Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) in Nigerian children 10 years post-adoption of artemisinin-based combination treatments

The emergence and spread of Plasmodium falciparum parasites resistant to artemisinin derivatives and their partners in southeastern Asia threatens malaria control and elimination efforts, and heightens the need for an alternative therapy. We have explored the distribution of P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) haplotypes 10 years following adoption of artemisinin-based combination therapies in a bid to investigate the possible re-emergence of Chloroquine-sensitive parasites in Nigeria, and investigated the effect of these P. falciparum haplotypes on treatment outcomes of patients treated with artemisinin-based combination therapies. A total of 271 children aged <5 years with uncomplicated falciparum malaria were included in this study. Polymorphisms on codons 72–76 of the Pfcrt gene and codon 86 and 184 of Pfmdr-1 were determined using the high resolution melting assay. Of 240 (88.6%) samples successfully genotyped with HRM for Pfcrt, wildtype C₇₂M₇₄N₇₅K₇₆ (42.9%) and mutant C₇₂I₇₄E₇₅T₇₆ (53.8%) were observed. Also, wildtype N₈₆Y₁₈₄ (62.9%) and mutant N₈₆F₁₈₄ (21.1%), Y₈₆Y₁₈₄ (6.4%), and Y₈₆F₁₈₄ (0.4%) haplotypes of Pfmdr-1 were observed. Measures of responsiveness to ACTs were similar in children infected with P. falciparum crt haplotypes (C₇₂I₇₄E₇₅T₇₆ and C₇₂M₇₄N₇₅K₇₆) and major mdr-1 haplotypes (N₈₆Y₁₈₄, N₈₆F₁₈₄ and Y₈₆Y₁₈₄). Despite a 10 year gap since the malaria treatment policy changed to ACTs, over 50% of the P. falciparum parasites investigated in this study harboured the Chloroquine-resistant C₇₂I₇₄E₇₅T₇₆ haplotype, however this did not compromise the efficacy of artemisinin-based combination therapies. Should complete artemisinin resistance emerge from or spread to Nigeria, chloroquine might not be a good alternative therapy.     

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads

Background

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3β (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT.

Methodology/Principal Findings

A subset of over 16,000 inhibitors of HsGSK-3 β from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed.

Conclusions/Significance

We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.     

Soil Metal Sorption Characteristics and its Influence on the Comparative Effectiveness of EDTA and Legume Intercrop on the Phytoremediative Abilities of Maize (Zea mays), Mucuna (Mucuna pruriens), Okra (Abelmoschus esculentus), and Kenaf (Hibiscus cannabinus)

Heavy metal pollution in soils and the high costs of remediation necessitate the evaluation of cheaper alternatives. The aim of this experiment was to evaluate Cd, Pb, Zn, and Cu sorption characteristics of three soils and their influence on the comparative effectiveness of EDTA and legume intercrop on the remediative abilities of maize, mucuna, okra, and kenaf. The sorption studies were done using standard procedures. The EDTA-assisted phytoextraction (6 mmol kg^?1) and the cowpea intercrop trials were conducted in triplicate. The metal-spiked soils were planted with maize, kenaf, and mucuna in the EDTA trial and maize, kenaf, and okra were planted in the cowpea intercrop experiment. Cadmium was prefentially sorbed in acid and alkaline soils and Cu in slightly acid soil. Cadmium uptake was significantly lower (P < 0.05) in all the plants. Bioconcentration factors of Pb, Cu, and Zn were higher (P < 0.05) in maize compared with other plants. Phytoremediative ability of the plants in trials were maize > kenaf > mucuna and okra > maize > kenaf, respectively. It was concluded that a legume intercrop can substitute EDTA- assisted phytoextraction to prevent groundwater contamination resulting from high solubility of metals by EDTA.     

Monitoring Copper Bioaccumulation in Cocoa from Copper-Based Pesticide–Treated Cocoa Farms Using Fuzzy Similarity Method

Cocoa production has been hindered by pests, leading to application of pesticides. These pesticide applications have raised health concerns. This study is therefore aimed at developing accumulation classifications for copper (Cu) in cocoa from plantations where Cu-based pesticides have been applied using fuzzy similarity method (FSM). Cocoa pods, seeds, leaves, and soil samples were collected from five different plantations from three states in Nigeria. The plant samples were digested using standard methods, whereas the Cu in soil samples was extracted with 1 M ammonium acetate. The products were analyzed for Cu using flame atomic absorption spectrophotometer. The Cu concentrations obtained were used to model the accumulation degree using FSM. The Cu concentrations were higher in the leaves than the seeds and the pods. The persistency of Cu was displayed by high concentrations (11.5–375 mg/kg) and enrichment factor in CRIN sites where this pesticide application has been stopped for over a decade. The leaves from Ondo sites had the highest Cu concentration, which indicates impact of recent applications. The FSM membership function and algorithm indicated that the plant parts had low degrees of accumulation for fresh leaves, pods, and seeds, the valued part applied in beverages, but for the dry leaves, the accumulation degree is high. It may be concluded that the applied Cu-based pesticide had more impact and accumulation on the leaves than the commercial valued seeds, but the Cu concentration in the seeds is above the recommended value.     

Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p paralogs results in catalytically active succinate dehydrogenase isoenzymes

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q(P)) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q(P) site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.     

In vitro morphogenic responses of African yam bean (<Emphasis Type="Italic">Sphenostylis stenocarpa</Emphasis> (Hochst ex. A. Rich.) Harms) accessions to plant growth regulators

Morphogenic responses of two accessions of African yam bean to different concentrations of plant growth regulator supplements to Murashige and Skoog basal medium was investigated to develop a more efficient regeneration system. Mature embryo explants were cultured on growth regulator-free and BAP + NAA supplemented media. Nodal cuttings excised from 4-week old shoots of the regenerated embryos were cultured on media containing varying concentrations and combinations of 6-benzyl aminopurine (BAP), kinetin and α-naphthalene acetic acid (NAA). Growth regulator-free medium favored embryo regeneration and growth over supplemented media and both enhanced shoot regeneration and rooting, but could not induce multiple shoot formation on embryo explants. Multiple shoots were produced by nodal explants and the highest average number of shoots (5.3 ± 2.3), leaves (7.7 ± 3.6), roots (3.7 ± 2.9) and root length (3.1 ± 0.0 cm) were obtained on a medium with 0.6 mg l^?1 BAP + 0.03 mg l^?1 NAA for accession TSs154, while in TSs5, highest number of shoots (3.2 ± 2.5) and leaves (5.9 ± 1.5) were induced by 2.0 mg l^?1 Kinetin + 0.05 mg l^?1 NAA. Such differential morphogenic responses to culture media underline the genotypic control of in vitro propagation of this crop. Embryo and nodal explants rooted directly on shoot regeneration media, and regenerated plantlets were successfully acclimatized. The efficient regeneration system obtained will enhance genetic improvement of African yam bean by facilitating molecular genetic transformation for advanced breeding.