Glycan Masking of Plasmodium vivax Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.     

Ca2+‐mediated exocytosis of subtilisin‐like protease 1: a key step in egress of Plasmodium falciparum merozoites

Egress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood‐stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra‐erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca²⁺ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca²⁺ just before egress, observed by time‐lapse video microscopy, suggested a role for intracellular Ca²⁺ in this process. Chelation of intracellular Ca²⁺ with chelators such as BAPTA‐AM or inhibition of Ca²⁺ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca²⁺ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin‐like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.     

Fitness Beats Truth in the Evolution of Perception

Acta Biotheoretica - Does natural selection favor veridical percepts—those that accurately (if not exhaustively) depict objective reality? Perceptual and cognitive scientists standardly claim...     

Molecular interactions and signaling mechanisms during erythrocyte invasion by malaria parasites

Invasion of erythrocytes by Plasmodium merozoites is a complex process that is mediated by specific molecular interactions. Here, we review recent studies on interactions between erythrocyte binding antigens (EBA) and PfRH proteins from the parasite and erythrocyte receptors involved in invasion. The timely release of these parasite ligands from internal organelles such as micronemes and rhoptries to the merozoite surface is critical for receptor-engagement leading to successful invasion. We review information on signaling mechanisms that control the regulated secretion of parasite proteins during invasion. Erythrocyte invasion involves the formation and movement of a junction between the invading merozoite and host erythrocyte. We review recent studies on the molecular composition of the junction and the molecular motor that drives movement of the junction.     

Association of severe malaria outcomes with platelet-mediated clumping and adhesion to a novel host receptor

Introduction

Severe malaria has been attributed partly to the sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature of vital host organs. Identification of P. falciparum cytoadherence phenotypes that are associated with severe malaria may lead to the development of novel strategies against life-threatening malaria.

Methods and Findings

Forty-six P. falciparum isolates from Mozambican children under 5 years of age with severe malaria (cases) were examined and compared to 46 isolates from sex and age matched Mozambican children with uncomplicated malaria (controls). Cytoadherence properties such as platelet-mediated clumping, rosetting and adhesion to purified receptors (CD36, ICAM1 and gC1qR), were compared between these matched pairs by non-parametric tests. The most common clinical presentation associated with severe malaria was prostration. Compared to matched controls, prevalence of platelet-mediated clumping was higher in cases (P = .019), in children presenting with prostration (P = .049) and in children with severe anaemia (P = .025). Prevalence of rosetting and gC1qR adhesion were also higher in isolates from cases with severe anemia and multiple seizures, respectively (P = .045 in both cases), than in controls.

Conclusions

These data indicate a role for platelet-mediated clumping, rosetting and adhesion to gC1qR in the pathogenesis of severe malaria. Inhibition of these cytoadherence phenotypes may reduce the occurrence or improve the prognosis of severe malaria outcomes.     

Gene expression profiling for mechanistic understanding of cellular aggregation in mammalian cell perfusion cultures

Aggregation of baby hamster kidney (BHK) cells cultivated in perfusion mode for manufacturing recombinant proteins was characterized. The potential impact of cultivation time on cell aggregation for an aggregating culture (cell line A) was studied by comparing expression profiles of 84 genes in the extracellular adhesion molecules (ECM) pathway by qRT‐PCR from 9 and 25 day shake flask samples and 80 and 94 day bioreactor samples. Significant up‐regulation of THBS2 (4.4‐ to 6.9‐fold) was seen in both the 25 day shake flask and 80 and 94 day bioreactor samples compared to the 9 day shake flask while NCAM1 was down‐regulated 5.1‐ to 8.9‐fold in the 80 and 94 day bioreactor samples. Subsequent comparisons were made between cell line A and a non‐aggregating culture (cell line B). A 65 day perfusion bioreactor sample from cell line B served as the control for 80 and 94 day samples from four different perfusion bioreactors for cell line A. Of the 84 genes in the ECM pathway, four (COL1A1, COL4A1, THBS2, and VCAN) were consistently up‐regulated in cell line A while two (NCAM1 and THBS1) were consistently down‐regulated. The magnitudes of differential gene expression were much higher when cell lines were compared (4.1‐ to 44.6‐fold) than when early and late cell line B samples were compared (4.4‐ to 6.9‐fold) indicating greater variability between aggregating and non‐aggregating cell lines. Based on the differential gene expression results, two mechanistic models were proposed for aggregation of BHK cells in perfusion cultures. Biotechnol. Bioeng. 2013; 110: 483–490. © 2012 Wiley Periodicals, Inc.     

Hindutva in the West: mapping the antinomies of diaspora nationalism

This introduction provides a historical background to Hindu nationalism and examines several theoretical and empirical themes that are important for its analysis both in India and the diaspora. It is argued that there has been a relative neglect within the research field of diaspora nationalist movements and the impact they can have on constituting antisecular and absolutist orientations to minorities and majorities both within the diaspora and in the “homeland”. The introduction examines the rise of the Hindutva movement in the 1920s and considers the debates about its relation to ethnic, nationalist, religious, racist and fascist ideologies. We consider how an examination of Hindu nationalism can modify many recent debates on “race” and ethnicity, multiculturalism and “diaspora”. Several themes relating to caste, gender and “Aryanism” are examined. The contents of this Special Issue are contextualized within these debates and a summary of the key themes of the contributions is provided.     

S6 Peptide Derived from KvAP Channel Shows Cooperativity in Gating on Bilayer Lipid Membrane

Collective behavior of S6 peptide channels derived from KvAP (a bacterial potassium channel) incorporated in lipid bilayer membrane, has been investigated at various applied potentials through multi-channel electrophysiological experiments. The current versus time traces at any particular membrane potential show clear steps for sequential opening of the multi-channels. The minimum current (representing one-channel current) was found out from the amplitude histograms. Accordingly, the number of open channels corresponding to a particular open state was calculated. It was observed that the above-mentioned one channel current is higher than the corresponding single-channel current at most of the applied membrane potentials. Moreover, the difference between the single and one channel conductances is a nonlinear function of the membrane potential. We conclude that the S6 multi-channels show co-operative gating. Voltage relaxation studies support the above-mentioned conclusion.