Molecular Identification of a Malaria Merozoite Surface Sheddase

Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface “sheddase,” but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite

Immune-mediated competition in rodent malaria is most likely caused by induced changes in innate immune clearance of merozoites

Malarial infections are often genetically diverse, leading to competitive interactions between parasites. A quantitative understanding of the competition between strains is essential to understand a wide range of issues, including the evolution of virulence and drug resistance. In this study, we use dynamical-model based Bayesian inference to investigate the cause of competitive suppression of an avirulent clone of Plasmodium chabaudi (AS) by a virulent clone (AJ) in immuno-deficient and competent mice. We test whether competitive suppression is caused by clone-specific differences in one or more of the following processes: adaptive immune clearance of merozoites and parasitised red blood cells (RBCs), background loss of merozoites and parasitised RBCs, RBC age preference, RBC infection rate, burst size, and within-RBC interference. These processes were parameterised in dynamical mathematical models and fitted to experimental data. We found that just one parameter μ, the ratio of background loss rate of merozoites to invasion rate of mature RBCs, needed to be clone-specific to predict the data. Interestingly, μ was found to be the same for both clones in single-clone infections, but different between the clones in mixed infections. The size of this difference was largest in immuno-competent mice and smallest in immuno-deficient mice. This explains why competitive suppression was alleviated in immuno-deficient mice. We found that competitive suppression acts early in infection, even before the day of peak parasitaemia. These results lead us to argue that the innate immune response clearing merozoites is the most likely, but not necessarily the only, mediator of competitive interactions between virulent and avirulent clones. Moreover, in mixed infections we predict there to be an interaction between the clones and the innate immune response which induces changes in the strength of its clearance of merozoites. What this interaction is unknown, but future refinement of the model, challenged with other datasets, may lead to its discovery

Centromere Plasmid: A New Genetic Tool for the Study of Plasmodium falciparum

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite

Attitudes to antipsychotic drugs and their side effects: a comparison between general practitioners and the general population

BACKGROUND: Attitudes towards antipsychotic medication play an important part in the treatment for schizophrenia and related disorders. We aimed measuring general practitioners' attitudes to antipsychotic drugs and their adverse side effects and comparing these with the attitudes of the general population. METHODS: Analysis and comparison of two representative samples, one comprising 100 General Practitioners (GPs), the other 791 individuals randomly selected from the general population. The setting was the German speaking cantons of Switzerland. RESULTS: General practitioners have significantly more positive attitudes towards anti-psychotic drugs than the general public. They reject widespread prejudices about the use of anti-psychotic medication significantly more than the general population. In particular the risk of dependency was assessed as 'low' by GP's (80%), in contrast to only 18% of the general population sample. In no instance did a majority of the GPs advise not tolerating any of the 10 possible adverse effects presented in this study. This is in marked contrast to the general population sample, where a majority recommended discontinuation for movement disorder (63%), strong tremor (59%), risk of dependency (55%) and feelings of unrest (54%). CONCLUSION: As well as effective management of side-effects being a vital aspect of patient and carer education, prescribing doctors need to be aware that their mentally ill patients are likely to be confronted with extremely negative public attitudes towards antipsychotic medication and with strong pressures to stop taking their medication in the event of side-effects

The application of foraging theory to the information searching behaviour of general practitioners

<p>Abstract</p> <p>Background</p> <p>General Practitioners (GPs) employ strategies to identify and retrieve medical evidence for clinical decision making which take workload and time constraints into account. Optimal Foraging Theory (OFT) initially developed to study animal foraging for food is used to explore the information searching behaviour of General Practitioners. This study is the first to apply foraging theory within this context.</p> <p>Study objectives were:</p> <p>1. To identify the sequence and steps deployed in identifiying and retrieving evidence for clinical decision making.</p> <p>2. To utilise Optimal Foraging Theory to assess the effectiveness and efficiency of General Practitioner information searching.</p> <p>Methods</p> <p>GPs from the Wellington region of New Zealand were asked to document in a pre-formatted logbook the steps and outcomes of an information search linked to their clinical decision making, and fill in a questionnaire about their personal, practice and information-searching backgrounds.</p> <p>Results</p> <p>A total of 115/155 eligible GPs returned a background questionnaire, and 71 completed their information search logbook.</p> <p>GPs spent an average of 17.7 minutes addressing their search for clinical information. Their preferred information sources were discussions with colleagues (38% of sources) and books (22%). These were the two most profitable information foraging sources (15.9 min and 9.5 min search time per answer, compared to 34.3 minutes in databases). GPs nearly always accessed another source when unsuccessful (95% after 1^stsource), and frequently when successful (43% after 2^ndsource). Use of multiple sources accounted for 41% of searches, and increased search success from 70% to 89%.</p> <p>Conclusions</p> <p>By consulting in foraging terms the most 'profitable' sources of information (colleagues, books), rapidly switching sources when unsuccessful, and frequently double checking, GPs achieve an efficient trade-off between maximizing search success and information reliability, and minimizing searching time. As predicted by foraging theory, GPs trade time-consuming evidence-based (electronic) information sources for sources with a higher information reward per unit time searched. Evidence-based practice must accommodate these 'real world' foraging pressures, and Internet resources should evolve to deliver information as effectively as traditional methods of information gathering.</p

Construction of Transgenic Plasmodium berghei as a Model for Evaluation of Blood-Stage Vaccine Candidate of Plasmodium falciparum Chimeric Protein 2.9

BACKGROUND:The function of the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1-19) expressed by Plasmodium has been demonstrated to be conserved across distantly related Plasmodium species. The green fluorescent protein (GFP) is a reporter protein that has been widely used because it can be easily detected in living organisms by fluorescence microscopy and flow cytometry. METHODOLOGY AND RESULTS:In this study, we used gene targeting to generate transgenic P. berghei (Pb) parasites (designated as PfMSP1-19Pb) that express the MSP1-19 of P. falciparum (Pf) and the GFP reporter protein simultaneously. The replacement of the PbMSP1-19 locus by PfMSP1-19 was verified by PCR and Southern analysis. The expression of the chimeric PbfMSP-1 and the GFP was verified by Western blot and fluorescence microscopy, respectively. Moreover, GFP-expressing transgenic parasites in blood stages can be readily differentiated from other blood cells using flow cytometry. A comparison of growth rates between wild-type and the PfMSP1-19Pb transgenic parasite indicated that the replacement of the MSP1-19 region and the expression of the GFP protein were not deleterious to the transgenic parasites. We used this transgenic mouse parasite as a murine model to evaluate the protective efficacy in vivo of specific IgG elicited by a PfCP-2.9 malaria vaccine that contains the PfMSP1-19. The BALB/c mice passively transferred with purified rabbit IgG to the PfCP-2.9 survived a lethal challenge of the PfMSP1-19Pb transgenic murine parasites, but not the wild-type P. berghei whereas the control mice passively transferred with purified IgG obtained from adjuvant only-immunized rabbits were vulnerable to both transgenic and wild-type infections. CONCLUSIONS:We generated a transgenic P. berghei line that expresses PfMSP1-19 and the GFP reporter gene simultaneously. The availability of this parasite line provides a murine model to evaluate the protective efficacy in vivo of anti-MSP1-19 antibodies, including, potentially, those elicited by the PfCP-2.9 malaria vaccine in human volunteers

Common Variants at 10 Genomic Loci Influence Hemoglobin A(1C) Levels via Glycemic and Nonglycemic Pathways

OBJECTIVE Glycated hemoglobin (HbA1c), used to monitor and diagnose diabetes, is influenced by average glycemia over a 2- to 3-month period. Genetic factors affecting expression, turnover, and abnormal glycation of hemoglobin could also be associated with increased levels of HbA1c. We aimed to identify such genetic factors and investigate the extent to which they influence diabetes classification based on HbA1c levels. RESEARCH DESIGN AND METHODS We studied associations with HbA1c in up to 46,368 nondiabetic adults of European descent from 23 genome-wide association studies (GWAS) and 8 cohorts with de novo genotyped single nucleotide polymorphisms (SNPs). We combined studies using inverse-variance meta-analysis and tested mediation by glycemia using conditional analyses. We estimated the global effect of HbA1c loci using a multilocus risk score, and used net reclassification to estimate genetic effects on diabetes screening. RESULTS Ten loci reached genome-wide significant association with HbA1c, including six new loci near FN3K (lead SNP/P value, rs1046896/P = 1.6 × 10−26), HFE (rs1800562/P = 2.6 × 10−20), TMPRSS6 (rs855791/P = 2.7 × 10−14), ANK1 (rs4737009/P = 6.1 × 10−12), SPTA1 (rs2779116/P = 2.8 × 10−9) and ATP11A/TUBGCP3 (rs7998202/P = 5.2 × 10−9), and four known HbA1c loci: HK1 (rs16926246/P = 3.1 × 10−54), MTNR1B (rs1387153/P = 4.0 × 10−11), GCK (rs1799884/P = 1.5 × 10−20) and G6PC2/ABCB11 (rs552976/P = 8.2 × 10−18). We show that associations with HbA1c are partly a function of hyperglycemia associated with 3 of the 10 loci (GCK, G6PC2 and MTNR1B). The seven nonglycemic loci accounted for a 0.19 (% HbA1c) difference between the extreme 10% tails of the risk score, and would reclassify ∼2% of a general white population screened for diabetes with HbA1c. CONCLUSIONS GWAS identified 10 genetic loci reproducibly associated with HbA1c. Six are novel and seven map to loci where rarer variants cause hereditary anemias and iron storage disorders. Common variants at these loci likely influence HbA1c levels via erythrocyte biology, and confer a small but detectable reclassification of diabetes diagnosis by HbA1c

Mutant Versions of the S. cerevisiae Transcription Elongation Factor Spt16 Define Regions of Spt16 That Functionally Interact with Histone H3

In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16 – the Spt16-M domain – is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation

A comprehensive 1000 Genomes-based genome-wide association meta-analysis of coronary artery disease

Existing knowledge of genetic variants affecting risk of coronary artery disease (CAD) is largely based on genome-wide association studies (GWAS) analysis of common SNPs. Leveraging phased haplotypes from the 1000 Genomes Project, we report a GWAS meta-analysis of 185 thousand CAD cases and controls, interrogating 6.7 million common (MAF>0.05) as well as 2.7 million low frequency (0.005<MAF<0.05) variants. In addition to confirmation of most known CAD loci, we identified 10 novel loci, eight additive and two recessive, that contain candidate genes that newly implicate biological processes in vessel walls. We observed intra-locus allelic heterogeneity but little evidence of low frequency variants with larger effects and no evidence of synthetic association. Our analysis provides a comprehensive survey of the fine genetic architecture of CAD showing that genetic susceptibility to this common disease is largely determined by common SNPs of small effect siz

Severe Plasmodium falciparum Malaria Is Associated with Circulating Ultra-Large von Willebrand Multimers and ADAMTS13 Inhibition

Plasmodium falciparum infection results in adhesion of infected erythrocytes to blood vessel endothelium, and acute endothelial cell activation, together with sequestration of platelets and leucocytes. We have previously shown that patients with severe infection or fulminant cerebral malaria have significantly increased circulatory levels of the adhesive glycoprotein von Willebrand factor (VWF) and its propeptide, both of which are indices of endothelial cell activation. In this prospective study of patients from Ghana with severe (n = 20) and cerebral (n = 13) P. falciparum malaria, we demonstrate that increased plasma VWF antigen (VWF∶Ag) level is associated with disproportionately increased VWF function. VWF collagen binding (VWF∶CB) was significantly increased in patients with cerebral malaria and severe malaria (medians 7.6 and 7.0 IU/ml versus 1.9 IU/ml; p<0.005). This increased VWF∶CB correlated with the presence of abnormal ultra-large VWF multimers in patient rather than control plasmas. Concomitant with the increase in VWF∶Ag and VWF∶CB was a significant persistent reduction in the activity of the VWF-specific cleaving protease ADAMTS13 (∼55% of normal; p<0.005). Mixing studies were performed using P. falciparum patient plasma and normal pooled plasma, in the presence or absence of exogenous recombinant ADAMTS13. These studies demonstrated that in malarial plasma, ADAMTS13 function was persistently inhibited in a time-dependent manner. Furthermore, this inhibitory effect was not associated with the presence of known inhibitors of ADAMTS13 enzymatic function (interleukin-6, free haemoglobin, factor VIII or thrombospondin-1). These novel findings suggest that severe P. falciparum infection is associated with acute endothelial cell activation, abnormal circulating ULVWF multimers, and a significant reduction in plasma ADAMTS13 function which is mediated at least in part by an unidentified inhibitor