Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain

The human soluble Epoxide Hydrolase (hsEH) is an enzyme involved in the hydrolysis of endogenous anti-inflammatory and cardio-protective signalling mediators known as epoxyeicosatrienoic acids (EETs). EETs’ conversion into the corresponding diols by hsEH generates non-bioactive molecules, thereby the enzyme inhibition would be expected to enhance the EETs bioavailability, and their beneficial properties. Numerous inhibitors have been developed to target the enzyme, some of which are showing promising antihypertensive and anti-inflammatory properties in vivo. Thus far, the preparation of the recombinant enzyme for enzymatic and structural in vitro studies has been performed mainly using a baculovirus expression system. More recently, it was reported that the enzyme could be exogenously expressed and isolated from E. coli, although limited amounts of active protein were obtained. We herein describe two novel methods to yield pure recombinant enzyme. The first describes the expression and purification of the full-length enzyme from eukaryotic cells HEK293-F, whilst the second concerns the C-terminal domain of hsEH obtained from the cost-effective and rapid E. coli prokaryotic system. The two methods successfully generated satisfactory amounts of functional enzyme, with virtually identical enzymatic activity. Overall, the protocols described in this paper can be employed for the recombinant expression and purification of active hsEH, to be used in future biomedical investigations and for high-throughput screening of inhibitors for potential use in the treatment of cardiovascular disease

Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1.

The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified 'β-clasp' structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets

The metabolic effects of intermittent versus continuous feeding in critically ill patients.

Intermittent (or bolus) feeding regimens in critically ill patients have been of increasing interest to clinicians and scientists. Changes in amino acid, fat and carbohydrate metabolites over time might yet deliver other benefits (e.g. modulation of the circadian rhythm and sleep, and impacts on ghrelin secretion, insulin resistance and autophagy). We set out to characterise these changes in metabolite concentration. The Intermittent versus Continuous Feeding in Critically Ill paitents study (NCT02358512) was an eight-centre single-blinded randomised controlled trial. Patients were randomised to received a continuous (control arm) or intermittent (6x/day, intervention arm) enteral feeding regimen. Blood samples were taken on trial days 1, 7 and 10 immediately before and 30 min after intermittent feeds, and at equivalent timepoints in the control arm. A pre-planned targeted metabolomic analysis was performend using Nuclear Resonance Spectroscopy. Five hundred and ninety four samples were analysed from 75 patients. A total of 24 amino acid-, 19 lipid based-, and 44 small molecule metabolite features. Across the main two axes of variation (40-60% and 6-8% of variance), no broad patterns distinguished between intermittent or continuous feeding arms, across intra-day sampling times or over the 10 days from initial ICU admission. Logfold decreases in abundance were seen in metabolites related to amino acids (Glutamine - 0.682; Alanine - 0.594), ketone body metabolism (Acetone - 0.64; 3-Hydroxybutyric Acid - 0.632; Acetonacetic Acid - 0.586), fatty acid (carnitine - 0.509) and carbohydrate metabolism ( Maltose - 0.510; Citric Acid - 0.485). 2-3 Butanediol, a by-product of sugar-fermenting microbial metabolism also decreased (- 0.489). No correlation was seen with change in quadriceps muscle mass for any of the 20 metabolites varying with time (all p > 0.05). Increasing severity of organ failure was related to increasing ketone body metabolism (3 Hydroxybutyric Acid-1 and - 3; p = 0.056 and p = 0.014), carnitine deficiency (p = 0.002) and alanine abundancy (p - 0.005). A 6-times a day intermittent feeding regimen did not alter metabolite patterns across time compared to continuous feeding in critically ill patients, either within a 24 h period or across 10 days of intervention. Future research on intermittent feeding regimens should focus on clinical process benefits, or extended gut rest and fasting

15-deoxy-Delta(12,14)-Prostaglandin J(2) inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors

The metabolic effects of intermittent versus continuous feeding in critically ill patients

Intermittent (or bolus) feeding regimens in critically ill patients have been of increasing interest to clinicians and scientists. Changes in amino acid, fat and carbohydrate metabolites over time might yet deliver other benefits (e.g. modulation of the circadian rhythm and sleep, and impacts on ghrelin secretion, insulin resistance and autophagy). We set out to characterise these changes in metabolite concentration. The Intermittent versus Continuous Feeding in Critically Ill paitents study (NCT02358512) was an eight-centre single-blinded randomised controlled trial. Patients were randomised to received a continuous (control arm) or intermittent (6x/day, intervention arm) enteral feeding regimen. Blood samples were taken on trial days 1, 7 and 10 immediately before and 30 min after intermittent feeds, and at equivalent timepoints in the control arm. A pre-planned targeted metabolomic analysis was performend using Nuclear Resonance Spectroscopy. Five hundred and ninety four samples were analysed from 75 patients. A total of 24 amino acid-, 19 lipid based-, and 44 small molecule metabolite features. Across the main two axes of variation (40–60% and 6–8% of variance), no broad patterns distinguished between intermittent or continuous feeding arms, across intra-day sampling times or over the 10 days from initial ICU admission. Logfold decreases in abundance were seen in metabolites related to amino acids (Glutamine − 0.682; Alanine − 0.594), ketone body metabolism (Acetone − 0.64; 3-Hydroxybutyric Acid − 0.632; Acetonacetic Acid − 0.586), fatty acid (carnitine − 0.509) and carbohydrate metabolism (Maltose − 0.510; Citric Acid − 0.485). 2–3 Butanediol, a by-product of sugar-fermenting microbial metabolism also decreased (− 0.489). No correlation was seen with change in quadriceps muscle mass for any of the 20 metabolites varying with time (all p > 0.05). Increasing severity of organ failure was related to increasing ketone body metabolism (3 Hydroxybutyric Acid-1 and − 3; p = 0.056 and p = 0.014), carnitine deficiency (p = 0.002) and alanine abundancy (p − 0.005). A 6-times a day intermittent feeding regimen did not alter metabolite patterns across time compared to continuous feeding in critically ill patients, either within a 24 h period or across 10 days of intervention. Future research on intermittent feeding regimens should focus on clinical process benefits, or extended gut rest and fasting

A thiol redox sensor in soluble epoxide hydrolase enables oxidative activation by intra-protein disulfide bond formation

Soluble epoxide hydrolase (sEH), an enzyme that broadly regulates the cardiovascular system, hydrolyses epoxyeicosatrienoic acids (EETs) to their corresponding dihydroxyeicosatrienoic acids (DHETs). We previously showed that endogenous lipid electrophiles adduct within the catalytic domain, inhibiting sEH to lower blood pressure in angiotensin II-induced hypertensive mice. As angiotensin II increases vascular H2O2, we explored sEH redox regulation by this oxidant and how this integrates with inhibition by lipid electrophiles to regulate vasotone. Kinetics analyses revealed that H2O2 not only increased the specific activity of sEH but increased its affinity for substrate and increased its catalytic efficiency. This oxidative activation was mediated by formation of an intra-disulfide bond between C262 and C264, as determined by mass spectrometry and substantiated by biotin-phenylarsinate and thioredoxin-trapping mutant assays. C262S/264S sEH mutants were resistant to peroxide-induced activation, corroborating the disulfide-activation mechanism. The physiological impact of sEH redox state was determined in isolated arteries and the effect of the pro-oxidant vasopressor angiotensin II on arterial sEH redox state and vasodilatory EETs indexed in mice. Angiotensin II induced the activating intra-disulfide in sEH, causing a decrease in plasma EET/DHET ratios that is consistent with the pressor response to this hormone. Although sEH C262–C264 disulfide formation enhances hydrolysis of vasodilatory EETs, this modification also sensitized sEH to inhibition by lipid electrophiles. This explains why angiotensin II decreases EETs and increases blood pressure, but when lipid electrophiles are also present, that EETs are increased and blood pressure lowered

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response.

The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank-Starling response

Reduced level of arousal and increased mortality in adult acute medical admissions: a systematic review and meta-analysis

Abstract Background Reduced level of arousal is commonly observed in medical admissions and may predict in-hospital mortality. Delirium and reduced level of arousal are closely related. We systematically reviewed and conducted a meta-analysis of studies in adult acute medical patients of the relationship between reduced level of arousal on admission and in-hospital mortality. Methods We conducted a systematic review (PROSPERO: CRD42016022048), searching MEDLINE and EMBASE. We included studies of adult patients admitted with acute medical illness with level of arousal assessed on admission and mortality rates reported. We performed meta-analysis using a random effects model. Results From 23,941 studies we included 21 with 14 included in the meta-analysis. Mean age range was 33.4 - 83.8 years. Studies considered unselected general medical admissions (8 studies, n=13,039) or specific medical conditions (13 studies, n=38,882). Methods of evaluating level of arousal varied. The prevalence of reduced level of arousal was 3.1%-76.9% (median 13.5%). Mortality rates were 1.7%-58% (median 15.9%). Reduced level of arousal was associated with higher in-hospital mortality (pooled OR 5.71; 95% CI 4.21-7.74; low quality evidence: high risk of bias, clinical heterogeneity and possible publication bias). Conclusions Reduced level of arousal on hospital admission may be a strong predictor of in-hospital mortality. Most evidence was of low quality. Reduced level of arousal is highly specific to delirium, better formal detection of hypoactive delirium and implementation of care pathways may improve outcomes. Future studies to assess the impact of interventions on in-hospital mortality should use validated assessments of both level of arousal and delirium

Patient-Reported Outcomes and Quality of Life Assessment. New Targets for New Targeted Therapy?

No abstract availabl