Erythrocyte membrane fluidity: A novel biomarker of residual cardiovascular risk in type 2 diabetes.

AIMS: Improving the composition of circulating fatty acids (FA) leads to a reduction in cardiovascular diseases (CVD) in high-risk individuals. The membrane fluidity of red blood cells (RBC), which reflects circulating FA status, may be a valid biomarker of cardiovascular (CV) risk in type 2 diabetes (T2D). METHODS: Red blood cell membrane fluidity, quantified as general polarization (GP), was assessed in 234 subjects with T2D, 86 with prior major CVD. Based on GP distribution, a cut-off of .445 was used to divide the study cohort into two groups: the first with higher GP, called GEL, and the second, defined as lower GP (LGP). Lipidomic analysis was performed to evaluate FA composition of RBC membranes. RESULTS: Although with comparable CV risk factors, the LGP group had a greater percentage of patients with major CVD than the GEL group (40% vs 24%, respectively, p < .05). Moreover, in a logistic regression analysis, a lower GP value was independently associated with the presence of macrovascular complications. Lipidomic analysis showed a clear shift of LGP membranes towards a pro-inflammatory condition due to higher content of arachidonic acid and increased omega 6/omega 3 index. CONCLUSIONS: Increased membrane fluidity is associated with a higher CV risk in subjects with T2D. If confirmed in prospective studies, membrane fluidity could be a new biomarker for residual CV risk assessment in T2D.

Fatty Acids Profile and the Relevance of Membranes as the Target of Nutrition-Based Strategies in Atopic Dermatitis: A Narrative Review.

Recently, the prevalence of atopic dermatitis has increased drastically, especially in urban populations. This multifactorial skin disease is caused by complex interactions between various factors including genetics, environment, lifestyle, and diet. In eczema, apart from using an elimination diet, the adequate content of fatty acids from foods (saturated, monounsaturated, and polyunsaturated fatty acids) plays an important role as an immunomodulatory agent. Different aspects regarding atopic dermatitis include connections between lipid metabolism in atopic dermatitis, with the importance of the MUFA levels, as well as of the omega-6/omega-3 balance that affects the formation of long-chain (C20 eicosanoic and C22 docosaenoic) fatty acids and bioactive lipids from them (such as prostaglandins). Impair/repair of the functioning of epidermal barrier is influenced by these fatty acid levels. The purpose of this review is to drive attention to membrane fatty acid composition and its involvement as the target of fatty acid supplementation. The membrane-targeted strategy indicates the future direction for dermatological research regarding the use of nutritional synergies, in particular using red blood cell fatty acid profiles as a tool for checking the effects of supplementations to reach the target and influence the inflammatory/anti-inflammatory balance of lipid mediators. This knowledge gives the opportunity to develop personalized strategies to create a healthy balance by nutrition with an anti-inflammatory outcome in skin disorders.

Geometrical isomerization of arachidonic acid during lipid peroxidation interferes with ferroptosis.

Geometrical mono-trans isomers of arachidonic acid (mtAA) are endogenous products of free radical-induced cis-trans double bond isomerization occurring to natural fatty acids during cell metabolism, including lipid peroxidation (LPO). Very little is known about the functional roles of mtAA and in general on the effects of mono-trans isomers of polyunsaturated fatty acids (mtPUFA) in various types of programmed cell death, including ferroptosis. Using HT1080 and MEF cell cultures, supplemented with 20 µM PUFA (i.e., AA, EPA or DHA) and their mtPUFA congeners, ferroptosis occurred in the presence of RSL3 (a direct inhibitor of glutathione peroxidase 4) only with the PUFA in their natural cis configuration, whereas mtPUFA showed an anti-ferroptotic effect. By performing the fatty acid-based membrane lipidome analyses, substantial differences emerged in the membrane fatty acid remodeling of the two different cell fates. In particular, during ferroptosis mtPUFA formation and their incorporation, together with the enrichment of SFA, occurred. This opens new perspectives in the role of the membrane composition for a ferroptotic outcome. While pre-treatment with AA promoted cell death for treatment with H2O2 and RSL3, mtAA did not. Cell death by AA supplementation was suppressed also in the presence of either ferroptosis inhibitors, such as the lipophilic antioxidant ferrostatin-1, or NADPH oxidase (NOX) inhibitors, including diphenyleneiodonium chloride and apocynin. Our results confirm a more complex scenario for ferroptosis than actually believed. While LPO processes are active, the importance of environmental lipid levels, balance among SFA, MUFA and PUFA in lipid pools and formation of mtPUFA influence the membrane phospholipid turnover, with crucial effects in the occurrence of cell death by ferroptosis.

Plasmalogens: Free Radical Reactivity and Identification of Trans Isomers Relevant to Biological Membranes.

Plasmalogens are membrane phospholipids with two fatty acid hydrocarbon chains linked to L-glycerol, one containing a characteristic cis-vinyl ether function and the other one being a polyunsaturated fatty acid (PUFA) residue linked through an acyl function. All double bonds in these structures display the cis geometrical configuration due to desaturase enzymatic activity and they are known to be involved in the peroxidation process, whereas the reactivity through cis-trans double bond isomerization has not yet been identified. Using 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (C18 plasm-20:4 PC) as a representative molecule, we showed that the cis-trans isomerization can occur at both plasmalogen unsaturated moieties, and the product has characteristic analytical signatures useful for omics applications. Using plasmalogen-containing liposomes and red blood cell (RBC) ghosts under biomimetic Fenton-like conditions, in the presence or absence of thiols, peroxidation, and isomerization processes were found to occur with different reaction outcomes due to the particular liposome compositions. These results allow gaining a full scenario of plasmalogen reactivity under free radical conditions. Moreover, clarification of the plasmalogen reactivity under acidic and alkaline conditions was carried out, identifying the best protocol for RBC membrane fatty acid analysis due to their plasmalogen content of 15-20%. These results are important for lipidomic applications and for achieving a full scenario of radical stress in living organisms.

Erythrocyte Plasma Membrane Lipid Composition Mirrors That of Neurons and Glial Cells in Murine Experimental In Vitro and In Vivo Inflammation.

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly.

Phospholipid fatty acid remodeling and carbonylated protein increase in extracellular vesicles released by airway epithelial cells exposed to cigarette smoke extract.

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

Critical Review on Fatty Acid-Based Food and Nutraceuticals as Supporting Therapy in Cancer.

Fatty acids have an important place in both biological and nutritional contexts and, from a clinical point of view, they have known consequences for diseases' onset and development, including cancer. The use of fatty acid-based food and nutraceuticals to support cancer therapy is a multidisciplinary subject, involving molecular and clinical research. Knowledge regarding polyunsaturated fatty acids essentiality/oxidizability and the role of lipogenesis-desaturase pathways for cell growth, as well as oxidative reactivity in cancer cells, are discussed, since they can drive the choice of fatty acids using their multiple roles to support antitumoral drug activity. The central role of membrane fatty acid composition is highlighted for the application of membrane lipid therapy. As fatty acids are also known as biomarkers of cancer onset and progression, the personalization of the fatty acid-based therapy is also possible, taking into account other important factors such as formulation, bioavailability and the distribution of the supplementation. A holistic approach emerges combining nutra- and pharma-strategies in an appropriate manner, to develop further knowledge and applications in cancer therapy.

Effects of Oxygen Tension for Membrane Lipidome Remodeling of Cockayne Syndrome Cell Models.

Oxygen is important for lipid metabolism, being involved in both enzymatic transformations and oxidative reactivity, and is particularly influent when genetic diseases impair the repair machinery of the cells, such as described for Cockayne syndrome (CS). We used two cellular models of transformed fibroblasts defective for CSA and CSB genes and their normal counterparts, grown for 24 h under various oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%) to examine the fatty acid-based membrane remodeling by GC analysis of fatty acid methyl esters derived from membrane phospholipids. Overall, we first distinguished differences due to oxygen tensions: (a) hyperoxia induced a general boost of desaturase enzymatic activity in both normal and defective CSA and CSB cell lines, increasing monounsaturated fatty acids (MUFA), whereas polyunsaturated fatty acids (PUFA) did not undergo oxidative consumption; (b) hypoxia slowed down desaturase activities, mostly in CSA cell lines and defective CSB, causing saturated fatty acids (SFA) to increase, whereas PUFA levels diminished, suggesting their involvement in hypoxia-related signaling. CSB-deprived cells are the most sensitive to oxidation and CSA-deprived cells are the most sensitive to the radical-based formation of trans fatty acids (TFA). The results point to the need to finely differentiate biological targets connected to genetic impairments and, consequently, suggest the better definition of cell protection and treatments through accurate molecular profiling that includes membrane lipidomes.

Extracellular circulating miRNAs as stress-related signature to search and rescue dogs.

Our research explores serum extracellular circulating miRNAs (ecmiRNAs) involved in dog stress response immediately after the search and rescue (SAR) of missing people. The experimental plan considers four arduous SAR simulations. The SAR dogs are trained by the Alpine School of the Military Force of Guardia di Finanza (Passo Rolle, Italy). The First SAR Trial analyzed dog serum samples at rest time (T0), and immediately after SAR performance (T1) using the miRNome-wide screening next-generation sequencing (NGS). T1 versus T0 NGS results revealed a different expression level of let-7a and let-7f. Subsequently, in a large sample size including: 1st (n = 6), 2nd (n = 6), 3rd (n = 6), and 4th (n = 4) trials, let-7a and let-7f were validated by qPCR. Bioinformatics analysis with TarBase (v.8) and the Diana-mirPath (v.3) revealed a functional role of let-7a and let-7f in the p53 pathway to restore cellular homeostasis. Let-7a and let-7f, highly expressed at T1, could stop MDMs-p53 inhibition inducing the p53 increase in level. In addition, let-7a and let-7f, via p53 post-transcriptional regulation, buffers p53 transcription spikes. During SAR stress, the possibility of p53 preconditioning could explain the phenomenon of "stress hardening" where the tolerance of particular stress increases after preconditioning.

Sapienic Acid Metabolism Influences Membrane Plasticity and Protein Signaling in Breast Cancer Cell Lines.

The importance of sapienic acid (6c-16:1), a monounsaturated fatty acid of the n-10 family formed from palmitic acid by delta-6 desaturase, and of its metabolism to 8c-18:1 and sebaleic acid (5c,8c-18:2) has been recently assessed in cancer. Data are lacking on the association between signaling cascades and exposure to sapienic acid comparing cell lines of the same cancer type. We used 50 µM sapienic acid supplementation, a non-toxic concentration, to cultivate MCF-7 and 2 triple-negative breast cancer cells (TNBC), MDA-MB-231 and BT-20. We followed up for three hours regarding membrane fatty acid remodeling by fatty acid-based membrane lipidome analysis and expression/phosphorylation of EGFR (epithelial growth factor receptor), mTOR (mammalian target of rapamycin) and AKT (protein kinase B) by Western blotting as an oncogenic signaling cascade. Results evidenced consistent differences among the three cell lines in the metabolism of n-10 fatty acids and signaling. Here, a new scenario is proposed for the role of sapienic acid: one based on changes in membrane composition and properties, and the other based on changes in expression/activation of growth factors and signaling cascades. This knowledge can indicate additional players and synergies in breast cancer cell metabolism, inspiring translational applications of tailored membrane lipid strategies to assist pharmacological interventions.

Targeted and untargeted metabolomic approach for GDM diagnosis.

Gestational diabetes mellitus (GDM) is a disorder which manifests itself for the first time during pregnancy and is mainly connected with glucose metabolism. It is also known that fatty acid profile changes in erythrocyte membranes and plasma could be associated with obesity and insulin resistance. These factors can lead to the development of diabetes. In the reported study, we applied the untargeted analysis of plasma in GDM against standard glucose-tolerant (NGT) women to identify the differences in metabolomic profiles between those groups. We found higher levels of 2-hydroxybutyric and 3-hydroxybutyric acids. Both secondary metabolites are associated with impaired glucose metabolism. However, they are products of different metabolic pathways. Additionally, we applied lipidomic profiling using gas chromatography to examine the fatty acid composition of cholesteryl esters in the plasma of GDM patients. Among the 14 measured fatty acids characterizing the representative plasma lipidomic cluster, myristic, oleic, arachidonic, and α-linoleic acids revealed statistically significant changes. Concentrations of both myristic acid, one of the saturated fatty acids (SFAs), and oleic acid, which belong to monounsaturated fatty acids (MUFAs), tend to decrease in GDM patients. In the case of polyunsaturated fatty acids (PUFAs), some of them tend to increase (e.g., arachidonic), and some of them tend to decrease (e.g., α-linolenic). Based on our results, we postulate the importance of hydroxybutyric acid derivatives, cholesteryl ester composition, and the oleic acid diminution in the pathophysiology of GDM. There are some evidence suggests that the oleic acid can have the protective role in diabetes onset. However, metabolic alterations that lead to the onset of GDM are complex; therefore, further studies are needed to confirm our observations.

Fatty-Acid-Based Membrane Lipidome Profile of Peanut Allergy Patients: An Exploratory Study of a Lifelong Health Condition.

Peanut allergy is a lifelong, increasingly prevalent, and potentially life-threatening disease burdening families and communities. Dietary, particularly polyunsaturated fatty acids (PUFAs), intakes can exert positive effects on immune and inflammatory responses, and the red blood cell (RBC) membrane lipidome contains stabilized metabolic and nutritional information connected with such responses. The fatty-acid-based membrane lipidome profile has been exploratorily evaluated in a small cohort of patients (eight males and one female, age range 4.1−21.7 years old, body mass index BMI < 25) with angioedema and/or anaphylaxis after peanut ingestion. This analysis was performed according to an ISO 17025 certified robotic protocol, isolating mature RBCs, extracting membrane lipids, and transforming them to fatty acid methyl esters for gas chromatography recognition and quantification. Comparison with a group of age- and BMI-matched healthy individuals and with benchmark interval values of a healthy population evidenced significant differences, such as higher levels of ω-6 (arachidonic acid), lower values of ω-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), together with an increased ω-6/ω-3 ratio in allergic patients. A significant inverse correlation was also found between specific immunoglobulin E (IgE) levels and ω-6 di-homo-gamma-linolenic acid (DGLA) and total PUFAs. Results of this preliminary study encourage screenings in larger cohorts, also in view of precision nutrition and nutraceuticals strategies, and stimulate interest to expand basic and applied research for unveiling molecular mechanisms that are still missing and individuating treatments in chronic allergic disorders.

Effect of a Fiber D-Limonene-Enriched Food Supplement on Intestinal Microbiota and Metabolic Parameters of Mice on a High-Fat Diet.

Several studies showed that D-Limonene can improve metabolic parameters of obese mice via various mechanisms, including intestinal microbiota modulation. Nevertheless, its effective doses often overcome the acceptable daily intake, rising concerns about toxicity. In this study we administered to C57BL/6 mice for 84 days a food supplement based on D-Limonene, adsorbed on dietary fibers (FLS), not able to reach the bloodstream, to counteract the metabolic effects of a high-fat diet (HFD). Results showed that daily administration of D-Limonene (30 and 60 mg/kg body weight) for 84 days decreased the weight gain of HFD mice. After 84 days we observed a statistically significant difference in weight gain in the group of mice receiving the higher dose of FLS compared to HFD mice (35.24 ± 4.56 g vs. 40.79 ± 3.28 g, p < 0.05). Moreover, FLS at both doses tested was capable of lowering triglyceridemia and also fasting glycemia at the higher dose. Some insights on the relevant fatty acid changes in hepatic tissues were obtained, highlighting the increased polyunsaturated fatty acid (PUFA) levels even at the lowest dose. FLS was also able to positively modulate the gut microbiota and prevent HFD-associated liver steatosis in a dose-dependent manner. These results demonstrate that FLS at these doses can be considered non-toxic and could be an effective tool to counteract diet-induced obesity and ameliorate metabolic profile in mice.

The Fatty Acid-Based Erythrocyte Membrane Lipidome in Dogs with Chronic Enteropathy.

Canine chronic enteropathies (CEs) are inflammatory processes resulting from complex interplay between the mucosal immune system, intestinal microbiome, and dietary components in susceptible dogs. Fatty acids (FAs) play important roles in the regulation of physiologic and metabolic pathways and their role in inflammation seems to be dual, as they exhibit pro-inflammatory and anti-inflammatory functions. Analysis of red blood cell (RBC) membrane fatty acid profile represents a tool for assessing the quantity and quality of structural and functional molecular components. This study was aimed at comparing the FA membrane profile, determined by Gas Chromatography and relevant lipid parameter of 48 CE dogs compared with 68 healthy dogs. In CE patients, the levels of stearic (p < 0.0001), dihomo-gamma-linolenic, eicosapentaenoic (p = 0.02), and docosahexaenoic (p = 0.02) acids were significantly higher, and those of palmitic (p < 0.0001) and linoleic (p = 0.0006) acids were significantly lower. Non-responder dogs presented higher percentages of vaccenic acid (p = 0.007), compared to those of dogs that responded to diagnostic trials. These results suggest that lipidomic status may reflect the "gut health", and the non-invasive analysis of RBC membrane might have the potential to become a candidate biomarker in the evaluation of dogs affected by CE.

The Erythrocyte Membrane Lipidome of Healthy Dogs: Creating a Benchmark of Fatty Acid Distribution and Interval Values.

Molecular-based approaches are rapidly developing in medicine for the evaluation of physiological and pathological conditions and discovery of new biomarkers in prevention and therapy. Fatty acid diversity and roles in health and disease in humans are topical subjects of lipidomics. In particular, membrane fatty acid-based lipidomics provides molecular data of relevance in the study of human chronic diseases, connecting metabolic, and nutritional aspects to health conditions. In veterinary medicine, membrane lipidomics, and fatty acid profiles have not been developed yet in nutritional approaches to health and in disease conditions. Using a protocol widely tested in human profiling, in the present study erythrocyte membrane lipidome was examined in 68 clinically healthy dogs, with different ages, sex, and sizes. In particular, a cluster composed of 10 fatty acids, present in membrane glycerophospholipids and representative of structural and functional properties of cell membrane, was chosen, and quantitatively analyzed. The interval values and distribution for each fatty acid of the cluster were determined, providing the first panel describing the healthy dog lipidomic membrane profile, with interesting correlation to bodyweight increases. This molecular information can be advantageously developed as benchmark in veterinary medicine for the evaluation of metabolic and nutritional status in healthy and diseased dogs.

The euploid blastocysts obtained after luteal phase stimulation show the same clinical, obstetric and perinatal outcomes as follicular phase stimulation-derived ones: a multicenter study.

STUDY QUESTION: Are the reproductive outcomes (clinical, obstetric and perinatal) different between follicular phase stimulation (FPS)- and luteal phase stimulation (LPS)-derived euploid blastocysts? SUMMARY ANSWER: No difference was observed between FPS- and LPS-derived euploid blastocysts after vitrified-warmed single embryo transfer (SET). WHAT IS KNOWN ALREADY: Technical improvements in IVF allow the implementation non-conventional controlled ovarian stimulation (COS) protocols for oncologic and poor prognosis patients. One of these protocols begins LPS 5 days after FPS is ended (DuoStim). Although, several studies have reported similar embryological outcomes (e.g. fertilization, blastulation, euploidy) between FPS- and LPS-derived cohort of oocytes, information on the reproductive (clinical, obstetric and perinatal) outcomes of LPS-derived blastocysts is limited to small and retrospective studies. STUDY DESIGN, SIZE, DURATION: Multicenter study conducted between October 2015 and March 2019 including all vitrified-warmed euploid single blastocyst transfers after DuoStim. Only first transfers of good quality blastocysts (≥BB according to Gardner and Schoolcraft's classification) were included. If euploid blastocysts obtained after both FPS and LPS were available the embryo to transfer was chosen blindly. The primary outcome was the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer in the two groups. To achieve 80% power (α = 0.05) to rule-out a 15% difference in the LBR, a total of 366 first transfers were required. Every other clinical, as well as obstetric and perinatal outcomes, were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Throughout the study period, 827 patients concluded a DuoStim cycle and among them, 339 did not identify any transferable blastocyst, 145 had an euploid blastocyst after FPS, 186 after LPS and 157 after both FPS and LPS. Fifty transfers of poor quality euploid blastocysts were excluded and 49 patients did not undergo an embryo transfer during the study period. Thus, 389 patients had a vitrified-warmed SET of a good quality euploid blastocyst (182 after FPS and 207 after LPS). For 126 cases (32%) where both FPS- and LPS-derived good quality blastocysts were available, the embryo transferred was chosen blindly with a 'True Random Number Generator' function where '0' stood for FPS-derived euploid blastocysts and '1' for LPS-derived ones (n = 70 and 56, respectively) on the website random.org. All embryos were obtained with the same ovarian stimulation protocol in FPS and LPS (GnRH antagonist protocol with fixed dose of rec-FSH plus rec-LH and GnRH-agonist trigger), culture conditions (continuous culture in a humidified atmosphere with 37°C, 6% CO2 and 5% O2) and laboratory protocols (ICSI, trophectoderm biopsy in Day 5-7 without assisted hatching in Day 3, vitrification and comprehensive chromosome testing). The women whose embryos were included had similar age (FPS: 38.5 ± 3.1 and LPS: 38.5 ± 3.2 years), prevalence of male factor, antral follicle count, basal hormonal characteristics, main cause of infertility and previous reproductive history (i.e. previous live births, miscarriages and implantation failures) whether the embryo came from FPS or LPS. All transfers were conducted after warming in an artificial cycle. The blastocysts transferred after FPS and LPS were similar in terms of day of full-development and morphological quality. MAIN RESULTS AND THE ROLE OF CHANCE: The positive pregnancy test rates for FPS- and LPS-derived euploid blastocysts were 57% and 62%, biochemical pregnancy loss rates were 10% and 8%, miscarriage rates were 15% and 14% and LBRs were 44% (n = 80/182, 95% CI 37-51%) and 49% (n = 102/207, 95% CI 42-56%; P = 0.3), respectively. The overall odds ratio for live birth (LPS vs FPS (reference)) adjusted for day of blastocyst development and quality, was 1.3, 95% CI 0.8-2.0, P = 0.2. Among patients with euploid blastocysts obtained following both FPS and LPS, the LBRs were also similar (53% (n = 37/70, 95% CI 41-65%) and 48% (n = 27/56, 95% CI 35-62%) respectively; P = 0.7). Gestational issues were experienced by 7.5% of pregnant women after FPS- and 10% of women following LPS-derived euploid single blastocyst transfer. Perinatal issues were reported in 5% and 0% of the FPS- and LPS-derived newborns, respectively. The gestational weeks and birthweight were similar in the two groups. A 5% pre-term delivery rate was reported in both groups. A low birthweight was registered in 2.5% and 5% of the newborns, while 4% and 7% showed high birthweight, in FPS- and LPS-derived euploid blastocyst, respectively. Encompassing the 81 FPS-derived newborns, a total of 9% were small and 11% large for gestational age. Among the 102 LPS-derived newborns, 8% were small and 6% large for gestational age. No significant difference was reported for all these comparisons. LIMITATIONS, REASONS FOR CAUTION: The LPS-derived blastocysts were all obtained after FPS in a DuoStim protocol. Therefore, studies are required with LPS-only, late-FPS and random start approaches. The study is powered to assess differences in the LBR per embryo transfer, therefore obstetric and perinatal outcomes should be considered observational. Although prospective, the study was not registered. WIDER IMPLICATIONS OF THE FINDINGS: This study represents a further backing of the safety of non-conventional COS protocols. Therefore, LPS after FPS (DuoStim protocol) is confirmed a feasible and efficient approach also from clinical, obstetric and perinatal perspectives, targeted at patients who need to reach the transfer of an euploid blastocyst in the shortest timeframe possible due to reasons such as cancer, advanced maternal age and/or reduced ovarian reserve and poor ovarian response. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Free-Radical-Mediated Formation of Trans-Cardiolipin Isomers, Analytical Approaches for Lipidomics and Consequences of the Structural Organization of Membranes.

Free-radical-mediated processes, such as peroxidation, isomerization and hydrogenation affecting fatty acid integrity and biological functions, have a trans-disciplinary relevance. Cardiolipins (CL, (1,3-diphosphatidyl-sn-glycerol)) and tetra-linoleoyl-CL are complex phospholipids, exclusively present in the Inner Mitochondrial Membrane (IMM) lipids, where they maintain membrane integrity and regulate enzyme functionalities. Peroxidation pathways and fatty acid remodeling are known causes of mitochondrial disfunctions and pathologies, including cancer. Free-radical-mediated isomerization with the change of the cis CL into geometrical trans isomers is an unknown process with possible consequences on the supramolecular membrane lipid organization. Here, the formation of mono-trans CL (MT-CL) and other trans CL isomers (T-CL) is reported using CL from bovine heart mitochondria and thiyl radicals generated by UV-photolysis from 2-mercaptoethanol. Analytical approaches for CL isomer separation and identification via 1H/13C NMR are provided, together with the chemical study of CL derivatization to fatty acid methyl esters (FAME), useful for lipidomics and metabolomics research. Kinetics information of the radical chain isomerization process was obtained using γ-irradiation conditions. The CL isomerization affected the structural organization of membranes, as tested by the reduction in unilamellar liposome diameter, and accompanied the well-known process of oxidative consumption induced by Fenton reagents. These results highlight a potential new molecular modification pathway of mitochondrial lipids with wide applications to membrane functions and biological consequences.

Fatty Acids and Membrane Lipidomics in Oncology: A Cross-Road of Nutritional, Signaling and Metabolic Pathways.

Fatty acids are closely involved in lipid synthesis and metabolism in cancer. Their amount and composition are dependent on dietary supply and tumor microenviroment. Research in this subject highlighted the crucial event of membrane formation, which is regulated by the fatty acids' molecular properties. The growing understanding of the pathways that create the fatty acid pool needed for cell replication is the result of lipidomics studies, also envisaging novel fatty acid biosynthesis and fatty acid-mediated signaling. Fatty acid-driven mechanisms and biological effects in cancer onset, growth and metastasis have been elucidated, recognizing the importance of polyunsaturated molecules and the balance between omega-6 and omega-3 families. Saturated and monounsaturated fatty acids are biomarkers in several types of cancer, and their characterization in cell membranes and exosomes is under development for diagnostic purposes. Desaturase enzymatic activity with unprecedented de novo polyunsaturated fatty acid (PUFA) synthesis is considered the recent breakthrough in this scenario. Together with the link between obesity and cancer, fatty acids open interesting perspectives for biomarker discovery and nutritional strategies to control cancer, also in combination with therapies. All these subjects are described using an integrated approach taking into account biochemical, biological and analytical aspects, delineating innovations in cancer prevention, diagnostics and treatments.

The n-10 Fatty Acids Family in the Lipidome of Human Prostatic Adenocarcinoma Cell Membranes and Extracellular Vesicles.

A new pathway leading to the n-10 fatty acid series has been recently evidenced, starting from sapienic acid, a monounsaturated fatty acid (MUFA) resulting from the transformation of palmitic acid by delta-6 desaturase. Sapienic acid has attracted attention as a novel marker of cancer cell plasticity. Here, we analyzed fatty acids, including the n-10 fatty acid contents, and for the first time, compared cell membranes and the corresponding extracellular vesicles (EV) of two human prostatic adenocarcinoma cell lines of different aggressiveness (PC3 and LNCaP). The n-10 components were 9-13% of the total fatty acids in both cancer cell lines and EVs, with total MUFA levels significantly higher in EVs of the most aggressive cell type (PC3). High sapienic/palmitoleic ratios indicated the preference for delta-6 versus delta-9 desaturase enzymatic activity in these cell lines. The expressions analysis of enzymes involved in desaturation and elongation by qRT-PCR showed a higher desaturase activity in PC3 and a higher elongase activity toward polyunsaturated fatty acids than toward saturated fatty acids, compared to LNCaP cells. Our results improve the present knowledge in cancer fatty acid metabolism and lipid phenotypes, highlighting EV lipidomics to monitor positional fatty acid isomer profiles and MUFA levels in cancer.

Effects of somatostatin, curcumin, and quercetin on the fatty acid profile of breast cancer cell membranes.

Breast cancer is a worldwide commonly found malignancy in women and effective treatment is regarded as a huge clinical challenge even in the presence of several treatment options. Extensive literature is available demonstrating polyphenols as phytopharmaceutical anticancer agents. Among the polyphenols, quercetin and curcumin have been reported to have a strong potential against breast cancer. However, so far, no comprehensive study has been performed to demonstrate the anticarcinogenic effects of curcumin, quercetin, and their combinations with somatostatin on the fatty acid profile of breast cancer cell membranes. We used MCF-7 and MDA-MB231 breast cancer cells incubated with curcumin and quercetin for 24 h, in the absence and presence of somatostatin, at their EC50 concentrations to evaluate membrane fatty acid based functional lipidomics together with the followup of EGFR and MAPK signaling pathways. The two cell lines gave different membrane free fatty acid reorganization. In MCF-7 cells, the following changes were observed: an increase of ω6 linoleic acid in the cells incubated with somatostatin + quercetin and quercetin and a decrease of ω3 acids in the cells incubated with somatostatin + curcumin compared to somatostatin and significant increases of monounsaturated fatty acid (MUFA), mono-trans arachidonic acid levels and docosapentaenoic acid for the cells incubated with somatostatin + quercetin compared to the control cells. In MDA-MB231 cells, incubations with curcumin, quercetin, and somatostatin + quercetin induced the most significant membrane remodeling with the increase of stearic acid, diminution of ω6 linoleic, arachidonic acids, and ω3 (docosapentaenoic and docosahexaenoic acids). Distinct signaling pathway changes were found for these cell lines. In MCF-7 cells, separate or combined incubations with somatostatin and quercetin, significantly decreased EGFR and incubation with curcumin decreased MAPK signaling. In MDA-MB231 cells, incubation with curcumin decreased AKT1 and p-AKT1 (Thr308) levels. Incubation with curcumin and quercetin decreased the EGFR levels. Our results showed that cytostatic and antioxidant treatments can be combined to induce membrane fatty acid changes, including lipid isomerization as specific free radical driven process, and to influence signaling pathways. This study aimed to contribute to the literature on these antioxidants in the treatment of breast cancer to clarify the effects and mechanisms in combination with somatostatin.