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Abstract Background Malaria caused by Plasmodium vivax is an experimentally neglected severe disease with a substantial burden on human health. Because of technical limitations, little is known about the biology of this important human pathogen. Whole genome analysis methods on patient-derived material are thus likely to have a substantial impact on our understanding of P. vivax pathogenesis and epidemiology. For example, it will allow study of the evolution and population biology of the parasite, allow parasite transmission patterns to be characterized, and may facilitate the identification of new drug resistance genes. Because parasitemias are typically low and the parasite cannot be readily cultured, on-site leukocyte depletion of blood samples is typically needed to remove human DNA that may be 1000X more abundant than parasite DNA. These features have precluded the analysis of archived blood samples and require the presence of laboratories in close proximity to the collection of field samples for optimal pre-cryopreservation sample preparation. Results Here we show that in-solution hybridization capture can be used to extract P. vivax DNA from human contaminating DNA in the laboratory without the need for on-site leukocyte filtration. Using a whole genome capture method, we were able to enrich P. vivax DNA from bulk genomic DNA from less than 0.5% to a median of 55% (range 20%-80%). This level of enrichment allows for efficient analysis of the samples by whole genome sequencing and does not introduce any gross biases into the data. With this method, we obtained greater than 5X coverage across 93% of the P. vivax genome for four P. vivax strains from Iquitos, Peru, which is similar to our results using leukocyte filtration (greater than 5X coverage across 96% ). Conclusion The whole genome capture technique will enable more efficient whole genome analysis of P. vivax from a larger geographic region and from valuable archived sample collections.

Abstract Long non-coding RNAs (lncRNA) are being increasingly recognized as important regulators of gene expression. A recent paper in Genome Biology reports the identification of a lncRNA family in Plasmodium falciparum, the cause of the most deadly form of malaria, that may help to explain the mechanism of antigenic variation in virulence genes of this important pathogen. See Research article: http://genomebiology.com/2011/12/6/R56/abstract

Abstract Background Epigenetic modifications of histones and regulation of chromatin structure have been implicated in regulation of virulence gene families in P. falciparum. To better understand chromatin-mediated gene regulation, we used a high-density oligonucleotide microarray to map the position and enrichment of nucleosomes across the entire genome of P. falciparum at three time points of the intra-erythrocytic developmental cycle (IDC) in vitro. We used an unmodified histone H4 antibody for chromatin immunoprecipitation of nucleosome-bound DNA. Results We observed generally low nucleosomal occupancy of intergenic regions and higher occupancy of protein coding regions. In contract to the overall small fluctuation of nucleosomal occupancy in most coding regions throughout the IDC, subtelomeric genes encoding surface proteins such as var and rif, as well as some core chromosomal genes such as transcription factors, showed large changes in chromatin structure. Telomeres harbored a region with the highest nucleosomal occupancy of the genome and also exhibited large changes with higher nucleosomal occupancy at schizont stages. While many of these subtelomeric genes were previously shown to be modified by H3K9 trimethylation, we also identified some housekeeping genes in core chromosome regions that showed extensive changes in chromatin structure but do not contain this modification. tRNA and basal transcription factor genes showed low nucleosomal occupancy at all times, suggesting of an open chromatin structure that might be permissive for constitutively high levels of expression. Generally, nucleosomal occupancy was not correlated with the steady-state mRNA levels. Several var genes were exceptions: the var gene with the highest expression level showed the lowest nucleosomal occupancy, and selection of parasites for var2CSA expression resulted in lower nucleosomal occupancy at the var2CSA locus. We identified nucleosome-free regions in intergenic regions that may serve as transcription start sites or transcription factor binding sites. Using the nucleosomal occupancy data as the baseline, we further mapped the genome-wide enrichment of H3K9 acetylation and detected general enrichment of this mark in intergenic regions. Conclusions These data on nucleosome enrichment changes add to our understanding of the influence of chromatin structure on the regulation of gene expression. Histones are generally enriched in coding regions, and relatively poor in intergenic regions. Histone enrichment patterns allow for identification of new putative gene-coding regions. Most genes do not show correlation between chromatin structure and steady-state mRNA levels, indicating the dominant roles of other regulatory mechanisms. We present a genome-wide nucleosomal occupancy map, which can be used as a reference for future experiments of histone modification mapping.

Abstract Background In recent years, a major increase in the occurrence of drug resistant falciparum malaria has been reported. Choline analogs, such as the bisthiazolium T4, represent a novel class of compounds with strong potency against drug sensitive and resistant P. falciparum clones. Although T4 and its analogs are presumed to target the parasite's lipid metabolism, their exact mechanism of action remains unknown. Here we have employed transcriptome and proteome profiling analyses to characterize the global response of P. falciparum to T4 during the intraerythrocytic cycle of this parasite. Results No significant transcriptional changes were detected immediately after addition of T4 despite the drug's effect on the parasite metabolism. Using the Ontology-based Pattern Identification (OPI) algorithm with an increased T4 incubation time, we demonstrated cell cycle arrest and a general induction of genes involved in gametocytogenesis. Proteomic analysis revealed a significant decrease in the level of the choline/ethanolamine-phosphotransferase (PfCEPT), a key enzyme involved in the final step of synthesis of phosphatidylcholine (PC). This effect was further supported by metabolic studies, which showed a major alteration in the synthesis of PC from choline and ethanolamine by the compound. Conclusion Our studies demonstrate that the bisthiazolium compound T4 inhibits the pathways of synthesis of phosphatidylcholine from choline and ethanolamine in P. falciparum, and provide evidence for post-transcriptional regulations of parasite metabolism in response to external stimuli.

Abstract Background With the sequence of the Plasmodium falciparum genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the P. falciparum genome sequence having revealed few cis-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of P. falciparum intergenic regions (~90% AT) presents significant challenges to in silico cis-regulatory element discovery. Results We have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich P. falciparum genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the Plasmodium literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted. Conclusion This GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that cis-regulatory elements play a role in the transcriptional control of many P. falciparum biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.

Abstract Background Both host and pathogen factors contribute to disease outcome in Plasmodium falciparum infection. The feasibility of studying the P. falciparum in vivo transcriptome to understand parasite transcriptional response while it resides in the human host is presented. Methods A custom made oligonucleotide array with probes based on the P. falciparum 3D7 laboratory strain chromosome 2 sequence was used to detect in vivo P. falciparum transcripts. This study analyzed transcripts from total RNA derived from small blood samples of P. falciparum infected patients and compared the in vivo expression profile to the in vitro cultivated 3D7 strain transcriptome. Results The data demonstrated that in vivo transcription can be studied from a small blood sample, despite the abundance of human RNA. The in vivo transcriptome is similar to the 3D7 ring stage transcriptome, but there are significant differences in genes encoding a sexual stage antigen and surface proteins. Conclusions Whole genome transcription analysis of P. falciparum can be carried out successfully and further studies in selected patient cohorts may provide insight into parasite in vivo biology and defense against host immunity.

Abstract Background To identify differentially expressed genes across experimental conditions in oligonucleotide microarray experiments, existing statistical methods commonly use a summary of probe-level expression data for each probe set and compare replicates of these values across conditions using a form of the t-test or rank sum test. Here we propose the use of a statistical method that takes advantage of the built-in redundancy architecture of high-density oligonucleotide arrays. Results We employ parametric and nonparametric variants of two-way analysis of variance (ANOVA) on probe-level data to account for probe-level variation, and use the false-discovery rate (FDR) to account for simultaneous testing on thousands of genes (multiple testing problem). Using publicly available data sets, we systematically compared the performance of parametric two-way ANOVA and the nonparametric Mack-Skillings test to the t-test and Wilcoxon rank-sum test for detecting differentially expressed genes at varying levels of fold change, concentration, and sample size. Using receiver operating characteristic (ROC) curve comparisons, we observed that two-way methods with FDR control on sample sizes with 2–3 replicates exhibits the same high sensitivity and specificity as a t-test with FDR control on sample sizes with 6–9 replicates in detecting at least two-fold change. Conclusions Our results suggest that the two-way ANOVA methods using probe-level data are substantially more powerful tests for detecting differential gene expression than corresponding methods for probe-set level data.

The precise mode of action of ganaplacide (KAF156), a phase III antimalarial candidate, remains elusive. Here we employ omics-based methods with the closely related chemical analog, GNF179, to search for potential Plasmodium targets. Ranking potential targets derived from chemical genetics and proteomic affinity chromatography methodologies identifies SEY1 , or Synthetic Enhancement of YOP1, which is predicted to encode an essential dynamin-like GTPase implicated in homotypic fusion of endoplasmic reticulum (ER) membranes. We demonstrate that GNF179 decreases Plasmodium SEY1 melting temperature. We further show that GNF179 binds to recombinant Plasmodium SEY1 and subsequently inhibits its GTPase activity, which is required for maintaining ER architecture. Using ultrastructure expansion microscopy, we find GNF179 treatment changes parasite ER and Golgi morphology. We also confirm that SEY1 is an essential gene in P. falciparum . These data suggest that SEY1 may contribute to the mechanism of action of imidazolopiperazines and is a new and attractive druggable target.

Assessment and proper management of sites contaminated with heavy metals require precise information on the spatial distribution of these metals. This study aimed to predict and map the distribution of Cd, Cu, Ni, Pb, and Zn across the conterminous USA using point observations, environmental variables, and Histogram-based Gradient Boosting (HGB) modeling. Over 9180 surficial soil observations from the Soil Geochemistry Spatial Database (SGSD) (n = 1150), the Geochemical and Mineralogical Survey of Soils (GMSS) (n = 4857), and the Holmgren Dataset (HD) (n = 3400), and 28 covariates (100 m × 100 m grid) representing climate, topography, vegetation, soils, and anthropic activity were compiled. Model performance was evaluated on 20 % of the data not used in calibration using the coefficient of determination (R 2 ), concordance correlation coefficient (ρ c ), and root mean square error (RMSE) indices. Uncertainty of predictions was calculated as the difference between the estimated 95 and 5 % quantiles provided by HGB. The model explained up to 50 % of the variance in the data with RMSE ranging between 0.16 (mg kg -1 ) for Cu and 23.4 (mg kg -1 ) for Zn, respectively. Likewise, ρ c ranged between 0.55 (Cu) and 0.68 (Zn), respectively, and Zn had the highest R 2 (0.50) among all predictions. We observed high Pb concentrations near urban areas. Peak concentrations of all studied metals were found in the Lower Mississippi River Valley. Cu, Ni, and Zn concentrations were higher on the West Coast; Cd concentrations were higher in the central USA. Clay, pH, potential evapotranspiration, temperature, and precipitation were among the model's top five important covariates for spatial predictions of heavy metals. The combined use of point observations and environmental covariates coupled with machine learning provided a reliable prediction of heavy metals distribution in the soils of the conterminous USA. The updated maps could support environmental assessments, monitoring, and decision-making with this methodology applicable to other soil databases, worldwide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Mycotoxin contamination of corn is a pervasive problem that negatively impacts human and animal health and causes economic losses to the agricultural industry worldwide. Historical aflatoxin (AFL) and fumonisin (FUM) mycotoxin contamination data of corn, daily weather data, satellite data, dynamic geospatial soil properties, and land usage parameters were modeled to identify factors significantly contributing to the outbreaks of mycotoxin contamination of corn grown in Illinois (IL), AFL >20 ppb, and FUM >5 ppm. Two methods were used: a gradient boosting machine (GBM) and a neural network (NN). Both the GBM and NN models were dynamic at a state-county geospatial level because they used GPS coordinates of the counties linked to soil properties. GBM identified temperature and precipitation prior to sowing as significant influential factors contributing to high AFL and FUM contamination. AFL-GBM showed that a higher aflatoxin risk index (ARI) in January, March, July, and November led to higher AFL contamination in the southern regions of IL. Higher values of corn-specific normalized difference vegetation index (NDVI) in July led to lower AFL contamination in Central and Southern IL, while higher wheat-specific NDVI values in February led to higher AFL. FUM-GBM showed that temperature in July and October, precipitation in February, and NDVI values in March are positively correlated with high contamination throughout IL. Furthermore, the dynamic geospatial models showed that soil characteristics were correlated with AFL and FUM contamination. Greater calcium carbonate content in soil was negatively correlated with AFL contamination, which was noticeable in Southern IL. Greater soil moisture and available water-holding capacity throughout Southern IL were positively correlated with high FUM contamination. The higher clay percentage in the northeastern areas of IL negatively correlated with FUM contamination. NN models showed high class-specific performance for 1-year predictive validation for AFL (73%) and FUM (85%), highlighting their accuracy for annual mycotoxin prediction. Our models revealed that soil, NDVI, year-specific weekly average precipitation, and temperature were the most important factors that correlated with mycotoxin contamination. These findings serve as reliable guidelines for future modeling efforts to identify novel data inputs for the prediction of AFL and FUM outbreaks and potential farm-level management practices., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Castano-Duque, Winzeler, Blackstock, Liu, Vergopolan, Focker, Barnett, Owens, van der Fels-Klerx, Vaughan and Rajasekaran.)

Introduction: Aflatoxin (AFL), a secondary metabolite produced from filamentous fungi, contaminates corn, posing significant health and safety hazards for humans and livestock through toxigenic and carcinogenic effects. Corn is widely used as an essential commodity for food, feed, fuel, and export markets; therefore, AFL mitigation is necessary to ensure food and feed safety within the United States (US) and elsewhere in the world. In this case study, an Iowa-centric model was developed to predict AFL contamination using historical corn contamination, meteorological, satellite, and soil property data in the largest corn-producing state in the US., Methods: We evaluated the performance of AFL prediction with gradient boosting machine (GBM) learning and feature engineering in Iowa corn for two AFL risk thresholds for high contamination events: 20-ppb and 5-ppb. A 90%-10% training-to-testing ratio was utilized in 2010, 2011, 2012, and 2021 ( n  = 630), with independent validation using the year 2020 ( n  = 376)., Results: The GBM model had an overall accuracy of 96.77% for AFL with a balanced accuracy of 50.00% for a 20-ppb risk threshold, whereas GBM had an overall accuracy of 90.32% with a balanced accuracy of 64.88% for a 5-ppb threshold. The GBM model had a low power to detect high AFL contamination events, resulting in a low sensitivity rate. Analyses for AFL showed satellite-acquired vegetative index during August significantly improved the prediction of corn contamination at the end of the growing season for both risk thresholds. Prediction of high AFL contamination levels was linked to aflatoxin risk indices (ARI) in May. However, ARI in July was an influential factor for the 5-ppb threshold but not for the 20-ppb threshold. Similarly, latitude was an influential factor for the 20-ppb threshold but not the 5-ppb threshold. Furthermore, soil-saturated hydraulic conductivity (Ksat) influenced both risk thresholds., Discussion: Developing these AFL prediction models is practical and implementable in commodity grain handling environments to achieve the goal of preventative rather than reactive mitigations. Finding predictors that influence AFL risk annually is an important cost-effective risk tool and, therefore, is a high priority to ensure hazard management and optimal grain utilization to maximize the utility of the nation's corn crop., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Branstad-Spates, Castano-Duque, Mosher, Hurburgh, Owens, Winzeler, Rajasekaran and Bowers.)

New antimalarial treatments with novel mechanism of action are needed to tackle Plasmodium falciparum infections that are resistant to first-line therapeutics. Here we report the exploration of MMV692140 (2) from the Pathogen Box, a collection of 400 compounds that was made available by Medicines for Malaria Venture (MMV) in 2015. Compound 2 was profiled in in vitro models of malaria and was found to be active against multiple life-cycle stages of Plasmodium parasites. The mode of resistance, and putatively its mode of action, was identified as Plasmodium falciparum translation elongation factor 2 (PfeEF2), which is responsible for the GTP-dependent translocation of the ribosome along mRNA. The compound maintains activity against a series of drug-resistant parasite strains. The structural motif of the tetrahydroquinoline (2) was explored in a chemistry program with its structure-activity relationships examined, resulting in the identification of an analog with 30-fold improvement of antimalarial asexual blood stage potency., (© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 ( pfmdr1 ). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

The neglected tropical disease, schistosomiasis, is caused by trematode blood flukes of the Schistosoma genus and infects approximately 200 million people worldwide. With just one partially effective drug available for disease treatment, new drugs are urgently needed. Herein, a series of 47 phthalimide (Pht) analogues possessing high-value bioactive scaffolds (i.e., benzimidazole and 1,2,3,-triazoles) was synthesized by click-chemistry. Compounds were evaluated for anti-schistosomal activity in culture against somules (post-infective larvae) and adults of Schistosoma mansoni, their predicted ADME (absorption, distribution, metabolism, and excretion) properties, and toxicity vs. HepG2 cells. The majority showed favorable parameters for surface area, lipophilicity, bioavailability and Lipinski score. Thirteen compounds were active at 10 µM against both somules and adults ( 6d , 6f , 6i - 6l , 6n - 6p , 6s , 6r' , 6t' and 6w ). Against somules, the majority caused degeneracy and/or death after 72 h; whereas against adult parasites, five compounds ( 6l , 6d , 6f , 6r' and 6s ) elicited degeneracy, tegumental (surface) damage and/or death. Strongest potency against both developmental stages was recorded for compounds possessing n-butyl or isobutyl as a linker, and a pentafluorophenyl group on triazole. Apart from five compounds for which anti-parasite activity tracked with toxicity to HepG2 cells, there was apparently no toxicity to HepG2 cells (EC 50 values ≥50 µM). The data overall suggest that phthaloyl-triazole compounds are favorable synthons for additional studies as anti-schistosomals., Competing Interests: The authors declare no competing financial interest.

Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target., Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: EI, SD, SB, JB, EO, ML, SG, JN, EW, AV, DG No competing interests declared, (© 2019, Istvan et al.)

Plasmodium parasites, the causative agent of malaria infections, rapidly evolve drug resistance and escape detection by the human immune response via the incredible mutability of its genome. Understanding the genetic mechanisms by which Plasmodium parasites develop antimalarial resistance is essential to understanding why most drugs fail in the clinic and designing the next generation of therapies. A systematic genomic analysis of 262 Plasmodium falciparum clones with stable in vitro resistance to 37 diverse compounds with potent antimalarial activity was undertaken with the main goal of identifying new drug targets. Despite several challenges inherent to this method of in vitro drug resistance generation followed by whole genome sequencing, the study was able to identify a likely drug target or resistance gene for every compound for which resistant parasites could be generated. Known and novel P falciparum resistance mediators were discovered along with several new promising antimalarial drug targets. Surprisingly, gene amplification events contributed to one-third of the drug resistance acquisition events. The study can serve as a model for drug discovery and resistance analyses in other similar microbial pathogens amenable to in vitro culture., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In this work we aimed to develop parasite-selective histone deacetylase inhibitors (HDAC) inhibitors with activity against the disease-causing asexual blood stages of Plasmodium as well as causal prophylactic and/or transmission blocking properties. We report the design, synthesis, and biological testing of a series of 13 terephthalic acid-based HDAC inhibitors. All compounds showed low cytotoxicity against human embryonic kidney (HEK293) cells (IC 50 : 8->51 μm), with 11 also having sub-micromolar in vitro activity against drug-sensitive (3D7) and multidrug-resistant (Dd2) asexual blood-stage P. falciparum parasites (IC 50 ≈0.1-0.5 μm). A subset of compounds were examined for activity against early- and late-stage P. falciparum gametocytes and P. berghei exo-erythrocytic-stage parasites. While only moderate activity was observed against gametocytes (IC 50 >2 μm), the most active compound (N 1 -((3,5-dimethylbenzyl)oxy)-N 4 -hydroxyterephthalamide, 1 f) showed sub-micromolar activity against P. berghei exo-erythrocytic stages (IC 50 0.18 μm) and >270-fold better activity for exo-erythrocytic forms than for HepG2 cells. This, together with asexual-stage in vitro potency (IC 50 ≈0.1 μm) and selectivity of this compound versus human cells (SI>450), suggests that 1 f may be a valuable starting point for the development of novel antimalarial drug leads with low host cell toxicity and multi-stage anti-plasmodial activity., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC 90 ) or 50% parasite killing (50% lethal dose [LD 50 ]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite's acidic digestive vacuole as the primary mode of both the bis -aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates. IMPORTANCE The global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite Plasmodium Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparum resistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparum chloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance., (Copyright © 2017 Dhingra et al.)

MMV007564 is a novel antimalarial benzimidazolyl piperidine chemotype identified in cellular screens. To identify the genetic determinant of MMV007564 resistance, parasites were cultured in the presence of the compound to generate resistant lines. Whole genome sequencing revealed distinct mutations in the gene named Plasmodium falciparum cyclic amine resistance locus (pfcarl), encoding a conserved protein of unknown function. Mutations in pfcarl are strongly associated with resistance to a structurally unrelated class of compounds, the imidazolopiperazines, including KAF156, currently in clinical trials. Our data demonstrate that pfcarl mutations confer resistance to two distinct compound classes, benzimidazolyl piperidines and imidazolopiperazines. However, MMV007564 and the imidazolopiperazines, KAF156 and GNF179, have different timings of action in the asexual blood stage and different potencies against the liver and sexual blood stages. These data suggest that pfcarl is a multidrug-resistance gene rather than a common target for benzimidazolyl piperidines and imidazolopiperazines.

Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties., (2010 Elsevier Ltd. All rights reserved.)

Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Background: Epigenetic modifications of histones and regulation of chromatin structure have been implicated in regulation of virulence gene families in P. falciparum. To better understand chromatin-mediated gene regulation, we used a high-density oligonucleotide microarray to map the position and enrichment of nucleosomes across the entire genome of P. falciparum at three time points of the intra-erythrocytic developmental cycle (IDC) in vitro. We used an unmodified histone H4 antibody for chromatin immunoprecipitation of nucleosome-bound DNA., Results: We observed generally low nucleosomal occupancy of intergenic regions and higher occupancy of protein coding regions. In contract to the overall small fluctuation of nucleosomal occupancy in most coding regions throughout the IDC, subtelomeric genes encoding surface proteins such as var and rif, as well as some core chromosomal genes such as transcription factors, showed large changes in chromatin structure. Telomeres harbored a region with the highest nucleosomal occupancy of the genome and also exhibited large changes with higher nucleosomal occupancy at schizont stages. While many of these subtelomeric genes were previously shown to be modified by H3K9 trimethylation, we also identified some housekeeping genes in core chromosome regions that showed extensive changes in chromatin structure but do not contain this modification. tRNA and basal transcription factor genes showed low nucleosomal occupancy at all times, suggesting of an open chromatin structure that might be permissive for constitutively high levels of expression. Generally, nucleosomal occupancy was not correlated with the steady-state mRNA levels. Several var genes were exceptions: the var gene with the highest expression level showed the lowest nucleosomal occupancy, and selection of parasites for var2CSA expression resulted in lower nucleosomal occupancy at the var2CSA locus. We identified nucleosome-free regions in intergenic regions that may serve as transcription start sites or transcription factor binding sites. Using the nucleosomal occupancy data as the baseline, we further mapped the genome-wide enrichment of H3K9 acetylation and detected general enrichment of this mark in intergenic regions., Conclusions: These data on nucleosome enrichment changes add to our understanding of the influence of chromatin structure on the regulation of gene expression. Histones are generally enriched in coding regions, and relatively poor in intergenic regions. Histone enrichment patterns allow for identification of new putative gene-coding regions. Most genes do not show correlation between chromatin structure and steady-state mRNA levels, indicating the dominant roles of other regulatory mechanisms. We present a genome-wide nucleosomal occupancy map, which can be used as a reference for future experiments of histone modification mapping.

Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.

Malaria sporozoites migrate through several cells prior to a productive invasion that involves the formation of a parasitophorous vacuole (PV) where sporozoites undergo transformation into Exo-erythorcytic forms (EEFs). The precise mechanism leading to sporozoite activation for invasion is unknown, but prior traversal of host cells is required. During cell migration sporozoites are exposed to large shifts in K(+) concentration. We report here that incubation of sporozoites to the intracellular K(+) concentration enhances 8-10 times the infectivity of Plasmodium berghei and 4-5 times the infectivity of Plasmodium yoelli sporozoites for a hepatocyte cell line, while simultaneously decreasing cell passage activity. The K(+) enhancing effect was time and concentration dependent, and was significantly decreased by K(+) channel inhibitors. Potassium-treated P. berghei sporozoites also showed enhanced numbers of EEFs in non-permissive cell lines. Treated sporozoites had reduced infectivity for mice, but infectivity was enhanced upon Kupffer cell depletion. Transcriptional analysis of K(+) treated and control sporozoites revealed a high degree of correlation in their levels of gene expression, indicating that the observed phenotypic changes are not due to radical changes in gene transcription. Only seven genes were upregulated by more than two-fold in K(+) treated sporozoites. The highest level was noted in PP2C, a phosphatase known to dephosphorylate the AKT potassium channel in plants.

A genomic approach was taken to study the effect of chloroquine (CQ) on Plasmodium falciparum cultures in multiple cell states, following short and long exposures to drug at varying concentrations. Six hundred genes from numerous functional groups were responsive to CQ amongst all cell states assayed in a micro-array analysis; however, the amplitude of fold-change was low in the majority of cases. Moreover, alterations in specific, functionally related cascades could not be discerned, leading us to believe there is no single signature response to CQ at the transcript level in P. falciparum. Instead, cell cycle changes appear to have a more pronounced effect on gene expression; only a fraction of the drug responsive loci (approximately 5%) were shared between two separate starting cultures that varied in staging profile in the current study, as well as a previous published analysis using SAGE technology [Gunasekera, A.M., Patankar, S., Schug, J., Eisen, G.,Wirth, D.F., 2003. Drug-induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum. Molecular Microbiology 50, 1229-1239]. These findings are important to report, given the striking contrast to similar studies in other model eukaryotic organisms.

Control of gene expression is poorly understood in the Plasmodium system, where relatively few homologues to known eukaryotic transcription factors have been uncovered. Recent evidence suggests that the parasite may utilize a combinatorial mode of gene regulation, with multiple cis-acting sequences contributing to overall activity at individual promoters [1]. To further probe this mechanism of control, we first searched for over-represented sequence motifs among gene clusters sharing similar expression profiles in Plasmodium falciparum. More specifically, we applied bioinformatic tools to a previously characterized micro-array data set from drug-treated asexual stage cultures (Gunasekera et al., submitted). Cluster analysis of 600 drug responsive genes identified only a single 5' motif, GAGAGAA. Two additional 5' motifs, ACTATAAAGA and TGCAC, were also shared among loci displaying patterns of coordinate expression across varying asexual growth stages. Secondly and most importantly, the functional relevance of each motif was tested in two independent assays-transient transfection and gel-retardation experiments. The GAGAGAA and TGCAC motifs were both active in the former. The GAGAGAA and ACTATAAAGA elements formed specific RNA-protein, but not DNA-protein complexes in gel shift assays, suggesting a key level of control at the RNA level. This is the first report of functionally characterized motifs in P. falciparum that were uncovered following clustering analysis of its asexual stage transcriptome. Together, both the bioinformatic and functional data reported here imply that multiple forms of gene regulation, including post-transcriptional control, may be important in the malarial system.

Microarray-based comparative genomic hybridization (CGH) provides a powerful tool for whole genome analyses and the rapid detection of genomic variation that underlies virulence and disease. In the field of Plasmodium research, many of the parasite genomes that one might wish to study in a high throughput manner are not laboratory clones, but clinical isolates. One of the key limitations to the use of clinical samples in CGH, however, is the miniscule amounts of genomic DNA available. Here we describe the successful application of multiple displacement amplification (MDA), a non-PCR-based amplification method that exhibits clear advantages over all other currently available methods. Using MDA, CGH was performed on a panel of NF54 and IT/FCR3 clones, identifying previously published deletions on chromosomes 2 and 9 as well as polymorphism in genes associated with disease pathology.

This study is motivated by two data sets which employ a custom Plasmodium falciparum version of the Affymetrix GeneChip, containing only perfect match (PM) oligonucleotides. A PM-only chip cannot be analysed using the standard Affymetrix-supplied software. We compared the performance of three match-only algorithms on these data: the Match Only Integral Distribution (MOID) algorithm, Robust Multichip Analysis (RMA), and the Model Based Expression Index (MBEI). We validated the differential expression of several genes using quantitative reverse transcriptase-PCR. We also performed a comparison using two publicly available 'benchmarking' data sets: the Latin Square spike-in data set generated by Affymetrix, and the Gene Logic dilution series. Since we know what the true fold changes are in these special data sets, they are helpful for assessment of expression algorithms.

Background: To identify differentially expressed genes across experimental conditions in oligonucleotide microarray experiments, existing statistical methods commonly use a summary of probe-level expression data for each probe set and compare replicates of these values across conditions using a form of the t-test or rank sum test. Here we propose the use of a statistical method that takes advantage of the built-in redundancy architecture of high-density oligonucleotide arrays., Results: We employ parametric and nonparametric variants of two-way analysis of variance (ANOVA) on probe-level data to account for probe-level variation, and use the false-discovery rate (FDR) to account for simultaneous testing on thousands of genes (multiple testing problem). Using publicly available data sets, we systematically compared the performance of parametric two-way ANOVA and the nonparametric Mack-Skillings test to the t-test and Wilcoxon rank-sum test for detecting differentially expressed genes at varying levels of fold change, concentration, and sample size. Using receiver operating characteristic (ROC) curve comparisons, we observed that two-way methods with FDR control on sample sizes with 2-3 replicates exhibits the same high sensitivity and specificity as a t-test with FDR control on sample sizes with 6-9 replicates in detecting at least two-fold change., Conclusions: Our results suggest that the two-way ANOVA methods using probe-level data are substantially more powerful tests for detecting differential gene expression than corresponding methods for probe-set level data.