Homology blocks of Plasmodium falciparum var genes and clinically distinct forms of severe malaria in a local population

Abstract Background The primary target of the human immune response to the malaria parasite Plasmodium falciparum, P. falciparum erythrocyte membrane protein 1 (PfEMP1), is encoded by the members of the hyper-diverse var gene family. The parasite exhibits antigenic variation via mutually exclusive expression (switching) of the ~60 var genes within its genome. It is thought that different variants exhibit different host endothelial binding preferences that in turn result in different manifestations of disease. Results Var sequences comprise ancient sequence fragments, termed homology blocks (HBs), that recombine at exceedingly high rates. We use HBs to define distinct var types within a local population. We then reanalyze a dataset that contains clinical and var expression data to investigate whether the HBs allow for a description of sequence diversity corresponding to biological function, such that it improves our ability to predict disease phenotype from parasite genetics. We find that even a generic set of HBs, which are defined for a small number of non-local parasites: capture the majority of local sequence diversity; improve our ability to predict disease severity from parasite genetics; and reveal a previously hypothesized yet previously unobserved parasite genetic basis for two forms of severe disease. We find that the expression rates of some HBs correlate more strongly with severe disease phenotypes than the expression rates of classic var DBLα tag types, and principal components of HB expression rate profiles further improve genotype-phenotype models. More specifically, within the large Kenyan dataset that is the focus of this study, we observe that HB expression differs significantly for severe versus mild disease, and for rosetting versus impaired consciousness associated severe disease. The analysis of a second much smaller dataset from Mali suggests that these HB-phenotype associations are consistent across geographically distant populations, since we find evidence suggesting that the same HB-phenotype associations characterize this population as well. Conclusions The distinction between rosetting versus impaired consciousness associated var genes has not been described previously, and it could have important implications for monitoring, intervention and diagnosis. Moreover, our results have the potential to illuminate the molecular mechanisms underlying the complex spectrum of severe disease phenotypes associated with malaria—an important objective given that only about 1% of P. falciparum infections result in severe disease.http://deepblue.lib.umich.edu/bitstream/2027.42/112650/1/12866_2013_Article_2116.pd

Structural Insight into Epitopes in the Pregnancy-Associated Malaria Protein VAR2CSA

Pregnancy-associated malaria is caused by Plasmodium falciparum malaria parasites binding specifically to chondroitin sulfate A in the placenta. This sequestration of parasites is a major cause of low birth weight in infants and anemia in the mothers. VAR2CSA, a polymorphic multi-domain protein of the PfEMP1 family, is the main parasite ligand for CSA binding, and identification of protective antibody epitopes is essential for VAR2CSA vaccine development. Attempts to determine the crystallographic structures of VAR2CSA or its domains have not been successful yet. In this study, we propose 3D models for each of the VAR2CSA DBL domains and we show that regions in the fold of VAR2CSA inter-domain 2 and a PfEMP1 CIDR domain seem to be homologous to the EBA-175 and Pkα-DBL fold. This suggests that ID2 could be a functional domain. We also identify regions of VAR2CSA present on the surface of native VAR2CSA by comparing reactivity of plasma containing anti-VAR2CSA antibodies in peptide array experiments before and after incubation with native VAR2CSA. By this method we identify conserved VAR2CSA regions targeted by antibodies that react with the native molecule expressed on infected erythrocytes. By mapping the data onto the DBL models we present evidence suggesting that the S1+S2 DBL sub-domains are generally surface-exposed in most domains, whereas the S3 sub-domains are less exposed in native VAR2CSA. These results comprise an important step towards understanding the structure of VAR2CSA on the surface of CSA-binding infected erythrocytes

Metoprolol compared to carvedilol deteriorates insulin-stimulated endothelial function in patients with type 2 diabetes - a randomized study

<p>Abstract</p> <p>Aim</p> <p>Studies of beta blockade in patients with type 2 diabetes have shown inferiority of metoprolol treatment compared to carvedilol on indices of insulin resistance. The aim of this study was to examine the effect of metoprolol versus carvedilol on endothelial function and insulin-stimulated endothelial function in patients with type 2 diabetes.</p> <p>Method</p> <p>24 patients with type 2 diabetes were randomized to receive either 200 mg metoprolol succinate or 50 mg carvedilol daily. Endothelium-dependent vasodilation was assessed by using venous occlusion plethysmography with increasing doses of intra-arterial infusions of the agonist serotonin. Insulin-stimulated endothelial function was assessed after co-infusion of insulin for sixty minutes. Vaso-reactivity studies were done before and after the two-month treatment period.</p> <p>Results</p> <p>Insulin-stimulated endothelial function was deteriorated after treatment with metoprolol, the percentage change in forearm blood-flow was 60.19% ± 17.89 (at the highest serotonin dosages) before treatment and -33.80% ± 23.38 after treatment (p = 0.007). Treatment with carvedilol did not change insulin-stimulated endothelial function. Endothelium-dependent vasodilation without insulin was not changed in either of the two treatment groups.</p> <p>Conclusion</p> <p>This study shows that vascular insulin sensitivity was preserved during treatment with carvedilol while blunted during treatment with metoprolol in patients with type 2 diabetes.</p> <p>Trial registration</p> <p>Current Controlled Trials NCT00497003</p

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens

Tissue microarray analysis of human FRAT1 expression and its correlation with the subcellular localisation of ?-catenin in ovarian tumours

The mechanisms involved in the pathogenesis of ovarian cancer are poorly understood, but evidence suggests that aberrant activation of Wnt/?-catenin signalling pathway plays a significant role in this malignancy. However, the molecular defects that contribute to the activation of this pathway have not been elucidated. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) is a candidate for the regulation of cytoplasmic ?-catenin. In this study, we developed in situ hybridisation probes to evaluate the presence of FRAT1 and used an anti-?-catenin antibody to evaluate by immunohistochemistry the expression levels and subcellular localisation of ?-catenin in ovarian cancer tissue microarrays. Expression of FRAT1 was found in some human normal tissues and 47% of ovarian adenocarcinomas. A total of 46% of ovarian serous adenocarcinomas were positive for FRAT1 expression. Accumulation of ?-catenin in the nucleus and/or cytoplasm was observed in 55% ovarian adenocarcinomas and in 59% of serous adenocarcinomas. A significant association was observed in ovarian serous adenocarcinomas between FRAT1 and ?-catenin expression (P&lt;0.01). These findings support that Wnt/?-catenin signalling may be aberrantly activated through FRAT1 overexpression in ovarian serous adenocarcinomas. The mechanism behind the overexpression of FRAT1 in ovarian serous adenocarcinomas and its significance is yet to be investigated

A combined metabolomic and phylogenetic study reveals putatively prebiotic effects of high molecular weight arabino-oligosaccharides when assessed by in vitro fermentation in bacterial communities derived from humans

AbstractPrebiotic oligosaccharides are defined by their selective stimulation of growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial for health. However, apart from the short chain fatty acids, little is known about bacterial metabolites created by fermentation of prebiotics, and the significance of the size of the oligosaccharides remains largely unstudied.By in vitro fermentations in human fecal microbial communities (derived from six different individuals), we studied the effects of high-mass (HA, >1 kDa), low-mass (LA, <1 kDa) and mixed (BA) sugar beet arabino-oligosaccharides (AOS) as carbohydrate sources. Fructo-oligosaccharides (FOS) were included as reference. The changes in bacterial communities and the metabolites produced in response to incubation with the different carbohydrates were analyzed by quantitative PCR (qPCR) and Liquid Chromatography–Mass Spectrometry (LC–MS), respectively.All tested carbohydrate sources resulted in a significant increase of Bifidobacterium spp. between 1.79 fold (HA) and 1.64 fold (FOS) in the microbial populations after fermentation, and LC–MS analysis suggested that the bifidobacteria contributed to decomposition of the arabino-oligosaccharide structures, most pronounced in the HA fraction, resulting in release of the essential amino acid phenylalanine. Abundance of Lactobacillus spp. correlated with the presence of a compound, most likely a flavonoid, indicating that lactobacilli contribute to release of such health-promoting substances from plant structures.Additionally, the combination of qPCR and LC–MS revealed a number of other putative interactions between intestinal microbes and the oligosaccharides, which contributes to the understanding of the mechanisms behind prebiotic impact on human health

Differential Analysis of Ovarian and Endometrial Cancers Identifies a Methylator Phenotype

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer

Epitope Mapping and Topographic Analysis of VAR2CSA DBL3X Involved in P. falciparum Placental Sequestration

Pregnancy-associated malaria is a major health problem, which mainly affects primigravidae living in malaria endemic areas. The syndrome is precipitated by accumulation of infected erythrocytes in placental tissue through an interaction between chondroitin sulphate A on syncytiotrophoblasts and a parasite-encoded protein on the surface of infected erythrocytes, believed to be VAR2CSA. VAR2CSA is a polymorphic protein of approximately 3,000 amino acids forming six Duffy-binding-like (DBL) domains. For vaccine development it is important to define the antigenic targets for protective antibodies and to characterize the consequences of sequence variation. In this study, we used a combination of in silico tools, peptide arrays, and structural modeling to show that sequence variation mainly occurs in regions under strong diversifying selection, predicted to form flexible loops. These regions are the main targets of naturally acquired immunoglobulin gamma and accessible for antibodies reacting with native VAR2CSA on infected erythrocytes. Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an α-helix. Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively. These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria

Improving virtual screening of G protein-coupled receptors via ligand-directed modeling

G protein-coupled receptors (GPCRs) play crucial roles in cell physiology and pathophysiology. There is increasing interest in using structural information for virtual screening (VS) of libraries and for structure-based drug design to identify novel agonist or antagonist leads. However, the sparse availability of experimentally determined GPCR/ligand complex structures with diverse ligands impedes the application of structure-based drug design (SBDD) programs directed to identifying new molecules with a select pharmacology. In this study, we apply ligand-directed modeling (LDM) to available GPCR X-ray structures to improve VS performance and selectivity towards molecules of specific pharmacological profile. The described method refines a GPCR binding pocket conformation using a single known ligand for that GPCR. The LDM method is a computationally efficient, iterative workflow consisting of protein sampling and ligand docking. We developed an extensive benchmark comparing LDM-refined binding pockets to GPCR X-ray crystal structures across seven different GPCRs bound to a range of ligands of different chemotypes and pharmacological profiles. LDM-refined models showed improvement in VS performance over origin X-ray crystal structures in 21 out of 24 cases. In all cases, the LDM-refined models had superior performance in enriching for the chemotype of the refinement ligand. This likely contributes to the LDM success in all cases of inhibitor-bound to agonist-bound binding pocket refinement, a key task for GPCR SBDD programs. Indeed, agonist ligands are required for a plethora of GPCRs for therapeutic intervention, however GPCR X-ray structures are mostly restricted to their inactive inhibitor-bound state

Serological Evidence of Discrete Spatial Clusters of Plasmodium falciparum Parasites

BACKGROUND: Malaria transmission may be considered to be homogenous with well-mixed parasite populations (as in the classic Ross/Macdonald models). Marked fine-scale heterogeneity of transmission has been observed in the field (i.e., over a few kilometres), but there are relatively few data on the degree of mixing. Since the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is highly polymorphic, the host's serological responses may be used to infer exposure to parasite sub-populations. METHODS AND FINDINGS: We measured the antibody responses to 46 individual PfEMP1 domains at four time points among 450 children in Kenya, and identified distinct spatial clusters of antibody responses to individual domains. 35 domains showed strongly significant sero-clusters at p = 0.001. Individuals within the high transmission hotspot showed the greatest diversity of anti-PfEMP1 responses. Individuals outside the hotspot had a less diverse range of responses, even if as individuals they were at relatively intense exposure. CONCLUSIONS: We infer that antigenically distinct sub-populations of parasites exist on a fine spatial scale in a study area of rural Kenya. Further studies should examine antigenic variation over longer periods of time and in different study areas