Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.     

Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion

Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.     

Physical activity patterns during weight maintenance following a low-energy density dietary intervention

Objective: The objective was to determine the role of physical activity (PA) and energy intake on weight maintenance among former University of Alabama at Birmingham EatRight Weight Management Program participants. Research Methods and Procedures: Eighty‐nine former participants completed follow‐up visits ≥1 year after completing EatRight. BMI was calculated using measured height and weight. Diet intake was estimated from 4‐day food records. PA was assessed using a module from the Behavioral Risk Factor Surveillance System Survey. Cut‐off points were chosen based on the distribution of minutes of activity. For moderate and total activity, cut‐off points were 0, 1 to 30, 31 to 60, and >60 minutes. For vigorous activity, the categories were 0, 1 to 20, 21 to 30, and >30 minutes. General linear models determined the association of PA with change in weight at follow‐up controlling for gender and total energy intake (kcal/d). Results: At follow‐up, 80% (n = 71) of participants maintained (regained <5% of program end weight) their body weight and 20% had gained weight. Mean weight change was 1.0 ± 6.5 kg. Maintainers consumed 384 fewer kcal/d on average. Maintainers had a lower energy density dietary pattern (1.58 vs. 2.01 kcal/g, p < 0.01). There was no significant difference in PA min/d reported by maintainers and gainers. Discussion: Our results suggest that, despite being minimally active, weight‐reduced individuals can successfully maintain their newly achieved body weight with appropriate caloric intake. Adopting a lower calorie, low energy density dietary pattern may reduce the amount of PA that is truly necessary for weight maintenance.     

MICROFAMILY (version 1): a computer program for detecting flanking‐region similarities among different microsatellite loci

Microsatellite flanking regions are not necessarily unique sequences, but they may group into sequence families. Microsatellites occurring within such families are likely to give multiple banding patterns during polymerase chain reaction amplifications. microfamily (version 1) is a program that detects flanking‐region similarities between different microsatellite‐containing sequences, thus allowing for potentially problematic sequences to be eliminated prior to primer design. The program also accomplishes some otherwise tedious sequence editing, such as checking for nonpermitted characters, and eliminates poorly readable extremities or potential vector/adapter contamination. microfamily is written in Perl and available for Linux and Windows systems at http://www.up.univ‐mrs.fr/local/egee/dir/meglecz/microfamily.html .     

Isolation and characterization of polymorphic microsatellites in Parnassius apollo and Euphydryas aurinia (Lepidoptera)

Microsatellite loci were developed from Parnassius apollo and Euphydryas aurinia, two endangered Palaearctic butterfly species. Respectively, six and five polymorphic loci were characterized from an enriched partial genomic library. Genetic diversity range from three to 25 alleles for the first species and from seven to 21 for the second. Although the presence of null alleles is suspected, these polymorphic loci are likely to provide important information on the fine scale genetic structure among populations of these species.     

Weight maintenance 2 years after participation in a weight loss program promoting low-energy density foods

Objective: Observational study designed to determine weight outcomes and associated dietary intake patterns for a sample of participants ≥1 year after completing the University of Alabama at Birmingham EatRight Weight Management Program. Research Methods and Procedures: Seventy‐four former participants (64% women) completed follow‐up visits ≥1 year after participating in EatRight, which promotes low‐energy density, high‐complex carbohydrate foods. Weight maintenance was defined as gaining <5% of body weight since completion of the EatRight program and staying below their program entry weight. Those who gained ≥5% of their body weight since completion were classified as gainers. Results: During EatRight, participants of the follow‐up study lost an average of 4.0 kg. After a mean follow‐up time of 2.2 years, the average weight change was +0.59 kg (mean BMI, 32.5 kg/m²). Seventy‐eight percent of participants gained <5% of their body weight; 46% had no weight regain or continued weight loss. Unadjusted mean intake for maintainers was 1608 kcal, whereas calorie intake for gainers was 1989 kcal. Despite eating slightly fewer calories (adjusted difference, 244; p = 0.058), maintainers ate a similar amount of food, resulting in a lower energy‐density pattern (p = 0.016) compared with those who regained ≥5% of body weight. Gainers also reported consuming larger portions of several food groups. Discussion: Our results indicate that low‐energy‐density eating habits are associated with long‐term weight maintenance. Those who maintain weight after the EatRight program consume a low‐energy‐density dietary pattern and smaller portions of food groups potentially high in energy density than those who regain weight.