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      Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum

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          Abstract

          Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro.

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          Most cited references26

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          Human malaria parasites in continuous culture.

          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.
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            Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.

            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.
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              Red cell membrane: past, present, and future.

              As a result of natural selection driven by severe forms of malaria, 1 in 6 humans in the world, more than 1 billion people, are affected by red cell abnormalities, making them the most common of the inherited disorders. The non-nucleated red cell is unique among human cell type in that the plasma membrane, its only structural component, accounts for all of its diverse antigenic, transport, and mechanical characteristics. Our current concept of the red cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network of skeletal proteins via transmembrane proteins. Structural and functional characterization of the many constituents of the red cell membrane, in conjunction with biophysical and physiologic studies, has led to detailed description of the way in which the remarkable mechanical properties and other important characteristics of the red cells arise, and of the manner in which they fail in disease states. Current studies in this very active and exciting field are continuing to produce new and unexpected revelations on the function of the red cell membrane and thus of the cell in health and disease, and shed new light on membrane function in other diverse cell types.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 June 2016
                2016
                : 11
                : 6
                : e0157873
                Affiliations
                [1 ]The Francis Crick Institute, Mill Hill Laboratory, Mill Hill, London, NW7 1AA, United Kingdom
                [2 ]Department of Microbiology & Molecular Medicine, University of Geneva, Rue Michel-Servet 1, CH-1211, Geneva 4, Switzerland
                [3 ]Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom
                Bernhard Nocht Institute for Tropical Medicine, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JAT CRC SD DB MJB. Performed the experiments: JAT CRC SD FH. Analyzed the data: JAT CRC SD FH MJB. Contributed reagents/materials/analysis tools: AG DB ED. Wrote the paper: JAT MJB.

                [¤]

                Current address: Wellcome Trust Centre for Molecular Parasitology, Glasgow, G12 8TA, United Kingdom

                Article
                PONE-D-16-13119
                10.1371/journal.pone.0157873
                4917082
                27332706
                67eb9e75-6db5-4954-8c3c-9db9c64f318a
                © 2016 Thomas et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 31 March 2016
                : 6 June 2016
                Page count
                Figures: 3, Tables: 1, Pages: 14
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000265, Medical Research Council;
                Award ID: U117532063
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004963, Seventh Framework Programme;
                Award ID: 242095
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 099950/Z/12/Z
                Award Recipient :
                This work was supported by the United Kingdom Medical Research Council ( http://www.mrc.ac.uk/) (U117532063 to MJB), the Francis Crick Institute ( https://www.crick.ac.uk/), the Wellcome Trust (099950/Z/12/Z to ED and ISSF2 funding to the London School of Hygiene and Tropical Medicine) and European Commission Framework Programme 7 contract no. 242095 (EviMalAR; http://www.evimalar.org/). JAT is in receipt of a Francis Crick Institute PhD studentship whilst SD was funded by an EviMalAR PhD studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                Red Blood Cells
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Plasmodium
                Biology and Life Sciences
                Parasitology
                Parasite Replication
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Malarial Parasites
                Medicine and Health Sciences
                Parasitic Diseases
                Medicine and Health Sciences
                Parasitic Diseases
                Malaria
                Medicine and Health Sciences
                Tropical Diseases
                Malaria
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Merozoites
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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                Uncategorized

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