A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

A Potent Kalihinol Analogue Disrupts Apicoplast Function and Vesicular Trafficking in P. falciparum Malaria.

Here we report the discovery of MED6-189, a new analogue of the kalihinol family of isocyanoterpene (ICT) natural products. MED6-189 is effective against drug-sensitive and -resistant P. falciparum strains blocking both intraerythrocytic asexual replication and sexual differentiation. This compound was also effective against P. knowlesi and P. cynomolgi. In vivo efficacy studies using a humanized mouse model of malaria confirms strong efficacy of the compound in animals with no apparent hemolytic activity or apparent toxicity. Complementary chemical biology, molecular biology, genomics and cell biological analyses revealed that MED6-189 primarily targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses in P. falciparum revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. The high potency of MED6-189 in vitro and in vivo, its broad range of efficacy, excellent therapeutic profile, and unique mode of action make it an excellent addition to the antimalarial drug pipeline.

PfMORC protein regulates chromatin accessibility and transcriptional repression in the human malaria parasite, P. falciparum.

The environmental challenges the human malaria parasite, Plasmodium falciparum, faces during its progression into its various lifecycle stages warrant the use of effective and highly regulated access to chromatin for transcriptional regulation. Microrchidia (MORC) proteins have been implicated in DNA compaction and gene silencing across plant and animal kingdoms. Accumulating evidence has shed light into the role MORC protein plays as a transcriptional switch in apicomplexan parasites. In this study, using CRISPR/Cas9 genome editing tool along with complementary molecular and genomics approaches, we demonstrate that PfMORC not only modulates chromatin structure and heterochromatin formation throughout the parasite erythrocytic cycle, but is also essential to the parasite survival. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments suggest that PfMORC binds to not only sub-telomeric regions and genes involved in antigenic variation but is also most likely a key modulator of stage transition. Protein knockdown experiments followed by chromatin conformation capture (Hi-C) studies indicate that downregulation of PfMORC induces the collapse of the parasite heterochromatin structure leading to its death. All together these findings confirm that PfMORC plays a crucial role in chromatin structure and gene regulation, validating this factor as a strong candidate for novel antimalarial strategies.

Impact of maternal mental health interventions on child-related outcomes in low- and middle-income countries: a systematic review and meta-analysis.

AIMS: Observational studies have shown a relationship between maternal mental health (MMH) and child development, but few studies have evaluated whether MMH interventions improve child-related outcomes, particularly in low- and middle-income countries. The objective of this review is to synthesise findings on the effectiveness of MMH interventions to improve child-related outcomes in low- and middle-income countries (LMICs). METHODS: We searched for randomised controlled trials conducted in LMICs evaluating interventions with a MMH component and reporting children's outcomes. Meta-analysis was performed on outcomes included in at least two trials. RESULTS: We identified 21 trials with 28 284 mother-child dyads. Most trials were conducted in middle-income countries, evaluating home visiting interventions delivered by general health workers, starting in the third trimester of pregnancy. Only ten trials described acceptable methods for blinding outcome assessors. Four trials showed high risk of bias in at least two of the seven domains assessed in this review. Narrative synthesis showed promising but inconclusive findings for child-related outcomes. Meta-analysis identified a sizeable impact of interventions on exclusive breastfeeding (risk ratio = 1.39, 95% confidence interval (CI): 1.13-1.71, ten trials, N = 4749 mother-child dyads, I2 = 61%) and a small effect on child height-for-age at 6-months (std. mean difference = 0.13, 95% CI: 0.02-0.24, three trials, N = 1388, I2 = 0%). Meta-analyses did not identify intervention benefits for child cognitive and other growth outcomes; however, few trials measured these outcomes. CONCLUSIONS: These findings support the importance of MMH to improve child-related outcomes in LMICs, particularly exclusive breastfeeding. Given, the small number of trials and methodological limitations, more rigorous trials should be conducted.

Lessons learned evaluating the baby friendly spaces program for south Sudanese refugees in Gambella, Ethiopia: strengthening research and programmatic partnerships to address maternal and child health and psychosocial needs in humanitarian emergencies.

BACKGROUND: During humanitarian crises, women and children are particularly vulnerable to morbidity and mortality. To address this problem, integrated child health interventions that include support for the well-being of mothers must be adapted and assessed in humanitarian settings. Baby Friendly Spaces (BFS) is a holistic program that aims to improve the health and wellbeing of pregnant and lactating women and their children under two years of age by providing psychosocial support and enhancing positive infant and young child-care practices. Using a mixed-methods, pre-post design, this study explored ways to strengthen the implementation and acceptability of the BFS program, and assess outcomes associated with participation among South Sudanese mothers and their children living in the Nguenyyiel refugee camp in Gambella, Ethiopia. DISCUSSION: A stronger evidence-base for integrated maternal and child health interventions, like BFS, in humanitarian emergencies is needed, but effectively conducting this type of research in unstable settings means encountering and working through myriad challenges. In this paper we discuss lessons learned while implementing this study, including, challenges related to ongoing local political and tribal conflicts and extreme conditions; implementation of a new digital data monitoring system; staff capacity building and turnover; and measurement were encountered. Strategies to mitigate such challenges included hiring and training new staff members. Regular weekly skype calls were held between Action Against Hunger Paris headquarters, the Action Against Hunger team in Gambella and Johns Hopkins' academic partners to follow study implementation progress and troubleshoot any emerging issues. Staff capacity building strategies included holding brief and focused trainings continuously throughout the study for both new and current staff members. Lastly, we engaged local Nuer staff members to help ensure study measures and interview questions were understandable among study participants. CONCLUSIONS: Research focused on strengthening program implementation is critically important for improving maternal and child health in humanitarian emergencies. Research in such settings demands critical problem-solving skills, strong supervision systems, flexibility in timeline and logistics, and tailor-made training for program and research staff members and context- adapted strategies for retaining existing staff.

PacBio assembly of a Plasmodium knowlesi genome sequence with Hi-C correction and manual annotation of the SICAvar gene family.

Plasmodium knowlesi has risen in importance as a zoonotic parasite that has been causing regular episodes of malaria throughout South East Asia. The P. knowlesi genome sequence generated in 2008 highlighted and confirmed many similarities and differences in Plasmodium species, including a global view of several multigene families, such as the large SICAvar multigene family encoding the variant antigens known as the schizont-infected cell agglutination proteins. However, repetitive DNA sequences are the bane of any genome project, and this and other Plasmodium genome projects have not been immune to the gaps, rearrangements and other pitfalls created by these genomic features. Today, long-read PacBio and chromatin conformation technologies are overcoming such obstacles. Here, based on the use of these technologies, we present a highly refined de novo P. knowlesi genome sequence of the Pk1(A+) clone. This sequence and annotation, referred to as the 'MaHPIC Pk genome sequence', includes manual annotation of the SICAvar gene family with 136 full-length members categorized as type I or II. This sequence provides a framework that will permit a better understanding of the SICAvar repertoire, selective pressures acting on this gene family and mechanisms of antigenic variation in this species and other pathogens.

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi - macaque model systems and summarizing exciting new progress in this area of research.

Genomics and integrated systems biology in Plasmodium falciparum: a path to malaria control and eradication.

The first draft of the human malaria parasite's genome was released in 2002. Since then, the malaria scientific community has witnessed a steady embrace of new and powerful functional genomic studies. Over the years, these approaches have slowly revolutionized malaria research and enabled the comprehensive, unbiased investigation of various aspects of the parasite's biology. These genome-wide analyses delivered a refined annotation of the parasite's genome, delivered a better knowledge of its RNA, proteins and metabolite derivatives, and fostered the discovery of new vaccine and drug targets. Despite the positive impacts of these genomic studies, most research and investment still focus on protein targets, drugs and vaccine candidates that were known before the publication of the parasite genome sequence. However, recent access to next-generation sequencing technologies, along with an increased number of genome-wide applications, is expanding the impact of the parasite genome on biomedical research, contributing to a paradigm shift in research activities that may possibly lead to new optimized diagnosis and treatments. This review provides an update of Plasmodium falciparum genome sequences and an overview of the rapid development of genomics and system biology applications that have an immense potential of creating powerful tools for a successful malaria eradication campaign.

Novel Gene Discovery in the Human Malaria Parasite using Nucleosome Positioning Data.

Recent genome-wide studies on nucleosome positioning in model organisms have shown strong evidence that nucleosome landscapes in the proximity of protein-coding genes exhibit regular characteristic patterns. Here, we propose a computational framework to discover novel genes in the human malaria parasite genome P. falciparum using nucleosome positioning inferred from MAINE-seq data. We rely on a classifier trained on the nucleosome landscape profiles of experimentally verified genes, and then used to discover new genes (without considering the primary DNA sequence). Cross-validation experiments show that our classifier is very accurate. About two thirds of the locations reported by the classifier match experimentally determined expressed sequence tags in GenBank, for which no gene has been annotated in the human malaria parasite.

Distinct physiological states of Plasmodium falciparum in malaria-infected patients.

Infection with the malaria parasite Plasmodium falciparum leads to widely different clinical conditions in children, ranging from mild flu-like symptoms to coma and death. Despite the immense medical implications, the genetic and molecular basis of this diversity remains largely unknown. Studies of in vitro gene expression have found few transcriptional differences between different parasite strains. Here we present a large study of in vivo expression profiles of parasites derived directly from blood samples from infected patients. The in vivo expression profiles define three distinct transcriptional states. The biological basis of these states can be interpreted by comparison with an extensive compendium of expression data in the yeast Saccharomyces cerevisiae. The three states in vivo closely resemble, first, active growth based on glycolytic metabolism, second, a starvation response accompanied by metabolism of alternative carbon sources, and third, an environmental stress response. The glycolytic state is highly similar to the known profile of the ring stage in vitro, but the other states have not been observed in vitro. The results reveal a previously unknown physiological diversity in the in vivo biology of the malaria parasite, in particular evidence for a functional mitochondrion in the asexual-stage parasite, and indicate in vivo and in vitro studies to determine how this variation may affect disease manifestations and treatment.

Influence of human p16(INK4) and p21(CIP1) on the in vitro activity of recombinant Plasmodium falciparum cyclin-dependent protein kinases.

The regulatory mechanisms of most cyclin dependent protein kinases (CDKs) are well understood and are highly conserved in eukaryotes. CDKs from the malaria parasite, Plasmodium falciparum, appear to be regulated in a similar manner with regard to cyclin binding and phosphorylation. In order to further understand their regulatory mechanisms, we examined two classes of cyclin dependent kinase inhibitors (CDIs) to inhibit a panel of plasmodial CDKs. We find that Pfmrk and PfPK5 are inhibited by heterologous p21(CIP1) with varying degrees of inhibition. In contrast, PfPK6, a kinase with sequence features characteristic of both a CDK and MAP kinase, is unaffected by this CDI. Furthermore, the CDK4/6 specific CDI, p16(INK4), fails to inhibit these plasmodial CDKs. Taken together, these results suggest that plasmodial CDKs may be regulated by the binding of inhibitory proteins in vivo.

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.

Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors.

BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.

Activation of a Plasmodium falciparum cdc2-related kinase by heterologous p25 and cyclin H. Functional characterization of a P. falciparum cyclin homologue.

Several Plasmodium falciparum genes encoding cdc2-related protein kinases have been identified, but the modalities of their regulation remains largely unexplored. In the present study, we investigated the regulation in vitro of PfPK5, a putative homologue of Cdk1 (cdc2) in P. falciparum. We show that (i) PfPK5 is efficiently activated by heterologous (human) cyclin H and p25, a cyclin-like molecule that specifically activates human Cdk5; (ii) the activated enzyme can be inhibited by chemical Cdk inhibitors; (iii) Pfmrk, a putative P. falciparum homologue of the Cdk-activating kinase, does neither activate nor phosphorylate PfPK5; and (iv) PfPK5 is able to autophosphorylate in the presence of a cyclin. Taken together, these results suggest that the regulation of Plasmodium Cdks may differ in important aspects from that of their human counterparts. Furthermore, we cloned an open reading frame encoding a novel P. falciparum protein possessing maximal homology to cyclin H from various organisms, and we show that this protein, called Pfcyc-1, is able to activate recombinant PfPK5 in vitro with an efficiency similar to that of human cyclin H and p25. This work opens the way to the development of screening procedures aimed at identifying compounds that specifically target the parasite Cdks.

[Quantified EEG and psychometric effects of 3 doses of dexfenfluramine in the young adult]. / Effets EEG quantifiés et psychométriques de 3 doses de dexfenfluramine chez l'adulte jeune.

In an acute, double-blind placebo-controlled study, three groups of nine healthy subjects were included. Each group received, in randomized and weekly intervals, a single oral dose of dexfenfluramine (15 mg, 30 mg or 60 mg) or placebo. Psychometric and EEG studies were carried out before as well as 1, 2, 3, 4, 5 and 6 h after drug administration. Changes from predrug to postdrug conditions for each time were determined by the ratio of absolute spectral power and compared to the evolution of power spectra under placebo conditions. Statistical evaluation was done with ANOVA. Dexfenfluramine induced a dose-dependent decrease of power spectra for theta and alpha 1 bands and an increase for the beta band. Topographic brain mapping of these significant changes displayed a central and posterior decrease for theta and alpha 1 bands, and a temporal localization for the beta-power. Maps of relative power-enhanced changes were seen in absolute power and provided false displays. Psychometric evaluation of dexfenfluramine effects only showed trends to extraversion and increased mood scores but no statistical significance was found. Arousal and performance tests were unchanged. These results suggest that qEEG variations are more sensitive to serotonergic drug effects than psychometric investigations. It appears that spontaneous EEG power spectra variations with time must be accounted for by drug evaluation. Absolute power variations are more reliable than relative power. Methodological implications are discussed.

Persistence of habituation deficits after neurological recovery from severe thiamine deprivation.

Rats were fed a thiamine-deficient diet for 4 weeks and injected daily with pyrithiamine during the last two weeks of the diet. This regime induced severe neurological anomalies such as ataxia, loss of righting reflex and visual place reflex, and finally full tonic-clonic seizures. These symptoms are reminiscent of the clinical Wernicke-Korsakoff syndrome. Injection of thiamine dramatically reversed these symptoms within one or two hours as seen in Wernicke patients. Six weeks later these rats showed a marked deficit in habituation of exploratory behavior and of the auditory orienting response. To what extent this chronic deficit in habituation contributes to the cognitive dysfunctions seen in Wernicke-Korsakoff patients is discussed. Histological examination of the brains of 6 of the rats revealed a heterogeneous pattern of damage to the brainstem, including mamillary bodies and several thalamic nuclei, reminiscent of that seen in Korsakoff patients. In addition there were many dark abnormal cells in limited fields of hippocampus neocortex and thalamus in almost all animals.

Age-related variation of EEG responses to clonidine, prazosin and yohimbine in rats.

The EEG-effects induced by intraperitoneal administration of clonidine, prazosin and yohimbine to 8 and 22 month old rats were compared. Clonidine (0.01 mg/kg) and prazosin (1 mg/kg) increased spectral powers, yohimbine (0.5 mg/kg) decreased them. In the older rats, EEG variations were smaller for prazosin and yohimbine, but larger for clonidine. These findings show that alpha receptor mediated influences on EEG are changed during aging and show that quantified EEG gives a picture of age related changes in the functional state of the neurotransmitter systems.