Aims/hypothesis To assess thiamine status by analysis of plasma, erythrocytes and urine in type 1 and type 2 diabetic patients and links to markers of vascular dysfunction. Methods Diabetic patients (26 type 1 and 48 type 2) with and without microalbuminuria and 20 normal healthy control volunteers were recruited. Erythrocyte activity of transketolase, the concentrations of thiamine and related phosphorylated metabolites in plasma, erythrocytes and urine, and markers of metabolic control and vascular dysfunction were determined. Results Plasma thiamine concentration was decreased 76% in type 1 diabetic patients and 75% in type 2 diabetic patients: normal volunteers 64.1 (95% CI 58.5-69.7) nmol/l, type 1 diabetes 15.3 (95% CI 11.5-19.1) nmol/l, p< 0.001, and type 2 diabetes 16.3 (95% CI 13.0-9.6) nmol/l, p<0.001. Renal clearance of thiamine was increased 24-fold in type 1 diabetic patients and 16-fold in type 2 diabetic patients. Plasma thiamine concentration correlated negatively with renal clearance of thiamine (r=−0.531, p< 0.001) and fractional excretion of thiamine (r=−0.616, p< 0.001). Erythrocyte transketolase activity correlated negatively with urinary albumin excretion (r=−0.232, p<0.05). Thiamine transporter protein contents of erythrocyte membranes of type 1 and type 2 diabetic patients were increased. Plasma thiamine concentration and urinary excretion of thiamine correlated negatively with soluble vascular adhesion molecule-1 (r=−0.246, p<0.05, and −0.311, p<0.01, respectively). Conclusions/interpretation Low plasma thiamine concentration is prevalent in patients with type 1 and type 2 diabetes, associated with increased thiamine clearance. The conventional assessment of thiamine status was masked by increased thiamine transporter content of erythrocytes.

Although laser desorption mass spectrometry was introduced in the 1960s, the potential of laser mass spectrometry was not realised until the introduction of matrix-assisted laser desorption/ionisation (MALDI) in the 1980s. The technique relies on light-absorbing compounds called matrices that are co-crystallised with the analyte to achieve high ionisation and desorption efficiencies. MALDI offers a lot of advantages and is an indispensable tool in macromolecule analysis. However, the presence of the matrix also produces a high chemical background in the region below m/z 700 in the mass spectrum. Surface-assisted laser desorption/ionisation (SALDI) substitutes the chemical matrix of MALDI for an active surface, which means that matrix interference can be eliminated. SALDI mass spectrometry has evolved in recent years into a technique with great potential to provide insight into many of the challenges faced in modern research, including the growing interest in "omics" and the demands of pharmaceutical science. A great variety of materials have been reported to work in SALDI. Examples include a number of nanomaterials and surfaces. The unique properties of nanomaterials greatly facilitate analyte desorption and ionisation. This article reviews recent advances made in relation to carbon- and semiconductor-based SALDI strategies. Examples of their environmental, chemical and biomedical applications are discussed with the aim of highlighting progression in the field and the robustness of the technique, as well as to evaluate the strengths and weaknesses of individual approaches. In addition, this article describes the physical and chemical processes involved in SALDI and explains how the unique physical and electronic properties of nanostructured surfaces allow them to substitute for the matrix in energy transfer processes.

Metabolic dysfunction of endothelial cells in hyperglycemia contributes to the development of vascular complications of diabetes where increased reactive glycating agent, methylglyoxal (MG), is involved. We assessed if increased MG glycation induced proteotoxic stress, identifying related metabolic drivers and protein targets. Human aortal endothelial cells (HAECs) were incubated in high glucose concentration (20 mM versus 5 mM control) in vitro for 3–6 days. Flux of glucose metabolism, MG formation and glycation and changes in cytosolic protein abundances, MG modification and proteotoxic responses were assessed. Similar studies were performed with human microvascular endothelial HMEC-1 cells where similar outcomes were observed. HAECs exposed to high glucose concentration showed increased cellular concentration of MG (2.27 ± 0.21 versus 1.28 ± 0.03 pmol/10 6 cells, P < 0.01) and formation of MG-modified proteins (24.0 ± 3.7 versus 14.1 ± 3.2 pmol/10 6 cells/day; P < 0.001). In proteomics analysis, high glucose concentration increased proteins of the heat shock response – indicating activation of the unfolded protein response (UPR) with downstream inflammatory and pro-thrombotic responses. Proteins susceptible to MG modification were enriched in protein folding, protein synthesis, serine/threonine kinase signalling, glycolysis and gluconeogenesis. MG was increased in high glucose by increased flux of MG formation linked to increased glucose metabolism mediated by proteolytic stabilisation and increase of hexokinase-2 (HK-2); later potentiated by proteolytic down regulation of glyoxalase 1 (Glo1) - the major enzyme of MG metabolism. Silencing of Glo1, selectively increasing MG, activated the UPR similarly. Silencing of HK-2 prevented increased glucose metabolism and MG formation. trans -Resveratrol and hesperetin combination (tRES-HESP) corrected increased MG and glucose metabolism by increasing expression of Glo1 and decreasing expression of HK-2. Increased MG glycation activates the UPR in endothelial cells and thereby may contribute to endothelial cell dysfunction in diabetic vascular disease where tRES-HESP may provide effective therapy.

Epithelial to mesenchymal transition (EMT), and the reverse mesenchymal to epithelial transition (MET), are known examples of epithelial plasticity that are important in kidney development and cancer metastasis. Here we identify ASPP2, a haploinsufficient tumour suppressor, p53 activator and PAR3 binding partner, as a molecular switch of MET and EMT. ASPP2 contributes to MET in mouse kidney in vivo. Mechanistically, ASPP2 induces MET through its PAR3-binding amino-terminus, independently of p53 binding. ASPP2 prevents β-catenin from transactivating ZEB1, directly by forming an ASPP2-β-catenin-E-cadherin ternary complex and indirectly by inhibiting β-catenin's N-terminal phosphorylation to stabilize the β-catenin-E-cadherin complex. ASPP2 limits the pro-invasive property of oncogenic RAS and inhibits tumour metastasis in vivo. Reduced ASPP2 expression results in EMT, and is associated with poor survival in hepatocellular carcinoma and breast cancer patients. Hence, ASPP2 is a key regulator of epithelial plasticity that connects cell polarity to the suppression of WNT signalling, EMT and tumour metastasis.

Pakistan Higher Education Commission.

This study describes a new tool for accurate and reliable high-throughput detection of copy number variation in the human genome. We have constructed a large-insert clone DNA microarray covering the entire human genome in tiling path resolution that we have used to identify copy number variation in human populations. Crucial to this study has been the development of a robust array platform and analytic process for the automated identification of copy number variants (CNVs). The array consists of 26,574 clones covering 93.7% of euchromatic regions. Clones were selected primarily from the published "Golden Path," and mapping was confirmed by fingerprinting and BAC-end sequencing. Array performance was extensively tested by a series of validation assays. These included determining the hybridization characteristics of each individual clone on the array by chromosome-specific add-in experiments. Estimation of data reproducibility and false-positive/negative rates was carried out using self-self hybridizations, replicate experiments, and independent validations of CNVs. Based on these studies, we developed a variance-based automatic copy number detection analysis process (CNVfinder) and have demonstrated its robustness by comparison with the SW-ARRAY method.

Hyperpolarised MRI with Dynamic Nuclear Polarisation overcomes the fundamental thermodynamic limitations of conventional magnetic resonance, and is translating to human studies with several early-phase clinical trials in progress including early reports that demonstrate the utility of the technique to observe lactate production in human brain cancer patients. Owing to the fundamental coupling of metabolism and tissue function, metabolic neuroimaging with hyperpolarised [1-13C]pyruvate has the potential to be revolutionary in numerous neurological disorders (e.g. brain tumour, ischemic stroke, and multiple sclerosis). Through the use of [1-13C]pyruvate and ethyl-[1-13C]pyruvate in naïve brain, a rodent model of metastasis to the brain, or porcine brain subjected to mannitol osmotic shock, we show that pyruvate transport across the blood-brain barrier of anaesthetised animals is rate-limiting. We show through use of a well-characterised rat model of brain metastasis that the appearance of hyperpolarized [1-13C]lactate production corresponds to the point of blood-brain barrier breakdown in the disease. With the more lipophilic ethyl-[1-13C]pyruvate, we observe pyruvate production endogenously throughout the entire brain and lactate production only in the region of disease. In the in vivo porcine brain we show that mannitol shock permeabilises the blood-brain barrier sufficiently for a dramatic 90-fold increase in pyruvate transport and conversion to lactate in the brain, which is otherwise not resolvable. This suggests that earlier reports of whole-brain metabolism in anaesthetised animals may be confounded by partial volume effects and not informative enough for translational studies. Issues relating to pyruvate transport and partial volume effects must therefore be considered in pre-clinical studies investigating neuro-metabolism in anaesthetised animals, and we additionally note that these same techniques may provide a distinct biomarker of blood-brain barrier permeability in future studies.

Objective: We tested whether it is possible to differentiate relapsing-remitting (RR) from secondary progressive (SP) disease stages in patients with multiple sclerosis (MS) using a combination of nuclear magnetic resonance (NMR) metabolomics and partial least squares discriminant analysis (PLS-DA) of biofluids, which makes no assumptions on the underlying mechanisms of disease.Methods: Serum samples were obtained from patients with primary progressive MS (PPMS), SPMS, and RRMS; patients with other neurodegenerative conditions; and age-matched controls. Samples were analyzed by NMR and PLS-DA models were derived to separate disease groups.Results: The PLS-DA models for serum samples from patients with MS enabled reliable differentiation between RRMS and SPMS. This approach also identified significant differences between the metabolite profiles of each of the MS groups (PP, SP, and RR) and the healthy controls, as well as predicting disease group membership with high specificity and sensitivity.Conclusions: NMR metabolomics analysis of serum is a sensitive and robust method for differentiating between different stages of MS, yielding diagnostic markers without a priori knowledge of disease pathogenesis. Critically, this study identified and validated a type II biomarker for the RR to SP transition in patients with MS. This approach may be of considerable benefit in categorizing patients for treatment and as an outcome measure in future clinical trials. Classification of evidence:This study provides Class II evidence that serum metabolite profiles accurately distinguish patients with different subtypes and stages of MS. Neurology ® 2014;83:1492-1499 GLOSSARY AD 5 Alzheimer disease; ALS 5 amyotrophic lateral sclerosis; AUC 5 area under the curve; MS 5 multiple sclerosis; NMR 5 nuclear magnetic resonance; PLS-DA 5 partial least squares discriminant analysis; PP 5 primary progressive; ROC 5 receiver operator characteristic; RR 5 relapsing-remitting; SP 5 secondary progressive.The transition from relapsing-remitting (RR) to secondary progressive (SP) multiple sclerosis (MS) occurs subtly and is difficult to define clinically.1 Identifying biomarkers that can distinguish between the different clinical phenotypes of MS is an important goal to ensure that the appropriate treatment regimens are adopted in a timely fashion.2-4 Furthermore, such biomarkers may provide new insight into the pathologic basis for the progressive process and lead to the development of effective treatments for disability prevention. Metabolomic profiling of biofluids with high-resolution proton nuclear magnetic resonance (NMR) spectroscopy and partial least squares discriminant analysis (PLS-DA), 6 a multivariate statistical pattern recognition technique, can be used to identify metabolites that vary in a correlated fashion within individual groups. 7,8 This approach enables patterns of metabolite variation that are characteristic of a specific disease to be defined, rather than requiring identification of a unique, candidate-led biomarke...