Phosphoenolpyruvate carboxylase dentified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in thePlasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery

Geometric least squares means ratios for the analysis of Plasmodium falciparum in vitro susceptibility to antimalarial drugs

<p>Abstract</p> <p>Background</p> <p>The susceptibility of microbes such as <it>Plasmodium falciparum </it>to drugs is measured in vitro as the concentration of the drug achieving 50% of maximum effect (IC₅₀); values from a population are summarized as geometric means. For antimalarial drugs, as well as for antibiotics, assessing changes in microbe susceptibility over time under drug pressure would help inform treatment policy decisions, but no standard statistical method exists as yet.</p> <p>Methods</p> <p>A mixed model was generated on log_e-transformed IC₅₀values and calculated geometric least squares means (GLSM) with 90% confidence intervals (CIs). In order to compare IC₅₀s between years, GLSM ratios (GLSMR) with 90%CIs were calculated and, when both limits of the 90%CIs were below or above 100%, the difference was considered statistically significant. Results were compared to those obtained from ANOVA and a generalized linear model (GLM).</p> <p>Results</p> <p>GLSMRs were more conservative than ANOVA and resulted in lower levels of statistical significance. The GLSMRs approach allowed for random effect and adjustment for multiple comparisons. GLM was limited in the number of year-to-year comparisons by the need for a single reference year. The three analyses yielded generally consistent results.</p> <p>Conclusion</p> <p>A robust analytical method can palliate inherent limitations of in vitro sensitivity testing. The random effects GLSMRs with adjustment for multiple comparisons and 90%CIs require only assumptions on the mixed model to be applied. Results are easy to display graphically and to interpret. The GLMSRs should be considered as an option for monitoring changes in drug susceptibility of <it>P. falciparum </it>malaria and other microbes.</p

Isothermal Microcalorimetry, a New Tool to Monitor Drug Action against Trypanosoma brucei and Plasmodium falciparum

Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly

Physical activity to improve cognition in older adults: can physical activity programs enriched with cognitive challenges enhance the effects? A systematic review and meta-analysis

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Effects of mefloquine and artesunate mefloquine on the emergence, clearance and sex ratio of Plasmodium falciparum gametocytes in malarious children

<p>Abstract</p> <p>Background</p> <p>The gametocyte sex ratio of <it>Plasmodium falciparum</it>, defined as the proportion of gametocytes that are male, may influence transmission but little is known of the effects of mefloquine or artesunate-mefloquine on gametocyte sex ratio and on the sex ratio of first appearing gametocytes.</p> <p>Methods</p> <p>350 children with uncomplicated <it>P. falciparum </it>malaria were enrolled in prospective treatment trial of mefloquine or artesunate-mefloquine between 2007 and 2008. Gametocytaemia was quantified, and gametocytes were sexed by morphological appearance, before and following treatment. The area under curve of gametocyte density <it>versus </it>time (AUC_gm) was calculated by linear trapezoidal method.</p> <p>Results</p> <p>91% and 96% of all gametocytes appeared by day 7 and day 14, respectively following treatment. The overall rate of gametocytaemia with both treatments was 31%, and was significantly higher in mefloquine than in artesunate-mefloquine treated children if no gametocyte was present a day after treatment began (25.3% <it>v </it>12.8%, P = 0.01). Gametocyte clearance was significantly faster with artesunate-mefloquine (1.8 ± 0.22 [sem] <it>v </it>5.6 ± 0.95 d; P = 0.001). AUC_gmwas significantly lower in the artesunate mefloquine group (P = 0.008). The pre-treatment sex ratio was male-biased, but post-treatment sex ratio or the sex ratio of first appearing gametocytes, was significantly lower and female-biased two or three days after beginning of treatment in children given artesunate-mefloquine.</p> <p>Conclusion</p> <p>Addition of artesunate to mefloquine significantly modified the emergence, clearance, and densities of gametocytes and has short-lived, but significant, sex ratio modifying effects in children from this endemic area.</p

A Comparison of Scent Marking between a Monogamous and Promiscuous Species of Peromyscus: Pair Bonded Males Do Not Advertise to Novel Females

Scent marking can provide behavioral and physiological information including territory ownership and mate advertisement. It is unknown how mating status and pair cohabitation influence marking by males from different social systems. We compared the highly territorial and monogamous California mouse (Peromyscus californicus) to the less territorial and promiscuous white-footed mouse (P. leucopus). Single and mated males of both species were assigned to one of the following arenas lined with filter paper: control (unscented arena), male scented (previously scent-marked by a male conspecific), or females present (containing females in small cages). As expected, the territorial P. californicus scent marked and overmarked an unfamiliar male conspecific's scent marks more frequently than P. leucopus. Species differences in responses to novel females were also found based on mating status. The presence of unfamiliar females failed to induce changes in scent marking in pair bonded P. californicus even though virgin males increased marking behavior. Pair bonding appears to reduce male advertisement for novel females. This is in contrast to P. leucopus males that continue to advertise regardless of mating status. Our data suggest that communication through scent-marking can diverge significantly between species based on mating system and that there are physiological mechanisms that can inhibit responsiveness of males to female cues

In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Recently, <it>Plasmodium falciparum </it>parasites bearing <it>Pfdhfr </it>I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves.</p> <p>Methods</p> <p>Thirty <it>Plasmodium falciparum </it>clinical isolates were collected in 2008 in the south-east of Madagascar. A part of <it>Pfdhfr </it>gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC_50sto pyrimethamine was performed on adapted isolates.</p> <p>Results</p> <p>Four different <it>Pfdhfr </it>alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of <it>P. falciparum </it>isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine <it>in vitro </it>susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC₅₀) < 10 nM. The geometric mean of IC₅₀of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC₅₀of the triple mutant parasite (13,804 nM).</p> <p>Conclusion</p> <p>The IC₅₀s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC₅₀s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the <it>Pfdhfr </it>I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.</p

Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia

<p>Abstract</p> <p>Background</p> <p>Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>malaria was assessed in Chumkiri, Kampot Province, Cambodia.</p> <p>Methods</p> <p>One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For <it>P. falciparum </it>infected subjects, PCR genotyping of <it>msp1</it>, <it>msp2</it>, and <it>glurp </it>was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the <it>pfmdr1 </it>gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC₅₀for anti-malarial drugs.</p> <p>Results</p> <p>Among <it>P. falciparum </it>infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased <it>pfmdr1 </it>copy number, higher initial parasitaemia, higher mefloquine IC₅₀, and longer time to parasite clearance. One <it>P. falciparum </it>isolate, from a treatment failure, had markedly elevated IC₅₀for both mefloquine (130 nM) and artesunate (6.7 nM). Among <it>P. vivax </it>infected subjects, 42.1% suffered recurrent <it>P. vivax </it>parasitaemia. None acquired new <it>P. falciparum </it>infection.</p> <p>Conclusion</p> <p>The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC₅₀suggest that artesunate resistance may be emerging on a background of mefloquine resistance.</p

Cue Integration in Categorical Tasks: Insights from Audio-Visual Speech Perception

Previous cue integration studies have examined continuous perceptual dimensions (e.g., size) and have shown that human cue integration is well described by a normative model in which cues are weighted in proportion to their sensory reliability, as estimated from single-cue performance. However, this normative model may not be applicable to categorical perceptual dimensions (e.g., phonemes). In tasks defined over categorical perceptual dimensions, optimal cue weights should depend not only on the sensory variance affecting the perception of each cue but also on the environmental variance inherent in each task-relevant category. Here, we present a computational and experimental investigation of cue integration in a categorical audio-visual (articulatory) speech perception task. Our results show that human performance during audio-visual phonemic labeling is qualitatively consistent with the behavior of a Bayes-optimal observer. Specifically, we show that the participants in our task are sensitive, on a trial-by-trial basis, to the sensory uncertainty associated with the auditory and visual cues, during phonemic categorization. In addition, we show that while sensory uncertainty is a significant factor in determining cue weights, it is not the only one and participants' performance is consistent with an optimal model in which environmental, within category variability also plays a role in determining cue weights. Furthermore, we show that in our task, the sensory variability affecting the visual modality during cue-combination is not well estimated from single-cue performance, but can be estimated from multi-cue performance. The findings and computational principles described here represent a principled first step towards characterizing the mechanisms underlying human cue integration in categorical tasks