Very Long Chain Polyunsaturated Fatty Acids Research Articles

Metabolic engineering of omega-3 long chain polyunsaturated fatty acids in plants using different ∆6- and ∆5-desaturases co-expressed with LPCAT from the marine diatom Phaeodactylum tricornutum

Continuous research on obtaining an even more efficient production of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in plants remains one of the main challenges of scientists working on plant lipids. Since crops are not able to produce these fatty acids due to the lack of necessary enzymes, genes encoding them must be introduced exogenously from native organisms producing VLC-PUFAs. In this study we reported, in tobacco leaves, the characterization of three distinct ∆6-desaturases from diatom Phaeodactylum tricornutum, fungi Rhizopus stolonifer and microalge Osterococcus tauri and two different ∆5-desaturases from P. tricornutum and single-celled saprotrophic eukaryotes Thraustochytrium sp. The in planta agroinfiltration of essential ∆6-desaturases, ∆6-elongases and ∆5-desaturases allowed for successful introduction of eicosapentaenoic acid (20:5∆5,8,11,14,17) biosynthesis pathway. However, despite the desired, targeted production of ω3-fatty acids we detected the presence of ω6-fatty acids, indicating and confirming previous results that all tested desaturases are not specifically restricted to neither ω3- nor ω6-pathway. Nevertheless, the additional co-expression of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) from Phaeodactylum tricornutum boosted the proportion of ω3-fatty acids in newly synthesized fatty acid pools. For the most promising genes combinations the EPA content reached at maximum 1.4% of total lipid content and 4.5% of all fatty acids accumulated in the TAG pool. Our results for the first time describe the role of LPCAT enzyme and its effectiveness in alleviating a bottleneck called ‘substrate dichotomy’ for improving the transgenic production of VLC-PUFAs in plants.

Regulation of meiotic telomere dynamics through membrane fluidity promoted by AdipoR2-ELOVL2

The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.

Abstract TMP109: Elovanoid Precursors Are Neuroprotective in Experimental Ischemic Stroke

Objective: Ischemic stroke triggers oxidative and biochemical responses that disrupt cell homeostasis. We previously demonstrated that Elovanoids (ELVs), a novel class of homeostatic lipid mediators, reduced infarct volumes and improved neurologic function when administered IV following middle cerebral artery occlusion (MCAo). The present study defines neuroprotection by very long-chain polyunsaturated fatty acid (VLC-PUFA) ELV precursors (C-32:6 and C-34:6) delivered intranasally following experimental ischemic stroke. Methods: Male SD rats underwent 2 hours of MCAo. Treatments with C-32:6, C-34:6 (1μg/μl, 20 μg per rat), or vehicle were administered at 1, 24, and 48h after onset of MCAo. Behavior testing was performed on days 1, 2, 3, and 7. Ischemic core and penumbra were computed from T2WI on day 7 followed by immunohistochemistry. RT-qPCR gene expression was investigated in a separate cohort of rats treated as above at 72 h. Results: C-32:6 and C-34:6 improved behavioral function on days three by (41% and 20%) and seven by (45% and 24%); T2WI total lesion volumes were reduced by (87% and 61%), respectively, compared to the vehicle. In the ischemic penumbra, C-32:6 and C-34:6 reduced TMEM119 + microglia by (38% and 29%), increased NeuN + neurons by (87% and 110%), reduced IgG staining by (54% and 25%), and increased the number of SMI-71 + vessels by (50% and 26%). In the core, C-32:6 and C-34:6 reduced TMEM119 + microglia by (12% and 15%), increased NeuN + neurons by (251% and 89%) and increased the number of SMI-71 + vessels by (31% and 70%). Anti-inflammatory marker genes for protective microglia and astrocyte phenotypes Tm4sf1, Thbs1, Ptx3, Gpc4, Fizz1, Cd14, Msr1, Manf , and Osmr were upregulated with C-32:6 and genes Fcrls, S1pr3, and Vim were downregulated with C-34:6. Conclusion: These data suggest that IN administered C-32:6 C-34:6 improved behavior, protected the ischemic penumbra, increased neurogenesis and angiogenesis, and reduced the number of microglia cells. The overall gene expression changes observed from free fatty acid C-32:6 indicate that its modulatory effects promote the biological activity of astrocytes, microglia, and cerebrovascular cells involved in plasticity.

Development of a high-performance thin-layer chromatography-based method for targeted glycerolipidome profiling of microalgae

The conditionally essential very-long-chain polyunsaturated fatty acids (VLC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5 n-3), play a vital role in human nutrition. Their biological activity is thereby greatly influenced by the distinct glycerolipid molecule that they are esterified to. Here, microalgae differ from the conventional source, fish oil, both in quantity and distribution of VLC-PUFAs among the glycerolipidome. Therefore, the aim of this study was to develop a fast and reliable one-dimensional high-performance thin-layer chromatography (HPTLC)-based method that allows the separation and quantification of the main microalgal glycerolipid classes (e.g., monogalactosyldiacylglycerol (MGDG), sulfoquinovosyl diacylglycerol (SQDG), phosphatidylglycerol (PG)), as well as the subsequent analysis of their respective fatty acid distribution via gas chromatography (GC) coupled to mass spectrometry (MS). Following optimization, method validation was carried out for 13 different lipid classes, based on the International Conference on Harmonization (ICH) guidelines. In HPTLC, linearity was effective between 100 and 2100 ng, with a limit of quantification between 62.99 and 90.09 ng depending on the glycerolipid class, with strong correlation coefficients (R2 > 0.995). The recovery varied between 93.17 and 108.12%, while the inter-day precision measurements showed coefficients of variation of less than 8.85%, close to the limit of detection. Applying this method to crude lipid extracts of four EPA producing microalgae of commercial interest, the content of different glycerolipid classes was assessed together with the respective FA distribution subsequent to band elution. The results showed that the described precise and accurate HPTLC method offers the possibility to be used routinely to follow variations in the glycerolipid class levels throughout strain screening, cultivation, or bioprocessing. Thus, additional quantitative analytical information on the complex lipidome of microalgae can be obtained, especially for n-3 and n-6 enriched lipid fractions.Graphical

A gram-scale synthesis of very-long chain polyunsaturated fatty acids (VLC-PUFAs).

This manuscript describes our third generation, gram-scale synthesis of very long chain-polyunsaturated fatty acids (VLC-PUFAs), a unique and increasingly important class of lipids. Critical to this work and what makes it different from our previous approach to this family was the avoidance of oxidation sequences. Central to accomplishing this involved the use of a Negishi coupling reaction between the acid chloride derived from DHA and a saturated alkyl zinc reaction. Overall, the general approach required 6 synthetic transformations from DHA and was accomplished with an overall yield of 40%.

Single-Cell Multiome Analysis Reveals Neuroprotection by Elovanoids in Experimental Ischemic Stroke

Ischemic stroke results in severe loss of neurological functions, and a large proportion of survivors display the onset of cognitive impairments. Administration of a novel class of very long-chain polyunsaturated fatty acids (VLC-PUFAs) termed elovanoids (ELV-N34) or its VLC-PUFA precursor (C34:6) are protective against both in vitro and in vivo models of uncompensated oxidative stress and ischemic stress. We found that ELV-N34 or C34:6 delivery attenuates sensorimotor deficits after ischemic stroke. Furthermore, we uncovered that ELV and its precursors protect against ischemic damage by modulating inflammatory signaling and increasing the expression of neuroprotective genes. We used 10X Single Cell Multiome (ATAC + Gene Expression) to compare genomic and epigenomic responses in naïve rats and after experimental ischemic treatment. Adult Sprague-Dawley rats underwent 2 hours of middle cerebral artery occlusion (MCAo) via intraluminal suture. Rats received vehicle, ELV-N34, or C34:6 delivered intranasally 1 hour, 1 day, and 2 days after 2 hours of MCAo. Brains were harvested on day 3 from the ipsilesional cortex (penumbra region) and subcortex. Nuclei were isolated and processed according to 10X Genomics Single Cell Multiome protocol. ELV-N34 or C34:6 treatment reduced neurological deficits after MCAo. Joint single-cell analysis revealed increases in microglia and leukocyte abundance as well as a loss of neuronal populations after MCAo. Differential gene expression revealed that ELV or its precursors protected against MCAo through complex mechanisms involving anti- inflammatory signaling and promoted neuroregeneration in microglia, astrocytes, neurons, and other neural cells. ELV/precursors exert their bioactivity by modulating conversion to neuroprotective cell phenotypes in neurodegenerative diseases such as stroke or Alzheimer's disease.

Elovanoid Precursor Inhibits Lipid Peroxidation in Retinal Cells: A Comprehensive Untargeted Lipidomic Approach

Age-related macular degeneration (AMD) accumulates amyloid-beta peptide as in senile plaques of Alzheimer's. Also, as in other neurodegenerative diseases, enhanced reactive oxygen species (ROS) and an overall uncompensated oxidative stress (UOS) environment evolve. Oxidized phospholipids (OxPLs) cause detrimental perturbations to photoreceptor cells (PRC) and to the retinal pigment epithelial cell (RPEC), a nondividing cell derived from the neuroectoderm, essential for PRC integrity. Elovanoids (ELVs) are lipid mediators that support PRC. They are oxygenated derivatives of very long-chain polyunsaturated fatty acids (VLC-PUFAs), specifically C32:6 and C34:6. PUFA-containing phospholipids in the cell membrane are susceptible to non-enzymatic (e.g., free radical and reactive oxygen species) and enzymatic oxidation of the unsaturated components, altering their structure and function. Studies have implicated oxidized phospholipids as relevant to the etiology of aging and neurodegeneration, including age-related macular degeneration and Alzheimer's. Due to high metabolic demands, RPE cells are under constant oxidative stress.

Deletion of Elovl5 leads to dyslipidemia and atherosclerosis in LDLR-deficient mice

Atherosclerosis is a chronic inflammatory disease for which hepatic steatosis and atherogenic dyslipidemia are significant risk factors. We investigated the effects of endogenously generated very-long-chain polyunsaturated fatty acids (VL-PUFAs) on dyslipidemia and atherosclerosis development using mice that lack ELOVL5, a PUFA elongase that is required for the synthesis of arachidonic acid, EPA, and DHA from the essential fatty acids linoleic and linolenic acids, and the LDL receptor (LDLR). Elovl5−/−;Ldlr−/− mice manifest increased liver triglyceride and cholesterol concentrations due to the activation of sterol regulatory element binding protein-1, a transcription factor that activates enzymes required for de novo lipogenesis. Plasma levels of triglycerides and cholesterol in VLDL, IDL, and LDL were markedly elevated in Elovl5−/−;Ldlr−/− mice fed a chow and the mice exhibited marked aortic atherosclerotic plaques. Bone marrow-derived monocytes from wild-type (WT) and Elovl5−/− mice were polarized to M1 and M2 macrophages, and the effects of ELOVL5 on inflammatory activity were determined. There were no differences in most of the markers tested for M1 and M2 polarized cells between WT and Elovl5−/− cells, except for a slight increase in PGE2 secretion in Elovl5−/− cells, likely due to elevated Cox-2 expression. These results suggest that the deletion of Elovl5 leads to hepatic steatosis and dyslipidemia, which are the major factors in severe atherosclerosis in Elovl5−/−;Ldlr−/− mice.

A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy.

The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina. We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods. This new approach can be used to detect in vitro the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.

Integrated inflammatory signaling landscape response after delivering Elovanoid free-fatty-acid precursors leading to experimental stroke neuroprotection

Despite efforts to identify modulatory neuroprotective mechanisms of damaging ischemic stroke cascade signaling, a void remains on an effective potential therapeutic. The present study defines neuroprotection by very long-chain polyunsaturated fatty acid (VLC-PUFA) Elovanoid (ELV) precursors C-32:6 and C-34:6 delivered intranasally following experimental ischemic stroke. We demonstrate that these precursors improved neurological deficit, decreased T2WI lesion volume, and increased SMI-71 positive blood vessels and NeuN positive neurons, indicating blood–brain barrier (BBB) protection and neurogenesis modulated by the free fatty acids (FFAs) C-32:6 and C-34:6. Gene expression revealed increased anti-inflammatory and pro-homeostatic genes and decreases in expression of pro-inflammatory genes in the subcortex. Additionally, the FFAs elicit a comprehensive downregulation of inflammatory microglia/monocyte-derived macrophages and astrocyte-associated genes in the subcortical region. Functional analysis reveals inhibition of immune-related pathways and production of upstream molecules related to detrimental signaling events in post-stroke acute and subacute phases.

Rod-specific downregulation of omega-3 very-long-chain polyunsaturated fatty acid pathway in age-related macular degeneration

Docosahexaenoic acid (DHA; 22:6) plays a key role in vision and is the precursor for very-long-chain polyunsaturated fatty acids (VLC-PUFAs). The release of 32- and 34-carbon VLC-PUFAs and DHA from sn-1 and sn-2 of phosphatidylcholine (PC) leads to the synthesis of cell-survival mediators, the elovanoids (ELVs) and neuroprotectin D1 (NPD1), respectively. Macula and periphery from age-related macular degeneration (AMD) donor retinas were assessed for the availability of DHA-related lipids by LC-MS/MS-based lipidomic analysis and MALDI-molecular imaging. We found reduced retina DHA and VLC-PUFA pathways to synthesize omega-3 ELVs from precursors that likely resulted in altered disks and photoreceptor loss. Additionally, we compared omega-3 (n-3) fatty acid with DHA (22:6) and omega-6 (n-6) fatty acid with arachidonic acid (AA; 20:4) pathways. n-3 PC(22:6/22:6, 44:12) and n-6 PC(20:4/20:4, 40:8) showed differences among male/female, macula/periphery, and normal/AMD retinas. Periphery of AMD retina males increased 44:12 abundance, while normal females increased 40:8 (all macula had an upward 40:8 tendency). We also showed that female AMD switched from n-3 to n-6 fatty acids; most changes in AMD occurred in the periphery of female AMD retinas. DHA and VLC-PUFA release from PCs leads to conversion in pro-survival NPD1 and ELVs. The loss of the neuroprotective precursors of ELVs in the retina periphery from AMD facilitates uncompensated stress and cell loss. In AMD, the female retina loses peripheral rods VLC-PUFAs to about 33% less than in males limiting ELV formation and its protective bioactivity.

A novel ELOVL4 variant, L168S, causes early childhood-onset Spinocerebellar ataxia-34 and retinal dysfunction: a case report

Spinocerebellar ataxia 34 (SCA34) is an autosomal dominant inherited disease characterized by age-related cerebellar degeneration and ataxia caused by mutations in the Elongation of Very Long Chain Fatty Acid-4 (ELOVL4) gene. The ELOVL4 enzyme catalyzes the biosynthesis of both very long chain saturated fatty acids (VLC-SFA) and very long chain polyunsaturated fatty acids (VLC-PUFA) that are important for neuronal, reproductive, and skin function. Several variants in ELOVL4 have been shown to cause different tissue-specific disorders including SCA34 with or without Erythrokeratodermia Variabilis (EKV), a skin condition characterized by dry, scaly skin, Autosomal Dominant Stargardt-Like Macular Dystrophy (STGD3), and seizures associated with neuro-ichthyotic disorders. What is puzzling is how different mutations in the same gene seem to cause different tissue-specific disorders. To date, no SCA34 patients have presented with both SCA34 and STGD3 pathology that is caused by ELOVL4 variants that cause truncation of ELOVL4. Here, we report a novel case of an early childhood onset and rapidly progressive cerebellar degeneration and retinal dysfunction in a Belgian-Italian girl who developed severe dysarthria and gait problems starting at about 3.5 years of age and progressed to immobility by 4.5 years of age. Brain magnetic resonance imaging (MRI) revealed progressive vermian, cerebellar, cortical atrophy, progressive corpus callosum slimming, and hot cross bun sign visible on the MRI. Ophthalmological examinations also revealed progressive macular dysfunction as measured by electroretinography. Using exome sequencing, we identified a novel heterozygous ELOVL4 variant, c.503 T > C (p. L168S) in the patient. To understand the enzymatic function of this novel ELOVL4 variant and how it alters the levels of VLC-PUFA and VLC-SFA biosynthesis to contribute to cerebellar and retinal dysfunction, we expressed wild-type ELOVL4 or the L168S ELOVL4 variant in cell culture and supplemented the cultures with VLC-PUFA or VLC-SFA precursors. We showed that the L168S ELOVL4 variant is deficient in the biosynthesis of VLC-SFA and VLC-PUFA. Our work suggests that differential depletion of these fatty acids may be a contributing factor to the pathogenic mechanism of SCA34 with or without EKV. Further studies will help further define how the different ELOVL4 variants cause different tissue-specific disorders with variable ages of onset.

Understanding the Roles of Very-Long-Chain Polyunsaturated Fatty Acids (VLC-PUFAs) in Eye Health.

Lipids serve many roles in the neural system, from synaptic stabilization and signaling to DNA regulation and neuroprotection. They also regulate inflammatory responses, maintain cellular membrane structure, and regulate the homeostatic balance of ions and signaling molecules. An imbalance of lipid subgroups is implicated in the progression of many retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, and diet can play a key role in influencing these diseases' onset, progression, and severity. A special class of lipids termed very-long-chain polyunsaturated fatty acids (VLC-PUFAs) is found exclusively in mammalian vertebrate retinas and a few other tissues. They comprise <2% of fatty acids in the retina and are depleted in the retinas of patients with diseases like diabetic retinopathy and AMD. However, the implications of the reduction in VLC-PUFA levels are poorly understood. Dietary supplementation studies and ELOVL4 transgene studies have had positive outcomes. However, much remains to be understood about their role in retinal health and the potential for targeted therapies against retinal disease.

EPA and DHA in microalgae: Health benefits, biosynthesis, and metabolic engineering advances

AbstractEicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are ω‐3 very long‐chain polyunsaturated fatty acids (VLC‐PUFAs) that offer a wide range of human health benefits impacting cardiovascular, anti‐inflammatory, and neurological health. It is widely known that humans inefficiently synthesize these compounds and as such rely on exogenous dietary sources, such as marine fish oils. Unfortunately, the production of marine fish oils is an unsustainable process and has suffered a dramatic fall in recent years due to overfishing and climate change, as the demand for EPA and DHA continues to rise. Therefore, there is an urgent need to develop alternative, sustainable sources for consumable EPA and DHA. Metabolic engineering of marine microalgae to improve their EPA and DHA productivity is regarded as a promising option that has received increasing commercial attention in recent years. In this mini‐review, we describe several notable health benefits of EPA and DHA, summarize the natural sources and biosynthesis of VLC‐PUFAS, as well as the recent advances in metabolic engineering of EPA and DHA production in representative microalgal and protist species, including Schizochytrium sp., Phaeodactylum tricornutum, and Nannochloropsis oceanica.

Differentiation-linked changes in the biosynthesis and turnover of sphingomyelins in rat male germ cells: Genes involved and effects of testosterone

In rodents, sphingomyelins (SMs) species with very-long-chain polyunsaturated fatty acid (VLCPUFA) are required for normal spermatogenesis. Data on the expression of enzymes with roles in their biosynthesis and turnover during germ cell differentiation and on possible effects on such expression of testosterone (Tes), known to promote this biological process, were lacking. Here we quantified, in isolated pachytene spermatocytes (PtS), round spermatids (RS), and later spermatids (LS), the mRNA levels from genes encoding ceramide (Cer), glucosylceramide (GlcCer), and SM synthases (Cers3, Gcs, Sms1, and Sms2) and sphingomyelinases (aSmase, nSmase) and assessed products of their activity in cells in culture using nitrobenzoxadiazole (NBD)-labeled substrates and [3H]palmitate as precursor. Transcript levels from Cers3 and Gcs were maximal in PtS. While mRNA levels from Sms1 increased with differentiation in the direction PtS→RS→LS, those from Sms2 increased between PtS and RS but decreased in LS. In turn, the nSmase transcript increased in the PtS→RS→LS order. During incubations with NBD-Cer, spermatocytes produced more GlcCer and SM than did spermatids. In total germ cells cultured for up to 25h with NBD-SM, not only abundant NBD-Cer but also NBD-GlcCer were formed, demonstrating SM→Cer turnover and Cer recycling. After 20h with [3H]palmitate, PtS produced [3H]SM and RS formed [3H]SM and [3H]Cer, all containing VLCPUFA, and Tes increased their labeling. In total germ cells, Tes augmented in 5h the expression of genes with roles in VLCPUFA synthesis, decreased the mRNA from Sms2, and increased that from nSmase. Thus, Tes enhanced or accelerated the metabolic changes occurring to VLCPUFA-SM during germ cell differentiation.

Sperm very long-chain polyunsaturated fatty acids: relation to semen parameters and live birth outcome in a multicenter trial

To determine whether the levels of sperm very long-chain polyunsaturated fatty acids (VLC-PUFAs) are correlated with sperm parameters and the outcome of live birth after conventional therapy for unexplained infertility. Cohort analysis of the Reproductive Medicine Network's Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation randomized controlled trial. Multicenter randomized controlled trial. Male partners from 185 couples with unexplained infertility who provided baseline semen samples for analysis. We determined the levels of VLC-PUFAs in total lipid isolated from sperm membranes using liquid chromatography-mass spectrometry/mass spectrometry analyses. Sperm concentration, motility, morphology, total motile count (TMC), and live birth after standard treatmentfor unexplained infertility. Total VLC-PUFA percentage was positively correlated with sperm concentration (Spearman's rank correlation (rs) 0.56, P<.0001), TMC (rs = 0.40, P<.0001), and morphology (rs = 0.26, P=.0005). After adjustment for male body mass index, age, and race, a one-standard-deviation increase in the percentage of total VLC-PUFA was associated with a 62% increase in the geometric mean (GM) of sperm concentration (GM Ratio: 1.62 [95% confidence intervals {CI}: 1.45, 1.82]) and a 43% increase in the geometric mean of TMC (GM Ratio: 1.43 [95% CI; 1.24, 1.63]). Although no evidence of association was observed for sperm motility, a positive relationship was also observed between the percentage of total VLC-PUFA and sperm morphology [adjusted incidence rate ratio (IRR) for one-standard-deviation increase in total VLC-PUFA: 1.18 (95% CI; 1.02, 1.36)]. After adjustment for female age and treatment group, the probability of a live birth outcome was 72% more likely among those in the third tertile of hydroxylated VLC-PUFA percentage than in the first tertile (RR 1.72 [95% CI; 1.01, 2.94]). The positive correlation between sperm VLC-PUFAs percentage and sperm parameters, as well as the significant association between hydroxylated VLC-PUFA percentage and the outcome of live birth, strongly suggest that this class of fatty liquid chromatography-mass spectrometry/mass spectrometry acids is essential for normal sperm structure and function.

Extending Omega 3/6 Fatty Acid Pathway in Arabidopsis thaliana using Microalgal Gene Δ6Des for Stearidonic acid and Dihomo-γ-linolenic acid Production

• Microalgal gene Δ6Des was cloned in a binary vector pCAMBIA1303. • F 1 transgenic plants having Δ6Des were viable at a concentration of 25 mg/L of hygromycin medium • Transgenic Arabidopsis thaliana showed novel ω3/6 fatty acids SDA (0.58%) and DGLA (25. 34%) To enhance the quality and quantity of oils in plant systems, specific candidate genes such as desaturase and elongase that can extend the biosynthetic pathways of very long chain poly unsaturated fatty acids (VLCPUFAs) are essential. In general, plant systems are capable of synthesizing essential fatty acids (EFAs). However, incorporation of a preliminary gene, Δ6Desaturase ( Δ6Des ), in the fatty acid biosynthetic pathway will elongate the pathway further to produce successive fatty acids. In this study, Δ6Des gene extracted from the marine microalga Isochrysis sp. was used to extend the omega (ω) 3/6 fatty acid biosynthetic pathway in a model plant Arabidopsis thaliana using Agrobacterium -mediated genetic transformation. Hygromycin-resistant plants were (25 mg/L) used for GUS and GFP assays and for molecular confirmation. GUS assay showed an intense blue color in F 1 transgenic A. thaliana plant tissues grown in the selection medium, which confirmed the presence of GUS gene in the T-DNA region, whereas non-transformed plant tissues such as leaf or stem did not show any blue color. GFP analysis also showed a green fluorescent color in tissues such as leaf, stem, and floral parts of F1 transgenic A. thaliana plants, which confirmed the presence of T-DNA. PCR and Southern blotting analyses also clearly showed the presence of Δ6Des in the T-DNA region of the F 1 transgenic plants. The fatty acid profile of F 1 transgenic plants showed the production of new fatty acids such as stearidonic acid (SDA-0.58%) and dihomo-γ-linolenic acid (DGLA-25.34%) that were not observed in non-transformed control plants. All these results showed the synthesis of new fatty acids in the transgenic plant, which proved that PUFA biosynthetic pathway can be extended in oilseed plants if specific genes are introduced through genetic transformation. This is the first study to report the productions of new fatty acids like SDA and DGLA in A. thaliana using Δ6Des gene from marine microalga Isochrysis sp.

[Construction of a testis Elovl4 gene knockout mouse model based on Cre/loxP system].

Very long chain polyunsaturated fatty acids (VLC-PUFAs) are unique fatty acids in tissues of mammals such as retina and testis, and the key enzyme of its biosynthesis is very long chain fatty acid elongase 4 (Elovl4). Development of an animal model of tissue-specific knockout of Elovl4 gene is conducive to the in-depth study of the biological function of VLC-PUFAs. Therefore, we constructed Stra8-Cre mice and Elovl4 floxed mice based on Cre/loxP system, and obtained the (Elovl4[flox/+], Stra8-Cre) heterozygous knockout mice by hybridization. Subsequently, female mice were selected to cross with male mice with homozygous Elovl4[flox/flox] to gain homozygous mice (Elovl4[flox/flox], Stra8-Cre) through genotype identification and screening. RT-PCR, qRT-PCR, Western blotting, immunohistochemistry and immunofluorescence techniques were used to detect the knock-out efficiency of Elovl4 in testis. The expression of Elovl4 in testis of both heterozygous and homozygous knockout mice were significantly down-regulated at mRNA and protein levels, but were not affected in other tissues. In summary, we constructed a mouse model with specific knockout of Elovl4 gene in testis, which provides a reliable animal model for studying the effect of VLC-PUFAs on the reproductive function of male mice and the underpinning molecular mechanisms.

Influence of very-long-chain polyunsaturated fatty acids on membrane structure and dynamics

The unique attributes of very-long-chain polyunsaturated fatty acids (VLC-PUFAs), their long carbon chains (n > 24) and high degree of unsaturation, impart unique chemical and physical properties to this class of fatty acids. The changes imparted by VLC-PUFA 32:6 n-3 on lipid packing and the compression moduli of model membranes were evaluated from π-A isotherms of VLC-PUFA in 1,2-distearoyl-sn-3-glycero-phosphocholine (DSPC) lipid monolayers. To compare the attractive or repulsive forces between VLC-PUFA and DSPC lipid monolayers, the measured mean molecular areas (MMAs) were compared with the calculated MMAs of an ideal mixture of VLC-PUFA and DSPC. The presence of 0.1, 1, and 10 mol % VLC-PUFA shifted the π-A isotherm to higher MMAs of the lipids comprising the membrane and the observed positive deviations from ideal behavior of the mixed VLC-PUFA:DSPC monolayers correspond to repulsive forces between VLC-PUFAs and DSPC. The MMA of the VLC-PUFA component was estimated using the measured MMAs of DSPC of 47.1 ± 0.7 Å2/molecule, to be 15,000, 1100, and 91 Å2/molecule at 0.1, 1, and 10 mol % VLC-PUFA:DSPC mixtures, respectively. The large MMAs of VLC-PUFA suggest that the docosahexaenoic acid tail reinserts into the membrane and adopts a nonlinear structure in the membrane, which is most pronounced at 0.1 mol % VLC-PUFA. The presence of 0.1 mol % VLC-PUFA:DSPC also significantly increased the compression modulus of the membrane by 28 mN/m compared with a pure DSPC membrane. The influence of VLC-PUFA on lipid “flip-flop” was investigated by sum-frequency vibrational spectroscopy. The incorporation of 0.1 mol % VLC-PUFA increased the DSPC flip-flop rate fourfold. The fact that VLC-PUFA promotes lipid translocation is noteworthy as retinal membranes require a high influx of retinoids which may be facilitated by lipid flip-flop.

Intrinsic differences in rod and cone membrane composition: implications for cone degeneration

In many retinal pathological conditions, rod and cone degeneration differs. For example, the early-onset maculopathy Stargardts disease type 1 (STGD1) is typified by loss of cones while rods are often less affected. We wanted to examine whether there exist intrinsic membrane differences between rods and cones that might explain such features. Abca4 mRNA and protein levels were quantified in rod- and cone-enriched samples from wild-type and Nrl-/- mice retinas; rod- and cone-enriched outer segments (ROS and COS respectively) were prepared from pig retinas, and total lipids were analyzed by flame ionization, chromatography, and tandem mass spectrometry. Immunohistochemical staining of cone-rich rodent Arvicanthis ansorgei retinas was conducted, and ultra-high performance liquid chromatography of lipid species in porcine ROS and COS was performed. Abca4 mRNA and Abca4 protein content was significantly higher (50-300%) in cone compared to rod-enriched samples. ROS and COS displayed dramatic differences in several lipids, including very long chain poly-unsaturated fatty acids (VLC-PUFAs), especially docosahexaenoic acid (DHA, 22:6n-3): ROS 20.6% DHA, COS 3.3% (p < 0.001). VLC-PUFAs (> 50 total carbons) were virtually absent from COS. COS were impoverished (> 6× less) in phosphatidylethanolamine compared to ROS. ELOVL4 ("ELOngation of Very Long chain fatty acids 4") antibody labelled Arvicanthis cones only very weakly compared to rods. Finally, there were large amounts (905 a.u.) of the bisretinoid A2PE in ROS, whereas it was much lower (121 a.u., ~ 7.5-fold less) in COS fractions. In contrast, COS contained fivefold higher amounts of all-trans-retinal dimer (115 a.u. compared to 22 a.u. in rods). Compared to rods, cones expressed higher levels of Abca4 mRNA and Abca4 protein, were highly impoverished in PUFA (especially DHA) and phosphatidylethanolamine, and contained significant amounts of all-trans-retinal dimer. Based on these and other data, we propose that in contrast to rods, cones are preferentially vulnerable to stress and may die through direct cellular toxicity in pathologies such as STGD1.