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.     

15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet

Lipoxygenases (LOXs) are a family of enzymes capable of peroxidizing phospholipids. A member of the LOX family of enzymes, 15-LOX, participates in the degradation of mitochondria and other organelles within differentiating red blood cells, the reticulocytes. The present study provides biochemical and immunocytochemical evidence for the presence of 15-LOX in the sperm cytoplasmic droplet (CD). Testicular, epididymal and ejaculated spermatozoa were evaluated for the presence of 15-LOX using an affinity-purified immune serum raised against a synthetic peptide corresponding to the C-terminal sequence of rabbit reticulocyte 15-LOX. Western blotting revealed an appropriate single band of approximately 81 kDa in boar spermatozoa but not in boar seminal plasma. When ejaculated boar spermatozoa were subjected to separation on a 45/90% Percoll gradient, 15-LOX co-migrated with the immotile sperm and cellular debris/CD fractions, but not with the motile sperm fraction containing morphologically normal spermatozoa without CDs. Varied levels of 15-LOX were expressed in ejaculated sperm samples from boars with varied semen quality. By immunofluorescence, prominent 15-LOX immunoreactivity was found within the residual body in the testis and within the CDs from caput, corpus and cauda epididymal and ejaculated spermatozoa. Components of the ubiquitin-dependent proteolytic pathway, which is thought to facilitate both spermiogenesis and reticulocyte organelle degradation, were also detected in the sperm CD. These included ubiquitin, the ubiquitin-conjugating enzyme E2, the ubiquitin C-terminal hydrolase PGP 9.5, and various 20S proteasomal core subunits of the alpha- and beta-type. The 15-LOX and various components of the ubiquitin-proteasome pathway were also detected in sperm CDs of other mammalian species, including the human, mouse, stallion and wild babirusa boar. We conclude that 15-LOX is prominently present in the mammalian sperm CD and thus may contribute to spermiogenesis, CD function or CD removal.     

普通烟草脂氧合酶基因家族鉴定及表达模式分析

脂氧合酶（LOX）是脂肪酸代谢途径的关键酶,在植物的生长发育及多种逆境胁迫响应过程中发挥重要作用。本研究对烟草基因组中的LOX家族成员进行鉴定,并利用进化分析、共线性分析和表达分析等方法解析LOX家族成员的潜在生物学功能。结果表明,在普通烟草中共鉴定得到18个LOX基因,其中12个LOX基因被锚定在染色体上。系统进化分析表明,普通烟草中新鉴定的LOX家族成员被分为9-LOX和13-LOX两个亚家族,其中13-LOX又可被分为Type I和Type II两个亚类。共线性分析表明,烟草NtLOX07、NtLOX10基因分别与拟南芥AtLOX05、AtLOX01基因形成同源基因对,并且大部分同源基因之间具有相似的表达模式。表达模式分析发现,普通烟草LOX基因表达具有一定的组织特异性,并且NtLOX06等基因能够被机械损伤和茉莉酸甲脂（MeJA）处理显著诱导。因此,普通烟草LOX家族成员可能在植物胁迫响应过程中发挥着重要的作用。     

Flocculation,cell surface hydrophobicity and 3‐OH oxylipins in the SMA strain of Saccharomyces pastorianus

Three‐hydroxy‐oxylipins (3‐OH oxylipins) have been previously detected in brewing yeast production strains at flocculation onset. In this work, the SMA strain of Saccharomyces pastorianus was characterized during growth in a miniature fermentation assay by measuring flocculation and cell surface hydrophobicity (CSH). Proportions of 3‐OH oxylipin were also measured concurrently during growth in the miniature fermentation assay and a defined 3‐OH oxylipin extraction protocol using ethyl acetate is presented along with a novel derivatization and gas chromatography–mass spectrometry (GC‐MS) detection approach. When the SMA strain was grown in the assay, near maximal CSH and flocculation levels were achieved by a 36 h fermentation time. Under the same culture conditions, the oxylipin 3‐OH decanoic acid (3‐OH 10:0) was identified. This oxylipin could not be detected early in the fermentation, but elevated relative levels of 3‐OH 10:0 were reached by 36 h, coinciding with increased CSH levels. It was previously presumed that the formation of 3‐OH oxylipins at flocculation onset might increase the CSH. However, results from this study suggest that 3‐OH 10:0 may not contribute to cell wall hydrophobicity. The flocculation behaviour of the SMA strain was also monitored in the presence of 3‐OH 10:0, but exposure to this oxylipin did not impact the sedimentation of this yeast, suggesting that 3‐OH oxylipins may not act as mediators of quorum sensing in this strain. Copyright © 2015 The Institute of Brewing & Distilling     

Recombinant Lipoxygenases and Oxylipin Metabolism in Relation to Food Quality

Lipoxygenases catalyze the conversion of polyunsaturated fatty acids into hydroperoxides, that are in turn converted to oxylipins, which play important roles in defence reactions in plants and animals. This review describes the distribution of lipoxygenases in Nature, their diversity in terms of structure and catalytic activity, and their significance for food biotechnology. The last includes the production of flavors and aromas, the destruction of vitamins, pigments and other anti-oxidants, the improvement of dough rheology during baking, and the potential of recombinant lipoxygenases, and other enzymes of oxylipin metabolism, for food biotechnology.     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Reduction of multiple reaction monitoring protein target list using correlation analysis

High mass resolution mass spectrometry provides hundreds to thousands of protein identifications per sample, and quantification is typically performed using label-free quantification. However, the gold standard of quantitative proteomics is multiple reaction monitoring (MRM) using triple quadrupole mass spectrometers and stable isotope reference peptides. This raises the question how to reduce a large data set to a small one without losing essential information. Here we present the reduction of such a data set using correlation analysis of bovine dairy ingredients and derived products. We were able to explain the variance in the proteomics data set using only 9 proteins across all major dairy protein classes: caseins, whey, and milk fat globule membrane proteins. We term this method Trinity-MRM. The reproducibility of the protein extraction and Trinity-MRM methods was shown to be below 5% in independent experiments (multi-day single-user and single-day multi-user) using double cream. Further application of this reductionist approach might include screening of large sample cohorts for biologically interesting samples before analysis by high-resolution mass spectrometry or other omics methodologies.     

Trans triacylglycerols from dairy products and industrial hydrogenated oil exhibit different effects on the function of human umbilical vein endothelial cells via modulating phospholipase A2/arachidonic acid metabolism pathways

Dairy fat intake has been considered as a risk factor for cardiovascular disease. Rodent models show that trans fatty acids in industrial hydrogenated oil and ruminant milk have different effects on cardiovascular diseases. One of the main reasons is that the distributions of trans fatty acids in triacylglycerols from dairy products and from industrial hydrogenated oil are different, which affects lipid absorption and metabolism. This study investigated the effects of 1,3-olein-2-elaidin (OEO, representing industrial hydrogenated oil triacylglycerols) and 1-vaccenic-2,3-olein (OOV, representing ruminant triacylglycerols in dairy products) on the function of human umbilical vein endothelial cells (HUVEC), including cell viability, lactate dehydrogenase (LDH) exudation rate, and nitric oxide secretory and nitric oxide synthase relative activity. We found that the detrimental effect of OEO on HUVEC was significantly greater than that of OOV. The results also showed that the absorption rate of OEO in HUVEC (78.25%) was significantly greater than that of OOV (63.32%). Mechanistically, based on phospholipidomics analysis, we found that calcium-independent phospholipase A2 (iPLA2) played a key role with regard to the OOV-mediated arachidonic acid (ARA)/COX-2/PG pathway, whereas secretory phospholipase A2 (sPLA2) and cytoplasmic phospholipase A2 (cPLA2) are responsible for the OEO-mediated ARA/COX-2/PG pathway. Moreover, OEO had a greater effect on the protein expression of COX-2 and PG secretion than OOV. In addition, iPLA2, sPLA2, and cPLA2 could mediate the ARA/CYP4A11 pathway in OOV-treated HUVEC, but only iPLA2 could mediate this pathway in HUVEC treated with OEO. We also found that sPLA2 could mediate the ARA/5-LOX pathway in HUVEC treated with OOV, but none of these 3 forms of PLA2 could mediate this pathway in HUVEC treated with OEO. On the other hand, after OOV treatment, trans-11 C18:1 was converted to beneficial forms of fatty acids in HUVEC, including conjugated linoleic acid (CLA) and trans-9 C16:1. In conclusion, we elucidated the potential mechanisms that might account for the diverse effects of triacylglycerols from industrial hydrogenated oil and ruminant milk on the function of HUVEC.     

Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis of Fusarium verticillioides and maize kernels

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize, even if frequently asymptomatic, producing a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins result as signals able to modulate the secondary metabolism in fungi. In keeping with this, a novel, quantitative LC-MS/MS method was designed to quantify up to 17 different oxylipins produced by F. verticillioides and maize kernels. By applying this method, we were able to quantify oxylipin production in vitro – F. verticillioides grown into Czapek–Dox/yeast extract medium amended with 0.2% w/v of cracked maize – and in vivo, i.e. during its growth on detached mature maize ears. This study pinpoints the role of oxylipins in a plant pathogen such as F. verticillioides and sets up a novel tool aimed at understanding the role oxylipins play in mycotoxigenic pathogens during their interactions with respective hosts.     

基于代谢组学辨析广西长寿饮食模式对机体的有益影响

为了深入探究饮食与机体健康状态的关系,本研究试图从代谢组学的角度,评估所构建的广西长寿饮食模式对人体的有益影响。筛选志愿者在经过2周习惯性饮食后,高依从性地遵循这种饮食模式2周,在习惯性饮食前,广西长寿饮食前（习惯性饮食后）和广西长寿饮食后3次分别采集志愿者的血液和粪便样品,进行代谢组学分析。归纳所得结果发现,广西长寿饮食干预后志愿者血清组氨酸、甘油、α-葡萄糖和β-葡萄糖相对丰度显著增加（P<0.05）,胆碱和乳酸的相对丰度显著降低（P<0.05）。此外还发现,粪便中的乙酸盐和丁酸盐含量显著上升（P<0.05）。代谢物组富集分析和途径分析结果表明,这些代谢物的变化可能与糖酵解/糖异生途径、丙酮酸代谢和半乳糖代谢3条途径密切相关。通过对差异代谢物的分析,认为广西长寿饮食模式对改善机体炎症水平、调节机体健康状态、预防机体罹患心血管疾病具有一定的作用。上述结果也从代谢组学角度对饮食促进机体健康长寿的关系提供了一定机制方面的阐释。     

多组学诠释食源性致病菌小菌落变种形成分子机制研究进展

摘 要:食源性致病菌在环境胁迫下可形成小菌落变种(small colony variants, SCVs), 被认为是其抗逆存活的重要调控方式之一。SCVs的形成及防治对于食品安全领域具有重要挑战 , 这是因为与正常细胞相比, SCVs通常具有较强的耐药性, 而且能够抵抗宿主细胞的清除并存活数十年。当条件适宜时，SCVs细胞甚至能够在保持耐药性的同时，重新转变为快速生长细胞。随着生物技术的高速发展, 多组学方法应用到微生物抗逆分子机制研究领域越来越常见。通过基因组学、转录组学、蛋白质组学、代谢组学和联合组学大大增加研究者对细胞中基因调控及蛋白质表达关键途径的理解。本文就近年来食源性致病菌SCVs诱导途径以及多组学诠释SCVs分子机制研究进展进行综述, 以期为建立针对性的安全控制技术, 抑制食品加工中食源性致病菌SCVs形成提供参考。     

The role of lipoxygenase-isoforms in atherogenesis

Lipoxygenases (LOXs) form a heterogeneous family of lipid-peroxidizing enzymes, and several LOX-isoforms (12/15-LOX, 5-LOX) have been implicated in atherogenesis. However, the precise role of these enzymes is still a matter of discussion. 12/15-LOXs are capable of oxidizing lipoproteins (low-density lipoprotein (LDL), high-density lipoprotein (HDL)) to atherogenic forms, and functional inactivation of this enzyme in murine atherosclerosis models slows down lesion formation. In contrast, rabbits that overexpress this enzyme were protected from lesion formation when fed a lipid-rich diet. To contribute to this discussion, we recently investigated the impact of 12/15-LOX overexpression on in vitro foam cell formation. When 12/15-LOX-transfected J774 cells were incubated in culture with modified LDL, we found that intracellular lipid deposition was reduced in the transfected cells when compared with the corresponding control transfectants. This paper briefly summarizes the current status of knowledge on the biological activity of different LOX-isoforms in atherogenesis and will also provide novel experimental data characterizing the role of 12/15-LOX in cellular LDL modification and for in vitro foam cell formation.     

鲜食玉米中脂氧合酶活力、脂肪酸与挥发性成分的相关性研究

研究了晶甜5号和晶甜紫花糯2号两种鲜食玉米在早于适采期、适采期和晚于适采期三个采收期脂氧合酶(LOX)的活力、脂肪酸和挥发性成分的组成及变化情况。结果显示样品的LOX活力为2.63~4.28 U/g FW,脂肪酸均以亚油酸的含量最高(32.40%~46.68%),而亚麻酸含量较低(0.97%~2.40%)。挥发性成分则以己醇或己醛的含量最高,早采和适采期的晶甜5号中己醇含量最高,晚采的晶甜5号和三个采收期的京甜紫花糯2号均以己醛含量最高。两种玉米的粗脂肪、脂氧合酶活力随采收期的延后而增加,但是亚油酸、己醛却表现为下降趋势。相关性分析表明LOX途径的底物脂肪酸亚油酸和亚麻酸与己醛呈现显著正相关关系,但与LOX活力的相关性未达显著水平。这说明鲜食玉米中来自LOX途径的挥发性成分可能主要取决于亚油酸或亚麻酸的量,而并非LOX活力的大小。另外,两种玉米挥发性成分尤其是C6醛醇含量的不同可能体现了样品间LOX途径上的差异。     

中国母乳组学队列研究与产业化应用

母乳为婴儿提供个性化、全面、均衡的营养成分以及免疫保护,是0～6月婴儿的最佳营养来源,是婴儿食物的金标准。母乳组学队列研究是全面解析中国母乳组成特征、建立中国婴儿食品规范与标准、制定婴儿营养指南、创制婴儿食品的必要前提。本文介绍了国家母婴乳品健康工程技术研究中心基于中国母婴营养健康出生队列研究（China maternal and infant nutrition health birth cohort study,MINC）的母乳脂质组学、糖组学、蛋白质组学和微生物组等组学检测技术、组成特征、与牛乳的差异及其在婴儿食品中的产业化应用等方面的研究进展,以期为母乳组学队列研究、模拟与产业化应用提供科技支撑与示范。     

生物合成己二酸的代谢工程研究进展

己二酸是一种重要的大宗化学品,主要用于合成尼龙和聚氨酯泡沫塑料,市场需求巨大,其高效生物合成至今还未实现。本文概括了酿酒酵母中构建和优化新的己二酸合成途径研究进展。首先,通过体内及体外活性测试,对催化每一步反应的酶进行筛选,构建初步的代谢途径。利用组学分析诊断和定位生物合成途径的瓶颈。对于途径中的限速酶采用蛋白质工程手段进行改造。其次,利用合成生物学和代谢工程手段优化代谢途径。优化手段具体包括:通过模块化优化,平衡各个基因之间的表达;利用蛋白支架,构建酶反应的流水线,减少中间产物的扩散,提高反应效率;通过RNA干扰技术抑制竞争代谢途径的流量,提高目标代谢途径的通量。最后,利用CRISPR/Cas9及全局转录机器工程(gTME)等最新技术进行基因组编辑、重排转录网络,最终获得己二酸的高产菌株及适用于高效生产其他芳香族化合物的底盘酵母菌株。     

Bioactive Oxylipins in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae (including strains used in fermentation processes) produce short chain (mainly 8 carbon) oxylipins and not potent inflammatory long chain (20 carbon) oxylipins such as prostaglandins. When acetylsalicylic acid (aspirin) was added to cultures of Sacch. cerevisiae UOFS Y‐2330, flocculation was significantly inhibited as well as the production of 3‐hydroxy 8:0 thereby linking flocculation and this oxylipin. Furthermore, no traces of 3‐hydroxy 8:0 could be detected at the start of flocculation in this yeast. This research is based on (i) reports that yeasts in general can produce bioactive prostaglandins, (ii) findings suggesting a link between aspirin‐sensitive prostaglandins and biofilm formation by Candida albicans, (iii) the discovery that the addition of low concentrations of aspirin abolish yeast biofilm formation and sexual cell aggregation and (iv) the recent discovery of a novel potent aspirin‐sensitive pro‐inflammatory 3‐hydroxy prostaglandin E₂ synthesized by Candida albicans in conjunction with mammalian cells probably during candidiasis.     

Regio– and Stereoselectivity of Malt Lipoxygenases LOX1 and LOX2

The characterisation of lipoxygenases LOX1 and LOX2 and hydroperoxyoctadecadienoic acid (HPODE) degrading enzymes from barley green malt is reported. Hydroxylapatite chromatography (HAC) and isoelectric focussing (IEF) were performed to separate and purify LOX isoenzymes. The regio‐ and stereo‐selectivity of LOX1 and LOX2 towards linoleic acid as substrate was characterised. HAC purified isoenzyme LOX1 showed a 9‐HPODE:13‐HPODE ratio of 75:25 and LOX2 a ratio of 39:61. IEF separated LOX1 and LOX2 transformed linoleic acid to 9‐:13‐HPODE ratios of 90:10, and 13:87, respectively. 9‐HPODE stereoisomers from LOX1 exhibited a S:R ratio of 93:7 and 13‐HPODE from LOX2 a S:R ratio of 89:11. However, the minor regioisomers were analysed with S:R = 48:52 (LOX1, 13‐HPODE) and 40:60 (LOX2, 9‐HPODE). These results indicate a complete LOX isoenzyme separation by IEF. Hydroperoxide‐metabolising enzymes, which were investigated in the IEF fractions, did not interfere with the dual position specificities of LOX isoenzymes.