The metabolism of the parasite Trypanosoma brucei has been the focus of numerous studies since the 1940s. Recently it was shown, using metabolomics coupled with heavy-atom isotope labelled glucose, that the metabolism of the bloodstream form parasite is more complex than previously thought. The present study also raised a number of questions regarding the origin of several metabolites, for example succinate, only a proportion of which derives from glucose. In order to answer some of these questions and explore the metabolism of bloodstream form T. brucei in more depth we followed the fate of five heavy labelled amino acids – glutamine, proline, methionine, cysteine and arginine – using an LC–MS based metabolomics approach. We found that some of these amino acids have roles beyond those previously thought and we have tentatively identified some unexpected metabolites which need to be confirmed and their function determined.
We have critically evaluated 3 techniques for the detection of mutations conferring drug resistance of Plasmodium falciparum, using samples containing known numbers of well-characterized parasites and artificial mixtures of these parasites at known proportions. We compared the sensitivity and specificity of mutation-specific polymerase chain reaction (MS-PCR), polymerase chain reaction followed by restriction enzyme digestion at polymorphic sites (PCR/RFLP), and a dot-blot/probe hybridization technique, for detection of point mutations at nucleotide 323 of the P. falciparum dihydrofolate reductase gene (dhfr) that confer resistance to the antimalarial drug pyrimethamine. We have also investigated the benefits in terms of sensitivity and reproducibility of the incorporation of radiolabelled nucleotides into the PCR/RFLP assay. We found that MS-PCR was very sensitive--at least 10 parasites could be detected in a sample--but non-specific amplification resulted in erroneous typing of some samples. PCR/RFLP was less sensitive; 10 parasites per sample could not always be detected, but the technique was specific. The addition of radiolabelled nucleotides to the assay did not markedly improve the sensitivity but the results were easier to read and there was less subjectivity in scoring the results. The dot-blot/probe hybridization technique was specific and sensitive, with similar levels of specificity and sensitivity to PCR/RFLP. On balance, the dot-blot/probe hybridization technique seems best suited to large-scale epidemiological surveys of genes associated with antimalarial drug resistance.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.