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Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-coenzyme A (acetyl-CoA) accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we show that β-hydroxybutyrate (β-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww and was secreted into the coronary effluent. Sodium oxamate, a lactate dehydrogenase (LDH) inhibitor, increased ischaemic β-OHB levels 5.3-fold and slowed contractile recovery. Inhibition of β-hydroxy-β-methylglutaryl (HMG)-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic β-OHB accumulation by 40%, despite increased flux through succinyl-CoA-3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.

Data supporting "β-Hydroxybutyrate Accumulates in the Rat Heart during Low-Flow Ischaemia with Implications for Functional Recovery". See README file for description.

Background Numerous pathologies result in multiple-organ failure, which is thought to be a direct consequence of compromised cellular bioenergetic status. Neither the nature of this phenotype nor its relevance to survival are well understood, limiting the efficacy of modern life-support. Methods To explore the hypothesis that survival from critical illness relates to changes in cellular bioenergetics, we combined assessment of mitochondrial respiration with metabolomic, lipidomic and redox profiling in skeletal muscle and blood, at multiple timepoints, in 21 critically ill patients and 12 reference patients. Results We demonstrate an end-organ cellular phenotype in critical illness, characterized by preserved total energetic capacity, greater coupling efficiency and selectively lower capacity for complex I and fatty acid oxidation (FAO)-supported respiration in skeletal muscle, compared to health. In survivors, complex I capacity at 48 h was 27% lower than in non-survivors (p = 0.01), but tended to increase by day 7, with no such recovery observed in non-survivors. By day 7, survivors’ FAO enzyme activity was double that of non-survivors (p = 0.048), in whom plasma triacylglycerol accumulated. Increases in both cellular oxidative stress and reductive drive were evident in early critical illness compared to health. Initially, non-survivors demonstrated greater plasma total antioxidant capacity but ultimately higher lipid peroxidation compared to survivors. These alterations were mirrored by greater levels of circulating total free thiol and nitrosated species, consistent with greater reductive stress and vascular inflammation, in non-survivors compared to survivors. In contrast, no clear differences in systemic inflammatory markers were observed between the two groups. Conclusion Critical illness is associated with rapid, specific and coordinated alterations in the cellular respiratory machinery, intermediary metabolism and redox response, with different trajectories in survivors and non-survivors. Unravelling the cellular and molecular foundation of human resilience may enable the development of more effective life-support strategies., Graphical abstract Image 1

Funder: Danish Council for Strategic Research; Grant(s): “LIFE-DNP: hyperpolarized magnetic resonance for in vivo quantification of lipid, sugar and amino acid metabolism in lifestyle related diseases”, The diabetic heart is energetically and metabolically abnormal, with increased fatty acid oxidation and decreased glucose oxidation. One factor contributing to the metabolic dysfunction in diabetes may be abnormal handling of acetyl and acyl groups by the mitochondria. L-carnitine is responsible for their transfer across the mitochondrial membrane, therefore, supplementation with L-carnitine may provide a route to improve the metabolic state of the diabetic heart. The primary aim of this study was to use hyperpolarized magnetic resonance imaging (MRI) to investigate the effects of L-carnitine supplementation on the in vivo metabolism of [1-13C]pyruvate in diabetes. Male Wistar rats were injected with either vehicle or streptozotocin (55 mg/kg) to induce type-1 diabetes. Three weeks of daily i.p. treatment with either saline or L-carnitine (3 g/kg/day) was subsequently undertaken. In vivo cardiac function and metabolism were assessed with CINE and hyperpolarized MRI, respectively. L-carnitine supplementation prevented the progression of hyperglycemia, which was observed in untreated streptozotocin injected animals and led to reductions in plasma triglyceride and ß-hydroxybutyrate concentrations. Hyperpolarized MRI revealed that L-carnitine treatment elevated pyruvate dehydrogenase flux by 3-fold in the diabetic animals, potentially through increased buffering of excess acetyl-CoA units in the mitochondria. Improved functional recovery following ischemia was also observed in the L-carnitine treated diabetic animals.

Deoxyribonucleic acid (DNA) has been shown to interact with poly(vinylidene fluoride) (PVDF), resulting in increased electroactive β-phase content and piezoelectric response under certain casting conditions. While the use of DNA as a self-poling agent has the potential to eliminate the need for additional stretching and poling steps in piezoelectric PVDF films, the mechanism through which DNA poles PVDF is not yet well understood, hindering the optimization of the process. Here, we performed a study to screen the effects of drying temperature, amount of nucleic acid additive, weight percentage of PVDF, spin-casting speed, and molecular weight of PVDF on spin-cast PVDF-DNA hybrid films. The phase composition and piezoelectric response of the resulting films were quantified using Fourier-transform infrared spectroscopy (FTIR) and a piezoelectric meter, respectively. We found that DNA has a significant influence on the PVDF films' β-phase content; however, this effect is masked at low drying temperatures. While DNA facilitates the formation of the electroactive β-phase of PVDF, we could not find evidence that DNA enhances PVDF's piezoelectric response. These results conflict with previous literature, which reported that DNA aligns the dipoles of PVDF such that the films exhibited a significant piezoelectric response. Overall, this study identifies that nucleic acid additives - under certain casting conditions - have an important effect on PVDF film phase composition.

Introduction: In a cohort of 1038 patients with pulmonary arterial hypertension (PAH) we identified 11 new mutations in ATP13A3. ATP13A3 is a poorly characterized ATPase, but recent studies suggest a role in polyamine (PA) transport. We sought to determine the impact of ATP13A3 deficiency on PA homeostasis in human pulmonary arterial endothelial cells (hPAECs). Further, we assessed if the introduction of a homolog of human disease-causing truncating mutation (P452Lfs) increased the susceptibility to PAH in a mouse model. Methods: ATP13A3 knockdown (KD) in hPAECs was achieved by siRNA transfection. P452Lfs mutant mice were generated via CRISPR-Cas9 (MRC Harwell). Echocardiography, invasive haemodynamic measurements, organ harvest were performed at 6 months of age. Polyamine content of the lungs and hPAECs was assessed by mass spectrometry. Results: ATP13A3 KD in hPAECs reduced intracellular PA content. mRNA expression of ornithine decarboxylase, rate-limiting enzyme of PA biosynthesis, was not altered, while its protein level was increased. ATP13A3 KD predisposed hPAECs to apoptosis, inhibited proliferation. P452Lfs mice developed PAH compared to wild type controls: exhibited shortened pulmonary artery acceleration time (12.1±0.8 vs 16.6±0.8ms), elevated right ventricular systolic pressure (30.2±0.5 vs 25.6±0.6mmHg) with no change in systemic pressures. Moreover, P452Lfs mice demonstrated right ventricular anterior wall thickening 0.65±0.05 vs 0.41±0.06mm and a decreased PA content in their lungs. Conclusions: Our initial findings provide further evidence that loss of function mutations in ATP13A3 are directly involved in causation of PAH and the mechanism involves alterations in cellular polyamine levels.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition, and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity and, in particular, how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid composition of cell membranes has been seldom investigated. Phospholipids (PLs) contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for membrane-associated proteins, and acting as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice which become obese either through genetic modification (ob/ob), diet (high fat diet), or a combination of the two, using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipids (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting that the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data are available at MetaboLights under the submission number: MTBLS1775.

Background: Large-scale, placebo-controlled trials established that nonsteroidal anti-inflammatory drugs confer a cardiovascular hazard: this has been attributed to depression of cardioprotective products of cyclooxygenase (COX)–2, especially prostacyclin. An alternative mechanism by which nonsteroidal anti-inflammatory drugs might constrain cardioprotection is by enhancing the formation of methylarginines in the kidney that would limit the action of nitric oxide throughout the vasculature. Methods: Targeted and untargeted metabolomics were used to investigate the effect of COX-2 deletion or inhibition in mice and in osteoarthritis patients exposed to nonsteroidal anti-inflammatory drugs on the l -arginine/nitric oxide pathway. Results: Analysis of the plasma and renal metabolome was performed in postnatal tamoxifen-inducible Cox-2 knockout mice, which exhibit normal renal function and blood pressure. This revealed no changes in arginine and methylarginines compared with their wild-type controls. Moreover, the expression of genes in the l -arginine/nitric oxide pathway was not altered in the renal medulla or cortex of tamoxifen inducible Cox-2 knockout mice. Therapeutic concentrations of the selective COX-2 inhibitors, rofecoxib, celecoxib, and parecoxib, none of which altered basal blood pressure or renal function as reflected by plasma creatinine, failed to elevate plasma arginine and methylarginines in mice. Finally, plasma arginine or methylarginines were not altered in osteoarthritis patients with confirmed exposure to nonsteroidal anti-inflammatory drugs that inhibit COX-1 and COX-2. By contrast, plasma asymmetrical dimethylarginine was increased in mice infused with angiotensin II sufficient to elevate blood pressure and impair renal function. Four weeks later, blood pressure, plasma creatinine, and asymmetrical dimethylarginine were restored to normal levels. The increase in asymmetrical dimethylarginine in response to infusion with angiotensin II in celecoxib-treated mice was also related to transient impairment of renal function. Conclusions: Plasma methylarginines are not altered by COX-2 deletion or inhibition but rather are elevated coincident with renal compromise.

This report outlines a case in which cranial gunshot trauma was suspected, but a large portion of the cranium was not recovered from the scene, so the precise nature and etiology of the fractures could not be determined with certainty. Scanning electron microscopy (SEM) coupled with energy dispersive x-ray spectroscopy (SEM-EDS) is widely accepted as a reliable method for detecting gunshot residue (GSR) on the hands of shooters and on fleshed remains, and experimental research has shown that the technique is applicable to skeletal remains. Therefore, SEM-EDS was utilized to analyze several cranial fragments and a control fragment from a thoracic vertebra for gunshot residue. The technique enables the examination of particle morphology and elemental composition of the inorganic compound. Barium, antimony, and lead were detected on four of the five cranial fragments, confirming the presence of gunshot residue. The fifth fragment had barium and lead, but no antimony. No trace elements were detected on the control fragment from the thoracic vertebra. SEM analysis of the bone surface revealed the presence of characteristic GSR spherical deposits on three of the fragments. The combined presence of signature trace elements and spherical surface deposits on the cranial fragments are consistent with the presence of gunshot residue. The cause of death was certified as disruptive head trauma, and given the circumstances of the case the manner of death was certified as homicide. The SEM-EDS analysis confirmed and supported the pathologist’s and anthropologist’s assessment of the circumstances surrounding the death. KEYWORDS: forensic anthropology, forensic science, gunshot wounds, skeletal trauma, gunshot residue, scanning electron microscopy, energy-dispersive x-ray spectroscopy

Introduction: Recently, our group identified 11 heterozygous mutations in ATP13A3 in a cohort of 1038 patients with pulmonary arterial hypertension (PAH). Of these, 6 variants were predicted to cause protein truncation. ATP13A3 is a poorly characterized P5-type ATPase, however studies in C. elegans suggest a role in polyamine transport and metabolism. Polyamines are known to be important in the regulation of basic cellular functions: cell proliferation, differentiation and apoptosis. In a genetically modified mouse, harbouring a human disease-associated truncating mutation (P452Lfs) of ATP13A3 we aimed to assess the development of PAH. Methods: P452Lfs knock-in mice were generated via CRISPR-Cas9. Wild type and mutant animals of both genders were assessed. Non-invasive echocardiography was carried out at 3 and 6 months of age. Invasive haemodynamic measurement followed by blood and organ harvesting for histological and molecular studies was performed at 6 months. Polyamine content of the lungs was assessed by mass spectrometry. Results: By 6 months male Atp13a3 mutant mice developed PAH compared to their wild type counterparts: they exhibited shortened pulmonary artery acceleration time (14.6±0.6 vs. 17.1±1.1 ms), elevated right ventricular systolic pressure (30.2±0.5 vs 25.6±0.6 mmHg) with no change in systemic pressures. In addition they are showing right ventricular dilatation as assessed by ECHO: right ventricular end-diastolic diameter of 1.86±0.17 vs 0.96±0.17 mm. In addition, male mutant mice demonstrated decreased polyamine content in their lungs. Conclusions: Our initial findings provide further evidence that loss of function mutations in ATP13A3 are directly involved in causation of PAH.

IntroductionRelative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances.13C labelled substrates have become a key tool for studying substrate use—yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle.ObjectivesWe aimed to generate a network model for the quantitative analysis of Krebs cycle intermediate isotopologue distributions measured by mass spectrometry, to determine the13C labelled proportion of acetyl-CoA entering the Krebs cycle.MethodsA model was generated, and validated ex vivo using isotopic distributions measured from isolated hearts perfused with buffer containing 11 mM glucose in total, with varying fractions of universally labelled with13C. The model was then employed to determine the relative oxidation of glucose and triacylglycerol by hearts perfused with 11 mM glucose and 0.4 mM equivalent Intralipid (a triacylglycerol mixture).ResultsThe contribution of glucose to Krebs cycle oxidation was measured to be 79.1 ± 0.9%, independent of the fraction of buffer glucose which was U-13C labelled, or of which Krebs cycle intermediate was assessed. In the presence of Intralipid, glucose and triglyceride were determined to contribute 58 ± 3.6% and 35.6 ± 0.8% of acetyl-CoA entering the Krebs cycle, respectively.ConclusionThese results demonstrate the accuracy of a functional model of Krebs cycle metabolism, which can allow quantitative determination of the effects of therapeutics and pathology on cardiac substrate metabolism.

Background Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are highly distinct disorders leading to LVH, but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. Method We undertook a detailed invasive (aortic root and coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison to non-LVH controls, to investigate cardiac fuel selection and metabolic remodelling. These patients were assessed under different physiological states (at rest and during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We then present findings from the clinical trial using perhexiline, a metabolic modulator of long-chain fatty acid β-oxidation, in patients with severe AS, and contextualise these with the up-stream metabolomic findings. Results We identified a highly discriminant metabolomic signature in severe AS characterised by striking accumulation of long-chain acylcarnitines, intermediates of long-chain transport and fatty acid metabolism, and validated this in a separate cohort. Mechanistically, we identify a down-regulation in the PPAR-α transcriptional network, including expression of genes regulating FAO. Evaluation of the symptomatic impact of perhexiline in severe AS, in contrast to HCM, revealed no significant beneficial effect, suggesting the underlying metabolic changes are attempts at metabolic adaptation. Conclusions We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate fundamental distinctions in substrate preference between AS and HCM, highlighting insufficient long-chain FAO, and the PPAR-α signalling network as a specific metabolic therapeutic target in AS.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity, and in particular how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid (PL) composition of cell membranes has been seldom investigated. PLs contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimised environment for membrane-associated proteins and as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice who become obese either through genetic modification (ob/ob), diet (high fat diet) or a combination of the two using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipid (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences.

Background The recent pandemic of obesity and the metabolic syndrome (MetS) has led to the realisation that new drug targets are needed to either reduce obesity or the subsequent pathophysiological consequences associated with excess weight gain. Certain nuclear hormone receptors (NRs) play a pivotal role in lipid and carbohydrate metabolism and have been highlighted as potential treatments for obesity. This realisation started a search for NR agonists in order to understand and successfully treat MetS and associated conditions such as insulin resistance, dyslipidaemia, hypertension, hypertriglyceridemia, obesity and cardiovascular disease. The most studied NRs for treating metabolic diseases are the peroxisome proliferator-activated receptors (PPARs), PPAR-α, PPAR-γ, and PPAR-δ. However, prolonged PPAR treatment in animal models has led to adverse side effects including increased risk of a number of cancers, but how these receptors change metabolism long term in terms of pathology, despite many beneficial effects shorter term, is not fully understood. In the current study, changes in male Sprague Dawley rat liver caused by dietary treatment with a PPAR-pan (PPAR-α, −γ, and –δ) agonist were profiled by classical toxicology (clinical chemistry) and high throughput metabolomics and lipidomics approaches using mass spectrometry. Results In order to integrate an extensive set of nine different multivariate metabolic and lipidomics datasets with classical toxicological parameters we developed a hypotheses free, data driven machine learning approach. From the data analysis, we examined how the nine datasets were able to model dose and clinical chemistry results, with the different datasets having very different information content. Conclusions We found lipidomics (Direct Infusion-Mass Spectrometry) data the most predictive for different dose responses. In addition, associations with the metabolic and lipidomic data with aspartate amino transaminase (AST), a hepatic leakage enzyme to assess organ damage, and albumin, indicative of altered liver synthetic function, were established. Furthermore, by establishing correlations and network connections between eicosanoids, phospholipids and triacylglycerols, we provide evidence that these lipids function as a key link between inflammatory processes and intermediary metabolism. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1292-2) contains supplementary material, which is available to authorized users.

We report the first example of a free-standing charged electret gate associated with a thermoelectric material. Hydrophilic polymers, the commercially available fluorocarbon polymer Novec and a cross-linked polystyrene, were charged before and after deposition of the prototypical thiophene semiconducting polymer poly(bisdodecylquaterthiophene) (PQT12), respectively. Conductivity and Seebeck coefficient were measured. The charging modulated both parameters with the expected inverse dependences on charging-induced static voltage. A tetrafluorotetracyanoquinodimethane (F4TCNQ)-doped sample and the polystyrene sample displayed Seebeck coefficients close to predictions of a general model recently proposed for polymer thermoelectrics. The freestanding gate is a possible approach to modulating polymer thermoelectrics without disrupting intermolecular packing or applying gate voltages during operation.

Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease.

A method of achieving a superhydrophobic surface based upon a highly filled polyurethane (PU) paint coating has been demonstrated through the use of a combined oxygen/argon plasma pretreatment and a fluoroalkyl silane (FAS) final treatment. The combined plasma-FAS treated PU surface has been investigated and characterised using: field emission gun secondary electron microscope (FEG-SEM); X-ray photoelectron spectroscopy (XPS); energy-dispersive X-ray spectroscopy (EDX); water contact angle analysis (WCA); atomic force microscopy (AFM), and; Fourier transform infrared spectroscopy (FTIR). It was found that the oxygen/argon plasma treatment increased both the surface roughness (Ra) and surface free energy (SFE) of the PU paint coating from approximately 60–320 nm, and, from ~52 to ~80 mN/m respectively. It was also found that the plasma process created a multiscale roughened texture through the process of differential ablation between the PU polymer and the barium sulphate solid content, which is present in the paint as an extender, and other additives. In addition, the process also imparted favourable polar groups into the PU surface from the ionised and radical oxygen species in the plasma. When the FAS coating was subsequently applied to the PU without prior plasma treatment, there was a significant increases in water contact angles. This parameter increased from approximately 60° on untreated PU to around 130° with FAS applied. In this case, the SFE decreased to ~7.5 mN/m and showed 42.0 at% fluorine present as indicated by XPS. However, subsequently applying the FAS polymer after plasma pretreatment takes advantage of the known synergistic relationship that exists between surface roughness and low surface free energy coatings. The two processes combined to create superhydrophobicity with a surface that exhibited water contact angles up to 153.1°. With this optimised process, the apparent SFE was 0.84 mN/m with a more highly fluorinated surface present. In this case 47.2 at% surface fluorine was observed by XPS. In addition to changes in SFE, plasma treatment was also observed to alter levels of surface gloss and colour. After exposure to 600 s of plasma gloss levels are shown to reduce from values of from ~50 to ~21 (GU), with small but significant corresponding increases in the lightness and yellowness of the surface.

The peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear receptors that regulate cellular homoeostasis and metabolism. PPARs control the expression of genes involved in fatty-acid and lipid metabolism. Despite evidence showing beneficial effects of their activation in the treatment of metabolic diseases, particularly dyslipidaemias and type 2 diabetes, PPAR agonists have also been associated with a variety of side effects and adverse pathological changes. Agonists have been developed that simultaneously activate the three PPAR receptors (PPARα, γ and δ) in the hope that the beneficial effects can be harnessed while avoiding some of the negative side effects. In this study, the hepatic effects of a discontinued PPAR-pan agonist (a triple agonist of PPAR-α, -γ, and -δ), was investigated after dietary treatment of male Sprague–Dawley (SD) rats. The agonist induced liver enlargement in conjunction with metabolomic and lipidomic remodelling. Increased concentrations of several metabolites related to processes of oxidation, such as oxo-methionine, methyl-cytosine and adenosyl-methionine indicated increased stress and immune status. These changes are reflected in lipidomic changes, and increased energy demands as determined by free fatty acid (decreased 18:3 n−3, 20:5 n−3 and increased ratios of n−6/n−3 fatty acids) triacylglycerol, phospholipid (decreased and increased bulk changes respectively) and eicosanoid content (increases in PGB2 and 15-deoxy PGJ2). We conclude that the investigated PPAR agonist, GW625019, induces liver enlargement, accompanied by lipidomic remodelling, oxidative stress and increases in several pro-inflammatory eicosanoids. This suggests that such pathways should be monitored in the drug development process and also outline how PPAR agonists induce liver proliferation., Highlights • The investigated PPAR-pan agonist increased oxidative damage in the rat liver. • Metabolomics can be used to follow the dose response nature of PPAR-pan agonists. • Metabolomics is a versatile tool in following oxidative stress.

This article contains mass spectrometry (MS) data investigating small molecule changes as an effect of a triple peroxisome proliferator-activated receptor (PPAR-pan) agonist GW625019 in the liver as described in the manuscript (Ament et al., 2016) [1]. Samples were measured using gas chromatography-mass spectrometry (GC–MS) for total fatty acid content, and liquid chromatography-mass spectrometry (LC–MS) to measure intact lipids, carnitines and selected aqueous metabolites and eicosanoids. Data files comprise of Excel (Microsoft, WA, USA) spreadsheets of identified metabolites and their area ratio values for total fatty acids, carnitines, aqueous metabolites, and eicosanoids where the intensity of the analytes were normalised to the intensity of the internal standard. In the case of open profiling intact lipid data, the Excel file contains area ratio values of retention time and mass to charge ratio pairs; again, the area ratio values were calculated by normalising to the intensity of the internal standard. It should be noted that several metabolic changes are potentially indirect (secondary, tertiary and ensuing changes). Keywords: Peroxisome proliferator activated receptors, PPAR, Metabolomics, Lipidomics, Mass spectrometry

This study demonstrates an energy harvester that combines a piezoelectric nanogenerator and an electret-based electrostatic generator. The device consists of an in-house fabricated nanocomposite (polydimethylsiloxane/barium titanate/carbon nanotube) as a piezoelectric layer and a monocharged Teflon fluorinated ethylene propylene as an electret electrostatic layer. The mechanical impedance of the structure can be altered easily by changing the nanocomposite monomer/cross-linker ratio and optimizing various mechanical energy sources. The energy harvester's performance was characterized by performing measurements with different frequencies (5–20 Hz) under applied dynamic loading. A total volumetric power density of ∼8.8 μW cm−3 and a total stored energy of ∼50.2 μJ min−1 were obtained. These findings indicate that this versatile, lightweight, and low-cost energy harvester can be employed as a power supply source for microelectronics in applications, such as wearables.

Polymers have a range of densities and stiffnesses that can be further tuned with the addition of dopants to control their acoustic impedance and attenuation, important properties that impact acoustic transduction in underwater, medical, and other non-airborne applications. Previous research on impedance-matched polymers is limited in scope, focusing on a specific application and varying a limited number of variables independently. Here, we perform a more generalized study to model the relationship between multiple fabrication and characterization conditions on the acoustic impedance and attenuation of biocompatible elastomers with ceramic nanoparticle dopants. Following an I-optimal design of experiments approach, fifty samples were characterized with factors of polymer type (PDMS, Ecoflex, and Polyurethane), sample thickness, dopant density, concentration, and size, and characterization frequency and temperature. Statistical analysis revealed the main variables, as well as interactions between variables, that have a significant affect on the acoustic properties of the elastomers. The resulting statistical model specifies the conditions necessary to match the polymers to any acoustic impedance in the range of 1 to 2.5 MRayls. We demonstrate how the model is used to fabricate an elastomer with an acoustic impedance matched to skin and minimum attenuation.

BACKGROUND Dietary changes have led to the growing prevalence of type 2 diabetes and nonalcoholic fatty liver disease. A hallmark of both disorders is hepatic lipid accumulation, derived in part from increased de novo lipogenesis. Despite the popularity of high-protein diets for weight loss, the effect of dietary protein on de novo lipogenesis is poorly studied. We aimed to characterize the effect of dietary protein on de novo lipid synthesis. METHODS We use a 3-way crossover interventional study in healthy males to determine the effect of high-protein feeding on de novo lipogenesis, combined with in vitro models to determine the lipogenic effects of specific amino acids. The primary outcome was a change in de novo lipogenesis–associated triglycerides in response to protein feeding. RESULTS We demonstrate that high-protein feeding, rich in glutamate, increases de novo lipogenesis–associated triglycerides in plasma (1.5-fold compared with control; P < 0.0001) and liver-derived very low-density lipoprotein particles (1.8-fold; P < 0.0001) in samples from human subjects (n = 9 per group). In hepatocytes, we show that glutamate-derived carbon is incorporated into triglycerides via palmitate. In addition, supplementation with glutamate, glutamine, and leucine, but not lysine, increased triglyceride synthesis and decreased glucose uptake. Glutamate, glutamine, and leucine increased activation of protein kinase B, suggesting that induction of de novo lipogenesis occurs via the insulin signaling cascade. CONCLUSION These findings provide mechanistic insight into how select amino acids induce de novo lipogenesis and insulin resistance, suggesting that high-protein feeding to tackle diabetes and obesity requires greater consideration. FUNDING The research was supported by UK Medical Research Council grants MR/P011705/1, MC_UP_A090_1006 and MR/P01836X/1. JLG is supported by the Imperial Biomedical Research Centre, National Institute for Health Research (NIHR)., A subset of amino acids may induce de novo lipogenesis in humans, suggesting that use of high-protein diets to tackle diabetes requires greater consideration.

BACKGROUND: Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn's disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression. OBJECTIVES: The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples. METHODS: A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis. RESULTS: Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A2 and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease. CONCLUSIONS: Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease., This study was funded by Agilent Technologies, Regione Autonoma della Sardegna (L.R.7/2007, Grant Number F71J12001180002), and the Medical Research Council UK (Grant Number MR/P011705/1).

The role of C17:0 and C15:0 in human health has recently been reinforced following a number of important biological and nutritional observations. Historically, odd chain saturated fatty acids (OCS-FAs) were used as internal standards in GC-MS methods of total fatty acids and LC-MS methods of intact lipids, as it was thought their concentrations were insignificant in humans. However, it has been thought that increased consumption of dairy products has an association with an increase in blood plasma OCS-FAs. However, there is currently no direct evidence but rather a casual association through epidemiology studies. Furthermore, a number of studies on cardiometabolic diseases have shown that plasma concentrations of OCS-FAs are associated with lower disease risk, although the mechanism responsible for this is debated. One possible mechanism for the endogenous production of OCS-FAs is α-oxidation, involving the activation, then hydroxylation of the α-carbon, followed by the removal of the terminal carboxyl group. Differentiation human adipocytes showed a distinct increase in the concentration of OCS-FAs, which was possibly caused through α-oxidation. Further evidence for an endogenous pathway, is in human plasma, where the ratio of C15:0 to C17:0 is approximately 1:2 which is contradictory to the expected levels of C15:0 to C17:0 roughly 2:1 as detected in dairy fat. We review the literature on the dietary consumption of OCS-FAs and their potential endogenous metabolism.

Summary Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5′-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling., Graphical Abstract, Highlights • An unbiased LC-MS screen reveals FAMIN as a purine nucleoside enzyme • FAMIN combines adenosine phosphorylase with ADA-, PNP-, and MTAP-like activities • FAMIN enables a purine nucleotide cycle (PNC) preventing cytoplasmic acidification • The FAMIN-dependent PNC balances the glycolysis-mitochondrial redox interface, Disease-linked, orphan FAMIN is an evolutionarily conserved, multifunctional purine nucleoside enzyme, with not only ADA-, PNP-, and MTAP-like activities but also adenosine phosphorylase activity. FAMIN enables a purine nucleotide cycle that balances the cytoplasmic-mitochondrial redox interface and prevents cytoplasmic acidification.

© The Author(s)., Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress., Authors received funding from Cancer Research UK - Senior Cancer Research Fellowship (C10701/A15181) and Cancer Research UK - PhD student stipend (C10701/A16134), and European Research Council - Consolidator Grant (m5C; 310360)

This study describes a recently developed and rapid method to measure bisphenol A (BPA), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) in bile of fish using enzymatic hydrolysis of samples followed by solid-phase extraction and ultra-performance liquid chromatography with tandem mass spectrometry. The limits of quantitation (LOQ) for BPA, EE2 and E2 were 6.3 ng mL−1, 12.5 ng mL−1 and 6.3 ng mL−1, respectively. These compounds were analyzed in bile of male English sole (Parophrys vetulus) collected from urban and non-urban sites in Puget Sound, WA, USA. The BPA and E2 concentrations (and occurrence) ranged from

Since the 1960s, the piezoelectricity in biopolymers (e.g. proteins and polynucleotides) has attracted considerable scientific attention. In particular, poly(glutamate)s have been one of the most popular targets for this research due to their well-defined helical structure and permanent polarity along the helical axis. To date, films of poly(glutamate)s have been shown to exhibit piezoelectricity only in shear mode (d14), mainly due to the limitation in fabricating electrically poled polymer samples. This paper describes a combined electrospinning and hot press method that allows production of poled poly(γ-benzyl-α,L-glutamate) (PBLG) films with piezoelectricity in all d33, d31 and d14 modes for the first time. It is found that this PBLG film belongs to the matrix structure of C∞v group, which is the same as that of poled PVDF film. The moderately high piezoelectric coefficients in both d33 and d14 modes as well as their thermal stability make the poled PBLG film an excellent candidate for use in flexible transducers and small energy harvesting devices.

The most common piezoelectric materials (PM) in use today are ceramic crystals which are heavy and brittle despite high piezoelectricity. Polymer-based PM is an alternative to ceramic crystals but they have lower piezoelectric coupling constants which deteriorates quickly at high temperature. In an effort to develop non-brittle and light PM with stable piezoelectric properties, we explored fabrication of composite materials comprising piezoelectric α-helical poly(α-amino acids), poly(γ-benzyl α, l -glutamate) (PBLG) and matrix polymer, poly(methylmethacrylate) (PMMA). Thick composite disks were created by contact charging of PBLG–MMA solution mixture followed by curing the MMA matrix in a designed mold. Compared to our prior method of corona discharge, this new method allowed the application of predefined electrical fields to the PMMA solution with little MMA evaporation. This communication presents the fabrication and characterization of a series of PBLG–PMMA composite disks with various PBLG compositions prepared under different poling conditions. The results show for the first time that all PBLGs can be poled in the direction normal to the disk surface and that the poled PBLGs within the PMMA matrix are directly responsible for the piezoelectricity of the composite materials. The two-polymer composite system allows independent modulation of film’s mechanical properties and piezoelectricity at a molecular level.

Polychlorinated biphenyl (PCB) levels and the factors affecting PCB accumulation in subadult and maturing Chinook salmon Oncorhynchus tshawytscha from Puget Sound were characterized. Specifically, we (1) determined PCB levels in Chinook salmon from Puget Sound and compared them with levels in Chinook salmon from other West Coast populations, (2) determined whether PCB accumulation mainly occurred in the freshwater or marine habitats, and (3) quantified the relative importance of fish age, fish size (fork length), lipid content, and saltwater age (the number of winters spent in saltwater) on PCB concentration. The average PCB concentration measured in skinless muscle tissue samples of subadult and maturing Chinook salmon collected from Puget Sound was 53 ng/g (wet weight), which was 3-5 times higher than those measured in six other populations of Chinook salmon on the West Coast of North America. Concentrations in the Puget Sound samples varied from 10 to 220 ng/g. A comparison of PCB body burdens...

A new class of molecular composite piezoelectric material was produced by simultaneous poling and curing of a homogeneous solution comprising poly(γ-benzyl α, l -glutamate) and methylmethacrylate via corona discharge methods. This film exhibited high piezoelectricity ( d 33 = 23 pC N −1 ), and its mechanical characteristics (modulus = 450 MPa) were similar to those of low molecular weight poly(methylmethacrylate). As it is produced via solution-based fabrication processes, the composite film is conducive to miniaturization for small sensors with integrated electronics, and could also potentially be used in piezoelectric coating applications.

The ectopic deposition of fat is thought to lead to lipotoxicity and has been associated with mitochondrial dysfunction and diabetic cardiomyopathy. We have measured mitochondrial respiratory capacities in the hearts of ob/ob and wild-type mice on either a regular chow (RCD) or high-fat (HFD) diet across four age groups to investigate the impact of diet and age on mitochondrial function alongside a comprehensive strategy for metabolic profiling of the tissue. Myocardial mitochondrial dysfunction was only evident in ob/ob mice on RCD at 14 months, but it was detectable at 3 months on the HFD. Liquid chromatography-mass spectrometry (LC-MS) was used to study the profiles of acylcarnitines and the accumulation of triglycerides, but neither class of lipid was associated with mitochondrial dysfunction. However, a targeted LC-MS/MS analysis of markers of oxidative stress demonstrated increases in GSSG/GSH and 8-oxoguanine, in addition to the accumulation of diacylglycerols, which are lipid species linked to lipotoxicity. Our results demonstrate that myocardial mitochondria in ob/ob mice on RCD maintained a similar respiratory capacity to that of wild type until a late stage in aging. However, on a HFD, unlike wild-type mice, ob/ob mice failed to increase mitochondrial respiration, which may be associated with a complex I defect following increased oxidative damage.

Background: In spite of the fact that drinking and smoking often occur together, little is known about the pharmacokinetic interaction between alcohol and nicotine. Previous research in neonatal rats demonstrated that nicotine reduces blood alcohol concentrations (BACs) if alcohol and nicotine are administered simultaneously. However, it is unclear whether such a phenomenon can be observed in adult subjects, given the fact that there is an ontogenetic difference in alcohol metabolism. Methods: A range of nicotine doses (0, 0.25, 0.5, 1, 2, 4, and 6 mg/kg) were administered individually with an alcohol dose (4 g/kg) via intragastric (IG) intubation to adult female rats, and the resultant BACs were measured at various time points following drug administration. Furthermore, the hypothesis that nicotine's role in reducing BACs is mediated through factors related to gastric function was examined by comparing the resultant BACs after an IG intubation or intraperitoneal (IP) injection of alcohol. Results: The results from this study showed significant nicotine dose–related decreases in BACs with 0.5, 1, 2, 4, and 6 mg/kg doses of nicotine at the various time points assessed. This effect, however, occurred only when alcohol was administered via IG intubation, but not after an IP injection of alcohol. Conclusions: These results suggest that the nicotine-induced decrease in BAC may be related to gastric function. One possible explanation was related to nicotine's action in delaying gastric emptying. The longer the alcohol was retained in the stomach, the more likely that the alcohol would be metabolized by gastric alcohol dehydrogenase before its absorption into the bloodstream by the small intestine (the major site of alcohol absorption).

Children with fetal alcohol syndrome (FAS) display altered performance in tasks of learning and memory, behaviours thought to be associated with the hippocampus. Altered hippocampal structure has been reported in some FAS children; therefore, a rat model system was used to determine whether the size and numbers of pyramidal cells in regions CA1 and CA3 of the hippocampal formation and granule cells in the dentate gyrus were altered by alcohol exposure during different periods of development. Rat pups were exposed to alcohol in utero during the second trimester-equivalent (E10-20), the first two trimesters-equivalent (E1-20), during the time of hippocampal pyramidal cell neurogenesis (E16-20), part of the third trimester-equivalent (P4-9), and all three trimesters-equivalent (E1-20+P4-9). Control animals (nutritional and untreated) were reared for all treatment conditions. All pups were perfused on P10. CA1 volume, pyramidal cell density, and number were reduced in pups treated with alcohol during the third trimester-equivalent, whether unique or as exposure during all three trimesters-equivalent. CA3 volume was reduced in alcohol-treated animals across all gestational ages; however, pyramidal cell density and number in this region were only reduced in animals treated with alcohol during the third trimester-equivalent. Volume of the dentate gyrus did not appear to be affected by alcohol treatment. Granule cell density and number in this region were reduced in animals treated with alcohol during the third trimester-equivalent. The third trimester-equivalent in the rat appears to be a developmental period during which the hippocampus is particularly susceptible to the effects of alcohol consumption. The resulting damage to the hippocampus may contribute to the behavioural deficits related to learning and memory noted in children with FAS.

It is important to select an appropriate model system for studies examining the mechanisms of ethanol-induced injury. The most common model systems use either mice or rats with ethanol administered by means of intragastric gavage or intraperitoneal injection, yet few studies have compared directly the blood ethanol concentration (BEC) profiles that result from each of these model systems. In the current study, Sprague-Dawley rats and C57BL/6J mice were given ethanol by means of intragastric gavage or intraperitoneal injection at 40 days of age. Blood samples were collected at consistent time intervals to determine BECs. Blood ethanol concentrations in mice were sharper, with a more rapid rise to a sharp peak BEC, followed by a relatively rapid decline. In contrast, rat BEC profiles showed an initial rapid rise, followed by a more gradual rise to peak concentrations, and, then, a relatively gradual decline. This difference was particularly evident in rats receiving ethanol intragastrically. The differences found in BEC profiles between rats and mice and between ethanol administration paradigms may yield differences in the extent or mechanism of damage induced by ethanol, an important consideration when selecting an appropriate model for the investigation of ethanol-induced tissue damage.

NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10-day-old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol-induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO-cyclic GMP pathway is essential for NMDA-mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with N(G)-nitro-L-arginine methyl ester eliminated both the NMDA-mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2'-(hydroxynitrosohydrazono) bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8-bromo-cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol-induced cell death by a mechanism(s) that involves the NO-cyclic GMP pathway.

Background Evidence from several recent metabolomic studies suggests that increased concentrations of triacylglycerols with shorter (14–16 carbon atoms), saturated fatty acids are associated with insulin resistance and the risk of type 2 diabetes. Although causality cannot be inferred from association studies, patients in whom the primary cause of insulin resistance can be genetically defined offer unique opportunities to address this challenge. Methods We compared metabolite profiles in patients with congenital lipodystrophy or loss-of-function insulin resistance (INSR gene) mutations with healthy controls. Results The absence of significant differences in triacylglycerol species in the INSR group suggest that changes previously observed in epidemiological studies are not purely a consequence of insulin resistance. The presence of triacylglycerols with lower carbon numbers and high saturation in patients with lipodystrophy suggests that these metabolite changes may be associated with primary adipose tissue dysfunction. The observed pattern of triacylglycerol species is indicative of increased de novo lipogenesis in the liver. To test this we investigated the distribution of these triacylglycerols in lipoprotein fractions using size exclusion chromatography prior to mass spectrometry. This associated these triacylglycerols with very low-density lipoprotein particles, and hence release of triacylglycerols into the blood from the liver. To test further the hepatic origin of these triacylglycerols we induced de novo lipogenesis in the mouse, comparing ob/ob and wild-type mice on a chow or high fat diet, confirming that de novo lipogenesis induced an increase in relatively shorter, more saturated fatty acids. Conclusions Overall, these studies highlight hepatic de novo lipogenesis in the pathogenesis of metabolic dyslipidaemia in states where energy intake exceeds the capacity of adipose tissue. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0179-6) contains supplementary material, which is available to authorized users.

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l(-1) NaCl (as control) or 0.7 mmol l(-1) NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.

Background : Our previous findings suggested that the intragastric coadministration of alcohol and nicotine to neonatal rats resulted in a significant decrease from the predicted peak blood alcohol concentration (BAC). We hypothesized that the coadministration of alcohol and nicotine would produce a nicotine dose–related decrease in peak BAC and a change in the BAC time curve profile. Methods: Sprague-Dawley rat pups were given alcohol and nicotine simultaneously via intragastric infusion. Two sets of nicotine doses were used in two independent studies. The low doses of nicotine were examined after the study of high doses of nicotine administration because of the possible ceiling effects from these nicotine doses on lowering BACs. Results: The results not only confirmed that the peak BAC was lowered by nicotine, but also generated new findings showing that the profile of BAC time curve was affected by these doses of nicotine. Concerns about possible ceiling effects led us to conduct another experiment to examine the effects of lower doses of nicotine on BACs. Those results showed a significant decline in BACs after cotreatment with 0.5 or 1 mg/kg nicotine and less robust changes on the BAC curve profiles. Although the nicotine dose at 0.25 mg/kg/day did not affect significantly the overall BAC profile, it did lower the peak BAC. Conclusions: Nicotine is capable of lowering the peak BAC among neonatal rat pups. Furthermore, the pattern of the BAC time curve seems to be more affected by high doses of nicotine.

Background: Prenatal alcohol exposure disrupts motor performance in affected offspring. The ventrolateral nucleus (VLN) of the thalamus functions to relay information between the cerebellum and motor cortex. Reductions in the size of the thalamus have been found in children with fetal alcohol syndrome, and therefore a rat model system was used to determine whether VLN size and neuronal number were altered by alcohol exposure during development. Methods: Rat pups were exposed to alcohol in utero during the first 10 days of gestation (first trimester equivalent), the second 10 days of gestation (second trimester equivalent), or the first two trimesters equivalent combined. Some pups were exposed to alcohol in utero during the time of VLN neurogenesis. In addition, offspring from some of the dams treated during the first two trimesters equivalent were reared artificially from postnatal day (P) 4 through P9 (part of the third trimester equivalent) and received binge-like alcohol during this time, resulting in offspring exposed to alcohol during all three trimesters equivalent. Other offspring from untreated dams were reared in the same manner but received alcohol only during the third trimester equivalent. Control animals (nutritional and untreated) were reared for all treatment conditions. All pups were perfused on P10. Results: A unique effect of alcohol treatment was not found for the VLN volume or the number of neural cells within the VLN. However, the period of VLN neurogenesis was found to be sensitive to both alcohol and nutritional control treatments, resulting in significant decreases in the VLN volume and neural cell number. Conclusions: Motor deficits seen in offspring exposed prenatally to alcohol do not seem to result from direct effects on the structure of the VLN of the thalamus.

Background: Alcohol-induced zinc deficiency is one of the mechanisms proposed as a cause of developmental brain damage associated with fetal alcohol syndrome. It is known that alcohol exposure during the brain growth spurt period leads to cerebellar Purkinje cell loss. Therefore, this study examined whether zinc supplementation was capable of preventing alcohol-induced Purkinje cell loss in the cerebellar vermis in a neonatal rat model system. Methods: Sprague-Dawley rat pups were given alcohol (EtOH; 4.5 g/kg/day), zinc (Zn; 0.54 mg/ml diet; [10 times the regular diet Zn concentration]), or both from postnatal days (PD) 4 through 9 using the artificial-rearing paradigm. A gastrostomy control (GC) and a suckle control group (SC) also were included. All pups were killed on PD 10. Following perfusion, the cerebellar vermis was dissected and processed for stereological cell counting. The total number of Purkinje cells and the volume of the cerebellar vermis were determined. Results: Alcohol produced a significant loss of Purkinje cells compared with that in the GC group (no EtOH and no Zn supplement). The zinc supplementation had no effect in attenuating alcohol-induced Purkinje cell loss in the cerebellar vermis. In fact, the serum zinc concentration data indicated higher zinc concentrations following either EtOH or Zn treatment. Interestingly, the GC group showed a significantly lower zinc concentration compared with the SC group, even though no significant difference in Purkinje cell numbers was observed between these two control groups. Conclusion: These findings indicate that alcohol exposure during the third trimester equivalent did not result in zinc deficiency in this neonatal rat model system, nor did zinc supplementation rescue the alcohol-induced Purkinje cell loss in the cerebellar vermis. These findings showed clearly that the serum zinc concentration was not correlated with Purkinje cell loss, suggesting that alcohol-induced loss of cerebellar Purkinje cells in this neonatal rat model system is independent of the availability of serum zinc.

Background: The mechanisms that mediate fetal brain injury which results from maternal alcohol consumption are not well understood. Although fetal hypoxia is a popularly proposed mechanism, it has been difficult to assess brain oxygenation in vivo. We measured intracellular high energy phosphate concentrations and estimated intracellular pH (pH i ) in brains of unanesthetized neonatal rat pups by using in vivo 31 P-NMR spectroscopy. We reasoned that decreases in brain oxygenation sufficient to result in brain injury would also reduce high energy phosphates and pH i . Methods: On postnatal day 4, before alcohol administration, pups were placed into a 20 mm diameter NMR probe, their heads were positioned carefully in the center of the 31 P detection coil, and spectra were collected over 20 min. Animals were then fed diet with or without 4.5 g/kg of ethanol in two (in succession) of 12 daily feedings via artificial rearing methods. A second spectrum was collected at 90 min after the beginning of the second alcohol feeding, at the time that coincided with the peak blood alcohol concentration (BAC). Identical feedings were performed daily until day 9, when pre- and postfeeding spectra were again obtained. Positive control groups were fed control diet and were studied in atmospheres of 5% oxygen, 95% nitrogen or 0% oxygen, 100% nitrogen. Results: Phosphocreatine (PCr), β-adenosine triphosphate (ATP), and pH i decreased and inorganic phosphate (P i ) increased in day 4 animals subjected to 0% oxygen (20 min) compared with pretreatment and all other treatment groups. Day 9 animals did not tolerate these conditions. There were no significant changes in response to 5% oxygen on day 4, but P, increased and β-ATP decreased compared with pretreatment values and compared with alcohol and control groups on day 9. There were no changes in PCr, β-ATP, or pH, in response to alcohol treatment at either age. PCr was significantly increased in the alcohol and 5% oxygen groups and apparently increased in the control group on day 9 compared with day 4, most likely due to increases in cranial muscle mass within the NMR coil. Conclusions: We conclude that acute alcohol exposure that results in peak BACs of 315 mg/dl does not alter brain high energy phosphate concentrations or pH i in neonatal rat pups, although these BACs are known to result in significant brain injury. These findings do not support hypoxia as a mechanism of alcohol-mediated brain injury during the third trimester equivalent in the rat pup model.

Introduction: Animal studies modeling fetal alcohol syndrome have demonstrated that developmental exposure to alcohol is associated with decreased brain weight and significant neuronal loss in multiple regions of the developing brain. Our previous data suggest that the Fas/Apo [apoptosis]-1 receptor is transiently expressed in the developing cerebral cortex during the peak period of naturally occurring apoptotic cell death and maximum sensitivity to alcohol. Therefore, we hypothesized that ethanol increases the expression of suicide receptors such as Fas/Apo-1 in the developing fetal cerebral cortex and leads to an upregulation or extension of the normal period of apoptosis and consequent disorganization of the neural circuitry. Methods: Ethanol was administered in one of four doses (120, 320, 630, and 950 mg/dl) to organotypic explant cultures of the developing cerebral cortex established from postnatal day 2 rats and maintained for 6 days in vitro. The number of cells expressing Fas/Apo-1 receptor mRNA was counted. Apoptosis was measured by the use of two independent assays; a cell death enzyme-linked immunosorbent assay for DNA fragmentation and flow cytometric analysis of Annexin-V binding to phosphatidylserine externalized to the outer leaflet of the plasma membrane. Necrosis was also estimated by two independent measures, the amount of lactate dehydrogenase released into culture medium and flow cytometric analysis of cells that were positive for both Annexin-V and propidium iodide. Results: A significantly larger number of developing cortical cells expressed Fas/Apo-1 mRNA at the lower doses (120 and 320 mg/dl) than at the higher doses (630 and 950 mg/dl). Furthermore, ethanol induced apoptosis in a dose-related manner, with peak apoptosis observed at a dose of 630 mg/dl in the case of DNA fragmentation and at 630 and 950 mg/dl in the case of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Ethanol did not induce necrosis at any of the administered doses of ethanol. Conclusions: Our data suggest that ethanol induces a susceptibility to apoptotic signals at low doses by upregulating the expression of mRNAs for cytotoxic receptors such as Fas/Apo-1 in the developing cerebral cortex. However, ethanol itself specifically induces apoptosis in the developing cerebral cortex only at higher doses.

To gain a better understanding into the mechanisms of damage incurred by neurons in periods following heavy alcohol exposure during development, we used an in vitro system to monitor the effects of alcohol and hypoxia on cell survival and DNA integrity. Samples representing the first few hours of exposure to alcohol and hypoxia were compared to those resulting from hypoxia alone. Measurements were taken from cell counts using Trypan blue exclusion and TUNEL assays as well as digital scans of the ethidium bromide fluorescence of genomic DNA isolated from the treated tissue. We found that DNA degradation from hypoxia was accelerated by several hours in the presence of 100 mM ethanol. This result depended on age, with adult animals (>8 months) having a similar response to 4-day postnatal animals, while the effect on 10-day postnatal animals and those of intermediate age (45 days postnatal) was increasingly delayed. Different methods of inducing the processive degradation of DNA produced laddering typical of apoptosis, a biphasic degradative process, or patterns usually associated with necrosis.

Previous research from our laboratory has shown that [ethanol (EtOH)] exposure during the brain growth spurt is detrimental to olfactory bulb development. This study extends those findings by examining the effects of EtOH, nicotine (NIC), and the combination of these drugs (EtOH/NIC) on olfactory bulb mitral cell numbers, as well as on various major neurotransmitter levels in neonatal rats. An artificial rearing paradigm was used in the present studies. These artificially reared pups were given 4 g/kg/day of EtOH and/or 6 mg/kg/day of NIC on postnatal day (PD) 4 to PD 9, except in the case of the acute neurochemistry study, in which the pups received treatment on PD 9 only. An artificially reared gastrostomy control group (GC) and a suckle control group were included. The mean total numbers of mitral cells in the EtOH and NIC groups were significantly reduced from that of the GC, as well as the volume of the left main olfactory bulb. There was no difference among any of the groups in mitral cell density. As for neurochemistry data, there was no difference in neurotransmitter levels among any of the groups in the repeat exposure regimen. There were, however, changes after the acute exposure (exposure on PD 9 only). Both serotonin and GABA levels were significantly increased only after NIC exposure. However, norepinephrine levels were significantly decreased after acute exposure in all three drug treatment groups, compared with that of the control group. Except for the GC control group, dopamine levels were not detected consistently after acute exposure to EtOH, NIC, or EtOH/NIC. Collectively, these findings demonstrate that exposure to EtOH or NIC individually during the brain growth spurt results in developmental deficits in the olfactory bulb, suggesting that both EtOH and NIC are neuroteratogens. Furthermore, this study demonstrated the capability of NIC to antagonize (protect) EtOH-induced mitral cell loss in the developing olfactory bulb.

Due to the significant species differences in the timing of different stages of brain development, to study the effects of drugs during the period equivalent to the third trimester in humans it is necessary to administer the drugs to neonatal rats, rather than in utero. In this study, we investigated the pharmacokinetic interactions between alcohol and cocaine. Such information is critical in understanding the roles of alcohol and cocaine in mediating neuroteratogenesis. Sprague–Dawley rat pups (10 days old) were given IP injections of alcohol (3.3 or 5.0 g/kg) and/or cocaine (40 mg/kg). At 20, 60, or 100 min (60 and 100 min for 3.3 g/kg alcohol only) after injections, 20 μl of tail blood was collected for blood alcohol concentration (BAC) determination. Immediately after tail blood collection, blood was collected and pooled for determinations of blood cocaine (BCC), benzoylecgonine (BBC), and cocaethylene concentration (BCEC). The slopes of the ascending BAC curves were unaffected in the presence of cocaine. BCC levels declined significantly as a function of time after the peak level at 20 min postinjection time. BCC levels were unchanged in pups receiving 3.3 g/kg alcohol, but the levels were significantly higher in 5.0 g/kg pups 20 min after injections. BBC concentrations were reduced to nearly 50% in the presence of alcohol (both doses) 20 min after injections. BCEC was detected at all time points when both alcohol and cocaine were injected. Taken together, these findings indicated that the enzymatic systems involved in converting cocaine to cocaethylene were functional at an early postnatal age, and the metabolism of cocaine was affected by the presence of alcohol in neonatal rats.

Neuronal cell loss is one of the most debilitating effects of fetal ethanol exposure. Cultures of cerebellar granule cells are a useful model to investigate ethanol neurotoxicity, because ethanol depletes cell numbers in these cultures, which also occurs in vivo. The primary goal of the present study was to identify and characterize agents that can ameliorate the ethanol-induced cell death that occurs in this culture system. Growth factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) can prevent neuronal degeneration after toxic insult in various experimental paradigms. These growth factors were investigated in the current study to determine whether or not they can mitigate ethanol-induced death of cerebellar granule cells in culture. Results indicate that NGF and bFGF significantly reduced the ethanol-induced cell loss. Both the NGF- and bFGF-mediated neuroprotection required protein and RNA synthesis, because actinomycin D (RNA synthesis inhibitor) and cycloheximide (protein synthesis inhibitor) blocked their neuroprotective effects. In addition to its neuroprotective effect, bFGF also had a neurotrophic effect and could enhance cell survival in the absence of ethanol exposure. NGF did not have a neurotrophic effect. Neither EGF nor IGF-I was neuroprotective, although the latter did have a substantial neurotrophic effect. In conclusion, bFGF and NGF have long been recognized for their role in enhancing neuronal cell survival and differentiation. This study suggests that these growth factors can also provide neuroprotection against ethanol-induced cell death.

The concurrent use of alcohol and cocaine has recently attracted attention in the medical research field due to the prevalence of this drug abuse pattern and the exclusive formation of a pharmacologically active substance, cocaethylene (CE). This is the first study to examine the neuroteratogenic effects of cocaethylene exposure during the brain growth spurt (part of the third trimester equivalent) on brain growth restrictions and neurochemical profiles. For the brain growth restrictions study, three groups of artificially reared rat pups were given daily injections of 0, 10 or 20 mg/kg cocaethylene (s.c.) from postnatal days (PDs) 4 through 9. One group of normally reared pups (suckle control) also was used. These pups were perfused on PD 10 and the brains were removed and weighed (forebrain, cerebellum and brainstem). For the neurochemistry study, five groups of artificially reared pups were used and were treated identically to those in the brain growth restrictions study, with the exceptions that animals assigned to acute cocaethylene treatment groups did not receive cocaethylene from PDs 4 through 8 and all animals in this study were sacrificed on PD 9 by decapitation. One suckle control group was included to control the possible artificial rearing effects on the neurochemical measures. Blood and fresh brain tissues (cortex, subcortical structures, cerebellum and brainstem) were collected for blood cocaethylene concentration and neurochemical analyses using GC/MS and HPLC techniques, respectively. The statistical analyses indicated that daily administration of 10 or 20 mg/kg cocaethylene, but not 0 mg/kg cocaethylene, significantly restricted the brain growth (brain weights) in all three brain regions assessed. Furthermore, cocaethylene administration from PDs 4 through 9 produced region-specific alterations in various neurotransmitter concentrations. The changes in neurotransmitter levels were not a function of the responses to the last cocaethylene injection on PD 9, since the outcomes between six days of cocaethylene treatment (PDs 4 to 9) and one day acute treatment (PD 9) were notably different. Furthermore, the artificial rearing procedure appeared to produce significant alterations in various neurotransmitter levels when compared with normally reared (suckle) controls. Collectively, these results suggest that cocaethylene is neuroteratogenic to the developing brain during the third trimester equivalent and the unique formation of cocaethylene resulting from the concurrent use of alcohol and cocaine may represent an increased risk to the developing brain beyond the intrinsic neuroteratogenic effects of cocaine and alcohol individually.

Prenatal alcohol exposure has been shown to damage the developing central nervous system (CNS) in a variety of ways, including neuroanatomical anomalies, neurochemical imbalance, and neuropharmacological dysfunction. The present study investigated one of the functional aspects of the dopaminergic system in neonatal rats exposed prenatally to a bingelike alcohol paradigm by measuring dopamine concentrations following a single postnatal cocaine challenge. Pregnant Sprague-Dawley rats were given daily intragastric intubations of 5.1 g/kg alcohol solution from embryonic day (E) 1 to 20. Pair-fed and ad lib-fed animals served as controls. On E33 (usually postnatal day 10), offspring from all groups were given injections (IP) of either 0, 20, or 40 mg/kg cocaine. Animals were sacrificed and the substantia nigra/ventral tegmental area (SN/VTA) and nucleus accumbens (NAc) were dissected for the determination of dopamine concentrations using HPLC. Basal dopamine levels (0 mg/kg cocaine group) did not alter as a function of prenatal alcohol treatment in either region. However, acute cocaine injection increased the dopamine content in NAc, but not in SN/VTA, in ad lib-fed animals, and this elevation in dopamine level was significantly attenuated by prenatal alcohol treatment in both female and male animals, and by prenatal pair-fed treatment in male animals. Taken together, these results indicate that there appears to be a regional difference in acute cocaine-induced dopamine elevation, and prenatal binge-like alcohol exposure significantly alters the functional responsiveness of dopaminergic system in NAc. Furthermore, these data suggest that male offspring may be more sensitive to stress-associated or nutritional influences during gestation.

Fast Na+ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in LIII-IV of the rat brain type IIA Na+ channel. We have analyzed macroscopic and single channel currents from Na+ channels with mutations within and adjacent to hydrophobic clusters in LIII-IV. Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in LIII-IV also affected the latency to first opening, indicating coupling between LIII-IV and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na+ channels.