Liver Tissue Extracts Research Articles

Hepatocellular Carcinoma Detection by Cell Sensor Based on Anti-GPC3 Single-Chain Variable Fragment

Precise identification of hepatocellular carcinoma (HCC) in early stages remains both a challenge and an opportunity for prevention and diagnosis of early liver cancer. Fast-detecting techniques like electrochemical sensing might provide more insight than current clinical methods owing to its high sensitivity, rapid detection, and convenient operation. Here, a single-chain variable fragment (scFv) was designed by retaining the antigen binding active sites of a recombinant humanized monoclonal antibody and modifying with histidine tags, and consequently used for the construction of cell sensor for HCC detection. The customized scFv showed specific binding signature on the membrane of HCC cell and positive correlation with cell concentration using fluorescence confocal microscopy and flow cytometry, respectively. A higher binding affinity of scFv over the original monoclonal antibody was also displayed. After the immobilization onto the screen-printed electrode through the high affinity between histidine and nickel-based nanomaterial, the constructed cell sensor exhibited a wide detection range of 10 2 to 10 7 cells ml −1 and a low detection limit of 2 cells ml −1 with a short incubation time of 5 min. The high selectivity in the liver tissue extract fluid derived from the design strategy was also displayed in the mouse samples. The customized scFv and related cell sensor demonstrated great potential for fast HCC analysis in early-stage cancer diagnosis.

Study on the mechanism of acute liver injury protection in Rhubarb anthraquinone by metabolomics based on UPLC-Q-TOF-MS.

As a traditional Chinese medicine, rhubarb has been used in a variety of liver diseases and it is widely used in clinic to prevent and treat acute liver injury. Anthraquinone, as the main medicinal component of rhubarb, can reverse the further development of liver fibrosis caused by acute liver injury. In this study, metabonomics was used to explore the mechanism of different doses of rhubarb anthraquinone on acute liver injury in rats. Rhubarb anthraquinone was administered intragastric to rats at doses of 3.9, 7.8 and 15.6mg/kg, respectively, for 7days, and then 30% CCl4 was injected intraperitoneally at the dose of 1ml/kg to replicate the acute liver injury model. The biochemical indicators content of ALT, AST, ALP, γ-GT, TG, TC, LDL, HDL in serum and GSH, Hyp, SOD, TNF-α, IL-6 and IL-8 in liver tissue extract were tested respectively, and liver tissue was histopathologically analysis. At the same time, UPLC-Q-TOF-MS combined with non-targeted metabolomics were used to study the metabolites and metabolic pathways of rhubarb anthraquinone in treating acute liver injury. Compared with normal rats, the contents of ALT, AST, ALP, TG, TC, LDL, γ-GT in serum and Hyp, MDA, IL-6, IL-8, TNF-α in the liver tissue extract were significantly increased in model rats (p < 0.05, p < 0.01), and the content of HDL in the serum was significantly decreased (p < 0.05); the activities of GSH and SOD in liver tissue extract were also significantly decreased (p < 0.05). After administration of rhubarb anthraquinone, compared with the model group, with the increase of dosage, some biochemical indexes showed opposite changes, and gradually approached to normal rats. 12 different metabolites were identified by metabonomics, and the biosynthesis and metabolism of phenylalanine, tyrosine and tryptophan, the metabolism of amino sugars, nucleotide sugars and pyrimidines metabolism, and the biosynthesis of steroid hormone were identified based on the biomarker analysis. Based on the biochemical analysis and metabonomics analysis of rats with acute liver injury treated with different doses of rhubarb anthraquinone, combined with histopathological observation, the results show that the protective effect of rhubarb anthraquinone on acute liver injury is related to the dosage; Meanwhile, the metabolic pathway analysis suggested that rhubarb anthraquinone alleviate acute liver injury by regulating inflammation, oxidative stress and fibrosis disorders. This study explained the therapeutic effect of rhubarb anthraquinone on acute liver injury from both material basis and action pathway, and provided safe and effective research ideas for clinical application of rhubarb.

Zinc deficiency and therapeutic role of sesame seeds on some physiological aspects of reproduction and growth in pregnant rats and its neonates

The present study aimed to find the Effect of zinc deficiency on some reproductive parameters among female rats and the growth of its neonates and to assess the therapeutic role of the sesame seeds. The research contained 35 pregnant females rats they were divided into seven groups, the first was control, the second was fed diet poor in zinc, the third group added sesame to diet poor in zinc and the fourth group was treated with diet poor in zinc added to it vitamin C. Groups five, six and seven were given poor zinc diet in the first, second and third trimester, respectively. The results showed a significant decrease of Prolactin (PRL) level in studied groups except in vitamin C group which showed no significant differences in value (P≤0.05) compared with a poor zinc diet group. While there were increasing in the FSH and LH levels in all studied groups except the first week which showed no significant difference compared with a poor zinc diet. Results indicated a significant decrease in the level of Estrogen(E2) in the first and third weeks, and increased its level in vitamin C and the second week groups, while sesame seeds group showed no significant difference compared with the poor zinc diet Group. The results showed an increased level of Progesterone (PRO) significantly in the second and the third week groups and a decrease in the sesame seeds and the first week groups, and the lack of significant difference in the vitamin C group compared with the poor zinc Group. The results showed a high level of superoxide dismutase( SOD) Glutathione (GSH) and low level of malondialdehyde (MDA) significantly in the studied groups except for the second week group appeared no significant difference in the level of GSH and MDA compared with a poor zinc diet group. The results of neonates showed significant increasing in the length of the body in sesame seeds and vitamin C groups, while showed no significant differences in the group of first, second and third weeks compared to poor zinc diet. Studied groups showed also a significant increase in body weight except for the second week group which showed a significant decrease, while the third week group did not show significant differences compared with the poor zinc diet Group. The results showed a significant decrease in mortality rate and the level of MDA in the brain, liver and placenta tissue extract in all groups except the second week group which showed a significant increase in the mortality rate and the level of liver MDA and placenta and in the first week in liver MDA only in comparison with a poor zinc diet group.

The phytochemical curcumin inhibits steroid sulfatase activity in rat liver tissue and NIH-3T3 mouse fibroblast cells

Curcumin is a phytochemical derived from the spice turmeric that is reported to have therapeutic effects. We are studying the enzyme steroid sulfatase (STS), which removes the sulfate group from inactive steroid hormones in peripheral tissues and we were interested in the effect of curcumin on STS activity due to its structural composition (polyphenolic). We sought to determine if curcumin affects STS activity in two model systems, rat liver and NIH-3T3 mouse fibroblast cells. STS assays were performed on tissue extracts of rat liver, and on NIH-3T3 microsomes and cells, with and without curcumin. Male and female rat liver extracts contained substantial amounts of STS activity, with males averaging higher (4–11 %) levels. Estradiol inhibited STS activity in livers of both sexes at 20 and 10 µM. Curcumin acted as a competitive inhibitor of STS activity in rat liver extracts, with a Ki of 19.8 µM in males and 9.3 µM in females. Curcumin also inhibited STS activity in NIH-3T3 microsomes at both 20 µM and 10 µM, and in whole NIH-3T3 cells at 20 µM. These data are the first to demonstrate STS inhibition by curcumin. Inhibition of STS results in lower active steroid hormone (estrogens and androgens) levels in tissues, possibly altering modulation of immune responses by these steroids.

Inhibition on XBP1s-driven lipogenesis by Qushi Huayu Decoction contributes to amelioration of hepatic steatosis induced by fructose

Ethnopharmacological relevanceQushi Huayu Decoction (QHD) is a traditional Chinese medicine formula consisting of five herbs, which has been used for non-alcoholic fatty liver disease (NAFLD) treatment in clinic for decades in China and validated in several NAFLD animal models. The hepatic de novo lipogenesis (DNL) is enhanced greatly to contribute to steatosis in NAFLD. The spliced form of X-box binding protein 1 (XBP1s) initiates DNL independently of sterol regulatory element-binding protein (SREBP) and carbohydrate-responsive element-binding protein (ChREBP). Aim of the studyTo disclose the mechanism of inhibition on hepatic DNL by QHD and the responsible compounds. MethodsThe effects of QHD on hepatic DNL were evaluated in mice induced by high-fructose diet (HFru). The effects of the serum-absorbed compounds of QHD on XBP1s were evaluated in HepG2 cells induced by tunicamycin. Hepatic histology, triglyceride (TG) and nonesterified fatty acids were observed. Hepatic apolipoprotein B100 and very low-density lipoprotein were measured to reflect lipid out-transport. The mRNA expression of XBP1s and its target genes were detected by real-time polymerase chain reaction. The protein expression of TG synthetases and DNL enzymes, and inositol requirement enzyme 1 alpha (IRE1α), phosphorylated IRE1α and XBP1s were detected in liver tissue and HepG2 cells by western-blot. The binding activity of SREBP1, protein expression of ChREBP and XBP1s were detected in the nuclear extracts of liver tissue. ResultsDynamical observing suggested feeding with HFru for 2 weeks was sufficient to induce hepatic lipogenesis and XBP1s. QHD ameliorated liver steatosis without enhancing out-transport of lipids, accompanied with more inhibitory effects on DNL enzymes than TG synthetases. QHD inhibits the nuclear XBP1s without affecting ChREBP and SREBP1. In QHD, chlorogenic acid, geniposide and polydatin inhibit lipogenesis initiated by XPB1s. ConclusionQHD probably decreases hepatic DNL by inhibiting XBP1s independent of SREBP1 and ChREBP. Chlorogenic acid, geniposide and polydatin are the potential responsible compounds.

Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

The Effect of Mycotoxins and Silymarin on Liver Lipidome of Mice with Non-Alcoholic Fatty Liver Disease.

Milk thistle-based dietary supplements have become increasingly popular. The extract from milk thistle (Silybum marianum) is often used for the treatment of liver diseases because of the presence of its active component, silymarin. However, the co-occurrence of toxic mycotoxins in these preparations is quite frequent as well. The objective of this study was to investigate the changes in composition of liver lipidome and other clinical characteristics of experimental mice fed by a high-fat methionine-choline deficient diet inducing non-alcoholic fatty liver disease. The mice were exposed to (i) silymarin, (ii) mycotoxins (trichothecenes, enniatins, beauvericin, and altertoxins) and (iii) both silymarin and mycotoxins, and results were compared to the controls. The liver tissue extracts were analyzed by ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry. Using tools of univariate and multivariate statistical analysis, we were able to identify 48 lipid species from the classes of diacylglycerols, triacylglycerols, free fatty acids, fatty acid esters of hydroxy fatty acids and phospholipids clearly reflecting the dysregulation of lipid metabolism upon exposure to mycotoxin and/or silymarin.

Profiling Carbohydrate Metabolism in Liver and Hepatocellular Carcinoma with [13C]-Glycerate Probes.

The interplay between glycolysis and gluconeogenesis is central to carbohydrate metabolism. Here, we describe novel methods to assess carbohydrate metabolism using [13C]-probes derived from glycerate, a molecule whose metabolic fate in mammals remains underexplored. Isotope-based studies were conducted via NMR and mass spectrometry analyses of freeze-clamped liver tissue extracts after [2,3-13C2]glycerate infusion. The ex vivo investigations were correlated with in vivo measurements using hyperpolarized [1-13C]glycerate. Application of [13C]glycerate to N-nitrosodiethylamine (DEN)-treated rats provided further assessments of intermediary carbohydrate metabolism in hepatocellular carcinoma. This method afforded direct analyses of control versus DEN tissues, and altered ratios of 13C metabolic products as well as unique glycolysis intermediates were observed in the DEN liver/tumor. Isotopomer studies showed increased glycerate uptake and altered carbohydrate metabolism in the DEN rats.

Proteomic consequences of the deletion of cytochrome P450 (CYP450) reductase in mice

Microsomal cytochrome P450 (CYP450) reductase enzymes play a major role in drug and xenobiotic metabolism. Mice which are deficient in hepatic CYP450 reductase serve as excellent models in understanding CYP450 drug metabolism and alterations in the underlying biology and function of these enzymes. A reversed-phase nano-bore UPLC-MS-based proteomic analysis, using an untargeted data independent approach (DIA), has been utilized for liver tissue extracts to evaluate differences between the proteomes of C57Bl6 wild type (WT) and hepatic P450 reductase mice (HRN™). Statistically curated, differentially expressed protein groups highlighted a variety of molecular and biological functions, including binding and catalytic related activities. Thus, elevations were seen for a number of CYP450 enzymes (Cyp2a5; Cyp2b10; Cyp2b19; Cyp2d26; Cyp2a5, Cyp2e1) in the liver extracts of HRN animals. In addition, the major urinary protein 2 (Mup2) was found to be present only in the livers of the HRN group, whilst enoyl-CoA hydratase domain-containing protein 2 (Echdc2) was similarly unique to the the WT livers.Pathway enrichment analysis of the WT liver data indicated perturbations of lipid and energy related pathways, which included bile acid biosynthesis, fatty acid omega oxidation and tricarboxylic acid (TCA) cycle as examples.

Alternariol exerts embryotoxic and immunotoxic effects on mouse blastocysts through ROS-mediated apoptotic processes.

Alternariol (AOH), a mycotoxin belonging to the genus Alternaria, has been shown to induce cytotoxicity, including apoptosis and cell cycle arrest, in several mammalian cell types. However, its effects on early-stage embryonic development require further investigation. Here, we have shown that AOH exerts embryotoxic effects on mouse blastocyst-stage embryos and long-term adverse effects on immunity in one-day-old newborn mice of the next generation. Significant apoptosis and decrease in total cell number, predominantly through loss of inner cell mass (ICM), and to a minor extent, trophectoderm (TE) cells, were observed in AOH-treated blastocysts. Moreover, AOH exerted detrimental effects on pre- and post-implantation embryo development potential and induced a decrease in fetal weight in in vitro development and embryo transfer assays. Injection of pregnant mice with AOH (1, 3 and 5mg/kg body weight/day) for 4days resulted in apoptosis of blastocyst-stage embryos and injurious effects on embryonic development from the zygote to blastocyst stage or embryo degradation and a further decrease in fetal weight. Furthermore, AOH exerted a long-term impact on the next generation, triggering a significant increase in total oxidative stress content and expression of genes encoding antioxidant proteins. Lower expression of CXCL1, IL-1β and IL-8 related to innate immunity was detected in liver tissue extracts obtained from one-day-old newborns of AOH-injected pregnant mice (5mg/kg body weight/day) relative to their non-treated counterparts. In addition, ROS served as an upstream regulator of AOH-triggered apoptotic processes and impairment of embryonic development. Our collective results highlight the potential of AOH as an embryotoxic and immunotoxic risk factor during embryo and infant development stages in mice.

NMR Analysis of Carboxylate Isotopomers of 13C-Metabolites by Chemoselective Derivatization with 15N-Cholamine.

A substantial fraction of common metabolites contains carboxyl functional groups. Their 13C isotopomer analysis by nuclear magnetic resonance (NMR) is hampered by the low sensitivity of the 13C nucleus, the slow longitudinal relaxation for the lack of an attached proton, and the relatively low chemical shift dispersion of carboxylates. Chemoselective (CS) derivatization is a means of tagging compounds in a complex mixture via a specific functional group. 15N1-cholamine has been shown to be a useful CS agent for carboxylates, producing a peptide bond that can be detected via 15N-attached H with high sensitivity in heteronuclear single quantum coherence experiments. Here, we report an improved method of derivatization and show how 13C-enrichment at the carboxylate and/or the adjacent carbon can be determined via one- and two-bond coupling of the carbons adjacent to the cholamine 15N atom in the derivatives. We have applied this method for the determination of 13C isotopomer distribution in the extracts of A549 cell culture and liver tissue from a patient-derived xenograft mouse.

Novel Insights into Mice Multi-Organ Metabolism upon Exposure to a Potential Anticancer Pd(II)-Agent

Pd(II)-compounds are presently regarded as promising anticancer drugs, as an alternative to Pt(II)-based drugs (e.g., cisplatin), which typically trigger severe side-effects and acquired resistance. Dinuclear Pd(II) complexes with biogenic polyamines such as spermine (Pd2Spm) have exhibited particularly beneficial cytotoxic properties, hence unveiling the importance of understanding their impact on organism metabolism. The present study reports the first nuclear magnetic resonance (NMR)-based metabolomics study to assess the in vivo impact of Pd2Spm on the metabolism of healthy mice, to identify metabolic markers with possible relation to biotoxicity/side-effects and their dynamics. The changes in the metabolic profiles of both aqueous and lipophilic extracts of mice kidney, liver, and breast tissues were evaluated, as a function of drug-exposure time, using cisplatin as a reference drug. A putative interpretation was advanced for the metabolic deviations specifically triggered by Pd2Spm, this compound generally inducing faster metabolic response and recovery to control levels for all organs tested, compared to cisplatin (except for kidney lipid metabolism). These results constitute encouraging preliminary metabolic data suggestive of potential lower negative effects of Pd2Spm administration.

The Application of a Nanomaterial Optical Fiber Biosensor Assay for Identification of Brucella Nomenspecies.

Bacteria in the genus Brucella are the cause of brucellosis in humans and many domestic and wild animals. A rapid and culture-free detection assay to detect Brucella in clinical samples would be highly valuable. Nanomaterial optical fiber biosensors (NOFS) are capable of recognizing DNA hybridization events or other analyte interactions with high specificity and sensitivity. Therefore, a NOFS assay was developed to detect Brucella DNA from cultures and in tissue samples from infected mice. An ionic self-assembled multilayer (ISAM) film was coupled to a long-period grating optical fiber, and a nucleotide probe complementary to the Brucella IS711 region and modified with biotin was bound to the ISAM by covalent conjugation. When the ISAM/probe duplex was exposed to lysate containing ≥100 killed cells of Brucella, or liver or spleen tissue extracts from Brucella-infected mice, substantial attenuation of light transmission occurred, whereas exposure of the complexed fiber to non-Brucella gram-negative bacteria or control tissue samples resulted in negligible attenuation of light transmission. Oligonucleotide probes specific for B. abortus, B. melitensis, and B. suis could also be used to detect and differentiate these three nomenspecies. In summary, the NOFS biosensor assay detected three nomenspecies of Brucella without the use of polymerase chain reaction within 30 min and could specifically detect low numbers of this bacterium in clinical samples.

Deuterium Depleted Water as an Adjuvant in Treatment ofCancer

For more than 20 years, since 1993, the Hungarian National Institute of Pharmacy and Nutrition has been conducting a research on the mechanisms of antitumor activity of light water (deuterium depleted water – DDW). Long-term randomized clinical trials in numerous treatment centers in Hungary in compliance with the Good Clinical Practice (GCP) indicate a high sensitivity of tumor cells to lower levels of deuterium (D) in the body. The replacement of the ordinary water (D)/H = 140 ppm) by the “light” one (D/H = 20-105 ppm) is accompanied by a decrease in the size of the tumor, gradual necrosis of tumor cells, an increase in the median survival of cancer patients (prostate cancer, breast cancer, lung cancer, etc.). The content of deuterium in the body at the level of 10-12 mmol is an order of magnitude higher compared to essential trace elements, which makes it possible to predict its key role in biochemical processes. Indeed, mass-spectrometric analysis shows that deuterium bioaccumulation in the human body depends on its phenotype. In contrast, when drinking water is replaced with DDW, the deuterium content in aqueous extracts of biological blood samples, muscle and liver tissues of laboratory animals goes down. The use of DDW as a prophylactic agent and many experimental results obtained in vitro and in vivo in China, Japan, the USA, Romania, and the Russian Federation make it possible to recommend DDW for use as a safe additional agent in the treatment of cancer.

Enrichment of intact phosphoproteins using immobilized titanium(IV) affinity chromatography microspheres

AbstractApproximately a third of eukaryotic proteins are estimated to be phosphorylated, and the protein phosphorylation is significant in regulating almost all aspects of cell life. Due to low stoichiometry of phosphorylation, efficient enrichment is often an essential step in phosphoproteomics study. Over the decades, lots of enrichment materials have been successfully developed and widely used for intact phosphoproeins. One of these materials is immobilized titanium(IV) affinity chromatography (Ti4+‐IMAC) microspheres, which has been widely studied and used for phosphopeptides enrichment. Application of Ti4+‐IMAC for intact phosphoprotein enrichment has also proven possible, where intact phosphoproteins (α‐casein and β‐casein) were successfully enrichment out of their mixture with BSA (1:1:10, w/w). Here we report our detailed investigation of intact phosphoprotein enrichment using Ti4+‐IMAC. With BSA and standard intact phosphoprotein β‐casein, selectivity with a wide range of mixing ratios of 1:1, 1:100, 1:500, 1:1000 and 1:2000 (β‐casein:BSA, w/w) and binding capacity were investigated; the enrichment capability of Ti4+‐IMAC was further investigated with real biological systems, including non‐fat milk, egg white and swine liver tissue extract. The results show that Ti4+‐IMAC is a promising material for intact phosphoprotein enrichment.

Alteration in the liver metabolome of rats with metabolic syndrome after treatment with Hydroxytyrosol. A Mass Spectrometry And Nuclear Magnetic Resonance - based metabolomics study

Metabolic syndrome (MetS) represents a group of abnormalities that enhances the risk for cardiovascular disease, diabetes and stroke. The Mediterranean diet seems to be an important dietary pattern, which reduces the incidence of MetS. Hydroxytyrosol (HT) - a simple phenol found in olive oil - has received increased attention for its antioxidant activity. Recently, the European Foods Safety Authority (EFSA) claimed that dietary consumption of HT exhibits a protective role against cardiovascular disease. In this study, an experimental protocol has been setup, including isolated HT administration in a diet induced model of MetS in young Wistar rats, in order to find out whether HT has a protective effect against MetS. Rats were randomly divided into two groups nurtured by high-carbohydrate high-fat (H) (MetS inducing diet) and high-carbohydrate high-fat + HT (HHT). HT (20mg/kg/d oral gavage, water vehicle) was administered for 8 weeks on the basal diet. Previous pharmacological evaluation of HT showed that hepatic steatosis was reduced and the inflammatory cells into the liver were infiltrated. These indicate that HT shows bioactivity against metabolic syndrome. Therefore, the metabolomics evaluation of liver extracts would indicate the putative biochemical mechanisms of HT activity. Thus, the extracts of liver tissues were analyzed using Ultra Performance Liquid Chromatography – High Resolution Mass Spectrometry (UPLC-HRMS, Orbitrap Discovery) and Nuclear Magnetic Resonance (NMR) spectroscopy (Bruker Avance III 600MHz). Multivariate analysis was performed in order to gain insight on the metabolic effects of HT administration on the liver metabolome. Normalization employing multiple internal standards and Quality Control–based Robust LOESS (LOcally Estimated Scatterplot Smoothing) Signal Correction algorithm (QC-RLSC) was added in the processing pipeline to enhance the reliability of metabolomic analysis by reducing unwanted information. Experimentally, HHT rats were clearly distinguished from H in PLS-DA, showing differences in the liver metabolome between the groups and specific biomarkers were determined supporting the pharmacological findings. More specifically, HT has shown to be effective towards the mobilization of lipids as various lipid classes being differentially regulated between the H and HHT groups. Interestingly branched fatty acid esters of hydroxy oleic acids (OAHSA) lipids have been shown to be up regulated to the HHT group, denoting the alleviation of the MetS to the animals administered with HT.

Multi-omic network-based interrogation of rat liver metabolism following gastric bypass surgery featuring SWATH proteomics.

Morbidly obese patients often elect for Roux-en-Y gastric bypass (RYGB), a form of bariatric surgery that triggers a remarkable 30% reduction in excess body weight and reversal of insulin resistance for those who are type II diabetic. A more complete understanding of the underlying molecular mechanisms that drive the complex metabolic reprogramming post-RYGB could lead to innovative non-invasive therapeutics that mimic the beneficial effects of the surgery, namely weight loss, achievement of glycemic control, or reversal of non-alcoholic steatohepatitis (NASH). To facilitate these discoveries, we hereby demonstrate the first multi-omic interrogation of a rodent RYGB model to reveal tissue-specific pathway modules implicated in the control of body weight regulation and energy homeostasis. In this study, we focus on and evaluate liver metabolism three months following RYGB in rats using both SWATH proteomics, a burgeoning label free approach using high resolution mass spectrometry to quantify protein levels in biological samples, as well as MRM metabolomics. The SWATH analysis enabled the quantification of 1378 proteins in liver tissue extracts, of which we report the significant down-regulation of Thrsp and Acot13 in RYGB as putative targets of lipid metabolism for weight loss. Furthermore, we develop a computational graph-based metabolic network module detection algorithm for the discovery of non-canonical pathways, or sub-networks, enriched with significantly elevated or depleted metabolites and proteins in RYGB-treated rat livers. The analysis revealed a network connection between the depleted protein Baat and the depleted metabolite taurine, corroborating the clinical observation that taurine-conjugated bile acid levels are perturbed post-RYGB.

Osteopontin in hepatocellular carcinoma: A possible biomarker for diagnosis and follow-up

Recently osteopontin (OPN), a protein of the extracellular matrix, has generated in hepatocellular carcinoma (HCC) a significant interest as a prognostic factor. Aim of this study was to confirm, in liver tissues of subjects with HCV-positive HCC undergoing liver transplantation (RL, n=10) and of donors (DL, n=14), the increase of OPN plasma and tissue concentration, the OPN splicing isoforms expression profiling together with those of thrombin, and to evaluate a possible association between OPN measurements. Their association with Notch-1, IV-Collagen-7s domain, IL-6 and TNF-α were also evaluated. Real-Time PCR experiments and immunometric assay were performed. mRNA expression resulted higher in RL than in DL for all analyzed genes and several correlations were found between them. The more relevant association were between OPN-a and OPN-b (p<0.0001), between thrombin and OPN-a (p=0.007), between 7s-collagen and OPN isoforms (p<0.05) and between Notch-1 with OPN-c (p=0.004). Both OPN plasma and liver tissue extract concentrations were assessed confirming the trend observed at the mRNA level. An important association was found between OPN plasma and protein (p<0.0001, r=0.96) even splitting patients in DL (p<0.0001, r=0.93) and RL (p<0.0001, r=0.96). A reduction of OPN plasma levels was found at 6months after transplantation. Considering MELD score as liver disease severity, the mRNA expression of our markers as well as of OPN plasma and tissue concentrations resulted increased as a function of clinical severity. Our results might be considered a useful starting point to validate OPN as a prognostic and diagnostic marker of HCC.

Environmental Chemicals Modulate Polar Bear (Ursus maritimus) Peroxisome Proliferator-Activated Receptor Gamma (PPARG) and Adipogenesis in Vitro.

We studied interactions between polar bear peroxisome proliferator-activated receptor gamma (pbPPARG) and selected compounds using a luciferase reporter assay and predictions through molecular docking. Furthermore, we studied adipogenesis by liver and adipose tissue extracts from a polar bear and three synthetic mixtures of contaminants in murine 3T3-L1 preadipocytes and polar bear adipose tissue-derived stem cells (pbASCs). PCB153 and p,p'-DDE antagonized pbPPARG, although their predicted receptor-ligand affinity was weak. PBDEs, tetrabromobisphenol A, and PCB170 had a weak agonistic effect on pbPPARG, while hexabromocyclododecane, bisphenol A, oxychlordane, and endosulfan were weak antagonists. pbPPARG-mediated luciferase activity was suppressed by synthetic contaminant mixtures reflecting levels measured in polar bear adipose tissue, as were transcript levels of PPARG and the PPARG target gene fatty acid binding protein 4 (FABP4) in pbASCs. Contaminant extracts from polar bear tissues enhanced triglyceride accumulation in murine 3T3-L1 cells and pbASCs, whereas triglyceride accumulation was not affected by the synthetic mixtures. Chemical characterization of extracts using nontarget methods revealed presence of exogenous compounds that have previously been reported to induce adipogenesis. These compounds included phthalates, tonalide, and nonylphenol. In conclusion, major legacy contaminants in polar bear adipose tissue exert antagonistic effects on PPARG, but adipogenesis by a mixture containing emerging compounds may be enhanced through PPARG or other pathways.

Simultaneous Analysis of Major Coenzymes of Cellular Redox Reactions and Energy Using ex Vivo (1)H NMR Spectroscopy.

Coenzymes of cellular redox reactions and cellular energy mediate biochemical reactions fundamental to the functioning of all living cells. Despite their immense interest, no simple method exists to gain insights into their cellular concentrations in a single step. We show that a simple 1H NMR experiment can simultaneously measure oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+ and NADH), oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH), and adenosine triphosphate (ATP) and its precursors, adenosine diphosphate (ADP) and adenosine monophosphate (AMP), using mouse heart, kidney, brain, liver, and skeletal muscle tissue extracts as examples. Combining 1D/2D NMR experiments, chemical shift libraries, and authentic compound data, reliable peak identities for these coenzymes have been established. To assess this methodology, cardiac NADH and NAD+ ratios/pool sizes were measured using mouse models with a cardiac-specific knockout of the mitochondrial Complex I Ndufs4 gene (cKO) and cardiac-specific overexpression of nicotinamide phosphoribosyltransferase (cNAMPT) as examples. Sensitivity of NAD+ and NADH to cKO or cNAMPT was observed, as anticipated. Time-dependent investigations showed that the levels of NADH and NADPH diminish by up to ∼50% within 24 h; concomitantly, NAD+ and NADP+ increase proportionately; however, degassing the sample and flushing the sample tubes with helium gas halted such changes. The analysis protocol along with the annotated characteristic fingerprints for each coenzyme is provided for easy identification and absolute quantification using a single internal reference for routine use. The ability to visualize the ubiquitous coenzymes fundamental to cellular functions, simultaneously and reliably, offers a new avenue to interrogate the mechanistic details of cellular function in health and disease.