The Ceramide Synthase Subunit Lac1 Regulates Cell Growth and Size in Fission Yeast

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Advances in Lipid Extraction Methods—A Review

Extraction of lipids from biological tissues is a crucial step in lipid analysis. The selection of appropriate solvent is the most critical factor in the efficient extraction of lipids. A mixture of polar (to disrupt the protein-lipid complexes) and nonpolar (to dissolve the neutral lipids) solvents are precisely selected to extract lipids efficiently. In addition, the disintegration of complex and rigid cell-wall of plants, fungi, and microalgal cells by various mechanical, chemical, and enzymatic treatments facilitate the solvent penetration and extraction of lipids. This review discusses the chloroform/methanol-based classical lipid extraction methods and modern modifications of these methods in terms of using healthy and environmentally safe solvents and rapid single-step extraction. At the same time, some adaptations were made to recover the specific lipids. In addition, the high throughput lipid extraction methodologies used for liquid chromatography-mass spectrometry (LC-MS)-based plant and animal lipidomics were discussed. The advantages and disadvantages of various pretreatments and extraction methods were also illustrated. Moreover, the emerging green solvents-based lipid extraction method, including supercritical CO2 extraction (SCE), is also discussed.

Alterations in health-related fatty acids in buffalo milk after processing to traditional dairy products

Milk provides some beneficial fatty acids which in dairy processing are subjected to pasteurization and fermentation. With the aim to assess such changes, aliquot parts of milk from 12 buffaloes were pooled and processed to germinated yoghurt and brined cheese, and to non-germinated curd – the respective samples of raw and dairy material subjected to lipid analysis. The results show that in cheese positive and negative changes are generally balanced, rumenic acid decreasing and other CLAs altered but not total CLA and PUFA; omega ratio and atherogenicity index worsened to little extent, due to adverse change in n-3, myristic and lauric acid. In yoghurt and curd CLA dramatically decreased, excluding rumenic acid; but vaccenic acid increased, though total trans isomers decreased; the worsened n-6/n-3 ratio and atherogenicity index is mostly because of the adverse effect on PUFAn-3 but also on myristic and lauric acid. In all products SFA and MUFA did not change, including palmitic, stearic, and oleic acid. It can be concluded that the decrease of CLA in yoghurt and curd is partially compensated by the increase in the vaccenic acid, while cheese making altered individual isomers but not groups of beneficial acids.

A new phenotypic classification system for dyslipidemias based on the standard lipid panel

Background Dyslipoproteinemias can be classified by their distinct lipoprotein patterns, which helps determine atherosclerotic cardiovascular disease (ASCVD) risk and directs lipid management but this has required advanced laboratory testing. Objective To develop a new algorithm for classifying lipoprotein disorders that only relies on the standard lipid panel. Methods Lipid thresholds for defining the different lipoprotein phenotypes were derived for Non-High-Density Lipoprotein-Cholesterol (NonHDL-C) and Triglycerides (TG) to be concordant when possible with the current US Multi-Society guidelines for blood cholesterol management. Results The new classification method categorizes patients into all the classical Fredrickson-like phenotypes except for Type III dysbetalipoproteinemia. In addition, a new hypolipidemic phenotype (Type VI) due to genetic mutations in apoB-metabolism is described. The validity of the new algorithm was confirmed by lipid analysis by NMR (N = 11,365) and by concordance with classification by agarose gel electrophoresis/beta-quantification (N = 5504). Furthermore, based on the Atherosclerosis Risk in Communities (ARIC) cohort (N = 14,742), the lipoprotein phenotypes differ in their association with ASCVD (TypeV>IIb > IVb > IIa > IVa > normolipidemic) and can be used prognostically as risk enhancer conditions in the management of patients. Conclusions We describe a clinically useful lipoprotein phenotyping system that is only dependent upon the standard lipid panel. It, therefore, can be easily implemented for increasing compliance with current guidelines and for improving the care of patients at risk for ASCVD.

Authenticity of Hay Milk vs. Milk from Maize or Grass Silage by Lipid Analysis

Hay milk is a traditional dairy product recently launched on the market. It is protected as “traditional specialty guaranteed” (TSG) and subjected to strict regulations. One of the most important restrictions is that the cow’s feed ration must be free from silage. There is the need for analytical methods that can discriminate milk obtained from a feeding regime including silage. This study proposes two analytical approaches to assess the authenticity of hay milk. Hay milk and milk from cows fed either with maize or grass silage were analyzed by targeted GC-MS for cyclopropane fatty acid (dihydrosterculic acid, DHSA) detection, since this fatty acid is strictly related to the bacterial strains found in silage, and by HPLC-HRMS. The presence of DHSA was correlated to the presence of maize silage in the feed, whereas it was ambiguous with grass silage. HPLC-HRMS analysis resulted in the identification of 14 triacylglycerol biomarkers in milk. With the use of these biomarkers and multivariate statistical analysis, we were able to predict the use of maize and grass silage in the cow’s diet with 100% recognition. Our findings suggest that the use of analytical approaches based on HRMS is a viable authentication method for hay milk.

Loss of hepatic Lgr4 and Lgr5 promotes nonalcoholic fatty liver disease

Background & Aims: The Rspo–Lgr4/5–Znrf3/Rnf43 module is a master regulator of hepatic Wnt/β–catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether liver–specific loss of the Wnt/β–catenin modulators Leucine–Rich Repeat–Containing G Protein–Coupled Receptor 4/5 (Lgr4/5) promotes nonalcoholic fatty liver disease (NAFLD). Methods: Mice with liver–specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were fed with normal diet (ND) or high fat diet (HFD). Livers of these mice were analyzed for lipid and fibrotic content by tissue staining and immunohistochemistry (IHC), and lipoproteins, inflammation and liver enzyme markers were measured in blood. Mechanistic insights into hepatic lipid accumulation were obtained by using ex vivo primary hepatocyte cultures derived from the Lgr4/5dLKO mice. Lipid analysis of mouse livers was performed by mass spectrometry (MS)–based untargeted lipidomic analysis. Results: We demonstrated that liver-specific ablation of Lgr4/5–mediated Wnt signaling resulted in hepatic steatosis, impaired bile acid (BA) secretion and predisposition to liver fibrosis. Under HFD conditions, we observed progressive intrahepatic fat accumulation, developing into macro–vesicular steatosis. Serum lipoprotein levels in HFD–fed Lgr4/5dLKO mice were decreased, rather than increased, suggesting that accumulation of fat in the liver was due to impaired lipid secretion by hepatocytes. Our lipidome analysis revealed a severe alteration of several lipid species in livers of Lgr4/5dLKO mice, including triacylglycerol estolides (TG–EST), a storage form of bioactive free fatty acid (FA) esters of hydroxy FAs (FAHFAs). Conclusions: Loss of hepatic Wnt/β–catenin activity by Lgr4/5 deletion led to deregulation of lipoprotein pathways, loss of BA secretion, intrinsic alterations of lipid homeostasis and the onset of NAFLD.

A Lipidomic Approach to Identify Potential Biomarkers in Exosomes From Melanoma Cells With Different Metastatic Potential

Melanoma, one of the most lethal cutaneous cancers, is characterized by its ability to metastasize to other distant sites, such as the bone. Melanoma cells revealed a variable in vitro propensity to be attracted toward bone fragments, and melanoma-derived exosomes play a role in regulating the osteotropism of these cells. We have here investigated the lipid profiles of melanoma cell lines (LCP and SK-Mel28) characterized by different metastatic propensities to colonize the bone. We have purified exosomes from cell supernatants by ultracentrifugation, and their lipid composition has been compared to identify potential lipid biomarkers for different migration and invasiveness of melanoma cells. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) lipid analysis has been performed on very small amounts of intact parental cells and exosomes by skipping lipid extraction and separation steps. Statistical analysis has been applied to MALDI mass spectra in order to discover significant differences in lipid profiles. Our results clearly show more saturated and shorter fatty acid tails in poorly metastatic (LCP) cells compared with highly metastatic (SK-Mel28) cells, particularly for some species of phosphatidylinositol. Sphingomyelin, lysophosphatidylcholine, and phosphatidic acid were enriched in exosome membranes compared to parental cells. In addition, we have clearly detected a peculiar phospholipid bis(monoacylglycero)phosphate as a specific lipid marker of exosomes. MALDI-TOF/MS lipid profiles of exosomes derived from the poorly and highly metastatic cells were not significantly different.