[Show abstract][Hide abstract] ABSTRACT: Materials discovery provides the opportunity to identify novel materials that are tailored to complex biological environments by using combinatorial mixing of monomers to form large libraries of polymers as micro arrays. The materials discovery approach is predicated on the use of the largest chemical diversity possible, yet previous studies into human pluripotent stem cell (hPSC) response to polymer microarrays have been limited to 20 or so different monomer identities in each study. Here we show that it is possible to print and assess cell adhesion of 141 different monomers in a microarray format. This provides access to the largest chemical space to date, allowing us to meet the regenerative medicine challenge to provide scalable synthetic culture ware. This study identifies new materials suitable for hPSC expansion that could not have been predicted from previous knowledge of cell-material interactions.
[Show abstract][Hide abstract] ABSTRACT: In this paper, our previously developed ambient LESA-MS methodology is implemented to analyze five types of thermally treated meat species, namely beef, pork, horse, chicken, and turkey meat, in order to select and identify heat-stable and species-specific peptide markers. In-solution tryptic digests of cooked meats were deposited onto a polymer surface, followed by LESA-MS analysis and evaluation using multivariate data analysis and tandem electrospray MS. The five types of cooked meat were clearly discriminated using principal component analysis and orthogonal partial least squares discriminant analysis. A number of 23 heat stable peptide markers unique to species and muscle protein were identified following data-dependent tandem LESA-MS analysis. Surface extraction and direct ambient MS analysis of mixtures of cooked meat species was performed for the first time and enabled detection of 10% (w/w) of pork, horse, and turkey meat, and 5% (w/w) of chicken meat in beef, using the developed LESA-MS/MS analysis. The study shows, for the first time, that ambient LESA-MS methodology displays specificity sufficient to be implemented effectively for the analysis of processed and complex peptide digests. The proposed approach is much faster and simpler than other measurement tools for meat speciation; it has potential for application in other areas of meat science or food production.
[Show abstract][Hide abstract] ABSTRACT: Polycystic ovary syndrome (PCOS) is a common disorder affecting between 5 and 18 % of females of reproductive age and can be diagnosed based on a combination of clinical, ultrasound and biochemical features, none of which on its own is diagnostic. A lipidomic approach using liquid chromatography coupled with accurate mass high-resolution mass-spectrometry (LC-HRMS) was used to investigate if there were any differences in plasma lipidomic profiles in women with PCOS compared with control women at different stages of menstrual cycle. Plasma samples from 40 women with PCOS and 40 controls aged between 18 and 40 years were analysed in combination with multivariate statistical analyses. Multivariate data analysis (LASSO regression and OPLS-DA) of the sample lipidomics datasets showed a weak prediction model for PCOS versus control samples from the follicular and mid-cycle phases of the menstrual cycle, but a stronger model (specificity 85 % and sensitivity 95 %) for PCOS versus the luteal phase menstrual cycle controls. The PCOS vs luteal phase model showed increased levels of plasma triglycerides and sphingomyelins and decreased levels of lysophosphatidylcholines and phosphatidylethanolamines in PCOS women compared with controls. Lipid biomarkers of PCOS were tentatively identified which may be useful in distinguishing PCOS from controls especially when performed during the menstrual cycle luteal phase.
[Show abstract][Hide abstract] ABSTRACT: We describe a novel liquid chromatography method for the simultaneous and quantitative profiling of forty-three oxylipins including eicosanoids, endocannabinoids and structurally-related bioactive lipids with modified acyl groups. The LC-MS/MS method uses switching at a defined time between negative and positive electrospray ionisation modes to achieve optimal detection sensitivity for all the lipids. The validated method is linear over a range 0.01 - 5 nmol/g (0.1 to 50 nmol/g for 2-arachidonoyl glycerol) with intra- and inter-day precision and accuracy was between 1.38 and 26.76% and 85.22 and 114.3% respectively. The method successfully quantified bioactive lipids in different tissue types in the rat, including spinal cord, dorsal root ganglia), knee joint, brain and plasma. Distinct regional differences in the pattern of lipid measured between tissue types were observed using principle component analysis (PCA). The method was applied to analyse tissue samples from an established pre-clinical rat model of osteoarthritis (OA) pain and showed that levels of 12-hydroxyeicosatetraenoic acid were significantly increased in the OA rat knee joint, compared to controls and that 15- hydroxyeicosatetraenoic acids was significantly increased in the dorsal root ganglia in model of osteoarthritis, compared to controls. The developed LC-MS/MS method has potential to provide detailed pathway profiling in tissues and biofluids where the disruption of bioactive oxylipins may be involved in disease states.
[Show abstract][Hide abstract] ABSTRACT: Current thinking, which is based mainly on rodent studies, is that physiologic doses of folic acid (pterylmonoglutamic acid), such as dietary vitamin folates, are biotransformed in the intestinal mucosa and transferred to the portal vein as the natural circulating plasma folate, 5-methyltetrahydrofolic acid (5-MTHF) before entering the liver and the wider systemic blood supply.OBJECTIVE: We tested the assumption that, in humans, folic acid is biotransformed (reduced and methylated) to 5-MTHF in the intestinal mucosa.DESIGN: We conducted a crossover study in which we sampled portal and peripheral veins for labeled folate concentrations after oral ingestion with physiologic doses of stable-isotope-labeled folic acid or the reduced folate 5-formyltetrahydrofolic acid (5-FormylTHF) in 6 subjects with a transjugular intrahepatic porto systemic shunt (TIPSS) in situ. The TIPSS allowed blood samples to be taken from the portal vein.RESULTS: Fifteen minutes after a dose of folic acid, 80 ± 12% of labeled folate in the hepatic portal vein was unmodified folic acid. In contrast, after a dose of labeled 5-FormylTHF, only 4 ± 18% of labeled folate in the portal vein was unmodified 5-FormylTHF, and the rest had been converted to 5-MTHF after 15 min (postdose).Conclusions: The human gut appears to have a very efficient capacity to convert reduced dietary folates to 5-MTHF but limited ability to reduce folic acid. Therefore, large amounts of unmodified folic acid in the portal vein are probably attributable to an extremely limited mucosal cell dihydrofolate reductase (DHFR) capacity that is necessary to produce tetrahydrofolic acid before sequential methylation to 5-MTHF. This process would suggest that humans are reliant on the liver for folic acid reduction even though it has a low and highly variable DHFR activity. Therefore, chronic liver exposure to folic acid in humans may induce saturation, which would possibly explain reports of systemic circulation of unmetabolized folic acid. This trial was registered at clinicaltrials.gov as NCT02135393.
American Journal of Clinical Nutrition 06/2014; · 6.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polymeric substrates are being identified that could permit translation of human pluripotent stem cells from laboratory-based research to industrial-scale biomedicine. Well-defined materials are required to allow cell banking and to provide the raw material for reproducible differentiation into lineages for large-scale drug-screening programs and clinical use. Yet more than 1 billion cells for each patient are needed to replace losses during heart attack, multiple sclerosis and diabetes. Producing this number of cells is challenging, and a rethink of the current predominant cell-derived substrates is needed to provide technology that can be scaled to meet the needs of millions of patients a year. In this Review, we consider the role of materials discovery, an emerging area of materials chemistry that is in large part driven by the challenges posed by biologists to materials scientists.
Nature Material 05/2014; 13(6):570-9. · 35.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alterations in the endocannabinoid system (ECS) are thought to play a role in learning and memory impairments observed in Alzheimer's disease (AD). We aimed to determine the status of the brain ECS in the AβPPswe/PS1ΔE9 model of AD. The ECS comprises the neuromodulatory lipid endocannabinoids, anandamide and 2-arachidonoyl glycerol (2AG), which interact with the G protein-coupled type-1 and type-2 cannabinoid receptors. Using mass spectrometry, we quantified endocannabinoid levels and assessed lipidomic profiles of the frontal cortex, hippocampus, and striatum of 4-8 month old wildtype and AβPPswe/PS1ΔE9 mice to determine whether regional variations in endocannabinoids and lipid metabolism are observed with age and disease progression. Additionally, open-field activity, performance in the contextual fear conditioning task, and various other tasks assessing spatial and recognition memory were examined to determine the influence of age and pathology on these parameters. At all ages, AβPPswe/PS1ΔE9 mice were significantly hyperactive in the open-field and acquired contextual fear as well as wildtype mice, reflecting intact associative learning. They, however, exhibited enhanced contextual fear memory and reduced contextual fear extinction regardless of age. Disturbances in striatal lipid metabolism were observed in 6 and 8 month old AβPPswe/PS1ΔE9 mice. Endocannabinoids increased significantly with age in the hippocampus and frontal cortex of both genotypes. 8 month old AβPPswe/PS1ΔE9 mice displayed significantly lower levels of striatal 2AG than wildtype mice, but greater cannabinoid receptor/effector coupling. This study shows that alterations in lipid metabolism and endocannabinoid signaling develop with age in AβPPswe/PS1ΔE9 mice, possibly contributing to the development of AD-like behavioral deficits.
Journal of Alzheimer's disease: JAD 05/2014; · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of ambient desorption electrospray ionization (DESI-MS) mass spectrometry and liquid extraction surface analysis mass spectrometry (LESA-MS) is explored for the first time to analyse skeletal muscle proteins obtained from mixture of standard proteins and raw meat. Single proteins and mixtures of up to five proteins (myoglobin, troponin C, actin, BSA, tropomyosin) were deposited onto a polymer surface, followed by in-situ tryptic digestion and comparative analysis using DESI-MS and LESA-MS using tandem electrospray MS. Peptide peaks specific to individual proteins were readily distinguishable with good signal-to-noise ratio in the five-component mixture. LESA-MS gave a more stable analysis and greater sensitivity compared with DESI-MS. Meat tryptic digests were subjected to peptidomics analysis by DESI-MS and LESA-MS. Bovine, horse, pig, chicken and turkey muscle digests were clearly discriminated using multivariate data analysis (MVA) of the peptidomic datasets. The most abundant skeletal muscle proteins were identified and correctly classified according to the species following MS/MS analysis. The study shows, for the first time, that ambient ionization techniques such as DESI-MS and LESA-MS have great potential for species-specific analysis and differentiation of skeletal muscle proteins by direct surface desorption.
[Show abstract][Hide abstract] ABSTRACT: Acylcarnitine accumulation in skeletal muscle and plasma has been observed in numerous models of mitochondrial lipid overload and insulin resistance. Fish oil omega-3 polyunsaturated fatty acids (n3PUFA) are thought to protect against lipid-induced insulin resistance. The present study tested the hypothesis that addition of n3PUFA to an intravenous lipid emulsion would limit muscle acylcarnitine accumulation and reduce the inhibitory effect of lipid overload on insulin action. On three occasions, six healthy young men underwent a 6-hour euglycaemic hyperinsulinaemic clamp accompanied by intravenous infusion of saline (Control), 10% Intralipid (n6PUFA), or 10% Intralipid + 10% Omegaven (2:1; n3PUFA). The decline in insulin-stimulated whole-body glucose infusion rate, muscle pyruvate dehydrogenase complex activation (PDCa), and glycogen storage associated with n6PUFA compared to Control, was prevented with n3PUFA. Muscle acetyl-CoA accumulation was greater following n6PUFA compared to Control and n3PUFA, suggesting that mitochondrial lipid overload was responsible for the lower insulin action observed. Despite these favourable metabolic effects of n3PUFA, accumulation of total muscle acylcarnitine was not attenuated when compared with n6PUFA. These findings demonstrate that n3PUFA exert beneficial effects on insulin-stimulated skeletal muscle glucose storage and oxidation independently of total acylcarnitine accumulation, which does not always reflect mitochondrial lipid overload.
[Show abstract][Hide abstract] ABSTRACT: Statins are lipid-lowering drugs widely used for prevention and treatment of cardiovascular and coronary heart diseases. These drugs are among the most commonly prescribed medicines intended for long term use. Statins are generally well tolerated. However, muscular adverse effects appear to be the most common obstacle that limits their use, resulting in poor patient compliance or even drug discontinuation. In addition, rare but potentially fatal cases of rhabdomyolysis have been reported with the use of these drugs, especially in the presence of certain risk factors. Previous reports have investigated statin-induced myotoxicity in vivo and in vitro using a number of cell lines, muscle tissues and laboratory animals, in addition to randomized clinical trials, observational studies and case reports. None of them have directly compared results from laboratory investigations with clinical observations of statin related muscular adverse effects. To the best of our knowledge this is the first review article that combines laboratory investigation with clinical aspects of statin-induced myotoxicity. By reviewing published literature of in vivo, in vitro and clinically relevant studies of statin myotoxicity, we aim at translating this important drug related problem in order to establish a clear picture of proposed mechanisms, to explain the risk factors and describe the diagnostic approaches currently employed for evaluating the degree of muscle damage induced by these agents. This review provides a baseline novel translational insight that can be used to enhance safety profile, minimize the chance of progression of these adverse effects to more severe and potentially fatal rhabdomyolysis and improve the overall patient compliance and adherence to long-term statin therapy.
[Show abstract][Hide abstract] ABSTRACT: Triterpene saponins are important bioactive constituents widely distributed in many plants. Saponins present in Caulophyllum (Berberidaceae) have not been fully characterized. In this study, we studied triterpene saponins from Caulophyllum robustum using liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-qTOF-MS). Rapid identification of Caulophyllum saponins was facilitated using low and high MS cone voltages to induce controlled fragmentation in positive mode. The full scan spectra at low cone voltage of 40V provided considerable structural information relating to aglycone skeletons, sugar types, and linked sequences for Caulophyllum saponins. Seven Caulophyllum aglycones were differentiated and identified by their diagnostic fragment ions combined with accurate mass measurements and characteristic fragmentation pathways. Peak intensity ratio of [aglycone+H-2H2O](+) to [aglycone+H-H2O](+) in full scan spectra acquired with low cone voltage is correlated with structural features of hederagenin and echinocystic acid and is useful for the discrimination of these positional isomers. However, at a high voltage of 200V, the saponin [M+H](+) ion and its fragmentation ions were not present; and the single saponin [M+Na](+) generated [Bα+Na](+) and [Y0α+Na](+) by in-source fragmentation, which provided structural information on the α- and β-sugar chains in the saponins. This approach enabled simultaneous acquisition of structural information on both aglycones and sugar chains from full scan spectra in one injection. Based on the developed strategy, 51 triterpene saponins of seven different classes were fully characterized or tentatively identified, of which 32 constituents were the first to be reported in genus Caulophyllum and 18 compounds were characterized as potentially new compounds.
Journal of Pharmaceutical and Biomedical Analysis. 01/2014; 100:109–122.
[Show abstract][Hide abstract] ABSTRACT: Biosensors are biological tools that can be used to assay bacterial cultures for quorum sensing signal molecules (QSSMs) both qualitatively and semiquantitatively. QSSMs can be extracted from Pseudomonas aeruginosa cultures using organic solvents and tentatively identified via thin layer chromatography in combination with biosensor overlays. Alternatively, QSSMs can be quantified in spent culture supernatants or solvent extracts using biosensor-based spectrophotometric, luminescence, or fluorescence assays.
[Show abstract][Hide abstract] ABSTRACT: Extracts taken from spent growth media from Pseudomonas aeruginosa can be analyzed for N-acyl-L-homoserine lactones and 2-alkyl-4-(1H)-quinolones (AQs), including the known quorum sensing signalling molecules of P. aeruginosa, in a specific and sensitive manner by liquid chromatography coupled with tandem mass spectrometric detection. This analysis can be conducted in a quantitative manner by comparison with matrix-matched calibration samples.
[Show abstract][Hide abstract] ABSTRACT: Current data indicate that there is a significant risk of endometrial cancer (EC) in women with polycystic ovarian syndrome (PCOS), although further research needed to clarify the exact molecular mechanisms. Endometrial hyperplasia is a premalignant condition that usually heralds EC and it shares identical risk factors with EC. Metabolic syndrome with a triad of obesity, hyperinsulinaemia and diabetes, which, is commonly observed in PCOS appears to be a key mechanism in EC pathogenesis. Measures to improve insulin resistance could therefore play a role in reducing the risk of EC in women with PCOS. Metformin is an insulin sensitizing agent which is safe, widely available and currently licensed for type-2 diabetes. It has been clearly shown in both animal and human studies that metformin is of value in reversing endometrial hyperplasia. Metformin may therefore prevent EC in PCOS. This article reviews the use of metformin in reducing EC risk in PCOS and makes a case for future research on this topic.