A mechanism of self-lipid endocytosis mediated by the receptor Mincle.

Cellular lipid uptake (through endocytosis) is a basic physiological process. Dysregulation of this process underlies the pathogenesis of diseases such as atherosclerosis, obesity, diabetes, and cancer. However, to date, only some mechanisms of lipid endocytosis have been discovered. Here, we show a previously unknown mechanism of lipid cargo uptake into cells mediated by the receptor Mincle. We found that the receptor Mincle, previously shown to be a pattern recognition receptor of the innate immune system, tightly binds a range of self-lipids. Moreover, we revealed the minimal molecular motif in lipids that is sufficient for Mincle recognition. Superresolution microscopy showed that Mincle forms vesicles in cytoplasm and colocalizes with added fluorescent lipids in endothelial cells but does not colocalize with either clathrin or caveolin-1, and the added lipids were predominantly incorporated in vesicles that expressed Mincle. Using a model of ganglioside GM3 uptake in brain vessel endothelial cells, we show that the knockout of Mincle led to a dramatic decrease in lipid endocytosis. Taken together, our results have revealed a fundamental lipid endocytosis pathway, which we call Mincle-mediated endocytosis (MiME), and indicate a prospective target for the treatment of disorders of lipid metabolism, which are rapidly increasing in prevalence.

Development of a rapid method for the analysis of trenbolone, nortestosterone, and zeranol in bovine liver using liquid chromatography tandem mass spectrometry.

A rapid liquid chromatography tandem mass spectrometry method has been developed and validated for the determination of α-trenbolone, ß-trenbolone, α-nortestosterone, ß-nortestosterone, zeranol, and taleranol in bovine liver. The impact of liquid-liquid extraction with methyl tert-butyl ether and optimized solid phase extraction on silica cartridges significantly reduced effort and time of sample preparation. Electrospray ionization gives a significant signal increase compared with atmospheric pressure chemical ionization and atmospheric pressure photoionization. The HPLC gradient was optimized to separate isobaric analytes and matrix constituents from the hormone molecules. The optimized time and temperature of enzymatic hydrolysis of conjugated trenbolone was 4 h at 52 °C. The method validated in the range of 0.5-30 µg kg(-1) for α-trenbolone, ß-trenbolone, zeranol, taleranol, and 2-30 µg kg(-1) for α-nortestosterone, ß-nortestosterone. Combined uncertainty of measurements was in the range of 4%-23%. The matrix effect was negligible (1%-5%) for all analytes except of α-nortestosterone (19%). The developed method with changes concerning sample size and hydrolysis was also applied for the analysis of meat, serum, and urine samples. Graphical Abstract Determination of trenbolone, nortestosterone and zeranol in bovine liver.

Suspension cell cultures of Panax vietnamensis as a biotechnological source of ginsenosides: growth, cytology, and ginsenoside profile assessment.

Introduction: Panax vietnamensis is a valuable medicinal plant and a source of a broad spectrum of biologically active ginsenosides of different structural groups. Overexploitation and low adaptability to planation cultivation have made this species vulnerable to human pressure and prompted the development of cell cultivation in vitro as a sustainable alternative to harvesting wild plants for their bioactive components. Despite high interest in biotechnological production, little is known about the main factors affecting cell growth and ginsenoside biosynthesis of this species under in vitro conditions. In this study, the potential of cell cultures of P. vietnamensis as a biotechnological source of ginsenosides was was assessed. Methods: Six suspension cell lines that were developed from different sections of a single rhizome through a multi-step culture optimization process and maintained for over 3 years on media with different mineral salt base and varying contents of auxins and cytokinins. These cell lines were evaluated for productivity parameters and cytological characteristics. Ginsenoside profiles were assessed using a combination of the reversed-phase ultra-high-performance liquid chromatography-Orbitrap-tandem mass spectrometry (UHPLC-Orbitrap-MS/MS) and ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC-TOF-MS). Results: All lines demonstrated good growth with a specific growth rate of 0.1-0.2 day-1, economic coefficient of 0.31-0.70, productivity on dry weight (DW) of 0.30-0.83 gDW (L·day)-1, and maximum biomass accumulation varying from 10 to 22 gDW L-1. Ginsenosides of the protopanaxadiol (Rb1, Rb2/Rb3, malonyl-Rb1, and malonyl-Rb2/Rb3), oleanolic acid (R0 and chikusetsusaponin IV), and ocotillol (vinaginsenoside R1) groups and their isomers were identified in cell biomass extracts. Chikusetsusaponin IV was identified in P. vietnamensis cell culture for the first time. Discussion: These results suggest that suspension cell cultures of Vietnamese ginseng have a high potential for the biotechnological production of biomass containing ginsenosides, particularly of the oleanolic acid and ocotillol groups.