A Potent Malaria Transmission Blocking Vaccine Based on Codon Harmonized Full Length Pfs48/45 Expressed in Escherichia coli

Malaria caused by Plasmodium falciparum is responsible for nearly 1 million deaths annually. Although much progress has been made in the recent past, the development of a safe, effective and affordable malaria vaccine has remained a challenge. A vaccine targeting sexual stages of the parasite will not only reduce malaria transmission by female Anopheles mosquitoes, but also reduce the spread of parasites able to evade immunity elicited by vaccines targeting pre-erythrocytic and erythrocytic asexual stages. We focused our studies on Pfs48/45, a protein expressed in the sexual stages developing within an infected person and one of the most promising transmission-blocking vaccine targets. Functional immunogenicity of Pfs48/45 protein requires proper disulfide bond formation, consequently evaluation of the immunogenicity of recombinant full-length Pfs48/45 has been hampered by difficulties in expressing properly folded protein to date. Here we present a strategy involving harmonization of codons for successful recombinant expression of full length Pfs48/45 in Escherichia coli. The purified protein, designated CH-rPfs48/45, was recognized by monoclonal antibodies directed against reduction-sensitive conformational epitopes in the native protein. Immunogenicity evaluation in mice revealed potent transmission blocking activity in membrane feeding assays of antisera elicited by CH-rPfs48/45 formulated in three different adjuvants, i.e. Alum, Montanide ISA-51 and complete Freund's adjuvant. More importantly, CH-rPfs48/45 formulated with Montanide ISA-51 when administered to nonhuman primates (Olive baboons, Papio anubis) resulted in uniformly high antibody responses (ELISA titers >2 million) in all five animals. Sera from these animals displayed greater than 93% blocking activity in membrane feeding assays after a single immunization, reaching nearly complete blocking after a booster dose of the vaccine. The relative ease of expression and induction of potent transmission blocking antibodies in mice and nonhuman primates provide a compelling rationale and basis for development of a CH-rPfs48/45 based malaria transmission blocking vaccine.     

Quorum-Quenching Acylase Reduces the Virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans Infection Model

The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-l-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-ΔpvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.Pseudomonas aeruginosa is an opportunistic gram-negative pathogen of vertebrates and a primary pathogen of insects (17). It mainly infects individuals who are immunocompromised, such as human immunodeficiency virus-infected patients, as well as those who have cystic fibrosis. In addition, those having disruptions in normal barriers caused by severe burns or indwelling medical devices are at risk. Hospital-acquired P. aeruginosa pneumonias and septicemias are frequently lethal (2, 3). To facilitate the establishment of infection, P. aeruginosa produces an impressive array of both cell-associated and extracellular virulence factors, such as proteases and phospholipases, and also small molecules, including rhamnolipid, phenazines, and cyanide (17). Expression of many of the extracellular factors is cell density controlled, does not occur until the late logarithmic phase of growth, and is mediated through specific quorum-sensing signal molecules (23). Two of these molecules, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), have been studied in great detail. In our laboratory, we previously demonstrated that PA2385(pvdQ) from P. aeruginosa PAO1 encodes an acyl-homoserine lactone (AHL) acylase. Analysis of the gene product showed that the posttranslational processing of the acylase as well as the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PvdQ protein is a member of the N-terminal nucleophile hydrolase superfamily. The main AHL signaling molecule of P. aeruginosa PAO1, 3-oxo-C12-HSL, is degraded by PvdQ (16). Addition of the purified protein to PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when pvdQ was overexpressed in P. aeruginosa (16). These results demonstrate that this protein has in situ quorum-quenching activity. This AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.To test our hypothesis that PvdQ can exert its beneficial functions also in vivo, we chose to study its effect on the infection of the nematode Caenorhabditis elegans. This model has been used before in multiple pathogenicity studies of Cryptococcus neoformans (13) and gram-positive (6) and gram-negative (9, 10, 11) bacteria. Infection with P. aeruginosa strain PA14 was found to result in fast (hours) or slow (days) killing, depending on the growth medium used (19, 20). When Darby and colleagues (2) used the system to study P. aeruginosa PAO1, a lethal paralysis of the worms was observed, indicating another mechanism by which P. aeruginosa can kill C. elegans. It was shown that quorum-sensing-dependent hydrogen cyanide production on rich medium by P. aeruginosa PAO1 is the causative agent for the fast paralysis (5). Under the same conditions, an attenuation of paralysis by an AHL acylase from Ralstonia sp. strain XJ12B upon expression in P. aeruginosa PAO1 was observed (12). Those authors performed the assays with a mixed population of worms only and did not test for slow killing. In this study we show that P. aeruginosa PAO1 can also elicit a slow-killing response when grown in low-nutrient medium. Moreover, we report that not only overexpression of the gene from its host but also external addition of the purified PvdQ renders P. aeruginosa PAO1 less pathogenic to C. elegans and increases the life span of the infected animals in both slow- and fast-killing assays. As the correlation between the bacterial virulence factors required for pathogenesis in mammals and in C. elegans has been shown to be very high (20), we propose that the external addition of purified AHL acylases may be developed into a novel quorum-quenching therapy.     

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4^1–157-Cernunnos^1–224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4^1–157 and Cernunnos^1–224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4–Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4–Cernunnos interface. We identified four X4 residues (Glu⁵⁵, Asp⁵⁸, Met⁶¹, and Phe¹⁰⁶) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.     

Ebola et autres,ces virus venus d’ailleurs

In the last decade, we faced a large number of emerging pathogens. As a consequence we had to adapt our medical practice as well as our health system. This review summarizes the main features of the recent emerging pathogens with a particular focus on the recent and ongoing Ebola outbreak, we tried to evaluate the consequences on our national health management.     

Integrating Wellness,Recovery, and Self-management for Mental Health Consumers

Three distinct, yet interrelated, terms—wellness, recovery, and self-management—have received increasing attention in the research, consumer, and provider communities. This article traces the origins of these terms, seeking to understand how they apply, individually and in conjunction with one another to mental health consumers. Each shares a common perspective that is health-centered rather than disease-centered and that emphasizes the role of consumers as opposed to professional providers as the central determinants of health and well-being. Developing approaches combining elements of each construct may hold promise for improving the overall health and well-being of persons with serious mental disorders.