Fatty Acyl Chains Research Articles

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

Photoinduced Online Enrichment-Deglycosylation of Glycolipids for Enhancing Lipid Coverage and Identification in Single-Cell Mass Spectrometry.

Single-cell lipidomics provides important information for molecular mechanisms of living processes and diseases at the individual cell level. However, single-cell lipidomic mass spectrometry (MS) techniques suffer from low lipid coverage and incomplete structural elucidation, especially for poorly ionizable glycosphingolipids (GSLs). Herein, a photoinduced enrichment-deglycosylation method of GSLs was developed and introduced into an ambient liquid extraction MS system for enhancing detection coverage and identification accuracy of GSLs in single-cell MS. GSL standards were selectively adsorbed on TiO2 in ammonia-added protic solvents. Under UV irradiation, the adsorbed GSLs would lose one hexosyl group (deglycosylation), and the products (>70% conversion efficiency) were desorbed from TiO2. By coating porous TiO2 into the capillary of the ambient liquid extraction MS system, online adsorption of GSLs and their separation from high-abundance phospholipids were achieved, largely reducing ion suppression. By UV irradiation, captured GSLs were rapidly deglycosylated and photodesorbed from TiO2 coating without solvent switching, resulting in 6-fold enrichment. With the new method, the detection coverage of GSLs was enhanced 9-fold without losing other lipidomes, compared with the conventional method. Moreover, deglycosylated GSLs from photodesorption had more MS/MS fragments than intact GSLs, facilitating detailed fatty acyl and sphingosine chain elucidation. Seven deglycosylated GSL peaks were identified with the confirmed hydroxyl group location in the fatty acyl chain, while only 1 was identified for intact GSL. The new method was applied to the single-cell lipidomics study of two types of nerve cells. Totally, 31 lipids including 11 GSLs were identified in a single cell, and 5 hexosylceramides were found significantly altered after neuron injury.

Radical-directed dissociation mass spectrometry for differentiation and relative quantitation of isomeric ether-linked phosphatidylcholines

BackgroundEther-linked phosphatidylcholines (PCs) include both plasmanyl and plasmenyl PCs, which contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. Profiling and quantifying ether PCs with accurate structural information is challenging because of the common presence of isomeric and isobaric species in a lipidome. ResultsIn the present study, radical directed dissociation (RDD) from collision-induced dissociation (CID) of the bicarbonate anion adduct of ether PCs has been investigated to differentiate and relatively quantify ether PCs. Alkyl- and alkenyl- PCs give diagnostic characteristic fragment patterns that enable their confident identification and isomer differentiation. Additionally, the sn-position specific product ions have proven effective for relative quantitation among isomers in ether PCs and their isobaric PC species. Using this methodology, we successfully identified a total of 30 PC-O species, 21 PC-P species at the chain composition level, and 22 species of isobaric PC at the sn-position level in the human plasma lipid extract. The quantitative analysis revealed that ether PCs with a 20:4 fatty acyl chain are relatively more abundant in human plasma. Finally, the profile of ether PCs in type 2 diabetic (T2D) groups compared to normal control groups revealed a significant decrease in PC-O 18:1/20:5. We also found it is the PC species containing a 17-carbon fatty acyl chain, rather than their isobaric ether PCs, that shows a decreasing trend in the T2D groups. Significanceether-linked PCs are firstly investigated by RDD mass spectrometry.

Discovering oxidized polar lipids in microalgae lipidome using liquid chromatography mass spectrometry approaches

Microalgae are rich in polar lipids, such as glycolipids, phospholipids and betaine lipids esterified with omega-3 polyunsaturated fatty acids (PUFA), which are prone to oxidation. Under stress conditions, the redox balance in microalgae is altered, leading to an abnormal production of reactive oxygen species (ROS) that can affect surrounding lipids. While few oxidized polar lipids have been described to play important roles in mammals and possess interesting bioactive properties, their occurrence in microalgae is poorly described, hindering their exploitation as novel bioactive compounds. To enhance our understanding of these lipids, we conducted a targeted analysis of oxidized polar lipids in lipid extracts obtained from five microalgae species: Chlorella vulgaris, Chlorococcum amblystomatis, Scendesmus obliquus, Nanochloropsis oceanica and Phaeodactylum ticornutum, using reverse-phase liquid chromatography mass spectrometry (RP-LC-MS). A detailed analysis of the LC-MS data identified 150 oxidized polar lipid species across different classes of phospholipids, glycolipids and betaine lipids in the five microalgae species. These modified oxidized lipids featured oxygenated species with 1–3 additional oxygen atoms in the fatty acyl chains. The predominant oxidized fatty acids esterified to these lipids were omega-3 eicosapentaenoic acid (EPA, 20:5 n-3) and α-linonelic acid (ALA, 18:3 n-3). Notably, most oxidized lipid species were identified in diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) in C. amblystomatis, S. obliquus and N. oceanica, monogalactosyldiacylglycerol (MGDG) in P. tricornutum, and phosphatidylethanolamine (PE) in C. vulgaris. Furthermore, distinct fragmentation patterns across lipid classes allowed the unequivocal identification of oxidized polar lipids. These findings reveal a diverse array of oxidized polar lipids in microalgae, predominantly enriched with oxidized omega-3 PUFA, highlighting microalgae as a natural source of oxidized polar lipids that may serve as a natural reservoir of bioactive omega-3 oxylipins.

Molecular dynamics simulations of lipid composition and its impact on structural and dynamic properties of skin membrane

The stratum corneum (SC) plays the most important role in the absorption of topical and transdermal drugs. In this study, we developed a multi-layered SC model using coarse-grained molecular dynamics (CGMD) simulations of ceramides, cholesterol, and fatty acids in equimolar proportions, starting from two different initial configurations. In the first approach, all ceramide molecules were initially in the hairpin conformation, and the membrane bilayers were pre-formed. In the second approach, ceramide molecules were introduced in either the hairpin or splayed conformation, with the lipid molecules randomly oriented at the start of the simulation. The aim was to evaluate the effects of lipid chain length on the structural and dynamic properties of SC. By incorporating ceramides and fatty acids of different chain lengths, we simulated the SC membrane in healthy and diseased states. We calculated key structural properties including the thickness, normalized lipid area, lipid tail order parameters, and spatial ordering of the lipids from each system. The results showed that systems with higher ordering and structural integrity contained an equimolar ratio of ceramides (chain length of 24 carbon atoms), fatty acids with chain lengths ≥ of 20 carbon atoms, and cholesterol. In these systems, strong apolar interactions between the ceramide and fatty acid long acyl chains restricted the mobility of the lipid molecules, thereby maintaining a compact lipid headgroup region and high order in the lipid tail region. The simulations also revealed distinct flip-flop mechanisms for cholesterol and fatty acid within the multi-layered membrane. Cholesterol is mostly diffused through the tail-tail interface region of the membrane and could flip-flop in the same bilayer. In contrast, fatty acids flip-flopped between adjacent leaflets of two bilayers in which the tails crossed the thinner headgroup region of the membrane. To conclude, our SC model provides mechanistic insights into lipid mobility and is flexible in its design and composition of different lipids, enabling studies of varying skin conditions.

Design, synthesis, and bioactivity investigation of novel cyclic lipopeptide antibiotics targeting top-priority multidrug-resistant gram-negative bacteria

ObjectivesPolymyxins are the last-line therapy for top-priority multidrug-resistant (MDR) gram-negative bacteria. However, polymyxin nephrotoxicity impedes its clinical application. This study aimed to design, synthesize, and identify a novel and promising polymyxin derivative with high efficacy and low toxicity. MethodsTo design polymyxin derivatives, we reduced the hydrophobicity of the two hydrophobic domains (fatty acyl chain and D-Phe6-L-Leu7) and modified the positive charged L-2,4-diaminobutyric acid (Dab) residues. Twenty-five derivatives were synthesized, and their antibacterial activities in vitro and renal cytotoxicities were determined. The nephrotoxicity and pharmacokinetic parameters of compound 12 were examined in rats. Antibacterial efficacy in vivo was evaluated using a mouse systemic infection model. Surface plasmon resonance analysis, compound 12-rifampicin combination therapy, and scanning electron microscopy were used to study the mechanism of action of compound 12. ResultsThis research found a new compound, identified as compound 12, which showed similar or increased antibacterial activity against all tested sensitive and carbapenem-resistant gram-negative bacteria. It exhibited reduced renal cytotoxicity and nephrotoxicity, a favorable pharmacokinetic profile, and maintained or improved antibacterial efficacy in vivo. Importantly, its anti-Pseudomonas aeruginosa activity significantly improved. Compound 12, when combined with rifampicin, enhanced the activity of rifampin against gram-negative bacteria. Compound 12 also showed a high affinity for lipopolysaccharide and disrupted cell membrane integrity. ConclusionReducing the hydrophobicity of the two domains reduced renal cytotoxicity and nephrotoxicity. Shortening the side chain of Dab3 by one carbon maintained or increased its antibacterial activity both in vitro and in vivo. Furthermore, only the length of the side chain of Dab9 could be shortened by one carbon among the Dab1,5 and Dab8,9 residues. The bactericidal effects of compound 12 were related to the disruption of cell membrane integrity. Compound 12 may be a promising candidate for combating sensitive and carbapenem-resistant gram-negative bacterial infections, especially Pseudomonas aeruginosa.

Fungal membrane determinants affecting sensitivity to antifungal cyclic lipopeptides from Bacillus spp.

Bacillus spp. produce numerous antimicrobial metabolites. Among these metabolites, cyclic lipopeptides (CLP) including fengycins, iturins, and surfactins are known to have varying antifungal activity against phytopathogenic fungi. The differential activities of CLP have been attributed to diverse mechanisms of action on fungal membranes. However, the precise biochemical determinants driving their antifungal modes of action have not been conclusively identified. In this study, three plant pathogenic fungi of varying lipopeptide sensitivities, Alternaria solani, Cladosporium cucumerinum, and Fusarium sambucinum, were studied to determine how their cell membrane lipid compositions may confer sensitivity and/or tolerance to fengycin, iturin, and surfactin. Results indicated that sensitivity to all three lipopeptides correlated with lower ergosterol content and elevated phospholipid fatty acid unsaturation. Fungal sensitivity to surfactin was also notably different than fengycin and iturin, as surfactin was influenced more by lower phosphatidylethanolamine amounts, higher levels of phosphatidylinositol, and less by phospholipid fatty acyl chain length. Results from this study provide insight into the fungal membrane composition of A. solani, F. sambucinum, and C. cucumerinum and the specific membrane characteristics influencing the antifungal effectiveness of fengycin, iturin, and surfactin. Understanding of these determinants should enable more accurate prediction of sensitivity-tolerance outcomes for other fungal species exposed to these important CLP.

Molecular Identification and Functional Characterization of LC-PUFA Biosynthesis Elongase (elovl2) Gene in Chinese Sturgeon (Acipenser sinensis).

Elongases of very-long-chain fatty acids (Elovls) are critical rate-limiting enzymes that are involved in LC-PUFA biosynthesis through catalyzing the two-carbon elongation of a pre-existing fatty acyl chain. Thus far, several Elovls have been extensively studied in teleost. However, the functional and physiological roles of Elovls in chondrichthyans have rarely been reported. In this study, we identified and characterized elovl2 from the endangered Chinese sturgeon (Acipenser sinensis) by whole genome scanning. The results show that the coding sequence of elovl2 was 894 bp in length, for a putative protein of 297 amnio acids. Comparative genomic analyses indicated that Chinese sturgeon elovl2 was evolutionarily conserved. Functional characterization in yeast demonstrated that the Chinese sturgeon Elovl2 could efficiently elongate C20 (ARA and EPA) and C22 (22:4n-6 and 22:5n-3) substrates, confirming its critical roles in LC-PUFA biosynthesis. Spatial and temporal expression analyses showed high elovl2 mRNA levels were detected in the liver and brain and showed an increase trend both in embryonic and post-hatching stages. Interestingly, diets with vegetable oils as lipid sources could significantly induce the high expression of elovl2 in Chinese sturgeon, implying that the endogenous LC-PUFA biosynthesis pathway was stimulated by lack of LC-PUFA in their diets. Our findings will enhance our understanding about the evolutionary and functional roles of elovl2 and provide novel insights into the LC-PUFA biosynthesis mechanism in vertebrates.

Quantitative determination of plasma cholesteryl ester levels in Japanese preadolescents from the Hokkaido study using liquid chromatography/tandem mass spectrometry

Cholesteryl esters (CE) are sterols comprising various fatty acyl chains attached to a cholesterol hydroxyl moiety. CEs are often considered plasma biomarkers of liver function; however, their absolute concentrations in the plasma of Japanese preadolescents have not been well explored. This study aimed to determine the plasma CE levels in Japanese preadolescents of different sexes, ages, and body weights living in Hokkaido, Japan using targeted liquid chromatography/tandem mass spectrometry. The analysis was performed on the non-fasting plasma of preadolescents aged 9–12 years (n = 339 healthy volunteers; 178 boys and 161 girls) from Sapporo, Hokkaido, Japan. The analysis results showed that the total CE levels in boys and girls were 871 ± 153 and 862 ± 96 pmol/μL, respectively. CE 18:2 (41 ± 2.9 %) was found to be the most abundant species followed by CE 18:1 (16 ± 1.5 %) and CE 16:0 (13 ± 1.1 %). The ω-3 fatty acid-containing CEs such as CE 18:3 and CE 20:5 were significantly lower in girls than in boys. Despite the different ages, CEs were tightly regulated in the plasma of children’s, and the total CEs ranged between 844 and 906 pmol/μL in boys and 824 and 875 pmol/μL in girls. The participants were further classified into three groups based on their body mass index underweight (n = 237), normal weight (n = 94), and overweight (n = 8). Most of the quantified CEs were accumulated in the overweight group. Interestingly, CE 18:3 was significantly upregulated in the overweight group compared to that in the normal range, and the area under the receiver operating characteristic curve was 0.73, suggesting that it could be a possible marker for obesity. This study marks the initial investigation of absolute CE levels in the plasma of children and can help elucidate the relationship between CEs and childhood obesity.

Integrated multi-omics analysis reveals molecular changes associated with chronic lipid accumulation following contusive spinal cord injury

Functional and pathological recovery after spinal cord injury (SCI) is often incomplete due to the limited regenerative capacity of the central nervous system (CNS), which is further impaired by several mechanisms that sustain tissue damage. Among these, the chronic activation of immune cells can cause a persistent state of local CNS inflammation and damage. However, the mechanisms that sustain this persistent maladaptive immune response in SCI have not been fully clarified yet.In this study, we integrated histological analyses with proteomic, lipidomic, transcriptomic, and epitranscriptomic approaches to study the pathological and molecular alterations that develop in a mouse model of cervical spinal cord hemicontusion.We found significant pathological alterations of the lesion rim with myelin damage and axonal loss that persisted throughout the late chronic phase of SCI. This was coupled by a progressive lipid accumulation in myeloid cells, including resident microglia and infiltrating monocyte-derived macrophages. At tissue level, we found significant changes of proteins indicative of glycolytic, tricarboxylic acid cycle (TCA), and fatty acid metabolic pathways with an accumulation of triacylglycerides with C16:0 fatty acyl chains in chronic SCI. Following transcriptomic, proteomic, and epitranscriptomic studies identified an increase of cholesterol and m6A methylation in lipid-droplet-accumulating myeloid cells as a core feature of chronic SCI.By characterizing the multiple metabolic pathways altered in SCI, our work highlights a key role of lipid metabolism in the chronic response of the immune and central nervous system to damage.

Effect of alterations in phospholipids and free fatty acids on aroma-active compounds in instant-boiled chuck tender, sirloin and silverside beef

Beef flavor profiles are strongly influenced by cooking methods and lipid composition. However, the effect of instant-boiling on the aroma of different beef slices was unclear. This study investigated the lipid profiles and instant-boiling volatile profiles of chuck tender (M. Supraspinatus), sirloin (M. Longissimus dorsi) and silverside (M. Biceps femoris). Quantitative lipidomics identified 336 lipid molecular species, of which 84–112 were quantitatively different among the three beef slices. Sirloin had lower phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol and free fatty acids than chuck tender and silverside. The unsaturated fatty acid acyl chains in phosphatidylethanolamine differed significantly. Solid phase microextraction-gas chromatography-olfactometry-mass spectrometry (SPME-GC-O-MS) identified hexanal, octanal, nonanal, decanal, (E)-2-octenal, (E)-2-nonenal, (E)-2-undecenal, (E,E)-2,4-nonadienal, (E,E)-2,4-decadienal, 1-octen-3-ol, 2-pentylfuran and acetoin as the aroma-active compounds of instant-boiled beef. Unsaturated free fatty acids and phosphatidylglycerols with unsaturated fatty acid residues positively correlated with the aroma-active compounds and might be crucial in flavor differences among the three beef slices. These findings provide greater understanding of the lipid and instant-boiling aroma-active compound profiles in chuck tender, sirloin and silverside, and reflect the suitability of different beef slices for instant-boiling from the aroma perspective.

Novel theoretical database-assisted UHPLC-Q-TOF/MS strategy for profiling and identifying oxidized triglycerides in pharmaceutical excipient soybean oil

Pharmaceutical excipient soybean oil is widely used in injections. Its main components, triglycerides, are easily oxidized due to their unsaturated fatty acyls, raising safety concerns. However, it is hard to analyze those oxidized triglycerides due to their diverse compositions and low abundance. In this study, all theoretical oxidized triglycerides were predicted and a database consisting of 329 oxidized triglycerides was constructed. Then, a novel theoretical database-assisted ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) strategy was developed to finely profile and identify oxidized triglycerides in soybean oil. A total of 106 and 116 oxidized triglycerides were identified and relatively quantified in oxidized soybean oil and long-term stored soybean oil and preparations. It was found that oxidized triglycerides containing carbonyl groups were significantly more prevalent than other forms and oxidized triglycerides with two oxidized fatty acyl chains had the highest relative abundance. Fifteen markers indicating the oxidation of soybean oil were discovered. This strategy could rapidly and directly analyze the oxidized triglycerides and assign their fatty acyl compositions for the first time. This study will improve the quality control of soybean oil and its preparations.

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer.

The onset and progression of cancer is associated with changes in the composition of the lipidome. Therefore, better understanding of the molecular mechanisms of these disease states requires detailed structural characterization of the individual lipids within the complex cellular milieu. Recently, changes in the unsaturation profile of membrane lipids have been observed in cancer cells and tissues, but assigning the position(s) of carbon-carbon double bonds in fatty acyl chains carried by membrane phospholipids, including the resolution of lipid regioisomers, has proven analytically challenging. Conventional tandem mass spectrometry approaches based on collision-induced dissociation of ionized glycerophospholipids do not yield spectra that are indicative of the location(s) of carbon-carbon double bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have emerged as alternative ion activation modalities wherein diagnostic product ions can enable de novo assignment of position(s) of unsaturation based on predictable fragmentation behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra are acquired on the same mass spectrometer to evaluate the relative performance of the two modalities for lipid identification and to interrogate the respective fragmentation pathways under comparable conditions. Based on investigations of lipid standards, fragmentation rules for each technique are expanded to increase confidence in structural assignments and exclude potential false positives. Parallel application of both methods to unsaturated phosphatidylcholines extracted from isogenic colorectal cancer cell lines provides high confidence in the assignment of multiple double bond isomers in these samples and cross-validates relative changes in isomer abundance.

Genetic and structural basis of colistin resistance in Klebsiella pneumoniae: Unravelling the molecular mechanisms

ObjectiveAntimicrobial resistance (AMR), together with multidrug resistance (MDR), mainly among Gram-negative bacteria, has been on the rise. Colistin (polymyxin E) remains one of the primary available last resorts to treat infections caused by MDR bacteria during the rapid emergence of global resistance. As the exact mechanism of bacterial resistance to colistin remains undetermined, this study warranted elucidation of the underlying mechanisms of colistin resistance and heteroresistance among carbapenem-resistant Klebsiella pneumoniae isolates. MethodsMolecular analysis was carried out on the resistant isolates using a genome-wide characterisation approach, as well as MALDI-TOF mass spectrometry, to identify lipid A. ResultsAmong the 32 carbapenem-resistant K. pneumoniae isolates, several isolates showed resistance and intermediate resistance to colistin. The seven isolates with intermediate resistance exhibited the “skip-well” phenomenon, attributed to the presence of resistant subpopulations. The three isolates with full resistance to colistin showed ions using MALDI-TOF mass spectrometry at m/z of 1840 and 1824 representing bisphosphorylated and hexa-acylated lipid A, respectively, with or without hydroxylation at position C’-2 of the fatty acyl chain. Studying the genetic environment of mgrB locus revealed the presence of two insertion sequences that disrupted the mgrB locus in the three colistin-resistant isolates: IS1R and IS903B. ConclusionsOur findings show that colistin resistance/heteroresistance was inducible with mutations in chromosomal regulatory networks controlling the lipid A moiety and insertion sequences disrupting the mgrB gene, leading to elevated minimum inhibitory concentration values and treatment failure. Different treatment strategies should be employed to avoid colistin heteroresistance-linked treatment failures, mainly through combination therapy using colistin with carbapenems, aminoglycosides, or tigecycline.

Structural characterization of sphingomyelins from tissue using electron-induced dissociation.

Sphingomyelins (SMs) and resulting metabolic products serve important functional and cell signaling roles and can act as potential biomarkers and therapeutic targets in many pathological disorders. SMs each contain a sphingoid base, an amide-linked fatty acyl chain, and a phosphocholine headgroup. Despite these simple building blocks, variations and modifications of both the sphingoid base and the fatty acyl chain result in a diverse array of structurally complicated SM compounds. Conventional tandem mass spectrometry (MS/MS) using the collision-induced dissociation (CID) method only provides limited structural information, necessitating other tools to unravel the structural complexity of these lipids. We utilize electron-induced dissociation (EID) and sequential CID/EID approaches to elucidate detailed structural features of SMs. Integrating the CID/EID method into an imaging MS workflow enables accurate identification of SMs directly from kidney tissue. The application of EID enables identification of SMs at the molecular species level, identifying the sphingosine base and the amide-linked fatty acyl chains. Furthermore, removal of the phosphocholine headgroup via CID followed by sequential EID in an MS3 analysis (CID/EID) enhances the structural information obtained. CID/EID provides diagnostic fragmentation patterns revealing the hydroxylation site and double bond position in both the sphingosine base and amide-linked fatty acyl chains. Detailed structural information of SMs from synthetic standards and biological tissue samples is obtained using an alternative electron-based dissociation method. Accurate characterization of SMs promises to better inform studies of tissue biochemistry, lipid metabolism, and molecular pathology.

Single-nucleus RNA sequencing and lipidomics reveal characteristics of transcriptional and lipid composition in porcine longissimus dorsi muscle

BackgroundGlobal per capita meat consumption continues to rise, especially pork. Meat quality is influenced by the content of intramuscular fat (IMF) as a key factor. The longissimus dorsi muscle of Dahe pigs (DHM, IMF: 7.98% ± 1.96%) and Dahe black pigs (DHBM, IMF: 3.30% ± 0.64%) was studied to explore cellular heterogeneity and differentially expressed genes (DEGs) associated with IMF deposition using single-nucleus RNA sequencing (snRNA-seq). The lipid composition was then analyzed using non-targeted lipidomics.ResultsA total of seven cell subpopulations were identified, including myocytes, fibroblast/fibro/adipogenic progenitors (FAPs), satellite cells, endothelial cells, macrophages, pericytes, and adipocytes. Among them, FAPs and adipocytes were more focused because they could be associated with lipid deposition. 1623 DEGs in the FAPs subpopulation of DHBM were up-regulated compared with DHM, while 1535 were down-regulated. These DEGs enriched in the glycolysis/gluconeogenesis pathway. 109 DEGs were up-regulated and 806 were down-regulated in the adipocyte subpopulation of DHBM compared with DHM, which were mainly enriched in the PPAR signaling pathway and fatty acid (FA) biosynthesis. The expression level of PPARG, ABP4, LEP, and ACSL1 genes in DHM was higher than that in DHBM. Lipidomics reveals porcine lipid composition characteristics of muscle tissue. A total of 41 lipid classes and 2699 lipid species were identified in DHM and DHBM groups. The top ten relative peak areas of lipid classes in DHM and DHBM were triglyceride (TG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), diglyceride (DG), cardiolipin (CL), ceramides (Cer), Simple Glc series (Hex1Cer), sphingomyelin (phSM), and phosphatidylinositol (PI). The relative peak areas of 35 lipid species in DHM were lower than DHBM, and 28 lipid species that were higher. There was a significant increase in the TG fatty acyl chains C6:0, C17:0, and C11:4, and a significant decrease in C16:0, C18:1, C18:2, and C22:4 in DHBM (p < 0.05).ConclusionsC16:0 FA may downregulate the expression level of PPARG gene, which leads to the downregulation of fat metabolism-related genes such as ACSL, PLIN2, and FABP4 in DHBM compared with DHM. This may be the reason that the lipid deposition ability of Dahe pigs is stronger than that of Dahe black pigs, which need further investigation.

Increased phosphatidylcholine and its hydroperoxides in serum low-density lipoproteins from patients with non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease is classified into simple steatosis (SS) and non-alcoholic steatohepatitis (NASH) according to histological findings from liver biopsies. Phosphatidylcholine (PC), the main component of phospholipids in serum lipoproteins, is easily oxidized to phosphatidylcholine hydroperoxide (PC-OOH). Although a lipid composition in the low-density lipoproteins (LDL) from patients with NASH could be abnormal, it remains unclear. Here, to better understand the characteristics of lipids in the LDL from NASH and SS, we compared the composition of PC and PC-OOH species in LDL particles (LDL-PC, LDL-PCOOH) from these patients, then clarified the association between these lipids and NASH severity. The serum samples from patients with NASH (female, n = 9) and SS (female, n = 4; male, n = 2) were used for isolation of LDL. Total lipids were extracted from isolated LDL, and the species of PC and PC-OOH were measured using liquid chromatography-mass spectrometry/mass spectrometry. The sum of LDL-PC and the sum of LDL-PCOOH were significantly higher in NASH than in SS. Several LDL-PC (PC 32:0, 32:1, 32:2, 34:3, 36:2, sum of PC with saturated fatty acyl chains and sum of LDL-PC with polyunsaturated fatty acyl chains) and several LDL-PCOOH (34:2, 36:2, 36:3 and total) were increased significantly with increasing fibrosis score. In particular, a series of LDL-PCOOH were more reflective of the severity of fibrosis score. LDL-PC and LDL-PCOOH species were strongly correlated with the fibrosis score in NASH, which suggests that abnormal LDL is involved in the development of liver fibrosis.

Effect of radio frequency roasting on the lipid profile of peanut oil and the mechanism of lipids transformation: Revealed by untargeted lipidomics approach

Radio frequency (RF) heating has been proved an alternative roasting method for peanuts, which could effectively degrade aflatoxins and possesses the advantages of greater heating efficiency and penetration depth. This study aimed to investigate the influences of RF roasting on the lipid profile of peanut oil under 150 °C target temperature with varied peanut moisture contents (8.29 % and 20 %) and holding times (0, 7.5, and 15 min), using ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS)-based lipidomics. In total, 2587 lipid species from 35 subclasses were identified. After roasting, the contents of sterol lipid (ST) and subclasses of glycerophospholipids (GPs) and glycoglycerolipids increased significantly, while fatty acid (FA), Oxidized (Ox-) FA, cholesterol (CE), and all subclasses of glycerolipids (GLs) decreased, and 1084 differential lipids were screened. The highest ST and lowest CE contents in peanut oil were achieved by medium roasting (7.5 min). The raise in moisture content of peanut simply affected a few GPs subclasses adversely. Compared with hot air (HA) roasting, RF decelerated lipid oxidation, showing higher levels of diacylglycerol, triacylglycerol and FA, with no additional negative impact and only 69 exclusive differential lipids. During RF roasting, hydrolysis and oxidation of fatty acyl chains into secondary oxides were the central behaviors of lipids transformation. This study could provide insights into the lipid changes and transformation mechanism of peanut oil by RF roasting processing.

Differentiating human phospholipase A2's activity toward phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate

Phospholipase A2's (PLA2's) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA2's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA2's toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P2). The in vitro assay revealed that Group IVA cPLA2 (GIVA cPLA2) showed relatively high activity toward PI and PI(4)P among the tested PLA2's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA2 and GVIA iPLA2 showed detectable activity toward PI(4,5)P2, but it appeared to be a poorer substrate for all of the PLA2's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA2 may best account for the PI and PIP hydrolysis in living cells.

Engineering of the start condensation domain with improved N-decanoyl catalytic activity for daptomycin biosynthesis.

Daptomycin, a lipopeptide comprising an N-decanoyl fatty acyl chain and a peptide core, is used clinically as an antimicrobial agent. The start condensation domain (dptC1) is an enzyme that catalyzes the lipoinitiation step of the daptomycin synthesis. In this study, we integrated enzymology, protein engineering, and computer simulation to study the substrate selectivity of the start condensation domain (dptC1) and to screen mutants with improved activity for decanoyl loading. Through molecular docking and computer simulation, the fatty acyl substrate channel and the protein-protein interaction interface of dptC1 are analyzed. Key residues at the protein-protein interface between dptC1 and the acyl carrier were mutated, and a single-point mutant showed more than three-folds improved catalytic efficiency of the target n-decanoyl substrate in comparing with the wild type. Moreover, molecular dynamics simulations suggested that mutants with increased catalytic activity may correlated with a more "open" and contracted substrate binding channel. Our work provides a new perspective for the elucidation of lipopeptide natural products biosynthesis, and also provides new resources to enrich its diversity and optimize the production of important components.