Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In <it>Plasmodium falciparum </it>an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.</p> <p>Methods</p> <p>Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.</p> <p>Results</p> <p>As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.</p> <p>Conclusions</p> <p>Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.</p

A human keratin 10 knockout causes recessive epidermolytic hyperkeratosis

Epidermolytic hyperkeratosis (EHK) is a blistering skin disease inherited as an autosomal-dominant trait. The disease is caused by genetic defects of the epidermal keratin K1 or K10, leading to an impaired tonofilament network of differentiating epidermal cells. Here, we describe for the first time a kindred with recessive inheritance of EHK. Sequence analysis revealed a homozygous nonsense mutation of the KRT10 gene in the affected family members, leading to a premature termination codon (p.Q434X), whereas the clinically unaffected consanguineous parents were both heterozygous carriers of the mutation. Semi-quantitative RT-PCR and western blot analysis demonstrated degradation of the KRT10 transcript, resulting in complete absence of keratin K10 protein in the epidermis and cultured keratinocytes of homozygous patients. This K10 null mutation leads to a severe phenotype, clinically resembling autosomal-dominant EHK, but differing in form and distribution of keratin aggregates on ultrastructural analysis. Strong induction of the wound-healing keratins K6, K16 and K17 was found in the suprabasal epidermis, which are not able to compensate for the lack of keratin 10. We demonstrate that a recessive mutation in KRT10 leading to a complete human K10 knockout can cause EHK. Identification of the heterogeneity of this disorder has a major impact for the accurate genetic counseling of patients and their families and also has implications for gene-therapy approache

Oligomeric interfaces as a tool in drug discovery:Specific interference with activity of malate dehydrogenase of Plasmodium falciparum in vitro

Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest. In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect-particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

Crude or purified, somatic or metabolic extracts of native antigens are routinely used for the serodiagnosis of human helminthic infections. These antigens are often cross-reactive, i.e., recognized by sera from patients infected with heterologous helminth species. To overcome limitations in antigen production, test sensitivity and specificity, chemically synthesized peptides offer a pure and standardized alternative, provided they yield acceptable operative characteristics. Ongoing genome and proteome work create new resources for the identification of antigens. Making use of the growing amount of genomic and proteomic data available in public databases, we tested a bioinformatic procedure for the selection of potentially antigenic peptides from a collection of protein sequences including conceptually translated nucleotide sequence data of Echinococcus multilocularis and E. granulosus (Plathyhelminthes, Cestoda). The in silico selection was combined with high-throughput screening of peptides on microarray and systematic validation of reactive candidates in enzyme-linked immunosorbent assay. Our study proved the applicability of this approach for selection of peptide antigens with good diagnostic characteristics. Our results suggested the pooling of several peptides to reach a high level of sensitivity required for reliable immunodiagnosis

Oligomeric protein interference validates druggability of aspartate interconversion in Plasmodium falciparum

The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

The Assembly of the Plasmodial PLP Synthase Complex Follows a Defined Course

Background: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers. Methodology/Principal Findings: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis. Conclusions/Significance: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows

Party rules, party resources, and the politics of parliamentary democracies: how parties organize in the 21st Century

This article introduces the first findings of the Political Party Database (PPDB) project, a major survey of party organizations in parliamentary and semi-presidential democracies. The project’s first round of data covers 122 parties in 19 countries. In this paper we describe the scope of the database, then investigate what it tells us about contemporary party organization in these countries, focussing on parties’ resources, structures and internal decision-making. We examine organizational patterns by country and party family, and where possible we make temporal comparisons with older datasets. Our analyses suggest a remarkable coexistence of uniformity and diversity. In terms of the major organizational resources on which parties can draw, such as members, staff and finance, the new evidence largely confirms the continuation of trends identified in previous research: i.e., declining membership, but enhanced financial resources and more paid staff. We also find remarkable uniformity regarding the core architecture of party organizations. At the same time, however, we find substantial variation between countries and party families in terms of their internal processes, with particular regard to how internally democratic they are, and in the forms that this democratization takes

High specificity of line-immunoassay based algorithms for recent HIV-1 infection independent of viral subtype and stage of disease

ABSTRACT: BACKGROUND: Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have shown that a patient's antibody reaction in a confirmatory line immunoassay (INNO-LIATM HIV I/II Score, Innogenetics) provides information on the duration of infection. Here, we sought to further investigate the diagnostic specificity of various Inno-Lia algorithms and to identify factors affecting it. METHODS: Plasma samples of 714 selected patients of the Swiss HIV Cohort Study infected for longer than 12 months and representing all viral clades and stages of chronic HIV-1 infection were tested blindly by Inno-Lia and classified as either incident (up to 12 m) or older infection by 24 different algorithms. Of the total, 524 patients received HAART, 308 had HIV-1 RNA below 50 copies/mL, and 620 were infected by a HIV-1 non-B clade. Using logistic regression analysis we evaluated factors that might affect the specificity of these algorithms. RESULTS: HIV-1 RNA <50 copies/mL was associated with significantly lower reactivity to all five HIV-1 antigens of the Inno-Lia and impaired specificity of most algorithms. Among 412 patients either untreated or with HIV-1 RNA ≥50 copies/mL despite HAART, the median specificity of the algorithms was 96.5% (range 92.0-100%). The only factor that significantly promoted false-incident results in this group was age, with false-incident results increasing by a few percent per additional year. HIV-1 clade, HIV-1 RNA, CD4 percentage, sex, disease stage, and testing modalities exhibited no significance. Results were similar among 190 untreated patients. CONCLUSIONS: The specificity of most Inno-Lia algorithms was high and not affected by HIV-1 variability, advanced disease and other factors promoting false-recent results in other STARHS. Specificity should be good in any group of untreated HIV-1 patients

Top-down contingent feature-specific orienting with and without awareness of the visual input

In the present article, the role of endogenous feature-specific orienting for conscious and unconscious vision is reviewed. We start with an overview of orienting. We proceed with a review of masking research, and the definition of the criteria of experimental protocols that demonstrate endogenous and exogenous orienting, respectively. Against this background of criteria, we assess studies of unconscious orienting and come to the conclusion that so far studies of unconscious orienting demonstrated endogenous feature-specific orienting. The review closes with a discussion of the role of unconscious orienting in action control