In Vitro Chemosensitization of Plasmodium falciparum to Antimalarials by Verapamil and Probenecid▿

We tested the effect of probenecid and verapamil in chemosensitizing Plasmodium falciparum to 14 antimalarials using the multidrug-resistant strain V1S and the drug-sensitive 3D7. Verapamil chemosensitizes V1S to quinine and chloroquine. Interestingly, probenecid profoundly chemosensitizes V1S to piperaquine. Thus, probenecid could be used to increase piperaquine efficacy in vivo

In Vitro Activity of Antifolate and Polymorphism in Dihydrofolate Reductase of Plasmodium falciparum Isolates from the Kenyan Coast: Emergence of Parasites with Ile-164-Leu Mutation▿

We have analyzed the activities of the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), WR99210, trimethoprim (TMP), methotrexate (MTX), and trimetrexate (TMX) against Kenyan Plasmodium falciparum isolates adapted in vitro for long-term culture. We have also assessed the relationship between these drug activities and mutations in dihydrofolate reductase (dhfr), a domain of the gene associated with antifolate resistance. As expected, WR99210 was the most potent drug, with a median 50% inhibitory concentration (IC50) of <0.075 nM, followed by TMX, with a median IC50 of 30 nM. The median IC50 of CCG was 37.80 nM, and that of MTX was 83.60 nM. PM and TMP were the least active drugs, with median IC50s of 733.26 nM and 29,656.04 nM, respectively. We analyzed parasite dhfr genotypes by the PCR-enzyme restriction technique. No wild-type dhfr parasite was found. Twenty-four of 33 parasites were triple mutants (mutations at codons 108, 51, and 59), and only 8/33 were double mutants (mutations at codons 108 and 51 or at codons 108 and 59). IC50s were 2.1-fold (PM) and 3.6-fold (TMP) higher in triple than in double mutants, though these differences were not statistically significant. Interestingly, we have identified a parasite harboring a mutation at codon 164 (Ile-164-Leu) in addition to mutations at codons 108, 51, and 59. This quadruple mutant parasite had the highest TMP IC50 and was in the upper 10th percentile against PM and CCG. We confirmed the presence of this mutation by sequencing. Thus, TMX and MTX are potent against P. falciparum, and quadruple mutants are now emerging in Africa

Adaptation and performance of rice genotypes in tropical and subtropical environments

Standardized field experiments were carried out to study the performance of five rice genotypes derived from different germplasm in terms of yield, harvest index (HI) and grain quality at eight agro-ecological sites of the tropics and subtropics across Asia during 2001 and 2002. Considering that indica and javanica genotypes adapt to warm climatic conditions, and japonica genotypes to cool agro-climatic conditions, it is hypothesized that indica × japonica hybrids may combine high yields and good quality traits under a wide range of agro-climatic conditions. Grain yield, HI, protein content and amylose content varied considerably among genotypes and environments. Mean rice yields of genotypes ranged from 1.5 to 11 t ha-1 across the eight sites; on average yields were 7.2 t ha-1 under subtropical and 2.7 t ha-1 under tropical conditions. The much lower yields in tropical environments resulted from a low biomass as well as a low HI. Among the genotypes, the indica × japonica hybrid showed the highest yield under subtropical conditions, and a higher yield than the japonica genotypes and the indica × javanica hybrid but lower than the indica genotype under tropical conditions. Phenology of genotypes varied strongly across environments. Low yields at tropical locations were associated with a low light capture due to short growth duration. Post-anthesis light-use efficiencies and the photothermal quotient explained much of the variation in yield. Protein content varied among genotypes depending on location and year. Variation in amylose content of rice grains was mainly associated with genotypic differences and much less with environmental conditions, but contents decreased with higher post-anthesis ambient temperatures. The indica × japonica hybrid combined high yields with a favourable amylose content and showed a better ability to adapt to cool and to warm agro-climatic conditions than the indica or japonica genotypes. Our study showed the magnitude of yield penalties associated with growing rice genotypes in environments to which they are not adapted. The consequences of these findings for improved adaptation of rice are discusse

Chromosomes 2 (A) and 10 (B) show over-expression of contiguous probes covering 21 and 22 CDS, respectively.

<p>Amplification of the signals for the left arms of chromosomes 2 (<b>A</b>) and 10 (<b>B</b>) are enlarged for each time point as indicated. Every single coloured dot corresponds to a 25-mer probe: red is for 0 h, blue for 12 h, green for 24 h and yellow for 36 h. Underneath every enlarged chromosomal arm are pink bars indicating 100% robustness of signal amplification at <i>p</i><0.01 (using SnoopCGH program with Smith–Waterman algorithm implementation). A normal distribution of the log ratios (y-axis) around the zero horizontal line is expected if the expression levels are the same along the chromosome (indicated as kilo base pair [kbp]). The CDS (represented under each chromosome by blue rectangles) contained within each amplified region are indicated on the right with their appropriate annotation (<a href="http://www.genedb.org" target="_blank">www.genedb.org</a>). The genes marked with an * have been found significant at B>0 in the pairwise comparisons of the microarray data in at least one time point, while the underlined genes have been double checked by qRT-PCR.</p

Correlation coefficients of log2 fold changes (FC) by RT-qPCR and microarray analysis.

<p>The linear regression is indicated on the plot, with an r² = 0.7389.</p

Lumefantrine drug selection regimen.

<p>Number of cycles during which V1S was cultured with varying Lumefantrine (LM) concentrations. In total, parasites were exposed to LM for 166 <i>P. falciparum</i> cycles, finally resulting in LM resistant V1S_LM.</p

Gene Ontology (GO) analysis at main time points of the <i>P. falciparum</i> asexual life cycle.

<p>The time points color legend is indicated at the top left corner of the barplot. GO enrichment is indicated as stacked percentages.</p

Changes in gene expression profiles between LM resistant V1S_LM and LM sensitive V1S <i>P. falciparum</i>.

<p><b>A.</b> Heatmap of 589 expressed genes showing Differential Expression (DE) in at least one time point (F adjusted <i>p</i><0.05). The 2 clusters highlighted with blue and red bars on the right hand side of the heatmap correspond to subtelomeric genes gradually switched off in the presence of LM, and transporters and cell cycle regulators gradually turned on in the presence of LM, respectively. Log2 ratio of V1S_LM vs. V1S expression is indicated by the color key ranging from −6 (blue, under-expression) to 6 (red, over-expression) <b>B.</b> Venn diagram showing the asexual life cycle distribution of DE genes. Analysis was based on linear modeling using Limma package of R/Bioconductor.</p