Design of a trial evaluating myocardial cell protection with cariporide, an inhibitor of the transmembrane sodium-hydrogen exchanger: the Guard During Ischemia Against Necrosis (GUARDIAN) trial

Background  

Direct myocardial cell protection in patients with unstable angina or evolving myocardial infarction (MI) could prevent cell necrosis or reduce its extent, and minimize the risk of MI and death associated with percutaneous coronary interventions (PCIs) and coronary artery bypass surgery. The myocardial NHE plays a critical role in mediating the progression of ischemia to necrosis by promoting intracellular accumulation of sodium and calcium in exchange for hydrogen. Blockage of the system in various experimental models of ischemia and reperfusion had a strong antinecrotic effect. The present paper describes a trial that was intended to investigate the potential clinical benefit of cariporide, a potent and selective inhibitor of the NHE, in a large spectrum of at-risk patients.     

The Vinculin-ΔIn20/21 Mouse: Characteristics of a Constitutive,Actin-Binding Deficient Splice Variant of Vinculin

Background

The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models.

Methodology/Principal Findings

Here, we investigate vinculin-ΔEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-ΔEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-ΔIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-ΔIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-ΔEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-ΔEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-ΔEx20 variant unveils vinculin''s dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites.

Conclusions/Significance

Vinculin-ΔEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development.     

Probing Neuroserpin Polymerization and Interaction with Amyloid-β Peptides Using Single Molecule Fluorescence

Neuroserpin is a member of the serine proteinase inhibitor superfamily. It can undergo a conformational transition to form polymers that are associated with the dementia familial encephalopathy with neuroserpin inclusion bodies and the wild-type protein can inhibit the toxicity of amyloid-β peptides in Alzheimer's disease. We have used a single molecule fluorescence method, two color coincidence detection, to determine the rate-limiting steps of the early stages of the polymerization of fluorophore-labeled neuroserpin and have assessed how this process is altered in the presence of Aβ_1-40. Our data show that neuroserpin polymerization proceeds first by the unimolecular formation of an active monomer, followed by competing processes of both polymerization and formation of a latent monomer from the activated species. These data are not in keeping with the recently proposed domain swap model of polymer formation in which the latent species and activated monomer are likely to be formed by competing pathways directly from the unactivated monomeric serpin. Moreover, the Aβ_1-40 peptide forms a weak complex with neuroserpin (dissociation constant of 10 ± 5 nM) that increases the amount of active monomer thereby increasing the rate of polymerization. The Aβ_1-40 is displaced from the complex so that it acts as a catalyst and is not incorporated into neuroserpin polymers.     

Synthesis and biological evaluation of new active For‐Met‐Leu‐Phe‐OMe analogues containing para‐substituted Phe residues

In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH₃ and ‐C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

An Immunochip-based interrogation of scleroderma susceptibility variants identifies a novel association at DNASE1L3

Introduction

The aim of the study was to interrogate the genetic architecture and autoimmune pleiotropy of scleroderma susceptibility in the Australian population.

Methods

We genotyped individuals from a well-characterized cohort of Australian scleroderma patients with the Immunochip, a custom array enriched for single nucleotide polymorphisms (SNPs) at immune loci. Controls were taken from the 1958 British Birth Cohort. After data cleaning and adjusting for population stratification the final dataset consisted of 486 cases, 4,458 controls and 146,525 SNPs. Association analyses were conducted using logistic regression in PLINK. A replication study was performed using 833 cases and 1,938 controls.

Results

A total of eight loci with suggestive association (P <10^-4.5) were identified, of which five showed significant association in the replication cohort (HLA-DRB1, DNASE1L3, STAT4, TNP03-IRF5 and VCAM1). The most notable findings were at the DNASE1L3 locus, previously associated with systemic lupus erythematosus, and VCAM1, a locus not previously associated with human disease. This study identified a likely functional variant influencing scleroderma susceptibility at the DNASE1L3 locus; a missense polymorphism rs35677470 in DNASE1L3, with an odds ratio of 2.35 (P = 2.3 × 10⁻¹⁰) in anti-centromere antibody (ACA) positive cases.

Conclusions

This pilot study has confirmed previously reported scleroderma associations, revealed further genetic overlap between scleroderma and systemic lupus erythematosus, and identified a putative novel scleroderma susceptibility locus.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0438-8) contains supplementary material, which is available to authorized users.     

Isolation and Identification of Aspergillus fumigatus Mycotoxins on Growth Medium and Some Building Materials

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions.     

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I–restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.     

The Complete Genome and Proteome of Laribacter hongkongensis Reveal Potential Mechanisms for Adaptations to Different Temperatures and Habitats

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish–borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors—such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases—are present. Proteomes of L. hongkongensis HLHK9 cultured at 37°C (human body temperature) and 20°C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)—NAGK-20 and NAGK-37—in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20°C, whereas NAGK-37 showed higher expression at 37°C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats.