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Journal ArticleDOI

Routine In Vitro Culture of Plasmodium falciparum: Experimental Consequences?

01 Jul 2018-Trends in Parasitology (Elsevier Current Trends)-Vol. 34, Iss: 7, pp 564-575
TL;DR: It is reasoned that culture conditions should be re-established as a primary consideration in in vitro malaria experimentation.
About: This article is published in Trends in Parasitology.The article was published on 2018-07-01. It has received 23 citations till now.
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Journal ArticleDOI
TL;DR: It is shown that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation.

52 citations


Cites methods from "Routine In Vitro Culture of Plasmod..."

  • ...REAGENT or RESOURCE SOURCE IDENTIFIER Peptide substrate GRTGRRNSI-NH2 Sigma-Aldrich Cat# SCP0212 Renilla-Glo(R) Luciferase Assay System Promega Cat# E2750 QiAmp DNA Blood Mini kit Qiagen https://www.qiagen.com/ Deposited Data PKG crystal structure (Baker et al., 2017) PDB ID: 5DYK Experimental Models: Organisms/Strains Anopheles stephensi mosquitoes Insectary Core Facility, New York University N/A Plasmodium falciparum asexual blood stage parasites Goldberg lab, Washington University 3D7-A10 clone P. falciparum asexual blood stage parasites Fidock lab, Columbia UniversityMedical Center Dd2-B2 clone P. falciparum NF54 CDPK1 T145M line Miller lab, Malaria Cell Biology, NIAID, NIH (Bansal et al., 2018) CDPK1T145M P. falciparum 3D7elo1-pfs16-CBG99 transgenic parasites Fidock lab, Columbia UniversityMedical Center (Cevenini et al., 2014) N/A BALB/c female mice The Jackson Laboratory https://www.jax.org/ P. berghei ANKA (Pb-Luc) Insectary Core Facility, New York University Pb-Luc, also known as P. bergheiANKA-GFP-Luc-SMCON Oligonucleotides All primers and cloning fragments are shown in Table S1 (J) and (K) N/A Recombinant DNA pSN054 vector (Nasamu et al., 2019) N/A pET28-PfTKL3-SAM(V54E)-KD (Abdi et al., 2010) N/A Software and Algorithms GraphPad Prism Version 8 GraphPad Software, San Diego, CA, USA www.graphpad.com ICY BioImage Analysis (Delves et al., 2016) http://icy.bioimageanalysis.org/ R Studio RStudio Team, 2015 http://www.rstudio.com/ Proteome Discoverer Thermo Scientific https://www.thermofisher.com/us/en/ home.html Mascot Matrix Science http://www.matrixscience.com/ Scaffold Q+S Proteome Software http://www.proteomesoftware.com/ ChemiDoc MP System and Image Lab 5.2.0 Bio-Rad https://www.bio-rad.com/ El- MAVEN software (Agrawal et al., 2019) https://elucidatainc.github.io/ElMaven/ Schrodinger molecular modeling suite Schrodinger http://www.schrodinger.com/ ll OPEN ACCESS Article...

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  • ...Dual Gamete Formation Assay Pf NF54 gametocytes were generated as reported elsewhere (Delves et al., 2016)....

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  • ...…et al., 2010) N/A Software and Algorithms GraphPad Prism Version 8 GraphPad Software, San Diego, CA, USA www.graphpad.com ICY BioImage Analysis (Delves et al., 2016) http://icy.bioimageanalysis.org/ R Studio RStudio Team, 2015 http://www.rstudio.com/ Proteome Discoverer Thermo Scientific…...

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  • ...Both time lapse images of exflagellation and fluorescence images of female gametes were automatically quantified using custom scripts in ICY BioImage Analysis (Delves et al., 2016)....

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  • ...Pf NF54 gametocytes were generated as reported elsewhere (Delves et al., 2016)....

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30 Apr 2015
Abstract: Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.

38 citations

Journal ArticleDOI
21 Oct 2020-eLife
TL;DR: Exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin at the trophozoite stage resulted in a ~ fourfold increase in sexual conversion, and no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion.
Abstract: Malaria transmission is dependent on the formation of gametocytes in the human blood. The sexual conversion rate, the proportion of asexual parasites that convert into gametocytes at each multiplication cycle, is variable and reflects the relative parasite investment between transmission and maintaining the infection. The impact of environmental factors such as drugs on sexual conversion rates is not well understood. We developed a robust assay using gametocyte-reporter parasite lines to accurately measure the impact of drugs on sexual conversion rates, independently from their gametocytocidal activity. We found that exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin (DHA) at the trophozoite stage resulted in a ~ fourfold increase in sexual conversion. In contrast, no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion. Our results reveal a complex relationship between antimalarial drugs and sexual conversion, with potential public health implications.

27 citations


Cites methods from "Routine In Vitro Culture of Plasmod..."

  • ...…used method to enhance sexual conversion and obtain large numbers of gametocytes in vitro relies on overgrowing blood-stage cultures (the ‘crash method’) (Delves et al., 2016) and/or maintaining the cultures with parasite-conditioned (spent) medium (Brancucci et al., 2015; Fivelman et al., 2007)....

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  • ...The most commonly used method to enhance sexual conversion and obtain large numbers of gametocytes in vitro relies on overgrowing blood-stage cultures (the ‘crash method’) (Delves et al., 2016) and/or maintaining the cultures with parasite-conditioned (spent) medium (Brancucci et al....

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Journal ArticleDOI
TL;DR: How epigenetic variation, directed transcriptional responses and also genetic changes that affect transcript levels can all contribute to transcriptional variation and, ultimately, parasite survival are discussed.
Abstract: Transcriptional differences enable the generation of alternative phenotypes from the same genome. In malaria parasites, transcriptional plasticity plays a major role in the process of adaptation to fluctuations in the environment. Multiple studies with culture-adapted parasites and field isolates are starting to unravel the different transcriptional alternatives available to Plasmodium falciparum and the underlying molecular mechanisms. Here we discuss how epigenetic variation, directed transcriptional responses and also genetic changes that affect transcript levels can all contribute to transcriptional variation and, ultimately, parasite survival. Some transcriptional changes are driven by stochastic events. These changes can occur spontaneously, resulting in heterogeneity within parasite populations that provides the grounds for adaptation by dynamic natural selection. However, transcriptional changes can also occur in response to external cues. A better understanding of the mechanisms that the parasite has evolved to alter its transcriptome may ultimately contribute to the design of strategies to combat malaria to which the parasite cannot adapt.

23 citations

Journal ArticleDOI
TL;DR: Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines.
Abstract: Malaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes. Analysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates. Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.

22 citations


Cites background from "Routine In Vitro Culture of Plasmod..."

  • ...ticularly in earlier trophic stages of the intra-erythrocytic cycle [47], and although we did not consider oxygen me-...

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References
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Journal ArticleDOI
20 Aug 1976-Science
TL;DR: Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen.
Abstract: Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.

7,496 citations

Journal ArticleDOI
03 Oct 2002-Nature
TL;DR: The genome sequence of P. falciparum clone 3D7 is reported, which is the most (A + T)-rich genome sequenced to date and is being exploited in the search for new drugs and vaccines to fight malaria.
Abstract: The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.

4,312 citations

Journal ArticleDOI
TL;DR: A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum, and results demonstrated that the method is sensitive and precise.
Abstract: A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum. Microtitration plates were used to prepare serial dilutions of the compounds to be tested. Parasites, obtained from continuous stock cultures, were subcultured in these plates for 42 h. Inhibition of uptake of a radiolabeled nucleic acid precursor by the parasites served as the indicator of antimalarial activity. Results of repeated measurements of activity with chloroquine, quinine, and the investigational new drug mefloquine demonstrated that the method is sensitive and precise. Several additional antimalarial drugs and compounds of interest were tested in vitro, and the results were consistent with available in vivo data. The use of P. falciparum isolates with known susceptibility to antimalarial drugs also permitted evaluation of the cross-resistance potential of each compound tested. The applications and expectations of this new test system within a drug development program are discussed.

2,474 citations

Journal ArticleDOI
02 Jan 2014-Nature
TL;DR: Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance.
Abstract: Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

1,639 citations

Journal ArticleDOI
20 May 2010-Nature
TL;DR: Chemical structures and associated data suggest several novel mechanisms of antimalarial action, such as inhibition of protein kinases and host–pathogen interaction related targets.
Abstract: Malaria is a devastating infection caused by protozoa of the genus Plasmodium. Drug resistance is widespread, no new chemical class of antimalarials has been introduced into clinical practice since 1996 and there is a recent rise of parasite strains with reduced sensitivity to the newest drugs. We screened nearly 2 million compounds in GlaxoSmithKline's chemical library for inhibitors of P. falciparum, of which 13,533 were confirmed to inhibit parasite growth by at least 80% at 2 microM concentration. More than 8,000 also showed potent activity against the multidrug resistant strain Dd2. Most (82%) compounds originate from internal company projects and are new to the malaria community. Analyses using historic assay data suggest several novel mechanisms of antimalarial action, such as inhibition of protein kinases and host-pathogen interaction related targets. Chemical structures and associated data are hereby made public to encourage additional drug lead identification efforts and further research into this disease.

953 citations