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Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
[Image: see text] New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes....
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical
Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151787/ https://www.ncbi.nlm.nih.gov/pubmed/25102353 http://dx.doi.org/10.1021/ac502098w |
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author | Cevenini, Luca Camarda, Grazia Michelini, Elisa Siciliano, Giulia Calabretta, Maria Maddalena Bona, Roberta Kumar, T. R. Santha Cara, Andrea Branchini, Bruce R. Fidock, David A. Roda, Aldo Alano, Pietro |
author_facet | Cevenini, Luca Camarda, Grazia Michelini, Elisa Siciliano, Giulia Calabretta, Maria Maddalena Bona, Roberta Kumar, T. R. Santha Cara, Andrea Branchini, Bruce R. Fidock, David A. Roda, Aldo Alano, Pietro |
author_sort | Cevenini, Luca |
collection | PubMed |
description | [Image: see text] New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z′ factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing d-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. |
format | Online Article Text |
id | pubmed-4151787 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American
Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-41517872015-08-07 Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites Cevenini, Luca Camarda, Grazia Michelini, Elisa Siciliano, Giulia Calabretta, Maria Maddalena Bona, Roberta Kumar, T. R. Santha Cara, Andrea Branchini, Bruce R. Fidock, David A. Roda, Aldo Alano, Pietro Anal Chem [Image: see text] New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z′ factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing d-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. American Chemical Society 2014-08-07 2014-09-02 /pmc/articles/PMC4151787/ /pubmed/25102353 http://dx.doi.org/10.1021/ac502098w Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) |
spellingShingle | Cevenini, Luca Camarda, Grazia Michelini, Elisa Siciliano, Giulia Calabretta, Maria Maddalena Bona, Roberta Kumar, T. R. Santha Cara, Andrea Branchini, Bruce R. Fidock, David A. Roda, Aldo Alano, Pietro Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites |
title | Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color
Assay and Single-Cell Imaging in Plasmodium falciparum Parasites |
title_full | Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color
Assay and Single-Cell Imaging in Plasmodium falciparum Parasites |
title_fullStr | Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color
Assay and Single-Cell Imaging in Plasmodium falciparum Parasites |
title_full_unstemmed | Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color
Assay and Single-Cell Imaging in Plasmodium falciparum Parasites |
title_short | Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color
Assay and Single-Cell Imaging in Plasmodium falciparum Parasites |
title_sort | multicolor bioluminescence boosts malaria research: quantitative dual-color
assay and single-cell imaging in plasmodium falciparum parasites |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151787/ https://www.ncbi.nlm.nih.gov/pubmed/25102353 http://dx.doi.org/10.1021/ac502098w |
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