Oxylipin Biosynthesis Research Articles

The Influence of Aspergillus fumigatus Fatty Acid Oxygenases PpoA and PpoC on Caspofungin Susceptibility

Aspergillus fumigatus can cause invasive pulmonary aspergillosis (IPA). Fungicidal azoles and fungistatic caspofungin (CAS) are the first- and second-line therapies, respectively, used to treat IPA. Treatment of A. fumigatus with CAS or micafungin induces the production of the oxylipin 5,8-diHODE by the fungal oxygenase PpoA. For this article, we investigated the influence of ppo genes, which encode the fatty acid oxygenases responsible for oxylipin biosynthesis, on CAS tolerance. The influence of PpoA and PpoC on CAS tolerance is mediated by MpkA phosphorylation and protein kinase A (PKA) activity. RNAseq transcriptional profiling and the label-free quantitative proteomics of the ppoA and ppoC mutants showed that differentially expressed genes and proteins are related to secondary metabolites and carbohydrate metabolism. We also characterized two clinical isolates, CM7555 and IFM61407, which decrease and increase susceptibility to CAS, respectively. CM7555 does not exhibit increased oxylipin production in the presence of CAS but oxylipin induction upon CAS exposure is increased in IFM61407, suggesting that oxylipins are not the only mechanism involved in CAS tolerance in these isolates. Upon CAS exposure, CM7555 has higher MpkA phosphorylation and PKA activity than IFM61407. Our results reveal the different aspects and genetic determinants involved in A. fumigatus CAS tolerance.

Development of a Desorption Electrospray Ionization-Multiple-Reaction-Monitoring Mass Spectrometry (DESI-MRM) Workflow for Spatially Mapping Oxylipins in Pulmonary Tissue.

Oxylipins are a class of low-abundance lipids formed via oxygenation of fatty acids. These compounds include potent signaling molecules (e.g., octadecanoids, eicosanoids) that can exert essential functions in the pathophysiology of inflammatory diseases including asthma. While some oxylipin signaling cascades have been unraveled using LC-MS/MS-based methods, measurements in homogenate samples do not represent the spatial heterogeneity of lipid metabolism. Mass spectrometry imaging (MSI) directly detects analytes from a surface, which enables spatial mapping of oxylipin biosynthesis and migration within the tissue. MSI has lacked the sensitivity to routinely detect low-abundance oxylipins; however, new multiple-reaction-monitoring (MRM)-based MSI technologies show increased sensitivity. In this study, we developed a workflow to apply desorption electrospray ionization coupled to a triple quadrupole mass spectrometer (DESI-MRM) to spatially map oxylipins in pulmonary tissue. The targeted MSI workflow screened guinea pig lung extracts using LC-MS/MS to filter oxylipin targets based on their detectability by DESI-MRM. A panel of 5 oxylipins was then selected for DESI-MRM imaging derived from arachidonic acid (TXB2, 11-HETE, 12-HETE), linoleic acid (12,13-DiHOME), and α-linolenic acid (16-HOTrE). To parse this new data type, a custom-built R package (quantMSImageR) was developed with functionality to label regions of interest as well as quantify and analyze lipid distributions. The spatial distributions quantified by DESI-MRM were supported by LC-MS/MS analysis, with both indicating that 16-HOTrE and 12-HETE were associated with airways, while 12,13-DiHOME and arachidonic acid were mapped to parenchyma. This study realizes the potential of targeted MSI to routinely map low-abundance oxylipins with high specificity at scale.

Oxylipin dynamics in dairy cows during clinical ketosis and after treatment with niacin and flunixin meglumine

Dairy cows with clinical ketosis (CK) exhibit metabolic changes, including intense adipose tissue (AT) lipolysis and systemic insulin resistance that increase plasma BHB and free fatty acids (FFA). Cows with CK also have systemic inflammation, predisposing them to inflammatory and infectious diseases. This inflammatory process is modulated in part by oxidized fatty acids (oxylipins) that regulate all aspects of inflammation. Oxylipin profiles have been characterized in healthy periparturient cows, but their dynamics during CK are unknown. CK is an acute metabolic disease requiring clinical therapy, commonly including propylene glycol (PG) as a gluconeogenic agent. Recently, we showed that including lipolysis inhibitors such as niacin (NIA) and flunixin meglumine (FM) improved CK recovery. These drugs may modulate oxylipin biosynthesis by regulating the release of PUFA (oxylipin substrates) and cyclooxygenase activity. However, their impact on oxylipin profiles in cows with CK is unknown. The objective of this study was to determine the dynamics of specific linoleic and arachidonic acid-derived oxylipins during CK and following therapy with PG, NIA, and FM. Multiparous Jersey cows (n = 72; 7.1 DIM) with CK from a commercial dairy were sampled. Inclusion criteria were CK symptoms (lethargy, depressed appetite, and reduced rumen fill) and blood BHB ≥ 1.2 mmol/L. CK cows (n = 24/treatment) were randomly assigned to one of the 3 treatments: 1) PG:310 g orally once daily for 5 d, 2) PG+NIA:24 g orally once daily for 3 d, 3) PG+NIA+FM:1.1 mg/kg IV once daily for 3 d. Healthy cows (HC; n = 24) matched by lactation and DIM (±2 d) were also included. Plasma oxylipins were quantified at enrollment and 7d (d7) later using HPLC-MS/MS. At enrollment, CK had higher concentrations of arachidonic acid (AA) derived 5- and 20-HETE, 8,9-, 11,12-, and 14–15-DHET, and lower concentrations of linoleic acid (LA) derived 12,13-EpOME, 13-oxoODE 9,10- and 12,13-DiHOME. Integrated analysis of biological pathways and oxylipin profiles using Ingenuity Pathway Analysis (IPA) revealed AA metabolism as the top pathway activated during CK. By d7, treatment with PGNIAFM restored plasma PUFAs and oxylipins to profiles similar to HC. IPA analysis showed that PGNIAFM activated the zinc transporter SLC30A7, associated with reduced activation of the AA pathway. Results indicate that higher FA availability during CK, driven in part by dysregulated lipolysis, increases the pool of substrates for oxylipin biosynthesis. These oxylipins may play a role in both metabolic dysregulation and restoring homeostasis during CK. Inhibiting lipolysis and cyclooxygenase activity with NIA and FM can alter AA and LA-derived oxylipins biosynthesis. These findings underscore the potential use of lipolysis inhibitors NIA and FM in CK therapeutics and highlight the importance of understanding oxylipin pathways in the pathogenesis of CK.

Abstract P220: Abdominal Aortic Perivascular Adipose Tissue Lipolysis Is Activated In Hypertensive Dahl SS Rats Fed a High-Fat Diet

Perivascular adipose tissues (PVAT) possess anti-contractile functions that are lost during hypertension (HTN). PVAT stores fatty acids (FA) and releases them through lipolysis upon adrenergic or inflammatory stimuli. Visceral AT lipolysis in humans with high abdominal adiposity and hypertension elevates FA release, promoting HTN via lipotoxicity and increased oxylipin synthesis. We hypothesize that lipolytic activity in abdominal aortic PVAT (aaPVAT) is increased during HTN. Plasma and aaPVAT were harvested from Dahl SS rats fed a high-fat (HF) or a control (CON) diet for 8 (♂=20, ♀=20), 16 (♂=10, ♀=10) and 24 (♂=14, ♀=14) wks. Mean arterial pressure (MAP±SEM; mmHg) was measured with sphyngomanometry. Free FA (FFA), β-hydroxybutyrate (BHB), glucose, and triglycerides were quantified with the CataChemWell-T analyzer, and individual FA using HPLC–MS/MS (reported as ng/dL). Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and phosphorylated pHSL(Ser563) were quantified in the Abby Western Blot. HF increased MAP compared to CON by 8 (131.5 vs 119.51±2.52), 16 (139.34 vs 118.26±5.7), and 24 wks (168.56 vs 121.94±3.93). HF at 24 wks had higher MAP compared to 8 and 16 wks. HF increased FFA and BHB at all time points and glucose at 24 wks. Plasma triglycerides were increased in HF vs. CON, and ♂ had higher levels than ♀ at all time points. ATGL, basal lipolysis rate-limiting enzyme, increased in aaPVAT from HF ♂ by 2.2, 2.53, and 1.82-fold at 8, 16, and 24 wks, respectively, compared to CON. In HF ♀, ATGL increased (1.95-fold change) at 16 wks vs. 8 wks. HSL content increased in HF vs. CON at 16 wks (2.1-fold change) but not at 24 wks. In contrast, pHSL abundance and the pHSL:HSL ratio were not affected by diet at 16 wks; however, both were reduced by 35.2% at 24 wks in HF ♂ and ♀. Lipolytic activity in HF coincided with higher plasma arachidonic (AA) and linoleic acid (LA) at 8 (AA HF=19.93, CON= 16.13±1.3; LA HF=29.22, CON=21.74±2.7) and 16 wks (AA HF=14.83, CON=11.61±1.34; LA HF=22.84, CON=11.66±2.9). Sex had no effect on AA or LA concentrations. Our results provide evidence that hypertensive Dahl SS rats fed HF exhibit higher aaPVAT basal lipolysis that may be driven by ATGL. In contrast, demand lipolysis, controlled by HSL activity, is reduced. Dysregulated basal lipolysis may lead to alterations in PVAT FA trafficking, enhance AA availability, and promote the biosynthesis of inflammatory oxylipins, thus impairing its vasoactive functions.

Oxylipin biosynthesis via an unprecedented 16-hydroperoxide lyase pathway in green tissues of cucumber (Cucumis sativus L.) plants

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.

The versatile CYP74 clan enzyme CYP440A19 from the European lancelet Branchiostoma lanceolatum biosynthesizes novel macrolactone, epoxydiene, and related oxylipins

The present work reports the detection and cloning of a new CYP74 clan gene of the European lancelet (Branchiostoma lanceolatum) and the biochemical characterization of the recombinant protein CYP440A19. CYP440A19 possessed epoxyalcohol synthase (EAS) activity towards the 13-hydroperoxides of linoleic and α-linolenic acids, which were converted into oxiranylcarbinols, i.e., (11S,12R,13S)-11-hydroxy-12,13-epoxy derivatives. The conversion of 9-hydroperoxides produced distinct products. Linoleic acid 9(S)-hydroperoxide (9-HPOD) was mainly converted into 9,14-diol (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid and macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In addition, (8Z)-colneleic acid was formed. Brief incubations of the enzyme with 9-HPOD in a biphasic system of hexane–water enabled the isolation of the short-lived 9,10-epoxydiene (9S,10R,11E,13E)-9,10-epoxy-11,13-octadecadienoic acid. The structure and stereochemistry of the epoxyalcohols, macrolactone, (8Z)-colneleic acid (Me), and 9,10-epoxydiene (Me) were confirmed by 1H-NMR, 1H-1H-COSY, 1H-13C-HSQC, and 1H-13C-HMBC spectroscopy. Macrolactone and cis-9,10-epoxydiene are novel products. The 9-hydroperoxide of α-linolenic acid was mainly converted into macrolactone 9(S),10(R)-epoxy-11(E),15(Z)-octadecadiene-13(S)-olide and a minority of divinyl ethers, particularly (8Z)-colnelenic acid. The versatility of enzyme catalysis, as well as the diversity of CYP74s and other enzymes involved in oxylipin biosynthesis, demonstrates the complexity of the lipoxygenase pathway in lancelets.

Emerging mechanisms of obesity-associated immune dysfunction.

Obesity is associated with a wide range of complications, including type 2 diabetes mellitus, cardiovascular disease, hypertension and nonalcoholic fatty liver disease. Obesity also increases the incidence and progression of cancers, autoimmunity and infections, as well as lowering vaccine responsiveness. A unifying concept across these differing diseases is dysregulated immunity, particularly inflammation, in response to metabolic overload. Herein, we review emerging mechanisms by which obesity drives inflammation and autoimmunity, as well as impairing tumour immunosurveillance and the response to infections. Among these mechanisms are obesity-associated changes in the hormones that regulate immune cell metabolism and function and drive inflammation. The cargo of extracellular vesicles derived from adipose tissue, which controls cytokine secretion from immune cells, is also dysregulated in obesity, in addition to impairments in fatty acid metabolism related to inflammation. Furthermore, an imbalance exists in obesity in the biosynthesis and levels of polyunsaturated fatty acid-derived oxylipins, which control a range of outcomes related to inflammation, such as immune cell chemotaxis and cytokine production. Finally, there is a need to investigate how obesity influences immunity using innovative model systems that account for the heterogeneous nature of obesity in the human population.

Plant Responses of Maize to Two formae speciales of Sporisorium reilianum Support Recent Fungal Host Jump.

Host jumps are a major factor for the emergence of new fungal pathogens. In the evolution of smut fungi, a putative host jump occurred in Sporisorium reilianum that today exists in two host-adapted formae speciales, the sorghum-pathogenic S. reilianum f. sp. reilianum and maize-pathogenic S. reilianum f. sp. zeae. To understand the molecular host-specific adaptation to maize, we compared the transcriptomes of maize leaves colonized by both formae speciales. We found that both varieties induce many common defense response-associated genes, indicating that both are recognized by the plant as pathogens. S. reilianum f. sp. reilianum additionally induced genes involved in systemic acquired resistance. In contrast, only S. reilianum f. sp. zeae induced expression of chorismate mutases that function in reducing the level of precursors for generation of the defense compound salicylic acid (SA), as well as oxylipin biosynthesis enzymes necessary for generation of the SA antagonist jasmonic acid (JA). In accordance, we found reduced SA levels as well as elevated JA and JA-Ile levels in maize leaves inoculated with the maize-adapted variety. These findings support a model of the emergence of the maize-pathogenic variety from a sorghum-specific ancestor following a recent host jump.

Identification of Lipoxygenase gene repertoire of Cannabis sativa and functional characterization of CsLOX13 gene

Lipoxygenase (LOX) enzymes play a pivotal role in the biosynthesis of oxylipins. The phyto-oxilipins have been implicated in diverse aspects of plant biology, from regulating plant growth and development to providing tolerance against biotic and abiotic stresses. C. sativa is renowned for its bioactive secondary metabolites, namely cannabinoids. LOX route is assumed to be involved in the biosynthesis of hexanoic acid, which is one of the precursors of cannabinoids of C. sativa. For obvious reasons, the LOX gene family deserves thorough investigation in the C. sativa. Genome-wide analysis revealed the presence of 21 LOX genes in C. sativa, which can be further grouped into 13-LOX and 9-LOX depending upon their phylogeny as well as the enzyme activity. The promoter regions of the CsLOX genes were predicted to contain cis-acting elements involved in phytohormones responsiveness and stress response. The qRT-PCR-based expression analysis of 21 LOX genes revealed their differential expression in different plant parts (root, stem, young leaf, mature leaf, sugar leaf, and female flower). The majority of CsLOX genes displayed preferential expression in the female flower, which is the primary site for the biosynthesis of cannabinoids. The highest LOX activity and expression level of a jasmonate marker gene were reported in the female flowers among all the plant parts. Several CsLOX genes were found to be upregulated by MeJA treatment. Based on the transient expression in Nicotiana benthamiana and the development of stable Nicotiana tabacum transgenic lines, we demonstrate that CsLOX13 encodes functional lipoxygenase and play an important role in the biosynthesis of oxylipins.

Volatilom und Transkriptom des Speisepilzes Agrocybe aegerita

Mushrooms are part of the human diet since time immemorial, appreciated for their nutritional value and especially for their delicious flavors. Hundreds of volatile organic compounds (VOCs) have been identified in fungi contributing to the unique aroma of each species. Generally, studies on mushroom VOCs are carried out with chopped fruiting bodies of more or less one developmental stage. For determine fungal aromas for assessment of the food quality this procedure might be adequate. Nonetheless, for analysis of the biological role of fungal VOCs in context of inter alia VOC biosynthesis or fungal communication this approach can suffer from drawbacks. First of all, damaging fruiting bodies can lead to VOC artefacts due to cell disruption and the occurrence of unnatural enzymatic reactions. Furthermore, fungal VOC profiles are dynamic, changing with ongoing development. For better understanding of the biological function of fungal VOCs it is therefore helpful to know which volatile patterns are characteristic for a certain developmental stage.Against this background, an approach was developed enabling on one hand the cultivation of fungi during different developmental stages, including the growth of fruiting bodies, and on the other hand the non‐invasive analysis of VOCs in the headspace (HS) of fungal cultures. These requirements were complied with modified crystallizing dishes for culture purposes and a HS‐ SPME‐GC‐MS approach to analyze the VOCs. This method was applied to analyze the volatilomes of the dikaryotic strain C. aegerita AAE‐3 and four monokaryotic offspring siblings with different fruiting phenotypes throughout ten life stages. At early stages, in the HS of all tested strains alcohols and ketones, such as oct‐1‐en‐3‐ol, 2‐methylbutan‐1‐ol and cyclopentanone, were the most prominent VOCs. Particularly counting for the dikaryon, the volatilome altered with continued fruiting body development exhibiting remarkable changes during sporulation. Here, sesquiterpenes, especially Δ6‐protoilludene, α‐cubebene and δ‐cadinene, were the most abundant VOCs in the HS of C. aegerita AAE‐3. After sporulation, the amount of sesquiterpenes decreased along with the appearance of other VOCs including octan‐3‐one. In contrast, less VOCs were present in the HS of the monokaryotic strains of which all were as well detectable in the HS of the dikaryon. The changes of the volatilome were the fundament for a subsequent transcriptome analysis aiming to identify enzymes involved in fungal VOC biosynthesis, especially regarding C8 VOC formation, which is, despite the fact that these substances are ubiquitous found in fungi, still barely understood. The transcriptomic study was carried out with seven developmental stages of C. aegerita AAE‐3, which during the volatilome study exhibited interesting volatile patterns. Additionally, fruiting bodies (five stages) and mycelia (seven stages) samples were harvested separately to get further insights about the putative origin of the VOCs observed in the HS of C. aegerita. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene‐ reductases could be identified. Especially the putative DOX AAE3_13098, the putative HPLs AAE3_05330 and AAE3_09203, the putative ADHs AAE3_00054 and AAE3_06559 as well as the putative ene‐reductase AAE3_15349 exhibit remarkable transcriptomic patterns making these enzymes highly interesting for future characterization studies. Furthermore, the study showed that the mycelium is probably the main source for sesquiterpenes observed during sporulation in the HS of C. aegerita AAE‐3 cultures whereas changes in the Cs profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies.

Harmonization of OF LC‐MS oxylipin analysis to investigate lipid mediator formation and signaling in immune cells

The oxidation products of polyunsaturated fatty acids (PUFA) ‐ oxylipins ‐ are formed via enzymatic conversion by cyclooxygenases, lipoxygenases (LOX) and cytochrome P450 monooxygenases or by autoxidation. Together with multiple PUFA substrates this leads to a large variety of oxylipins, whose physiological functions are ‐ besides those of well‐studied prostaglandins and leukotrienes ‐ little understood. The interplay of several PUFA‐oxidizing enzymes, as in the case of multihydroxylated oxylipins, additionally complicates the investigation of their biosynthesis. Due to regulatory crosstalk and complex interactions of different oxylipin classes during physiological processes, such as inflammation, a study of the whole oxylipin profile rather than individual compounds is necessary. Therefore, in order to understand the functions of these lipid mediators potent and reliable biochemical methods and (molecular) biological models are required, in combination with sensitive and sound preanalytical, analytical and post‐analytical procedures for their investigation.The first part of this thesis investigated the relevance of harmonizing such analytical methods for the quantitative determination of oxylipins in biological samples. Based on the outcome of an international laboratory comparison, comparable and reproducible results were obtained across independent laboratories when these harmonized methods were used for sample preparation and analysis by means of liquid chromatography coupled with tandem mass spectrometry. The examination of the effect of different storage times and temperatures as a parameter of pre‐analytics demonstrated that oxylipin levels in plasma samples are robust to long‐term storage and, furthermore, to minor variations during plasma generation. As accurate quantification of oxylipin concentrations rises and falls with the quality of analytical standards, a strategy for the verification of oxylipin standard concentrations with mass‐ spectrometry and UV spectroscopy based approaches using certified standard compounds was presented.In the second part of the thesis the established methods were applied to study the biosynthesis of oxylipins in immune cells with a special attention to the involved LOX. Stimulation of primary human M2‐like macrophages with the liver X receptor (LXR) agonist T09 led to a massive induction of ALOX15 gene expression and thus, 15‐LOX abundance and activity. Cholesterol derivatives were identified as potent endogenous ligands of LXR yielding increased 15‐LOX abundance and activity. The examination of 5‐LOX induced synthesis of multihydroxylated metabolites through supplementation of neutrophils with 15‐HETE, 18‐ HEPE and 17‐HDHA primary leads to formation of double oxygenated oxylipins. In intact cells 5‐LOX preferred DHA‐derived 17‐HDHA as substrate, whereas upon cell integrity destruction the formation of 15‐HETE derived multihydroxylated oxylipins increased.With the combination of harmonized protocols for sampling and analysis this thesis sets the basis for the reliable quantification of oxylipins. The use of the optimized methodologies allowed to further characterize regulatory pathways of the ARA cascade in human immune cells, contributing to a more thorough understanding of inflammation regulation.

The Effect of Increasing Concentrations of Omega-3 Fatty Acids from either Flaxseed Oil or Preformed Docosahexaenoic Acid on Fatty Acid Composition, Plasma Oxylipin, and Immune Response of Laying Hens

BackgroundThere is a lack of nutrition guidelines for the feeding of omega-3 polyunsaturated fatty acids (PUFA) to laying hens. Knowledge as to whether the type and concentrations of α-linolenic acid (ALA) and/or docosahexaenoic acid (DHA) in the diet can make a difference to the birds’ immune responses when subjected to a lipopolysaccharide (LPS) challenge is limited. ObjectivesThe study was designed to determine the potential nutritional and health benefits to laying hens when receiving dietary omega-3 PUFA from either ALA or DHA. MethodsA total of 80 Lohmann LSL-Classic (white egg layer, 20 wk old) were randomly assigned to 1 of 8 treatment diets (10 hens/treatment), provided 0.2%, 0.4%, 0.6%, or 0.8% of total dietary omega-3 PUFA, provided as either ALA-rich flaxseed oil or DHA-enriched algal biomass. After an 8-wk feeding period, the birds were challenged with Escherichia coli-derived LPS (8 mg/kg; i.v. injection), with terminal sample collection 4 h after challenge. Egg yolk, plasma, liver, and spleen samples were collected for subsequent analyses. ResultsIncreasing dietary omega-3 supplementation yielded predictable responses in egg yolk, plasma, and liver fatty acid concentrations. Dietary intake of ALA contributed mainly to ALA-derived oxylipins. Meanwhile, eicosapentaenoic acid- and DHA-derived oxylipins were primarily influenced by DHA dietary intake. LPS increased the concentrations of almost all the omega-6 PUFA-, ALA-, and DHA-derived oxylipins in plasma and decreased hepatic mRNA expression of COX-2 and 5-LOX (P < 0.001) involved in the biosynthesis of oxylipins. LPS also increased mRNA expression of proinflammatory cytokine IFN-γ and receptor TLR-4 (P < 0.001) in the spleen. ConclusionsThese results revealed that dietary intake of ALA and DHA had unique impacts on fatty acid deposition and their derived oxylipins and inflammatory responses under the administration of LPS in laying hens.

Oxylipin biosynthetic gene families of Cannabis sativa.

Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.

Genome-wide analysis of oxylipins and oxylipin profiles in a pediatric population.

Oxylipins are inflammatory biomarkers derived from omega-3 and-6 fatty acids implicated in inflammatory diseases but have not been studied in a genome-wide association study (GWAS). The aim of this study was to identify genetic loci associated with oxylipins and oxylipin profiles to identify biologic pathways and therapeutic targets for oxylipins. We conducted a GWAS of plasma oxylipins in 316 participants in the Diabetes Autoimmunity Study in the Young (DAISY). DNA samples were genotyped using the TEDDY-T1D Exome array, and additional variants were imputed using the Trans-Omics for Precision Medicine (TOPMed) multi-ancestry reference panel. Principal components analysis of 36 plasma oxylipins was used to capture oxylipin profiles. PC1 represented linoleic acid (LA)- and alpha-linolenic acid (ALA)-related oxylipins, and PC2 represented arachidonic acid (ARA)-related oxylipins. Oxylipin PC1, PC2, and the top five loading oxylipins from each PC were used as outcomes in the GWAS (genome-wide significance: p < 5×10-8). The SNP rs143070873 was associated with (p < 5×10-8) the LA-related oxylipin 9-HODE, and rs6444933 (downstream of CLDN11) was associated with the LA-related oxylipin 13 S-HODE. A locus between MIR1302-7 and LOC100131146, rs10118380 and an intronic variant in TRPM3 were associated with the ARA-related oxylipin 11-HETE. These loci are involved in inflammatory signaling cascades and interact with PLA2, an initial step to oxylipin biosynthesis. Genetic loci involved in inflammation and oxylipin metabolism are associated with oxylipin levels.

Mutational Analysis of Aspergillus fumigatus Volatile Oxylipins in a Drosophila Eclosion Assay.

Aspergillus fumigatus is a ubiquitous opportunistic pathogen. We have previously reported that volatile organic compounds (VOCs) produced by A. fumigatus cause delays in metamorphosis, morphological abnormalities, and death in a Drosophila melanogaster eclosion model. Here, we developed A. fumigatus deletion mutants with blocked oxylipin biosynthesis pathways (∆ppoABC) and then exposed the third instar larvae of D. melanogaster to a shared atmosphere with either A. fumigatus wild-type or oxylipin mutant cultures for 15 days. Fly larvae exposed to VOCs from wild-type A. fumigatus strains exhibited delays in metamorphosis and toxicity, while larvae exposed to VOCs from the ∆ppoABC mutant displayed fewer morphogenic delays and higher eclosion rates than the controls. In general, when fungi were pre-grown at 37 °C, the effects of the VOCs they produced were more pronounced than when they were pre-grown at 25 °C. GC-MS analysis revealed that the wild-type A. fumigatus Af293 produced more abundant VOCs at higher concentrations than the oxylipin-deficient strain Af293∆ppoABC did. The major VOCs detected from wild-type Af293 and its triple mutant included isopentyl alcohol, isobutyl alcohol, 2-methylbutanal, acetoin, and 1-octen-3-ol. Unexpectedly, compared to wild-type flies, the eclosion tests yielded far fewer differences in metamorphosis or viability when flies with immune-deficient genotypes were exposed to VOCs from either wild-type or ∆ppoABC oxylipin mutants. In particular, the toxigenic effects of Aspergillus VOCs were not observed in mutant flies deficient in the Toll (spz6) pathway. These data indicate that the innate immune system of Drosophila mediates the toxicity of fungal volatiles, especially via the Toll pathway.

Metabolomic analysis of methyl jasmonate treatment on phytocannabinoid production in Cannabis sativa.

Cannabis sativa is a multi-use and chemically complex plant which is utilized for food, fiber, and medicine. Plants produce a class of psychoactive and medicinally important specialized metabolites referred to as phytocannabinoids (PCs). The phytohormone methyl jasmonate (MeJA) is a naturally occurring methyl ester of jasmonic acid and a product of oxylipin biosynthesis which initiates and regulates the biosynthesis of a broad range of specialized metabolites across a number of diverse plant lineages. While the effects of exogenous MeJA application on PC production has been reported, treatments have been constrained to a narrow molar range and to the targeted analysis of a small number of compounds. Using high-resolution mass spectrometry with data-dependent acquisition, we examined the global metabolomic effects of MeJA in C. sativa to explore oxylipin-mediated regulation of PC biosynthesis and accumulation. A dose-response relationship was observed, with an almost two-fold increase in PC content found in inflorescences of female clones treated with 15 mM MeJA compared to the control group. Comparison of the inflorescence metabolome across MeJA treatments coupled with targeted transcript analysis was used to elucidate key regulatory components contributing to PC production and metabolism more broadly. Revealing these biological signatures improves our understanding of the role of the oxylipin pathway in C. sativa and provides putative molecular targets for the metabolic engineering and optimization of chemical phenotype for medicinal and industrial end-uses.

Functional Study of Lipoxygenase-Mediated Resistance against Fusarium verticillioides and Aspergillus flavus Infection in Maize.

Mycotoxin contamination of maize kernels by fungal pathogens like Fusarium verticillioides and Aspergillus flavus is a chronic global challenge impacting food and feed security, health, and trade. Maize lipoxygenase genes (ZmLOXs) synthetize oxylipins that play defense roles and govern host-fungal interactions. The current study investigated the involvement of ZmLOXs in maize resistance against these two fungi. A considerable intraspecific genetic and transcript variability of the ZmLOX family was highlighted by in silico analysis comparing publicly available maize pan-genomes and pan-transcriptomes, respectively. Then, phenotyping and expression analysis of ZmLOX genes along with key genes involved in oxylipin biosynthesis were carried out in a maize mutant carrying a Mu transposon insertion in the ZmLOX4 gene (named UFMulox4) together with Tzi18, Mo17, and W22 inbred lines at 3- and 7-days post-inoculation with F. verticillioides and A. flavus. Tzi18 showed the highest resistance to the pathogens coupled with the lowest mycotoxin accumulation, while UFMulox4 was highly susceptible to both pathogens with the most elevated mycotoxin content. F. verticillioides inoculation determined a stronger induction of ZmLOXs and maize allene oxide synthase genes as compared to A. flavus. Additionally, oxylipin analysis revealed prevalent linoleic (18:2) peroxidation by 9-LOXs, the accumulation of 10-oxo-11-phytoenoic acid (10-OPEA), and triglyceride peroxidation only in F. verticillioides inoculated kernels of resistant genotypes.

The wound-activated ERF15 transcription factor drives Marchantia polymorpha regeneration by activating an oxylipin biosynthesis feedback loop.

The regenerative potential in response to wounding varies widely among species. Within the plant lineage, the liverwort Marchantia polymorpha displays an extraordinary regeneration capacity. However, its molecular pathways controlling the initial regeneration response are unknown. Here, we demonstrate that the MpERF15 transcription factor gene is instantly activated after wounding and is essential for gemmaling regeneration following tissue incision. MpERF15 operates both upstream and downstream of the MpCOI1 oxylipin receptor by controlling the expression of oxylipin biosynthesis genes. The resulting rise in the oxylipin dinor-12-oxo-phytodienoic acid (dn-OPDA) levels results in an increase in gemma cell number and apical notch organogenesis, generating highly disorganized and compact thalli. Our data pinpoint MpERF15 as a key factor activating an oxylipin biosynthesis amplification loop after wounding, which eventually results in reactivation of cell division and regeneration. We suggest that the genetic networks controlling oxylipin biosynthesis in response to wounding might have been reshuffled over evolution.

Lipoxygenase pathway in brown algae: The biosynthesis of novel oxylipins ‘ectocarpins’ by hydroperoxide bicyclase CYP5164A3 of Ectocarpus siliculosus

The sequence encoding the CYP5164A3 of the brown alga Ectocarpus siliculosus (Stramenopiles, SAR) was heterologously expressed in E. coli cells. The resulting recombinant CYP74 clan-related protein CYP5164A3 possessed a selective activity towards the α-linolenic acid 13(S)-hydroperoxide (13-HPOTE) and eicosapentaenoic acid 15(S)-hydroperoxide (15-HPEPE). The major products were the heterobicyclic oxylipins. For instance, the 13-HPOTE was converted into plasmodiophorols A, B, and C formed at about 14:3:2 ratio. Plasmodiophorols A-C have been recently described as the products of enzyme hydroperoxide bicyclase CYP50918A1 of cercozoan Plasmodiophora brassicae (Rhizaria, SAR). Furthermore, an unknown compound 1 was detected. Purified product 1 (Me) was identified as a novel substituted 3-propenyl-6-oxabicyclo[3.1.0]hexane based on its MS and NMR spectral data. Conversion of 15-HPEPE by CYP5164A3 resulted in products 7 and 8, analogous to plasmodiophorols A and B. This work uncovered the CYP5164A3 as the first hydroperoxide bicyclase in brown algae. Apparently, this enzyme plays a crucial role in the biosynthesis of heterobicyclic oxylipins like hybridalactone, ecklonilactones, and related natural products, widespread in brown algae.

Pseudomonas aeruginosa Secretes the Oxylipin Autoinducer Synthases OdsA and OdsB via the Xcp Type 2 Secretion System.

The oxylipin-dependent quorum-sensing system (ODS) of Pseudomonas aeruginosa relies on the production and sensing of two extracellular oxylipins, 10S-hydroxy-(8E)-octadecenoic acid (10-HOME) and 7S,10S-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Here, we implemented a genetic screen of P. aeruginosa strain PAO1 aimed to identify genes required for 10-HOME and 7,10-DiHOME production. Among the 14 genes identified, four encoded previously known components of the ODS and 10 encoded parts of the Xcp type II secretion system (T2SS). We subsequently created a clean xcpQ deletion mutant, which encodes the necessary outer membrane component of Xcp, and found it recapitulated the impaired functionality of the T2SS transposon mutants. Further studies showed that the ΔxcpQ mutant was unable to secrete the oxylipin synthase enzymes across the outer membrane. Specifically, immunoblotting for OdsA, which is responsible for the generation of 10-HOME from oleic acid, detected the enzyme in supernatants from wild-type PAO1 but not ΔxcpQ cultures. Likewise, chromatography of supernatants found that 10-HOME was not in supernatants collected from the ΔxcpQ mutant. Accordingly, diol synthase activity was increased in the periplasm of ΔxcpQ mutant consistent with a stoppage in its transport. Importantly, after exposure of the ΔxcpQ mutant to exogenous 10-HOME and 7,10-DiHOME, the ODS effector genes become active; thus, the sensing component of the ODS does not involve the T2SS. Finally, we observed that Xcp contributed to robust in vitro and in vivo biofilm formation in oleic acid availability- and ODS-dependent manner. Thus, T2SS-mediated transport of the oxylipin synthase enzymes to outside the bacterial cell is required for ODS functionality. IMPORTANCE We previously showed that the ODS of P. aeruginosa produces and responds to oxylipins derived from host oleic acid by enhancing biofilm formation and virulence. Here, we developed a genetic screen strategy to explore the molecular basis for oxylipins synthesis and detection. Unexpectedly, we found that the ODS autoinducer synthases cross the outer membrane using the Xcp type 2 secretion system (T2SS) of P. aeruginosa, and so the biosynthesis of oxylipins occurs extracellularly. T2SS promoted biofilm formation in the presence of oleic acid as a result of ODS activation. Our results identify two new T2SS secreted proteins in P. aeruginosa and reveal a new way by which this important opportunistic pathogen interacts with the host environment.