RISK and SAFE Signaling Pathway Involvement in Apolipoprotein A-I-Induced Cardioprotection

Recent findings indicate that apolipoprotein A-I (ApoA-I) may be a protective humoral mediator involved in remote ischemic preconditioning (RIPC). This study sought to determine if ApoA-I mediates its protective effects via the RISK and SAFE signaling pathways implicated in RIPC. Wistar rats were allocated to one of the following groups. Control: rats were subjected to myocardial ischemia/reperfusion (I/R) without any further intervention; RIPC: four cycles of limb I/R were applied prior to myocardial ischemia; ApoA-I: 10 mg/Kg of ApoA-I were intravenously injected prior to myocardial ischemia; ApoA-I + inhibitor: pharmacological inhibitors of RISK/SAFE pro-survival kinase (Akt, ERK1/2 and STAT-3) were administered prior to ApoA-I injection. Infarct size was significantly reduced in the RIPC group compared to Control. Similarly, ApoA-I injection efficiently protected the heart, recapitulating RIPC-induced cardioprotection. The ApoA-I protective effect was associated with Akt and GSK-3β phosphorylation and substantially inhibited by pretreatment with Akt and ERK1/2 inhibitors. Pretreatment with ApoA-I in a rat model of I/R recapitulates RIPC-induced cardioprotection and shares some similar molecular mechanisms with those of RIPC-involved protection of the heart.     

Molecular diagnosis of neonatal diabetes mellitus using next-generation sequencing of the whole exome

Background

Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.

Methodology/Principal Findings

We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient''s mother, father and young brother, all of whom are normoglycemic.

Conclusions/Significance

WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.     

Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase

The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate.     

clonality V.0.4: a randomization-based program to test for heterozygosity-genet size relationships in clonal organisms

clonality V.0.4 is a program for testing heterozygosity-genet size relationships in clonal organisms using a randomization procedure. The software has been developed under the Borland Delphi developing environment and a Windows-executable version is freely downloadable from http://gemi.mpl.ird.fr/SiteSGASS/Prugnolle/ClonalityPage.html. The program compares the observed F(IS) of the population with the F(IS) expected if genets (multilocus genotypes present in multiple copies within the population) were chosen randomly from the set of different multilocus genotypes. The randomization procedure is performed with the same number of genets and the same number of repetitions per genet as what is observed in the original data set.     

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune‐dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species‐dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA‐dependent secretory pathway. This led to a general inhibition of protein secretion by PDT‐treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin‐based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro‐apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.     

Evaluation of the Effectiveness of Malaria Vector Control Measures in Urban Settings of Dakar by a Specific Anopheles Salivary Biomarker

Standard entomological methods for evaluating the impact of vector control lack sensitivity in low-malaria-risk areas. The detection of human IgG specific to Anopheles gSG6-P1 salivary antigen reflects a direct measure of human–vector contact. This study aimed to assess the effectiveness of a range of vector control measures (VCMs) in urban settings by using this biomarker approach. The study was conducted from October to December 2008 on 2,774 residents of 45 districts of urban Dakar. IgG responses to gSG6-P1 and the use of malaria VCMs highly varied between districts. At the district level, specific IgG levels significantly increased with age and decreased with season and with VCM use. The use of insecticide-treated nets, by drastically reducing specific IgG levels, was by far the most efficient VCM regardless of age, season or exposure level to mosquito bites. The use of spray bombs was also associated with a significant reduction of specific IgG levels, whereas the use of mosquito coils or electric fans/air conditioning did not show a significant effect. Human IgG response to gSG6-P1 as biomarker of vector exposure represents a reliable alternative for accurately assessing the effectiveness of malaria VCM in low-malaria-risk areas. This biomarker tool could be especially relevant for malaria control monitoring and surveillance programmes in low-exposure/low-transmission settings.     

Modulation of PAI-1 and proMMP-9 syntheses by soluble TNFalpha and its receptors during differentiation of the human monocytic HL-60 cell line

During phorbol ester-induced differentiation of HL-60 monocytic cells, tumor necrosis factoralpha (TNFalpha) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFalpha-responsive genes. We investigated how, during phorbol ester-induced differentiation of HL-60 cells, the secreted TNFalpha modulated plasminogen activator inhibitor type I (PAI-1) and gelatinase B (MMP-9) syntheses, two proteins involved in pericellular proteolysis. The differentiation-induced release of TNFalpha, was abolished by the hydroxamate-based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti-TNFalpha significantly down-regulated PAI-1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic-like cells), which underlined the activating role of autocrine TNFalpha during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFalpha, but RU36156 or anti-TNFalpha no longer had an effect on PAI-1 synthesis. This lack of effect was not due to a default of TNFalpha signaling since PAI-1 synthesis was still stimulated in response to exogenous TNFalpha. TNFalpha receptor RI was also actively released and was shown to reduce TNFalpha activity which may account for the inability of soluble TNFalpha to up-regulate PAI-1 synthesis. In later mature stage, cells became susceptible to exogenous TNFalpha-induced apoptosis and rapidly lost their ability to respond to TNFalpha. The MMP-9 synthesis followed similar regulation as PAI-1. Isolated human blood monocytes-derived macrophages behave like HL-60-derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFalpha is active and then down-regulated by RI, which may temper a continuous up-regulation of the synthesis of proteins involved in pericellular proteolysis.     

Viral fusion peptides and identification of membrane-interacting segments

Viral envelope glycoproteins promote infection by mediating fusion between viral and cellular membranes. Fusion occurs after dramatic conformational changes within fusion proteins, leading to the exposure of a short stretch of mostly apolar residues, termed the fusion peptide, which is presumed to insert into the membrane and initiate the fusion process. The typical global composition of fusion peptides, rich in hydrophobic but also in small amino acids such as alanine and glycine, was used here as bait to detect other peptidic segments that can insert into membranes. We so evidenced a similar composition in several cytotoxic peptides, which promote pore formation such as peptides involved in amyloidoses and hydrophobic alpha-hairpins of pore-forming toxins. It is suggested that the structural plasticity observed for several membrane active peptides can be conferred by this particular global amino acid composition, which could be thus used to predict such functional behavior from genome data.