Mounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the nutrient-rich media used for standard in vitro culture contains supra-physiological nutrient concentrations, pathogens encounter a... more
Mounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the nutrient-rich media used for standard in vitro culture contains supra-physiological nutrient concentrations, pathogens encounter a relatively restrictive environment in vivo. We assessed the effect of nutrient limitation on the protozoan parasite that causes malaria and demonstrated that short-term growth under physiologically-relevant mild nutrient stress (or metabolic priming) triggers increased tolerance of the potent antimalarial drug dihydroartemisinin (DHA). We observed beneficial effects using both short-term survival assays and longer-term proliferation studies, where metabolic priming increases parasite survival to a level previously defined as DHA resistant (>1% survival). We performed these assessments by either decreasing single nutrients that have distinct roles in parasite metabolism or using a media formulation with reductions in many nutrients that simulates ...
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Table S1. Reactions modified to curate the Plasmodium falciparum genome-scale metabolic model. (ST2A and ST2B) Table S2. Down-regulated Differentially Expressed Genes in (A) Short and (B) Long-term Chloroquine-treated Parasites. The gene... more
Table S1. Reactions modified to curate the Plasmodium falciparum genome-scale metabolic model. (ST2A and ST2B) Table S2. Down-regulated Differentially Expressed Genes in (A) Short and (B) Long-term Chloroquine-treated Parasites. The gene IDs, corresponding gene names, and fold changes of significantly down-regulated genes in response to chloroquine treatment. Table S3. Up-regulated Differentially Expressed Genes in (A) Short and (B) Long-term Chloroquine-treated Parasites. The gene IDs, corresponding gene names, and fold changes of significantly up-regulated genes in response to chloroquine treatment. Table S4. Significant Down Regulation of Metabolic Genes Arising from Chloroquine Treatment. A: Metabolic DEGs Common to Long and Short-term Treated Conditions. B: Metabolic DEGs Unique to Short-term Treated Condition. The gene IDs, corresponding gene names, and metabolic subsystems of genes with significant expression changes in response to chloroquine treatment. C: Metabolic DEGs Uni...
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iPfal17 in SBML format.ďťż (XML 2000Â kb)
Research Interests: Pharmacology, Genetics, Hematology, Developmental Biology, Molecular Biology, and 12 moreMetabolism, Computational Biology, Cancer, Malaria, Virology, Cell Biology, Infectious Diseases, Transcriptional Network Reconstruction, Medicine, Plasmodium falciparum, Flux Balance Analysis, and artemisinin resistance
Artemisinin sensitive and resistant parasites utilize different metabolic genes and pathways. Enrichment analysis of gene utilization in sensitive and resistant parasite models demonstrates functional differences in expression data... more
Artemisinin sensitive and resistant parasites utilize different metabolic genes and pathways. Enrichment analysis of gene utilization in sensitive and resistant parasite models demonstrates functional differences in expression data integration. Consensus gene utilization from resistant and sensitive models (both Cambodian and Vietnamese datasets) were used and compared to unconstrained model. Black = p
Research Interests: Pharmacology, Biochemistry, Genetics, Microbiology, Physiology, and 13 moreInorganic Chemistry, Hematology, Metabolism, Computational Biology, Cancer, Malaria, Virology, Ecology, Transcriptional Network Reconstruction, Medicine, Plasmodium falciparum, Flux Balance Analysis, and artemisinin resistance
Functional differences in data-driven sensitive and resistant models. Gene states from four condition-specific models, the results of MADE integration, cluster by sensitivity not by location. Active genes in red/blue, with genes removed... more
Functional differences in data-driven sensitive and resistant models. Gene states from four condition-specific models, the results of MADE integration, cluster by sensitivity not by location. Active genes in red/blue, with genes removed from expression-constrained models in white. (TIFF 1795Â kb)
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Modifications and reaction additions in iPfal17 curation. Table S2. Reactions deleted from Plata et al. model (iTH366) in generating iPfal17. Table S3. Predicted lethal reactions in wild-type blood-stage Plasmodium falciparum. Table S4:... more
Modifications and reaction additions in iPfal17 curation. Table S2. Reactions deleted from Plata et al. model (iTH366) in generating iPfal17. Table S3. Predicted lethal reactions in wild-type blood-stage Plasmodium falciparum. Table S4: Predicted lethal genes in wild-type blood-stage Plasmodium falciparum. Table S5. Consensus predicted lethal reactions across 4 expression-constrained models. Table S6. PlasmoGem orthologous results. Table S7: Extended Table 4. Table S8. Microarray platform blast results. Table S9: Metabolites in the in silico extracellular environment. (XLSX 1104Â kb)
Research Interests: Pharmacology, Evolutionary Biology, Genetics, Microbiology, Inorganic Chemistry, and 11 moreMetabolism, Computational Biology, Plant Biology, Malaria, Infectious Diseases, Space Science, Transcriptional Network Reconstruction, Medicine, Plasmodium falciparum, Flux Balance Analysis, and artemisinin resistance
Artemisinin resistance is better predicted by metadata classifier than expression classifier. Using full expression profile (A) or metadata (B), including patient and parasite features (see Methods), we can classify samples as artemisinin... more
Artemisinin resistance is better predicted by metadata classifier than expression classifier. Using full expression profile (A) or metadata (B), including patient and parasite features (see Methods), we can classify samples as artemisinin sensitive or resistant by Random forest analysis. Of the top 25 most important variables (gene probes) in the expression classifier, 12 encoded exported proteins, four genes of complete unknown function, three encoded a putative kinase and putative phosphatases, one encoded a component of dynein, four were uncharacterized genes though to be involved in protein folding or trafficking, and one encoded a transcription factor. Abbreviation key: (all from [49] unless noted otherwise). aprs_mutation = apicoplast ribosomal protein S10 (PF3D7_1460900.1) mutation [14]; fd_mutation = ferredoxin (PF3D7_1318100) mutation [14]; Field_site = location at which blood sample was collected; mdr_mutation = multidrug resistance protein 2 (PF3D7_1447900) mutation; part...
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Distribution of genome-wide expression data demonstrates moderate differential expression between sensitive and resistant parasites. Fold change values from differential expression between sensitive and resistant parasites from Cambodia... more
Distribution of genome-wide expression data demonstrates moderate differential expression between sensitive and resistant parasites. Fold change values from differential expression between sensitive and resistant parasites from Cambodia (A) and Vietnam (B) with significantly differentially expressed genes in red. Fold change is the ratio of mean expression in resistant parasites to mean expression in sensitive parasites, for each respective country. (PDF 820Â kb)
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Self-aligned sequential lateral field non-uniformities extending uniformly over the sample channel depth are fabricated using a single lithography step for enabling phenotype-specific dielectrophoretic separation of cells.
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Single cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a... more
Single cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed base content. Through optimization of a quasi-linear genome amplification method, we achieve more even genome coverage and better targeting of the parasite genome over contaminants. These improvements are particularly important for expanding the accessibility of single cell approaches to new organisms and cell types and for improving the study of genetic mechanisms of adaptation.
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Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently... more
Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently extrapolated to infer knowledge about another parasite, across and within genera. Modelin vitroorin vivosystems are frequently used to enhance experimental manipulability, but these systems generally use species related to, yet distinct from, the clinically relevant causal pathogen. Characterization of functional differences among parasite species is confined topost hocor single target studies, limiting the utility of this extrapolation approach. To address this challenge and to accelerate parasitology research broadly, we present a functional comparative analysis of 192 genomes, representing every high-quality, publicly-available protozoan parasite genome includingPlasmodium, Toxoplasma, Cryptosporidium, Entamoeba, Trypanosoma, Leishmania, Giardia, an...
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Plasmodiumprotozoan parasites undergo rounds of asexual replication inside human erythrocytes, progressing from ring stage, to trophozoites and schizonts, before egress and reinvasion. Given the discovery of ring-specific artemisinin... more
Plasmodiumprotozoan parasites undergo rounds of asexual replication inside human erythrocytes, progressing from ring stage, to trophozoites and schizonts, before egress and reinvasion. Given the discovery of ring-specific artemisinin tolerance and quiescence inPlasmodium falciparum, there is great urgency to better understand ring stage biology. However, the lack of an effective enrichment method has left rings and related parasite stages understudied compared to their late stage counterparts, which can be easily isolated due to their paramagnetic properties. Here, a method for separatingall Plasmodiuminfected erythrocytes from uninfected erythrocytes is presented. This approach takes advantage of streptolysin-O (SLO) to preferentially lyse uninfected erythrocytes as previously shown by Jackson,et al.Following lytic treatment, Percoll gradient centrifugation removes lysed cells, leaving an intact cell population enriched in infected erythrocytes. ThisSLO-Percoll (SLOPE) method is ef...
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Background. Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for polymerase chain reaction (PCR) testing to confirm... more
Background. Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for polymerase chain reaction (PCR) testing to confirm Plasmodium infection or for epidemiologic studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of Plasmodium species using real-time PCR.Methods. We extracted DNA from archived blood smears and corresponding red blood cell pellets collected from asymptomatic children in Southwestern Uganda in 2010. We then performed real-time PCR followed by high resolution melting (HRM) to identify Plasmodium species and compared our results to those of microscopy.Results. We analysed a total of 367 blood smears and corresponding red blood cell pellets that included 185 (50.4%) positive smears by microscopy. Compared to microscopy, PCR-HRM from smear DNA had a sensitivity of 93.0% (Confidence interva...
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BACKGROUND: Malaria remains a major public health burden and resistance has emerged to every antimalarial on the market, including the frontline drug artemisinin. Our limited understanding of Plasmodium biology hinders the elucidation of... more
BACKGROUND: Malaria remains a major public health burden and resistance has emerged to every antimalarial on the market, including the frontline drug artemisinin. Our limited understanding of Plasmodium biology hinders the elucidation of resistance mechanisms. In this regard, systems biology approaches can facilitate the integration of existing experimental knowledge and further understanding of these mechanisms. RESULTS: Here, we developed a novel genome-scale metabolic network reconstruction, iPfal17, of the asexual blood-stage P. falciparum parasite to expand our understanding of metabolic changes that support resistance. We identified 11 metabolic tasks to evaluate iPfal17 performance. Flux balance analysis and simulation of gene knockouts and enzyme inhibition predict candidate drug targets unique to resistant parasites. Moreover, integration of clinical parasite transcriptomes into the iPfal17 reconstruction reveals patterns associated with antimalarial resistance. These resul...
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Due to improved instrument sensitivity and access, the use of metabolomics is gaining traction for the study of many organisms and pathogens. For the intracellular malaria parasite, Plasmodium falciparum, both targeted and untargeted... more
Due to improved instrument sensitivity and access, the use of metabolomics is gaining traction for the study of many organisms and pathogens. For the intracellular malaria parasite, Plasmodium falciparum, both targeted and untargeted metabolite detection has improved our understanding of pathogenesis, host-parasite interactions, parasite response to antimalarials, and impacts of resistance. However, protocols for purification are not optimized for investigations of intracellular pathogens and noise-limiting analysis parameters are not well defined. To explore influential parameters, we purified a diverse set of in vitro grown intra-erythrocytic P. falciparum parasites for untargeted metabolomics studies. Following metabolite identification, data processing included normalization to double stranded DNA, total protein, or parasite number to correct for different sample sizes and stage differences. We found that parasite-derived variables were most appropriate for normalization as they...
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Summary Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as... more
Summary Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome‐mediated respiration. This reduction was explained by RNAi‐mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometr...
Research Interests: Biochemistry, Mass Spectrometry, Biology, Phospholipids, Molecular Microbiology, and 15 moreMitochondria, Medicine, Biological Sciences, RNA interference, Fatty Acid Synthesis, Fatty acids, Animals, Oxygen, Fatty Acid, Protozoan Proteins, Mitochondrion, Mitochondrial Proteins, Complex I, Acyl carrier protein, and Medical and Health Sciences
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Research Interests: Biochemistry, Biology, Mitochondria, Medicine, Saccharomyces cerevisiae, and 15 moreHumans, Sequence alignment, Fatty Acid Synthesis, Fatty acids, Animals, Enzyme, Electrophoresis, Fatty Acid, Amino Acid Sequence, Knock out, Protein Transport, Biochemistry and cell biology, Dehydratase, Mitochondrion, and Molecular Sequence Data
Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of... more
Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesiz...
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Atovaquone is a component of Malarone, a widely prescribed antimalarial combination, that targets malaria respiration. Here we show that parasites with high-level resistance to an inhibitor of dihydroorotate dehydrogenase demonstrate... more
Atovaquone is a component of Malarone, a widely prescribed antimalarial combination, that targets malaria respiration. Here we show that parasites with high-level resistance to an inhibitor of dihydroorotate dehydrogenase demonstrate unexpected atovaquone tolerance. Fortunately, the tolerance is diminished with proguanil, the second partner in Malarone. It is important to understand such "genetic cross talk" between respiration and pyrimidine biosynthesis since many antimalarial drug development programs target these two seemingly independent pathways.
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Research Interests: Biology, Malaria, International Cooperation, Research, Medicine, and 13 morePlasmodium, India, Biological Sciences, Humans, Animals, Host-parasite interactions, Mosquito Control, Culicidae, Genetic variation, Communicable disease control, Insect Vectors, Severity of Illness Index, and Medical and Health Sciences
Background Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a... more
Background Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries. Methods Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-G...
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Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in malaria-endemic countries, we developed a suite of TaqMan assays for known... more
Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in malaria-endemic countries, we developed a suite of TaqMan assays for known resistance markers and compartmentalized them into a single array card (TAC). We included 87 assays for species identification, the detection of Plasmodium falciparum mutations associated with chloroquine, atovaquone, pyrimethamine, sulfadoxine, and artemisinin resistance, and neutral single nucleotide polymorphism (SNP) genotyping. Assay performance was first optimized using DNA from common laboratory parasite lines and plasmid controls. The limit of detection was 0.1-10 pg of DNA and yielded 100% accuracy when compared to sequencing. The tool was then evaluated on 87 clinical blood samples from around the world and the malaria-TAC once again achieved 100% accuracy when compared to sequencing, plus detected the presence of mixed infections in clinical...