Lipidomic signature of Bacillus licheniformis I89 during the different growth phases unravelled by high-resolution liquid chromatography-mass spectrometry.

Bacillus licheniformis I89 is a non-pathogenic, Gram-positive bacterium, frequently found in soil. It has several biotechnological applications as producer of valuable compounds such as proteases, amylases, surfactants, and lantibiotics. Herein, it is reported the identification of the polar lipidome of B. licheniformis I89 during the different growth phases (lag, exponential and stationary) at 37 °C. The analytical approach relied on hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry (HILIC-ESI-MS), accurate mass measurements and tandem mass spectrometry (MS/MS). In the lipidome of B. licheniformis I89 were identified four phospholipid classes: phosphatidylethanolamine, phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin; two glycolipid classes: monoglycosyldiacylglycerol and diglycosyldiacylglycerol; and two phosphoglyceroglycolipid classes: mono-alanylated lipoteichoic acid primer and lipoteichoic acid primer. The same lipid species were identified at the different growth phases, but there were significant differences on the relative abundance of some molecular species. There was a significant increase in the 30:0 lipid species and a significant decrease in the 32:0 lipid species, between exponential and stationary phases, when compared to lag phase. No differences were observed between exponential and stationary phases. The lipidomic-based approach used herein is a very promising tool to be employed in the study of bacterial lipid composition, which is a requirement to understand its metabolism and response to growth conditions.

Lipid remodelling in human melanoma cells in response to UVA exposure.

Extensive exposure to UVA is thought to increase the risk of malignancy and the progression of melanoma, the most serious type of skin cancer. It is well known that alterations in lipid metabolism represent an early event in carcinogenesis, but the impact of UVA exposure on the lipid composition of cancer cells is still largely unknown. In this study we aimed at investigating lipid remodeling in human melanoma cells in response to UVA exposure. After UVA irradiation, lipid extracts were either immediately collected from SK-MEL-28 cells or collected after a recovery period of 2 h or 24 h. The lipid profiles for each event were determined by liquid chromatography or gas chromatography coupled to mass spectrometry. UVA exposure led to major alterations in both fatty acids (FA) and phospholipid profiles. An increase of monounsaturated FA (MUFA) and FA18:0, as well as a decrease of FA16:0, were observed 24 h after irradiation. Moreover, phosphatidylcholine (PC) decreased and phosphatidylinositol (PI) increased after UVA exposure. Molecular alterations in the PC, lysoPC, PI, phosphatidylethanolamine (PE), ether-linked PE and phosphatidylglycerol (PG) profiles were also observed. The absence of cleaved caspase-3 after 2 h and 24 h of re-incubation is correlated with impairment of apoptosis. Overall, these data showed changes in membrane lipids, which may be associated with lipogenesis after UVA exposure which, in turn, is usually a determinant for cell survival.

Lipidomics of Mesenchymal Stromal Cells: Understanding the Adaptation of Phospholipid Profile in Response to Pro-Inflammatory Cytokines.

Mesenchymal stromal cells (MSCs) present anti-inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. In clinical applications MSCs are subjected to a strong pro-inflammatory environment, essential to their immunosuppressive action. Despite the wide clinical use of these cells, how MSCs exert their effect remains unclear. Several lipids are known to be involved in cell's signaling and modulation of cellular functions. The aim of this paper is to examine the variation in lipid profile of MSCs under pro-inflammatory environment, induced by the presence of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), using the most modern lipidomic approach. Major changes in lipid molecular profile of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysoPC (LPC), and sphingomyelin (SM) classes were found. No changes were observed in the phosphatidylinositol (PI) profile. The levels of PC species with shorter fatty acids (FAs), mainly C16:0, decreased under pro-inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of LPC(18:0), which is known to be an anti-inflammatory LPC, observed in MSCs subjected to TNF-α and IFN-γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF-α and IFN-γ also enhanced the levels of PE(40:6) and decreased the levels of PE(O-38:6). Higher expression of PS(36:1) and SM(34:0) along with a decrease in PS(38:6) levels were observed. These results indicate that lipid metabolism and signaling are modulated during MSCs activation, which suggests that lipids may be involved in MSCs functional and anti-inflammatory activities.

Alteration in Phospholipidome Profile of Myoblast H9c2 Cell Line in a Model of Myocardium Starvation and Ischemia.

Myocardium infarction is one of the most deathly cardiovascular diseases. It is characterized by myocardium ischemia as a result of nutrients depletion and hypoxia. The cell can respond to this injury by autophagy or apoptosis, which determines the evolution and possible recovery of the myocardium infarction. Lipids play an important role in cardiovascular disease. However reports stating lipidome variations in cardiovascular disease are scarce and the role that lipids play in this pathological condition is not completely understood. The aim of this work was to identify changes in lipid profile of a myoblast H9c2 cell line under starvation and ischemia, to better understand and recognize new biomarkers for myocardial infarction. Lipidomic profile was evaluated by HILIC-LC-MS and GC-MS. Cardiac cells showed alterations in phosphatidylcholines PC (34:1) and PC (36:2), lysophosphatidylcholines lyso PC(16:0), lysoPC(18:1) and lysoPC(18:0), phosphatidylethanolamine PE (34:1), phosphatidylserine PS (36:1), phosphatidylinositol PI (36:2), PI (38:3) and PI (38:5), sphingomyelin SM (34:1) and cardiolipins CL(68:4), CL(72:5) and CL(74:7) in ischemia and/or starvation, in comparison with control. Specific differences observed only in starvation were decrease of SM (34:1) and FA (20:4), and increase of PS (36:1). Differences observed only in ischemia were decrease of PC (36:2), lyso PC (16:0) and FA (18:1) and simultaneous increase of FA (16:0), and FA (18:0). Interestingly, PC (34:1) increased in ischemia and decreased in starvation. In conclusion, our work suggests that lipids are potential markers for evaluation of cell fate, either cell death or recovery, which will be useful to improve diagnosis and prognostic of cardiovascular diseases. J. Cell. Physiol. 231: 2266-2274, 2016. © 2016 Wiley Periodicals, Inc.

Phospholipidomic Profile Variation on THP-1 Cells Exposed to Skin or Respiratory Sensitizers and Respiratory Irritant.

Occupational exposure to low molecular weight reactive chemicals often leads to development of allergic reactions such as allergic contact dermatitis and respiratory allergies. Further insights into the interaction of these chemicals with physiopathological relevant cellular models might provide the foundations for novel non-animal approaches to safety assessment. In this work we used the human THP-1 cell line to determine phospholipidome changes induced by the skin sensitizer 1-fluoro-2,4-dinitrobenzene (DNFB), the respiratory allergen hexamethylene diisocyanate (HDI), and the irritant methyl salicylate (MESA). We detected that these chemicals differently induce lipid peroxidation and modulate THP-1 IL-1ß, IL-12B, IL-8, CD86, and HMOX1 transcription. Decreased phosphatidylethanolamine content was detected in cells exposed to MESA, while profound alterations in the relative abundance of cardiolipin species were observed in cells exposed to DNFB. All chemicals tested induced a decrease in the relative abundance of plasmanyl phosphatidylcholine species PC (O-16:0e/18:1) and phosphatidylinositol species PI (34:1), while increasing PI (38:4). An increased abundance of oleic acid was observed in the phospholipids of cells exposed to DNFB while a decreased abundance of palmitic acid was detected in cells treated with MESA or DNFB. We conclude that both specific and common alterations at phospholipidome levels are triggered by the different chemicals, while not allowing a complete distinction between them using a Canonical Analysis of Principal Coordinates (CAP). The common effects observed at phospholipids level with all the chemicals tested might be related to unspecific cell cytotoxic mechanisms that nevertheless may contribute to the elicitation of specific immune responses. J. Cell. Physiol. 231: 2639-2651, 2016. © 2016 Wiley Periodicals, Inc.

Bioprospecting of Marine Macrophytes Using MS-Based Lipidomics as a New Approach.

The marine environment supports a remarkable diversity of organisms which are a potential source of natural products with biological activities. These organisms include a wide variety of marine plants (from micro- to macrophytes), which have been used in the food and pharmaceutical industry. However, the biochemistry and biological activities of many of these macrophytes (namely macroalgae and halophytes, including seagrasses) are still far from being fully explored. Most popular bioactive components include polysaccharides, peptides, phenolics and fatty acids (FAs). Polar lipids (glycolipids, phospholipids and betaine lipids) are emerging as novel value-added bioactive phytochemicals, rich in n-3 FA, with high nutritional value and health beneficial effects for the prevention of chronic diseases. Polar lipids account various combinations of polar groups, fatty acyl chains and backbone structures. The polar lipidome of macrophytes is remarkably diverse, and its screening represents a significant analytical challenge. Modern research platforms, particularly mass spectrometry (MS)-based lipidomic approaches, have been recently used to address this challenge and are here reviewed. The application of lipidomics to address lipid composition of marine macrophytes will contribute to the stimulation of further research on this group and foster the exploration of novel applications.

Molecular insights into mitochondrial dysfunction in cancer-related muscle wasting.

Alterations in muscle mitochondrial bioenergetics during cancer cachexia were previously suggested; however, the underlying mechanisms are not known. So, the goal of this study was to evaluate mitochondrial phospholipid remodeling in cancer-related muscle wasting and its repercussions to respiratory chain activity and fiber susceptibility to apoptosis. An animal model of urothelial carcinoma induced by exposition to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and characterized by significant body weight loss due to skeletal muscle mass decrease was used. Morphological evidences of muscle atrophy were associated to decreased respiratory chain activity and increased expression of mitochondrial UCP3, which altogether highlight the lower ability of wasted muscle to produce ATP. Lipidomic analysis of isolated mitochondria revealed a significant decrease of phosphatidic acid, phosphatidylglycerol and cardiolipin in BBN mitochondria, counteracted by increased phosphatidylcholine levels. Besides the impact on membrane fluidity, this phospholipid remodeling seems to justify, at least in part, the lower oxidative phosphorylation activity observed in mitochondria from wasted muscle and their increased susceptibility to apoptosis. Curiously, no evidences of lipid peroxidation were observed but proteins from BBN mitochondria, particularly the metabolic ones, seem more prone to carbonylation with the consequent implications in mitochondria functionality. Overall, data suggest that bladder cancer negatively impacts skeletal muscle activity specifically by affecting mitochondrial phospholipid dynamics and its interaction with proteins, ultimately leading to the dysfunction of this organelle. The regulation of phospholipid biosynthetic pathways might be seen as potential therapeutic targets for the management of cancer-related muscle wasting.

Detection of phosphatidylserine with a modified polar head group in human keratinocytes exposed to the radical generator AAPH.

Phosphatidylserine (PS) is preferentially located in the inner leaflet of the cell membrane, and translocation of PS oxidized in fatty acyl chains to the outside of membrane has been reported as signaling to macrophage receptors to clear apoptotic cells. It was recently shown that PS can be oxidized in serine moiety of polar head-group. In the present work, a targeted lipidomic approach was applied to detecting OxPS modified at the polar head-group in keratinocytes that were exposed to the radical generator AAPH. Glycerophosphoacetic acid derivatives (GPAA) were found to be the major oxidation products of OxPS modified at the polar head-group during oxidation induced by AAPH-generated radicals, similarly to previous observations for the oxidation induced by OH radical. The neutral loss scan of 58Da and a novel precursor ion scan of m/z 137.1 (HOPO3CH2COOH) allowed the recognition of GPAA derivatives in the total lipid extracts obtained from HaCaT cells treated with AAPH. The positive identification of serine head group oxidation products in cells under controlled oxidative conditions opens new perspectives and justifies further studies in other cellular environments in order to understand fully the role of PS polar head-group oxidation in cell homeostasis and disease.

Evaluation of the photooxidation of galactosyl- and lactosylceramide by electrospray ionization mass spectrometry.

RATIONALE: Glycosphingolipids are important lipid molecules namely as constituents of the plasma membrane organized in lipid rafts, in signal transduction, and cell-cell communication. Although many human diseases are associated with oxidative stress and lipid oxidation, a link between oxidative stress and modification of glycosphingolipids has never been addressed. METHODS: In this study, the structural changes caused by UVA-induced photooxidation of galactosyl- (GalCer) and lactosylceramide (LacCer) molecular species were studied by electrospray ionization mass spectrometry (ESI-MS and MS/MS), using a quadrupole time-of-flight (QTOF) mass spectrometer and high-performance liquid chromatography/tandem mass spectrometry with a C5 stationary phase (C5 HPLC/MS/MS) using a linear ion trap. RESULTS: ESI-MS spectra of GalCer and LacCer after photooxidation showed new ions with a mass shift of +32 Da when compared with the ions of the non-modified glycosphingolipids. These new species were assigned as hydroperoxyl derivatives, confirmed by HPLC/MS/MS and through FOX 2 assay. In the ESI-MS and LC/MS of lactosylceramide a new ion with lower m/z value, assigned as glucosylceramide (GlcCer) + 32 Da, was also detected and proposed to be formed due to oxidative cleavage of lactosyl moieties. ESI-MS/MS of the oxidized species allowed us to infer the presence of isomeric hydroperoxyl derivatives, with the hydroperoxyl moiety either linked to the sphingosine backbone or in the unsaturated acyl chain. Oxidation in the sugar moieties was observed in the case of LacCer, suggesting an oxidation via radical reactive oxygen species that can induce the oxidative cleavage of the lactosyl moiety. CONCLUSIONS: This study shows that glycosphingolipids are prone to oxidation and the identified mass spectrometry fingerprint of oxidized galactosyl- and lactosylceramide species will support their future identification in lipidomic studies of biological samples under oxidative conditions.

Fatty Acid Profiling as a Tool for Fostering the Traceability of the Halophyte Plant Salicornia ramosissima and Contributing to Its Nutritional Valorization.

Salicornia ramosissima, commonly known as glasswort or sea asparagus, is a halophyte plant cultivated for human consumption that is often referred to as a sea vegetable rich in health-promoting n-3 fatty acids (FAs). Yet, the effect of abiotic conditions, such as salinity and temperature, on the FA profile of S. ramosissima remains largely unknown. These factors can potentially shape its nutritional composition and yield unique fatty acid signatures that can reveal its geographical origin. In this context, samples of S. ramosissima were collected from four different locations along the coastline of mainland Portugal and their FAs were profiled through gas chromatography-mass spectrometry. The lipid extracts displayed a high content of essential FAs, such as 18:2n-6 and 18:3n-3. In addition to an epoxide fatty acid exclusively identified in samples from the Mondego estuary, the relative abundance of FAs varied between origin sites, revealing that FA profiles can be used as site-specific lipid fingerprints. This study highlights the role of abiotic conditions on the nutritional profile of S. ramosissima and establishes FA profiling as a potential avenue to trace the geographic origin of this halophyte plant. Overall, the present approach can make origin certification possible, safeguard quality, and enhance consumers' trust in novel foods.

Infodemic and sources of information about COVID-19 in a Brazilian population: what are the associated factors?

BACKGROUND: With the COVID-19 pandemic, social isolation, and information search have increased dramatically. This increased search for information about the Coronavirus, called infodemic, was greatly affected by fake news and information without scientific evidence. This article aimed to assess the infodemic amid the COVID-19 pandemic and its association with sociodemographic and pandemic-related variables, as well as describe the main sources from which people obtained information about COVID-19. METHODS: A cross-sectional population-based study was performed in Criciúma, Brazil. All individuals aged 18 years or older, residing in the 607 households systematically selected, were invited to answer the questionnaire. Infodemic and sources to seek information about COVID-19 were evaluated, as well as sociodemographic and pandemic-related variables. Adjusted Poisson regression with robust variance was used to evaluate associations. RESULTS: A total of 863 individuals participated in the study. The prevalence of infodemic was 22.1%, and television was the main source of information (58.9%). Three groups presented a higher prevalence of infodemic: older adults (PR: 1.65), individuals with low income (PR: 2.97), and those who had had contact with someone infected (PR: 2.20). CONCLUSIONS: The findings reflect how some groups are more exposed to infodemic, and underline the responsibility and importance of intersectoral actions for dissemination of information about COVID-19.

Prospective phospholipid markers for skin sensitization prediction in keratinocytes: a phospholipidomic approach.

Prevalence of skin inflammatory disorders has increased in recent years being estimated that 15-20% of the general population suffers from allergic contact dermatitis (ACD). Currently, the sensitizing potential of chemicals is assessed through animal tests; however growing ethical concerns and actual legislative framework impose the development of new alternative tests. Several genomic and proteomic approaches have already indicated some potential biomarkers, but lipidomic analysis was not so far explored with this purpose. A growing body of data suggests that phospholipids (PLs) play important roles in the modulation of immune responses. Therefore, this work focused in identifying changes in the PLs profile of human keratinocytes (KCs). For that, HaCaT cell line was exposed to two immune stimulators: the strong skin allergen 2,4-dinitrofluorobenzene (DNFB) and the non-allergenic stimulus LPS, and to the irritant benzalkonium chloride (BC), using off line TLC-ESI-MS, HPLC-MS and MS/MS. LPS and DNFB reduced PS class relative content, corroborating with consistent changes observed in its molecular profile. PC profile was also altered by immune stimulators. These findings suggest that PC and PS molecular species may discriminate immunogenic compounds from irritants. Analysis of such alterations may be therefore valuable in a future in vitro test platform for skin sensitization prediction.

Photodynamic oxidation of Escherichia coli membrane phospholipids: new insights based on lipidomics.

RATIONALE: The irreversible oxidation of biological molecules, such as lipids, can be achieved with a photosensitizing agent and subsequent exposure to light, in the presence of molecular oxygen. Although lipid peroxidation is an important toxicity mechanism in bacteria, the alterations caused by the photodynamic therapy on bacterial phospholipids are still unknown. In this work, we studied the photodynamic oxidation of Escherichia coli membrane phospholipids using a lipidomic approach. METHODS: E. coli ATCC 25922 were irradiated for 90 min with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of a tricationic porphyrin [(5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF]. Lipids were extracted and separated by thin-layer chromatography. Phospholipid classes were quantified by phosphorus assay and analyzed by electrospray ionization tandem mass spectrometry. Fatty acids were analyzed by gas chromatography. Quantification of lipid hydroperoxides was performed by FOX2 assay. Analysis of the photodynamic oxidation of a phospholipid standard was also performed. RESULTS: Our approach allowed us to see that the photodynamic treatment induced the formation of a high amount of lipid hydroperoxides in the E. coli lipid extract. Quantification of fatty acids revealed a decrease in the unsaturated C16:1 and C18:1 species suggesting that oxidative modifications were responsible for their variation. It was also observed that photosensitization induced the oxidation of phosphatidylethanolamines with C16:1, C18:1 and C18:2 fatty acyl chains, with formation of hydroxy and hydroperoxy derivatives. CONCLUSIONS: Membrane phospholipids of E. coli are molecular targets of the photodynamic effect induced by Tri-Py(+) -Me-PF. The overall change in the relative amount of unsaturated fatty acids and the formation of PE hydroxides and hydroperoxides evidence the damages in bacterial phospholipids caused by this lethal treatment.

Rapeseed oil-rich diet alters hepatic mitochondrial membrane lipid composition and disrupts bioenergetics.

Diet is directly related with physiological alterations occurring at a cell and subcellular level. However, the role of diet manipulation on mitochondrial physiology is still largely unexplored. Aiming at correlating diet with alterations of mitochondrial membrane composition and bioenergetics, Wistar-Han male rats were fed for 11, 22 and 33 days with a rapeseed oil-based diet and mitochondrial bioenergetics, and membrane composition were compared at each time point with a standard diet group. Considerable differences were noticed in mitochondrial membrane lipid composition, namely in terms of fatty acyl chains and relative proportions of phospholipid classes, the modified diet inducing a decrease in the saturated to unsaturated molar ratio and an increase in the phosphatidylcholine to phosphatidylethanolamine molar ratio. Mass spectrometry lipid analysis showed significant differences in the major species of cardiolipin, with an apparent increased incorporation of oleic acid as a result of exposure to the modified diet. Rats fed the modified diet during 22 days showed decreased hepatic mitochondrial state 3 respiration and were more susceptible to Ca(2+)-induced transition pore opening. Rapeseed oil-enriched diet also appeared to promote a decrease in hydroperoxide production by the respiratory chain, although a simultaneous decrease in vitamin E content was detected. In conclusion, our data indicate that the rapeseed oil diet causes negative alterations on hepatic mitochondrial bioenergetics, which may result from membrane remodeling. Such alterations may have an impact not only on energy supply to the cell, but also on drug-induced hepatic mitochondrial liabilities.

Tacrine and its analogues impair mitochondrial function and bioenergetics: a lipidomic analysis in rat brain.

Tacrine is an acetylcholinesterase (AChE) inhibitor used as a cognitive enhancer in the treatment of Alzheimer's disease (AD). However, its low therapeutic efficiency and a high incidence of side effects have limited its clinical use. In this study, the molecular mechanisms underlying the impact on brain activity of tacrine and two novel tacrine analogues (T1, T2) were approached by focusing on three aspects: (i) their effects on brain cholinesterase activity; (ii) perturbations on electron transport chain enzymes activities of non-synaptic brain mitochondria; and (iii) the role of mitochondrial lipidome changes induced by these compounds on mitochondrial bioenergetics. Brain effects were evaluated 18 h after the administration of a single dose (75.6 µmol/kg) of tacrine or tacrine analogues. The three compounds promoted a significant reduction in brain AChE and butyrylcholinesterase (BuChE) activities. Additionally, tacrine was shown to be more efficient in brain AChE inhibition than T2 tacrine analogue and less active than T1 tacrine analogue, whereas BuChE inhibition followed the order: T1 > T2 > tacrine. The studies using non-synaptic brain mitochondria show that all the compounds studied disturbed brain mitochondrial bioenergetics mainly via the inhibition of complex I activity. Furthermore, the activity of complex IV is also affected by tacrine and T1 treatments while FoF(1) -ATPase is only affected by tacrine. Therefore, the compounds' toxicity as regards brain mitochondria, which follows the order: tacrine >> T1 > T2, does not correlate with their ability to inhibit brain cholinesterase enzymes. Lipidomics approaches show that phosphatidylethanolamine (PE) is the most abundant phospholipids (PL) class in non-synaptic brain mitochondria and cardiolipin (CL) present the greatest diversity of molecular species. Tacrine induced significant perturbations in the mitochondrial PL profile, which were detected by means of changes in the relative abundance of phosphatidylcholine (PC), PE, phosphatidylinositol (PI) and CL and by the presence of oxidized phosphatidylserines. Additionally, in both the T1 and T2 groups, the lipid content and molecular composition of brain mitochondria PL are perturbed to a lesser extent than in the tacrine group. Abnormalities in CL content and the amount of oxidized phosphatidylserines were associated with significant reductions in mitochondrial enzymes activities, mainly complex I. These results indicate that tacrine and its analogues impair mitochondrial function and bioenergetics, thus compromising the activity of brain cells.

Evaluation of the capacity of oxidized phosphatidylserines to induce the expression of cytokines in monocytes and dendritic cells.

Oxidized phospholipids are known to be key signaling molecules in the onset of several diseases involving inflammation. The aim of this study was to evaluate the effect of oxidized phosphatidylserines (oxPS) in modulating the immune system, through cytokine production. Flow cytometry analysis was used to evaluate the oxPS capacity to induce the expression of different cytokines by monocytes, myeloid dendritic cells (mDCs) and DCs CD14(-/low)CD16(+). oxPS were formed during oxidation induced by the hydroxyl radical. Among the four families of oxPS studied, only oxPS modified in the polar head with formation of a terminal hydroperoxyacetaldehyde upregulated the production of cytokines IL-8 and TNF-α by monocytes and DCs subsets (mDCs and CD14(-/low)CD16(+) DCs). This family of oxPS showed the capacity to upregulate the production of IL-1ß, IL-6, and MIP-1ß from the same type of cells. A significant raise in the percentage of monocytes and dendritic cells producing the studied cytokines was observed, when compared with basal control. Oxidation products modified in the fatty acyl chain did not upregulate TNF and IL-8. oxPS with terminal hydroperoxyacetaldehyde has pro-inflammatory properties. This outcome may help to understand the biological role of phosphatidylserine oxidation products in inflammatory processes and in dysfunctions of immune system.

Profiling changes triggered during maturation of dendritic cells: a lipidomic approach.

Lipids are important in several biological processes because they act as signalling and regulating molecules, or, locally, as membrane components that modulate protein function. This paper reports the pattern of lipid composition of dendritic cells (DCs), a cell type of critical importance in inflammatory and immune responses. After activation by antigens, DCs undergo drastic phenotypical and functional transformations, in a process known as maturation. To better characterize this process, changes of lipid profile were evaluated by use of a lipidomic approach. As an experimental model of DCs, we used a foetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). The results showed that LPS treatment increased ceramide (Cer) and phosphatidylcholine (PC) levels and reduced sphingomyelin (SM) and phosphatidylinositol (PI) content. Mass spectrometric analysis of a total lipid extract and of each class of lipids revealed that maturation promoted clear changes in ceramide profile. Quantitative analysis enabled identification of an increase in the total ceramide content and enhanced Cer at m/z 646.6, identified as Cer(d18:1/24:1), and at m/z 648.6, identified as Cer(d18:1/24:0). The pattern of change of these lipids give an extremely rich source of data for evaluating modulation of specific lipid species triggered during DC maturation.

Liquid chromatography/tandem mass spectrometry analysis of long-chain oxidation products of cardiolipin induced by the hydroxyl radical.

The anionic phospholipid cardiolipin (CL) is found almost exclusively in the inner membrane of mitochondria, playing an important role in energy metabolism. Oxidation of CL has been associated with apoptotic events and various pathologies. In this study, electrospray ionization mass spectrometry coupled with liquid chromatography (LC/ESI-MS) was used to identify tetralinoleoyl-cardiolipin (TLCL) modifications induced by the OH(·) radical generated under Fenton reaction conditions (H(2)O(2) and Fe(2+)). The identified oxidation products of TLCL contained 2, 4, 6 and 8 additional oxygen atoms. These long-chain oxidation products were characterized by LC/ESI-MS/MS as doubly [M-2H](2-) and singly charged [M-H](-) ions. A detailed analysis of the fragmentation pathways of these precursor ions allowed the identification of hydroperoxy derivatives of CL. MS/MS analysis indicated that CL oxidation products with 4, 6 and 8 oxygen atoms have one fatty acyl chain bearing 4 oxygen atoms ([RCOO+4O](-)). Even when the TLCL molecule was oxidized by the addition of eight oxygen atoms, one of the acyl chains remained non-modified and one fatty acyl chain contained three or four oxygen atoms. This led us to conclude that under oxidative conditions by the OH(·) radical, the distribution of oxygens/peroxy groups in the CL molecule is not random, even when CL has the same fatty acyl chains in all the positions. Using mass spectrometry, the oxidation products have been unequivocally assigned, which may be useful for their detection in biological samples.

Influence of amino acid relative position on the oxidative modification of histidine and glycine peptides.

The radical oxidation of isomeric peptides containing one reactive amino acid [histidine (H)] and another less reactive amino acid [glycine (G)] in the form of dipeptides (HG and GH) and tripeptides (HGG, GHG, and GGH) was studied by mass spectrometry coupled to liquid chromatography (LC-MS) for detection and LC-MS(n) for structural characterization. The oxidation products identified were keto, hydroxy, keto-hydroxy, and hydroperoxide derivatives for both di- and tripeptides. Among these, it was found that insertion of oxygen atoms occurred at histidine for HG and HGG, and both histidine and glycine for GH, GHG, and GGH. In addition, oxidation products formed by alkoxyl rearrangement reactions with cleavage of the peptide chain were also identified for GH, GHG, and GGH, corroborating hydrogen abstraction step in G residues. These findings were supported through the identification of radical intermediate species formed and trapped with 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) spin trap. The observation of DMPO adducts bearing two spin trap molecules reinforced the abstraction of two hydrogen atoms from the same molecule. Overall, modification sites identified showed that the outcome of oxidative behavior of peptides is influenced not only by the reactivity of the amino acid in the peptide sequence but also by its relative position within the sequence.

Unravelling the fatty acid profiles of different polychaete species cultured under integrated multi-trophic aquaculture (IMTA).

Polychaetes can be successfully employed to recover otherwise wasted nutrients present in particulate organic matter (POM) of aquaculture effluents. The present study describes the fatty acid (FA) profile of four different polychaete species cultured in sand filters supplied with effluent water from a marine fish farm. The FA profile of cultured and wild Hediste diversicolor was compared and revealed a ≈ 24.2% dissimilarity, with cultured biomass displaying a higher content in two essential n-3 highly unsaturated FA (HUFA) (EPA [20:5 n-3] and DHA [22:6 n-3]-eicosapentaenoic and docosahexaenoic acid, respectively). The comparison of the FA profile of cultured H. diversicolor with that of other polychaete species whose larvae successfully settled on the sand filters (Diopatra neapolitana, Sabella cf. pavonina and Terebella lapidaria) revealed that their FA profile, which is here described for the first time, displayed high levels of EPA and DHA (≈ 1.5-4.8 and 1.0-1.1 µg mg-1 DW, respectively). The highest concentration of total FA per biomass of polychaete was recorded in H. diversicolor and T. lapidaria, with both species being the ones whose FA profiles revealed a lowest level of dissimilarity and more closely resembled that of the aquafeed used in the fish farm. In the present work it was demonstrated that it is possible to produce polychaetes biomass with high nutritional value through an eco-design concept such as integrated multi-trophic aquaculture (IMTA). Indeed, this framework promotes a cleaner production and, in this specific case, allowed to recover essential fatty acids that are commonly wasted in aquaculture effluents.