Delta-6 Desaturase Research Articles

Effects of mineral methionine hydroxy analog chelate in sow diets on epigenetic modification and growth of progeny.

The study was conducted to determine the effects of mineral methionine hydroxy analog chelate (MMHAC) partially replacing inorganic trace minerals in sow diets on epigenetic and transcriptional changes in the muscle and jejunum of progeny. The MMHAC is zinc (Zn), manganese (Mn), and copper (Cu) chelated with methionine hydroxy analog (Zn-, Mn-, and Cu-methionine hydroxy analog chelate [MHAC]). On day 35 of gestation, 60 pregnant sows were allotted to two dietary treatments in a randomized completed block design using parity as a block: 1) ITM: inorganic trace minerals with zinc sulfate (ZnSO4), manganese oxide (MnO), and copper sulfate (CuSO4) and 2) CTM: 50% of ITM was replaced with MMHAC (MINTREX trace minerals, Novus International Inc., St Charles, MO). Gestation and lactation diets were formulated to meet or exceed NRC requirements. On days 1 and 18 of lactation, milk samples from 16 sows per treatment were collected to measure immunoglobulins (immunoglobulin G, immunoglobulin A, and immunoglobulin M) and micromineral concentrations. Two pigs per litter were selected to collect blood to measure the concentration of immunoglobulins in the serum, and then euthanized to collect jejunal mucosa, jejunum tissues, and longissimus muscle to measure global deoxyribonucleic acid methylation, histone acetylation, cytokines, and jejunal histomorphology at birth and day 18 of lactation. Data were analyzed using Proc MIXED of SAS. Supplementation of MMHAC tended to decrease (P = 0.059) body weight (BW) loss of sows during lactation and tended to increase (P = 0.098) piglet BW on day 18 of lactation. Supplementation of MMHAC increased (P < 0.05) global histone acetylation and tended to decrease myogenic regulatory factor 4 messenger ribonucleic acid (mRNA; P = 0.068) and delta 4-desaturase sphingolipid1 (DEGS1) mRNA (P = 0.086) in longissimus muscle of piglets at birth. Supplementation of MMHAC decreased (P < 0.05) nuclear factor kappa B mRNA in the jejunum and DEGS1 mRNA in longissimus muscle and tended to decrease mucin-2 (MUC2) mRNA (P = 0.057) and transforming growth factor-beta 1 (TGF-β1) mRNA (P = 0.057) in the jejunum of piglets on day 18 of lactation. There were, however, no changes in the amounts of tumor necrosis factor-alpha, interleukin-8, TGF-β, MUC2, and myogenic factor 6 in the tissues by MMHAC. In conclusion, maternal supplementation of MMHAC could contribute to histone acetylation and programming in the fetus, which potentially regulates intestinal health and skeletal muscle development of piglets at birth and weaning, possibly leading to enhanced growth of their piglets.

ω-6/ω-3 fatty acid ratio as an essential predictive biomarker in the management of type 2 diabetes mellitus.

Intake of dietary fatty acid may play a major role in the prevention and management of lifestyle-related diseases such as type 2 diabetes mellitus (T2DM). Therefore, the aim of this study was to find an association between ω-6 to ω-3 fatty acid ratio and T2DM. Fasting plasma glucose, glycated hemoglobin, and insulin were measured using commercially available kits. Fatty acid methyl esters were prepared using standard protocols. Delta-5 desaturase (D5D) and delta-6 desaturase (D6D) activities were determined from product-to-precursor ratios of individual fatty acids in plasma. Statistical analysis was performed using SPSS version 20. The ratio of ω-6 to ω-3 was higher in the group with diabetes (13:1) when compared with the group without diabetes (4:1) and was statistically significant (P < 0.0001). Further association studies showed that univariate model with the ω-6 to ω-3 ratio and a multivariate model with D5D, D6D, and ω-6 to ω-3 ratio could serve as predictive polyunsaturated fatty acid pathway models for T2DM. From the study results, it is evident that ω-6 to ω-3 fatty acid ratios can serve as essential predictive biomarkers in the management of patients with T2DM. This would not only help in management but would also aid in prevention of increased T2DM incidence in India. These results potentiate the need to maintain an ideal balance of ω-6 to ω-3, as prevention is always better than cure.

Cold stress regulates lipid metabolism via AMPK signalling in Cherax quadricarinatus.

Lipids play an important role in protecting poikilotherms from cold stress, but relatively little is known about the regulation of lipid metabolism under cold stress, especially in crustaceans. In the present study, red-clawed crayfish Cherax quadricarinatus was employed as a model organism. Animals were divided into four temperature groups (25, 20, 15 and 9°C) and treated for 4 weeks, with the 25°C group serving as a control. The total lipid content in the hepatopancreas as well as the triglyceride, cholesterol and free fatty acid levels in the hemolymph were determined. Lipids stored in the hepatopancreas and hemolymph decreased with decreasing temperature, with changes in the 9°C group most pronounced, indicating that lipids are the main energy source for crayfish at low temperatures. Furthermore, enzyme activity of lipase, fatty acid synthase, acetyl-CoA carboxylase, and lipoprotein esterase, and gene expression analysis of fatty acid synthase gene, acetyl-CoA carboxylase gene and carnitine palmitoyltransferase gene showed that the digestion, synthesis and oxidation of lipids in the hepatopancreas were inhibited under low temperature stress, but expression of sphingolipid delta-4 desaturase (DEGS) was increased, indicating an increase in the demand for highly unsaturated fatty acids at low temperatures. Analysis of the expression of genes related to the AMP-activated protein kinase (AMPK) signalling pathway revealed that the adiponectin receptor gene was rapidly upregulated at low temperatures, which may in turn activate the expression of the downstream AMPKα gene, thereby inhibiting lipid anabolism.

Dual expression of transgenic delta-5 and delta-6 desaturase in tilapia alters gut microbiota and enhances resistance to Vibrio vulnificus infection.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit antibacterial and anti-inflammatory activities. Furthermore, diets rich in n-3 PUFAs are known to improve disease resistance and limit pathogen infection in commercial aquaculture fishes. In this study, we examined the effects of transgenic overexpression of n-3 PUFA biosynthesis genes on the physiological response to bacterial infection in tilapia. We first established tilapia strains with single or dual expression of salmon delta-5 desaturase and/or delta-6 desaturase and then challenged the fish with Vibrio vulnificus infection. Interestingly, our data suggest that n-3 PUFA-mediated alterations in gut microbiota may be important in determining disease outcome via effects on immune response of the host. Both liver- and muscle-specific single and dual expression of delta-5 desaturase and delta-6 desaturase resulted in higher n-3 PUFA content in transgenic fish fed with a LO basal diet. The enrichment of n-3 PUFAs in dual-transgenic fish is likely responsible for their improved survival rate and comparatively reduced expression of inflammation- and immune-associated genes after V. vulnificus infection. Gut microbiome analysis further revealed that dual-transgenic tilapia had high gut microbiota diversity, with low levels of inflammation-associated microbiota (i.e., Prevotellaceae). Thus, our findings indicate that dual expression of transgenic delta-5 and delta-6 desaturase in tilapia enhances disease resistance, an effect that is associated with increased levels of n-3 PUFAs and altered gut microbiota composition.

Genetic variants in FADS1 and ELOVL2 increase level of arachidonic acid and the risk of Alzheimer's disease in the Tunisian population

Polyunsaturated fatty acids (PUFAs) are closely related to various physiological conditions. In several age-related diseases including Alzheimer's disease (AD) altered PUFAs metabolism has been reported. However, the mechanism behind PUFAs impairment and AD developpement remains unclear. In humans, PUFAs biosynthesis requires delta-5 desaturase (D5D), delta-6 desaturase (D6D) and elongase 2 activities; which are encoded by fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), and elongation of very-long-chain fatty acids-like 2 (ELOVL2) genes, respectively. In the present work, we aim to assess whether genetic variants in FADS1, FADS2 and ELOVL2 genes influence plasma and erythrocyte PUFA composition and AD risk. A case-control study was carried out in 113 AD patients and 161 healthy controls.Rs174556, rs174617, and rs3756963 of FADS1, FADS2, and ELOVL2 genes, respectively were genotyped using PCR-RFLP. PUFA levels were quantified using Gas Chromatography. Genotype distributions of rs174556 (FADS1) and rs3756963 (ELOVL2) were different between case and control groups. The genotype TT of rs174556 and rs3756963 single nucleotide polymorphism (SNP) increases significantly the risk of AD in our population. PUFA analysis showed higher plasma and erythrocyte arachidonic acid (AA) level in patients with AD, whereas only plasma docosahexaenoic acid (DHA) was significantly decreased in AD patients. The indexes AA/Dihomo-gamma-linolenic acid (DGLA) and C24:4n-6/Adrenic acid (AdA) were both higher in the AD group. Interestingly, patients with TT genotype of rs174556 presented higher AA level and AA/DGLA index in both plasma and erythrocyte. In addition, higher AA and AA/DGLA index were observed in erythrocyte of TT genotype ofrs3756963 carrier's patients. Along with, positive correlation between AA/DGLA index, age or Gamma-linolenic acid (GLA)/ Linoleic acid (LA) index was seen in erythrocyte and /or plasma of AD patients. After adjustment for confounding factors, the genotype TT of rs174556, erythrocyte AA and AA/DGLA index were found to be predictive risk factors for AD while plasma DHA was found associated with lower AD risk. Both rs174556 and rs3756963 influence AD risk in the Tunisian population and they are likely associated with high AA level. The combination of the two variants increases further the susceptibility to AD. We suggest that FADS1 and ELOVL2 variants could likely regulate the efficiency of AA biosynthesis which could be at the origin of inflammatory derivate.

Dietary Supplementation with Omega-6 LC-PUFA-Rich Microalgae Regulates Mucosal Immune Response and Promotes Microbial Diversity in the Zebrafish Gut.

The effect of dietary omega-6 long-chain polyunsaturated fatty acid (LC-PUFA) on host microbiome and gut associated immune function in fish is unexplored. The effect of dietary supplementation with the omega-6 LC-PUFA-rich microalga Lobosphaera incisa wild type (WT) and its delta-5 desaturase mutant (MUT), rich in arachidonic-acid and dihomo-gamma-linolenic acid (DGLA), respectively, on intestinal gene expression and microbial diversity was analyzed in zebrafish. For 1 month, fish were fed diets supplemented with broken biomass at 7.5% and 15% (w/w) of the two L. incisa strains and a control nonsupplemented commercial diet. Dietary supplementation resulted in elevated expression of genes related to arachidonic acid metabolism-cyclooxygenase 2 (cox-2), lipoxygenase 1(lox-1), anti-inflammatory cytokine-interleukin 10 (il-10), immune defense-lysozyme (lys), intestinal alkaline phosphatase (iap), complement (c3b), and antioxidants-catalase (cat), glutathione peroxidase (gpx). Microbiome analysis of the gut showed higher diversity indices for microbial communities in fish that were fed the supplemented diets compared to controls. Different treatment groups shared 237 operational taxonomic units (OTUs) that corresponded to the core microbiome, and unique OTUs were evident in different dietary groups. Overall, the zebrafish gut microbiome was dominated by the phylum Fusobacteria and Proteobacteria (averaging 38.4% and 34.6%, respectively), followed by Bacteroidetes (12.9%), Tenericutes, Planctomycetes, and Actinobacteria (at 3.1–1.3%). Significant interaction between some of the immune-related genes and microbial community was demonstrated.

Fatty acid desaturation in red blood cell membranes of patients with type 2 diabetes is improved by zinc supplementation

Background/ObjectiveMembrane flexibility can be a determining factor in pathophysiological mechanisms of type 2 diabetes (T2D). As a cofactor of delta-5 desaturase (D5D) and delta-6 desaturase (D6D), and gene expression regulator, zinc may play a role modulating membrane flexibility by increasing membrane polyunsaturated fatty acids (PUFA) abundance. The objective of this study was to evaluate the effect of a 24-month zinc supplementation (30 mg elemental zinc) on membrane fatty acid composition in patients with T2D. Subjects/methodsSixty patients with T2D were evaluated. Thirty were randomly assigned to the zinc supplemented group and thirty to the placebo group. Fatty acid composition in red blood cell (RBC) membranes was determined by gas chromatography. Expression of gene encoding for D5D (FADS1), and D6D (FADS2) were evaluated in peripheral blood mononuclear cells by real-time polymerase chain reaction. ResultsAfter 24 months of supplementation, a greater abundance of docosapentaenoic acid (C22:5 n-3), arachidonic acid (C20:4 n-6), adrenic acid (C22:4 n-6), and total n-6 PUFA was found (p = 0.001, p = 0.007, p = 0.033, p = 0.048, respectively). The unsaturated fatty acids/saturated fatty acids ratio, and unsaturation index was increased in the zinc supplemented group at month 24 (p = 0.003 and p = 0.000, respectively). FADS1 gene was upregulated in the zinc group in relation to placebo at month 12 (p = 0.020). ConclusionsSupplementation with 30 mg/d elemental zinc during 24 months in patients with T2D had an effect on the composition of RBC membranes increasing PUFA abundance and in turn, improving membrane flexibility. This effect may be mediated by induction of D5D gene expression.

Circulating and Adipose Tissue Fatty Acid Composition in Black South African Women with Obesity: A Cross-Sectional Study.

Background and Aims: During positive energy balance, excess lipid storage in subcutaneous adipose tissue (SAT) is associated with increased lipolysis. Elevated circulating fatty acid (FA) concentrations from both SAT lipolysis and dietary fat intake may result in visceral adipose tissue (VAT) accumulation, impairment of glucose metabolism, altogether increasing obesity-associated metabolic risks. We aimed to test the hypothesis that FA composition of red blood cell total phospholipids (RBC-TPL) and SAT is associated with body fat centralisation (VAT/SAT ratio) and insulin sensitivity (SI) in black South African women with obesity. Methods: Participants’ (n = 41) body fat composition and distribution, SI, and RBC-TPL, abdominal and gluteal SAT (gSAT) FA composition (gas-liquid chromatography) were measured. Results: RBC-TPL contained higher proportions of saturated fatty acids (SFAs) than SAT (p < 0.001), which were associated with lower SI (p < 0.05). Mono-unsaturated fatty acids (MUFAs) and stearoyl-CoA desaturase-1 (SCD1)-16 were lower, while poly-unsaturated fatty acids (PUFAs), and delta-5 and delta-6 desaturase indices were higher in RBC-TPL than SAT (p < 0.001). Interestingly, FA profiles differed between SAT depots with higher SFAs and lower MUFAs, SCD1-16 and SCD1-18 indices in abdominal compared to gluteal SAT (p < 0.01). In both SAT depots, higher SFAs and lower PUFAs (n-3 and n-6) correlated with lower VAT/SAT ratio; and lower PUFAs (n-3 and n-6) and higher total MUFA correlated with higher SI. Conclusion: Our findings confirm the relationships between the FA composition of RBC-TPL and SAT and metabolic risk in black women with obesity, which are dependent on both the FA class, and the tissue type/blood compartment in which they are distributed.

Fatty Acid Profile and Desaturase Activities in 7-10-Year-Old Children Attending Primary School in Verona South District: Association between Palmitoleic Acid, SCD-16, Indices of Adiposity, and Blood Pressure.

In previous studies, dietary and circulating fatty acids (FA) and desaturases activity (delta-5 desaturase [D5D], delta-6 desaturase [D6D], and stearoyl-CoA desaturase [SCD-16]) involved in their metabolism were associated with metabolic and cardiovascular disorders. The aim of the study was to assess the association between different FAs and desaturases activity (estimated as product:precursor ratios) with individual cardiovascular risk factors (in particular, anthropometric measurements and blood pressure [BP]) in children. The FA profile was determined on a whole-blood drop in 243 children (age: 8.6 ± 0.72 years) participating in a school-based cross-sectional study. Docosahexaenoic acid (DHA) inversely correlated with indices of adiposity, glucose, and triglycerides. Palmitoleic acid and SCD-16 were directly associated with markers of adiposity and BP, even after adjustment for main confounders. D6D correlated directly with the waist/height ratio. Children with excess weight (>85th percentile; that is overweight plus obese ones) showed higher palmitic acid, palmitoleic acid, and higher SCD-16 activity as compared to normal-weight children. Most of the associations were confirmed in the excess-weight group. Omega-3 FAs, particularly DHA, but not omega-6 FA, showed a potentially beneficial association with metabolic parameters, whereas palmitoleic acid and SCD-16 showed a potentially harmful association with indices of adiposity and BP, especially in obese children.

The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells

ABSTRACT ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer. Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

THE EFFECT OF THIOTRIAZOLINE ON ENERGY PRODUCTION IN CONDITIONS OF CHRONIC MYOCARDIAL ISCHEMIA

Objective: to study molecular effects of thiotriasoline of the patients with coronary heart disease (CHD). Materials and methods: 60 patients with stable angina of I – III functional classes were compared regarding the effects of thiotriasoline via standard therapy regime (STR), taking into account the activity of basic products level free radical oxidation (FRO) and antioxidant systems (AOS), proteomic protein profile. Results: in 24 weeks on the background of STR and thiotriasoline there is a revealed statistically reliable growth of glutathione reductase content, reduction of malonic dialdehyde, increase of endothelial lipase detection rate, phosphomevalonatekinase, γ-butyrobetaine hydroxylase, linoleil CoA-desaturase, reduction of delta-5 desaturase. Conclusion: thiotriasoline possesses cardioprotective activity with antioxidant and cytoprotective effects, reducing free radical products content and raising the activity of antioxidant systems ferments with regard to patients with CHD.

FADS1 and ELOVL2 polymorphisms reveal associations for differences in lipid metabolism in a cross-sectional population-based survey of Brazilian men and women

Cardiometabolic risk involves environmental and genetic factors. We aimed to investigate the relationship between plasma fatty acids and single nucleotide polymorphisms (SNPs), located in elongase and desaturases genes, and cardiometabolic parameters in a cross-sectional population-based survey. A sample of 226 adults who participated in the Health Survey of Sao Paulo, Brazil, was selected. Clinical and anthropometric variables, plasma lipoprotein, and fatty acid were evaluated. We hypothesized that differences in SNPs could lead to changes in plasma long-chain polyunsaturated fatty acids. We analyzed the relationship between SNPs in FADS1 (rs174546) and ELOVL2 (rs953413) genes, plasma fatty acid profiles, and cardiometabolic-related phenotypes using multiple linear regression, which was adjusted for confounders. Plasma high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels were significantly lower in carriers of the T allele for the FADS1 SNP. Plasma oleic acid levels were statistically higher in individuals with CT/TT genotypes in the FADS1 and AG/GG genotypes in the ELOVL2 SNPs in comparison to the CC and AA genotypes, respectively. Higher levels of linoleic and linolenic acid were found for T-allele carriers of FADS1 SNP. The estimated activity of the stearoyl CoA desaturase enzyme (SDC_18) was higher in the CT/TT genotypes (FADS1). Delta-5 desaturase estimated activity was statistically lower in the presence of the minor FADS1 allele. The estimated activity of the enzyme delta-6 desaturase was statistically lower for FADS1 CT and TT genotypes. SNPs in FADS1 and ELOVL2 genes showed protective associations for lipid metabolism and could be markers of lower cardiometabolic risk.

The n-10 Fatty Acids Family in the Lipidome of Human Prostatic Adenocarcinoma Cell Membranes and Extracellular Vesicles.

A new pathway leading to the n-10 fatty acid series has been recently evidenced, starting from sapienic acid, a monounsaturated fatty acid (MUFA) resulting from the transformation of palmitic acid by delta-6 desaturase. Sapienic acid has attracted attention as a novel marker of cancer cell plasticity. Here, we analyzed fatty acids, including the n-10 fatty acid contents, and for the first time, compared cell membranes and the corresponding extracellular vesicles (EV) of two human prostatic adenocarcinoma cell lines of different aggressiveness (PC3 and LNCaP). The n-10 components were 9–13% of the total fatty acids in both cancer cell lines and EVs, with total MUFA levels significantly higher in EVs of the most aggressive cell type (PC3). High sapienic/palmitoleic ratios indicated the preference for delta-6 versus delta-9 desaturase enzymatic activity in these cell lines. The expressions analysis of enzymes involved in desaturation and elongation by qRT-PCR showed a higher desaturase activity in PC3 and a higher elongase activity toward polyunsaturated fatty acids than toward saturated fatty acids, compared to LNCaP cells. Our results improve the present knowledge in cancer fatty acid metabolism and lipid phenotypes, highlighting EV lipidomics to monitor positional fatty acid isomer profiles and MUFA levels in cancer.

New Delta‐5‐Desaturase Inhibitor Suppress Lung Cancer Progression: A Paradigm Shift on COX‐2 Biology in Lung Cancer Treatment

ObjectiveAs the first leading cause of cancer deaths in the United States, lung cancer still attracts much research attention. Cyclooxygenase‐2 (COX‐2) is considered a classic target due to overexpression in nearly 80% non‐small‐cell lung cancer (NSCLC, major type) patients. The traditional concept of the COX‐2 inhibitor was designed to limit the COX‐2‐catalyzed arachidonic acid (AA) peroxidation and the formation of the PGE2 (subsequent cancer promoter). However, many COX‐2 inhibitors have failed to show clinical efficacy. Instead of inhibiting COX‐2, our previous studies have demonstrated that the genetically knocking down the delta‐5‐desaturase (D5D) by siRNA in cancer cells can lead to the inhibition of AA and PGE2 formation. This phenomenon promotes the COX‐2 catalyzed DGLA peroxidation to form 8‐hydroxyoctanoic acid (8‐HOA), which could be served as an anti‐cancer free radical byproduct in cancer therapy. However, the application of siRNA is still hindered by its in vivo instability and off‐target effect. Therefore, continued research into a new D5D small molecule inhibitor for lung cancer is required to expand the clinical benefit to the patient and to improve outcomes in NSCLC. In this study, we have first proposed the 10,11‐Dihydro‐5H‐dibenz[b,f]azepine as a newly synthesized D5D inhibitor and evaluated its effect on lung cancer by both in vitro and in vivo model.MethodAfter the treatment of novel D5D inhibitor in vitro, the DGLA, AA, PGE2, and 8‐HOA profile was measured by LC/MS or GC/MS to assess the D5D activity in A549 NSCLC cells. Colony formation assay, wound‐healing assay, transwell assay, flow cytometry analysis, and western blot analysis was performed on A549 cells to evaluate the effect of D5D inhibitor on cancer cell proliferation, migration, and apoptosis. The in vivo effect of the D5D inhibitor was determined by xenograft mouse models in a 4 weeks treatment period along with supplementation of DGLA.ResultsThe fatty acid profile results indicated that the treatment of D5D inhibitor (10 μM) significantly suppressed the AA and PGE2 but promoted 8‐HOA formation in A549 cells. The cell survival, migration, and proliferation of A549 cells were improved by the DGLA (100 μM) and D5D inhibitor treatment. Consistent with our in vitro data, the in vivo results suggested that the D5D inhibitor (15 mg/kg) along with DGLA supplementation could promote 8‐HOA formation to a threshold level, resulting in significant tumor reduction in both A549 and LLC xenograft mouse (nu/nu or C57B6) models. D5D inhibitor also suppressed the metastasis of LLC cells from the site of injection to the lung. Additionally, western blotting and immunohistochemistry results indicated that the D5D inhibitor also promoted apoptosis in tumor tissues.ConclusionOur new concept makes active use of the commonly overexpressed COX‐2 in cancer cells (no longer to be considered a problem, but instead a benefit), and concurrently targets D5D to also elicit DGLA’s anti‐cancer effect; these two efforts will act in concert to result in more effective and safer therapeutic outcomes for lung cancer treatment.Support or Funding InformationNIHR15CA195499

Development of a Novel ω‐6 Polyunsaturated Fatty Acid Based Treatment Strategy for Non‐Melanoma Skin Cancer by Inhibiting Delta‐5‐Desaturase

BackgroundCyclooxygenase‐2 (COX‐2) overexpression has been considered as one of the culprits for cancer progression, due to precancerous prostaglandin production from downstream ω‐6 polyunsaturated fatty acid (PUFA), arachidonic acid (AA), in the majority of the cancers including, Nonmelanoma skin cancer (NMSC). However, COX‐2 inhibitors have failed to provide protection against cancer in clinics. As per the recent studies from our lab, manipulation of ω‐6 PUFA metabolism by genetically inhibiting delta‐5‐desaturase (D5D), requires for metabolism of an upstream ω‐6 PUFA, dihomo gamma‐linolenic acid (DGLA), to AA, results in accumulation of DGLA which undergo peroxidation by COX‐2 to produce an anti‐cancer product, 8‐Hydroxyoctanoic acid (8‐HOA) in pancreatic and colon cancer model. However, this approach was associated with limitations, such as the degradation of D5D siRNA by endonuclease and the inability to cross the cell membrane. Hence in order to overcome the limitation and to get a sustained production of 8‐HOA inside the tumor, we developed a novel D5D inhibitor (D5Di) and hypothesized that D5Di could stimulate 8‐HOA production, which exerts anti‐cancer activity in NMSC.MethodsIn vitro studies like colony formation assay for proliferation analysis and PI Annexin V double staining assay for apoptosis analysis using A431 cell line and in vivo studies using xenograft mouse model evaluating the protective effect of D5Di against NMSC were performed. In order to dissect the molecular pathway, Western blot analysis, and immunohistochemistry studies were performed.ResultsD5Di treatment in the presence of DGLA resulted in a significant increase in apoptosis by 14.23±1.01% and reduction in survival fraction by 29.91±0.70%. Similar to the in vitro study, treatment with D5Di in animals receiving the DGLA supplement displayed a significant reduction in tumor size. The reduction in tumor size was due to a significantly high level of 8‐HOA (1.32±0.122μM) and a low PGE2/PGE1 ratio. In addition, an increase in cleaved PARP and reduction in Ki‐67 were observed by immunohistochemistry, which were indicating inhibitory effect of D5Di on tumor growth. Moreover, treatment with D5Di also caused downregulation of anti‐apoptotic factors such as procaspase‐3, procaspase‐9, and increased DNA damage marker, γH2Ax, both in vitro and in vivo.ConclusionThese results concluded that the novel strategy of providing D5Di with DGLA could be a new therapeutic approach for advanced and high‐risk NMSC.Support or Funding InformationThis research work is supported by NIH R15CA195499.

The global epidemiology of emerging Histoplasma species in recent years.

Histoplasmosis is a serious infectious disease in humans caused by Histoplasma spp. (Onygenales), whose natural reservoirs are thought to be soil enriched with bird and bat guano. The true global burden of histoplasmosis is underestimated and frequently the pulmonary manifestations are misdiagnosed as tuberculosis. Molecular data on epidemiology of Histoplasma are still scarce, even though there is increasing recognition of histoplasmosis in recent years in areas distant from the traditional endemic regions in the Americas. We used multi-locus sequence data from protein coding loci (ADP-ribosylation factor, H antigen precursor, and delta-9 fatty acid desaturase), DNA barcoding (ITS1/2+5.8s), AFLP markers and mating type analysis to determine the genetic diversity, population structure and recognise the existence of different phylogenetic species among 436 isolates of Histoplasma obtained globally. Our study describes new phylogenetic species and the molecular characteristics of Histoplasma lineages causing outbreaks with a high number of severe outcomes in Northeast Brazil between 2011 and 2015. Genetic diversity levels provide evidence for recombination, common ancestry and clustering of Brazilian isolates at different geographic scales with the emergence of LAm C, a new genotype assigned to a separate population cluster in Northeast Brazil that exhibited low diversity indicative of isolation. The global survey revealed that the high genetic variability among Brazilian isolates along with the presence of divergent cryptic species and/or genotypes may support the hypothesis of Brazil being the center of dispersion of Histoplasma in South America, possibly with the contribution of migratory hosts such as birds and bats. Outside Brazil, the predominant species depends on the region. We confirm that histoplasmosis has significantly broadened its area of occurrence, an important feature of emerging pathogens. From a practical point of view, our data point to the emergence of histoplasmosis caused by a plethora of genotypes, and will enable epidemiological analysis focused on understanding the processes that lead to histoplasmosis. Further, the description of this diversity opens avenues for comparative genomic studies, which will allow progress toward a consensus taxonomy, improve understanding of the presence of hybrids in natural populations of medically relevant fungi, test reproductive barriers and to explore the significance of this variation.

Effect of JAK-STAT pathway in regulation of fatty liver hemorrhagic syndrome in chickens.

ObjectiveTo explore the molecular mechanisms of fatty liver hemorrhagic syndrome (FLHS) in laying hens, an experiment was conducted to reveal the differences in histopathological observation and gene expression between FLHS group and normal group.MethodsWe compared the histopathological difference using hematoxylin and eosin staining and proceeded with RNA sequencing of adipose tissue to search differentially expressed genes and enriched biological processes and pathways. Then we validated the mRNA expression levels by real-time polymerase chain reaction and quantified protein levels in the circulation by enzyme-linked immunosorbent assay.ResultsWe identified 100 differentially expressed transcripts corresponding to 66 genes (DEGs) were identified between FLHS-affected group and normal group. Seven DEGs were significantly enriched in the immune response process and lipid metabolic process, including phospholipase A2 group V, WAP kunitz and netrin domain containing 2, delta 4-desaturase sphingolipid 2, perilipin 3, interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF), and suppressor of cytokine signaling 3 (SOCS3). And these genes could be the targets of immune response and be involved in metabolic homeostasis during the process of FLHS in laying hens. Based on functional categories of the DEGs, we further proposed a model to explain the etiology and pathogenesis of FLHS. IL-6 and SOCS3 mediate inflammatory responses and the satiety hormone of leptin, induce dysfunction of Jak-STAT signaling pathway, leading to insulin resistance and lipid metabolic disorders. Conversely, CNTF may reduce tissue destruction during inflammatory attacks and confer protection from inflammation-induced insulin resistance in FLHS chickens.ConclusionThese findings highlight the therapeutic implications of targeting the JAK-STAT pathway. Inhibition of IL6 and SOCS3 and facilitation of CNTF could serve as a favorable strategy to enhance insulin action and improve glucose homoeostasis, which are of importance for treating obesity-related disorders for chickens.

Milk fatty acid composition, rumen microbial population and animal performance in response to diets rich in linoleic acid supplemented with

Context Rumen biohydrogenation is an important way to produce conjugated linoleic acid (CLA), especially the rumenic acid isomer. However, CLA is principally synthesised endogenously in lactating mammals by delta 9-desaturase in breast tissue. Aims The aim of the study was to evaluate milk fatty acid profile, rumen microbial population and animal performance in response to diets containing sunflower oil either supplemented with or without flavonoids and essential oils from Piper betle L. powder (PP) in dairy goats. Method Twelve multiparous Saanen goats (42 ± 1.00 kg; mean ± s.d.) were randomly assigned to two treatment groups in an experiment that lasted for 6 weeks. The two experimental diets formulated as total mixed ration were: control (CTH) diet (containing 0% PP) and DPB diet (CTH diet containing 1.3% PP on a dry-matter basis). Key results Inclusion of flavonoids and essential oils from PP in the diet (DPB) did not affect dry-matter intake but resulted in a greater milk yield and altered the composition of milk. Compared with the control diet (CTH), the DPB diet decreased the saturated fatty acid concentration and increased the unsaturated fatty acid concentration in milk. Inclusion of PP decreased the C18:0 production (P &lt; 0.05), resulting in higher C18:1 trans11 and C18:2 cis9 trans11 (P &lt; 0.05) concentrations. Overall, DPB diet increased the total CLA by 1.5-fold, from 1.77 to 2.62 g/100 g fatty acid. The desaturase rate (except desaturase for carbon 18, P &lt; 0.05), and atherogenic and thrombogenic indices were not affected by inclusion of PP in the DPB diet. Moreover, the DPB diet escalated total volatile fatty acid production and altered the volatile fatty acid profile. Compared with goats fed with CTH diet, PP supplementation increased the presence of ruminal Butyrivibrio fibrisolvens by ~5-fold, but the presence of B. proteoclasticus decreased to about 1/11 of the control. Conclusions The use of sunflower oil at 17.6 g/kg diet and inclusion of a practical dose of flavonoids and essential oils from Piper betle L. leaves in the diet of dairy goats can be an efficient method to improve milk yield and milk composition, including increasing the CLA concentration of milk. Implications These results constitute an alternative strategy to improve milk quality, without negatively affecting animal performance.

Expression of enzymes involved in polyunsaturated fatty acid synthesis in the stallion testis and epididymis.

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions.

Improved SDA Production in High Lipid Accumulating Strain of Mucor circinelloides WJ11 by Genetic Modification

Stearidonic Acid (SDA; 18:4, n-3) is a ω-3 polyunsaturated fatty acid which is nutritionally important and has pharmaceutical applications. Hence, scientists are trying to construct SDA producing oleaginous microorganisms by genetic modification. Two enzymes, Delta-6 Desaturase (D6D) and ω-3 desaturase catalyze the reactions to produce SDA from Linoleic Acid (LA; 18:2, n-6). But the key enzyme, ω-3 desaturase converts LA to ALA and GLA to SDA is absent in high lipid accumulating Mucor circinelloides WJ11, retarding its ability to produce SDA. Therefore, in this study, we overexpressed fad3 gene from Arabidopsis thaliana in M. circinelloides WJ11 to produce SDA. Overexpression of fad3 gene in M. circinelloides WJ11 resulted in the production of 340 mg/l SDA. This research opened a new opportunity to make use of this fungus for industrial production of SDA.