IL-10 constrains sphingolipid metabolism to limit inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.

Pathophysiological Involvement of Mast Cells and the Lipid Mediators in Pulmonary Vascular Remodeling.

Mast cells are responsible for IgE-dependent allergic responses, but they also produce various bioactive mediators and contribute to the pathogenesis of various cardiovascular diseases, including pulmonary hypertension (PH). The importance of lipid mediators in the pathogenesis of PH has become evident in recent years, as exemplified by prostaglandin I2, the most central therapeutic target in pulmonary arterial hypertension. New bioactive lipids other than eicosanoids have also been identified that are associated with the pathogenesis of PH. However, it remains largely unknown how mast cell-derived lipid mediators are involved in pulmonary vascular remodeling. Recently, it has been demonstrated that mast cells produce epoxidized n-3 fatty acid (n-3 epoxides) in a degranulation-independent manner, and that n-3 epoxides produced by mast cells regulate the abnormal activation of pulmonary fibroblasts and suppress the progression of pulmonary vascular remodeling. This review summarizes the role of mast cells and bioactive lipids in the pathogenesis of PH. In addition, we introduce the pathophysiological role and therapeutic potential of n-3 epoxides, a mast cell-derived novel lipid mediator, in the pulmonary vascular remodeling in PH. Further knowledge of mast cells and lipid mediators is expected to lead to the development of innovative therapies targeting pulmonary vascular remodeling.

Identification and Evolution Analysis of the Complete Methyl Farnesoate Biosynthesis and Related Pathway Genes in the Mud Crab, Scylla paramamosain.

The sesquiterpenoid hormone methyl farnesoate (MF) plays a vital role during crustacean development, which is mainly evidenced by its varied titers during different developmental stages. However, the biosynthesis pathways of MF remain obscure to some extent. In this study, we identified the complete MF biosynthesis and related pathway genes in Scylla paramamosain, including three involved in acetyl-CoA metabolism, eight in the mevalonate pathway, five in the sesquiterpenoids synthesis pathway, and five in the methionine cycle pathway. Bioinformatics, genomic structure, and phylogenetic analysis indicated that the JH biosynthesis genes might have experienced evolution after species differentiation. The mRNA tissue distribution analysis revealed that almost all genes involving in or relating to MF syntheses were highly expressed in the mandibular organ (MO), among which juvenile hormone acid methyltransferase was exclusively expressed in the MO, suggesting that most of these genes might mainly function in MF biosynthesis and that the methionine cycle pathway genes might play a crucial regulatory role during MF synthesis. In addition, the phylogenetic and tissue distribution analysis of the cytochrome P450 CYP15-like gene suggested that the epoxidized JHs might exist in crustaceans, but are mainly synthesized in hepatopancreas rather than the MO. Finally, we also found that betaine-homocysteine S-methyltransferase genes were lost in insects while methionine synthase was probably lost in most insects except Folsomia candida, indicating a regulatory discrepancy in the methionine cycle between crustaceans and insects. This study might increase our understanding of synthetic metabolism tailored for sesquiterpenoid hormones in S. paramamosain and other closely related species.

Title: Involvement of unsaturated fatty acid biosynthesis in CRC progression based on in vitro and in silico studies.

Obesity is one of the risk factors concerns of colorectal cancer (CRC), the most common type of gastrointestinal cancer, due to the changing lifestyle and especially diet. There are various molecular pathways associated with obesity and the risk of CRC incidence, such as insulin resistance or elevated plasma free fatty acids, which alter the signaling pathways of intestinal epithelial cells. The aim of this study was to better understand the significance of unsaturated fatty acid biosynthesis on pathogenesis of colon cancer in obese. Based on GSE20931 dataset, obese individuals affected by CRC had higher increased gene expression than non-obese individuals. The analysis showed that in obese individuals, the 16 signaling pathway genes were activated and increased (FDR <0.05) significantly. The biosynthetic pathway of unsaturated fatty acids showed a cross-talk with the arachidonic acid metabolism pathway and the PPAR signaling pathway is influenced and regulated via these pathways. The biosynthetic pathway of unsaturated fatty acids consisting of 22 genes, were analyzed using GEO data and revealed that 4 genes (HSD17B12, TECR, FADS2, ELOVL5) from this pathway were significantly increased (FDR <0.05). These data were validated based on TCGA data (Adj.p.value <0.001). The expression level of candidate genes in HT-29 cells decreased significantly (P.value <0.01), and PPARγ expression increased under linoleic acid treatment (200 µM) compared to control cells. Moreover, in presence of linoleic acid treatment, migration, colony formation, and proliferation decreased (P.value <0.01) in presence of treatment. In summary, the Biosynthesis pathway of unsaturated fatty acids is an interesting and critical pathway in CRC.

Biochemical, metabolic, and immune responses of mud crab (Scylla paramamosain) after mud crab reovirus infection.

Mud crab reovirus (MCRV) is a serious pathogen that leads to large economic losses in the mud crab farming. However, the molecular mechanism of the immune response after MCRV infection is unclear. In the present study, physiological, transcriptomic, and metabolomic responses after MCRV infection were investigated. The results showed that MCRV infection could increase lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities. MCRV infection decreased antioxidant enzyme activity levels, induced oxidative stress, and caused severe histological damage. Transcriptome analysis identified 416 differentially expressed genes, including 354 up-regulated and 62 down-regulated genes. The detoxification, immune response, and metabolic processes-related genes were found. The results showed that two key pathways including phagocytosis and apoptosis played important roles in response to MCRV infection. The combination of transcriptomic and metabolomic analyses showed that related metabolic pathways, such as glycolysis, citrate cycle, lipid, and amino acid metabolism were also significantly disrupted. Moreover, the biosynthesis of unsaturated fatty acids was activated in response to MCRV infection. This study provided a novel insight into the understanding of cellular mechanisms in crustaceans against viral invasion.

The MAP-Kinase HOG1 Controls Cold Adaptation in Rhodosporidium kratochvilovae by Promoting Biosynthesis of Polyunsaturated Fatty Acids and Glycerol.

The aim of this study was to investigate the role of RKHog1 in the cold adaptation of Rhodosporidium kratochvilovae strain YM25235 and elucidate the correlation of biosynthesis of polyunsaturated fatty acids (PUFAs) and glycerol with its cold adaptation. The YM25235 strain was subjected to salt, osmotic, and cold stress tolerance analyses. mRNA levels of RKhog1, Δ12/15-fatty acid desaturase gene (RKD12), RKMsn4, HisK2301, and RKGPD1 in YM25235 were detected by reverse transcription quantitative real-time PCR. The contents of PUFAs, such as linoleic acid (LA) and linolenic acid (ALA) was measured using a gas chromatography-mass spectrometer, followed by determination of the growth rate of YM25235 and its glycerol content at low temperature. The RKHog1 overexpression, knockout, and remediation strains were constructed. Stress resistance analysis showed that overexpression of RKHog1 gene increased the biosynthesis of glycerol and enhanced the tolerance of YM25235 to cold, salt, and osmotic stresses, respectively. Inversely, the knockout of RKHog1 gene decreased the biosynthesis of glycerol and inhibited the tolerance of YM25235 to different stresses. Fatty acid analysis showed that the overexpression of RKHog1 gene in YM25235 significantly increased the content of LA and ALA, but RKHog1 gene knockout YM25235 strain had decreased content of LA and ALA. In addition, the mRNA expression level of RKD12, RKMsn4, RKHisK2301, and RKGPD1 showed an increase at 15 °C after RKHog1 gene overexpression but were unchanged at 30 °C. RKHog1 could regulate the growth adaptability and PUFA content of YM25235 at low temperature and this could be helpful for the cold adaptation of YM25235.

Metabolic and molecular evidence for long-chain PUFA biosynthesis capacity in the grass carp Ctenopharyngodon idella.

There is a growing interest to understand the capacity of farmed fish species to biosynthesise the physiologically important long-chain (≥C20) n-3 and n-6 polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA), from their C18 PUFA precursors available in the diet. In fish, the LC-PUFA biosynthesis pathways involve sequential desaturation and elongation reactions from α-linolenic acid (ALA) and linoleic acid (LA), catalysed by fatty acyl desaturases (Fads) and elongation of very long-chain fatty acids (Elovl) proteins. Our current understanding of the grass carp (Ctenopharyngodon idella) LC-PUFA biosynthetic capacity is limited despite representing the most farmed finfish produced worldwide. To address this knowledge gap, this study first aimed at characterising molecularly and functionally three genes (fads2, elovl5 and elovl2) with putative roles in LC-PUFA biosynthesis. Using an in vitro yeast-based system, we found that grass carp Fads2 possesses ∆8 and ∆5 desaturase activities, with ∆6 ability to desaturase not only the C18 PUFA precursors (ALA and LA) but also 24:5n-3 to 24:6n-3, a key intermediate to obtain DHA through the "Sprecher pathway". Additionally, the Elovl5 showed capacity to elongate C18 and C20 PUFA substrates, whereas Elovl2 was more active over C20 and C22. Collectively, the molecular cloning and functional characterisation of fads2, elovl5 and elovl2 demonstrated that the grass carp has all the enzymatic activities required to obtain ARA, EPA and DHA from LA and ALA. Importantly, the hepatocytes incubated with radiolabelled fatty acids confirmed the yeast-based results and demonstrated that these enzymes are functionally active.

Puerarin improves OVX-induced osteoporosis by regulating phospholipid metabolism and biosynthesis of unsaturated fatty acids based on serum metabolomics.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a serious problem for the women over 50 years old. Natural product puerarin (PUE) has been proven to improve PMOP with high safety. PMOP is a metabolic disorder affecting bone metabolism, indicating that endogenous metabolites amelioration may be a novel strategy for PMOP therapy. However, what the metabolic profile of POMP will be after PUE treatment is still obscure. PURPOSE: We purpose to figure out the metabolic characteristics of PMOP and to explore the intrinsic mechanism on the anti-osteoporosis efficacy after PUE treatment based on the serum metabolomics. METHODS: We established OVX rats as osteoporosis model, and the animals were distributed into Sham, OVX, and OVX+PUE (100 mg/kg/d) group. The femurs were analyzed by µ-CT and three-point bending test. Serum metabolomics was performed by UPLC/Q-TOF-MS. We also determined the body weight, liver weight, and the levels of serum TC, TG, LDL-C, and HDL-C. The key proteins of the PPARγ pathway and Wnt pathway were analyzed by Western blot and qPCR experiments. RESULTS: PUE treatment for 14 weeks both improved the bone structure and ameliorated lipid metabolism in ovariectomized rats. By determination and further analysis of serum metabolomics, we revealed that the endogenous metabolites was significantly changed in ovariectomized rats, and PUE treatment adjusted 23 differential metabolites, which were involved in phospholipid metabolism metabolism and PUFAs metabolic pathways. Close correlationships were futher found between the indexes of bone metabolism, lipid metabolism and the differential metabolites, particularly LysoPA, S1P and n-3/n-6 PUFAs. Further, we discovered that PUE regulated differentiation of BMSCs to elicit anti-osteoporosis efficacy, attributing to Wnt/ß-catenin signaling activation and PPARγ pathway inhibition initiated by metabolomics. CONCLUSION: PUE improves OVX-induced osteoporosis and lipid metabolism by regulating phospholipid metabolism and biosynthesis of PUFAs, resulting in reducing the adipogenic differentiation and promoting osteogenic differentiation of BMSCs via Wnt pathway activation and PPARγ pathway inhibition in ovariectomized rats. The study provides us a novel mechanism to explain the improvement of osteoporosis by PUE, depicts a metabolic profile of PMOP, and gives us another point cut for further exploring the pathogenesis of PMOP and looking for biomarkers of osteoporosis.

Expression of genes and localization of enzymes involved in polyunsaturated fatty acid synthesis in rabbit testis and epididymis.

The metabolism of polyunsaturated fatty acids (PUFAs) plays an important role in male reproduction. Linoleic and alpha-linolenic acids need to be provided in the diet and they are converted into long chain polyunsaturated fatty acids by steps of elongation and desaturation, exerted by elongases 2 (ELOVL2) and 5 (ELOVL5) and Δ5- (FADS1) and Δ6-desaturase (FADS2). This study aims to assess the gene expression and localization of enzymes involved in the synthesis of n-3 and n-6 long-chain PUFAs in control rabbits and those fed diets containing 10% extruded flaxseed. Enzyme and PUFA localization were assessed in the testes and epididymis by immunofluorescence. Testes showed high gene expression of FADS2, ELOVL2 and ELOVL5 and low expression of FADS1. Intermediate metabolites, enzymes and final products were differently found in Leydig, Sertoli and germinal cells. FADS2 was localized in interstitial cells and elongated spermatids; ELOVL5 in meiotic cells; FADS1 was evident in interstitial tissue, Sertoli cells and elongated spermatids; ELOVL2 in interstitial cells. Epididymal vesicles were positive for FADS1, ELOVL2 and ELOVL5 as well as docosahexaenoic, eicosapentaenoic, and arachidonic acids. This knowledge of fatty acids (FA) metabolism in spermatogenesis and the influence of diet on FA profile could help identify causes of male infertility, suggesting new personalized therapy.

Highly Valuable Polyunsaturated Fatty Acids from Microalgae: Strategies to Improve Their Yields and Their Potential Exploitation in Aquaculture.

Microalgae have a great potential for the production of healthy food and feed supplements. Their ability to convert carbon into high-value compounds and to be cultured in large scale without interfering with crop cultivation makes these photosynthetic microorganisms promising for the sustainable production of lipids. In particular, microalgae represent an alternative source of polyunsaturated fatty acids (PUFAs), whose consumption is related to various health benefits for humans and animals. In recent years, several strategies to improve PUFAs' production in microalgae have been investigated. Such strategies include selecting the best performing species and strains and the optimization of culturing conditions, with special emphasis on the different cultivation systems and the effect of different abiotic factors on PUFAs' accumulation in microalgae. Moreover, developments and results obtained through the most modern genetic and metabolic engineering techniques are described, focusing on the strategies that lead to an increased lipid production or an altered PUFAs' profile. Additionally, we provide an overview of biotechnological applications of PUFAs derived from microalgae as safe and sustainable organisms, such as aquafeed and food ingredients, and of the main techniques (and their related issues) for PUFAs' extraction and purification from microalgal biomass.

Helicobacter pylori FabX contains a [4Fe-4S] cluster essential for unsaturated fatty acid synthesis.

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa.

Lack of ∆5 Desaturase Activity Impairs EPA and DHA Synthesis in Fish Cells from Red Sea Bream and Japanese Flounder.

Long-chain (≥ C20) polyunsaturated fatty acids (LC-PUFA), such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are necessary for human health and are obtained from marine fish-derived oils. Marine fish are LC-PUFA-rich animals; however, many of them require LC-PUFA for growth. Therefore, it is suggested that they do not have sufficient ability to biosynthesize LC-PUFA. To evaluate in vivo LC-PUFA synthetic activity in fish cells, fish-derived cell lines from red sea bream (Pagrus major, PMS and PMF), Japanese flounder (Paralichthys olivaceus, HINAE), and zebrafish (Danio rerio, BRF41) were incubated with n-3 fatty acids labeled by radioisotopes or stable isotopes, and then, n-3 PUFA were analyzed by thin-layer chromatography or liquid chromatography-mass spectrometry. Labeled EPA and DHA were biosynthesized from labeled α-linolenic acid (18:3n-3) in BRF41, whereas they were not detected in PMS, PMF, or HINAE cells. We next cloned the fatty acid desaturase 2 (Fads2) cDNAs from PMF cells and zebrafish, expressed in budding yeasts, and then analyzed the substrate specificities of enzymes. As a result, we found that Fads2 from PMF cells was a ∆6/∆8 desaturase. Collectively, our study indicates that cell lines from red sea bream and Japanese flounder were not able to synthesize EPA or DHA by themselves, possibly due to the lack of ∆5 desaturase activity. Furthermore, this study provides a sensitive and reproducible non-radioactive method for evaluating LC-PUFA synthesis in fish cells using a stable isotope and liquid chromatography-mass spectrometry.

A cryptic long-chain 3-ketoacyl-ACP synthase in the Pseudomonas putida F1 unsaturated fatty acid synthesis pathway.

The Pseudomonas putida F1 genome contains five genes annotated as encoding 3-ketoacyl-acyl carrier protein (ACP) synthases. Four are annotated as encoding FabF (3-ketoacyl-ACP synthase II) proteins, and the fifth is annotated as encoding a FabB (3-ketoacyl-ACP synthase I) protein. Expression of one of the FabF proteins, FabF2, is cryptic in the native host and becomes physiologically important only when the repressor controlling fabF2 transcription is inactivated. When derepressed, FabF2 can functionally replace FabB, and when expressed from a foreign promoter, had weak FabF activity. Complementation of Escherichia coli fabB and fabF mutant strains with high expression showed that P. putida fabF1 restored E. coli fabF function, whereas fabB restored E. coli fabB function and fabF2 restored the functions of both E. coli fabF and fabB. The P. putida ΔfabF1 deletion strain was almost entirely defective in synthesis of cis-vaccenic acid, whereas the ΔfabB strain is an unsaturated fatty acid (UFA) auxotroph that accumulated high levels of spontaneous suppressors in the absence of UFA supplementation. This was due to increased expression of fabF2 that bypasses loss of fabB because of the inactivation of the regulator, Pput_2425, encoded in the same operon as fabF2. Spontaneous suppressor accumulation was decreased by high levels of UFA supplementation, whereas competition by the P. putida ß-oxidation pathway gave increased accumulation. The ΔfabB ΔfabF2 strain is a stable UFA auxotroph indicating that suppressor accumulation requires FabF2 function. However, at low concentrations of UFA supplementation, the ΔfabF2 ΔPput_2425 double-mutant strain still accumulated suppressors at low UFA concentrations.

PsrA positively regulates the unsaturated fatty acid synthesis operon fabAB in Azotobacter vinelandii.

In Pseudomonas spp. PsrA, a transcriptional activator of the rpoS gene, regulates fatty acid catabolism by repressing the fadBA5 ß-oxidation operon. In Azotobacter vinelandii, a soil bacterium closely related to Pseudomonas species, PsrA is also an activator of rpoS expression, although its participation in the regulation of lipid metabolism has not been analyzed. In this work we found that inactivation of psrA had no effect on the expression of ß-oxidation genes in this bacterium, but instead decreased expression of the unsaturated fatty acid biosynthetic operon fabAB (3-hydroxydecanoyl-ACP dehydratase/isomerase and 3-ketoacyl-ACP synthase I). This inactivation also reduced the unsaturated fatty acid content, as revealed by the thin-layer chromatographic analysis, and confirmed by gas chromatography; notably, there was also a lower content of cyclopropane fatty acids, which are synthesized from unsaturated fatty acids. The absence of PsrA has no effect on the growth rate, but showed loss of cell viability during long-term growth, in accordance with the role of these unsaturated and cyclopropane fatty acids in the protection of membranes. Finally, an electrophoretic mobility shift assay revealed specific binding of PsrA to the fabA promoter region, where a putative binding site for this regulator was located. Taken together, our data show that PsrA plays an important role in the regulation of unsaturated fatty acids metabolism in A. vinelandii by positively regulating fabAB.

Expression of long-chain polyunsaturated fatty acids biosynthesis genes during the early life-cycle stages of the tropical gar Atractosteus tropicus.

Long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), including eicosapentaenoic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), are essential in multiple physiological processes, especially during early development of vertebrates. LC-PUFA biosynthesis is achieved by two key families of enzymes, fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl). The present study determined the expression patterns of genes encoding desaturases (fads1 and fads2) and elongases (elovl2 and elovl5) involved in the LC-PUFA biosynthesis during early life-stages of the tropical gar Atractosteus tropicus. We further analyzed the fatty acid profiles during early development of A. tropicus to evaluate the impact of Fads and Elovl enzymatic activities. Specific oligonucleotides were designed from A. tropicus transcriptome to perform qPCR (quantitative polymerase chain reaction) on embryonic and larval stages, along with several organs (intestine, white muscle, brain, liver, heart, mesenteric adipose, kidney, gill, swim bladder, stomach, and spleen) collected from juvenile specimens. Fatty acid content of feeds and embryonic and larval stages were analyzed. Results show that fads1, fads2, elovl2 and elovl5 expression was detected from embryonic stages with expression peaks from day 15 post hatching, which could be related to transcriptional and dietary factors. Moreover, fads1, fads2 and elovl2 showed a higher expression in intestine, while elovl5 showed a higher expression in liver, suggesting that the tropical gar activates its LC-PUFA biosynthetic machinery to produce ARA, EPA and DHA to satisfy physiological demands at crucial developmental milestones during early development.

Biosynthesis of Long-Chain Polyunsaturated Fatty Acids in Marine Gammarids: Molecular Cloning and Functional Characterisation of Three Fatty Acyl Elongases.

Long-chain (C20-24) polyunsaturated fatty acids (LC-PUFAs) are essential nutrients that are mostly produced in marine ecosystems. Previous studies suggested that gammarids have some capacity to endogenously produce LC-PUFAs. This study aimed to investigate the repertoire and functions of elongation of very long-chain fatty acid (Elovl) proteins in gammarids. Our results show that gammarids have, at least, three distinct elovl genes with putative roles in LC-PUFA biosynthesis. Phylogenetics allowed us to classify two elongases as Elovl4 and Elovl6, as they were bona fide orthologues of vertebrate Elovl4 and Elovl6. Moreover, a third elongase was named as "Elovl1/7-like" since it grouped closely to the Elovl1 and Elovl7 found in vertebrates. Molecular analysis of the deduced protein sequences indicated that the gammarid Elovl4 and Elovl1/7-like were indeed polyunsaturated fatty acid (PUFA) elongases, whereas Elovl6 had molecular features typically found in non-PUFA elongases. This was partly confirmed in the functional assays performed on the marine gammarid Echinogammarus marinus Elovl, which showed that both Elovl4 and Elovl1/7-like elongated PUFA substrates ranging from C18 to C22. E. marinus Elovl6 was only able to elongate C18 PUFA substrates, suggesting that this enzyme does not play major roles in the LC-PUFA biosynthesis of gammarids.

Characterization of ω3 fatty acid desaturases from oomycetes and their application toward eicosapentaenoic acid production in Mortierella alpina.

ω3 Polyunsaturated fatty acids are currently obtained mainly from fisheries; thus, sustainable alternative sources such as oleaginous microorganisms are required. Here, we describe the isolation, characterization, and application of 3 novel ω3 desaturases with ω3 polyunsaturated fatty acid-producing activity at ordinary temperatures (28 °C). First, we selected Pythium sulcatum and Plectospira myriandra after screening for oomycetes with high eicosapentaenoic acid/arachidonic acid ratios and isolated the genes psulω3 and pmd17, respectively, which encode ω3 desaturases. Subsequent characterization showed that PSULω3 exhibited ω3 desaturase activity on both C18 and C20 ω6 polyunsaturated fatty acids while PMD17 exhibited ω3 desaturase activity exclusively on C20 ω6 polyunsaturated fatty acids. Expression of psulω3 and pmd17 in the arachidonic acid-producer Mortierella alpina resulted in transformants that produced eicosapentaenoic acid/total fatty acid values of 38% and 40%, respectively, at ordinary temperatures. These ω3 desaturases should facilitate the construction of sustainable ω3 polyunsaturated fatty acid sources.

Hyperpolarized 13 C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in†vivo Study.

Hyperpolarized [1-13 C]fumarate is a promising magnetic resonance imaging (MRI) biomarker for cellular necrosis, which plays an important role in various disease and cancerous pathological processes. To demonstrate the feasibility of MRI of [1-13 C]fumarate metabolism using parahydrogen-induced polarization (PHIP), a low-cost alternative to dissolution dynamic nuclear polarization (dDNP), a cost-effective and high-yield synthetic pathway of hydrogenation precursor [1-13 C]acetylenedicarboxylate (ADC) was developed. The trans-selectivity of the hydrogenation reaction of ADC using a ruthenium-based catalyst was elucidated employing density functional theory (DFT) simulations. A simple PHIP set-up was used to generate hyperpolarized [1-13 C]fumarate at sufficient 13 C polarization for ex vivo detection of hyperpolarized 13 C malate metabolized from fumarate in murine liver tissue homogenates, and in vivo 13 C MR spectroscopy and imaging in a murine model of acetaminophen-induced hepatitis.

Biosynthesis of LC-PUFAs and VLC-PUFAs in Pampus argenteus: Characterization of Elovl4 Elongases and Regulation under Acute Salinity.

Salinity has been demonstrated to influence the biosynthesis of long-chain (C20-24) polyunsaturated fatty acids (LC-PUFAs) in teleost fish. Since LC-PUFAs are essential nutrients for vertebrates, it is central to understand how fish cope with an acute change in salinity associated with natural events. We herein report on the cloning and functional characterization of two elongation of very-long-chain fatty acid (Elovl)4 proteins, namely, Elovl4a and Elovl4b, and study the roles that these enzymes play in the biosynthesis of LC-PUFAs and very-long-chain (>C24) polyunsaturated fatty acids (VLC-PUFAs) in marine teleost Pampus argenteus. The P. argenteus Elovl4 displayed all of the typical features of Elovl-like enzymes and have eyes and brain as major sites through which they exert their functions. Moreover, functional studies showed that the P. argenteus Elovl4 can effectively elongate C18-22 substrates to C36 VLC-PUFA. Because both P. argenteus Elovl4 are able to produce 24:5n - 3 from shorter precursors, we tested whether the previously reported Δ6 Fads2 from P. argenteus was able to desaturate 24:5n - 3 to 24:6n - 3, a key step for docosahexaenoic acid (DHA) synthesis. Our results showed that P. argenteus can indeed bioconvert 24:5n - 3 into 24:6n - 3, suggesting that P. argenteus has the enzymatic capacity required for DHA biosynthesis through the coordinated action of both Elovl4 and Fads2. Furthermore, an acute salinity test indicated that low-salinity stress (12 ppt) upregulated genes involved in LC-PUFA biosynthesis, with 12 ppt salinity treatment showing the highest hepatic LC-PUFA content. Overall, our results unveiled that the newly characterized Elovl4 enzymes have indispensable functions in LC- and VLC-PUFA biosynthesis. Moreover, acute salinity change influenced the biosynthesis of LC-PUFA in P. argenteus. This study provided new insight into the biosynthesis of LC- and VLC-PUFAs in vertebrates and the physiological responses that teleosts have under acute salinity stress.

A 13CO2 Enrichment Experiment to Study the Synthesis Pathways of Polyunsaturated Fatty Acids of the Haptophyte Tisochrysis lutea.

The production of polyunsaturated fatty acids (PUFA) in Tisochrysis lutea was studied using the gradual incorporation of a 13C-enriched isotopic marker, 13CO2, for 24 h during the exponential growth of the algae. The 13C enrichment of eleven fatty acids was followed to understand the synthetic pathways the most likely to form the essential polyunsaturated fatty acids 20:5n-3 (EPA) and 22:6n-3 (DHA) in T. lutea. The fatty acids 16:0, 18:1n-9 + 18:3n-3, 18:2n-6, and 22:5n-6 were the most enriched in 13C. On the contrary, 18:4n-3 and 18:5n-3 were the least enriched in 13C after long chain polyunsaturated fatty acids such as 20:5n-3 or 22:5n-3. The algae appeared to use different routes in parallel to form its polyunsaturated fatty acids. The use of the PKS pathway was hypothesized for polyunsaturated fatty acids with n-6 configuration (such as 22:5n-6) but might also exist for n-3 PUFA (especially 20:5n-3). With regard to the conventional n-3 PUFA pathway, Δ6 desaturation of 18:3n-3 appeared to be the most limiting step for T. lutea, "stopping" at the synthesis of 18:4n-3 and 18:5n-3. These two fatty acids were hypothesized to not undergo any further reaction of elongation and desaturation after being formed and were therefore considered "end-products". To circumvent this limiting synthetic route, Tisochrysis lutea seemed to have developed an alternative route via Δ8 desaturation to produce longer chain fatty acids such as 20:5n-3 and 22:5n-3. 22:6n-3 presented a lower enrichment and appeared to be produced by a combination of different pathways: the conventional n-3 PUFA pathway by desaturation of 22:5n-3, the alternative route of ω-3 desaturase using 22:5n-6 as precursor, and possibly the PKS pathway. In this study, PKS synthesis looked particularly effective for producing long chain polyunsaturated fatty acids. The rate of enrichment of these compounds hypothetically synthesized by PKS is remarkably fast, making undetectable the 13C incorporation into their precursors. Finally, we identified a protein cluster gathering PKS sequences of proteins that are hypothesized allowing n-3 PUFA synthesis.