Experimental Evolution of Resistance to Artemisinin Combination Therapy Results in Amplification of the mdr1 Gene in a Rodent Malaria Parasite

Background: Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT): resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated. Methodology/Principal Findings: We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN) + mefloquine (MF) version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN + MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN + MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes. Conclusion: Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s), as ye

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

<p>Abstract</p> <p>Background</p> <p>Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the <it>hMLH1 </it>gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all <it>MLH1 </it>alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, <it>in vivo </it>assays for functional characterization of <it>MLH1 </it>mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of <it>hMLH1 </it>mutations <it>in vivo</it>, based on co-expression of human MLH1 and PMS2 in yeast cells.</p> <p>Methods</p> <p>Yeast <it>MLH1 </it>and <it>PMS1 </it>genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.</p> <p>Results</p> <p>The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related <it>MLH1 </it>variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.</p> <p>Conclusion</p> <p>Results of our <it>in vivo </it>yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of <it>MLH1 </it>variants in cancer patients found throughout the entire coding region of the gene.</p

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation-a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design

Investigation of heavy metal pollution in eastern Aegean Sea coastal waters by using Cystoseira barbata, Patella caerulea, and Liza aurata as biological indicators

In order to have an extensive contamination profile of heavy metal levels (Cd, Cu, Fe, Mn, Ni, Pb, and Zn), seawater, sediment, Patella caerulea, Cystoseira barbata, and Liza aurata were investigated by using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Samples were collected from five coastal stations along the eastern Aegean Sea coast (Turkey) on a monthly basis from July 2002 through May 2003. According to the results of this study, heavy metal levels were arranged in the following sequence: Fe > Pb > Zn > Mn > Ni > Cu > Cd for water, Fe > Cu > Mn > Ni > Zn > Pb > Cd for sediment, Fe > Zn > Mn > Pb > Ni > Cd > Cu for C. barbata, Fe > Zn > Mn > Ni > Pb > Cu > Cd for P. caerulea, and Fe > Zn > Mn > Cu > Ni > Pb > Cd for L. aurata. Moreover, positive relationships between Fe in water and Mn in water, Fe in sediment and Mn in sediment, Fe in C. barbata and Mn in C. barbata, Fe in P. caerulea and Mn in P. caerulea, and Fe in L. aurata and Mn in L. aurata may suggest that these metals could be originated from the same anthropogenic source. C. barbata represented with higher bioconcentration factor (BCF) values, especially for Fe, Mn, and Zn values. This observation may support that C. barbata can be used as an indicator species for the determinations of Fe, Mn, and Zn levels. Regarding Turkish Food Codex Regulation's residue limits, metal values in L. aurata were found to be lower than the maximum-permissible levels issued by Turkish legislation and also the recommended limits set by FAO/WHO guidelines. The results of the investigation indicated that P. caerulea, L. aurata, and especially C. barbata are quantitative water-quality bioindicators and biomonitoring subjects for biologically available metal accumulation for Aegean Sea coastal waters