Trials and tribulations of statistical significance in biochemistry and omics.

Over recent years many statisticians and researchers have highlighted that statistical inference would benefit from a better use and understanding of hypothesis testing, p-values, and statistical significance. We highlight three recommendations in the context of biochemical sciences. First recommendation: to improve the biological interpretation of biochemical data, do not use p-values (or similar test statistics) as thresholded values to select biomolecules. Second recommendation: to improve comparison among studies and to achieve robust knowledge, perform complete reporting of data. Third recommendation: statistical analyses should be reported completely with exact numbers (not as asterisks or inequalities). Owing to the high number of variables, a better use of statistics is of special importance in omic studies.

Urinary Leukotriene E4 and Prostaglandin D2 Metabolites Increase in Adult and Childhood Severe Asthma Characterized by Type 2 Inflammation. A Clinical Observational Study.

Rationale: New approaches are needed to guide personalized treatment of asthma.Objectives: To test if urinary eicosanoid metabolites can direct asthma phenotyping.Methods: Urinary metabolites of prostaglandins (PGs), cysteinyl leukotrienes (CysLTs), and isoprostanes were quantified in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study including 86 adults with mild-to-moderate asthma (MMA), 411 with severe asthma (SA), and 100 healthy control participants. Validation was performed internally in 302 participants with SA followed up after 12-18 months and externally in 95 adolescents with asthma.Measurement and Main Results: Metabolite concentrations in healthy control participants were unrelated to age, body mass index, and sex, except for the PGE2 pathway. Eicosanoid concentrations were generally greater in participants with MMA relative to healthy control participants, with further elevations in participants with SA. However, PGE2 metabolite concentrations were either the same or lower in male nonsmokers with asthma than in healthy control participants. Metabolite concentrations were unchanged in those with asthma who adhered to oral corticosteroid treatment as documented by urinary prednisolone detection, whereas those with SA treated with omalizumab had lower concentrations of LTE4 and the PGD2 metabolite 2,3-dinor-11ß-PGF2α. High concentrations of LTE4 and PGD2 metabolites were associated with lower lung function and increased amounts of exhaled nitric oxide and eosinophil markers in blood, sputum, and urine in U-BIOPRED participants and in adolescents with asthma. These type 2 (T2) asthma associations were reproduced in the follow-up visit of the U-BIOPRED study and were found to be as sensitive to detect T2 inflammation as the established biomarkers.Conclusions: Monitoring of urinary eicosanoids can identify T2 asthma and introduces a new noninvasive approach for molecular phenotyping of adult and adolescent asthma.Clinical trial registered with www.clinicaltrials.gov (NCT01976767).

Anabolic androgenic steroids exert a selective remodeling of the plasma lipidome that mirrors the decrease of the de novo lipogenesis in the liver.

INTRODUCTION: The abuse of anabolic androgenic steroids (AASs) is a source of public concern because of their adverse effects. Supratherapeutic doses of AASs are known to be hepatotoxic and regulate the lipoproteins in plasma by modifying the metabolism of lipids in the liver, which is associated with metabolic diseases. However, the effect of AASs on the profile of lipids in plasma is unknown. OBJECTIVES: To describe the changes in the plasma lipidome exerted by AASs and to discuss these changes in the light of previous research about AASs and de novo lipogenesis in the liver. METHODS: We treated male Wistar rats with supratherapeutic doses of nandrolone decanoate and testosterone undecanoate. Subsequently, we isolated the blood plasma and performed lipidomics analysis by liquid chromatography-high resolution mass spectrometry. RESULTS: Lipid profiling revealed a decrease of sphingolipids and glycerolipids with palmitic, palmitoleic, stearic, and oleic acids. In addition, lipid profiling revealed an increase in free fatty acids and glycerophospholipids with odd-numbered chain fatty acids and/or arachidonic acid. CONCLUSION: The lipid profile presented herein reports the imprint of AASs on the plasma lipidome, which mirrors the downregulation of de novo lipogenesis in the liver. In a broader perspective, this profile will help to understand the influence of androgens on the lipid metabolism in future studies of diseases with dysregulated lipogenesis (e.g. type 2 diabetes, fatty liver disease, and hepatocellular carcinoma).

Linoleic acid-derived lipid mediators increase in a female-dominated subphenotype of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality; however, the role of inflammatory mediators in its pathobiology remains unclear. The aim of this study was to investigate the influence of gender in COPD on lipid mediator levels.Bronchoalveolar lavage fluid (BALF) and serum were obtained from healthy never-smokers, smokers and COPD patients (Global Initiative for Chronic Obstructive Lung Disease stage I-II/A-B) (n=114). 94 lipid mediators derived from the cytochrome-P450, lipoxygenase, and cyclooxygenase pathways were analysed by liquid chromatography-mass spectrometry.Multivariate modelling identified a 9-lipid panel in BALF that classified female smokers with COPD from healthy female smokers (p=6×10(-6)). No differences were observed for the corresponding male population (p=1.0). These findings were replicated in an independent cohort with 92% accuracy (p=0.005). The strongest drivers were the cytochrome P450-derived epoxide products of linoleic acid (leukotoxins) and their corresponding soluble epoxide hydrolase (sEH)-derived products (leukotoxin-diols). These species correlated with lung function (r=0.87; p=0.0009) and mRNA levels of enzymes putatively involved in their biosynthesis (r=0.96; p=0.003). Leukotoxin levels correlated with goblet cell abundance (r=0.72; p=0.028).These findings suggest a mechanism by which goblet cell-associated cytochrome-P450 and sEH activity produce elevated leukotoxin-diol levels, which play a putative role in the clinical manifestations of COPD in a female-dominated disease sub-phenotype.

Effects of selective COX-2 inhibition on allergen-induced bronchoconstriction and airway inflammation in asthma.

BACKGROUND: Prostaglandins that constrict and relax airways are synthesized in reactions catalyzed by either COX-1 or COX-2. It is not known whether selective inhibition of COX-2 makes asthmatic responses better or worse. OBJECTIVE: To determine the effects of the selective COX-2 inhibitor, etoricoxib, on allergen-induced bronchoconstriction in asthmatic subjects. METHODS: Sixteen subjects with mild atopic asthma underwent rising dose inhalation challenges with allergen or methacholine to determine PD20 FEV1 during a control study period or after 10 to 13 days of treatment with etoricoxib (90 mg once daily). The order of study periods was randomized with at least 2-week washout periods. Induced sputum cells and fractional exhaled nitric oxide levels were used to assess airway inflammation and blood assays for COX-1 and COX-2 activity to assess enzyme inhibition. Urinary excretion of lipids was used to assess prostaglandin biosynthesis. RESULTS: Etoricoxib did not change baseline lung function, nor airway responsiveness to allergen or to methacholine. Neither were the allergen-induced increase in sputum eosinophils and fractional exhaled nitric oxide levels affected by treatment. The biochemical effectiveness of the treatment was established both in the blood assays and by the distinct inhibitory effect of etoricoxib on urinary excretion of tetranor-prostaglandin E2 (P < .001). CONCLUSIONS: This first study of COX-2 inhibition in provoked asthma found no negative effects of etoricoxib on allergen-induced airflow obstruction and sputum eosinophils, basal lung function, or methacholine responsiveness. The study suggests that short-term use of COX-2 inhibitors is safe in subjects with asthma.

Ammonium bicarbonate buffers combined with hybrid surface technology columns improve the peak shape of strongly tailing lipids.

BACKGROUND: Lipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column. RESULTS: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation. SIGNIFICANCE: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.

Inhibiting the endoplasmic reticulum stress response enhances the effect of doxorubicin by altering the lipid metabolism of liver cancer cells.

Hepatocellular carcinoma (HCC) is characterized by a low and variable response to chemotherapeutic treatments. One contributing factor to the overall pharmacodynamics is the activation of endoplasmic reticulum (ER) stress pathways. This is a cellular stress mechanism that becomes activated when the cell's need for protein synthesis surpasses the ER's capacity to maintain accurate protein folding, and has been implicated in creating drug-resistance in several solid tumors. OBJECTIVE: To identify the role of ER-stress and lipid metabolism in mediating drug response in HCC. METHODS: By using a chemically-induced mouse model for HCC, we administered the ER-stress inhibitor 4µ8C and/or doxorubicin (DOX) twice weekly for three weeks post-tumor initiation. Histological analyses were performed alongside comprehensive molecular biology and lipidomics assessments of isolated liver samples. In vitro models, including HCC cells, spheroids, and patient-derived liver organoids were subjected to 4µ8C and/or DOX, enabling us to assess their synergistic effects on cellular viability, lipid metabolism, and oxygen consumption rate. RESULTS: We reveal a pivotal synergy between ER-stress modulation and drug response in HCC. The inhibition of ER-stress using 4µ8C not only enhances the cytotoxic effect of DOX, but also significantly reduces cellular lipid metabolism. This intricate interplay culminates in the deprivation of energy reserves essential for the sustenance of tumor cells. CONCLUSIONS: This study elucidates the interplay between lipid metabolism and ER-stress modulation in enhancing doxorubicin efficacy in HCC. This novel approach not only deepens our understanding of the disease, but also uncovers a promising avenue for therapeutic innovation. The long-term impact of our study could open the possibility of ER-stress inhibitors and/or lipase inhibitors as adjuvant treatments for HCC-patients.

Dynamics of arachidonic acid mobilization by inflammatory cells.

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites.

Quantification of lipid mediator metabolites in human urine from asthma patients by electrospray ionization mass spectrometry: controlling matrix effects.

Eicosanoids (e.g., prostaglandins and leukotrienes) are inflammatory signaling molecules that are metabolized and excreted in urine. The quantification of eicosanoid metabolites in human urine has been demonstrated to provide insight into the inflammatory and oxidative stress status of the individual. However, urine is a complex matrix that can exhibit profound matrix effects for quantification via liquid chromatography coupled to mass spectrometry (LC-MS/MS). This phenomenon can lead to impairment and biasing of results, because the sample background is dependent on the fluid intake and water-salt balance. Herein we describe an analytical methodology to address these limitations via the normalization of extracted urine volume by the ratio of absorbance at 300 nm to an optimized reference material. The platform is composed of 4 LC-MS/MS methods that collectively quantify 26 lipid mediators and their metabolites, with on-column limits of detection between 0.55 and 15 fmol. Prior to optimization, internal standards exhibited strong matrix effects with up to 50% loss of signal. Notably, the accuracy of exact deuterated structural analogues was found to vary based upon the number of incorporated deteurium. The platform was used to analyze urine from 16 atopic asthmatics under allergen provocation, showing increases in metabolites of prostaglandin D2, cysteinyl leukotrienes, and isoprostanes following the challenge. This method presents a functional and reproducible approach to addressing urine-specific matrix effects that can be readily formatted for quantifying large numbers of samples.

Application of 'omics technologies to biomarker discovery in inflammatory lung diseases.

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an "unbiased" approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the 'omics field. The contributions of the individual 'omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease.

Altered arachidonate distribution in macrophages from caveolin-1 null mice leading to reduced eicosanoid synthesis.

In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E(2) and LTB(4) production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response.

Influence of cellular arachidonic acid levels on phospholipid remodeling and CoA-independent transacylase activity in human monocytes and U937 cells.

The availability of free arachidonic acid (AA) constitutes a limiting step in the synthesis of biologically active eicosanoids. Free AA levels in cells are regulated by a deacylation/reacylation cycle of membrane phospholipids, the so-called Lands cycle, as well as by further remodeling reactions catalyzed by CoA-independent transacylase. In this work, we have comparatively investigated the process of AA incorporation into and remodeling between the various phospholipid classes of human monocytes and monocyte-like U937 cells. AA incorporation into phospholipids was similar in both cell types, but a marked difference in the rate of remodeling was appreciated. U937 cells remodeled AA at a much faster rate than human monocytes. This difference was found not to be related to the differentiation state of the U937 cells, but rather to the low levels of esterified arachidonate found in U937 cells compared to human monocytes. Incubating the U937 cells in AA-rich media increased the cellular content of this fatty acid and led to a substantial decrease of the rate of phospholipid AA remodeling, which was due to reduced CoA-independent transacylase activity. Collectively, these findings provide the first evidence that cellular AA levels determine the amount of CoA-independent transacylase activity expressed by cells and provide support to the notion that CoA-IT is a major regulator of AA metabolism in human monocytes.

Markers of monocyte activation revealed by lipidomic profiling of arachidonic acid-containing phospholipids.

Stimulated human monocytes undergo an intense trafficking of arachidonic acid (AA) among glycerophospholipidclasses. Using HPLC coupled to electrospray ionization mass spectrometry, we have characterized changes in the levels of AA-containing phospholipid species in human monocytes. In resting cells, AA was found esterified into various molecular species of phosphatidylinositol (PI), choline glycerophospholipids (PCs), and ethanolamine glycerophospholipids (PEs). All major AA-containing PC and PI molecular species decreased in zymosan-stimulated cells; however, no PE molecular species was found to decrease. In contrast, the levels of three AA-containing species increased in zymosan-activated cells compared with resting cells: 1,2-diarachidonyl-glycero-3-phosphoinositol [PI(20:4/20:4)]; 1,2-diarachidonyl-glycero-3-phosphocholine [PC(20:4/20:4)]; and 1-palmitoleoyl-2-arachidonyl-glycero-3-phosphoethanolamine [PE(16:1/20:4)]. PI(20:4/20:4) and PC(20:4/20:4), but not PE(16:1/20:4), also significantly increased when platelet-activating factor or PMA were used instead of zymosan to stimulate the monocytes. Analysis of the pathways involved in the synthesis of these three lipids suggest that PI(20:4/20:4) and PC(20:4/20:4) were produced in a deacylation/reacylation pathway via acyl-CoA synthetase-dependent reactions, whereas PE(16:1/20:4) was generated via a CoA-independent transacylation reaction. Collectively, our results define the increases in PI(20:4/20:4) and PC(20:4/20:4) as lipid metabolic markers of human monocyte activation and establish lipidomics as a powerful tool for cell typing under various experimental conditions.

Ammonia Concentration in the Eluent Influences Fragmentation Pattern of Triacylglycerols in Mass Spectrometry Analysis.

Correct assessment of the fatty acyl at the glycerol sn-2 position in triacylglycerol (TAG) analysis by liquid chromatography and mass spectrometry (LC-MS) is challenging. Ammonium hydroxide (NH4OH) is the preferred choice for the solvent additive for the formation of the ammonium adduct ([M + NH4]+). In this study, the influence of different NH4OH concentrations in the eluents on TAG adduct formation and fragmentation under LC-MS analysis was assessed. Increasing NH4OH concentrations delayed the chromatographic elution time according to a power function. The [M + NH4]+ and [M + ACN + NH4]+ adducts (where ACN means acetonitrile) were formed at all ammonium concentrations assayed. [M + ACN + NH4]+ predominated above 18.26 mM [NH4OH], and the intensity of [M + NH4]+ dropped. TAG fragmentation for fatty acyl release in the MSE was reduced with increasing [M + ACN + NH4]+ adduct, which suggests that ACN stabilizes the adduct in a way that inhibits the rupture of the ester bonds in TAGs. A linear equation (Hsn-I = a × H[M+NH4]+, where sn-I refers to the sn position of the glycerol (I = 1, 2, or 3) and H is the peak height) was deduced to quantify the dehydroxydiacylglycerol fragment intensity in relation to [M + NH4]+ intensity in the full scan. This equation had a slope mean value of 0.369 ± 0.058 for the sn-1 and sn-3 positions, and of 0.188 ± 0.007 for the sn-2 position.

Orthogonality in Principal Component Analysis Allows the Discovery of Lipids in the Jejunum That Are Independent of Ad Libitum Feeding.

Ad libitum feeding of experimental animals is preferred because of medical relevance together with technical and practical considerations. In addition, ethical committees may require ad libitum feeding. However, feeding affects the metabolism so ad libitum feeding may mask the effects of drugs on tissues directly involved in the digestion process (e.g., jejunum and liver). Despite this effect, principal component analysis has the potential of identifying metabolic traits that are statistically independent (orthogonal) to ad libitum feeding. Consequently, we used principal component analysis to discover the metabolic effects of doxorubicin independent of ad libitum feeding. First, we analyzed the lipidome of the jejunum and the liver of rats treated with vehicle or doxorubicin. Subsequently, we performed principal component analysis. We could identify a principal component associated to the hydrolysis of lipids during digestion and a group of lipids that were orthogonal. These lipids in the jejunum increased with the treatment time and presented a polyunsaturated fatty acid as common structural trait. This characteristic suggests that doxorubicin increases polyunsaturated fatty acids. This behavior agrees with our previous in vitro results and suggests that doxorubicin sensitized the jejunum to ferroptosis, which may partially explain the toxicity of doxorubicin in the intestines.

Oogenesis and lipid metabolism in the deep-sea sponge Phakellia ventilabrum (Linnaeus, 1767).

Sponges contain an astounding diversity of lipids that serve in several biological functions, including yolk formation in their oocytes and embryos. The study of lipid metabolism during reproduction can provide information on food-web dynamics and energetic needs of the populations in their habitats, however, there are no studies focusing on the lipid metabolism of sponges during their seasonal reproduction. In this study, we used histology, lipidome profiling (UHPLC-MS), and transcriptomic analysis (RNA-seq) on the deep-sea sponge Phakellia ventilabrum (Demospongiae, Bubarida), a key species of North-Atlantic sponge grounds, with the goal to (i) assess the reproductive strategy and seasonality of this species, (ii) examine the relative changes in the lipidome signal and the gene expression patterns of the enzymes participating in lipid metabolism during oogenesis. Phakellia ventilabrum is an oviparous and most certainly gonochoristic species, reproducing in May and September in the different studied areas. Half of the specimens were reproducing, generating two to five oocytes per mm2. Oocytes accumulated lipid droplets and as oogenesis progressed, the signal of most of the unsaturated and monounsaturated triacylglycerides increased, as well as of a few other phospholipids. In parallel, we detected upregulation of genes in female tissues related to triacylglyceride biosynthesis and others related to fatty acid beta-oxidation. Triacylglycerides are likely the main type of lipid forming the yolk in P. ventilabrum since this lipid category has the most marked changes. In parallel, other lipid categories were engaged in fatty acid beta-oxidation to cover the energy requirements of female individuals during oogenesis. In this study, the reproductive activity of the sponge P. ventilabrum was studied for the first time uncovering their seasonality and revealing 759 lipids, including 155 triacylglycerides. Our study has ecological and evolutionary implications providing essential information for understanding the molecular basis of reproduction and the origins and formation of lipid yolk in early-branching metazoans.

Comprehensive Peak Characterization (CPC) in Untargeted LC-MS Analysis.

LC-MS-based untargeted metabolomics is heavily dependent on algorithms for automated peak detection and data preprocessing due to the complexity and size of the raw data generated. These algorithms are generally designed to be as inclusive as possible in order to minimize the number of missed peaks. This is known to result in an abundance of false positive peaks that further complicate downstream data processing and analysis. As a consequence, considerable effort is spent identifying features of interest that might represent peak detection artifacts. Here, we present the CPC algorithm, which allows automated characterization of detected peaks with subsequent filtering of low quality peaks using quality criteria familiar to analytical chemists. We provide a thorough description of the methods in addition to applying the algorithms to authentic metabolomics data. In the example presented, the algorithm removed about 35% of the peaks detected by XCMS, a majority of which exhibited a low signal-to-noise ratio. The algorithm is made available as an R-package and can be fully integrated into a standard XCMS workflow.

Etherglycerophospholipids and ferroptosis: structure, regulation, and location.

Two pioneering studies by Zou et al. and Cui et al. have reported that the synthesis of etherglycerophospholipids (etherPLs) sensitizes cells to ferroptosis. The location and regulation of etherPLs suggest that: (i) lipid peroxidation in the inner leaflet of the plasma membrane might be of importance in ferroptosis, and (ii) different etherPLs may differently sensitize cells to ferroptosis.

Anthracyclins Increase PUFAs: Potential Implications in ER Stress and Cell Death.

Metabolic and personalized interventions in cancer treatment require a better understanding of the relationship between the induction of cell death and metabolism. Consequently, we treated three primary liver cancer cell lines with two anthracyclins (doxorubicin and idarubin) and studied the changes in the lipidome. We found that both anthracyclins in the three cell lines increased the levels of polyunsaturated fatty acids (PUFAs) and alkylacylglycerophosphoethanolamines (etherPEs) with PUFAs. As PUFAs and alkylacylglycerophospholipids with PUFAs are fundamental in lipid peroxidation during ferroptotic cell death, our results suggest supplementation with PUFAs and/or etherPEs with PUFAs as a potential general adjuvant of anthracyclins. In contrast, neither the markers of de novo lipogenesis nor cholesterol lipids presented the same trend in all cell lines and treatments. In agreement with previous research, this suggests that modulation of the metabolism of cholesterol could be considered a specific adjuvant of anthracyclins depending on the type of tumor and the individual. Finally, in agreement with previous research, we found a relationship across the different cell types between: (i) the change in endoplasmic reticulum (ER) stress, and (ii) the imbalance between PUFAs and cholesterol and saturated lipids. In the light of previous research, this imbalance partially explains the sensitivity to anthracyclins of the different cells. In conclusion, our results suggest that the modulation of different lipid metabolic pathways may be considered for generalized and personalized metabochemotherapies.

Prostaglandin D2 inhibits mediator release and antigen induced bronchoconstriction in the Guinea pig trachea by activation of DP1 receptors.

The mechanism by which cyclooxygenase (COX) inhibition increases antigen-induced responses in airways remains unknown. Male albino guinea pigs were sensitized to ovalbumin (OVA). Intact rings of the trachea were isolated and mounted in organ baths for either force measurements or lipid mediator release analysis by UPLC-MS/MS or EIA following relevant pharmacological interventions. First, challenge with OVA increased the release of all primary prostanoids (prostaglandin (PG) D2/E2/F2α/I2 and thromboxanes). This release was eliminated by unselective COX inhibition (indomethacin) whereas selective inhibition of COX-2 (lumiracoxib) did not inhibit release of PGD2 or thromboxanes. Additionally, the increased levels of leukotriene B4 and E4 after OVA were further amplified by unselective COX inhibition. Second, unselective inhibition of COX and selective inhibition of the prostaglandin D synthase (2-Phenyl-Pyrimidine-5-Carboxylic Acid (2,3-dihydro-indol-1-yl)-amide) amplified the antigen-induced bronchoconstriction which was reversed by exogenous PGD2. Third, a DP1 receptor agonist (BW 245c) concentration-dependently reduced the antigen-induced constriction as well as reducing released histamine and cysteinyl-leukotrienes, a response inhibited by the DP1 receptor antagonist (MK-524). In contrast, a DP2 receptor agonist (15(R)-15-methyl PGD2) failed to modulate the OVA-induced constriction. In the guinea pig trachea, endogenous PGD2 is generated via COX-1 and mediates an inhibitory effect of the antigen-induced bronchoconstriction via DP1 receptors inhibiting mast cell release of bronchoconstrictive mediators. Removal of this protective function by COX-inhibition results in increased release of mast cell mediators and enhanced bronchoconstriction.