The Contribution of Amino Acid Region ASP695-TYR698 of Factor V to Procofactor Activation and Factor VA Function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K(i) values of approximately 6.3 and approximately 5.3 microm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp-Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC(50) of 1.6 microm (with a K(D) for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --\u3e Lys, Tyr(696) --\u3e Phe, Asp(697) --\u3e Lys, and Tyr(698) --\u3e Phe (factor V(2K2F)) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa (factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase

Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening

High mobility group box 1 (HMGB1), a highly conserved, ubiquitous protein, is released into the circulation during sterile inflammation (e.g. arthritis, trauma) and circulatory shock. It participates in the pathogenesis of delayed inflammatory responses and organ dysfunction. While several molecules have been identified that modulate the release of HMGB1, less attention has been paid to identify pharmacological inhibitors of the downstream inflammatory processes elicited by HMGB1 (C23-C45 disulfide C106 thiol form). In the current study, a cell-based medium-throughput screening of a 5000+ compound focused library of clinical drugs and drug-like compounds was performed in murine RAW264.7 macrophages, in order to identify modulators of HMGB1-induced tumor-necrosis factor alpha (TNFα) production. Clinically used drugs that suppressed HMGB1-induced TNFα production included glucocorticoids, beta agonists, and the anti-HIV compound indinavir. A re-screen of the NIH clinical compound library identified beta-agonists and various intracellular cAMP enhancers as compounds that potentiate the inhibitory effect of glucocorticoids on HMGB1-induced TNFα production. The molecular pathways involved in this synergistic anti-inflammatory effect are related, at least in part, to inhibition of TNFα mRNA synthesis via a synergistic suppression of ERK/IκB activation. Inhibition of TNFα production by prednisolone+salbutamol pretreatment was also confirmed in vivo in mice subjected to HMGB1 injection; this effect was more pronounced than the effect of either of the agents administered separately. The current study unveils several drug-like modulators of HMGB1-mediated inflammatory responses and offers pharmacological directions for the therapeutic suppression of inflammatory responses in HMGB1-dependent diseases. © 2013 Gerö et al

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies

Position Paper on Water, Energy, Food and Ecosystem (WEFE) Nexus and Sustainable development Goals (SDGs)

The EU and the international community is realising that the Water, Energy, Food and Ecosystem components are interlinked and require a joint planning in order to meet the daunting global challenges related to Water, Energy and Food security and maintaining the ecosystem health and in this way, reach the SDGs. If not dealt with, the world will not be able to meet the demand for water, energy and food in a not too far future and, in any case, in a not sustainable way. The strain on the ecosystems resulting from unsustainable single-sector planning will lead to increasing poverty, inequality and instability. The Nexus approach is fully aligned with and supportive of the EU Consensus on Development. Key elements of the Consensus will require collaborative efforts across sectors in ways that can be supported/implemented by a Nexus approach. In this way, transparent and accountable decision-making, involving the civil society is key and common to the European Consensus on Development and the Nexus approach. The Nexus approach will support the implementation of the SDG in particular SDG 2 (Food), SDG 6 (Water) and SDG 7 (Energy), but most SDGs have elements that link to food, water and energy in one or other way, and will benefit from a Nexus approach. The SDGs are designed to be cross-cutting and be implemented together, which is also reflected in a WEFE Nexus approach. A Nexus approach offers a sustainable way of addressing the effects of Climate Change and increase resilience. The WEFE Nexus has in it the main drivers of climate change (water, energy and food security) and the main affected sectors (water and the environment). Decisions around policy, infrastructure, … developed based on the WEFE Nexus assessments will be suitable as elements of climate change mitigation and adaptation. In fact, it is difficult to imagine solutions to the climate change issue that are not built on a form of Nexus approach. The Nexus approach is being implemented around the world, as examples in the literature demonstrate. These examples together with more examples from EU and member state development cooperation will help build experience that can be consolidated and become an important contribution to a Toolkit for WEFE Nexus Implementation. From the expert discussions, it appears that because of the novelty of the approach, a Toolkit will be an important element in getting the Nexus approach widely used. This should build on experiences from practical examples of NEXUS projects or similar inter-sectorial collaboration projects; and, there are already policy, regulation and practical experience to allow institutions and countries to start applying the Nexus concept.JRC.D.2-Water and Marine Resource

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Addictive Behaviour of Adolescents in Secondary Schools in Hungary

Objective: To investigate drug use among secondary schoolchildren aged 14 - 18 years in the city of Szeged in Hungary in order to observe and describe drug use, smoking and alcohol intake and to find risk factors for these behaviours. Methods: The questionnaire was concerned with recreational drug use, smoking and drinking habits as well as family structure and life-style based upon the European School Project on Alcohol and Other Drugs questionnaire. Statistical methods used were the chi(2) test and multiple logistic regression analysis. The standard errors were estimated using the Huber-White procedure. Results: Overall 20% of schoolchildren reported experience of drug usage. Boys were significantly more likely to report drug use than girls ( chi(2) = 4.65; p = 0.031). Cannabis was the most commonly reported drug used (96%). The proportion of drug users was significantly higher among those who had smoked (13%) or were current smokers (65%) compared to those who never smoked (1%; chi(2) = 164.7; p < 0.001). Moreover, the risk of drug use was significantly higher in those children who reported a drug user in the family (OR 9.3; 95% CI 4.3 - 19.9; p < 0.001). Conclusions: Our findings support the hypothesis that strategies for the prevention of drug use should consider the association between drug and tobacco use