A novel UHPLC-ESI-MS/MS method and automatic calculation software for regiospecific analysis of triacylglycerols in natural fats and oils

Regioisomeric analysis of triacylglycerols (TAGs) in natural oils and fats is a highly challenging task in analytical chemistry. Here we present a software (TAG Analyzer) for automatic calculation of regioisomeric composition of TAGs based on the mass spectral data from recently reported ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC−ESI−MS/MS) method for analyzing TAG regioisomers. The software enables fast and accurate processing of complex product ion spectra containing structurally informative diacylglycerol [M+NH4−RCO2H–NH3]+ and fatty acid ketene [RCO]+ fragment ions. Compared to manual processing, the developed software offers higher throughput with faster calculation as well as more accurate interpretation of chromatographically overlapping isobaric TAGs. The software determines results by constructing a synthetic spectrum to match the measured fragment ion spectrum, and by reporting the optimal concentrations of TAGs used to create the synthetic spectrum. This type of calculation is often extremely challenging for manual interpretation of the fragment ion spectra of isobaric TAGs with shared fragments, hence the need for automated data processing. The developed software was validated by analyzing a wide range of mixtures of regiopure TAG reference compounds of known composition and a commercial olive oil sample. Additionally, the method was also applied for regiospecific analysis of TAGs in human milk as an example of natural fats and oils with a highly complex TAG profile. The results indicate that the software is capable of resolving regioisomeric composition of natural TAGs even of the most complex composition. This novel calculation software combined with our existing UHPLC-ESI-MS/MS method form a highly efficient tool for regioisomeric analysis of TAGs in natural fats and oils.</p

The SNARE Protein SNAP23 and the SNARE-Interacting Protein Munc18c in Human Skeletal Muscle Are Implicated in Insulin Resistance/Type 2 Diabetes

OBJECTIVE-Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS-We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6. RESULTS-We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS-We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23. Diabetes 59: 1870-1878, 201

β-Aminoisobutyric Acid Induces Browning of White Fat and Hepatic β-Oxidation and Is Inversely Correlated with Cardiometabolic Risk Factors

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) regulates metabolic genes in skeletal muscle and contributes to the response of muscle to exercise. Muscle PGC-1α transgenic expression and exercise both increase the expression of thermogenic genes within white adipose. How the PGC-1α-mediated response to exercise in muscle conveys signals to other tissues remains incompletely defined. We employed a metabolomic approach to examine metabolites secreted from myocytes with forced expression of PGC-1α, and identified β-aminoisobutyric acid (BAIBA) as a small molecule myokine. BAIBA increases the expression of brown adipocyte-specific genes in white adipocytes and β-oxidation in hepatocytes both in vitro and in vivo through a PPARα-mediated mechanism, induces a brown adipose-like phenotype in human pluripotent stem cells, and improves glucose homeostasis in mice. In humans, plasma BAIBA concentrations are increased with exercise and inversely associated with metabolic risk factors. BAIBA may thus contribute to exercise-induced protection from metabolic diseases

Creating sequential programs from Event-B models

The original publication is available at www.springerlink.com.International audienceEvent-B is an emerging formal method with good tool support for various kinds of system modelling. However, the control flow in Event-B consists only of non-deterministic choice of enabled events. In many applications, notably in sequential program construction, more elaborate control flow mechanisms would be convenient. This paper explores a method, based on a scheduling language, for describing the flow of control. The aim is to be able to express schedules of events; to reason about their correctness; to create and verify patterns for introducing correct control flow. The conclusion is that using patterns, it is feasible to derive efficient sequential programs from event-based specifications in many cases

The assembly of cytosolic lipid droplets and its effect on insulin sensitivity

Accumulation of neutral lipids, in particular triglycerides, in non-adipocytes is highly related to the development of insulin resistance and its consequences, type-2 diabetes and cardiovascular diseases. The accumulation of triglycerides occurs in so-called lipid droplets in the cytosol. The lipid droplet is a highly dynamic organelle consisting of a core of neutral lipids surrounded by a monolayer of amphipathic lipids and proteins. The mechanism of assembly of these droplets is poorly understood and the main aim of this thesis was to investigate this mechanism at the molecular level. Another aim was to determine the relationship between lipid storage and insulin sensitivity of the cell. In paper I, gain- and loss-of-function experiments showed that phospholipase D1 promotes the formation of lipid droplets. In addition, a cytosolic protein required for assembly of the droplets was isolated and identified as the extracellular regulated kinase 2 (ERK2). The importance of ERK2 in the formation of lipid droplets was confirmed in intact cells using gain- and loss-of-function experiments. Both PLD1 and ERK2 were shown to be necessary for the effect of insulin on lipid droplet biosynthesis. Finally, ERK2 was shown to exert its effects through phosphorylation of the motor protein dynein. Lipid droplets are formed as primordial structures with a diameter of 0.1–0.4 µm. In paper II, it was found that these primordial droplets grow in size by a fusion process that is independent of triglyceride biosynthesis. This conclusion was based on investigations in a cell-free system, on pulse-chase experiments in intact cells, and by 3D reconstructions of time-lapse studies of fluorescent droplets in intact cells. Intact microtubules and dynein were found to be essential for fusion between the droplets. The mechanism behind the fusion process was investigated further in paper III. The SNARE proteins SNAP23, VAMP4, and syntaxin5 were shown to be present on lipid droplets and to mediate their fusion. Previously described co-factors for SNARE-mediated fusion events (NSF and -SNAP) were also found to be present on droplets. It is well known that SNAP23 also mediates the insulin-stimulated fusion between transport vesicles containing the glucose transporter 4 (GLUT4) and the plasma membrane—a process that is essential for insulin-stimulated glucose uptake. Treatment of cells with oleic acid caused massive accumulation of lipid droplets, and also translocation of SNAP23 from the plasma membrane to sites within the cell, including lipid droplets. This was paralleled by an ablation of insulin-stimulated glucose uptake—an effect that was totally reversed by overexpression of SNAP23. Thus, SNAP23 may be a molecular link between insulin resistance and neutral lipid storage

Community Management - The Role Community Managers Play in the Video Game Industry in Sweden

The video game industry has developed into a multibillion dollar industry. As the rise of social media and other online networks has enabled consumers to more easily express their opinions and critique about the products, these platforms become important when it comes to obtaining customer knowledge. The responsibilities of a community manager includes to obtain this knowledge and share it further with the game developers. Hence, a shared effort between the firm and the online communities of the firm can help create value for the customers in the long run. This study aims to gain a deeper understanding of the community manager's role within the process of transferring information from the communities into knowledge for the company, and also to explore how it differs in different sized companies. Theory regarding knowledge transfer and conversion, as well as customer knowledge management theory have be covered and in addition also theories on online communities. To achieve the aim of the study a qualitative and exploratory research was undertaken by selecting four case companies. Gathering the empirical findings, it became clear that one company had the most interesting community out of the four cases and therefore it became the main focus in the analysis. A community manager’s role is different depending on the company and its size. Larger firms are more concerned with a social media presence and smaller firms are more concerned with maintaining close relationships. Reaching a conclusion, it was clear that the community manager at Expansive Worlds, had several different roles within the process of knowledge transfer, in terms of acquiring, combining, and sharing knowledge.

Modular Verification of Finite Blocking in Non-terminating Programs

Most multi-threaded programs synchronize threads via blocking operations such as acquiring locks or joining other threads. An important correctness property of such programs is for each thread to make progress, that is, not to be blocked forever. For programs in which all threads terminate, progress essentially follows from deadlock freedom. However, for the common case that a program contains non-terminating threads such as servers or actors, deadlock freedom is not sufficient. For instance, a thread may be blocked forever by a non-terminating thread if it attempts to join that thread or to acquire a lock held by that thread. In this paper, we present a verification technique for finite blocking in non-terminating programs. The key idea is to track explicitly whether a thread has an obligation to perform an operation that unblocks another thread, for instance, an obligation to release a lock or to terminate. Each obligation is associated with a measure to ensure that it is fulfilled within finitely many steps. Obligations may be used in specifications, which makes verification modular. We formalize our technique via an encoding into Boogie, which treats different kinds of obligations uniformly. It subsumes termination checking as a special case.ISSN:1868-896