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Contrast-induced nephropathy (CIN) is a major complication of imaging in patients with chronic kidney disease (CKD). The publication of an academic randomized controlled trial (RCT; n = 83) reporting oral (N)-acetylcysteine (NAC) to reduce CIN led to 70 clinical trials, 23 systematic reviews, and 2 large RCTs showing no benefit. However, no mechanistic studies were conducted to determine how NAC might work; proposed mechanisms included renal artery vasodilatation and antioxidant boosting. We evaluated the proposed mechanisms of NAC action in participants with healthy and diseased kidneys. Four substudies were performed. Two randomized, double-blind, placebo-controlled, three-period crossover studies (n = 8) assessed the effect of oral and intravenous (i.v.) NAC in healthy kidneys in the presence/absence of iso-osmolar contrast (iodixanol). A third crossover study in patients with CKD stage III (CKD3) (n = 8) assessed the effect of oral and i.v. NAC without contrast. A three-arm randomized, double-blind, placebo-controlled parallel-group study, recruiting patients with CKD3 (n = 66) undergoing coronary angiography, assessed the effect of oral and i.v. NAC in the presence of contrast. We recorded systemic (blood pressure and heart rate) and renal (renal blood flow (RBF) and glomerular filtration rate (GFR)) hemodynamics, and antioxidant status, plus biomarkers of renal injury in patients with CKD3 undergoing angiography. Primary outcome for all studies was RBF over 8 hours after the start of i.v. NAC/placebo. NAC at doses used in previous trials of renal prophylaxis was essentially undetectable in plasma after oral administration. In healthy volunteers, i.v. NAC, but not oral NAC, increased blood pressure (mean area under the curve (AUC) mean arterial pressure (MAP): mean difference 29 h⋅mmHg, P = 0.019 vs. placebo), heart rate (28 h⋅bpm, P 0.001), and RBF (714 h⋅mL/min, 8.0% increase, P = 0.006). Renal vasodilatation also occurred in the presence of contrast (RBF 917 h⋅mL/min, 12% increase, P = 0.005). In patients with CKD3 without contrast, only a rise in heart rate (34 h⋅bpm, P = 0.010) and RBF (288 h⋅mL/min, 6.0% increase, P = 0.001) occurred with i.v. NAC, with no significant effect on blood pressure (MAP rise 26 h⋅mmHg, P = 0.156). Oral NAC showed no effect. In patients with CKD3 receiving contrast, i.v. NAC increased blood pressure (MAP rise 52 h⋅mmHg, P = 0.008) but had no effect on RBF (151 h⋅mL/min, 3.0% increase, P = 0.470), GFR (29 h⋅mL/min/1.73m², P = 0.122), or markers of renal injury. Neither i.v. nor oral NAC affected plasma antioxidant status. We found oral NAC to be poorly absorbed and have no reno-protective effects. Intravenous, not oral, NAC caused renal artery vasodilatation in healthy volunteers but offered no protection to patients with CKD3 at risk of CIN. These findings emphasize the importance of mechanistic clinical studies before progressing to RCTs for novel interventions. Thousands were recruited to academic clinical trials without the necessary mechanistic studies being performed to confirm the approach had any chance of working.

Background Macrophages play a central role in inflammation by phagocytosing invading pathogens, apoptotic cells and debris, as well as mediating repair of tissues damaged by trauma. In order to do this, these dynamic cells generate a variety of inflammatory mediators including eicosanoids such as prostaglandins, leukotrienes and hydroxyeicosatraenoic acids (HETEs) that are formed through the cyclooxygenase, lipoxygenase and cytochrome P450 pathways. The ability to examine the effects of eicosanoid production at the protein level is therefore critical to understanding the mechanisms associated with macrophage activation. Results This study presents a stable isotope labelling with amino acids in cell culture (SILAC) -based proteomics strategy to quantify the changes in macrophage protein abundance following inflammatory stimulation with Kdo2-lipid A and ATP, with a focus on eicosanoid metabolism and regulation. Detailed gene ontology analysis, at the protein level, revealed several key pathways with a decrease in expression in response to macrophage activation, which included a promotion of macrophage polarisation and dynamic changes to energy requirements, transcription and translation. These findings suggest that, whilst there is evidence for the induction of a pro-inflammatory response in the form of prostaglandin secretion, there is also metabolic reprogramming along with a change in cell polarisation towards a reduced pro-inflammatory phenotype. Conclusions Advanced quantitative proteomics in conjunction with functional pathway network analysis is a useful tool to investigate the molecular pathways involved in inflammation.

Arteriosclerosis is an important age-dependent disease that encompasses atherosclerosis, in-stent restenosis (ISR), pulmonary hypertension, autologous bypass grafting and transplant arteriosclerosis. Endothelial dysfunction and the proliferation of vascular smooth muscle cell (vSMC)-like cells is a critical event in the pathology of arteriosclerotic disease leading to intimal-medial thickening (IMT), lipid retention and vessel remodelling. An important aspect in guiding clinical decision-making is the detection of biomarkers of subclinical arteriosclerosis and early cardiovascular risk. Crucially, relevant biomarkers need to be good indicators of injury which change in their circulating concentrations or structure, signalling functional disturbances. Extracellular vesicles (EVs) are nanosized membraneous vesicles secreted by cells that contain numerous bioactive molecules and act as a means of intercellular communication between different cell populations to maintain tissue homeostasis, gene regulation in recipient cells and the adaptive response to stress. This review will focus on the emerging field of EV research in cardiovascular disease (CVD) and discuss how key EV signatures in liquid biopsies may act as early pathological indicators of adaptive lesion formation and arteriosclerotic disease progression. EV profiling has the potential to provide important clinical information to complement current cardiovascular diagnostic platforms that indicate or predict myocardial injury. Finally, the development of fitting devices to enable rapid and/or high-throughput exosomal analysis that require adapted processing procedures will be evaluated.

New readily accessible systemic redox biomarkers are needed to understand the biological roles reactive oxygen species (ROS) play in humans because overtly flawed, technically fraught, and unspecific assays severely hamper translational progress. The antibody-linked oxi-state assay (ALISA) makes it possible to develop valid ROS-sensitive target-specific protein thiol redox state biomarkers in a readily accessible microplate format. Here, we used a maximal exercise bout to disrupt redox homeostasis in a physiologically meaningful way to determine whether the catalytic core of the serine/threonine protein phosphatase PP2A is a candidate systemic redox biomarker in human erythrocytes. We reasoned that: constitutive oxidative stress (e.g., haemoglobin autoxidation) would sensitise erythrocytes to disrupted ion homeostasis as manifested by increased oxidation of the ion regulatory phosphatase PP2A. Unexpectedly, an acute bout of maximal exercise lasting ~16 min decreased PP2A-specific reversible thiol oxidation (redox ratio, rest: 0.46; exercise: 0.33) without changing PP2A content (rest: 193 pg/ml; exercise: 191 pg/ml). The need for only 3-4 μl of sample to perform ALISA means PP2A-specific reversible thiol oxidation is a capillary-fingertip blood-compatible candidate redox biomarker. Consistent with biologically meaningful redox regulation, thiol reductant-inducible PP2A activity was significantly greater (+10%) at rest compared to exercise. We establish a route to developing new readily measurable protein thiol redox biomarkers for understanding the biological roles ROS play in humans.

Authors gratefully thank the following funding sources for financial support: Scottish Enterprise (POC13), EPSRC (EP/K005499/1 and Capital for Great Technologies grant EP/L017008/1), European Social Fund Studentship and the University of St Andrews School of Chemistry. The formulation and antibacterial efficacy of nitric oxide (NO)-releasing MOF–polyurethane films are reported for the first time. Uniform standalone films were successfully prepared containing 1, 5, 10, and 15 wt% CPO-27 (Ni). The MOF within each film was successfully activated and loaded with NO. Adsorption and release profiles are reported for the films and show that while the polymer influences the quantity of NO adsorbed and stored by the MOF and the time scale of release; the proportion of stored NO that is released is dependent on wt% loading of MOF in the film. In a cytotoxicity assay, the formulations exhibited very low toxicity (

Hyperglycaemia is known to induce endothelial dysfunction and changes in metabolic function, which could be implicated in diabetes-induced cardiovascular disease. To date, however, little is known about the impact of physiologically relevant concentrations of fructose on endothelial cells. A novel in vitro model was devised to establish the impact of substitution of a small proportion of glucose with an equal concentration (0.1 mM or 1 mM) of fructose on EA.hy926 endothelial cells during periodic carbohydrate “meals” superimposed on a normoglycaemic (5.5 mM) background. Parallel experiments were conducted using meals consisting of normoglycaemic glucose, intermediate glucose (12.5 mM) or profound hyperglycaemia (25 mM), each delivered for 2 h, with and without substituted fructose over 50 h. Outcome measures included nitrite as a surrogate marker of the mediator of healthy endothelial function, nitric oxide (NO), and a range of bioenergetic parameters using a metabolic analyser. Despite its relatively low proportion of carbohydrate load, intermittent fructose induced a substantial reduction (approximately 90%) in NO generation in cells treated with either concentration of fructose. Cell markers of oxidative stress were not altered by this treatment regimen. However, the cells experienced a marked increase in metabolic activity induced by fructose, irrespective of the glucose concentration delivered simultaneously in the “meals”. Indeed, glucose alone failed to induce any metabolic impact in this model. Key metabolic findings were a 2-fold increase in basal oxygen consumption rate and a similar change in extracellular acidification rate–a marker of glycolysis. Non-metabolic oxygen consumption also increased substantially in cells exposed to fructose. There was no difference between results with 0.1 mM fructose and those with 1 mM fructose. Low, physiologically relevant concentrations of fructose, delivered in a pattern that mimics mealtime consumption, had a profound impact on endothelial function and bioenergetics in an in vitro cell model. The results suggest that endothelial cells are exquisitely sensitive to circulating fructose; the potential ensuing dysfunction could have major implications for development of atherosclerotic disease associated with high fructose consumption.

Percutaneous coronary intervention for the treatment of coronary artery disease is most commonly performed in the UK through the radial artery, as this is safer than the femoral approach. However, despite improvements in technology and techniques, complications can occur. The most common complication, arterial spasm, can cause intense pain and, in some cases, procedural failure. The incidence of spasm is dependent on several variables, including operator experience, artery size, and equipment used. An anti-spasmolytic cocktail can be applied to reduce spasm, which usually includes an exogenous nitric oxide (NO) donor (glyceryl trinitrate). NO is an endogenous local vasodilator and therefore is a potential target for anti-spasm intervention. However, systemic administration can result in unwanted side-effects, such as hypotension. A method that adopts local delivery of NO might be advantageous. This review article describes the mechanisms involved in radial artery spasm, discusses the advantages and disadvantages of current strategies to reduce spasm, and highlight the potential of NO-loaded nanoporous materials for use in this setting.

Background Accurate, continuous heart rate measurements are important for health assessment, physical activity, and sporting performance, and the integration of heart rate measurements into wearable devices has extended its accessibility. Although the use of photoplethysmography technology is not new, the available data relating to the validity of measurement are limited, and the range of activities being performed is often restricted to one exercise domain and/or limited intensities. Objective The primary objective of this study was to assess the validity of the Polar OH1 and Fitbit Charge 3 devices for measuring heart rate during rest, light, moderate, vigorous, and sprint-type exercise. Methods A total of 20 healthy adults (9 female; height: mean 1.73 [SD 0.1] m; body mass: mean 71.6 [SD 11.0] kg; and age: mean 40 [SD 10] years) volunteered and provided written informed consent to participate in the study consisting of 2 trials. Trial 1 was split into 3 components: 15-minute sedentary activities, 10-minute cycling on a bicycle ergometer, and incremental exercise test to exhaustion on a motorized treadmill (18-42 minutes). Trial 2 was split into 2 components: 4 × 15-second maximal sprints on a cycle ergometer and 4 × 30- to 50-m sprints on a nonmotorized resistance treadmill. Data from the 3 devices were time-aligned, and the validity of Polar OH1 and Fitbit Charge 3 was assessed against Polar H10 (criterion device). Validity was evaluated using the Bland and Altman analysis, Pearson moment correlation coefficient, and mean absolute percentage error. Results Overall, there was a very good correlation between the Polar OH1 and Polar H10 devices (r=0.95), with a mean bias of −1 beats·min-1 and limits of agreement of −20 to 19 beats·min-1. The Fitbit Charge 3 device underestimated heart rate by 7 beats·min-1 compared with Polar H10, with a limit of agreement of −46 to 33 beats·min-1 and poor correlation (r=0.8). The mean absolute percentage error for both devices was deemed acceptable ( Conclusions Compared with our criterion device, Polar OH1 was accurate at assessing heart rate, but the accuracy of Fitbit Charge 3 was generally poor. Polar OH1 performed worse during trial 2 compared with the activities in trial 1, and the validity of the Fitbit Charge 3 device was particularly poor during our cycling exercises.

Meta-analyses have indicated that individuals with type 1 or type 2 diabetes are at increased risk of suffering a severe form of COVID-19 and have a higher mortality rate than the non-diabetic population. Patients with diabetes have chronic, low-level systemic inflammation, which results in global cellular dysfunction underlying the wide variety of symptoms associated with the disease, including an increased risk of respiratory infection. While the increased severity of COVID-19 amongst patients with diabetes is not yet fully understood, the common features associated with both diseases are dysregulated immune and inflammatory responses. An additional key player in COVID-19 is the enzyme, angiotensin-converting enzyme 2 (ACE2), which is essential for adhesion and uptake of virus into cells prior to replication. Changes to the expression of ACE2 in diabetes have been documented, but they vary across different organs and the importance of such changes on COVID-19 severity are still under investigation. This review will examine and summarise existing data on how immune and inflammatory processes interplay with the pathogenesis of COVID-19, with a particular focus on the impacts that diabetes, endothelial dysfunction and the expression dynamics of ACE2 have on the disease severity.

BACKGROUND Accurate, continuous heart rate measurements are important for health assessment, physical activity, and sporting performance, and the integration of heart rate measurements into wearable devices has extended its accessibility. Although the use of photoplethysmography technology is not new, the available data relating to the validity of measurement are limited, and the range of activities being performed is often restricted to one exercise domain and/or limited intensities. OBJECTIVE The primary objective of this study was to assess the validity of the Polar OH1 and Fitbit Charge 3 devices for measuring heart rate during rest, light, moderate, vigorous, and sprint-type exercise. METHODS A total of 20 healthy adults (9 female; height: mean 1.73 [SD 0.1] m; body mass: mean 71.6 [SD 11.0] kg; and age: mean 40 [SD 10] years) volunteered and provided written informed consent to participate in the study consisting of 2 trials. Trial 1 was split into 3 components: 15-minute sedentary activities, 10-minute cycling on a bicycle ergometer, and incremental exercise test to exhaustion on a motorized treadmill (18-42 minutes). Trial 2 was split into 2 components: 4 × 15-second maximal sprints on a cycle ergometer and 4 × 30- to 50-m sprints on a nonmotorized resistance treadmill. Data from the 3 devices were time-aligned, and the validity of Polar OH1 and Fitbit Charge 3 was assessed against Polar H10 (criterion device). Validity was evaluated using the Bland and Altman analysis, Pearson moment correlation coefficient, and mean absolute percentage error. RESULTS Overall, there was a very good correlation between the Polar OH1 and Polar H10 devices (r=0.95), with a mean bias of −1 beats·min-1 and limits of agreement of −20 to 19 beats·min-1. The Fitbit Charge 3 device underestimated heart rate by 7 beats·min-1 compared with Polar H10, with a limit of agreement of −46 to 33 beats·min-1 and poor correlation (r=0.8). The mean absolute percentage error for both devices was deemed acceptable ( CONCLUSIONS Compared with our criterion device, Polar OH1 was accurate at assessing heart rate, but the accuracy of Fitbit Charge 3 was generally poor. Polar OH1 performed worse during trial 2 compared with the activities in trial 1, and the validity of the Fitbit Charge 3 device was particularly poor during our cycling exercises. CLINICALTRIAL

This project has been funded by the EU’s INTERREG VA programme, managed by the special EU Programmes Body (SEUPB) Background and Aims N-acetyl-L-cysteine (NAC) has been proposed as a prophylactic for the prevention of contrast induced nephropathy (CIN), a serious, adverse complication following administration of contrast media in patients with diabetes and chronic kidney disease (CKD). However, clinical trial results have failed to demonstrate consistently the beneficial qualities of NAC in this setting. The suggested mechanism by which NAC has been proposed to be effective is via antioxidant activity, but NAC itself appears not to be a powerful antioxidant; intracellular conversion of NAC to glutathione (GSH) is essential for antioxidant effects. We tested the hypothesis that oral or intravenous NAC fails to increase antioxidant activity in the plasma of healthy volunteers or CKD stage III patients undergoing elective coronary angiography. Method 16 healthy volunteers (8 receiving contrast media) and 8 patients with chronic renal failure were recruited to 3 separate groups as a part of a crossover design study. 66 patients with CKD stage III undergoing elective coronary angiography were recruited to a parallel group randomized controlled trial. Each volunteer or patient was randomised to receive NAC (i.v.; 50 mg/kg/hr infusion for 2 h, followed by 20 mg/kg/hr infusion for a further 5 h), or NAC (oral; 1200 mg twice daily, commencing the day before the study visit) or placebo. Participants attended the Clinical Research Facility on three occasions for the crossover groups, but only once in the parallel group trial. Patients were infused with para-aminohippurate (PAH) and inulin to enable measurement of renal function parameters. Plasma and paired buffy coat samples were collected at several time points for 2 hours before contrast infusion and for 6 hours after. Samples were used to measure various clinical parameters, along with intracellular and plasma thiol concentrations and the plasma antioxidant capacity (oxygen radical antioxidant capacity; ORAC). Results Plasma NAC concentrations peaked at 222 µM (healthy volunteers), 354 µM (CKD patients), 271 µM (healthy volunteers receiving contrast) and 348 µM (CKD patients undergoing elective coronary angiography) following infusion of NAC. The corresponding peak concentrations were ∼2 µM following oral NAC across all groups. No comparable increase in the plasma compartment ORAC antioxidant capacity was measured in any of the treatment groups. Equally, contrast infusion itself did not significantly reduce antioxidant capacity in plasma, even in the placebo group. Buffy coat measures showed that there was a significant increase of intracellular GSH in some patients. Also, no clear additional benefit was witnessed by increasing plasma NAC ∼100-fold through infusion of NAC over oral administration. Conclusion NAC fails to influence ORAC antioxidant capacity in the plasma compartment of CKD stage III patients receiving contrast media. Our results question the importance of oxidative stress in contrast-induced nephropathy. Furthermore, the findings fail to demonstrate a direct antioxidant impact of NAC in the plasma compartment and question the antioxidant hypothesis related to contrast-induced nephropathy and NAC.

Introduction N-acetyl-L-cysteine (NAC) has been proposed as a prophylactic for the prevention of contrast induced nephropathy (CIN), a common, undesirable complication following the administration of contrast media in patients with diabetes and chronic kidney disease (CKD).1 However, clinical trial results have failed to consistently demonstrate beneficial qualities of NAC in this setting. The proposed mechanism by which NAC has been proposed to be effective is via antioxidant activity, but NAC itself is not a powerful antioxidant. Intracellular conversion of NAC to glutathione (GSH) is essential for antioxidant effects (Treweeke, Winterburn et al. 2012). We tested the hypotheses that whilst oral or intravenous NAC fails to increase antioxidant activity in the plasma of CKD stage III patients undergoing elective coronary angiography, intracellular conversion of NAC to GSH can occur in buffy coat cells deficient in GSH. Methods 66 patients with CKD stage III undergoing elective coronary angiography were recruited to a parallel-group randomized controlled trial. Each patient was randomised to receive NAC (i.v.; 50 mg/kg/hr infusion for 2 h, followed by 20 mg/kg/hr infusion for a further 5 h), or NAC (oral; 1200 mg twice daily, commencing the day before contrast exposure) or placebo upon visiting the Clinical Research Facility. On the day of the study, patients were infused with PAH and inulin to regulate several renal function parameters and infused with contrast media prior to the coronary angiography (Sandilands, Cameron et al. 2012). Plasma and paired buffy coat samples were collected at several time points during the trial in order to measure various clinical parameters, along with intracellular and plasma thiol concentrations and the plasma antioxidant capacity (oxygen radical antioxidant capacity; ORAC). Results Plasma NAC concentrations peaked at 348 µM following infusion of NAC of the 11 data sets included; the corresponding peak concentrations were 2.8 µM following oral NAC treatment from 7 data sets. No comparable increase in the plasma compartment ORAC antioxidant capacity was measured in any of the treatment groups. Equally, contrast infusion itself did not significantly reduce antioxidant capacity in plasma, even in the placebo group. Buffy coat measures showed that there was a significant increase of intracellular GSH some patients, although the effect was variable. Also, no clear additional benefit was witnessed by increasing plasma NAC >10-fold through infusion of NAC over oral administration. Conclusion NAC fails to influence ORAC antioxidant capacity in the plasma compartment of CKD stage III patients receiving contrast media. Our results question the importance of oxidative stress in contrast-induced nephropathy. Furthermore, the findings fail to demonstrate a direct antioxidant impact of NAC in the plasma compartment. Instead, we found that NAC can increase intracellular GSH in some deficient individuals, which might carry some benefits in terms of detoxification and question the antioxidant hypothesis related to contrast-induced nephropathy and NAC. References Sandilands EA, Cameron S, Paterson F, Donaldson S, Briody L, Crowe J, Donnelly J, Thompson A, Johnston NR, Mackenzie I, Uren N, Goddard J, Webb DJ, Megson IL, Bateman N, Eddleston M. ‘Mechanisms for an effect of acetylcysteine on renal function after exposure to radio-graphic contrast material: study protocol.’ BMC Clin Pharmacol 2012;12:3. Treweeke AT, Winterburn TJ, Mackenzie I, Barrett F, Barr C, Rushworth GF, Dransfield I, MacRury SM, Megson IL. ‘N-Acetylcysteine inhibits platelet-monocyte conjugation in patients with type 2 diabetes with depleted intraplatelet glutathione: a randomised controlled trial.’Diabetologia 2012;55(11):2920–2928.

Introduction: Organophosphorus (OP) insecticide self-poisoning is a global problem, killing tens of thousands of people every year. Oxidative stress has been proposed to play a pathological role in...

Developmental synapse pruning refines burgeoning connectomes. The basic mechanisms of mitochondrial reactive oxygen species (ROS) production suggest they select inactive synapses for pruning: whether they do so is unknown. To begin to unravel whether mitochondrial ROS regulate pruning, we made the local consequences of neuromuscular junction (NMJ) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; and (2) chemically heterogeneous antioxidants rescue synaptic inactivity induced motor deficits. Regardless of whether it was achieved with muscle (α-bungarotoxin), nerve (α-latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial ROS in vivo. The manganese porphyrins MnTE-2-PyP5+ and/or MnTnBuOE-2-PyP5+ blocked mitochondrial ROS to significantly reduce neurotoxin and endogenous pruning cue induced motor deficits. Selectively inducing mitochondrial ROS—using mitochondria-targeted Paraquat (MitoPQ)—recapitulated synaptic inactivity induced motor deficits; which were significantly reduced by blocking mitochondrial ROS with MnTnBuOE-2-PyP5+. We unveil mitochondrial ROS as synaptic activity sentinels that regulate the phenotypical consequences of forced synaptic inactivity at the NMJ. Our novel results are relevant to pruning because synaptic inactivity is one of its defining features., Graphical abstract fx1, Highlights • Exogenous and endogenous synaptic inactivity models increase mitochondrial ROS. • Manganese porphyrins rescue synaptic inactivity induced motor deficits. • Mitochondrial ROS may select inactive synapses for pruning during development.

Antioxidants have been the subject of intense research interest mainly due to their beneficial properties associated with human health and wellbeing. Phenolic molecules, such as naturally occurring Resveratrol and Vanillin, are well known for their anti-oxidant properties, providing a starting point for the development of new antioxidants. Here we report, for the first time, the synthesis of a number of new vanillin through the reductive amination reaction between vanillin and a selection of amines. All the compounds synthesised, exhibited strong antioxidant properties in DPPH, FRAP and ORAC assays, with compounds 1b and 2c being the most active. The latter also demonstrated the ability to protect plasmid DNA from oxidative damage in the presence of the radical initiator AAPH. At cellular level, neuroblastoma SH-SY5Y cells were protected from oxidative damage (H2O2, 400 μM) with both 1b and 2c. The presence of a tertiary amino group, along with the number of vanillin moieties in the molecule contribute for the antioxidant activity. Furthermore, the delocalization of the electron pair of the nitrogen and the presence of an electron donating substituent to enhance the antioxidant properties of this new class of compounds. In our opinion, vanillin derivatives 1b and 2c described in this work can provide a viable platform for the development of antioxidant based therapeutics.

Polyphenols are widely regarded to have a wide range of health-promoting qualities, including beneficial effects on cardiovascular disease. Historically, the benefits have been linked to their well-recognized powerful antioxidant activity. However, the concept that the beneficial effects are attributable to direct antioxidant activity in vivo does not pay sufficient heed to the fact that polyphenols degrade rapidly, are poorly absorbed and rapidly metabolized, resulting in very low bioavailability. This review explores alternative mechanisms by which polyphenols, or their metabolites, exert biological activity via mechanisms that can be activated by physiologically relevant concentrations. Evidence is presented to support the action of phenolic derivatives on receptors and signalling pathways to induce adaptive responses that drive changes in endogenous antioxidant, antiplatelet, vasodilatory and anti-inflammatory effects. The implications are that in vitro antioxidant measures as predictors of polyphenol protective activity in vivo hold little relevance and that closer attention needs to be paid to bioavailable metabolites to understand the mode of action of these diet-derived components. Linked Articles This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc

Eating a high calorie meal is known to induce endothelial dysfunction and it is reported that consuming drinks rich in antioxidants may be protective against this. In this study we assessed the effects of three antioxidant drinks with considerable disparity in their antioxidant content on endothelial function. Seven apparently healthy overweight and older adults (BMI 25–35; mean age 57 ± 3 years; one male, six females) completed four trials in a randomized counterbalanced design. Water (control), orange juice, green tea, or red wine were consumed with a high calorie meal (>900 kcal). Endothelial function was measured by flow-mediated dilatation immediately before (fasted, baseline) and 2 h after the meal. Blood samples were also obtained for lipid and glucose analysis, plasma nitrite (NO2-) and oxidized low-density lipoprotein (ox-LDL). Participants returned after a minimum 3 days washout to complete the remaining arms of the study. The results found that the high calorie meal induced a substantial increase in triglycerides, but not cholesterol or glucose, at 2 h after meal ingestion. FMD was significantly reduced by ∼35% at this timepoint, but the effect was not attenuated by co-ingestion of any of the antioxidant drinks. Reduced FMD was mirrored by a reduction in NO2-, but ox-LDL was not increased at 2 h after the meal. None of the undertaken measures were influenced by the antioxidant drinks. We conclude that co-ingestion of none of our test antioxidant drinks protected against the substantial post-prandial endothelial dysfunction induced by an unhealthy meal challenge in our sample population at a 2 h timepoint.

Cardiovascular disease (CVD) is the primary cause of morbidity and mortality in patients with diabetes. Endothelial cell dysfunction is a factor in the atherosclerotic process that underpins CVD, but the link between diabetes and endothelial dysfunction is not fully understood. In this study, the effects of glucose exposure on a human-derived endothelial cell line, EA.Hy926, was examined. EA.hy926 cells were grown in two distinctive pre-treatment glucose conditions during the cell growth phase: 5.5 mM (‘normal glucose’; NG) or 25 mM glucose (‘high glucose’; HG). Cells were subsequently exposed to three different test glucose concentrations for 48 hours (5.5, 12.5 and 25 mM). The effect of the glucose exposure on cell viability, growth rate, metabolism, and cell functionality were assessed. The study showed that the glucose exposure in the growth phase (HG and NG conditions) does not affect EA.hy926 cell viability, membrane integrity or growth rate. However, EA.hy926 metabolism and mitochondrial activity were modulated by the glucose concentration in the growth phase; cells cultured in NG medium showed a more energetic profile, a greater NAD/NADH ratio and ADP/ATP ratio. Increasing the test glucose concentration was also found to reduce endothelial nitric oxide (NO) synthase-derived NO generation from NG cells, as indicated by an increase in the surrogate NO marker, nitrite, in the supernatant. In conclusion, the present study found that exposure of EA.hy926 endothelial cells to hyperglycaemic conditions induced a rapid, reversible change in metabolism. In addition, acute exposure of cells to hyperglycaemic conditions had a substantial impact on mediators that are key to endothelial modulation of vascular tone and the atherosclerotic process. The results lend weight to the hypothesis that modulation of endothelial cell metabolism and function by glucose mediates the dysfunction that underpins atherogenesis.

Introduction N-acetyl-l-cysteine (NAC) has been proposed as a prophylactic for the prevention of contrast induced nephropathy (CIN), a severe, undesirable consequence following angiography that accounts for ∼12% of all hospital-acquired acute renal injury. However, results from clinical trials of NAC in this setting to date have been inconclusive. The potential benefits of NAC in CIN have been attributed to its antioxidant potential, but NAC itself is a very weak antioxidant; intracellular conversion to the detoxifying agent, glutathione (GSH) is a pre-requisite for antioxidant benefits.1 We tested the hypothesis that neither oral nor intravenous NAC conveys antioxidant activity in the plasma of healthy volunteers or patients with chronic renal disease (CKD), but that intracellular conversion to GSH was achieved in those patients depleted of intracellular GSH. Materials and methods 8 healthy volunteers and 8 patients with chronic renal failure were recruited to separate studies conducted in parallel. Each volunteer or patient was randomised to receive NAC (i.v.; 50 mg/kg/hr infusion for 2 hour, followed by 20 mg/kg/hr infusion for a further 5 hour), or NAC (oral; 1200 mg twice daily, commencing the day before the study visit) or placebo on each of three visits to the Clinical Research Facility. On study days, volunteers were infused with inulin and PAH to facilitate a number of renal function measures.2 Plasma samples and matched buffy coat samples were obtained at intervals throughout the study for measurement of clinical parameters, together with plasma antioxidant capacity, plasma and intracellular thiol concentrations. Results Plasma NAC concentrations peaked at 187 µM (healthy volunteers) and 280 µM (CKD patients) when infused with NAC; the equivalent peak concentrations were 2 µM in both groups after oral administration of NAC. There was no equivalent increase in plasma antioxidant capacity in any of the treatment groups. Intracellular measures from buffy coat cells indicated that there was a significant increase in GSH in some healthy volunteers and some patients, but the effect was variable. In addition, there was no added benefit of increasing plasma NAC ∼10 fold through the i.v. delivery route over oral administration. Conclusion NAC is ineffective in altering antioxidant capacity in the plasma compartment, but can be converted to GSH in the intracellular environment in subjects with low intracellular GSH at baseline. Subsequent studies will determine the impact of contrast medium on both plasma and intracellular thiol concentrations and antioxidant capacity, as well as the extent of the capability of NAC to protect against the effects. Our results, however, call into question whether NAC could be effective in this setting and highlight the complexity of the concept of antioxidant capacity in vivo. This project has been funded by the EU’s INTERREG VA programme, managed by the Special EU Programmes Body (SEUPB). References Sandilands, E. A., S. Cameron, F. Paterson, S. Donaldson, L. Briody, J. Crowe, J. Donnelly, A. Thompson, N. R. Johnston, I. Mackenzie, N. Uren, J. Goddard, D. J. Webb, I. L. Megson, N. Bateman and M. Eddleston (2012). ‘Mechanisms for an effect of acetylcysteine on renal function after exposure to radio-graphic contrast material: study protocol.’ BMC Clin Pharmacol12:3. Treweeke, A. T., T. J. Winterburn, I. Mackenzie, F. Barrett, C. Barr, G. F. Rushworth, I. Dransfield, S. M. MacRury and I. L. Megson (2012). ‘N-Acetylcysteine inhibits platelet-monocyte conjugation in patients with type 2 diabetes with depleted intraplatelet glutathione: a randomised controlled trial.’Diabetologia55(11):2920–2928.

Endothelial dysfunction is key factor in atherosclerotic cardiovascular disease. Post-prandial increase in glucose, triglycerides, fatty acids, protein and insulin leads to endothelial dysfunction and an increase in intracellular reactive oxygen species generation on account of metabolic overload has been implicated. Supplementation with vitamin C has been shown to reverse the negative effects of postprandial endothelial dysfunction, lending weight to the suggestion that oxidative stress might play a crucial role in induced endothelial dysfunction. The aim of this study was to test the hypothesis that co-consumption of antioxidant-rich drinks with a high-calorie meal ameliorates post-prandial endothelial dysfunction. This study was an open-label crossover design. 7 healthy volunteers (BMI 25–35; mean age 57±3 years; 1 male, 6 females) co-ingested a high-fat and high-carbohydrate meal (∼50 g,∼35 g, respectively;≥900 kcal) with one of a range of drinks (250 ml) with different antioxidant profiles; water (control), orange juice, green tea or red wine. Endothelial function was measured immediately before (baseline) and 2 hours after the meal challenge by flow mediated dilatation (FMD) of the branchial artery. Additionally, various blood parameters including glucose, cholesterol, oxidised low density lipoprotein (oxLDL), triglycerides, ascorbate and oxygen radical antioxidant capacity (ORAC) were measured pre- and post- (2 hour) meal consumption. FMD results showed substantial decrease in endothelial function (∼−30%) 2 hour after the meal challenge. However, none of the antioxidant drinks protected against meal-induced endothelial dysfunction at this time point. Significant increase in triglycerides (≥100%, p In conclusion, none of the antioxidant drinks protected against meal-induced endothelial dysfunction. This, together with the lack of change in oxLDL concentrations or plasma antioxidant capacity, suggest that oxidative stress might not play a leading role in endothelial dysfunction at this time point and/or that co-ingesting antioxidant drinks with unhealthy meals is unlikely to prevent endothelial dysfunction in the acute phase. This study was part-funded by the BBC ‘Trust me I’m a doctor’ series. KG and DC are funded by Highlands and Islands Enterprise; DM is supported by the European Union’s INTERREG VA Programme, managed by the Special EU Programmes Body (SEUPB).

Alzheimer's disease (AD) is the most common cause of dementia worldwide, normally affecting people aged over 65. Due to the multifactorial nature of this disease, a “multi-target-directed ligands” (MTDLs) approach for the treatment of this illness has generated intense research interest in the past few years. Vanillin is a natural antioxidant and it provides a good starting point for the synthesis of new compounds with enhanced antioxidant properties, together with many biological activities, including β-amyloid peptide aggregating and acetylcholinesterase inhibiting properties. Here we report novel vanillin derivatives, bearing a tacrine or a naphthalimido moiety. All compounds exhibited improved antioxidant properties using DPPH assay, with IC(50) as low as 19.5 μM, FRAP and ORAC assays, with activities up to 1.54 and 6.4 Trolox equivalents, respectively. In addition, all compounds synthesized showed inhibitory activity toward acetylcholinesterase enzyme at μmolar concentrations using the Ellman assay. Computational docking studies of selected compounds showed interactions with both the catalytic anionic site and the peripheral anionic site of the enzyme. Furthermore, these compounds inhibited Aβ((1–42)) amyloid aggregation using the fluorometric ThT assay, with compound 4 showing comparable inhibitory activity to the positive control, curcumin. At cellular level compound 4 (1 μM) showed significant protective effects in neuroblastoma SH-SY5Y cell line when treated with hydrogen peroxide (400 μM). In our opinion, vanillin derivatives could provide a viable platform for future development of multi-targeted ligands against AD.

Background Reflexology is a widely used complementary therapy. The effects of reflexology on the cardiovascular system are not well characterised. Arterial stiffness (compliance) is a marker of vascular health. This study aimed to evaluate the effects of reflexology on arterial compliance in healthy volunteers. Methods 12 healthy volunteers (1 male; 11 female; mean age 44.8 ± 10.8 yrs) received 10 min of reflexology on each foot in a single-blind randomised study. The main outcome measures were measurements of cardiovascular parameters including heart rate, blood pressure and arterial compliance (augmentation index). Results Reflexology had no significant effect on heart rate, blood pressure or augmentation index (all p > 0.05). Conclusions In healthy volunteers, there were no consistent changes in haemodynamic parameters with a single brief reflexology treatment. Thus from a cardiovascular point of view, reflexology (as delivered) would appear to have a limited (if any) effect on the cardiovascular system.

Objectives: Acute coronary syndrome (ACS) is the major cause of mortality worldwide and caused mainly by atherosclerosis of coronary arteries. Apolipoprotein B100 (ApoB100) is a major component of low-density lipoprotein (LDL) and its oxidation can trigger inflammation in vascular endothelial cells leading to atherosclerosis. The association between antibodies to ApoB100-derived antigens and atherosclerotic diseases has been studied in recent years, but the findings appear to be controversial. The present study developed an ELISA in-house with ApoB100-derived peptide antigens to circulating anti-ApoB100 IgG antibodies in patients with ACS. Methods: Fifteen ApoB100-derived peptide antigens (Ag1–Ag15) were designed to develop an in-house ELISA for the detection of circulating anti-ApoB100 IgG levels in 350 patients with ACS and 201 control subjects amongst a Chinese population. Binary logistic regression was applied to examine the differences in anti-ApoB IgG levels between the patient group and the control group with adjustment for a number of confounding factors; the correlation between anti-ApoB100 IgG levels and clinical characteristics was also tested. Results: Patients with ACS had significantly higher levels of plasma IgG for Ag1 (adjusted P

Highlights • Anti-ApoB IgG is involved in the development of acute myocardial infarction (AMI). • We looked for biomarkers for the prediction of acute myocardial infarction. • An ELISA antibody test was developed to detect anti-ApoB IgG. • The test was used in clinical screening for anti-ApoB IgG in patients with AMI., A number of studies have reported an association between increased levels of antibodies against oxidized low-density lipoprotein (oxLDL) and cardiovascular disease, but the anti-oxLDL antibody has not been confirmed to serve as an effective biomarker for prediction of acute myocardial infarction (AMI). Apolipoprotein B100 (ApoB100)-derived peptide fragments generated by proteolytic degradation and aldehyde modification are the major antigens in oxLDL, and so the present work was undertaken to detect circulating IgG for Apo-B100-derived peptide antigens. An in-house enzyme-linked immunosorbent assay (ELISA) was developed with eight ApoB100-derived peptide antigens (Ag1–Ag8) to detect circulating anti-ApoB100 IgG levels in 267 patients with AMI and 201 control subjects. Binary logistic regression analysis revealed that circulating IgG for Ag1 was significantly higher in the patient group than the control group (P

Despite limited bioavailability and rapid degradation, dietary anthocyanins are antioxidants with cardiovascular benefits. This study tested the hypothesis that the antioxidant protection conferred by the anthocyanin, delphinidin, is mediated by modulation of endogenous antioxidant defences, driven by its degradation product, gallic acid. Delphinidin was found to degrade rapidly (t1/2 ~ 30 min), generating gallic acid as a major degradation product. Both delphinidin and gallic acid generated oxygen-centred radicals at high (100 μM) concentrationsin vitro. In a cultured human umbilical vein endothelial cell model of oxidative stress, the antioxidant protective effects of both delphinidin and gallic acid displayed a hormesic profile; 100 μM concentrations of both were cytotoxic, but relatively low concentrations (100 nM–1 μM) protected the cells and were associated with increased intracellular glutathione. We conclude that delphinidin is intrinsically unstable and unlikely to confer any direct antioxidant activityin vivoyet it offered antioxidant protection to cells at low concentrations. This paradox might be explained by the ability of the degradation product, gallic acid, to confer benefit. The findings are important in understanding the mode of protection conferred by anthocyanins and reinforce the necessity to conductin vitroexperiments at biologically relevant concentrations.

Background Evidence points to activation of pro-inflammatory and pro-thrombotic stimuli during the haemodialysis process in end-stage renal disease (ESRD) with potential to predispose to cardiovascular events. Diabetes is associated with a higher incidence of cardiovascular disease in haemodialysis patients. We tested the hypothesis that a range of mediators and markers that modulate cardiovascular risk are elevated in haemodialysis patients with diabetes compared to those without. Methods Men and women with diabetes (n = 6) and without diabetes (n = 6) aged 18–90 years receiving haemodialysis were recruited. Blood samples were collected and analysed pre- and post-haemodialysis sessions for (platelet-monocyte conjugates (PMC), oxidised LDL (Ox-LDL), endothelin 1 (ET-1) and vascular endothelial growth factor (VEGF-A). Results PMC levels significantly increased after haemodialysis in both groups (diabetes p = 0.047; non-diabetes p = 0.005). Baseline VEGF-A was significantly higher in people with diabetes (p = 0.009) and post-dialysis levels were significantly reduced in both groups (P = 0.002). Ox-LDL and CRP concentrations were not significantly different between groups nor affected in either group post-dialysis. Similarly, ET-1 concentrations were comparable in all patients at baseline, with no change post-dialysis in either group. Conclusions In this pilot study, we have confirmed that circulating PMCs are increased following dialysis irrespective of diabetes status. This is likely to be a mechanistic process and offers a potential explanation for high rates of vascular events associated with haemodialysis. The higher VEGF-A concentrations between patients with and without diabetes is a previously unreported finding in diabetic ESRD. Further research is merited to establish whether VEGF-A is a marker or mediator (or both) of cardiovascular risk in haemodialysis.

Atherothrombotic disease is a well-recognized complication of diabetes and is a major contributor to the high morbidity and mortality associated with diabetes. Although there is substantial evidence linking diabetes with cardiovascular disease, the specific effect of hyper- (or hypo-) glycaemia is less well understood. The present review focuses on the impact that glycaemic dysregulation has on respiratory function and ROS (reactive oxygen species) generation in the endothelial cells that are critical in preventing several key steps in the atherothrombotic process. Endothelial cells are particularly susceptible to ROS-mediated dysfunction not only because of reduced cell viability and increased senescence, but also because one of the major endothelium-derived factors that help to protect against atherosclerosis, nitric oxide, is rapidly deactivated by superoxide radicals.

N-acetyl-l-cysteine (NAC) has long been used therapeutically for the treatment of acetaminophen (paracetamol) overdose, acting as a precursor for the substrate (l-cysteine) in synthesis of hepatic glutathione (GSH) depleted through drug conjugation. Other therapeutic uses of NAC have also emerged, including the alleviation of clinical symptoms of cystic fibrosis through cysteine-mediated disruption of disulfide cross-bridges in the glycoprotein matrix in mucus. More recently, however, a wide range of clinical studies have reported on the use of NAC as an antioxidant, most notably in the protection against contrast-induced nephropathy and thrombosis. The results from these studies are conflicting and a consensus is yet to be reached regarding the merits or otherwise of NAC in the antioxidant setting. This review seeks to re-evaluate the mechanism of action of NAC as a precursor for GSH synthesis in the context of its activity as an "antioxidant". Results from recent studies are examined to establish whether the pre-requisites for effective NAC-induced antioxidant activity (i.e. GSH depletion and the presence of functional metabolic pathways for conversion of NAC to GSH) have received adequate consideration in the interpretation of the data. A key conclusion is a reinforcement of the concept that NAC should not be considered to be a powerful antioxidant in its own right: its strength is the targeted replenishment of GSH in deficient cells and it is likely to be ineffective in cells replete in GSH.

The neuromuscular junction (NMJ) defines the functional interface between nerve and muscle. Reactive oxygen species (ROS) are linked to pathological denervation in multiple neurodegenerative diseases, but whether and how ROS cause denervation is unclear. Our hypothesis is that ROS reiteratively repeat the signaling pathways of developmental denervation to cause pathological denervation in multiple diseases. The stage 37-38 Xenopus Laevis model allows us to “hi-jack” the systems of functional denervation and track cellular changes in vivo. To address our hypothesis, we used SPARC to induce developmental denervation. SPARC induced both denervation and increased ROS levels. MnTE-2-PyP5+ and MnTnBuOE -2-PyP5+, two antioxidants, block ROS to abolish SPARC induced denervation. Next, we used α-Latrotoxin, α-Bungarotoxin and Mito-Paraquat to mimic pathological nerve, muscle and environmental toxin induced denervation, respectively. Analogous to SPARC, each pathological denervation model increases ROS. Strikingly, MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+ block ROS to abolish pathological denervation in each of the three models. We conclude that ROS are a shared biochemical feature of pathological denervation, recapitulating an endogenous redox regulated developmental denervation pathway.

Oxidative stress, the unbalance between the occurrence of oxygen/nitrogen reactive species (ROS and RNS) and the ability of the organism to inactivate these harmful molecules is linked with several disorders such neurodegenerative diseases, cardiovascular diseases, cancer and immune disarray. In the last decades, antioxidants have been the subject of intense research for their possible use as treatment or for prevention of these pathologies. Here we report a series of new vanillin derivatives obtained through reductive amination reaction. These derivatives showed strong antioxidant properties in different antioxidant assays, such as: DPPH, FRAP and ORAC assays. In addition, they exhibited DNA protection from oxidative damage in the presence of the radical initiator AAPH. At cellular level the most promising compound protects SH-SY5Y neuroblastoma cell line from oxidative damage induced by H2O2 (400 µM) and with a mixture rotenone/oligomycin (3 and 1 mM respectively). Furthermore, it showed a decrease in ROS formation (using DCFDA as fluorescent probe), when the cells were exposed to H2O2 (1 mM) for 30 minutes.

This study investigated the impact of either type 2 diabetes or obesity, separately or in combination, on the absolute amounts of microparticles (MP) and the pathways by which these are associated with either condition. The concentrations of circulating MP derived from platelets (PMP), leukocytes (LMP) and monocytes (MMP), together with their specific activation markers, were compared in 30 subjects who were characterised across 4 cohorts as obese or type 2 diabetes. The subjects with type 2 diabetes had elevated concentrations of total PMP (P = 0.003), and PMP that were fibrinogen-positive (P = 0.04), tissue factor-positive (P

Acute renal failure (ARF) has high mortality and no effective treatment. Nitric oxide (NO) delivery represents a credible means of preventing the damaging effects of vasoconstriction, central to ARF, but design of drugs with the necessary renoselectivity is challenging. Here, we developed N-hydroxyguanidine NO donor drugs that were protected against spontaneous NO release by linkage to glutamyl adducts that could be cleaved by γ-glutamyl transpeptidase (γ-GT), found predominantly in renal tissue. Parent NO donor drug activity was optimized in advance of glutamyl adduct prodrug design. A lead compound that was a suitable substrate for γ-GT-mediated deprotection was identified. Metabolism of this prodrug to the active parent compound was confirmed in rat kidney homogenates, and the prodrug was shown to be an active vasodilator in rat isolated perfused kidneys (EC50 ~50 μM). The data confirm that glutamate protection of N-hydroxyguanidines is an approach that might hold promise in ARF.

Aims In the UK, lifestyle intervention is first-line management in Type 2 diabetes. It is unclear what type of diet is most efficacious for improving glycaemic control. This study investigated the effects of an oat-enriched diet on glycaemic control, postprandial glycaemia, inflammation and oxidative stress compared with standard dietary advice. Methods In a randomized crossover design, 27 volunteers with Type 2 diabetes, managed on diet and lifestyle only, were observed for two consecutive 8-week periods following either the oat-enriched diet or re-enforced standard dietary advice. Volunteers attended at baseline (habitual intake) and 8 and 16 weeks. Measurements included basic clinical measurements and fasted and postprandial (3-h) glucose and insulin in response to a healthy test meal. Markers of inflammation and oxidative stress, including high-sensitivity C-reactive protein, interleukin 6, interleukin 18, tumour necrosis factor-alpha, adiponectin, thiobarbituric acid reactive substances, oxygen radical antioxidant capacity, oxidized LDL and urinary isoprostanes, were also measured at fasting and in the postprandial period. Results There were no diet-related effects on glycaemic control or glycaemic or insulinaemic responses to the test meal. Total cholesterol (5.1 ± 1.0 vs. 4.9 ± 0.8 mmol/l, P = 0.019) concentrations declined following the oat-enriched diet compared with standard dietary advice. There was a postprandial decline in adiponectin concentration (P = 0.009), but no effect of dietary intervention. None of the measures of oxidative stress or inflammation were altered by the oat-enriched diet compared with standard dietary advice. Conclusion The oat-enriched diet had a modest impact on lipid lowering, but did not impact on oxidative stress or inflammation in these volunteers with Type 2 diabetes.

The beneficial health properties of dietary omega-3 polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have long been known and their metabolic dysfunction has been linked to a range of diseases including various inflammatory disorders, cardiovascular diseases, and cancer. However, the molecular mechanisms underlying their health benefits have remained unclear. Recent technological advances in lipidomic analytical strategies have resulted in the discovery of a range of bioactive mediators derived from EPA and DHA that possess potent anti-inflammatory and pro-resolving properties and that may be responsible, at least in part, for the beneficial effects observed. These mediators include resolvins, protectins and maresins, as well as EPA derivatives of classical arachidonic acid derived eicosanoids, such as prostaglandin E3 . The aim of this review is to provide an overview of the biosynthetic pathways and biological properties of these omega-3 mediators, with a particular focus on the emerging importance of the counter-regulatory role of omega-3 and -6 fatty acids in the spatial and temporal regulation of the inflammatory response. It will also provide an insight into a range of lipidomic approaches, which are currently available to analyse these fatty acids and their metabolites in biological matrices.

The Seven Countries Study suggested an association between serum cholesterol and cardiovascular disease (CVD). However, the association was not consistent across the various cohorts of participants in different countries; while it was very clear in US and Northern European cohorts, it was weak in Southern European and Japanese cohorts. Nevertheless, the study triggered research into cholesterol-lowering drug strategies, ultimately leading to the development of statins amongst others. Clinical evidence in support of statins is strong and the vast majority of the medical community advocate these drugs as highly effective first-line therapeutics in primary and secondary prevention of CVD. However, growing evidence of side-effects associated with statins in a significant proportion of patients suggests that these drugs are not a universal solution to CVD. There is a need, therefore, to revisit the evidence and to re-appraise the relative importance of cholesterol amongst many other lipids as potential modulators of atherogenesis. In this review, we assess the relative merits of statin therapy in CVD versus dietary interventions that impact on lipids other than cholesterol, including omega-3 fatty acids and polar lipid fractions of various foods (e.g. fish and olive oil). We conclude that careful design around the lipid components of dietary interventions presents a credible alternative in patients who are intolerant to statins or averse to taking such drugs.

BACKGROUND:There is no consensus and a limited evidence base for choice of contrast agents (CA) in angiography. This study evaluated the impact of iohexol and iodixanol CA on fibrinolytic factors (tissue plasminogen activator [t-PA] and plasminogen activator inhibitor-1 [PAI-1]), as well as platelet-monocyte conjugates in cardiac patients undergoing elective angiography in a double-blind, randomised parallel group study. METHODS:Patients (men, 50-70 years old; n = 12) were randomised to receive either iohexol (Omnipaque; n = 6) or iodixanol (Visipaque; n = 6) during elective angiography at Raigmore Hospital, Inverness, UK. Arterial and venous blood samples were drawn prior to CA delivery and following angiography. Assessment of platelet-monocyte conjugation, t-PA and PAI-1 antigen and activity was conducted in samples pre- and post-angiography. OUTCOME:Plasma t-PA antigen was depressed equally in the study groups after angiography, but there was a greater reduction in PAI-1 antigen in the group receiving iodixanol. These findings corresponded to a substantial reduction in t-PA activity in patients receiving iohexol, with no change in those receiving iodixanol (P = 0.023 between the CA groups). Both CAs caused a reduction in platelet-monocyte conjugation, with no difference between the groups. No adverse events were reported during the trial. CONCLUSION:Avoiding reduced plasma t-PA activity might be an important consideration in choosing iodixanol over iohexol in patients at risk of thrombosis following angiography. The trial is registered on the ISRCTN register (ISRCTN51509735) and funded by the Coronary Thrombosis Trust and National Health Service (Highland) R&D Endowments. The funders had no influence over study design or reporting. TRIAL REGISTRATION:Controlled-Trials.com ISRCTN51509735.

The authors gratefully acknowledge the funding of this work from Scottish Enterprise (POC13), the EPSRC (EP/K025112/1), the British Heart Foundation (NH/11/8/29253) and the Royal Society (Brian Mercer Award for Innovation - MI120033). Date of Acceptance: 17/01/2016 Nitric Oxide (NO) storage and release measurements have been recorded for Ni-doped CPO-27 (Mg) and CPO-27 (Zn), and the biological effect of the released NO was assessed in porcine coronary artery relaxation tests. The results indicate that the doping strategy leads to increased levels of NO storage and delivery compared to the parent materials and that NO dosage and biological response can be tuned via this approach to suit the requirements of particular applications. Publisher PDF

Schizophrenia is a complex mental disorder with unknown aetiology. Both candidate gene and genome-wide association (GWA) studies suggest that the human leukocyte antigen (HLA) system may play a part in development of the illness, but the causal HLA variant(s) remain(s) unclear. Previous studies showed that the DRB1*0101 and DRB1*13 alleles might be associated with a high risk of schizophrenia. Therefore, the present study was undertaken to test their association with the disease by genotyping seven DRB1-tagging single nucleotide polymorphisms (SNPs) in a British population. The results showed that, of the previously reported variants that were associated with schizophrenia, the DRB1*1303 allele was the only one marginally associated with a protective effect on the illness in our sample set (χ² = 4.138, P = 0.042, odds ratio (OR) = 0.42, 95 % confidence interval (CI) 0.27-0.66). Interestingly, a significant association was found for rs424232 (χ² = 9.404, P = 0.002, OR = 0.69, 95 % CI 0.54-0.88), which is a tag SNP for the DRB1*1303 allele and located near to the NOTCH4 gene that is a schizophrenia susceptibility locus confirmed by GWA studies. Analysis with the Haploview program demonstrated that rs424232 was in complete linkage disequilibrium with rs3130297 and rs3131296 present in the NOTCH4 locus. While we have failed to confirm association of the candidate alleles in the DRB1 gene with a high risk of schizophrenia, the present work suggests that the association signal detected in the HLA class II locus may extend a relatively long distance, and more work is needed in order to identify the true causal variants within this region or nearby.

Aims/hypothesis The aim of this study was to determine whether oral dosing with N-acetylcysteine (NAC) increases intraplatelet levels of the antioxidant, glutathione (GSH), and reduces platelet–monocyte conjugation in blood from patients with type 2 diabetes. Methods In this placebo-controlled randomised crossover study, the effect of oral NAC dosing on platelet–monocyte conjugation and intraplatelet GSH was investigated in patients with type 2 diabetes (eligibility criteria: men or post-menopausal women with well-controlled diabetes (HbA1c

Background Non-alcoholic fatty liver disease (NAFLD) can lead to cirrhosis and hepatocellular carcinoma and is strongly associated with obesity and insulin resistance. The aim of this study was to assess if plasma markers associated with NAFLD are increased in people with concomitant diabetes compared with those without. Methods A total of 68 participants were recruited from diabetes and liver clinics. Fatty liver disease was indicated by routine blood tests and ultrasonography. Forty-seven participants had type 2 diabetes; of them, 18 had no fatty liver disease as defined previously (DNoFLD) and 29 had fatty liver disease (DFLD); the remaining 21 had fatty liver disease but no diabetes (NonDFLD). Serum samples were analyzed for adiponectin (APN), alanine and aspartate aminotransferases and plasma for cholesterol, triglyceride, hyaluronic acid (HA), procollagen peptide III, alkaline phosphatase and fibrinogen. Results Hyaluronic acid and procollagen peptide III were significantly higher and adiponectin significantly lower in DFLD than NonDFLD and DNoFLD, the difference being particularly marked for hyaluronic acid and APN. There was no difference in these markers between NonDFLD and DNoFLD and no association between any plasma or serum marker and ultrasound grade of steatosis. Conclusion We have identified markers of hepatic steatosis that appear to be specific for people with type 2 diabetes. A further longitudinal study is merited to assess the role of these markers in understanding the progression of hepatic steatosis and fibrosis in people with and without diabetes. Copyright © 2012 John Wiley & Sons, Ltd.

A recent Cochrane review concluded that low glycaemic index (GI) diets are beneficial in glycaemic control for patients with type 2 diabetes mellitus (T2DM). There are limited UK data regarding the dietary GI in free-living adults with and without T2DM. We measured the energy and macronutrient intake and the dietary GI in a group (n = 19) of individuals with diet controlled T2DM and a group (n = 19) without diabetes, matched for age, BMI and gender. Subjects completed a three-day weighed dietary record. Patients with T2DM consumed more daily portions of wholegrains (2.3 vs. 1.1, P = 0.003), more dietary fibre (32.1 vs. 20.9 g, P < 0.001) and had a lower diet GI (53.5 vs. 57.7, P = 0.009) than subjects without T2DM. Both groups had elevated fat and salt intake and low fruit and vegetable intake, relative to current UK recommendations.CONCLUSIONS:Patients with T2DM may already consume a lower GI diet than the general population but further efforts are needed to reduce dietary GI and achieve other nutrient targets.

The inflammatory response is an integral part of the innate immune mechanism that is triggered in response to a real or perceived threat to tissue homeostasis, with a primary aim of neutralizing infectious agents and initiating repair to damaged tissue. By design, inflammation is a finite process that resolves as soon as the threat of infection abates and sufficient repair to the tissue is complete. Resolution of inflammation involves apoptosis and subsequent clearance of activated inflammatory cells – a tightly regulated event. Chronic inflammation is a characteristic feature in virtually all inflammatory diseases, including atherosclerosis, and it is becoming increasingly clear that derangement of the processes usually involved in resolution of inflammation is an underlying feature of chronic inflammatory conditions. This review will draw on evidence from a range of diseases in which dysregulated inflammation is important, with particular emphasis on cardiovascular disease.

Apoptosis of neutrophils and their subsequent phagocytosis is critical to the successful resolution of inflammation. During inflammation, activated inflammatory cells generate reactive oxygen and nitrogen species, including nitric oxide (NO) and superoxide anion (O(2)(•-)), which rapidly combine to generate peroxynitrite (ONOO(-)). NO and ONOO(-) are proapoptotic in human neutrophils. This study examines the effects of NO and ONOO(-) on caspase activation and mitochondrial permeability in human neutrophils and determines the ability of these species to evoke apoptosis in human monocyte-derived macrophages (MDMs). NO or ONOO(-) release from donor compounds was characterized by electrochemistry and electron paramagnetic resonance. Neutrophils and MDMs isolated from the peripheral blood of healthy volunteers were exposed to NO or ONOO(-) before analysis of apoptosis by caspase activation, mitochondrial permeability, and annexin V binding. Both NO and ONOO(-) induced apoptosis via rapid activation of caspases 2 and 3 in neutrophils. In contrast, only ONOO(-) promoted apoptosis in MDMs, whereas a variety of NO donors were ineffective at inducing apoptosis in this cell type. We propose that human macrophages are refractory to NO-stimulated apoptosis in order that they persist long enough within the inflammatory focus to phagocytose apoptotic neutrophils, thereby ensuring successful resolution of inflammation.

BACKGROUND: Metal oxide nanoparticles (NPs) have been widely used in industry, cosmetics, and biomedicine.OBJECTIVES: We examined hazards of several well-characterized high production volume NPs because of increasing concern about occupational exposure via inhalation.METHODS: A panel of well-characterized NPs [cerium oxide (CeO2NP), titanium dioxide (TiO2NP), carbon black (CBNP), silicon dioxide (SiO2NP), nickel oxide (NiONP), zinc oxide (ZnONP), copper oxide (CuONP), and amine-modified polystyrene beads] was instilled into lungs of rats. We evaluated the inflammation potencies of these NPs 24 hr and 4 weeks post-instillation. For NPs that caused significant inflammation at 24 hr, we then investigated the characteristics of the inflammation. All exposures were carried out at equal-surface-area doses.RESULTS: Only CeO2NP, NiONP, ZnONP, and CuONP were inflammogenic to the lungs of rats at the high doses used. Strikingly, each of these induced a unique inflammatory footprint both acutely (24 hr) and chronically (4 weeks). Acutely, patterns of neutrophil and eosinophil infiltrates differed after CeO2NP, NiONP, ZnONP, and CuONP treatment. Chronic inflammatory responses also differed after 4 weeks, with neutrophilic, neutrophilic/lymphocytic, eosinophilic/fibrotic/granulomatous, and fibrotic/granulomatous inflammation being caused respectively by CeO2NP, NiONP, ZnONP, and CuONP.CONCLUSION: Different types of inflammation imply different hazards in terms of pathology, risks, and risk severity. In vitro testing could not have differentiated these complex hazard outcomes, and this has important implications for the global strategy for NP hazard assessment. Our results demonstrate that NPs cannot be viewed as a single hazard entity and that risk assessment should be performed separately and with caution for different NPs.

Objectives A number of studies have reported a genetic association of the AKT1 gene with schizophrenia, although some have failed to replicate the AKT1 association. This study was undertaken to further explore the AKT1 association with more single nucleotide polymorphisms in a British sample. Methods A total of 221 families, consisting of 148 fathers, 204 mothers and 222 offspring affected with schizophrenia, were recruited for genetic analysis. Analysis for allelic and haplotypic associations was performed with the UNPHASED program, using likelihood-based association analysis for nuclear families with missing parental genotype data. Results Allelic association was detected at rs1130214 (chi(2)=6.28, P=0.012) and at rs11847866 (chi(2)=4.64, P=0.031), although the remaining single nucleotide polymorphisms did not show allelic association with schizophrenia. The global P value of overall associations was 0.059 after 10000 permutations. Assessment using the Haploview program revealed rs1130214, rs2494746 and rs11847866 in the same linkage disequilibrium block and haplotype analysis showed disease association for the rs1130214-rs2494746-rs11847866 haplotypes (chi(2)=10.18, d.f.=4, P=0.037), of which the T-G-A haplotype was excessively transmitted (chi(2)=6.93, uncorrected P=0.008) and this haplotypic association survived Bonferroni correction (P=0.04). Conclusion The present results provide further evidence to support the AKT1 association with schizophrenia.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • There are conflicting views in the literature as to whether cannabinoids have an impact on platelet activity and to what extent cannabinoid receptors are involved. This is an important issue to resolve because platelet effects of putative therapeutic cannabinoid inhibitors and stimulators will have an impact on their potential benefits and safety. WHAT THIS PAPER ADDS • The data presented in this manuscript clearly show that the endocannabinoid 2-arrachidonyl glycerol can activate platelet activity, but that the effects are mediated through an aspirin-sensitive pathway that is not affected by cannabinoid receptor antagonists or FAAH inhibition, but is abolished by MAGL inhibition. The findings question the role of cannabinoid receptors in platelet function and suggest that platelet function is unlikely to be directly affected by cannabinoid receptor antagonists, at least in the acute phase. AIMS Cannabinoid receptor-1 (CB1) antagonists suppress appetite and induce weight loss. Direct antagonism of CB1 receptors on platelets might be an additional benefit for CB1 antagonists, but the role of CB1 receptors in platelets is controversial. We tested the hypothesis that the endocannabinoid, 2-arachidonyl glycerol (2-AG), induces platelet aggregation by a COX-mediated mechanism rather than through CB1 receptor activation, in blood obtained from healthy volunteers and patients with coronary artery disease receiving low dose aspirin. METHODS Aggregatory responses to the cannabinoids 2-AG and Δ9-THC were examined in blood sampled from healthy volunteers (n= 8) and patients (n= 12) with coronary artery disease receiving aspirin using whole blood aggregometry. The effects of CB1 (AM251) and CB2 (AM630) antagonists, as well as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) inhibitors and aspirin on 2-AG-induced aggregation were also assessed. RESULTS AM251 (100 nm–30 µm) had no effect on platelet aggregation induced by either ADP (P= 0.90) or thrombin (P= 0.86). 2-AG, but not Δ9-THC, induced aggregation. 2-AG-induced aggregation was unaffected by AM251 and AM630 but was abolished by aspirin (P 75%, P < 0.001) in blood from patients with coronary artery disease receiving aspirin compared with that from healthy volunteers. CONCLUSIONS 2-AG-mediated activation of platelets is via metabolism to arachidonic acid by MAGL, and not through direct action on CB1 or CB2 receptors, at least in the acute phase.

Nitric oxide (NO) is important for the regulation of a number of diverse biological processes, including vascular tone, neurotransmission, inflammatory cell responsiveness, defence against invading pathogens and wound healing. Transition metal exchanged zeolites are nanoporous materials with high-capacity storage properties for gases such as NO. The NO stores are liberated upon contact with aqueous environments, thereby making them ideal candidates for use in biological and clinical settings. Here, we demonstrate the NO release capacity and powerful bactericidal properties of a novel NO-storing Zn 2+ -exchanged zeolite material at a 50 wt.% composition in a polytetrafluoroethylene polymer. Further to our published data showing the anti-thrombotic effects of a similar NO-loaded zeolite, this study demonstrates the anti-bacterial properties of NO-releasing zeolites against clinically relevant strains of bacteria, namely Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Clostridium difficile . Thus our study highlights the potential of NO-loaded zeolites as biocompatible medical device coatings with anti-infective properties.