Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum: Biochemical properties and possible involvement in MAPK regulation

We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials

Marine Actinomycetes: A New Source of Compounds against the Human Malaria Parasite

Background Malaria continues to be a devastating parasitic disease that causes the death of 2 million individuals annually. The increase in multi-drug resistance together with the absence of an efficient vaccine hastens the need for speedy and comprehensive antimalarial drug discovery and development. Throughout history, traditional herbal remedies or natural products have been a reliable source of antimalarial agents, e.g. quinine and artemisinin. Today, one emerging source of small molecule drug leads is the world's oceans. Included among the source of marine natural products are marine microorganisms such as the recently described actinomycete. Members of the genus Salinispora have yielded a wealth of new secondary metabolites including salinosporamide A, a molecule currently advancing through clinical trials as an anticancer agent. Because of the biological activity of metabolites being isolated from marine microorganisms, our group became interested in exploring the potential efficacy of these compounds against the malaria parasite.[br/] Methods We screened 80 bacterial crude extracts for their activity against malaria growth. We established that the pure compound, salinosporamide A, produced by the marine actinomycete, Salinispora tropica, shows strong inhibitory activity against the erythrocytic stages of the parasite cycle. Biochemical experiments support the likely inhibition of the parasite 20S proteasome. Crystal structure modeling of salinosporamide A and the parasite catalytic 20S subunit further confirm this hypothesis. Ultimately we showed that salinosporamide A protected mice against deadly malaria infection when administered at an extremely low dosage.[br/] Conclusion These findings underline the potential of secondary metabolites, derived from marine microorganisms, to inhibit Plasmodium growth. More specifically, we highlight the effect of proteasome inhibitors such as salinosporamide A on in vitro and in vivo parasite development. Salinosporamide A (NPI-0052) now being advanced to phase I trials for the treatment of refractory multiple myeloma will need to be further explored to evaluate the safety profile for its use against malaria

Assessing the effectiveness and observing fidelity of a psychosocial support program for Rohingya refugee mothers and their children in Cox’s Bazar, Bangladesh

Introduction: Despite the well-recognized risk poor maternal mental health poses to early child development, it is still rarely addressed in global health programming, especially in humanitarian settings where access to health and mental health infrastructures may be limited. Recognizing the critical role of maternal psychosocial wellness in addressing the health and development of children in conflict, Action contre La Faim/Action Against Hunger (ACF) developed the Baby Friendly Spaces (BFS) program. BFS is a holistic, evidenced-based psychosocial support program that aims to enhance mothers’ wellbeing, internal resources, and child caring skills in order to create a buffer against the deleterious health and developmental impacts of conflict on children. Objectives: In Bangladesh, we sought to evaluate the effectiveness of a psychosocial support program for Rohingya refugee mothers and their malnourished children under two years old living in Cox’s Bazar’s camps. Methods: For this study, we used a matched pair randomization, where ten BFS program sites were allocated to either continue providing services “as usual” or to an “enhanced BFS program” after re-training and providing continuous supportive supervision of the BFS staff throughout the trial period. 600 mothers and their children were enrolled in the study and attended psychosocial stimulation activities related to child care practices and care for women. Data were collected at baseline and 8-week follow-up. Primary outcomes included maternal distress and wellbeing, functioning, and coping. For implementation purpose, a survey was administered on confidence at work for all BFS staff and a fidelity observation assessment was conducted. Results: Relative to “as usual” sites, mothers in enhanced implementation sites reported greater reductions in distress (B=-.30) and improvement in wellbeing (B=.58). These differences were small, but marginally significant (p=.058; p=.038) with standard estimation; There was no significant difference between the two groups for daily functioning and coping. BFS providers in “enhanced BFS program” reported higher confidence in service delivery than their colleagues (p=.01). Fidelity varied widely across different components, with some very high and some very low adherence. There tended to be better adherence to procedures in group versus individual sessions and for some specific activities across domains, for enhanced versus standard BFS. Conclusions: Findings highlight the value of innovative study approaches for real-world evidence generation. Small but feasible adjustments to implementation can both improve program delivery for maximizing impact. Consequently, low-intensity psychosocial support activities holds potential for reducing distress and improving subjective well-being of conflict affected mothers

Quantum Phase Transition in a Resonant Level Coupled to Interacting Leads

An interacting one-dimensional electron system, the Luttinger liquid, is distinct from the "conventional" Fermi liquids formed by interacting electrons in two and three dimensions. Some of its most spectacular properties are revealed in the process of electron tunneling: as a function of the applied bias or temperature the tunneling current demonstrates a non-trivial power-law suppression. Here, we create a system which emulates tunneling in a Luttinger liquid, by controlling the interaction of the tunneling electron with its environment. We further replace a single tunneling barrier with a double-barrier resonant level structure and investigate resonant tunneling between Luttinger liquids. For the first time, we observe perfect transparency of the resonant level embedded in the interacting environment, while the width of the resonance tends to zero. We argue that this unique behavior results from many-body physics of interacting electrons and signals the presence of a quantum phase transition (QPT). In our samples many parameters, including the interaction strength, can be precisely controlled; thus, we have created an attractive model system for studying quantum critical phenomena in general. Our work therefore has broadly reaching implications for understanding QPTs in more complex systems, such as cold atoms and strongly correlated bulk materials.Comment: 11 pages total (main text + supplementary

PlasmoDraft: a database of Plasmodium falciparum gene function predictions based on postgenomic data

<p>Abstract</p> <p>Background</p> <p>Of the 5 484 predicted proteins of <it>Plasmodium falciparum</it>, the main causative agent of malaria, about 60% do not have sufficient sequence similarity with proteins in other organisms to warrant provision of functional assignments. Non-homology methods are thus needed to obtain functional clues for these uncharacterized genes.</p> <p>Results</p> <p>We present PlasmoDraft <url>http://atgc.lirmm.fr/PlasmoDraft/</url>, a database of Gene Ontology (GO) annotation predictions for <it>P. falciparum </it>genes based on postgenomic data. Predictions of PlasmoDraft are achieved with a <it>Guilt By Association </it>method named Gonna. This involves (1) a predictor that proposes GO annotations for a gene based on the similarity of its profile (measured with transcriptome, proteome or interactome data) with genes already annotated by GeneDB; (2) a procedure that estimates the confidence of the predictions achieved with each data source; (3) a procedure that combines all data sources to provide a global summary and confidence estimate of the predictions. Gonna has been applied to all <it>P. falciparum </it>genes using most publicly available transcriptome, proteome and interactome data sources. Gonna provides predictions for numerous genes without any annotations. For example, 2 434 genes without any annotations in the Biological Process ontology are associated with specific GO terms (<it>e.g</it>. Rosetting, Antigenic variation), and among these, 841 have confidence values above 50%. In the Cellular Component and Molecular Function ontologies, 1 905 and 1 540 uncharacterized genes are associated with specific GO terms, respectively (740 and 329 with confidence value above 50%).</p> <p>Conclusion</p> <p>All predictions along with their confidence values have been compiled in PlasmoDraft, which thus provides an extensive database of GO annotation predictions that can be achieved with these data sources. The database can be accessed in different ways. A global view allows for a quick inspection of the GO terms that are predicted with high confidence, depending on the various data sources. A gene view and a GO term view allow for the search of potential GO terms attached to a given gene, and genes that potentially belong to a given GO term.</p

A Systematic Map of Genetic Variation in Plasmodium falciparum

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified ≈500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs

A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites

Transcriptional Profiling of Plasmodium falciparum Parasites from Patients with Severe Malaria Identifies Distinct Low vs. High Parasitemic Clusters

Background: In the past decade, estimates of malaria infections have dropped from 500 million to 225 million per year; likewise, mortality rates have dropped from 3 million to 791,000 per year. However, approximately 90% of these deaths continue to occur in sub-Saharan Africa, and 85% involve children less than 5 years of age. Malaria mortality in children generally results from one or more of the following clinical syndromes: severe anemia, acidosis, and cerebral malaria. Although much is known about the clinical and pathological manifestations of CM, insights into the biology of the malaria parasite, specifically transcription during this manifestation of severe infection, are lacking. Methods and Findings: We collected peripheral blood from children meeting the clinical case definition of cerebral malaria from a cohort in Malawi, examined the patients for the presence or absence of malaria retinopathy, and performed whole genome transcriptional profiling for Plasmodium falciparum using a custom designed Affymetrix array. We identified two distinct physiological states that showed highly significant association with the level of parasitemia. We compared both groups of Malawi expression profiles with our previously acquired ex vivo expression profiles of parasites derived from infected patients with mild disease; a large collection of in vitro Plasmodium falciparum life cycle gene expression profiles; and an extensively annotated compendium of expression data from Saccharomyces cerevisiae. The high parasitemia patient group demonstrated a unique biology with elevated expression of Hrd1, a member of endoplasmic reticulum-associated protein degradation system. Conclusions: The presence of a unique high parasitemia state may be indicative of the parasite biology of the clinically recognized hyperparasitemic severe disease syndrome