Csilla Pelyhe

Background: Extracellular vesicles (EVs) isolated from blood plasma are extensively investigated both as biomarkers and therapeutics. However, efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in-vivo experiments is a great challenge. Thus, the aim of this study was to develop an isolation method to separate the majority of EVs from major impurities such as lipoprotein particles and abundant plasma proteins albumin and fibrinogen. Methods: Rat blood samples were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30 and 10% iodixanol layers and centrifuged at 120,000×g for 24 hours. Ten fractions (F1-10) were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion-, or bind-elute chromatography. Efficiency and purity were assessed by Western blots. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5±10.4%; 48.1±5.8%; 41.9±3.8%, respectively) at a density of 1.128-1.174g/mL, where the presence of vesicles with a mean diameter of 38±2nm was confirmed by EM and DLS. Only 1.4±0.5% of LDL and chylomicron marker, 3.0±1.3% of HDL marker, and 9.9±0.4% of albumin remained in the EV-rich F6. However, 32.8±1.5% of the total fibrinogen beta was found in this fraction. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein contamination were below detection limit in EV-rich fractions. However, BEC decreased efficiency of EV isolation, and fibrinogen was still present in EV-rich fractions. Conclusion: This is the first demonstration that DGUC is able to markedly reduce the lipoprotein content of EV isolates while it separates EVs with high efficiency. Moreover, isolation of lipoprotein- and albumin-free EVs from blood plasma can be achieved by DGUC followed by BEC, however, on the expense of reduced EV yield.

BACKGROUND: Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. METHODS AND RESULTS: Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. CONCLUSION: Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.

Selenium plays important roles in antioxidant defence systems, prevents cell damage and is necessary for growth, fertility, and immune system in farm animals. Recently, nano-elemental Se has attracted wide spread attention due to its high bioavailability and low toxicity, because nanometer particulates exhibit novel characteristics, such as great specific surface area, high surface activity, a lot of surface active centres, high catalytic efficiency and strong adsorbing ability and over and above the character of low toxicity of Se0. Nano-Se had similar or higher bioavailability and much less toxicity in broiler chicken and goat as compared to selenite, while in contrast some studies suggested that nano-Se was more toxic than selenite to Medaka fish. Feeding trials have proven that nano-Se supplementation had a positive impact on the growth, feed efficiency of the rumen, fertility and on the antioxidant status of the animals. However, the bioactivity of nano-Se depends on the size, while heat treatment has measurable effect on the size, structure, and bioactivity of selenium nanoparticles. This suggests that if extensive heat exposure is unavoidable during the feed processing, smaller sized selenium nanoparticles should be used. A basic understanding of the absorption, distribution, and clearance and the whole metabolic pathways and mechanisms of nano-Se should be modelled and next to this the understand of negative effects should be investigated and demonstrated in parallel with the novel, promising characteristics as well before using it in common in the animal nutrition.

BACKGROUND AND PURPOSE: Incidence and severity of obesity are increasing worldwide, however, efficient and safe pharmacological treatments are not yet available. Certain MAO inhibitors reduce body weight, although their effects on metabolic parameters have not been investigated. Here, we have assessed effects of a widely used, selective MAO-B inhibitor, selegiline, on metabolic parameters in a rat model of diet-induced obesity. EXPERIMENTAL APPROACH: selegiline (CON + S and HFS + S) or vehicle (CON, HFS) once daily. Whole body, subcutaneous and visceral fat was measured by CT, and glucose and insulin tolerance were tested. Expression of glucose transporters and chemokines was assessed by quantitative RT-PCR. KEY RESULTS: Selegiline decreased whole body fat, subcutaneous- and visceral adiposity, measured by CT and epididymal fat weight in the HFS group, compared with HFS placebo animals, without influencing body weight. Oral glucose tolerance and insulin tolerance tests showed impaired glucose homeostasis in HFS and HFS + S groups, although insulin levels in plasma and pancreas were unchanged. HFS induced expression of Srebp-1c, Glut1 and Ccl3 in adipose tissue, which were alleviated by selegiline. CONCLUSIONS AND IMPLICATIONS: Selegiline reduced adiposity, changes in adipose tissue energy metabolism and adipose inflammation induced by HFS diet without affecting the increased body weight, impairment of glucose homeostasis, or behaviour. These results suggest that selegiline could mitigate harmful effects of visceral adiposity.

Abstract The effects of a single oral dose of 1.82 mg kg −1 bw of T-2 and HT-2 toxin (T-2), 1.75 mg kg −1 bw deoxynivalenol (DON) and 15-acetyl DON, 1.96 mg kg −1 bw fumonisin B 1 (FB 1 ) or 1.85 mg kg −1 bw ochratoxin A (OTA) were investigated in common carp juveniles on lipid peroxidation, the parameters of the glutathione redox system including the expression of their encoding genes in a short-term (24 h) experiment. Markers of the initiation phase of lipid peroxidation, conjugated dienes, and trienes, were slightly affected by DON and OTA treatment at 16-h sampling. The termination marker, malondialdehyde, concentration increased only as an effect of FB 1 . Glutathione content and glutathione peroxidase activity showed significantly higher levels in the T-2 and FB 1 groups at 8 h, and in the DON and FB 1 groups at 16 h. The expression of glutathione peroxidase genes ( gpx4a, gpx4b ) showed a dual response. Downregulation of gpxa was observed at 8 h, as the effect of DON, FB 1 , and OTA, but an upregulation in the T-2 group. At 16 h gpx4a upregulated as an effect of DON, T-2, and FB 1, and at 24 h in the DON and T-2 groups. Expression of gpx4b downregulated at 8 h, except in the T-2 group, and upregulation observed as an effect of T-2 at 24 h. The lack of an increase in the expression of nrf2, except as the effect of DON at 8 h, and a decrease in the keap1 expression suggests that the antioxidant defence system was activated at gene and protein levels through Keap1–Nrf2 independent pathways.