Identification of lipid derivatives in Hep G2 cells

Metabolism of polyunsaturated fatty acids results in biosynthesis of mediators with different physiological effects. These metabolites include prostaglandins, prostacyclins, isoprostanes and others that are important signalling molecules and regulate a variety of physiological and pathophysiological processes including inflammation. Prostaglandins and isoprostanes are produced by either non-enzymatic lipid peroxidation or by enzyme-induced peroxidation (cyclooxygenases and lipoxygenases). They are used as biomarkers of oxidative stress. The aim of our study was to assess the effect of eicosapentaenoic acid (EPA) supplementation with added benzo(a)pyrene (BaP) on HepG2 cells by using a UHPLC/MS-TOF method. This rapid and simple method was developed for the identification, separation and quantification of 8-iPGF3α, PGF3α, 8-isoPGF2α and 5-iPF2α in cultured cells. The UHPLC/MS-TOF method was validated. The calculated limit of detection was in the range of 0.16-0.50 ng/mL, precision (% RSD): 1.2-2.1% and recoveries better than 88%. This method empowered qualitative and quantitative analysis of the selected individual prostaglandins derived from arachidonic acid and eicosapentaenoic acid from cell extracts

N-3 fatty acids as resolvents of inflammation in the A549 cells

Background Fatty acids and their derivatives are one of the most crucial inflammation mediators. The aim of our study was to evaluate the impact of polyunsaturated fatty acids as eicosanoids precursors on the A549 cell line. Methods Cells were incubated with 40 μM of arachidonic, eicosapentaenoic or docosahexaenoic acid for 24 h, then activated with LPS. Fatty acids content in the cell membranes were determined using gas chromatography. COX-2, cPGES and FP-receptor quantities were determined by Western blot. 8-Isoprostane F2α concentrations were determined by EIA. Maresin and protectin D1 contents were analyzed by UHPLC/MS-TOF method. Results Significant differences in membrane fatty acids and levels of 8-isoPGF2α in the activated cells were detected. Elevated expression of COX-2 and FP-receptor was observed in cells treated with AA and activated with LPS. Moreover, compared to AA and AA + LPS groups, cells incubated with EPA, DHA, EPA + LPS and DHA + LPS showed decreased expression of COX-2, cPGES and FP-receptor. In cells incubated with EPA or DHA and activated with LPS maresin and protectin D1 were detected. Conclusions The results of the study have revealed the pro-inflammatory properties of AA, while the EPA and DHA had the opposite, resolving effect. Interestingly, FP-receptor inhibition by EPA and DHA demonstrated the unique role of the FP-receptor as a potential target for antagonists, in the diseases of inflammatory character. This study provides new information about n-3 fatty acids and their pro-resolving mediators, which can be used in the process of developing new anti-inflammatory drugs

Resolvin D2 plays a protective role in RAW 264.7 Cells treated with polycyclic aromatic hydrocarbons

Eicosanoids are signaling molecules that control the immune processes and might have effects on inflammatory diseases. The aim of our study was to evaluate the effect of added resolvin D2 (RvD2), after treatment with polycyclic aromatic hydrocarbons (PAHs), on RAW 264.7 cells by using a UHPLC/MS-TOF method for the quantification of eicosanoids: 8-iPGF3α, PGF3α, 8-isoPGF2α, PGF2α and 5-iPF2α as well as cyclooxygenase 2 (COX-2), prostaglandin E synthase (cPGES) and prostaglandin F2α (FP) receptor protein expression by Western blot. The levels of PGF3α, PGF2α 8-iPGF3α 8-isoPGF2α and 5-iPF2α were decreased in RAW 264.7 cells after the exposure to PAHs and treatment with RvD2. It was observed that COX-2, cPGES and FP-receptor expression was decreased after co-treatment of the cells with PAHs and RvD2. Our findings suggest that RvD2 has anti-oxidant, anti-inflammatory and pro-resolving properties that may contribute significantly to alleviation of the harmful effects caused by PAHs in macrophages. Moreover, these results suggest that a diet rich in n-3 fatty acids might be helpful in resolving the inflammation and mitigating the effects of environmental stress in macrophages

Plant expression, lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation

Current immunisation programmes against hepatitis B virus (HBV) increasingly often involve novel tri-component vaccines containing—together with the small (S-HBsAg)—also medium and large surface antigens of HBV (M- and L-HBsAg). Plants producing all HBsAg proteins can be a source of components for a potential oral ‘triple’ anti-HBV vaccine. The objective of the presented research was to study the potential of M/L-HBsAg expression in leaf tissue and conditions of its processing for a prototype oral vaccine. Tobacco and lettuce carrying M- or L-HBsAg genes and resistant to the herbicide glufosinate were engineered and integration of the transgenes was verified by PCR and Southern hybridizations. M- and L-HBsAg expression was confirmed by Western blot and assayed by ELISA at the level of micrograms per g of fresh weight. The antigens displayed a common S domain and characteristic domains preS2 and preS1 and were assembled into virus-like particles (VLPs). Leaf tissues containing M- and L-HBsAg were lyophilised to produce a starting material of an orally administered vaccine formula. The antigens were distinctly sensitive to freeze-drying conditions and storage temperature, in the aspect of stability of S and preS domains and formation of multimeric particles. Efficiency of lyophilisation and storage depended also on the initial antigen content in plant tissue, yet M-HBsAg appeared to be approximately 1.5–2 times more stable than L-HBsAg. The results of the study provide indications concerning the preparation of two other constituents, next to S-HBsAg, for a plant-derived prototype oral tri-component vaccine against hepatitis B

Empirical Models for Surface Roughness and Topography in 5-Axis Milling Based on Analysis of Lead Angle and Curvature Radius of Sculptured Surfaces

The orientation of the tool axis and the variable curvature of the machined profile of a sculptured surface have a significant impact on the roughness and topography of the surface in the process of 5-axis milling by means of a toroidal milling cutter. The selection of the orientation of the toroidal milling cutter axis relative to the radius of curvature of the machined surface profile is very important as it can provide a better surface quality and an even distribution of roughness parameters. In this paper, an attempt to carry out model tests to obtain mathematical relationships was made. These relationships were to determine the impact of the tool axis orientation and the variable curvature radius of the machined profile on the surface roughness and its topography in the 5-axis milling process of sculptured surfaces. The tests were conducted on an example of a turbine blade made of Inconel 718 alloy. A measurable effect of the work undertaken was the development of model relationships that can be applied in specialized modules of CAM (Computer Aided Manufacturing) systems supporting the programming of 5-axis machining of sculptured surfaces. The models developed will also make it possible to obtain an evenly distributed roughness on the machined sculptured surface, especially on the surface of the turbine blades of the Inconel 718 alloy, as indicated by the results of the tests carried out

A new method of the positioning and analysis of the roughness deviation in five-axis milling of external cylindrical gear

Five-axis milling is a modern, flexible and constantly developing manufacturing process, which can be used for the machining of external cylindrical gears by means of cylindrical end mills and special disc mills on universal multi-axis machining centres. The article pre-sents a new method of positioning the tip and the axis of the end mill and the disc cutter in order to ensure a constant value of deviation of the theoretical roughness Rth along the entire length of the tooth profile. The first part presents a mathematical model of the five-axis milling process of the cylindrical gear and an algorithm for calculating the Rth deviation values. The next section describes the positioning of the end mill and the disc cutter. Then, a new method for the empirical determination of the distribution of the involute root angle Δui and the da-ta description by means of the interpolation function are presented and described. In the conducted numerical tests, the influence of the geometrical parameters of the cylindrical gear on the deviation Rth is determined, assuming a constant Rth value in the five-axis milling pro-cess