31 nM and 81 nM against 3D7 and Dd2 strains, respectively. These results highlight the first time use of clindamycin and mortiamide D as antimalarial molecular hybrids and establish these valuable hits for future optimization.

Chemical Composition and Antiplasmodial Activity of the Essential Oil of Rhododendron subarcticum Leaves from Nunavik, Québec, Canada.

Dwarf Labrador tea, Rhododendron subarcticum Harmaja, is a popular medicinal plant in use by First Nations of Northern Canada, but its phytochemistry has remained largely unexplored. We have isolated and characterized the essential oil from a population of this species harvested near the treeline in Nunavik, Québec. Analyses by gas chromatography-mass spectrometry (GC-MS) and gas chromatography/flame-ionization detection (GC/FID) led to the identification of 53 compounds; the main secondary metabolites were ascaridole (64.7% of the total FID area) and p-cymene (21.1%). Such a composition resembles a chemotype observed for R. tomentosum, a close relative found mainly in Europe and Asia, but has never been attributed to R. subarcticum. Growth inhibition assays against different strains of Plasmodium falciparum (3D7, Dd2), the parasite responsible for the most severe form of malaria, were conducted with either the R. subarcticum's essential oil or the isolated ascaridole. Our results show that the essential oil's biological activity can be attributed to ascaridole as its IC50 is more than twice that of ascaridole [ascaridole's IC50 values are 147.3 nM (3D7) and 104.9 nM (Dd2)].

Updating normative cross-sectional values and secular trends in body mass, body height and body mass index among Québec children and adolescents.

OBJECTIVE: The main objective of this study was to examine secular trends in body mass, body height and body mass index (BMI) from measured rather than self-reported values between 1972 and 2017. METHODS: A total of 4500 students (males = 51%) were selected from a stratified sampling. The age range varied between 6.0 and 17.9 years. The sample came from 24 elementary schools and 12 high schools located in six urban cities from the province of Québec. All the tests selected were based on standardized procedures that are recognized as valid and reliable. Standardization and modeling of smoothed percentile curves for each variable for both sexes were produced. RESULTS: Regional variations between youth from the province of Québec and those from all other Canadian provinces confirm the relevance of using data specific to the target population. Comparisons with the 1972 and 1982 data show an important increase in body mass (~ 7 kg, i.e. 16.4%) and BMI (~ 1.4 kg·m-2, i.e. 19.9%) with minor change in body height (~ 1.8 cm, i.e. 3.9%). Youth from low-income backgrounds (p = 0.001) as well as those living in large urban cities (p = 0.002) see their probability of developing overweight or obesity greatly increase (low-income = 2.1 times; large urban cities = 1.3 times). However, overweight and obesity rates seem to have stabilized at around 21% since 2004. CONCLUSION: This study provides up-to-date data on factors that contribute to the prevalence of overweight and obesity in youth in urban settings of Québec, and will be instrumental in guiding public health strategies designed to optimize growth outcomes.

RéSUMé: OBJECTIF: L'objectif principal de cette étude était d'examiner les tendances séculaires de la masse corporelle, de la taille et de l'indice de masse corporelle (IMC) de 1972 à 2017 à partir de valeurs mesurées plutôt que de valeurs autodéclarées. MéTHODE: Un total de 4 500 étudiants (garçons = 51 %) ont été sélectionnés à partir d'un échantillon stratifié. La fourchette d'âge variait entre 6,0 et 17,9 ans. L'échantillon provenait de 24 écoles primaires et 12 écoles secondaires situées dans six villes de la province de Québec (Canada). Tous les tests sélectionnés reposaient sur des procédures standardisées, reconnues comme valides et fidèles. Une standardisation et une modélisation des courbes centiles lissées ont été produites pour chaque variable pour les deux sexes. RéSULTATS: Les variations régionales entre les jeunes du Québec et ceux du reste du Canada confirment la pertinence d'utiliser des données spécifiques à la population cible. Les comparaisons entre les données de 1972 et de 1982 montrent une augmentation importante de la masse corporelle et de l'IMC (p ≤ 0,05) sans changement important de la taille corporelle. Les jeunes issus de milieux à faibles revenus (p = 0,001) ainsi que ceux vivant dans les grandes villes (p = 0,002) voient leurs risques de développer un surpoids ou de l'obésité augmenter considérablement (faibles revenus = 2,1 fois; grandes villes urbaines = 1,3 fois). Toutefois, les taux de surpoids et d'obésité semblent s'être stabilisés autour de 21 % depuis 2004. CONCLUSION: Cette étude fournit des données à jour sur les facteurs qui contribuent à la prévalence de l'embonpoint et de l'obésité chez les jeunes des milieux urbains du Québec et contribuera à orienter les stratégies de santé publique afin d'optimiser le suivi sur la croissance physique des jeunes.

A Phosphoinositide-Binding Protein Acts in the Trafficking Pathway of Hemoglobin in the Malaria Parasite Plasmodium falciparum.

Phosphoinositide lipids play key roles in a variety of processes in eukaryotic cells, but our understanding of their functions in the malaria parasite Plasmodium falciparum is still very much limited. To gain a deeper comprehension of the roles of phosphoinositides in this important pathogen, we attempted gene inactivation for 24 putative effectors of phosphoinositide metabolism. Our results reveal that 79% of the candidates are refractory to genetic deletion and are therefore potentially essential for parasite growth. Inactivation of the gene coding for a Plasmodium-specific putative phosphoinositide-binding protein, which we named PfPX1, results in a severe growth defect. We show that PfPX1 likely binds phosphatidylinositol-3-phosphate and that it localizes to the membrane of the digestive vacuole of the parasite and to vesicles filled with host cell cytosol and labeled with endocytic markers. Critically, we provide evidence that it is important in the trafficking pathway of hemoglobin from the host erythrocyte to the digestive vacuole. Finally, inactivation of PfPX1 renders parasites resistant to artemisinin, the frontline antimalarial drug. Globally, the minimal redundancy in the putative phosphoinositide proteins uncovered in our work supports that targeting this pathway has potential for antimalarial drug development. Moreover, our identification of a phosphoinositide-binding protein critical for the trafficking of hemoglobin provides key insight into this essential process. IMPORTANCE Malaria represents an enormous burden for a significant proportion of humanity, and the lack of vaccines and problems with drug resistance to all antimalarials demonstrate the need to develop new therapeutics. Inhibitors of phosphoinositide metabolism are currently being developed as antimalarials but our understanding of this biological pathway is incomplete. The malaria parasite lives inside human red blood cells where it imports hemoglobin to cover some of its nutritional needs. In this work, we have identified a phosphoinositide-binding protein that is important for the transport of hemoglobin in the parasite. Inactivation of this protein decreases the ability of the parasite to proliferate. Our results have therefore identified a potential new target for antimalarial development.

Fragment-Based Phenotypic Lead Discovery To Identify New Drug Seeds That Target Infectious Diseases.

Fragment-based lead discovery has emerged over the last decades as one of the most powerful techniques for identifying starting chemical matter to target specific proteins or nucleic acids in vitro. However, the use of such low-molecular-weight fragment molecules in cell-based phenotypic assays has been historically avoided because of concerns that bioassays would be insufficiently sensitive to detect the limited potency expected for such small molecules and that the high concentrations required would likely implicate undesirable artifacts. Herein, we applied phenotype cell-based screens using a curated fragment library to identify inhibitors against a range of pathogens including Leishmania, Plasmodium falciparum, Neisseria, Mycobacterium, and flaviviruses. This proof-of-concept shows that fragment-based phenotypic lead discovery (FPLD) can serve as a promising complementary approach for tackling infectious diseases and other drug-discovery programs.

Optimization of a highly standardized carboxyfluorescein succinimidyl ester flow cytometry panel and gating strategy design using discriminative information measure evaluation.

The design of a panel to identify target cell subsets in flow cytometry can be difficult when specific markers unique to each cell subset do not exist, and a combination of parameters must be used to identify target cells of interest and exclude irrelevant events. Thus, the ability to objectively measure the contribution of a parameter or group of parameters toward target cell identification independent of any gating strategy could be very helpful for both panel design and gating strategy design. In this article, we propose a discriminative information measure evaluation (DIME) based on statistical mixture modeling; DIME is a numerical measure of the contribution of different parameters towards discriminating a target cell subset from all the others derived from the fitted posterior distribution of a Gaussian mixture model. Informally, DIME measures the "usefulness" of each parameter for identifying a target cell subset. We show how DIME provides an objective basis for inclusion or exclusion of specific parameters in a panel, and how ranked sets of such parameters can be used to optimize gating strategies. An illustrative example of the application of DIME to streamline the gating strategy for a highly standardized carboxyfluorescein succinimidyl ester (CFSE) assay is described.

Total synthesis and antimalarial activity of mortiamides A-D.

Mortiamides A-D (1-4) are head-to-tail cyclic heptapeptides that were identified from a novel Mortierella sp. isolate obtained from marine sediments from Northern Canada. Herein we report the first total synthesis of mortiamides A-D (1-4) on a solid support by concomitant cyclization/cleavage without any oligomerization side reactions, and overall yields up to 48%. We also report on the antiplasmodial activity of mortiamides A-D (1-4). We show that three out of the four tested mortiamides (A, B and D) have moderate antiplasmodial activity, while mortiamide D (4) exhibits low micromolar activity.

Mass spectra alignment using virtual lock-masses.

Mass spectrometry is a valued method to evaluate the metabolomics content of a biological sample. The recent advent of rapid ionization technologies such as Laser Diode Thermal Desorption (LDTD) and Direct Analysis in Real Time (DART) has rendered high-throughput mass spectrometry possible. It is used for large-scale comparative analysis of populations of samples. In practice, many factors resulting from the environment, the protocol, and even the instrument itself, can lead to minor discrepancies between spectra, rendering automated comparative analysis difficult. In this work, a sequence/pipeline of algorithms to correct variations between spectra is proposed. The algorithms correct multiple spectra by identifying peaks that are common to all and, from those, computes a spectrum-specific correction. We show that these algorithms increase comparability within large datasets of spectra, facilitating comparative analysis, such as machine learning.

Stage specific gene expression and cellular localization of two isoforms of the serine hydroxymethyltransferase in the protozoan parasite Leishmania.

Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions.

Pre-vaccination inflammation and B-cell signalling predict age-related hyporesponse to hepatitis B vaccination.

Aging is associated with hyporesponse to vaccination, whose mechanisms remain unclear. In this study hepatitis B virus (HBV)-naive older adults received three vaccines, including one against HBV. Here we show, using transcriptional and cytometric profiling of whole blood collected before vaccination, that heightened expression of genes that augment B-cell responses and higher memory B-cell frequencies correlate with stronger responses to HBV vaccine. In contrast, higher levels of inflammatory response transcripts and increased frequencies of pro-inflammatory innate cells correlate with weaker responses to this vaccine. Increased numbers of erythrocytes and the haem-induced response also correlate with poor response to the HBV vaccine. A transcriptomics-based pre-vaccination predictor of response to HBV vaccine is built and validated in distinct sets of older adults. This moderately accurate (area under the curve≈65%) but robust signature is supported by flow cytometry and cytokine profiling. This study is the first that identifies baseline predictors and mechanisms of response to the HBV vaccine.

An in vitro co-infection model to study Plasmodium falciparum-HIV-1 interactions in human primary monocyte-derived immune cells.

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission. The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells, but that it decreased HIV-1 infection of macrophages(8). To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed. Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.