Intact Phospholipids Research Articles

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is a genetic disorder, diagnosed according to the presence of phenotypical traits of the myocardium. A specific biomarker which can associate with the severity of HCM is lacking. Extracellular Vesicles (EVs), nanoparticles released by cells into biological fluids, hold promise as accessible diagnostic tools, as their abundance and molecular composition can reflect the severity of cardiac diseases (Rizzuto 2024). We aim at characterising plasma-derived EVs isolated from 28 HCM patients and 18 healthy volunteers (CTR). Methods The whole cohort underwent transthoracic echocardiography, whereas magnetic resonance and exome sequencing was performed on HCM patients. EVs were isolated via ultracentrifugation from plasma of both groups. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis, transmission electron microscopy, Western blot, targeted (Olink) and untargeted (nLC-MS/MS) proteomics and FACS analysis. Plasma-derived EV subpopulations were characterised according to their cellular origin. Data are expressed as median and interquartile range (25th, 75th). Results Most patients were male (68% HCM and 66% CTR) with a median age of 58 (49-69) years (HCM) and 47 (44-52) (CTR). Among HCM patients, missense mutations in the MYH7 gene were the most identified. The median maximum wall thickness (WTMax) in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. The isolated EVs expressed tetraspanins (CD9, CD63 and CD81), Alix and β-integrin and showed an intact phospholipid bilayer of 100 nm in size. EV concentration was reduced in HCM vs CTRL, respectively 2.8*109 EV/ml/cell count (2*109-4*109) and 4.6*10⁹ EV/ml/cell count (3*109-6*109). Among 15 plasma-derived EV subpopulations studied, those released from platelets (CD41a) and activated platelets (CD62P and CD40L) were increased in HCM patients vs CTR by 1.7 fold. Negative associations were found between E/e’, parameter of diastolic function, and cardiomyocyte-derived EVs (r= -0.2658), and between left ventricle ejection fraction and platelet-derived EVs (r=-0.2189); a positive correlation was found between WTMax and EVs derived from activated endothelial cells (r=0.2330). The proteomic analysis of EVs shows an enrichment in proteins related to platelets adhesion and inflammation in HCM patients. Conclusions HCM patients present a peculiar phenotypic pattern of EVs that may associate with diagnostic clinical features of HCM.

LC–MS/MS-based phospholipid profiling of plant-pathogenic bacteria with tailored separation of methyl-branched species

Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC–MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.Graphical

Variations of intact phospholipid compositions in the digestive system of Antarctic krill, Euphausia superba, between summer and autumn.

The biochemical composition of Antarctic krill, Euphausia superba, is largely determined by their feeding behaviour. As they supply energy for animals of a higher trophic level and are also commercialized for human consumption, the interest in research on the species is high. Lipids, especially phospholipids, make up a high proportion of dry weight in krill. Seasonal changes are well documented in the fingerprint of free fatty acids analysed after hydrolysis of phospholipids, but the underlying intact polar lipids are rarely considered. In this study, we evaluated the compositions of intact phospholipids (IPLs) in the stomach, digestive gland and hind gut of Antarctic krill caught in summer and autumn at the Antarctic Peninsula region. Using high-resolution mass spectrometry, the fatty acid composition of 179 intact phospholipids could be resolved. Most IPLs were phosphatidylcholines, followed by phosphatidylethanolamines. Several very long chain polyunsaturated fatty acids up to 38:8, which have not been reported in krill before, were identified. The composition shifted to higher molecular weight IPLs with a higher degree of unsaturation for summer samples, especially for samples of the digestive gland. The data supplied in this paper provides new insights into lipid dynamics between summer and autumn usually described by free fatty acid biomarkers.

Chain-locked precursor ion scanning based HPLC–MS/MS for in-depth molecular analysis of lipase-catalyzed transesterification of structured phospholipids containing ω-3 fatty acyl chains

Lipase-catalyzed transesterification of structured phospholipids (sPLs) is a hot topic, but the structural variation of the fatty acyl chains in intact phospholipids at the molecular level remains unclear to date. The present study explored the detailed characteristics of synthesized phospholipids through high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in precursor ion scan mode. The optimal conditions were in-depth inspected and determined for the reaction system, including phospholipase A1 as catalyst, 15% lipase loading, and 1% water content. The sPLs enriched with EPA/DHA were structurally and quantitatively characterized by focusing on the fragments of m/z 301.6 (eicosapentaenoic acid, EPA) and m/z 327.6 (docosahexaenoic acid, DHA), and the results were statistically analyzed using partial least squares discriminant analysis and clustered heatmap hierarchical clustering analysis. PC 38:6 (18:1/20:5), PC 38:7 (18:2/20:5), PC o-40:6 (o-18:0/22:6), and PE 40:8 (18:2/22:6) etc. were revealed as the main variables that were active in the reaction.

Effects of Soybean Lecithin on Growth Performance, Intestine Morphology, and Liver Tissue Metabolism in Rock Bream (Oplegnathus fasciatus) Larvae

Investigations have demonstrated a strong and positive association between dietary intact phospholipid (PL) inclusion and aquatic larval growth, nevertheless, the precise molecular mechanism underlying PL inclusion on growth performance has not been well elucidated. This study aimed to investigate the effects of dietary soybean lecithin (SL) inclusion on growth performance, liver metabolism, resistance to hypoxia stress, and potential molecular mechanisms in rock bream (Oplegnathus fasciatus) larvae. Four types of equal-protein and equal-lipid content microdiets (MDs) were formulated with graded levels of SL to achieve phospholipid levels of (PLs, dry matter) 3.84% (SL0), 6.71% (SL4), 9.38% (SL8), and 12.21% (SL12). Rock bream larvae (25 days post-hatching) were fed the respective MDs for 30 days with three replicates. We found that dietary SL inclusion promoted growth performance, survival rate, and stress resistance to hypoxia stress. The increased dietary SL inclusion improved intestinal structure, as shown by the increased perimeter ratio, muscular thickness, and mucosal fold height of the mid-intestinal tissue. Moreover, a high SL inclusion diet (SL12) increased the activity of the key lipolysis-related enzyme (lipase [LP]) in liver tissue but decreased the activity of amino acid catabolism-related enzymes (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]). RNA sequencing results in liver tissue revealed that the SL12 diet increased the transcriptional level of fatty acid activation-related genes (acsl6 and acsbg2), phospholipid catabolism-related genes (acat2, lpin2, and crls), and amino acid synthesis-related genes (gs, csb, aldh18a1, and oct), but decreased the expression of amino acid catabolism-related gene gprt2. Notably, the SL12 diet significantly increased the expression of ribosome biogenesis-related genes (pes1, nop56, nop58, and rpf2) in liver tissue. The ribosome protein-related pathways were the most enriched pathways mapped in the GO database. Collectively, this study demonstrated the necessity of dietary SL for survival, growth performance, promotion of mid-intestinal morphology, and hypoxia stress during the rock bream larval stage. The SL-induced growth performance promotion was likely attributed to increasing nutrient acquisition by intestinal morphology improvement and to increasing SL catabolism and thereby sparing amino acids for protein synthesis.

PD36-08 BLOOD TESTIS BARRIER-MIMETIC DROPLET INTERFACE BILAYERS (DIBS) FOR PASSIVE PERMEABILITY PREDICTION

You have accessJournal of UrologyCME1 May 2022PD36-08 BLOOD TESTIS BARRIER-MIMETIC DROPLET INTERFACE BILAYERS (DIBS) FOR PASSIVE PERMEABILITY PREDICTION Jaime Korner, Meghan Robinson, Katherine Elvira, Ryan Flannigan, and Stephanie Willerth Jaime KornerJaime Korner More articles by this author , Meghan RobinsonMeghan Robinson More articles by this author , Katherine ElviraKatherine Elvira More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Stephanie WillerthStephanie Willerth More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002594.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The blood testis barrier (BTB) serves as a “gatekeeper” for the apical compartment of the seminiferous epithelium where spermatogenesis takes place. Tight junctions between adjacent Sertoli cells create the BTB, regulate transport of molecules, and protect the luminal compartment from toxins and pathogens. Investigating the impact of toxins and drugs, and the regulation of human spermatogenesis requires realistic in vitro models. The purpose of this study was to develop an artificial cellular membrane to mimic the BTB using droplet interface bilayers (DIBs) derived from human Sertoli cell lipid extracts. METHODS: Human Sertoli cells were collected from a dissociated human testicular biopsy using magnetic activated sorting for Follicle-Stimulating Hormone Receptor (FSHR). Sertoli cells were expanded in vitro and lipids were extracted using a Lipid Extraction Kit (Cell Biolabs). Permeability experiments were conducted using a poly(dimethylsiloxane) (PDMS) microfluidic device which allows for the controlled formation of pairs of differing droplet compartments connected by a phospholipid bilayer. A solution of Sertoli lipids in squalene (10 mg/mL) was used as the outer phase. Donor droplets contained fluorophore (100 µM in buffer) and acceptor droplets contained only buffer (both pH 7.4). The DIB-connected droplet pairs were formed at 50 °C and the temperature was decreased to 37 °C for observation. RESULTS: These results represent the first DIBs to be made using human lipid extracts and the first use of DIBs to model permeability of the BTB. We found that the BTB-mimetic DIBs were impermeable to both fluorophores tested: fluorescein and calcein. Calcein is normally membrane-impermeable and serves as a control experiment to confirm the presence of an intact phospholipid bilayer. Fluorescein has been observed to permeate DIBs made using single, synthetic lipids and intestine-mimetic DIBs but not most blood-brain barrier-mimetic DIBs. CONCLUSIONS: DIBs can be made from human Sertoli cell lipid extracts and show great potential to more accurately model BTB permeability. Source of Funding: Collaborative Healthy Grant © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e635 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jaime Korner More articles by this author Meghan Robinson More articles by this author Katherine Elvira More articles by this author Ryan Flannigan More articles by this author Stephanie Willerth More articles by this author Expand All Advertisement PDF DownloadLoading ...

Antiphospholipid autoantibodies in Lyme disease arise after scavenging of host phospholipids by Borrelia burgdorferi.

A close association with its vertebrate and tick hosts allows Borrelia burgdorferi, the bacterium responsible for Lyme disease, to eliminate many metabolic pathways and instead scavenge key nutrients from the host. A lipid-defined culture medium was developed to demonstrate that exogenous lipids are an essential nutrient of B. burgdorferi, which can accumulate intact phospholipids from its environment to support growth. Antibody responses to host phospholipids were studied in mice and humans using an antiphospholipid ELISA. Several of these environmentally acquired phospholipids including phosphatidylserine and phosphatidic acid, as well as borrelial phosphatidylcholine, are the targets of antibodies that arose early in infection in the mouse model. Patients with acute infections demonstrated antibody responses to the same lipids. The elevation of antiphospholipid antibodies predicted early infection with better sensitivity than did the standardized 2-tier tests currently used in diagnosis. Sera obtained from patients with Lyme disease before and after antibiotic therapy showed declining antiphospholipid titers after treatment. Further study will be required to determine whether these antibodies have utility in early diagnosis of Lyme disease, tracking of the response to therapy, and diagnosis of reinfection, areas in which current standardized tests are inadequate.

Hybrid bilayer membranes on metallurgical polished aluminum

In this work we describe the functionalization of metallurgically polished aluminum surfaces yielding biomimetic electrodes suitable for probing protein/phospholipid interactions. The functionalization involves two simple steps: silanization of the aluminum and subsequent fusion of multilamellar vesicles which leads to the formation of a hybrid bilayer lipid membrane (hBLM). The vesicle fusion was followed in real-time by fast Fourier transform electrochemical impedance spectroscopy (FFT EIS). The impedance-derived complex capacitance of the hBLMs was approximately 0.61 µF cm−2, a value typical for intact phospholipid bilayers. We found that the hBLMs can be readily disrupted if exposed to > 400 nM solutions of the pore-forming peptide melittin. However, the presence of cholesterol at 40% (mol) in hBLMs exhibited an inhibitory effect on the membrane-damaging capacity of the peptide. The melittin-membrane interaction was concentration dependent decreasing with concentration. The hBLMs on Al surface can be regenerated multiple times, retaining their dielectric and functional properties essentially intact.

Using stable isotope tracers to monitor membrane dynamics in C. elegans

Membranes within an animal are composed of phospholipids, cholesterol, and proteins that together form a dynamic barrier. The types of lipids that are found within a membrane bilayer impact its biophysical properties including its fluidity, permeability, and susceptibility to damage. While membrane composition is very stable in healthy adults, aberrant membrane structure is seen in a wide and varied array of diseases as well as during natural aging. Despite the wide-reaching impacts of membrane composition, there is relatively little known about how membrane landscape is established and maintained over time. In vivo biochemical modeling of membrane lipids is needed to understand how these molecules interact in their natural configurations. Here, we have described analytical methods that increase the capacity to map the dynamics of individual membrane phospholipids using different types of mass spectrometry. Specifically, we describe novel stable isotope (13C and 15N) strategies to quantify the turnover of dozens of fatty acid tails and intact phospholipids simultaneously.

Identification and quantification of phospholipids in strawberry seeds and pulp (Fragaria × ananassa cv San Andreas) by liquid chromatography with electrospray ionization and tandem mass spectrometry.

An extensive characterization and quantification of intact phospholipids (PLs) in strawberry (Fragaria × ananassa cv San Andreas) seed and pulp was carried out by hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS). More than 150 intact polar lipids including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), lysophosphatidylcholines (LPCs), and lysophosphatidylethanolamines (LPEs) were identified in negative ESI mode. PC 18:2/18:2 and 18:2/18:3 were found to be the major components of strawberry lipid extracts at concentrations of 230 ± 36 and 189 ± 32 μg/g, respectively, in seeds and at concentrations of 330 ± 50 and 140 ± 22 μg/g, respectively, in pulp. The lipidic extracts of both strawberry seeds and pulp exhibited the dominance of LPC 16:0/0:0 at a content of 132 ± 19 and 114 ± 16 μg/g, respectively, and LPC 0:0/18:2 at 236 ± 20 and 150 ± 20 μg/g, respectively. The other most abundant species of strawberry seeds and pulp were PE 18:2/18:2, 40 ± 9 and 190 ± 40 μg/g, followed by PI 16:0/18:2, 51 ± 15 and 24 ± 8 μg/g, respectively, while PG, PA, and LPE show comparable abundance below 10 μg/g. The most recurrent fatty acyl substituents of PLs were C18:3 (α-linolenic acid), C18:2 (linoleic acid), C18:1 (oleic acid), C18:0 (stearic acid), C16:0 (palmitic acid), and relatively high contents of a shorter chain such as C14:0 (myristic acid).

HILIC-ESI-FTMS with All Ion Fragmentation (AIF) Scans as a Tool for Fast Lipidome Investigations.

Lipidomics suffers from the lack of fast and reproducible tools to obtain both structural information on intact phospholipids (PL) and fatty acyl chain composition. Hydrophilic interaction liquid chromatography with electrospray ionization coupled to an orbital-trap Fourier-transform analyzer operating using all ion fragmentation mode (HILIC-ESI-FTMS-AIF MS) is seemingly a valuable resource in this respect. Here, accurate m/z values, HILIC retention times and AIF MS scan data were combined for PL assignment in standard mixtures or real lipid extracts. AIF scans in both positive and negative ESI mode, achieved using collisional induced dissociation for fragmentation, were applied to identify both the head-group of each PL class and the fatty acyl chains, respectively. An advantage of the AIF approach was the concurrent collection of tandem MS-like data, enabling the identification of linked fatty acyl chains of precursor phospholipids through the corresponding carboxylate anions. To illustrate the ability of AIF in the field of lipidomics, two different types of real samples, i.e., the lipid extracts obtained from human plasma and dermal fibroblasts, were examined. Using AIF scans, a total of 253 intact lipid species and 18 fatty acids across 4 lipid classes were recognized in plasma samples, while FA C20:3 was confirmed as the fatty acyl chain belonging to phosphatidylinositol, PI 38:3, which was found to be down-regulated in fibroblast samples of Parkinson’s disease patients.

Interplay between Cocaine, Drug Removal, and Methylphenidate Reversal on Phospholipid Alterations in Drosophila Brain Determined by Imaging Mass Spectrometry.

Cocaine dependence displays a broad impairment in cognitive performance including attention, learning, and memory. To obtain a better understanding of the action of cocaine in the nervous system, and the relation between phospholipids and memory, we have investigated whether phospholipids recover in the brain following cocaine removal using the fly model, Drosophila melanogaster. In addition, the effects of methylphenidate, a substitute medication for cocaine dependence, on fly brain lipids after cocaine abuse are also determined to see if it can rescue the lipid changes caused by cocaine. Time of flight secondary ion mass spectrometry with a (CO2)6000+ gas cluster ion beam was used to detect intact phospholipids. We show that cocaine has persistent effects, both increasing and decreasing the levels of specific phosphatidylethanolamines and phosphatidylinositols. These changes remain after cocaine withdrawal and are not rescued by methylphenidate. Cocaine is again shown to generally increase the levels of phosphatidylcholines in the fly brain; however, after drug withdrawal, the abundance of these lipids returns to the original level and methylphenidate treatment of the flies following cocaine exposure enhances the reversal of the lipid level reducing them below the original control. The study provides insight into the molecular effects of cocaine and methylphenidate on brain lipids. We suggest that phosphatidylcholines could be a potential target for the treatment of cocaine abuse as well as be a significant hallmark of cognition and memory loss with cocaine.

High turnover rate of free phospholipids in soil confirms the classic hypothesis of PLFA methodology

Phospholipid fatty acid (PLFA) analysis has been widely used to study soil microbial community structure. Soil PLFAs are taken to be indicative of living organisms since phospholipids are assumed to rapidly degrade after cell death. However, the turnover rate of phospholipids has never been accurately quantified. For this purpose, a short incubation experiment was conducted by amending paddy soil with extracted phospholipids, using the 13C labeling technique and PLFA analysis to quantify phospholipid degradation. Both bacterial (Methylocystis sp. and Escherichia coli) and fungal (Simplicillium subtropicum) phospholipids had high turnover rates. The half-life (t½) for different phospholipids ranged from 14 to 27 h and the average t½ for total phospholipids was about 20 h at 25 °C but nearly double that at 15 °C. However, phospholipids had a similar turnover rate in a soil with lower microbial biomass (84 mg C kg−1) compared to a soil with higher microbial biomass (305 mg C kg−1). Assimilation of 13C into other phospholipids was very low but followed the same timescale. Overall, this provides for the first time direct evidence for high turnover rates in soil through the analysis of specific 13C-labelled PLFAs and confirms the classic hypothesis that intact phospholipids represent living cells, necessary for the validity of the established PLFA methodology.

Modified Lipid Extraction Methods for Deep Subsurface Shale.

Growing interest in the utilization of black shales for hydrocarbon development and environmental applications has spurred investigations of microbial functional diversity in the deep subsurface shale ecosystem. Lipid biomarker analyses including phospholipid fatty acids (PLFAs) and diglyceride fatty acids (DGFAs) represent sensitive tools for estimating biomass and characterizing the diversity of microbial communities. However, complex shale matrix properties create immense challenges for microbial lipid extraction procedures. Here, we test three different lipid extraction methods: modified Bligh and Dyer (mBD), Folch (FOL), and microwave assisted extraction (MAE), to examine their ability in the recovery and reproducibility of lipid biomarkers in deeply buried shales. The lipid biomarkers were analyzed as fatty acid methyl esters (FAMEs) with the GC-MS, and the average PL-FAME yield ranged from 67 to 400 pmol/g, while the average DG-FAME yield ranged from 600 to 3,000 pmol/g. The biomarker yields in the intact phospholipid Bligh and Dyer treatment (mBD + Phos + POPC), the Folch, the Bligh and Dyer citrate buffer (mBD-Cit), and the MAE treatments were all relatively higher and statistically similar compared to the other extraction treatments for both PLFAs and DGFAs. The biomarker yields were however highly variable within replicates for most extraction treatments, although the mBD + Phos + POPC treatment had relatively better reproducibility in the consistent fatty acid profiles. This variability across treatments which is associated with the highly complex nature of deeply buried shale matrix, further necessitates customized methodological developments for the improvement of lipid biomarker recovery.

Abstract 1152: Lipid elongation: an unexplored therapeutic target in prostate cancer

Abstract Dysregulated lipid metabolism is one of the hallmarks of cancer, particularly for prostate cancer (PCa). PCa cells exhibit distinctive metabolic features such as upregulation of enzymes involved in de novo synthesis, uptake and beta-oxidation of lipids, which promote prostate cancer growth, metastasis, and drug resistance. Androgen signalling is a major driver of both PCa growth and lipid metabolism in PCa cells, however the precise effects of androgens on cellular lipid composition and the molecular pathways by which androgens regulate lipid metabolism in PCa cells are yet to be elucidated. In this study we investigated the effect of androgens on the lipid composition of PCa cell membranes and the enzymes involved in lipid metabolism, and explored the influence of these enzymes on tumour cell behaviour such as cell migration, proliferation and attachment. PCa cell lines (AR positive and negative) were cultured in the absence or presence of androgens or the anti-androgen enzalutamide, and changes in intact phospholipid species were assessed by ESI-MS/MS-based lipidomics. This analysis revealed a complexity of changes in phospholipid profiles in response to androgen treatment. Strikingly, elongation of the fatty acyl chains was consistently observed for multiple phospholipid classes in response to the androgens mibolerone or 5α-dihydotestosterone, whereas inhibition of elongation was observed in the presence of enzalutamide. Transcriptional analysis of critical lipid metabolism pathways revealed that the enzymes that catalyse lipid elongation (ELOVLs) were markedly induced by androgens in multiple PCa cell lines, and siRNA depletion of these enzymes, either alone or in combination, reversed the androgen-induced fatty acyl elongation phenotype. The androgenic regulation of ELOVL enzymes was confirmed in clinical PCa cohorts and in primary tumours cultured as explants. Targeting ELOVL gene expression also significantly attenuated the tumorigenic properties of PCa cells. ELOVL downregulation decreased LNCaP cell migration, and adhesion to fibronectin. Furthermore, ELOVL knock down significantly decreased three-dimensional spheroid growth of LNCaP cells using a hang drop assay. The impact of these enzymes on the lipid profile of PCa cell membrane and cell viability, adhesion and migration suggests that they may represent promising and previously unexplored therapeutic targets. Citation Format: Zeyad D. Nassar, Margaret M. Centenera, Jelle Machiels, Samuel J. Polacek, Katarzyna Bloch, Wayne D. Tilley, Luke A. Selth, Johannes V. Swinnen, Lisa Butler. Lipid elongation: an unexplored therapeutic target in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1152. doi:10.1158/1538-7445.AM2017-1152

Microbial lipid signatures and substrate potential of organic matter in permafrost deposits: Implications for future greenhouse gas production

AbstractA terrestrial permafrost core from Buor Khaya in northern Siberia comprising deposits of Late Pleistocene to Early Holocene age has been investigated to characterize living and past microbial communities with respect to modern and paleoclimate environmental conditions and to evaluate the potential of the organic matter (OM) for greenhouse gas generation. Microbial life markers—intact phospholipids and phospholipid fatty acids—are found throughout the entire core and indicate the presence of living microorganisms also in older permafrost deposits. Biomarkers for past microbial communities (branched and isoprenoid glycerol dialkyl glycerol tetraether as well as archaeol) reveal links between increased past microbial activity and intervals of high OM accumulation accompanied by increased OM quality presumably caused by local periods of moister and warmer environmental conditions. Concentrations of acetate as an excellent substrate for methanogenesis are used to assess the OM quality with respect to microbial degradability for greenhouse gas production. For this purpose two acetate pools are determined: the pore water acetate and OM bound acetate. Both depth profiles reveal similarities to the OM content and quality indicating a link between the amount of the stored OM and the potential to provide substrates for microbial greenhouse gas production. The data suggest that OM stored in the permafrost deposits is not much different in terms of OM quality than the fresh surface organic material. Considering the expected increase of permafrost thaw due to climate warming, this implies a potentially strong impact on greenhouse gas generation from permafrost areas in future with positive feedback on climate variation.

13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans.

Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining 13C and 15N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism.

Influence of Development and Dietary Phospholipid Content and Composition on Intestinal Transcriptome of Atlantic Salmon (Salmo salar).

The inclusion of intact phospholipids in the diet is essential during larval development and can improve culture performance of many fish species. The effects of supplementation of dietary phospholipid from marine (krill) or plant (soy lecithin) sources were investigated in Atlantic salmon, Salmo salar. First feeding fry were fed diets containing either krill oil or soybean lecithin supplying phospholipid at 2.6%, 3.2%, 3.6% and 4.2% of diet. Fish were sampled at ~ 2.5 g (~1,990°day post fertilization, dpf) and ~10 g (2,850°dpf). By comparison of the intestinal transcriptome in specifically chosen contrasts, it was determined that by 2,850°dpf fish possessed a profile that resembled that of mature and differentiated intestinal cell types with a number of changes specific to glycerophospholipid metabolism. It was previously shown that intact phospholipids and particularly phosphatidylcholine are essential during larval development and that this requirement is associated with the inability of enterocytes in young fry to endogenously synthesize sufficient phospholipid for the efficient export of dietary lipid. In the immature phase (~1,990°dpf), the dietary phospholipid content as well as its class composition impacted on several biochemical and morphological parameters including growth, but these differences were not associated with differences in intestinal transcriptomes. The results of this study have made an important contribution to our understanding of the mechanisms associated with lipid transport and phospholipid biosynthesis in early life stages of fish.

Analysis of phosphate and phosphate containing headgroups enzymatically cleaved from phospholipids of Bacillus subtilis by capillary electrophoresis.

A new, fast, selective, and reliable capillary electrophoresis method has been developed for analysis of selected phosphoesters (phosphoserine, phosphoethanolamine, phosphoglycerol) and phosphate. The method is based on separation of specific phosphate containing headgroups (phosphoesters) which are cleaved from the glycerol skeleton of a phospholipid by a regioselective enzyme (phospholipase C). Analysis of intact phospholipids with the same polar headgroup but different fatty acids shows that fatty acid composition has a high impact on separation of phospholipids, so analysis of separated polar headgroups, which avoids this influence, represents a much more suitable approach for phospholipid class research. Optimization of method parameters results in running buffers of relatively narrow pH interval (pH about 10) where all phosphoesters are separated. Further method validation has shown that direct UV detection has a sufficient detection limit for all analytes to perform suitable analyses of cell membrane lipids. The optimized method was tested on the lysate of cell membrane of Bacillus subtilis, where all analytes were determined.

Improved fatty acid analysis of conjugated linoleic acid rich egg yolk triacylglycerols and phospholipid species.

Reports from chicken conjugated linoleic acid (CLA) feeding trials are limited to yolk total fatty acid composition, which consistently described increased saturated fatty acids and decreased monounsaturated fatty acids. However, information on CLA triacylglycerol (TAG) and phospholipid (PL) species is limited. This study determined the fatty acid composition of total lipids in CLA-rich egg yolk produced with CLA-rich soy oil, relative to control yolks using gas chromatography with flame ionization detection (GC-FID), determined TAG and PL fatty acid compositions by thin-layer chromatography-GC-FID (TLC-GC-FID), identified intact PL and TAG species by TLC-matrix-assisted laser desorption/ionization mass spectrometry (TLC-MALDI-MS), and determined the composition of TAG and PL species in CLA and control yolks by direct flow infusion electrospray ionization MS (DFI ESI-MS). In total, 2 lyso-phosphatidyl choline (LPC) species, 1 sphingomyelin species, 17 phosphatidyl choline species, 19 TAG species, and 9 phosphatidyl ethanolamine species were identified. Fifty percent of CLA was found in TAG, occurring predominantly in C52:5 and C52:4 TAG species. CLA-rich yolks contained significantly more LPC than did control eggs. Comprehensive lipid profiling may provide insight on relationships between lipid composition and the functional properties of CLA-rich eggs.