Single-nucleotide polymorphism,linkage disequilibrium and geographic structure in the malaria parasite <Emphasis Type="Italic">Plasmodium vivax</Emphasis>: prospects for genome-wide association studies

Background

The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.

Results

We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.

Conclusion

These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/90

     

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A.     

Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor

A new specific and sensitive method for determination of tissue plasminogen activator (t-PA) in plasma samples has been used to demonstrate the presence of a fast inhibitor to t-PA in plasma. By adding [35S]Met internally labeled t-PA (Mr approximately 70,000) to plasma, we were able to demonstrate the rapid formation of a stable complex with an apparent molecular weight of about 115,000 as estimated by gel filtration. The complex was partially purified by immunoadsorbtion chromatography on insolubilized antibodies against porcine t-PA, and a molecular weight of about 120,000 was found by dodecyl sulfate-polyacrylamide gel electrophoresis. From the apparent molecular weight of the complex (120,000) and the molecular weight of t-PA (70,000), a molecular weight of about 50,000 would be expected for the inhibitor. However, gel filtration of inhibitor-rich plasma resulted in the appearance of a symmetrical peak of t-PA inhibitory activity with an apparent molecular weight of about 205,000. The reason for this discrepancy is not known, but several different models are possible.     

Purification of high and low molecular weight plasminogen activator inhibitor 1 from fibrosarcoma cell-line HT 1080 conditioned medium

Functionally active (high-Mr) and inactive (low-Mr) plasminogen activator inhibitor 1 (PAI) have been purified from fibrosarcoma cell-line HT 1080 conditioned medium, containing 1% fetal calf serum. The two forms were first purified by affinity chromatography on heparin-Sepharose and then separated from each other by gel filtration on Sephadex G-150. The final purification was achieved by affinity chromatography on insolubilized monoclonal antibodies towards human PAI. Alternatively, the low-Mr form was purified by chromatography on carboxymethyl-cellulose. Low-Mr PAI purified in this way, could be almost fully reactivated by treatment with guanidinium chloride. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting revealed that the low-Mr form contained nothing but PAI at an Mr of about 50,000. In addition to PAI, the high-Mr form contained a component, which was not antigenically related to PAI. This compound had a molecular weight of about 75,000 and its NH2-terminal amino acid sequence corresponded to that of human vitronectin. We conclude that the high-Mr form of PAI constitutes a complex between 50,000 Mr PAI and vitronectin from fetal calf serum.     

Parasitism of frozen Halyomorpha halys eggs by Trissolcus japonicus: applications for rearing and experimentation

The brown marmorated stink bug, Halyomorpha halys (Stål), has become a well-known pest to growers and homeowners since its 1996 introduction to the United States. A classical biocontrol programme is under development using the egg parasitoid Trissolcus japonicus. Widespread implementation of biocontrol requires efficient mass rearing, which is constrained by the availability of fresh H. halys eggs. In this study, parasitism rate, developmental time, sex ratio and size were compared between wasps reared on fresh versus frozen, newly laid (<1 d old) versus variably aged (0–3 d old), and frozen egg masses stored ≤4 y. Frozen eggs yielded 56–65% fewer wasps, with parasitism rate decreasing 1–3% per month stored. Parasitism rate, sex ratio and developmental time were comparable between newly laid and variably aged eggs. Freezing eggs for any duration did not affect sex ratio or weight of emerged wasps, but delayed emergence 5–6 d. To simulate deployment of sentinel eggs in the field, we incubated frozen eggs at 20°C and 30°C for 1–9 d before exposing them to T. japonicus, then evaluated parasitism trends. Trissolcus japonicus parasitism rate decreased 5–8% per day incubated, unhatched wasps increased 9% per day incubated and sex ratio was not impacted. Variably aged, frozen and longer stored eggs can be used for T. japonicus rearing and experimentation without affecting emerged wasp sex ratio or size within one generation, but have lower parasitism and slower development. Frozen sentinel eggs are effective <3–5 d, especially in hot conditions.     

Inactivation of Myc-induced p53-dependent apoptosis in human tumors

The Myc family of oncoproteins promote cell growth and are frequently overexpressed in human tumors. However, Myc can also trigger cell death by apoptosis. This is at least in part mediated via the ARF-p53 pathway. Myc activation leads to a selection for inactivation of ARF or p53, allowing cell survival and tumor progression. Restoration of p53-dependent apoptosis by various means is an attractive approach for new cancer therapy.