Person: Van tyne, Daria
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Van tyne
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Daria
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Van tyne, Daria
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Publication Changes in drug sensitivity and anti-malarial drug resistance mutations over time among Plasmodium falciparum parasites in Senegal(BioMed Central, 2013) Van tyne, Daria; Dieye, Baba; Valim, Clarissa; Daniels, Rachel; Sène, Papa Diogoye; Lukens, Amanda; Ndiaye, Mouhamadou; Bei, Amy; Ndiaye, Yaye Die; Hamilton, Elizabeth; Ndir, Omar; Mboup, Souleymane; Volkman, Sarah K; Wirth, Dyann; Ndiaye, DaoudaBackground: Malaria treatment efforts are hindered by the rapid emergence and spread of drug resistant parasites. Simple assays to monitor parasite drug response in direct patient samples (ex vivo) can detect drug resistance before it becomes clinically apparent, and can inform changes in treatment policy to prevent the spread of resistance. Methods: Parasite drug responses to amodiaquine, artemisinin, chloroquine and mefloquine were tested in approximately 400 Plasmodium falciparum malaria infections in Thiès, Senegal between 2008 and 2011 using a DAPI-based ex vivo drug resistance assay. Drug resistance-associated mutations were also genotyped in pfcrt and pfmdr1. Results: Parasite drug responses changed between 2008 and 2011, as parasites became less sensitive to amodiaquine, artemisinin and chloroquine over time. The prevalence of known resistance-associated mutations also changed over time. Decreased amodiaquine sensitivity was associated with sustained, highly prevalent mutations in pfcrt, and one mutation in pfmdr1 – Y184F – was associated with decreased parasite sensitivity to artemisinin. Conclusions: Directly measuring ex vivo parasite drug response and resistance mutation genotyping over time are useful tools for monitoring parasite drug responses in field samples. Furthermore, these data suggest that the use of amodiaquine and artemisinin derivatives in combination therapies is selecting for increased drug tolerance within this population.Publication Structure, Function, and Biology of the Enterococcus faecalis Cytolysin(MDPI, 2013) Van tyne, Daria; Martin, Melissa Janet; Gilmore, MichaelEnterococcus faecalis is a Gram-positive commensal member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large class of lanthionine-containing bacteriocins produced by Gram-positive bacteria. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics as well. The current understanding of cytolysin biosynthesis, structure/function relationships, and contribution to the biology of E. faecalis are reviewed, and opportunities for using emerging technologies to advance this understanding are discussed.Publication Sequence-Based Association and Selection Scans Identify Drug Resistance Loci in the Plasmodium Falciparum Malaria Parasite(Proceedings of the National Academy of Sciences, 2012) Park, Daniel John; Lukens, Amanda; Neafsey, Daniel; Schaffner, Stephen; Chang, Hsiao-Han; Valim, Clarissa; Ribacke, Ulf; Van tyne, Daria; Galinsky, Kevin; Galligan, Meghan; Becker, Justin S.; Ndiaye, Daouda; Mboup, Souleymane; Wiegand, Roger; Hartl, Daniel; Sabeti, Pardis; Wirth, Dyann; Volkman, SarahThrough rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite—the deadliest of those that cause malaria—is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites’ in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.Publication Enrichment of Reticulocytes from Whole Blood using Aqueous Multiphase Systems of Polymers(Wiley-Blackwell, 2014) Kumar, Ashok Ashwin; Lim, Caeul; Moreno, Yovany; Mace, Charles R.; Syed, Abeer; Van tyne, Daria; Wirth, Dyann; Duraisingh, Manoj; Whitesides, GeorgeThis paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)—immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.Publication Dual defensin strategy for targeting Enterococcus faecalis(Proceedings of the National Academy of Sciences, 2013) Gilmore, Michael; Lebreton, Francois; Van tyne, DariaPublication SNP Genotyping Identifies New Signatures of Selection in a Deep Sample of West African Plasmodium falciparum Malaria Parasites(Oxford University Press, 2012) Amambua-Ngwa, Alfred; Barnes, Kayla G.; Sene, Papa; Conway, David J.; Park, Daniel John; Volkman, Sarah; Bei, Amy; Lukens, Amanda; Van tyne, Daria; Ndiaye, Daouda; Wirth, Dyann; Neafsey, Daniel; Schaffner, StephenWe used a high-density single-nucleotide polymorphism array to genotype 75 Plasmodium falciparum isolates recently collected from Senegal and The Gambia to search for signals of selection in this malaria endemic region. We found little geographic or temporal stratification of the genetic diversity among the sampled parasites. Through application of the iHS and REHH haplotype-based tests for positive selection, we found evidence of recent selective sweeps at a known drug resistance locus, at several known antigenic loci, and at several genomic regions not previously identified as sites of recent selection. We discuss the value of deep population-specific genomic analyses for identifying selection signals within sampled endemic populations of parasites, which may correspond to local selection pressures such as distinctive therapeutic regimes or mosquito vectors.Publication Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology(Public Library of Science, 2012) Martincová, Eva; Voleman, Luboš; Najdrová, Vladimíra; De Napoli, Maximiliano; Eshar, Shiri; Gualdron, Melisa; Hopp, Christine S.; Sanin, David E.; Tembo, Dumizulu L.; Walker, Dawn; Marcinčiková, Michaela; Tachezy, Jan; Doležal, Pavel; Van tyne, DariaHydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes.Publication Genetic Surveillance Detects Both Clonal and Epidemic Transmission of Malaria following Enhanced Intervention in Senegal(Public Library of Science, 2013) Daniels, Rachel; Chang, Hsiao-Han; Séne, Papa Diogoye; Park, Danny C.; Neafsey, Daniel; Schaffner, Stephen; Hamilton, Elizabeth; Lukens, Amanda; Van tyne, Daria; Mboup, Souleymane; Sabeti, Pardis; Ndiaye, Daouda; Wirth, Dyann; Hartl, Daniel; Volkman, SarahUsing parasite genotyping tools, we screened patients with mild uncomplicated malaria seeking treatment at a clinic in Thiès, Senegal, from 2006 to 2011. We identified a growing frequency of infections caused by genetically identical parasite strains, coincident with increased deployment of malaria control interventions and decreased malaria deaths. Parasite genotypes in some cases persisted clonally across dry seasons. The increase in frequency of genetically identical parasite strains corresponded with decrease in the probability of multiple infections. Further, these observations support evidence of both clonal and epidemic population structures. These data provide the first evidence of a temporal correlation between the appearance of identical parasite types and increased malaria control efforts in Africa, which here included distribution of insecticide treated nets (ITNs), use of rapid diagnostic tests (RDTs) for malaria detection, and deployment of artemisinin combination therapy (ACT). Our results imply that genetic surveillance can be used to evaluate the effectiveness of disease control strategies and assist a rational global malaria eradication campaign.Publication Identification and Characterization of Novel Drug Resistance Loci in Plasmodium falciparum(2013-03-15) Van tyne, Daria; Wirth, Dyann Fergus; Rubin, Eric; Duraisingh, Manoj; Hartl, Daniel; Sabeti, Pardis; Hanage, William; Ferdig, Michael; Marti, MatthiasMalaria has plagued mankind for millennia. Antimalarial drug use over the last century has generated highly drug-resistant parasites, which amplify the burden of this disease and pose a serious obstacle to control efforts. This dissertation is motivated by the simple fact that malaria parasites have become resistant to nearly every antimalarial drug that has ever been used, yet the precise genetic mechanisms of parasite drug resistance remain largely unknown. Our work pairs genomics-age technologies with molecular biology, genetics and molecular epidemiology in order to identify and characterize novel genes that contribute to drug resistance in P. falciparum. In the Introduction, we highlight relevant opportunities and challenges in trying to identify and understand the genetic basis of malaria drug resistance as it emerges to currently used therapies. In Chapter One, we demonstrate how genome-wide association studies (GWAS) can be applied to P. falciparum in order to identify novel drug resistance loci. Functional follow-up revealed that overexpression of the novel candidate gene PF10_0355 made parasites more resistant to the drugs halofantrine, lumefantrine and mefloquine. These findings show that PF10_0355 plays a role in parasite drug response, as well as provides validation of our GWAS approach. In Chapter Two, we further characterize PF10_0355 and show that modulation of the gene by either knockout or allelic replacement changes parasite drug sensitivity. Furthermore, we show that moderate changes in drug response measured in the short-term can have dramatic effects when parasites are competed with one another under drug pressure. In Chapter Three, we use an overexpression approach to functionally follow up other novel drug resistance genes generated by GWAS in P. falciparum. We find that overexpression is a useful way to begin to screen candidate drug resistance loci in the malaria parasite. In Chapter Four, we use a DAPI-based ex vivo drug assay to monitor drug resistance among parasites circulating in Thiés, Senegal. In the future, we will look for genetic markers of parasite drug resistance in this population by GWAS. Finally, in the Discussion we present an essay about malaria evolution and eradication written for non-specialists. Our hope is that the work presented in this dissertation furthers understanding of drug resistance in the malaria parasite, both within and beyond the malaria research community.Publication A Global Transcriptional Analysis of Plasmodium Falciparum Malaria Reveals A Novel Family of Telomere-Associated lncRNAs(BioMed Central, 2011) Broadbent, Kate Mariel; Park, Daniel John; Wolf, Ashley Robin; Van tyne, Daria; Sims, Jennifer Sung; Ribacke, Ulf; Volkman, Sarah; Duraisingh, Manoj; Wirth, Dyann; Sabeti, Pardis; Rinn, JohnBackground: Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results: We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions: We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation.