Isolation,cloning and transgenic expression of hepatitis B surface antigen (HBsAg) in Solanum lycopersicum L

The Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans. HBV causes acute and chronic hepatitis. Chronic hepatitis leads to hepatocellular carcinoma, which is a significant cause of death. DNA-based immunization programs to control the spread of Hepatitis B in developing countries are costly and require special storage and transportation. The alternative way is to express Hepatitis B surface antigen (HBsAg) in plants to develop oral vaccines. In this study, HBsAg gene was isolated, cloned, and then transformed in tomato plants. The transgenic tomato plants were confirmed through RT-qPCR. HBsAg expression was analysed in mature green and red stages of tomato fruit through quantitative real-time PCR. It was observed that expression of HBsAg was high in matured red tomato as compared to mature green. The present study is the first step to developing Solanum lycopersicum as an edible vaccine production system in this world region.     

SCD1 deficiency protects mice against ethanol-induced liver injury

Stearoyl-CoA desaturase 1 (SCD1) is a delta-9 fatty acid desaturase that catalyzes the synthesis of mono-unsaturated fatty acids (MUFA). SCD1 is a critical control point regulating hepatic lipid synthesis and β-oxidation. Scd1 KO mice are resistant to the development of diet-induced non-alcoholic fatty liver disease (NAFLD). Using a chronic-binge protocol of ethanol-mediated liver injury, we aimed to determine if these KO mice are also resistant to the development of alcoholic fatty liver disease (AFLD).Mice fed a low-fat diet (especially low in MUFA) containing 5% ethanol for 10 days, followed by a single ethanol (5 g/kg) gavage, developed severe liver injury manifesting as hepatic steatosis. This was associated with an increase in de novo lipogenesis and inflammation. Using this model, we show that Scd1 KO mice are resistant to the development of AFLD. Scd1 KO mice do not show accumulation of hepatic triglycerides, activation of de novo lipogenesis nor elevation of cytokines or other pro-inflammatory markers. Incubating HepG2 cells with a SCD1 inhibitor induced a similar resistance to the effect of ethanol, confirming a role for SCD1 activity in mediating ethanol-induced hepatic injury.Taken together, our study shows that SCD1 is a key player in the development of AFLD and associated deleterious effects, and suggests SCD1 inhibition as a therapeutic option for the treatment of this hepatic disease.     

Monocytes from Chronic HBV Patients React In Vitro to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease

Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the in vitro effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected in vivo. We observed that in vitro HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier in vivo exposure to HBsAg does not affect the in vitro response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production in vitro. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive in vivo monocyte activation.     

PPAR�� isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation

Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithelial cells. Expression and activation of either PPARγ 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARγ isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARγ1. In contrast, PPARγ2 expression significantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in confirmation of in vitro data, a PPARγ agonist versus high-fat diet (HFD) regimen in vivo confirmed that PPARγ agonization increased prostatic differentiation markers, whereas HFD downregulated PPARγ-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress.     

Scd1 mammary-specific vector constructed and overexpressed in goat fibroblast cells resulting in an increase of palmitoleic acid and oleic acid

Stearoyl-CoA desaturase-1 (Scd1) is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Overexpression of Scd1 in transgenic animals would modify the nutritional value of ruminant-derived foods by increasing the monounsaturated fatty acid (MUFA) and decreasing the saturated fatty acid (SFA) content. The aim of this study was to develop an effective Scd1 vector that is specifically expressed in dairy goat mammary glands. We successfully amplified the goat full length Scd1 cDNA and evaluated its activity in goat ear skin-derived fibroblast cells (GEFCs) by lipid analysis. In addition, we constructed a mammary gland-specific expression vector and confirmed efficient expression of Scd1 in goat mammary epithelial cells (GMECs) by qRT-PCR and Western blot analysis. Fatty acid analysis showed that Scd1-overexpression resulted in an increase in levels of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), from 1.73 ± 0.02% to 2.54 ± 0.02% and from 27.25 ± 0.13% to 30.37 ± 0.04%, respectively (both p < 0.01) and the ratio of MUFA to SFA was increased. This work lays a foundation for the generation of Scd1 transgenic goats.     

Role of Stearoyl-CoA Desaturase-1 in Skin Integrity and Whole Body Energy Balance

The skin is the single largest organ in humans, serving as a major barrier to infection, water loss, and abrasion. The functional diversity of skin requires the synthesis of large amounts of lipids, such as triglycerides, wax esters, squalene, ceramides, free cholesterol, free fatty acids, and cholesterol and retinyl esters. Some of these lipids are used as cell membrane components, signaling molecules, and a source of energy. An important class of lipid metabolism enzymes expressed in skin is the Δ⁹-desaturases, which catalyze the synthesis in Δ⁹-monounsaturated lipids, primarily oleoyl-CoA (18:1n-9) and palmitoyl-CoA (16:1n-7), the major monounsaturated fatty acids in cutaneous lipids. Mice with a deletion of the Δ⁹-desaturase-1 isoform (SCD1) either globally (Scd1^−/−) or specifically in the skin (skin-specific Scd1-knockout; SKO) present with marked changes in cutaneous lipids and skin integrity. Interestingly, these mice also exhibit increased whole body energy expenditure, protection against diet-induced adiposity, hepatic steatosis, and glucose intolerance. The increased energy expenditure in skin-specific Scd1-knockout (SKO) mice is a surprising phenotype, as it links cutaneous lipid homeostasis with whole body energy balance. This minireview summarizes the role of skin SCD1 in regulating skin integrity and whole body energy homeostasis and offers a discussion of potential pathways that may connect these seemingly disparate phenotypes.     

Tissue specific expression of hepatitis B virus surface antigen in transgenic plant cells and tissue culture

The tobacco plants (Nicotiana tabacum L.) carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh1, maize alcohol dehydrogenase gene intron were obtained. The presence of the adh1 gene intron did not significantly change the level of expression of the HBsAg gene in plants. The analysis of expression of hepatitis B virus surface antigen (HBs-antigen) in transformed plants expressing the HBsAg under the control of different promoters was made. The level of HBs-antigen in plants carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to 0.01% of total amount of soluble protein. The level of HBs-antigen in plants carrying the HBsAg gene controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant and made up to 0.06% of the total amount of soluble protein. Hairy root and callus cultures of plants carrying the HBsAg gene and expressing the HBs-antigen were obtained.     

COH-SR4 Reduces Body Weight,Improves Glycemic Control and Prevents Hepatic Steatosis in High Fat Diet-Induced Obese Mice

Obesity is a chronic metabolic disorder caused by imbalance between energy intake and expenditure, and is one of the principal causative factors in the development of metabolic syndrome, diabetes and cancer. COH-SR4 (“SR4”) is a novel investigational compound that has anti-cancer and anti-adipogenic properties. In this study, the effects of SR4 on metabolic alterations in high fat diet (HFD)-induced obese C57BL/J6 mice were investigated. Oral feeding of SR4 (5 mg/kg body weight.) in HFD mice for 6 weeks significantly reduced body weight, prevented hyperlipidemia and improved glycemic control without affecting food intake. These changes were associated with marked decreases in epididymal fat mass, adipocyte hypertrophy, increased plasma adiponectin and reduced leptin levels. SR4 treatment also decreased liver triglycerides, prevented hepatic steatosis, and normalized liver enzymes. Western blots demonstrated increased AMPK activation in liver and adipose tissues of SR4-treated HFD obese mice, while gene analyses by real time PCR showed COH-SR4 significantly suppressed the mRNA expression of lipogenic genes such as sterol regulatory element binding protein-1c (Srebf1), acetyl-Coenzyme A carboxylase (Acaca), peroxisome proliferator-activated receptor gamma (Pparg), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr), as well as gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) in the liver of obese mice. In vitro, SR4 activates AMPK independent of upstream kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Together, these data suggest that SR4, a novel AMPK activator, may be a promising therapeutic compound for treatment of obesity, fatty liver disease, and related metabolic disorders.     

Dietary fish oil differentially ameliorates high-fructose diet-induced hepatic steatosis and hyperlipidemia in mice depending on time of feeding

Chrononutrition is the science of nutrition based on chronobiology. Numerous epidemiological studies have shown that fish oil (FO) reduces the risk of cardiovascular events through various actions such as lowering triglycerides. The present study aimed to determine the time of day when the hypertriglyceridemia-decreasing ability of FO is optimal in mice. A high-fructose diet (HFrD) that induces hyperlipidemia in mice was replaced with the same diet containing 4% FO (HFrD-4% FO) at different times of the day for 2 weeks as described below. Mice were fed with HFrD alone (CTRL) or with HFrD containing 4% FO for 12 h around the time of activity onset [breakfast (BF)-FO] or offset [dinner (DN)-FO]. Plasma and liver concentrations of triglycerides and total cholesterol were reduced in BF-FO but not in DN-FO mice compared with CTRL mice. The temporal expression of genes associated with fatty acid synthesis such as Fasn, Acaca, Scd1 and Acly in the liver was significantly suppressed in both BF-FO and DN-FO mice. Expression levels of Scd1 in epididymal adipose tissue were significantly suppressed only in the BF-FO mice. Plasma concentrations of docosahexaenoic acid and eicosapentaenoic acid were far more increased in BF-FO than in DN-FO mice. Significantly more of these n-3 polyunsaturated fatty acids (PUFAs) were excreted in the feces of DN-FO than of BF-FO mice. These findings suggest that dietary FO exerts more hypolipidemic activity at the time of breakfast than dinner because the intestinal absorption of n-3 PUFAs is more effective at that time.     

ApoB100-LDL Acts as a Metabolic Signal from Liver to Peripheral Fat Causing Inhibition of Lipolysis in Adipocytes

Background

Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown.

Methods and Findings

We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr^−/−Apob ^100/100).

Conclusions

Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome.     

Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen

The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01–0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.     

Endothelial lipase mediates efficient lipolysis of triglyceride-rich lipoproteins

Triglyceride-rich lipoproteins (TRLs) are circulating reservoirs of fatty acids used as vital energy sources for peripheral tissues. Lipoprotein lipase (LPL) is a predominant enzyme mediating triglyceride (TG) lipolysis and TRL clearance to provide fatty acids to tissues in animals. Physiological and human genetic evidence support a primary role for LPL in hydrolyzing TRL TGs. We hypothesized that endothelial lipase (EL), another extracellular lipase that primarily hydrolyzes lipoprotein phospholipids may also contribute to TRL metabolism. To explore this, we studied the impact of genetic EL loss-of-function on TRL metabolism in humans and mice. Humans carrying a loss-of-function missense variant in LIPG, p.Asn396Ser (rs77960347), demonstrated elevated plasma TGs and elevated phospholipids in TRLs, among other lipoprotein classes. Mice with germline EL deficiency challenged with excess dietary TG through refeeding or a high-fat diet exhibited elevated TGs, delayed dietary TRL clearance, and impaired TRL TG lipolysis in vivo that was rescued by EL reconstitution in the liver. Lipidomic analyses of postprandial plasma from high-fat fed Lipg^-/- mice demonstrated accumulation of phospholipids and TGs harboring long-chain polyunsaturated fatty acids (PUFAs), known substrates for EL lipolysis. In vitro and in vivo, EL and LPL together promoted greater TG lipolysis than either extracellular lipase alone. Our data positions EL as a key collaborator of LPL to mediate efficient lipolysis of TRLs in humans and mice.     

Analysis of Transgenic Tobacco Plants Carrying the Gene for the Surface Antigen of the Hepatitis B Virus

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBs antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HBsAg gene under a single 35S promoter was 0.0001–0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002–0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F₁ plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F₀ plants.     

Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

Defective Adipose Lipolysis and Altered Global Energy Metabolism in Mice with Adipose Overexpression of the Lipolytic Inhibitor G0/G1 Switch Gene 2 (G0S2)

Biochemical and cell-based studies have identified the G0S2 (G₀/G₁ switch gene 2) as a selective inhibitor of the key intracellular triacylglycerol hydrolase, adipose triglyceride lipase. To better understand the physiological role of G0S2, we constructed an adipose tissue-specific G0S2 transgenic mouse model. In comparison with wild type animals, the transgenic mice exhibited a significant increase in overall fat mass and a decrease in peripheral triglyceride accumulation. Basal and adrenergically stimulated lipolysis was attenuated in adipose explants isolated from the transgenic mice. Following fasting or injection of a β3-adrenergic agonist, in vivo lipolysis and ketogenesis were decreased in G0S2 transgenic mice when compared with wild type animals. Consequently, adipose overexpression of G0S2 prevented the “switch” of energy substrate from carbohydrates to fatty acids during fasting. Moreover, G0S2 overexpression promoted accumulation of more and larger lipid droplets in brown adipocytes without impacting either mitochondrial morphology or expression of oxidative genes. This phenotypic change was accompanied by defective cold adaptation. Furthermore, feeding with a high fat diet caused a greater gain of both body weight and adiposity in the transgenic mice. The transgenic mice also displayed a decrease in fasting plasma levels of free fatty acid, triglyceride, and insulin as well as improved glucose and insulin tolerance. Cumulatively, these results indicate that fat-specific G0S2 overexpression uncouples adiposity from insulin sensitivity and overall metabolic health through inhibiting adipose lipolysis and decreasing circulating fatty acids.     

Antibody response to preS1 in hepatitis-B-virus-induced liver disease and after immunization

Antibodies to the preS1-encoded sequence of hepatitis B virus (HBV) envelope were detected by ELISA using a synthetic peptide analogue of preS1 proteins, in different groups of HBV-infected subjects and also in hepatitis B vaccine recipients. Such antibodies were specifically found in only 1 % of HBsAg chronic carriers including patients with cirrhosis and primary liver cancer. Anti-preS1 were detected in patients with acute hepatitis; in 13 % of the HBsAg⁺ sera obtained before recovery and in 37 % of the sera obtained after recovery.Anti-preS1 antibodies were detected in recipients of a plasma-derived vaccine, but not in those receiving a recombinant vaccine. The results indicate that anti-preS1 is an earlier serum marker of HBV clearance than anti-preS2 and anti-S antibodies.