Polymorphism detection of <i>DGAT1</i> and <i>Lep</i> genes in Anatolian water buffalo (<i>Bubalus bubalis</i>) populations in Turkey

Acyl-CoA: diacylglycerol–acyltransferase 1 (DGAT1) enzyme plays a key role in controlling the synthesis rate triglyceride from diacylglycerol. Leptin (LP, OB, obese) is an important hormone that synthesizes mostly from adipose tissue and regulates glucose metabolism and homeostasis. DGAT1 and Lep genes are closely related to reproduction, growth, milk yield and composition in water buffalo breeds. This study aimed to identify genetic variation in the DGAT1 and Lep gene regions in 150 water buffalo individuals from five different provinces of Turkey using DNA sequencing. A total of 38 nucleotide variations and indels have identified 761 bp long partial intron 2 and exon 3 and 5′ UTR regions of the Lep gene in Anatolian water buffalo populations; 422 bp long partial exon 7–9 and exon 8 regions of DGAT1 gene were amplified and two mutations were defined in the point of 155 and 275 nucleotide that is three genotypes for S allele and Y allele of DGAT1 gene in intron 7 in Anatolian buffalo populations, respectively. These SNPs may have an effect on reproduction, growth, milk yield and composition in water buffalo populations and may prove to be useful for water buffalo breeding.

Salicylic Acid-Mediated Diacylglycerol/Triacylglycerol Conversion Affects the Freezing Tolerance of Arabidopsis

Diacylglycerol (DAG) is likely converted to triacylglycerol (TAG) by the enzyme diacylglycerol acyltransferase (DGAT), and this conversion is important in freezing tolerance of Arabidopsis. The phytohormone salicylic acid (SA) plays important roles in the chilling and freezing tolerance of plants. In our study, we analysed the chilling phenotype, proline and sugar accumulation, phytohormone measurement, and lipid profiling of dgat1 mutants during chilling or freezing stress. We found that dgat1-1 mutants exhibited higher sensitivity to long exposure to cold stress and showed lower proline and sugar accumulation under cold acclimation conditions. The freezing-sensitive phenotype of dgat1 mutants can be ameliorated by mutations of key salicylic acid (SA) signalling components SAG101, EDS1, and PAD4 through phenotyping analysis of double mutants. Dgat1 mutants accumulated more SA, ABA, JA-Ile (jasmonate isoleucine) and OPDA (12- oxyphytodienoic acid) during freezing stress and after recovery. In addition, the DAG/TAG content in the SA-deficient mutant sid2 was lower than that in the wild type, while the SA-excessive accumulated mutant siz1 showed the opposite trend. In summary, SA could mediate the freezing tolerance of Arabidopsis by regulating the ratio of DAG and TAG, which influences the integrity of the membrane.

Characterization of a Haematococcus pluvialis Diacylglycerol Acyltransferase 1 and Its Potential in Unsaturated Fatty Acid-Rich Triacylglycerol Production

The unicellular green alga Haematococcus pluvialis has been recognized as an industry strain to produce simultaneously esterified astaxanthin (EAST) and triacylglycerol (TAG) under stress induction. It is necessary to identify the key enzymes involving in synergistic accumulation of EAST and TAG in H. pluvialis. In this study, a novel diacylglycerol acyltransferase 1 was systematically characterized by in vivo and in silico assays. The upregulated expression of HpDGAT1 gene was positively associated with the significant increase of TAG and EAST contents under stress conditions. Functional complementation by overexpressing HpDGAT1 in a TAG-deficient yeast strain H1246 revealed that HpDGAT1 could restore TAG biosynthesis and exhibited a high substrate preference for monounsaturated fatty acyl-CoAs (MUFAs) and polyunsaturated fatty acyl-CoAs (PUFAs). Notably, heterogeneous expression of HpDGAT1 in Chlamydomonas reinhardtii and Arabidopsis thaliana resulted in a significant enhancement of total oils and concurrently a high accumulation of MUFAs- and PUFAs-rich TAGs. Furthermore, molecular docking analysis indicated that HpDGAT1 contained AST-binding sites. These findings evidence a possible dual-function role for HpDGAT1 involving in TAG and EAST synthesis, demonstrating that it is a potential target gene to enrich AST accumulation in this alga and to design oil production in both commercial algae and oil crops.

Lipid droplets are beneficial for rabies virus replication by facilitating viral budding

Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets have been reported to play a role in pathogenesis of several viruses. However, its role on RABV infection remains unclear. Here, we initially found that RABV infection upregulated lipid droplet (LD) production in multiple cells and mouse brains. After the treatment of atorvastatin, a specific inhibitor of LD, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhance the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglycerides synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication and their biogenesis is regulated via NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but its role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier and transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.

An integrated computational framework to design a multi-epitopes vaccine against Mycobacterium tuberculosis

Tuberculosis (TB) is a highly contagious disease that mostly affects the lungs and is caused by a bacterial pathogen, Mycobacterium tuberculosis. The associated mortality rate of TB is much higher compared to any other disease and the situation is more worrisome by the rapid emergence of drug resistant strains. Bacillus Calmette–Guerin (BCG) is the only licensed attenuated vaccine available for use in humans however, many countries have stopped its use as it fails to confer protective immunity. Therefore, urgent efforts are required to identify new and safe vaccine candidates that are not only provide high immune protection but also have broad spectrum applicability. Considering this, herein, I performed an extensive computational vaccine analysis to investigate 200 complete sequenced genomes of M. tuberculosis to identify core vaccine candidates that harbor safe, antigenic, non-toxic, and non-allergic epitopes. To overcome literature reported limitations of epitope-based vaccines, I carried out additional analysis by designing a multi-epitopes vaccine to achieve maximum protective immunity as well as to make experimental follow up studies easy by selecting a vaccine that can be easily analyzed because of its favorable physiochemical profile. Based on these analyses, I identified two potential vaccine proteins that fulfill all required vaccine properties. These two vaccine proteins are diacylglycerol acyltransferase and ESAT-6-like protein. Epitopes: DSGGYNANS from diacylglycerol acyltransferase and AGVQYSRAD, ADEEQQQAL, and VSRADEEQQ from ESAT-6-like protein were found to cover all necessary parameters and thus used in a multi-epitope vaccine construct. The designed vaccine is depicting a high binding affinity for different immune receptors and shows stable dynamics and rigorous van der Waals and electrostatic binding energies. The vaccine also simulates profound primary, secondary, tertiary immunoglobulin production as well as high interleukins and interferons count. In summary, the designed vaccine is ideal to be evaluated experimentally to decipher its real biological efficacy in controlling drug resistant infections of M. tuberculosis.

Study on the Regulation Mechanism of Lipopolysaccharide on Oxidative Stress and Lipid Metabolism of Bovine Mammary Epithelial Cells

The long-term feeding of a high-concentrate diet (the concentrate ratio is greater than 60 %) leads to mammary gland inflammatory response in ruminants and decreased quality in dairy cows and affects the robust development of the dairy industry. The main reason is closely related to elevated lipopolysaccharide (LPS) in the body. In this experiment, a bovine mammary epithelial cell line (MAC-T) was used as a model, and LPS at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, 10000 ng/ml) was added to the cells. The cell survival rate, oxidative stress indicators, total lipid droplet area, triglyceride content and key genes regulating lipid metabolism were detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), assay kit, microscope observation and RT-PCR methods to explore the regulatory mechanism of mammary health and milk fat synthesis. The results showed that compared with those of the control group, the survival rates of cells were significantly decreased after 9 h of stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.01). The contents of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in cells were significantly decreased (P<0.05). Compared with that of the control group, the content of malondialdehyde (MDA) in cells was significantly increased (P<0.05) after stimulation with 10000 ng/ml LPS for 9 h. After 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS, the total lipid drop area and triglyceride (TG) content of MAC-T cells were significantly decreased (P<0.05). The expression levels of fatty acid synthesis-related genes Acetyl-CoA carboxylase (ACC) and Stearoyl-CoA desaturase 1 (SCD-1) were significantly decreased after 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS (P<0.05), while the expression levels of Fatty Acid synthetase (FAS) were significantly decreased after stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.05). TG synthesis by the related gene Diacylglycerol acyltransferase-1 (DGAT1) was significantly lower than that of the control group after stimulation with 1000 ng/ml and 10000 ng/ml LPS for 9 h (P<0.05), and Diacylglycerol acyltransferase-2 (DGAT2) also showed a significant decrease after 10000 ng/ml LPS stimulation (P<0.05). In conclusion, adding different concentrations of LPS to MAC-T cells not only led to a decrease in cell activity, resulting in oxidative damage, but also affected fatty acid and TG synthesis, which may ultimately be closely related to the decrease in milk fat synthesis.

Histone Demethylase KDM7A Contributes to the Development of Hepatic Steatosis by Targeting Diacylglycerol Acyltransferase 2

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. While the development of NAFLD is correlated with aberrant histone methylation, modifiers of histone methylation involved in this event remain poorly understood. Here, we studied the functional role of the histone demethylase KDM7A in the development of hepatic steatosis. KDM7A overexpression in AML12 cells upregulated diacylglycerol acyltransferase 2 (DGAT2) expression and resulted in increased intracellular triglyceride (TG) accumulation. Conversely, KDM7A knockdown reduced DGAT2 expression and TG accumulation, and significantly reversed free fatty acids-induced TG accumulation. Additionally, adenovirus-mediated overexpression of KDM7A in mice resulted in hepatic steatosis, which was accompanied by increased expression of hepatic DGAT2. Furthermore, KDM7A overexpression decreased the enrichment of di-methylation of histone H3 lysine 9 (H3K9me2) and H3 lysine 27 (H3K27me2) on the promoter of DGAT2. Taken together, these results indicate that KDM7A overexpression induces hepatic steatosis through upregulation of DGAT2 by erasing H3K9me2 and H3K27me2 on the promoter.