Paced QRS morphology predicts incident left ventricular systolic dysfunction and atrial fibrillation

Background

The prognostic significance of paced QRS complex morphology on surface ECG remains unclear. This study aimed to assess long-term outcomes associated with variations in the paced QRS complex.

Sorting and pooling strategy: a novel tool to map a virus proteome for CD8 T-cell epitopes

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.     

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.     

Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity

Glutathione (GSH) is the major intracellular thiol present in 1-10-mm concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.     

Cellular recognition of trimyristoylated peptide or enterobacterial lipopolysaccharide via both TLR2 and TLR4

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.     

A multimedia tool for the informed consent of patients prior to gastric banding

Objective: Severe obesity is a clear indication for appropriate, effective weight loss therapy. One option is operative intervention, e.g., gastric banding. Risks of the operation and therapeutic alternatives need to be comprehensibly presented to the patient. The literature has shown that better informed consent is obtained using information presented in a multimedia/video‐based format. The current study developed and evaluated a multimedia program aimed at obtaining informed consent from obese patients before gastric banding. Research Methods and Procedure: An interactive multimedia program was developed with information about preoperative examinations, the operation itself, hospital stay, operative risks, alternative therapies, and the pathophysiology and health risks of obesity. Two groups (Group 1, n = 20, mean age 38 years, informed consent attained with conventional document information; Group 2, n = 20, mean age 37 years, informed consent attained with additional multimedia information) were interviewed regarding comprehensibility of the information presented, personal satisfaction, and anxiety levels during the informed consent process. Results: Group 2 showed significantly better (p < 0.05) understanding of the presented information and higher levels of satisfaction with the informed consent process. Anxiety levels did not significantly differ between the two groups. Discussion: Because patient satisfaction with the informed consent process and understanding of the presented information significantly improved, the multimedia program clearly benefits both surgeons and patients. Personal contact from the surgeon remains essential. High volumes of information presented in multimedia format do not alleviate patient anxiety, and personal contact may be beneficial.     

Using likelihood-free inference to compare evolutionary dynamics of the protein networks of H. pylori and P. falciparum

Gene duplication with subsequent interaction divergence is one of the primary driving forces in the evolution of genetic systems. Yet little is known about the precise mechanisms and the role of duplication divergence in the evolution of protein networks from the prokaryote and eukaryote domains. We developed a novel, model-based approach for Bayesian inference on biological network data that centres on approximate Bayesian computation, or likelihood-free inference. Instead of computing the intractable likelihood of the protein network topology, our method summarizes key features of the network and, based on these, uses a MCMC algorithm to approximate the posterior distribution of the model parameters. This allowed us to reliably fit a flexible mixture model that captures hallmarks of evolution by gene duplication and subfunctionalization to protein interaction network data of Helicobacter pylori and Plasmodium falciparum. The 80% credible intervals for the duplication–divergence component are [0.64, 0.98] for H. pylori and [0.87, 0.99] for P. falciparum. The remaining parameter estimates are not inconsistent with sequence data. An extensive sensitivity analysis showed that incompleteness of PIN data does not largely affect the analysis of models of protein network evolution, and that the degree sequence alone barely captures the evolutionary footprints of protein networks relative to other statistics. Our likelihood-free inference approach enables a fully Bayesian analysis of a complex and highly stochastic system that is otherwise intractable at present. Modelling the evolutionary history of PIN data, it transpires that only the simultaneous analysis of several global aspects of protein networks enables credible and consistent inference to be made from available datasets. Our results indicate that gene duplication has played a larger part in the network evolution of the eukaryote than in the prokaryote, and suggests that single gene duplications with immediate divergence alone may explain more than 60% of biological network data in both domains.     

Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum

Lipoic acid (LA) is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.     

GROmaρs: A GROMACS-Based Toolset to Analyze Density Maps Derived from Molecular Dynamics Simulations

We introduce a computational toolset, named GROmaρs, to obtain and compare time-averaged density maps from molecular dynamics simulations. GROmaρs efficiently computes density maps by fast multi-Gaussian spreading of atomic densities onto a three-dimensional grid. It complements existing map-based tools by enabling spatial inspection of atomic average localization during the simulations. Most importantly, it allows the comparison between computed and reference maps (e.g., experimental) through calculation of difference maps and local and time-resolved global correlation. These comparison operations proved useful to quantitatively contrast perturbed and control simulation data sets and to examine how much biomolecular systems resemble both synthetic and experimental density maps. This was especially advantageous for multimolecule systems in which standard comparisons like RMSDs are difficult to compute. In addition, GROmaρs incorporates absolute and relative spatial free-energy estimates to provide an energetic picture of atomistic localization. This is an open-source GROMACS-based toolset, thus allowing for static or dynamic selection of atoms or even coarse-grained beads for the density calculation. Furthermore, masking of regions was implemented to speed up calculations and to facilitate the comparison with experimental maps. Beyond map comparison, GROmaρs provides a straightforward method to detect solvent cavities and average charge distribution in biomolecular systems. We employed all these functionalities to inspect the localization of lipid and water molecules in aquaporin systems, the binding of cholesterol to the G protein coupled chemokine receptor type 4, and the identification of permeation pathways through the dermicidin antimicrobial channel. Based on these examples, we anticipate a high applicability of GROmaρs for the analysis of molecular dynamics simulations and their comparison with experimentally determined densities.