Eicosapentaenoic acid (EPA, C20:5, n-3) is a long chain crucial unsaturated fatty acid, essential for the regulation of critical biological functions in humans. Its benefits include the therapeutic treatment of cardiovascular disease, schizophrenia and Alzheimer's disease. The fungus Pythium irregulare (ATCC 10951) has great potential as a natural EPA producer. In this study, the electroporation conditions for P. irregulare were determined. The auxotrophic selectable genes ura, trp and his were respectively cloned into the plasmid pESC to construct shuttle vectors. Electroporation with 2.0kV and a 0.2cm cuvette was applied as the most effective condition for heterogeneous genes transformation. The yield and content of EPA and other components of total fatty acids (TFA) were further determined by the FAME approach with GC, as well as the analysis of biomass. The EPA content in P. irregulare with heterologous pESC-TRP vector reached 16.68 mg/g if cultured in auxotrophic medium, which showed a 52.33% increase compared to the wild-type P. irregulare. The maximum of EPA yield was 98.52 mg/L from P. irregulare containing the pESC-URA plasmid, a 32.28% increase over the wild-type. However, the maximum cell dried weight of these two organisms were respectively 6.13g/L and 5.3g/L, significantly less than the 6.80g/L of the wild-type. Not only was a feasible approach detected to electro-transform and increase the EPA yield of P. irregulare, this study also inferred that Ï -6 route was mainly involved in the EPA biosynthesis in this organism. An antisense RNA (asRNA) thermodynamic model was developed to design new asRNA constructs capable of fine-tuning gene expression knockdown. The asRNA technology is now identified as an effective and specific method for regulating microbial gene expression at the posttranscriptional level. This is done by targeting mRNA molecules. Although the study of regulation by small RNAs is advanced in eukaryotes, the regulation of expression through artificially introducing antisense oligodeoxynucleotides into host is still being developed in prokaryotes. To study the thermodynamics of asRNA and mRNA binding, (i) the fluorescence protein genes GFP and mCherry were separately cloned into the common pUC19 vector and (ii) antisense GFP and antisense mCherry DNA fragments were randomly amplified and inserted into the constructed plasmid under the control of an additional plac promoter and terminator. The expression level of fluorescence reporter proteins was determined by plate reader in this combinatorial study. A thermodynamic model to describe the relationship between asRNA binding and observed expression level was created. The study indicates two factors that minimum binding energy of the asRNA-mRNA complex and the percentage of asRNA binding mRNA were crucial for regulating the expression level. The correlation relationship between gene expression level and binding percentage multiplied by the minimum binding energy was found to show a good correlation between the thermodynamic parameters and the observed level of gene expression. The model has the potential to predict the sequence of asRNA and the approach will ultimately be applied to cyanobacteria to increase lipids production. Here, the long-term approach is to build metabolic switches from asRNA that can turn "on/off" various cellular programs and metabolic pathways at will in a fine-tuned manner. This will allow engineers to control metabolic activity in response to reactor conditions. / Master of Science

Yes / Background: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. Objectives: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. Methods: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC–tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. Results: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). Conclusions: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy.

Yes / There remains an unmet need for safe and cost-effective adjunctive treatment of advanced colorectal cancer (CRC). The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and has anti-inflammatory as well as antineoplastic properties. A phase 2 randomised trial of preoperative EPA free fatty acid 2 g daily in patients undergoing surgery for CRC liver metastasis showed no difference in the primary endpoint (histological tumour proliferation index) compared with placebo. However, the trial demonstrated possible benefit for the prespecified exploratory endpoint of postoperative disease-free survival. Therefore, we tested the hypothesis that EPA treatment, started before liver resection surgery (and continued postoperatively), improves CRC outcomes in patients with CRC liver metastasis. Methods and analysis: The EPA for Metastasis Trial 2 trial is a randomised, double-blind, placebo-controlled, phase 3 trial of 4 g EPA ethyl ester (icosapent ethyl (IPE; Vascepa)) daily in patients undergoing liver resection surgery for CRC liver metastasis with curative intent. Trial treatment continues for a minimum of 2 years and maximum of 4 years, with 6monthly assessments, including quality of life outcomes, as well as annual clinical record review after the trial intervention. The primary endpoint is CRC progression-free survival. Key secondary endpoints are overall survival, as well as the safety and tolerability of IPE. A minimum 388 participants are estimated to provide 247 CRC progression events during minimum 2-year follow-up, allowing detection of an HR of 0.7 in favour of IPE, with a power of 80% at the 5% (two sided) level of significance, assuming drop-out of 15%. Ethics and dissemination: Ethical and health research authority approval was obtained in January 2018. All data will be collected by 2025. Full trial results will be published in 2026. Secondary analyses of health economic data, biomarker studies and other translational work will be published subsequently. Trial registration number NCT03428477. / The EMT2 trial is funded by Yorkshire Cancer Research (L387) and is sponsored by the University of Leeds. The EMT2 biospecimen collection is funded by the National Institutes of Health (1R01CA243454-01A1) and is sponsored by the University of Leeds ( governance-ethics@ leeds. ac. uk). Both studies have been adopted to the NIHR Clinical Research Network (CRN) Portfolio (CPMS ID 34700 and 47372, respectively) and have benefited from CRN research staff support.

Purpose: To 1) critically appraise available randomized controlled trials (RTCs) addressing the efficacy of long-chain ω-3 fatty acids as a secondary prevention agent of hypertriglyceridemia, and 2) make recommendations for clinical practice. Data Sources: All RCTs identified from several databases from 1993-2003 were reviewed by two independent reviewers who extracted data from each study and used the previously tested Boyack and Lookinland Methodological Quality Index (MQI) to determine study quality. Results: Ten studies reported long-chain ω-3 fatty acids to be effective in the treatment of hypertriglyceridemia. The average decrease in triglycerides (TG) was 29%, total cholesterol (TC) 11.6%, very low density lipoprotein (VLDL) 30.2%, and low-density lipoprotein (LDL) 32.5%. One study found LDLs to increase by 25%. The average increase in high-density lipoprotein (HDL) was 10%. The overall average MQI score was 36% (26%-54%). Many of the RCTs had serious shortcomings including short duration, lack of a power analysis, no intention to treat analysis, no report of blind assessment of outcome, and lack of dietary control as a confounding variable. Conclusions/Implications: Overall study methodology was weak. Although the evidence supporting the use of long-chain ω-3 fatty acids in the secondary prevention of hypertriglyceridemia is reasonably strong, until there are larger RCTs of stronger methodological quality, it is not recommended to treat hypertriglyceridemia with ω-3 fatty acid supplementation in lieu of lipid lowering medications.

Yes / Abstract Objective Omega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22). Design A randomised, open-label, cross-over trial of 8 weeks’ treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week ‘washout’ period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry. Results Both omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or β diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects. Conclusion Omega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure. / NIHR/EME Yorkshire Cancer Research (YCR)

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

The gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) require n-3 long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for optimal growth and health. Due to the rapid growth of global aquaculture the quantity of marine oils used in aquafeeds has been limited, yet the overall quantity of oil in an aquafeed has increased by the addition of vegetable oil (VO) to supply dietary energy. For aquaculture to continue to grow more fish must be produced with less marine ingredients, yet EPA and DHA must be maintained at levels above fish requirements. This project set out to re-evaluate the requirement for EPA and DHA in gilthead seabream and European seabass. Two dose-response studies were designed and executed where juvenile seabream and seabass were fed one of six levels of EPA+DHA (0.2 – 3.2 % as fed). Biometric data were collected and analysed to determine new requirement estimates for EPA+DHA for fish of two weight ranges (24 – 80 g and 80 – 200 g). The effects of the dietary LC-PUFA gradient on lipid composition and metabolism were also considered.

L’hypertension artérielle pulmonaire (HTAP) est une pathologie affectant les artères pulmonaires de petit calibre entraînant une augmentation des résistances artérielles pulmonaires aboutissant à une défaillance cardiaque droite. La vasoconstriction, le remodelage des artères pulmonaires distales, l’inflammation et le stress oxydant sont de facteurs clés de la pathogénèse de l'HTAP. Nous avons testé les potentiels de la formulation EPA:DHA 6:1 à prévenir l’hypertension pulmonaire et les altérations cardiovasculaires et pulmonaires induites par l’injection de monocrotaline chez le rat. Le traitement des rats monocrotaline avec l’EPA:DHA 6:1 (500 mg/kg/j, p.o) prévient significativement l’élévation de la pression artérielle pulmonaire moyenne, la pression systolique du ventricule droit, diminue le débit cardiaque, l’hypertrophie et la dilatation du ventricule droit. L’EPA:DHA 6:1 réduit également les résistances vasculaires pulmonaires, le remodelage des artérioles pulmonaires et les infiltrations lymphocytaires et macrophagiques. De plus, l’EPA:DHA 6:1 inhibe la production des espèces réactives de l’oxygène, diminue la surexpression des sous-unités p22phox et p47phox de la NADPH oxydase, des cyclooxygénases 1 et 2, des récepteurs ETA et ETB de l’endothéline-1, de la eNOS découplée et améliore la dysfonction endothéliale des artères pulmonaires. L’EPA:DHA 6:1 exercent des effets anti-inflammatoires, antioxydants et vasoprotecteurs et prévient le développement de l’HTAP induite par l’injection de monocrotaline chez le rat. / Pulmonary arterial hypertension (PAH) is characterized by remodeling of the small pulmonary arteries leading to a progressive increase in pulmonary vascular resistance and right ventricular failure. Pulmonary endothelial dysfunction, inflammation and oxidative stress promote the development of pulmonary hypertension. Omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) have been shown to protect the cardiovascular system and reduce inflammation and oxidative stress. The present study evaluate the potential of EPA:DHA 6:1 to prevent monocrotaline-induced PAH in rats. EPA:DHA 6:1 treatment (500 mg/kg/d, p.o) prevented the MCT-induced mean pulmonary arterial pressure, right ventricular systolic pressure and decreased cardiac output. EPA:DHA 6:1 also attenuated right ventricular hypertrophy by reducing Fulton’s index and pulmonary arterial remodeling, decreased lymphocytes T and macrophages infiltration. EPA:DHA 6:1 treatment significantly reduced MCT-induced vascular oxidative stress and improved endothelial function in pulmonary arteries. The protective effect of EPA:DHA 6:1 was associated with the prevention of the MCT-induced upregulation of NADPH oxidase subunits (p22phox and p47phox), COX-1 and COX-2, endothelin A and B receptors and uncoupled eNOS in pulmonary arterioles. Our studies show that the EPA:DHA 6:1 formulation exerts anti-inflammatory, anti-oxidant and has a protective vascular effect in the pulmonary arteries, which may contribute to prevent and potentially cure pulmonary hypertension induced by MCT in rat.

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

L’optimisation du statut tissulaire en acides gras polyinsaturés à longues chaines n-3 (AGPILC n-3) fait partie des objectifs actuels en nutrition humaine. En effet, de nombreux effets bénéfiques sur la santé humaine ont été démontrés pour ces différents AGPILC n-3. Néanmoins, la bioconversion de ces dérivés à partir de leur précurseur alimentaire, l’acide α-linolénique, est limitée chez l’Homme et ne permet pas de subvenir aux besoins. Parmi cette famille d’acides, l’acide docosahexaénoïque (DHA) et de l’acide eicosapentaénoïque (EPA) ont été largement étudiés à l’inverse de ceux de l’acide docosapentaénoïque n-3 (DPA). Le premier objectif de ces travaux a été d’étudier l’impact d’une supplémentation nutritionnelle en DPA sur le métabolisme des acides gras et le métabolisme lipidique chez le rat sain. Pour cela, une méthode de purification du DPA à haute pureté et en grande quantité par chromatographie liquide a tout d’abord été mise en place. Ce résultat participe à faciliter la recherche future des effets in vivo du DPA dont la disponibilité commerciale est faible. Après sa supplémentation nutritionnelle, le DPA a été incorporé dans de multiples tissus et a impacté les compositions tissulaires en AGPILC n-3 de certains organes spécifiques comme le foie, le cœur, le poumon, la rate et le rein. Ainsi, la supplémentation en DPA pourrait être une source d’EPA, de DPA, et dans une moindre mesure de DHA. Enfin, comparé à une supplémentation en EPA ou en DHA, le DPA a été l’AGPILC n-3 le plus puissant pour améliorer la triglycéridémie et la cholestérolémie des animaux. Ces informations participeront certainement à une prise en compte plus importante du DPA tant dans les études nutritionnelles ainsi que dans l’installation des futures recommandations nutritionnelles. Le deuxième axe de recherche a porté sur la capacité d’une incorporation partielle de lipides laitiers dans le régime à augmenter le statut tissulaire en AGPI-LC n-3. L’incorporation de lipides laitiers a augmenté le statut en DHA cérébral et rétinien et le statut en DPA dans les autres tissus étudiés. De plus, l’induction de la conversion des AGPILC n-3 à partir de leur précurseur par les lipides laitiers a été complémentaire avec la supplémentation en DPA pour augmenter le statut tissulaire en AGPI-LC n-3. Ces résultats s’insèrent dans un contexte de nutrition infantile, période pendant laquelle l’incorporation des AGPI-LC n-3 est maximale dans les tissus nerveux. Ainsi, ces résultats apportent des données intéressantes tant pour les entreprises que pour les professionnels de santé pour recommander à la consommation des formules infantiles contenant des lipides laitiers. / The optimization of tissue status in n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) is one of the current goals in human nutrition. Indeed, many beneficial effects on human health have been demonstrated for these different n-3 LCPUFA. However, the bioconversion of these derivatives from their food precursor, α-linolenic acid, is limited in humans and it does not meet the needs of the organism. Among this family of fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have been widely studied in contrast to those of n-3 docosapentaenoic acid (DPA). The first objective of this work was to study the impact of dietary supplementation with DPA on fatty acid metabolism and lipid metabolism in healthy rats. For this, a method for purifying DPA with high purity and in large quantities by liquid chromatography was first established. This result helps future research of the in vivo effects of DPA, poorly commercially available. After its nutritional supplementation, DPA was incorporated into multiple tissues and it has affected the n-3 LCPUFA tissue compositions of specific organs such as the liver, heart, lung, spleen and the kidney. Thus, supplementation with DPA could be a source of EPA, DPA, and to a lesser extent DHA. Finally, compared to supplementation with EPA or DHA, DPA was the most potent n-3 LCPUFA to improve triglyceridemia and cholesterolemia in animals. This information will certainly contribute to a greater consideration of DPA both in nutritional studies and in the direction of future nutritional recommendations. The second area of ​​research focused on the ability of a partial incorporation of dairy lipids in the diet to increase the tissue status in n-3 LCPUFA. Incorporation of dairy lipids in the diet increased DHA status in brain and retina and DPA status in the other studied tissues. In addition, the induction of conversion of n-3 LCPUFA from their precursor by dairy lipid was complementary with the DPA supplementation to increase n-3 LCPUFA status in tissues. These results fit into a context of infant nutrition, during which the incorporation of n-3 LCPUFA is maximal in nervous tissues. Thus, these results provide interesting data for both companies and health professionals to recommend infant formulas containing dairy lipids for consumption.