Liver mRNA Research Articles

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT-/- newborn pup.

Effect of piperine and quercetin alone or in combination with marbofloxacin on CYP3A37 and MDR1 mRNA expression levels in broiler chickens

After oral route of administration, drug absorption is unpredictable and is governed by various factors such as multi drug resistance-1 (MDR1) an efflux transporter and drug metabolizing enzymes (like CYP3A4, CYP3A37, CYP2D6) at intestine and liver. Naturally available phyto chemicals like piperine and quercetin as well as some floroquinolones are known to inhibit MDR1 and CYP3A37 activity and increases bioavailability of co-administered drugs. This study was carried out to investigate the effect of piperine and quercetin alone or in combination with marbofloxacin on CYP3A37 and MDR1 mRNA expression levels in liver and intestine of broiler chicken. After oral administration of piperine and quercetin for 3 consecutive days followed by with or without oral administration of marbofloxacin for 5 days, CYP3A37 and MDR1 mRNA expression levels were determined using quantitative real-time PCR. Total of 36 broiler chickens in seven individual groups were treated with different regimen and the mRNA expression levels at duodenum and liver were analyzed with apt statistical tools. After piperine and quercetin combined treatment with marbofloxacin, CYP3A37 mRNA expression levels were significantly down regulated by 20.57 (p = .034) and 25.95 (p = .003) folds; and MDR1 mRNA expression levels were also significantly down regulated by 11.33 (p = .012) and 33.59 (p = .006) folds in liver and duodenum, respectively. Down regulation of CYP3A37 and MDR1 mRNA in liver and duodenum indicate the combined pretreatment of piperine and quercetin may be useful for improving the pharmacokinetics of orally administered drugs which are substrates for CYP3A37 and MDR1.

P: 14 Non-alcoholic Fatty Liver Disease Alters Expression of Genes Governing Hepatic Nitrogen Conversion

BACKGROUND: We recently showed that the functional capacity for ureagenesis is deficient in patients with NAFLD. The aim of this study was to assess the expression of urea cycle and related genes to elucidate whether there may be a gene regulatory basis to the functional problem. METHODS: Liver mRNA expression analyses within the gene pathway governing hepatic nitrogen conversion were performed in 20 non-diabetic, biopsy-proven NAFLD patients (8 simple steatosis; 12 non-alcoholic steatohepatitis (NASH)), and compared with 12 obese and 14 lean healthy control persons. The relationship between gene expressions and functional capacity for ureagenesis was described. RESULTS: Gene expression of most urea cycle-related enzymes were downregulated in NAFLD vs. both control groups and markedly so for the urea cycle flux-generating carbamoyl phosphate synthetase (CPS1) (∼3.5-fold, P < 0.0001). In the NASH patients, the reduction of CPS1 expression paralleled the deficit in functional ureagenesis (P = 0.03). Additionally, the expression of several genes involved in amino acid uptake and degradation, and the glucagon receptor gene, were downregulated in NAFLD. Conversely, gene expression of glutamine synthetase (GS) increased more than 1.5-fold (P ≤ 0.03), inversely related to CPS1 expression (P = 0.004). CONCLUSIONS: NAFLD downregulated the expression of urea cycle-related genes. Downregulation of the urea cycle flux-generating enzyme CPS1 correlated with loss of functional capacity for urea synthesis in patients with NASH. On gene level, these changes coincided with an increase in the major ammonia scavenging enzyme GS. The effects seemed to be related to a fatty liver as such rather than NASH or obesity. The findings support that gene regulatory mechanisms are involved in the deficient urea synthesis of NAFLD, but it remains unexplained how the hepatocyte fat accumulation exerts these

Effects of dietary Antrodia cinnamomea fermented product supplementation on metabolism pathways of antioxidant, inflammatory, and lipid metabolism pathways-a potential crosstalk.

ObjectiveThis study was conducted to fathom the underlying mechanisms of nutrition intervention and redox sensitive transcription factors regulated by Antrodia cinnamomea fermented product (FAC) dietary supplementation in broiler chickens.MethodsFour hundreds d-old broilers (41±0.5 g/bird) assigned to 5 groups were examined after consuming control diet, or control diet replaced with 5% wheat bran (WB), 10% WB, 5% FAC, and 10% FAC. Liver mRNA expression of antioxidant, inflammatory and lipid metabolism pathways were analyzed. Prostaglandin E2 (PGE2) concentration in each group were tested in the chicken peripheral blood mononuclear cells (cPBMCs) of 35-d old broilers to represent the stress level of the chickens. Furthermore, these cells were stimulated with 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) and lipopolysaccharide (LPS) to evaluate the cell stress tolerance by measuring cell viability and oxidative species.ResultsHeme oxygenase-1, glutathione S-transferase, glutamate-cysteine ligase, catalytic subunit, and superoxide dismutase, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) that regulates the above antioxidant genes were all up-regulated significantly in FAC groups. Reactive oxygen species modulator protein 1 and NADPH oxygenase 1 were both rather down-regulated in 10% FAC group as comparison with two WB groups. Despite expressing higher level than control group, birds receiving diet containing FAC had significantly lower expression level in nuclear factor-kappa B (NF-κB) and other genes (inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1β, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and cyclooxygenase 2) involving in inflammatory pathways. Additionally, except for 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase that showed relatively higher in both groups, the WB, lipoprotein lipase, Acetyl-CoA carboxylase, fatty acid synthase, fatty acid binding protein, fatty acid desaturase 2 and peroxisome proliferator-activated receptor alpha genes were expressed at higher levels in 10% FAC group. In support of above results, promoted Nrf2 and inhibited NF-κB nuclear translocation in chicken liver were found in FAC containing groups. H2O2 and NO levels induced by LPS and AAPH in cPBMCs were compromised in FAC containing diet. In 35-d-old birds, PGE2 production in cPBMCs was also suppressed by the FAC diet.ConclusionFAC may promote Nrf2 antioxidant pathway and positively regulate lipid metabolism, both are potential inhibitor of NF-κB inflammatory pathway.

Cloning and identification of antimicrobial peptide, hepcidin from freshwater carp, Catla catla on pathogen challenge and PAMPs stimulation.

Hepcidin, a cationic cysteine-rich antimicrobial peptide (AMP) acts in hormone regulation and iron homeostasis in the host body. However, the biological property of hepcidin in immune reaction remains unexplored. In aquatic milieu, environmental and pathogenic stressors cause detrimental infections, which are defended by various immunological cells and antimicrobial peptides. In this study, hepcidin gene has been cloned from freshwater carp, Catla catla. The partially cloned hepcidin consists of 200bp nucleotide sequence encoding 66 amino acids. Nucleotide sequence showed 97% and 91% similarity with Labeo rohita and Cyprinus carpio, respectively. Expression profile revealed significant up-regulation (P ≤ 0.0001) in liver as compared to other tissues in different conditions. In Aeromonas hydrophila challenged C. catla, liver showed higher expression level of hepcidin at 72h as compared to other tissues. In skin, hepcidin expression showed significant upraise during 24h in Streptococcus uberis infection. In Argulus sp. infected fishes, up-regulation of hepcidin expression was noted in liver, intestine and skin. The inactivated viral antigen-stimulated fishes, a substantial rise in liver was observed implying hepcidin as an important molecule in combating the pathogenic infections in freshwater carp, C. catla. Fishes stimulated with pathogen-associated molecular patterns (PAMPs) triggered the increased expression of hepcidin mRNA in liver, kidney and skin. This study indicates the presence of hepcidin as antimicrobial peptide in neutralizing the pathogenic infection in fishes.

MicroRNAs hsa-miR-495-3p and hsa-miR-486-5p suppress basal and rifampicin-induced expression of human sulfotransferase 2A1 (SULT2A1) by facilitating mRNA degradation

Drug metabolizing enzymes mediate biotransformation of drugs and play an essential role in drug efficacy and toxicity. Human sulfotransferases are a superfamily of Phase II detoxification enzymes that metabolize a wide spectrum of endogenous compounds and xenobiotics. SULT2A1 is one of the most abundant hepatic sulfotransferases and it catalyzes the sulfate conjugation of many endogenous substrates, such as bile acids and steroids. In the current study, we utilized a systematic approach by combining a series of computational analyses and in vitro methods to identify miRNAs that repress SULT2A1 expression post-transcriptionally. Our in silico analyses predicted miRNA response elements for hsa-miR-495-3p and hsa-miR-486-5p within the 3′-UTR of SULT2A1 mRNA and the levels of these miRNAs were inversely correlated with that of SULT2A1 mRNA in human liver. Using fluorescence-based RNA electrophoretic mobility shift assays, we found that hsa-miR-495-3p and hsa-miR-486-5p interacted directly with the SULT2A1 3′-UTR. The activity of a luciferase reporter gene construct containing sequences from the SULT2A1 3-UTR was suppressed by hsa-miR-486-5p and hsa-miR-495-3p. Furthermore, gain- and loss-of-function assays demonstrated that hsa-miR-486-5p and hsa-miR-495-3p negatively modulate basal and rifampicin-induced expression of SULT2A1 in HepG2 cells by decreasing mRNA stability.

The different expression of caspase-1 in HBV-related liver disease and acts as a biomarker for acute-on-chronic liver failure

BackgroundCaspase-1 is an evolutionarily conserved enzyme that proteolytically cleaves the precursors of the inflammatory cytokines interleukin 1β and interleukin 18. However, the role of caspase-1 in determining the severity of acute-on-chronic liver failure (ACLF) has yet to be elucidated. We evaluated the expression levels of caspase-1 in HBV-related liver disease and assessed its utility as a biomarker predicting the severity of ACLF.MethodsThe gene, protein and activity levels of caspase-1 were measured in the liver and/or serum of subjects with HBV-related disease. We also analysed the correlation between the expression levels of caspase-1 and liver injury of ACLF.ResultsCompared with the values observed in normal subjects, the relative caspase-1 mRNA and protein levels in livers were decreased in patients with CHB, LC, and HCC but increased in those with ACLF; moreover, ACLF patients had the lowest serum level and hepatic activity of caspase-1 among the five groups. The serum caspase-1 levels in ACLF patients showed a negative correlation with total serum bilirubin and a positive correlation with serum total protein and albumin. Importantly, the serum caspase-1 levels in the surviving group with ACLF were higher than those in the non-surviving group and showed different dynamic trends. Analyses of the area under the receiver operating characteristic curve indicated that caspase-1 (AUC = 0.84, AUC of MELD score = 0.72) may be a useful marker for independently predicting ACLF.ConclusionCaspase-1 is a potential non-invasive biomarker of disease progression and prognosis in ACLF.

Effects of Portulaca Oleracea Extract on Acute Alcoholic Liver Injury of Rats.

The present study was envisaged to investigate the chemical constituents and the intervention effects of Portulaca oleracea extract (POE) on acute alcoholic liver injury of rats. The chemical composition of POE was detected by high performance liquid chromatography (HPLC). Sixty male Wistar rats were divided into 6 groups: Normal control (NC) group, acute alcoholic liver injury model group (ALI), low, medium and high dose of POE (25, 50, 100 mg/kg) groups and bifendate (BF, 3.75 mg/kg) group. Each group was given by intragastrical administration for 7 days. Alcoholic liver injury was induced in the experimental model by administering 50% ethanol at 8 mL/kg and repeated administration after 6 h, for a period of 7 days. The results showed that pretreatment with POE significantly reduced the ethanol-elevated serum level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and triglyceride (TG). The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in liver were enhanced followed by administration of POE, while the content of nitric oxide (NO) and malondialdehyde (MDA) was found to decrease. Hepatic content of tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was also reduced by POE treatment. These results indicated that POE could increase the antioxidant capacity and relieve the inflammatory injury of the liver cells induced by ethanol. Meanwhile, in our study, POE reduced the expression of miR-122, acetyl coenzyme A carboxylase (ACC) 1 mRNA and protein and increased the expression of lipoprotein lipase (LPL) mRNA and protein in liver, which indicated that POE could improve the lipid metabolism disorder induced by ethanol. Our findings suggested that POE had protective effects on acute alcoholic liver injury of rats.

Estrogen Receptor β2 Oversees Germ Cell Maintenance and Gonadal Sex Differentiation in Medaka, Oryzias latipes

SummaryIn vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor β2 (ERβ2) during embryogenesis of medaka, and here we investigated the functional importance of ERβ2 in female gonad development and maintenance using a transgenerational ERβ2-knockdown (ERβ2-KD) line and ERβ2-null mutants. We found that ERβ2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERβ2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERβ2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

Exploration of the temporal-spatial expression pattern and DNA methylation-related regulation of the duck telomerase reverse transcriptase gene

Telomerase reverse transcriptase (TERT) is a catalytic subunit of telomerase that adds TTAGGG repeats to the 3'-overhang of telomeres. In the present study, we detected that the duck TERT (dTERT) gene was highly expressed in small intestine and kidney, followed by heart, leg muscle, spleen, pancreas, gonad, and liver at neonatal stage. From embryonic to neonatal stage, the highest dTERT mRNA in liver appeared at stage E19 (19 days at embryonic stage), while for the leg muscle the maximum expression occurred at E26. We also measured the relative telomerase activity (RTA) and relative telomere length (RTL) in the examined tissues and found that the changed tendency of RTA and RTL was not very consistent with that of TERT. In silico analysis revealed that there were three CpG islands (S1, S2, and S3) within the 5' regulatory region of the dTERT gene. Bisulfite sequencing PCR (BSP) assay showed that liver (D7, 7 days after birth) which expressed significantly lower dTERT mRNA had an obviously higher methylation level of S1 compared with small intestine (D7) or liver (E19). Quantitative real-time PCR analysis revealed that the expression of DNA methyltransferase DNMT1 in liver (D7) was significantly higher than that in small intestine (D7) or in liver (E19). In vitro, dTERT expression was upregulated and the methylation status of S1 decreased in both duck embryonic fibroblasts and small intestinal epithelial cells following treatment with the demethylation reagent, 5-aza-2'-deoxycytidine (5-aza-dC), further suggesting that dTERT is epigenetically regulated by DNA methylation. This work lays a solid foundation for further study of TERT function and regulation in avian species.

AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.

Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency

Sodium taurocholate cotransporting polypeptide (NTCP, encoded by Slc10a1/SLC10A1) deficiency can result in hypercholanemia but no obvious symptoms in both mice and humans. However, the consequence of and response to long-term hypercholanemia caused by NTCP deficiency remain largely unexplored. Here, we analyzed lifelong dynamics of serum total bile acid (TBA) levels in Slc10a1-/- mice, and we also assessed changes of TBA levels in 33 young individuals with SLC10A1 loss-of-function variant p.Ser267Phe. We found that overall serum TBA levels tended to decrease gradually with age in both Slc10a1-/- mice and p.Ser267Phe individuals. Liver mRNA profiling revealed notable transcription alterations in hypercholanemic Slc10a1-/- mice, including inhibition of bile acid (BA) synthesis, enhancement of BA detoxification, and altered BA transport. Members of the sulfotransferase (SULT) family showed the most dramatic increases in livers of hypercholanemic Slc10a1-/- mice, and one of their BA sulfates, taurolithocholic acid 3-sulfate, significantly increased. Importantly, consistent with the mouse studies, comprehensive profiling of 58 BA species in sera of p.Ser267Phe individuals revealed a markedly increased level of BA sulfates. Together, our findings indicate that the enhanced BA sulfation is a major mechanism for BA detoxification and elimination in both mice and humans with Slc10a1/SLC10A1 deficiency.

A Network Involving Gut Microbiota, Circulating Bile Acids, and Hepatic Metabolism Genes That Protects Against Non-Alcoholic Fatty Liver Disease.

Gut microbiota contributes to non-alcoholic fatty liver disease (NAFLD) pathogenesis by multiple mechanisms not yet completely understood. Novel differential features between germ-free mice (GFm) transplanted with protective or non-protective cecal microbiota against NAFLD are investigated. Gut microbiota composition, plasma, and fecal bile acids (BAs) and liver mRNAs are quantified in GFm recipients from four donor mice differing in NAFLD severity (control diet, high-fat diet [HFD]-responder, HFD-non-responder, and quercetin-supplemented HFD). Transplanted GFm are on control or HFD for 16-weeks. Multivariate analysis shows that GFm colonized with microbiota from HFD-non-responder and quercetin supplemented-HFD donors (protected against NAFLD) clusters together, whereas GFm colonized with microbiota from control and HFD-responder mice (non-protected against NAFLD) establishes another cluster. Protected phenotype is associated with increased gut Desulfovibrio and Oscillospira, reduced gut Bacteroides and Oribacterium, lower primary and higher secondary BAs in plasma and feces, induction of hepatic BA transporters, and repression of hepatic lipogenic and BA synthesis genes. Protective gut microbiota associates with increased specific secondary BAs, which likely inhibit lipogenic pathways and enhance bile flow in the liver. This novel cross-talk between gut and liver, via plasma BAs, that promotes protection against NAFLD may have clinical and nutritional relevance.

Individual liver plasmacytoid dendritic cells are capable of producing IFNα and multiple additional cytokines during chronic HCV infection.

Plasmacytoid dendritic cells (pDCs) are “natural” interferon α (IFNα)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFNα mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFNα. We used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze DC frequencies/functions in liver and peripheral blood mononuclear cells (PBMCs) of hepatitis C virus (HCV)-infected patients, to examine correlations between DC function and gene expression of matched whole liver tissue and liver mononuclear cells (LMCs), and to determine if pDCs can produce multiple cytokines. T cells often produce multiple cytokines/chemokines but until recently technical limitations have precluded tests of polyfunctionality in individual pDCs. Mass cytometry (CyTOF) revealed that liver pDCs are the only LMC that produces detectable amounts of IFNα in response TLR-7/8 stimulation. Liver pDCs secreted large quantities of IFNα (~2 million molecules of IFNα/cell/hour) and produced more IFNα than PBMCs after stimulation, p = 0.0001. LMCs secreted >14-fold more IFNα than IFNλ in 4 hours. Liver pDC frequency positively correlated with whole liver expression of “IFNα-response” pathway (R2 = 0.58, p = 0.007) and “monocyte surface” signature (R2 = 0.54, p = 0.01). Mass cytometry revealed that IFNα-producing pDCs were highly polyfunctional; >90% also made 2–4 additional cytokines/chemokines of our test set of 10. Liver BDCA1 DCs, but not BDCA3 DCs, were similarly polyfunctional. pDCs from a healthy liver were also polyfunctional. Our data show that liver pDCs retain the ability to make abundant IFNα during chronic HCV infection and produce many other immune modulators. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation during chronic HCV infection.

338 Effect of eicosapentaenoic acid and docosahexaenoic acid supplementation on placenta and fetal liver mRNA, and fetal liver and brain fatty acids profile on early gestation in sheep

Abstract Polyunsaturated fatty acids supplementation in late gestation change offspring metabolism; however, their effect is not well known on early gestation in ewes. The objectives of this study were to determine the effect of dietary supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in pregnant ewes on the concentration of EPA and DHA on fetal liver (FL) and fetal central nervous system (FCNS), and to evaluate the effect of the supplementation with EPA+ DHA on mRNA expression of genes associated with transport and metabolism of fatty acids (FA) in FL and placenta (caruncles and cotyledons). Twelve ewes (4 pens, three per pen) were blocked by pregnancy day. The ewes were assigned during the first 45 d of gestation to diet with an addition of 1.5% (dry matter bases) monounsaturated FA (MUFA) or EPA+DHA. A C-section was conducted at d 45 of gestation to collect FL, FCNS, caruncle and cotyledon. Data were analyzed using a mixed procedure (SAS). For the placenta mRNA concentration, a 2x2 factorial was used considering caruncle and cotyledon as the second main factor. Isomers of C18:1 (t6,8 and t12) increase (P < 0.05) in FL and FCNS with MUFA supplementation, fatty acids C20:3 (n-6), C20:3 (n-3), C22:1, C22:5 and C22:6 increase (P < 0.05) in FL and FCNS with EPA+DHA supplementation. In FL there was a tendency to increase for mRNA expression of FATP-1 (P = 0.10) with EPA+DHA supplementation, while mRNA concentration for LPL was greater (P = 0.02) for MUFA supplementation. In placenta DNMT3b and FFAR-4 showed a significant FA x tissue interaction (P < 0.05). These results suggest that FA supplementation during early gestation alters the FA profile in FL and FCNS and changed mRNA concentration of genes involved in the transport of FA and cell metabolism.

Characterization of a classical 2-cysteine peroxiredoxin1 gene from Chinese soft-shelled turtle Pelodiscus sinensis with its potent antioxidant properties and putative immune response

Peroxiredoxin family members could function in host defense against oxidative stress, and modulate immune response. In the present study, a 2-cysteine peroxiredoxin gene named PsPrx1 was isolated from Chinese soft-shelled turtle Pelodiscus sinensis. The PsPrx1 cDNA was composed of 1130 bp, consisted of 199 amino acid residues and included a Redoxin and AphC-TSA domain. As detected by qPCR, PsPrx1 was ubiquitously expressed in the examined tissues with the higher levels in liver and spleen. Upon the immune challenge with A. jandaei bacteria and oxidative stress with ammonia pressure, both mRNA and protein expression level in liver could be significantly enhanced. The results of immunohistochemical examinations showed PsPrx1 was mainly distributed at the junction between the hepatic cells. The general functional properties of PsPrx1 were confirmed using purified rPsPrx1 protein. From the results, rPsPrx1 protein was confirmed to exhibit antioxidant activity and antibacterial properties. The potential for scavenging extracellular H2O2 was evidenced by the purified rPsPrx1 protein in vitro system. In the mixed-function oxidase assay, rPsPrx1 also exhibited a dose-dependent inhibition of DNA damage. These results suggest that rPsPrx1 was implicated defense against microbial pathogens and oxidants, and would provide important information to further understand the functional mechanism of Prx1 in P. sinensis immunity.

Total liver phosphatidylcholine content associates with non-alcoholic steatohepatitis and glycine N-methyltransferase expression.

Alterations in liver phosphatidylcholine (PC) metabolism have been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Although genetic variation in the phosphatidylethanolamine N-methyltransferase (PEMT) enzyme synthesizing PC has been associated with disease, the functional mechanism linking PC metabolism to the pathogenesis of non-alcoholic steatohepatitis (NASH) remains unclear. Serum PC levels and liver PC contents were measured using proton nuclear magnetic resonance (NMR) spectroscopy in 169 obese individuals [age 46.6±10 (mean±SD) years, BMI 43.3±6kg/m2 , 53 men and 116 women] with histological assessment of NAFLD; 106 of these had a distinct liver phenotype. All subjects were genotyped for PEMT rs7946 and liver mRNA expression of PEMT and glycine N-methyltransferase (GNMT) was analysed. Liver PC content was lower in those with NASH (P=1.8x10-6 ) while serum PC levels did not differ between individuals with NASH and normal liver (P=0.591). Interestingly, serum and liver PC did not correlate (rs =-0.047, P=0.557). Serum PC and serum cholesterol levels correlated strongly (rs =0.866, P=7.1x10-49 ), while liver PC content did not correlate with serum cholesterol (rs =0.065, P=0.413). Neither PEMT V175M genotype nor PEMT expression explained the association between liver PC content and NASH. Instead, liver GNMT mRNA expression was decreased in those with NASH (P=3.8x10-4 ) and correlated with liver PC content (rs =0.265, P=0.001). Decreased liver PC content in individuals with the NASH is independent of PEMT V175M genotype and could be partly linked to decreased GNMT expression.

Hepatic transcriptomics analysis reveals that fructose intervention down-regulated xenobiotics-metabolising enzymes through aryl hydrocarbon receptor signalling suppression in C57BL/6N mice.

For decades, fructose intake has been recognised as an environmental risk for metabolic syndromes and diseases. Here we comprehensively examined the effects of fructose intake on mice liver transcriptomes. Fructose-supplemented water (34 %; w/v) was fed to both male and female C57BL/6N mice at their free will for 6 weeks, followed by hepatic transcriptomics analysis. Based on our criteria, differentially expressed genes (DEG) were selected and subjected to further computational analyses to predict key pathways and upstream regulator(s). Subsequently, predicted genes and pathways from the transcriptomics dataset were validated via quantitative RT-PCR analyses. As a result, we identified eighty-nine down-regulated and eighty-eight up-regulated mRNA in fructose-fed mice livers. These DEG were subjected to bioinformatics analysis tools in which DEG were mainly enriched in xenobiotic metabolic processes; further, in the Ingenuity Pathway Analysis software, it was suggested that the aryl hydrocarbon receptor (AhR) is an upstream regulator governing overall changes, while fructose suppresses the AhR signalling pathway. In our quantitative RT-PCR validation, we confirmed that fructose suppressed AhR signalling through modulating expressions of transcription factor (AhR nuclear translocator; Arnt) and upstream regulators (Ncor2, and Rb1). Altogether, we demonstrated that ad libitum fructose intake suppresses the canonical AhR signalling pathway in C57BL/6N mice liver. Based on our current observations, further studies are warranted, especially with regard to the effects of co-exposure to fructose on (1) other types of carcinogens and (2) inflammation-inducing agents (or even diets such as a high-fat diet), to find implications of fructose-induced AhR suppression.

Effects of lysine deficiency or excess on growth and the expression of lipid metabolism genes in slow-growing broilers

This experiment was conducted to evaluate the effects of lysine deficiency or excess on growth and the expression of lipid metabolism genes in slow-growing birds. A total of 360 one-day-old chicks were randomly divided into 3 groups, with 6 replicates of 20 birds each. The birds fed the basal diet with a total lysine 0.60% (LL), 1.00% (ML), or 1.40% (HL). The amount of lysine (ML) as the control group, LL and HL as the experimental group, the trial period last 3 wk. The results showed that compared with ML, LL significantly decreased average daily gain and average daily feed intake and remarkably increased feed conversion ratio of birds at 21 day old (P < 0.01), while the above indices in HL had no significant effects (P > 0.05). Besides, LL reduced the pectoral muscle rate (P < 0.01) and decreased the percentage of abdominal fat significantly (P < 0.05). In addition, compared with ML, the expression of fatty acid binding protein 1 (FABP1), acetyl-CoA carboxylase (ACC), malic enzyme (ME), and sterol regulatory element binding protein 1 (SREBP1c) mRNA of liver in LL was significantly decreased (P < 0.05), and the expression of cholesteryl ester transfer protein (CETP) mRNA was significantly increased (P < 0.01), whereas LL had no significant effects on the expression of peroxisome proliferator activated receptor alpha (PPARα) mRNA (P > 0.05). Moreover, compared with ML, HL significantly reduced the expression of FABP1, ACC, ME, SREBP-1c, and PPARα mRNA in the liver (P < 0.05), and had no significant effects on the expression of CETP mRNA (P > 0.05). The results of current research suggest that dietary lysine deficiency could reduce the growth and fat deposition of slow-growing broilers mainly by downregulating the expression of lipid synthesis genes.

The archaeology of coding RNA.

Different theories concerning the origin of RNA (and, in particular, mRNA) point to the concatenation and expansion of proto-tRNA-like structures. Different biochemical and biophysical tools have been used to search for ancient-like RNA elements with a specific structure in genomic viral RNAs, including that of the hepatitis C virus, as well as in cellular mRNA populations, in particular those of human hepatocytes. We define this method as "archaeological," and it has been designed to discover evolutionary patterns through a nonphylogenetic and nonrepresentational strategy. tRNA-like elements were found in structurally or functionally relevant positions both in viral RNA and in one of the liver mRNAs examined, the antagonist interferon-alpha subtype 5 (IFNA5) mRNA. Additionally, tRNA-like elements are highly represented within the hepatic mRNA population, which suggests that they could have participated in the formation of coding RNAs in the distant past. Expanding on this finding, we have observed a recurring dsRNA-like motif next to the tRNA-like elements in both viral RNAs and IFNA5 mRNA. This suggested that the concatenation of these RNA motifs was an activity present in the RNA pools that might have been relevant in the RNA world. The extensive alteration of sequences that likely triggered the transition from the predecessors of coding RNAs to the first fully functional mRNAs (which was not the case in the stepwise construction of noncoding rRNAs) hinders the phylogeny-based identification of RNA elements (both sequences and structures) that might have been active before the advent of protein synthesis. Therefore, our RNA archaeological method is presented as a way to better understand the structural/functional versatility of a variety of RNA elements, which might represent "the losers" in the process of RNA evolution as they had to adapt to the selective pressures favoring the coding capacity of the progressively longer mRNAs.