Total Liver RNA Research Articles

DIFFERENTIAL EXPRESSION AND CYCLE THRESHOLD OF MYOSTATIN IN NIGERIAN LOCALLY ADAPTED AND EXOTIC TURKEYS

Myostatin (MSTN) gene plays a key role in the regulation of myogenesis. This study focused on the relative and differential expression of MSTN gene in Nigerian locally adapted and exotic turkeys. A total of two hundred turkeys comprising one hundred and fifty Nigerian locally adapted (fifty each of black, lavender and white colour variants) and fifty Hybrid converter (exotic) day-old poults were used for this study. The total RNA in liver, breast and thigh muscle tissues was extracted at six weeks (grower stage) and sixteen weeks (finisher stage) of age. The cycle threshold (CT), delta CT and relative expression (R) values of MSTN gene in Nigerian locally adapted and exotic turkeys were analyzed by real-time PCR and calculated using 2-△△Ct. The highest (p<0.05) ΔCT (12.82) and R (1.38) were respectively found in the breast and liver tissues of local black turkey at 6 weeks while the highest (p<0.05) ΔCT (10.60) and R (1.24) of MSTN gene were obtained from the breast and liver tissues of exotic turkey at 16 weeks. Generally, there was reduction in relative expression at finisher phase in the breast and thigh tissues of exotic and Nigerian locally adapted (black and lavender plumage variants) turkeys. This expression pattern provided useful information on the genetic basis for growth variation in exotic and Nigerian locally adapted turkeys and could be employed for genetic improvement of Nigeria locally adapted turkeys in a short period of time.

Multi‑omics‑based analysis of the regulatory mechanism of gypenosides on bile acids in hypercholesterolemic mice.

Gynostemma pentaphyllum is a traditional medicine used by ethnic minorities in southwest China and gypenosides are currently recognized as essential components of the pharmacological substances of Gynostemma pentaphyllum, which are effective in regulating metabolic syndrome, especially in improving hepatic metabolic disorders. The present study randomly divided C57BL/6J male mice into the normal diet control group (ND), high-fat diet modeling group (HFD) and gypenosides group (GP). Liquid chromatography-mass spectrometry (UPLC-MS) was applied to quantify bile acids in the liver, bile and serum of mice in ND, HFD and GP groups. Liver proteins were extracted for trypsin hydrolysis and analyzed quantitatively using UPLC-MS + MS/MS (timsTOF Pro 2). Total mouse liver RNA was extracted from ND, HFD and GP groups respectively, cDNA sequencing libraries constructed and sequenced using BGISEQ-500 sequencing platform. The expression of key genes Fxr, Shp, Cyp7a1, Cyp8b1, and Abab11 was detected by RT-qPCR. The results showed that gypenosides accelerated free bile acid synthesis by promoting the expression of bile acid synthase CYP7A1 and CYP8B1 genes and proteins and accelerating the secretion of conjugated bile acids from the liver to the bile ducts. GP inhibited the bile acid transporters solute carrier organic anion transporter family member (SLCO) 1A1 and SLCO1A4, reducing the reabsorption of free bile acids and accelerating the excretion of free bile acids from the blood to the kidneys. It also promoted the metabolic enzyme CYP3A11, which accelerated the metabolism and clearance of bile acids, thus maintaining the balance of the bile acid internal environment.

Metabonomic and transcriptomic analyses of Tripterygium glycosides tablet-induced hepatotoxicity in rats

We aimed to explore novel biomarkers involved in alterations of metabolism and gene expression related to the hepatotoxic effects of Tripterygium glycosides tablet (TGT) in rats. Rats were randomly divided into groups based on oral administration of TGTs for 6 weeks: control, low-dose (9.5 mg/kg), and high-dose (18.9 mg/kg). Serum samples and total liver RNA were subjected to metabonomic and transcriptomic analyses. Thirteen metabolites were significantly up-regulated by liver injury induced by Tripterygium glycosides. Five potential biomarkers were more sensitive than Alanine aminotransferase (ALT) for accurate and timely prediction of hepatic damage. The four metabolic pathways most obviously regulated by hepatotoxicity were D-glutamine and D-glutamate metabolism, alanine, aspartate and glutamate metabolism, ether lipid metabolism, and tryptophan metabolism. Transcriptomics revealed significant differences in 1792 mRNAs and 400 long non-coding (lnc) RNAs. Dysregulated lncRNAs in the TGT-induced hepatotoxicity group were associated with genes involved in amino acid metabolism using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Up-regulated expression of Ehhadh, Gpt, and Got1, and down-regulated expression of dopa decarboxylase (Ddc), Cyp1a2, Ido2, Aldh1b1, and asparagine synthetase (Asns) was validated by quantitative real-time PCR. This multiomics study has elucidated the relationship between amino metabolism and liver injury, revealing potential biomarkers.

Abstract 417: Elevation Of Serum Hdl And Apoe Level In A Novel Vps35l Deficient Mouse Model

Aim: Defect of the endosomal protein sorting factor VPS35L causes novel congenital anomalies syndrome, in which cholesterol dysmetabolism is one of the symptoms. To reveal the biochemical mechanisms, the a model mouse was developed and characterized. Methods: Loss-of-function mutation, N995del, VPS35L knock-in (KI) mice were generated. Serum lipoprotein profiles were assessed by LipoSEARCH™ (Immuno-Biological Laboratories, Japan). For biochemical assays, 70 micro-L of serum was sub-fractionated with Superose 6 column in PBS and total and free cholesterol detected using LabAssay™cholesterol (Fujifilm Wako Chemicals, Japan) and Pureauto™S F-CHO-N (SEKISUI MEDICAL, Japan) assay kits. Negative images of fractioned lipoproteins were observed by TEM (JEM-1400Plus, JEOL, Japan). ApoA-I and apoE in the fractions were determined by use of a mouse apoA1 ELISA PRO kit and a mouse apoE ELISA PRO kit (Mabtech AB, Sweden). Mouse liver total RNA was isolated by ISOGEN (Nippon Gene, Japan) and first-strand cDNA generated using the Invitrogen™ SuperScript™IV First-Strand Synthesis system with random hexamers. For quantitative real-time PCR, the StepOnePlus™ real-time PCR system (Thermo Fisher) was used, and the PCR end product of each well confirmed by the melt curve analysis. Results: Lipoprotein analysis indicated the KI mice showed 1.9-fold higher total cholesterol than WT mice. LDL-C and HDL-C were increased 2.9- and 1.9-fold compared to WT mice, respectively. ApoA-I levels in the large HDL and HDL fractions were all significantly increased 1.5-, and 1.6-fold, whereas apoE levels were elevated 25-, and 8.4-fold, respectively. Conclusions: Ablation of VPS35L in MEF(3T3) cells caused loss of endocytosis of the LDL receptor and LRP1, and perhaps even SR-BI, which may lead to increased serum LDL and HDL levels in the loss-of-function KI model mice. Surprisingly. a high level of apoE was detected in KI mice, especially in the HDL fraction. In this case, apoE catabolism became abnormal due to the lack of LRP1 at the cell surface. Abnormal metabolism of apoE may affect the central nervous system or neuron generation. Further examination of the phenotype of this this model mouse line are warranted.

Revealing potential anti-fibrotic mechanism of Ganxianfang formula based on RNA sequence

BackgroundGanxianfang (GXF) formula as a traditional Chinese medicine (TCM) is used for liver fibrosis in clinical practice while its mechanism is unclear. The aim of this study is to explore the molecular mechanism of GXF against CCl4-induced liver fibrosis rats.MethodsDetected the main compounds of GXF by UPLC-MS/MS. Evaluated the efficacy of GXF (1.58, 3.15, 4.73 g/kg/day) and Fuzheng Huayu (FZHY, positive control, 0.47 g/kg/day) through serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels and histopathological changes. Explored the underlying mechanisms by integrating our total liver RNA sequencing (RNA-seq) data with recent liver single-cell sequencing (scRNA-seq) studies. Verified potential pharmacodynamic substances of GXF by hepatic stellate cell (HSC)-T6 line.ResultsMain compounds were identified in GXF by UPLC-MS/MS, including baicalin, wogonoside and matrine etc. With GXF-high dose treatment, the elevation of ALT and AST induced by CCl4 were significantly reduced, and the protective effect of GXF-high dose treatment was better than FZHY. Liver histopathological changes were alleviated by GXF-high dose treatment, the ISHAK scoring showed the incidence of liver cirrhosis (F5/F6) decreased from 76.5 to 55.6%. The results of liver hydroxyproline content were consistent with the histopathological changes. RNA-seq analysis revealed the differential genes (DEGs) were mainly enriched in ECM-receptor interaction and chemokine signaling pathway. GXF effectively inhibited collagen deposition and significantly downregulated CCL2 to inhibit the recruitment of macrophages in liver tissue. Integrating scRNA-seq data revealed that GXF effectively inhibited the expansion of scar-associated Trem2+CD9+ macrophages subpopulation and PDGFRα+PDGFRβ+ scar-producing myofibroblasts in the damaged liver, and remodeled the fibrotic niche via regulation of ligand-receptor interactions including TGFβ/EGFR, PDGFB/PDGFRα, and TNFSF12/TNFRSF12a signaling. In vitro experiments demonstrated that baicalin, matrine and hesperidin in GXF inhibited the activation of hepatic stellate cells.ConclusionsThis study clarified the potential anti-fibrotic effects and molecular mechanism of GXF in CCl4-induced liver fibrosis rats, which deserves further promotion and application.

Curc-mPEG454, a PEGylated curcumin derivative, as a multi-target anti-fibrotic prodrug

Our previous studies demonstrated that Curc-mPEG454, a curcumin derivative modified with short-chain polyethylene glycol (PEG), not only increased the blood concentration of curcumin, but also retained its anti-inflammatory activity. Here, we aimed to evaluate the anti-fibrotic effect of Curc-mPEG454 on a rat liver fibrosis model induced by carbon tetrachloride (CCl4), and to explore the underlying mechanisms by integrating our total liver RNA sequencing (RNA-seq) data with recent liver single-cell sequencing (scRNA-seq) studies. 50 mg/kg and 100 mg/kg Curc-mPEG454 treatment significantly reduced the elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) induced by CCl4, and the incidence of liver cirrhosis decreased from 75% to 37% and 35%, respectively. RNA-seq analysis revealed that Curc-mPEG454 significantly upregulated aldehyde oxidase 1 (AOX1) while downregulated cytochrome p450 26A1 (CYP26A1) and cytochrome p450 26B1 (CYP26B1) resulting in restoring liver retinoic acid (RA) level, increased glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) expression to synthesize hepatic glutathione (GSH), and inhibited liver inflammation via down-regulating the Prostaglandin E Synthase 2 (PTGES2)/prostacyclin E2 (PGE2) signaling. Integrating scRNA-seq data revealed that Curc-mPEG454 effectively inhibited the expansion of scar-associated macrophage subpopulation and scar-producing myofibroblasts in the damaged liver, and remodeled the fibrotic niche via regulation of ligand-receptor interactions including platelet-derived growth factor-B (PDGF-B)/platelet-derived growth factor receptor-α (PDGFR-α) signaling. As a multi-target prodrug, PEGylated curcumin deserves further attention and research.

NCOA4 Acts in Hepatocytes to Regulate Ferritin Subunit Levels and Hepatic Iron Storage

The intracellular protein nuclear receptor coactivator 4 (NCOA4) has been shown to mediate the degradation of the iron storage protein ferritin (Mancias et al., Nature 2014; Dowdle et al., Nat Cell Biol 2014). Accordingly, mice with global disruption of Ncoa4 show ferritin accumulation in multiple organs (Bellelli R et al., Cell Rep 2016). How NCOA4 activity in different tissues contributes to local and systemic iron homeostasis, however, has not yet been clearly defined. Here we selectively dissect the role of NCOA4 in the liver, the body's major iron depot.To facilitate targeted Ncoa4 knockdown in hepatocytes, we utilized Ncoa4 -targeting siRNA that was conjugated to triantennary N-acetylgalactosamine (GalNAc- Ncoa4 siRNA) to promote uptake by the hepatocyte asialoglycoprotein receptor. We first validated the ability of GalNAc- Ncoa4 siRNA treatment to target endogenous murine Ncoa4 mRNA for degradation in murine Hepa1-6 hepatoma cells. We next assessed the ability of GalNAc- Ncoa4 siRNA to acutely lower Ncoa4 expression in mouse liver. Eight-week-old C57BL/6N female mice were injected subcutaneously with 1, 3, or 5 mg/kg body weight of a sterile, endotoxin-tested solution of GalNAc- Ncoa4 siRNA in saline or with vehicle (saline) alone, and tissues were collected for analysis 24 hrs after injection. Compared to vehicle alone, GalNAc- Ncoa4 siRNA effectively lowered Ncoa4 expression in total liver RNA; because the degree of Ncoa4 knockdown that was achieved by the 3 mg/kg and 5 mg/kg doses was similar, the 3mg/kg dose was selected for our further experiments to reduce the possibility of off-target effects.We next examined the durability of hepatocyte Ncoa4 knockdown achieved by a single injection of GalNAc -Ncoa4 siRNA as well as the resulting effects on hepatic and systemic iron parameters. Eight-week-old C57BL/6N female mice were injected once with GalNAc- Ncoa4 siRNA or saline vehicle, and blood and tissues were collected for analysis at 1, 2, 3, or 4 weeks after injection. Compared to saline treatment, a single injection of GalNAc -Ncoa4 siRNA produced a significant, sustained reduction in hepatic Ncoa4 mRNA; Ncoa4 mRNA remained maximally suppressed at the 1, 2, and 3-week timepoints and began to rise slightly 4 weeks after injection. Saline-treated mice showed a slight, progressive increase in mean liver non-heme iron concentration (LIC) over the 4 weeks following injection, consistent with the known effect of age on murine hepatic iron stores. However, in mice injected with a single dose of GalNAc- Ncoa4 siRNA, the rise in LIC over the 4-week time period was significantly greater, consistent with increased hepatic iron storage. Immunoblotting of liver lysates revealed that levels of the ferritin heavy (FTH) and light (FTL) subunits increased in mice treated with GalNAc- Ncoa4 siRNA compared to saline-treated controls. These increases in FTH and FTL were evident 1 week after GalNAc- Ncoa4 siRNA injection, and FTH and FTL levels continued to rise with additional weeks of Ncoa4 knockdown. In mice treated with saline, spleen non-heme iron concentration (SIC) increased over the 4 weeks following injection, consistent with the known effect of age on murine splenic iron stores. However, at the 3 and 4-week time points, mice treated with GalNAc- Ncoa4 siRNA showed a significant reduction in SIC compared to saline-treated controls, which correlated histologically with reduced iron retention in splenic macrophages. Compared to saline treatment, GalNAc -Ncoa4 siRNAtreatment did not produce significant changes in spleen weight, serum iron, complete blood count, or non-heme iron concentration of heart, pancreas, or muscle.Collectively, our findings indicate that NCOA4 acts in hepatocytes to limit LIC as well as hepatic levels of FTH and FTL. Furthermore, local NCOA4 activity in hepatocytes impacts iron distribution to the spleen. Our results are compatible with a model in which degradation of the ferritin protein complex in hepatocytes occurs as a constitutive process that is mediated by NCOA4. Importantly, our findings demonstrate that in the setting of hepatocyte Ncoa4 knockdown, other cellular pathways cannot compensate to maintain normal LIC and normal hepatic levels of FTH and FTL. Additionally, our findings raise the possibility that enhancement of NCOA4 activity in hepatocytes might serve as a therapeutic approach to promote hepatic iron mobilization in iron overload disorders. DisclosuresNo relevant conflicts of interest to declare.

Gene Expression Profiling in Liver of Mice Fed a High-Fat Diet Supplemented With Watermelon Flesh, Arginine, or Citrulline Shows Similar Pattern Changes

Abstract Objectives Watermelon is a nutrient-dense fruit known to contain high levels or arginine and citrulline; these two compounds may influence the nitric oxide pathway, vasodilation, and thus be hypotensive. We tested the hypothesis that when C57BL/6J male mice fed a high-fat diet (HF) had additions to the HF diet of either watermelon flesh (WF), arginine (ARG) or citrulline (CIT), changes in gene expression patterns would occur vs. those seen in HF. Further, we hypothesize that patterns of expression seen in WF, ARG, and CIT groups would be somewhat similar based on increased dietary levels of ARG and CIT in all three groups. Methods Following prior work (Becraft et al.; 2018, Egea et al. 2020), groups of mice were provided either a low-fat diet (LF, 10% kcal fat), high-fat diet (HF, 45% kcal fat), HF plus Watermelon Flesh (WF), HF plus 1% (w/w) arginine (ARG) or 1% (w/w citrulline (CIT) for 10 weeks. Watermelon flesh was provided at 10% of total energy. After ten weeks, animals were euthanized, and liver total RNA was isolated using Trizol. Total RNA was then used for gene expression analysis (N = 4 per group) using Clariom S microarrays and TAC analysis software (ThermoFisher). Results Mice fed WF, ARG, and CIT had several shared canonical pathways of gene expression, including eicosanoid metabolism via cytochrome P450 monooxygenases and exercise-induced circadian rhythm (All P < 0.05). Intake of WF and ARG significantly up-regulated both Cyp2c9 and Cyp2c38 mRNA levels (P < 0.05). The Bst2 gene was significantly down-regulated in all three groups compared to HF mice (P < 0.05). The Cyp2b9 gene was upregulated ∼10.7 fold in WF, and > 1000-fold in ARG mice (P < 0.05). Conclusions We demonstrated that when added to a HF diet, WF, ARG, and CIT all produced a change in hepatic gene expression in male mice. Possibly due to the close relationship of ARG and CIT metabolism, and high content of ARG and CIT in WF, expression patterns observed in all three groups demonstrated a high degree of similarity. Several genes, including Cyp2c9, Cyp2c38, and Elvol5 were up-regulated; these genes may be involved in modifying steroids and arachidonic acid and other long-chain fatty acids. Funding Sources National Watermelon Promotion Board.

Reduced Cleavage of Von Willebrand Factor By ADAMTS13 Exacerbates Acute Kidney Injury Secondary to Traumatic Injury

Introduction: Increases in plasma von Willebrand Factor (vWF) antigen levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated following severe injury. We hypothesized that following traumatic injury, endothelial damage and activation would result in the release of high molecular weight multimeric (HMWM) vWF forms into circulation with a concominant decrease in ADAMTS13. We further hypothesized that the alterations in circulating levels and functions of these factors, following traumatic injury, promote an increased thrombogenic and inflammatory response, which exacerbates the risk of developing post traumatic acute kidney injury (AKI). Methods: Plasma samples from a cohort of patients with trauma induced AKI, collected at 0-, 24-, and 72-hours after admission, were evaluated against plasma samples from trauma patients that were matched for injury characteristics, but were without AKI. VWF and ADAMTS13 activity and antigen levels were evaluated. Circulating vWF multimeric composition was determined by vertical agarose gel electrophoresis. We further evaluated the effects of traumatic injury on ADAMTS13 and vWF by means of a pre-clinical model of trauma and hemorrhage, which is comprised of a liver laceration, cardiac puncture, and soft tissue injury. Following this polytrauma model, hepatic ADAMTS13 RNA expression was evaluated by qRT-PCR of total liver RNA; analyzed liver samples were taken immediately before, 24-, and 72-hours after injury. The impact of the polytrauma model on renal function was evaluated via changes in plasma cystatin C, while the effect on the renal microvasculature was evaluated by means of immunofluorescent staining of vWF and fibrinogen in kidney sections. Results: VWF antigen was increased in trauma patients when compared to healthy controls upon admission (11.0 ± 3.72 ug/mL vs. 4.64 ± 0.91 ug/mL), persisting through 72-hours (13.11 ± 3.16 ug/mL). The percentage of HMWM vWF forms in trauma patient samples was elevated at all time points compared to pooled normal plasma (PNP) (27.9 ± 4.5% and 15.9 ± 0.8). Trauma patient ADAMTS13 activity was decreased at all time points (0.48 ± 0.14 IU/mL vs. 1.27 ± 0.26 IU/mL). AKI patients showed a 1.54 ± 0.48-fold increase in vWF antigen levels over the 72-hour period (p=0.016), while there was no change in the control trauma group. Those who developed AKI demonstrated a larger proportion of HMWM vWF at 72-hours than their non-AKI counterparts (32.9 ± 2.4% vs 28.8 ± 3.2%). qRT-PCR of murine total liver RNA, following the polytrauma model, exhibited ADAMTS13 RNA expression levels that were 35% of normal at 24-hours and 60% of normal at 72 hours post-injury. A matched analysis of murine plasma cystatin C levels demonstrated a marked elevation 24-hours post polytrauma model (sham: 510ng/mL (435-561) vs trauma: 550 (479-701)). H&E staining of kidney sections, which were harvested following the polytrauma model, demonstrated generalized renal tubule dilation and denudation, suggestive of post-traumatic acute tubular necrosis. Immunofluorescent studies of kidney sections from the same mice exhibited 8-15µm microvascular plugs that were positive for fibrinogen with a vWF core; renal immunofluorescent studies of control mice demonstrated no microvascular depositions. Conclusions: These data indicate that traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes inflammation and thrombosis. In the immediate moments following injury, these mechanisms support life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to a burden that may produce microvascular clot deposition, exacerbating pathologic states such as acute kidney injury. Disclosures No relevant conflicts of interest to declare.

Prolonged Circulation of Ultra-Large Von Willebrand Factor and a Reduction in ADAMTS13 Activity Promotes Microvascular Disease Following Traumatic Injury

Introduction: Increases in plasma von Willebrand Factor (vWF) levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated in diseases of microvascular injury. We hypothesized that following severe trauma, a burst of ultra-large vWF (UL-vWF) is released into the bloodstream by damaged endothelium, resulting in increased thrombogenicity due to circulating vWF multimers. We further hypothesized that traumatic injury would lead to a deficit of ADAMTS13, promoting the accumulation of UL-vWF forms and, ultimately, the increased risk of microvascular disease, such as acute kidney injury (AKI). Methods: A cohort of 37 severely injured trauma patients was analyzed for antigen levels of plasma vWF and ADAMTS13 at 0- and 24-hours after admission. Circulating vWF multimeric composition from both time points was determined by vertical agarose gel electrophoresis. Multivariate analyses were performed with data abstracted from the electronic medical records to identify further dependences. A similar analysis was also performed on plasma from a cohort of 8 patients with trauma induced AKI at 0-, 24-, and 72-hours after admission; these patients were well matched against trauma patients without AKI. Finally, we utilized a murine model of polytrauma and hemorrhage, in conjunction with qRT-PCR of ADAMTS13 in total liver RNA, to specifically address how the expression of ADAMTS13 is altered by the systemic effects of traumatic injury. Results: Circulating vWF levels were increased in severe trauma patients when compared to healthy controls at presentation (189% (110-263) vs. 95% (74-120)) and persisted through 24-hours (213% (146-257) vs. 132% (57-160)). Ultra-large vWF forms were elevated at both 0- and 24-hours when compared to pooled normal plasma ((10.0% (8.9-14.3) and 11.3% (9.1-21.2), respectively, vs 0.6%). The largest vWF forms within trauma patient plasma circulated at 33±4 dimers vs 18±1 dimer in length within pooled normal plasma. Severe trauma patient ADAMTS13 activity was decreased at 0-hours (66% (47-86) vs. 100% (98-100)) and at 24-hours (72.5% (56-87.3) vs 103% (103-103)) when compared to healthy patients. Furthermore, the proportion of circulating low molecular weight multimeric (LMWM) vWF to total circulating vWF forms was directly dependent upon ADAMTS13 activity at 24-hours (Decreased ADAMTS13 Activity: 20.4% (15.0-22.7) LMWM vWF vs Normal Activity: 25.8% (22.7-35.2) LMWM vWF). Strikingly, ADAMTS13 activity independently predicted the development of coagulopathy, correlating with presentation INR (ρ =-0.63), activated clotting time of thromboelastography (TEG) (ρ=-0.36), and TEG maximum amplitude (ρ=0.36). ADAMTS13 activity also closely correlated with injury severity (ISS) (ρ=-0.34) and blood product transfusion (ρ =-.45). The cohort of 8 trauma patients who went on to develop AKI showed a 1.54-fold (1.02-2.05) increase in plasma vWF antigen levels between 0 and 72 hours, while those who did not develop AKI showed no change in vWF levels over this time period. Furthermore, those who developed AKI demonstrated a smaller proportion of LMWM vWF in plasma than those who did not (25.4% (23.4-28.0) vs 31.2% (27.2-35.6)), suggesting the increased thrombogenicity of their circulating vWF forms. Finally, qRT-PCR of total liver RNA in 6 mice demonstrated a 2-fold decrease in ADAMTS13 RNA expression levels between the times immediately before and 24-hours after trauma. Altogether, these data indicate that both circulating ADAMTS13 and its production are deficient in the days following severe injury. Conclusions: Severe traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes thrombosis. Not only is the concentration of circulating ADAMTS13 decreased following traumatic injury, but hepatic expression of the enzyme is lacking as well. In the immediate moments following injury, these mechanisms contribute to life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to burden that may exacerbate microvascular disease. Disclosures Ragni: Bioverativ/Sanofi: Consultancy, Research Funding; Sangamo: Research Funding; Shire/Takeda: Consultancy, Other: Study drug; Alnylam/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; Spark Therapeutics: Consultancy, Research Funding; ICER: Consultancy; OPKO: Research Funding; Biomarin: Consultancy, Research Funding. Rollins-Raval:Bayer, Inc: Membership on an entity's Board of Directors or advisory committees. Raval:Sanofi: Membership on an entity's Board of Directors or advisory committees; Bayer, Inc: Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

A multiplex RNA quantification method to determine the absolute amounts of mRNA without reverse transcription

We have developed a highly sensitive microarray-based method that determines the absolute amounts of mRNA in a total RNA sample in a multiplex manner without reverse transcription. This direct mRNA measurement promotes high-throughput testing and reduces bias in transcriptome analyses. Furthermore, quantification of the absolute amount of mRNA allows transcriptome analysis without common controls or additional, complicated normalization. The method, called Photo-DEAN, was validated using chemically synthesized RNAs of known quantities and mouse liver total RNA samples. We found that the absolute amounts of mRNA were successfully measured without the cDNA synthesis step, with a sensitivity of 15 zmol achieved in 7 h.

Conspecific vitellogenin induces the expression of vg gene in the Indian male walking catfish, Clarias batrachus (Linn.)

To understand the regulatory mechanism of Vg induced vitellogenin synthesis the vg gene expression in Indian male walking catfish, Clarias batrachus was investigated. Semipurified conspecific Vg containing Vg1 and Vg2 in a ratio of 2.7:1.0 was administered into male catfish and vg cDNA (1.1kb) was amplified from total RNA in liver by reverse transcriptase polymerase chain reaction (RT-PCR) using primers designed from published sequence of Clarias macrocephalus. The nucleotide and deduced amino acid sequence of the cDNA shared maximum similarities (98–100%) with the corresponding sequences of known E2-induced Vg of C. batrachus and C. macrocephalus in the database. A 0.178kb cDNA fragment nested within the 1.1kb DNA was then amplified by real time PCR to evaluate the role of Vg on relative expression of vg gene. The result revealed a significant upregulation (1.79 fold) of vg mRNA at 6h reaching maximum level (9.78 fold) at 24h post Vg injection as compared to the saline control. Similarly, E2-treatment also showed maximum mRNA expression (7.73 fold) at 24h post injection. The findings suggest that like E2, Vg itself can induce vg gene expression resulting in a significant increase in plasma Vg levels (9.11±0.73mg/ml for Vg1 and 3.02±0.28mg/ml for Vg 2) in male catfish where E2 is lacking. The work provides further opportunity to study the regulatory mechanism of vg gene expression by Vg.

Characterization and mechanism of phosphoinositide 3-kinases (PI3Ks) members in insulin-induced changes of protein metabolism in yellow catfish Pelteobagrus fulvidraco

In the present study, seven phosphoinositide 3-kinase (PI3K) members (PI3KCa, PI3KCb, PI3KCd, PI3KCg, PI3KC2a, PI3KC2b and PI3KC3, respectively) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco, and their roles in insulin-induced changes of protein metabolism were determined. These seven PI3Ks can be divided into three classes, class I (including PI3KCa, PI3KCb, PI3KCd and PI3KCg), class II (including PI3KC2a and PI3KC2b) and class III (only including PI3KC3). Compared with mammals, all of these members share similar domain structure. Their mRNAs were widely expressed across ten tested tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, intestine, heart, kidney and ovary), but at variable levels. In the in vivo study, insulin treatment significantly increased hepatic protein content at 3h, accompanied with reduced plasma total amino acid contents and liver ALT activity, and with increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in liver. At 6h and 12h, insulin injection showed no significant effect on liver protein content and plasma total amino acid, but reduced liver ALT activity and increased liver total RNA and the mRNA levels of AKT2, mTORC1 and S6K1 in liver at 6h. In the in vitro study, insulin incubation also tended to increase protein content of hepatocytes, accompanied with reduced cell medium total amino acid contents and hepatocytes ALT activity, and increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in hepatocytes. However, insulin treatment showed no significant effect on GDH activity and mRNA expression of PI3KCa, PI3KCd, PI3KCg, PI3KC2b, PI3KC3 and eEF2 both in the in vivo and in vitro studies. Effects of insulin on the mRNA levels of eIF-4E and 4E-BP1 were different between the in vivo and in vitro studies, and also time-dependent. Compared to single insulin group, insulin+wortmannin group increased ALT activity at 6h but reduced T-RNA content at 6 and 12h. AKT2 and S6K1 mRNA levels at 6 and 12h, mRNA levels of mTORC1, 4E-BP1 and eEF2 at 3 and 6h, and EIF-4E mRNA levels at 3 and 12h, PI3KCb and PI3KC2a mRNA levels were significantly lower in insulin+wortmannin group than those in single insulin group. Thus, our study demonstrated that among seven PI3K members, PI3KCb and PI3KC2a were more sensitive to the insulin signaling pathway, and insulin stimulated hepatic protein synthesis in yellow catfish through PI3K signaling pathway.

Effects of RNAs on chromatin accessibility and gene expression suggest RNA-mediated activation

The study of the interaction between RNA and DNA sequences in activating genes has important significance for understanding the mechanisms of RNA-mediated activation. Here, we used in vitro chromatin reconstitution approach to observe whether RNAs increase DNase I digestion, plasmid transfection to observe whether RNAs promote gene expression, and bioinformatics analysis to predict the binding ability of RNAs to centromere DNA (constitutive heterochromatin). Synthetic RNAs (23nt) that were complementary to mouse albumin gene and total liver RNA increased DNase I digestion sensitivity of mouse albumin gene, suggesting that RNAs can increase chromatin accessibility. Transcribed sense-antisense tandem Alu elements activated an enhanced green fluorescent protein reporter gene after stable transfection. Bioinformatics analysis showed that the binding strength of RNA population to centromere DNAs is significantly lower than that of their flanking sequences, which suggests that the centromere is not easily affected by RNAs produced from other transcribed regions and may be the reason why centromeres consist of constitutive heterochromatin. The results in this paper illustrate that RNAs complementary to DNA sequences play roles in activating genes. Since RNA is mainly produced from the cell’s own DNA, the work presented in this paper suggests that RNAs transcribed from DNA create feedback that activates DNA transcription.

688. AAV8-Mediated Liver Gene Targeting Without Nucleases Rescues Lethality in a Mouse Model of the Crigler-Najjar Syndrome

The Crigler-Najjar Syndrome Type I (CNSI) is a rare monogenic disease caused by null mutations in the UGT1A1 gene, resulting in bilirubin conjugation deficiency and unconjugated hyperbilirubinemia, with lifelong risk of neurological damage, kernicterus and death. Current clinical practice consists of phototherapy treatment (PT) for more than 10-12 h/day, profoundly affecting (social) life quality. However, PT becomes less effective with age, leaving liver transplantation as the only therapeutic option, which is associated with significant morbidity and mortality (90% survival, 10 years after transplantation).rAAV-mediated liver gene therapy in neonates is associated with the loss of viral genomes over time, and insertional mutagenesis due to integration of the expression cassette into genomic oncogenic loci. Gene targeting/editing strategies using co-expression of a nuclease increase the probability of off-targeting and mutagenesis, as well as an immune-response directed against the nuclease.To overcome these issues, we applied a novel and safer in vivo gene targeting approach recently developed and successfully applied to a hemophilia B model, based on the targeted insertion of a promoterless version of the Factor IX cDNA into the albumin locus, without the use of nucleases.To implement this approach for Crigler-Najjar, we used a lethal mouse model of the disease previously developed in our laboratory. The strategy is based in the in frame knock-in of the human UGT1a1 cDNA into the albumin locus using a promoterless rAAV8 vector. The hUGT1a1 cDNA is inserted downstream of the albumin ORF, with a ribosomal skipping 2A sequence. This results in a single chimeric mRNA driven from the endogenous albumin locus that produces two proteins: albumin and UGT1a1.We IP injected mutant pups, temporarily treated with PT up to P15 (a procedure that rescues all mutant mice), at different ages with a rAAV8 donor vector to determine the most effective timepoint. We observed a therapeutic reduction in plasma bilirubin of AAV8-treated mice (n=4-6 per timepoint, p£0.001, ANOVA). RT-PCR analysis of total liver RNA showed the presence of the hybrid Albumin-Ugt1a1 mRNA, which was confirmed by DNA cloning and sequencing, proving that the Ugt1a1 transcript was derived from the endogenous albumin locus.More importantly, when we injected P4 mice and treated them with PT up to P8 (a procedure that alone results in 95%-100% mortality), we rescued lethality in all rAAV-treated mutant mice, while all untreated animals died (n=7, p£0.001, Log-rank test).Future steps will involve the improvement of the therapeutic efficiency by transducing a higher number of viral genomes, and mRNA translatability by using codon optimized versions of the hUGT1a1 cDNA.The results presented here support the conclusion that the promoterless gene targeting therapy without nucleases, already effective for hemophilia B, proved to be efficient for CNSI, strongly suggesting that this approach is applicable for providing therapeutic levels of an intracellular enzyme responsible for a lethal liver metabolic disease.

The possible protective effect of Coriandrum sativum seeds methanolic extract on hepato-renal toxicity induced by sodium arsenite in albino rats

Article history: This work has been carried out in order to investigate the possible ameliorative effect of Coriander seeds methanolic extract (CSE) on sodium arsenite (As) - induced toxicity in albino rats. 2,3 Dimercaptosuccinic acid (DMSA) was used as a standerd chelating agent. The experiment lasted for 8 weeks and blood samples were withdrawn 4 and 8 weeks after As and other treatments administrations. As was used to induce hepatotoxicity in rats in a dose equivalent to 100 p.p.m . In-vivo studies using the different biochemical techniques employed in hepato-renal functions as well as liver apoptotic DNA and total RNA alterations proved that As caused a significant increase in parameters concerned to hepato-renal toxicity while treatment of CSE or DMSA caused an ameliorative effect on this toxicity. Administration of CSE and DMSA together along with arsenite proves the synergistic effects of these chelating agents on arsenite toxicity.

Icariin is a PPARα activator inducing lipid metabolic gene expression in mice.

Icariin is effective in the treatment of hyperlipidemia. To understand the effect of icariin on lipid metabolism, effects of icariin on PPARα and its target genes were investigated. Mice were treated orally with icariin at doses of 0, 100, 200, and 400 mg/kg, or clofibrate (500 mg/kg) for five days. Liver total RNA was isolated and the expressions of PPARα and lipid metabolism genes were examined. PPARα and its marker genes Cyp4a10 and Cyp4a14 were induced 2-4 fold by icariin, and 4-8 fold by clofibrate. The fatty acid (FA) binding and co-activator proteins Fabp1, Fabp4 and Acsl1 were increased 2-fold. The mRNAs of mitochondrial FA β-oxidation enzymes (Cpt1a, Acat1, Acad1 and Hmgcs2) were increased 2-3 fold. The mRNAs of proximal β-oxidation enzymes (Acox1, Ech1, and Ehhadh) were also increased by icariin and clofibrate. The expression of mRNAs for sterol regulatory element-binding factor-1 (Srebf1) and FA synthetase (Fasn) were unaltered by icariin. The lipid lysis genes Lipe and Pnpla2 were increased by icariin and clofibrate. These results indicate that icariin is a novel PPARα agonist, activates lipid metabolism gene expressions in liver, which could be a basis for its lipid-lowering effects and its beneficial effects against diabetes.

Effect of Panax notoginseng saponins on liver drug metablic enzyme activity, mrna and protein expressions in rats

To study the effect of Panax notoginseng saponins (PNS) on liver drug metabolic enzyme activity, mRNA and protein expressions in rats. Male Wistar rats were randomly divided into nine groups. After administration of the test drugs, their liver microsomes, liver total RNA and total protein were extracted to detect the regulating effect of PNS on liver drug metabolic enzyme activity-related subtype enzymatic activity, mRNA and protein expression by substrate probe, quantitative PCR and Western Blot technology. The result of this experiment was that PNS could significantly induce CYP1A2 and CYP2E1 enzyme activity, mRNA expression, CYP2E1 protein expression level. PNS significantly induced CYP3A mRNA expression, but with no significant effect in CYP3A enzyme activity level. PNS had no significant effect CYP1A1 and CYP2B mRNA expressions and enzyme activity levels. PNS had selective regulations on different P450 subtypes, and the major subtypes were CYP1A2 and CYP2E1. In clinical practice, particularly in the combination with CYP1A2 and CYP2E1 metabolism-related drugs, full consideration shall be given to the possible drug interactions in order to avoid potential toxic and side effects. Meanwhile, whether the induction effect of CYP2E1 gets involved in ginsenoside's effect incavenging free radicals deserves further studies.

Characterization of the native C-reactive protein (cCRP) and the corresponding liver mRNA in dogs

C-reactive protein (CRP) plays an important role in the acute phase reaction in humans and dogs. For the canine CRP (cCRP) only an in silico deduced preliminary transcript and amino acid sequence is available. The objective of this study was to further characterize the native cCRP protein and its corresponding liver mRNA. Furthermore, immunological similarities of serum CRP in related animal species were investigated. Native cCRP protein was isolated from dog-sera by affinity chromatography and further analyzed by immunodetection, protein sequencing (mass spectrometry and N-terminal Edman sequencing), 2D-gel electrophoresis, and glycoprotein analysis. Furthermore, cCRP cDNA sequence was determined from dog liver total RNA by RT-PCR. Gel electrophoresis, immunodetection and glycoprotein detection revealed two cCRP isotypes with different molecular weights (22 and 25kDa) with the upper band being glycosylated. Selective glycoprotein analysis showed sialic acid terminally linked (2–6) to galactose or N-acetylgalactosamine and subsequent PNGase F treatment identified N-terminal linkage. Mass spectrometry confirmed approximately 45% of the cCRP predicted amino acid sequence and N-terminal amino acid sequencing revealed a shorter native cCRP than expected (204 amino acids). The new canine CRP mRNA sequence confirms 100% of the formerly deduced sequence. Immunological homologies to the canine CRP protein were found in selected dog-related species.This study contributes major molecular details to the knowledge about canine CRP. Such structural information may assist in developing new diagnostic tools for inflammatory-based diseases in dogs as well as other dog-related species.

EVALUATION OF THE TOXIC EFFECTS OF DIFENOCONAZOLE FUNGICIDE (SCORE) ON MALE ALBINO RATS

This study was designed to estimate the LD50 of Score (difenoconazole) fungicide and evaluate its toxic effects on body weight, liver weight, some biochemical parameters and hematological profiles of male albino rats after chronic oral daily doses (1/20, 1/10 and 1/5 LD.50) for 8 weeks. Rats were divided into 4 groups, the first group (G1) as control group, the other groups (G2, G3 and G4) treated daily with 20,40 and 80 mg/100 g.b.w. for 1, 4 and 8 weeks respectively The medium lethal dose (LD.50) of Score was 400 mg /100g. body weight of adult male rat. Significant decreases in body weight, serum total protein, albumin, globulin, liver DNA and liver RNA, RBCs count, hemoglobin and blood Platelets count However, significantly increase observed in hepatosomatic index, serum enzymes (ALAT, ASAT, ALP and CK), cholesterol, triglyceride, total lipids, serum creatinine and serum bilirubin and WBC's count