Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine

Control of pyrethroid and DDT-resistant Anopheles gambiae by application of indoor residual spraying or mosquito nets treated with a long-lasting organophosphate insecticide, chlorpyrifos-methyl.

BACKGROUND: Scaling up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) with support from the Global Fund and President's Malaria Initiative is providing increased opportunities for malaria control in Africa. The most cost-effective and longest-lasting residual insecticide DDT is also the most environmentally persistent. Alternative residual insecticides exist, but are too short-lived or too expensive to sustain. Dow Agrosciences have developed a microencapsulated formulation (CS) of the organophosphate chlorpyrifos methyl as a cost-effective, long-lasting alternative to DDT. METHODS: Chlorpyrifos methyl CS was tested as an IRS or ITN treatment in experimental huts in an area of Benin where Anopheles gambiae and Culex quinquefasiactus are resistant to pyrethroids, but susceptible to organophosphates. Efficacy and residual activity was compared to that of DDT and the pyrethroid lambdacyalothrin. RESULTS: IRS with chlorpyrifos methyl killed 95% of An. gambiae that entered the hut as compared to 31% with lambdacyhalothrin and 50% with DDT. Control of Cx. quinquefasciatus showed a similar trend; although the level of mortality with chlorpyrifos methyl was lower (66%) it was still much higher than for DDT (14%) or pyrethroid (15%) treatments. Nets impregnated with lambdacyhalothrin were compromised by resistance, killing only 30% of An. gambiae and 8% of Cx. quinquefasciatus. Nets impregnated with chlorpyrifos methyl killed more (45% of An gambiae and 15% of Cx. quinquefasciatus), but its activity on netting was of short duration. Contact bioassays on the sprayed cement-sand walls over the nine months of monitoring showed no loss of activity of chlorpyrifos methyl, whereas lambdacyhalothrin and DDT lost activity within a few months of spraying. CONCLUSION: As an IRS treatment against pyrethroid resistant mosquitoes chlorpyrifos methyl CS outperformed DDT and lambdacyhalothrin. In IRS campaigns, chlorpyrifos methyl CS should show higher, more-sustained levels of malaria transmission control than conventional formulations of DDT or pyrethroids. The remarkable residual activity indicates that cost-effective alternatives to DDT are feasible through modern formulation technology

Protein Sci

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes

Subunit Interface in <i>GSTE2</i> variants.

<p>a. Close-up detail of interface groups in the <i>GSTE2</i> dimer. Phenylalanine residues F113 contributed by the respective helices H4 as well as tyrosines Y133 from neighbouring helices pack together to form a linear stack. b. Electron density map (contoured at 1.0 σ) for the <i>GSTE2</i> ZAN/U variant. The mutated residue T114 is shown. The preceding residue F113 is poorly ordered and has been modeled as adopting two alternate conformations (towards the front and back of the paper plane).</p

Crystal structure of <i>GSTE2</i> ZAN/U variant.

<p>a. Superposition of the crystal structure of ZAN/U determined in this study (orange) and the Kimusu 1B variant (grey; PDB entry IMI). A high degree of local and overall structural agreement is clearly noticeable. The location of the docked DDT is based on the computational prediction of Wang et al.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092662#pone.0092662-Wang1" target="_blank">[15]</a>. Some manual adjustments were made to relieve steric clashes and to better superimpose the DDT on the position of the hexyl group of bound S-hexylglutathione. b. Close-up detail of the ZAN/U active site. c. Superposition of structure of ZAN/U and Kimusu 1B variant local to position 114 (colour code as in a. A superimposition of ZAN/U from <i>An. gambiae</i> with the <i>GSTE2</i> from <i>An. funestus</i> is provided in Figure S3).</p

Enzyme kinetic parameters of three GSTE2 alleles with substrate DDT.

<p>Three variant GSTE2 proteins were expressed from a DDT resistant (ZAN/U) and susceptible (Kisumu) strain of <i>An. gambiae</i> and assayed with substrate DDT over a range of concentrations. The maximum enzyme rate (V_max), substrate concentration at half the maximum rate (K_M) and catalytic turn-over (K_cat) were calculated for each protein from a Michaelis-Menten or substrate inhibition equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092662#pone-0092662-g001" target="_blank">Figure 1</a>).</p

Dose-response curves for <i>Drosophila melanogaster</i> adults transformed with <i>Anopheles gambiae Gste2</i> alleles.

<p>The left panel shows survival of control (CyO x UAS+<i>Gste2</i>-Kisumu1B; black circles) and Kisumu allele expressing lines (<i>Actin</i>-Gal4 x UAS+<i>Gste2</i>-Kisumu1B; open circle) together with 95% confidence intervals. The right panel shows survival of control (CyO x UAS+<i>Gste2</i>-ZANU; black circles) and ZAN/U allele (Actin-Gal4 x UAS+<i>Gste2</i>-ZANU; open circle) together with 95% confidence intervals.</p

Comparison of GSTE2 catalysed DDT metabolism for three variant recombinant proteins over a DDT dilution series.

<p>Three allelic variants of enzyme GSTE2 from <i>An. gambiae</i> are compared over a range of DDT concentrations and the mean production of DDE plotted from three replicate assays. Fitted curves used the Michaelis-Menten equation for the ZAN/U allele and a substrate inhibition equation for the two Kisumu alleles</p

Geographical variation in frequency of <i>Gste2</i>-I114T in the S and M molecular forms of <i>An. gambiae</i> across Africa.

<p>Blue represents the I114 and red the T114 frequency. The molecular form of the collection is indicated by the letter overlaid on each chart. Samples were from: Benin S-form n = 111; M-form n = 223. Burkina Faso S-form n = 115; M-form n = 216. Cameroon S-form n = 55; M-form n = 652. Ghana S-form n = 29; M-form n = 758. Guinea-Bissau S-form n = 38; M-form n = 39. Mali S-form n = 31; M-form n = 26. Uganda S-form n = 207. The base map was obtained from <a href="http://en.wikipedia.org/wiki/File:Africa_satellite_orthographic.jpg" target="_blank">http://en.wikipedia.org/wiki/File:Africa_satellite_orthographic.jpg</a> and was created by NASA. Details of the locations are given in Table S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092662#pone.0092662.s001" target="_blank">File S1</a>.</p