The Repeat Region of the Circumsporozoite Protein is Critical for Sporozoite Formation and Maturation in Plasmodium

The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH₂-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH₂-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.     

Regulation of membrane-bound PKC in adult cardiac ventricular myocytes

Activation of protein kinase C (PKC) is thought to involve translocation to the particulate fraction. The present study demonstrates a membrane-associated, inactive pool of PKC in adult rat ventricular myocytes. Membranes were isolated from stimulated (phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1)) or control myocytes and PKC activity determined in the absence (active PKC) or presence (total PKC) of PMA. An inactive, PMA-responsive, pool of PKC was detected. In intact myocytes, PMA or ET-1 induced a translocation of PKC epsilon from the cytosol into the particulate fraction. In contrast, ET-1 decreased both total and active PKC in the membranes: this decrease was associated with a loss of PKC epsilon immunoreactivity. PMA increased the amount of membrane-associated, inactive PKC. Our results demonstrate the presence of a membrane-associated pool of PKC in cardiac myocytes that is differentially modulated by ET-1 or PMA.     

Identification of relevant properties for epitopes detection using a regression model

A B-cell epitope is a part of an antigen that is recognized by a specific antibody or B-cell receptor. Detecting the immunogenic region of the antigen is useful in numerous immunodetection and immunotherapeutics applications. The aim of this paper is to find relevant properties to discriminate the location of potential epitopes from the rest of the protein surface. The most relevant properties, identified using two evaluation approaches, are the geometric properties, followed by the conservation score and some chemical properties, such as the proportion of glycine. The selected properties are used in a patch-based epitope localization method including a Single-Layer Perceptron for regression. The output of this Single-Layer Perceptron is used to construct a probability map on the antigen surface. The predictive performances of the method are assessed by computing the AUC using cross validation on two benchmark data sets and by computing the AUC and the precision for a third independent test set.     

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.     

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.     

Neurotoxic Sequelae of Mercury Exposure: An Intervention and Follow-up Study in the Brazilian Amazon

Since 1995, the Caruso Project used an Ecosystem Approach to Human Health to examine mercury (Hg) exposure in fish-eating communities in the Brazilian Amazon and develop interventions to maximise nutrition from traditional diet and minimise toxic risk. In 1995, 2000 and 2006, this study followed fish consumption, Hg levels, and visual and motor functions in 31 villagers. Questionnaires gathered information on socio-demographics and diet. Hair Hg (H-Hg) levels were measured. Visual acuity, colour vision, manual dexterity and grip strength were assessed. Data was analysed using general linear models of repeated measures. Total fish consumption, similar in 1995 and 2000, decreased in 2006. Carnivorous fish consumption initially decreased and then remained stable, whereas non-carnivorous fish consumption first increased and then decreased. H-Hg declined from 17.6 to 7.8 μg/g. Visual functions showed a significant decrease over time, with those with H-Hg ≥ 20 μg/g in 1995 showing greater loss. Motor functions showed initial improvement and then returned to the 1995 performance level. Decrease in Hg exposure is attributed to the intervention and socio-economic changes in the village. While there may be a certain reversibility of motor deficits, visual capacities may decrease progressively with respect to exposure prior to the intervention.     

Increased FAT/CD36 cycling and lipid accumulation in myotubes derived from obese type 2 diabetic patients

Background

Permanent fatty acid translocase (FAT/)CD36 relocation has previously been shown to be related to abnormal lipid accumulation in the skeletal muscle of type 2 diabetic patients, however mechanisms responsible for the regulation of FAT/CD36 expression and localization are not well characterized in human skeletal muscle.

Methodology/Principal Findings

Primary muscle cells derived from obese type 2 diabetic patients (OBT2D) and from healthy subjects (Control) were used to examine the regulation of FAT/CD36. We showed that compared to Control myotubes, FAT/CD36 was continuously cycling between intracellular compartments and the cell surface in OBT2D myotubes, independently of lipid raft association, leading to increased cell surface FAT/CD36 localization and lipid accumulation. Moreover, we showed that FAT/CD36 cycling and lipid accumulation were specific to myotubes and were not observed in reserve cells. However, in Control myotubes, the induction of FAT/CD36 membrane translocation by the activation of (AMP)-activated protein kinase (AMPK) pathway did not increase lipid accumulation. This result can be explained by the fact that pharmacological activation of AMPK leads to increased mitochondrial beta-oxidation in Control cells.

Conclusion/Significance

Lipid accumulation in myotubes derived from obese type 2 diabetic patients arises from abnormal FAT/CD36 cycling while lipid accumulation in Control cells results from an equilibrium between lipid uptake and oxidation. As such, inhibiting FAT/CD36 cycling in the skeletal muscle of obese type 2 diabetic patients should be sufficient to diminish lipid accumulation.     

Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.     

Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila

In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues.