1 The present results support the link between BT and systemic circulatory dysfunction find more in cirrhosis, suggesting that intestinal decontamination could enhance the hemodynamic effects of terlipressin and contribute to a decrease in rebleeding events in patients with variceal bleeding taking antibiotic prophylaxis.7 Georgios N. Kalambokis M.D.*, Athanasia Mouzaki M.D., Ph.D.‡, Maria Rodi M.D.‡, Konstantinos Pappas M.D.†, Epameinondas V. Tsianos M.D., Ph.D.*, * First Division of Internal Medicine and Hepato-Gastroenterology Unit, Ioannina, Greece, † Department of Cardiology, Medical School of Ioannina, Ioannina, Greece, ‡ Division of Hematology, Department of

Internal Medicine, Medical School, University of Patras, Patras, Greece. “
“We read with great interest the article entitled “Toll-like receptor 4 is involved in the development

of fructose-induced hepatic steatosis in mice”, published in a recent issue of HEPATOLOGY.1 In this study, Spruss et al. verified the hypothesis that Toll-like receptor 4 (TLR-4) may play a central role in the onset of fructose-induced nonalcoholic fatty liver disease (NAFLD). To this aim, the authors used wild-type (C3H/HouJ) mice and TLR-4 mutant (C3H/HeJ) mice, both fed plain water or 30% fructose-enriched solution for 8 weeks. As already described by other studies,2, 3 chronic intake of 30% fructose solution leads to hepatic steatosis and some features of metabolic syndrome in wild-type animals, including the increase of body and liver weight, hepatic triglyceride levels, and www.selleckchem.com/products/BI6727-Volasertib.html plasma levels of alanine aminotransferase (ALT). Interestingly, TLR-4 mutants fed water presented only a weak decrease of SB-3CT liver weight and hepatic triglycerides with respect to wild-type animals fed water, and the enrichment with fructose exclusively caused the restoration of the significantly increased levels of these two parameters. These results clearly suggest that the presence of TLR-4 is essential to explain liver damage, body weight gain, and ALT impairment due to the fructose intake. Furthermore, the

authors found that plasma endotoxin levels were significantly increased both in wild-type and mutant mice fed chronically with a 30% fructose solution, in comparison to water-fed controls. The role of fructose in NAFLD development was not entirely unknown to researchers. In particular, a recent work4 demonstrates that patients with NAFLD have a significantly greater consumption of fructose than controls, and an increased hepatic expression of fructokinase messenger RNA. Although the role of TLR-4 in carbohydrate-dependent NAFLD has been only recently suggested by Thuy and colleagues,5 they have pinpointed one of the potential mechanisms through which fructose could participate in NAFLD development and progression in humans: a carbohydrate-rich diet may produce ethanol when intestinal stasis favors bacterial overgrowth in the upper parts of the gastrointestinal tract.

1 The present results support the link between BT and systemic circulatory dysfunction check details in cirrhosis, suggesting that intestinal decontamination could enhance the hemodynamic effects of terlipressin and contribute to a decrease in rebleeding events in patients with variceal bleeding taking antibiotic prophylaxis.7 Georgios N. Kalambokis M.D.*, Athanasia Mouzaki M.D., Ph.D.‡, Maria Rodi M.D.‡, Konstantinos Pappas M.D.†, Epameinondas V. Tsianos M.D., Ph.D.*, * First Division of Internal Medicine and Hepato-Gastroenterology Unit, Ioannina, Greece, † Department of Cardiology, Medical School of Ioannina, Ioannina, Greece, ‡ Division of Hematology, Department of

Internal Medicine, Medical School, University of Patras, Patras, Greece. “
“We read with great interest the article entitled “Toll-like receptor 4 is involved in the development

of fructose-induced hepatic steatosis in mice”, published in a recent issue of HEPATOLOGY.1 In this study, Spruss et al. verified the hypothesis that Toll-like receptor 4 (TLR-4) may play a central role in the onset of fructose-induced nonalcoholic fatty liver disease (NAFLD). To this aim, the authors used wild-type (C3H/HouJ) mice and TLR-4 mutant (C3H/HeJ) mice, both fed plain water or 30% fructose-enriched solution for 8 weeks. As already described by other studies,2, 3 chronic intake of 30% fructose solution leads to hepatic steatosis and some features of metabolic syndrome in wild-type animals, including the increase of body and liver weight, hepatic triglyceride levels, and this website plasma levels of alanine aminotransferase (ALT). Interestingly, TLR-4 mutants fed water presented only a weak decrease of Astemizole liver weight and hepatic triglycerides with respect to wild-type animals fed water, and the enrichment with fructose exclusively caused the restoration of the significantly increased levels of these two parameters. These results clearly suggest that the presence of TLR-4 is essential to explain liver damage, body weight gain, and ALT impairment due to the fructose intake. Furthermore, the

authors found that plasma endotoxin levels were significantly increased both in wild-type and mutant mice fed chronically with a 30% fructose solution, in comparison to water-fed controls. The role of fructose in NAFLD development was not entirely unknown to researchers. In particular, a recent work4 demonstrates that patients with NAFLD have a significantly greater consumption of fructose than controls, and an increased hepatic expression of fructokinase messenger RNA. Although the role of TLR-4 in carbohydrate-dependent NAFLD has been only recently suggested by Thuy and colleagues,5 they have pinpointed one of the potential mechanisms through which fructose could participate in NAFLD development and progression in humans: a carbohydrate-rich diet may produce ethanol when intestinal stasis favors bacterial overgrowth in the upper parts of the gastrointestinal tract.

In the study, we aim to detail anti-metastatic effects and molecular mechanisms of baicalein on HCC cells. Methods: The anti-metastatic effect of baicalein was determined using wound healing assay and transwell invasive model. selleck The expression of MMP-2, MMP-9 and u-PA mRNA and protein in MHCC97H cells was determined by quantitative RT-PCR and western blot. The activity of MMP-2, MMP-9 and u-PA was determined by zymography.

The expression of TIMP-1 and TIMP-2 was determined by Western blot. The expression of MEK1 and ERK1/2 was measured by Western blot. Expression Plasmids (pcDNA3.1(+)-MEK1)were constructed and transfected Results: The migration and invasion of MHCC97H cells were markedly suppressed by baicalein in a dose-dependent manner. MMP-2, -9 and u-PA have been considered to be important in cancer cell invasion and metastasis because they play important PD0332991 ic50 roles in the degradation of the ECM. In our study, we found that treatment with baicalein on MHCC97H for 24h resulted in a decrease in MMP-2, -9 and u-PA expression, as well as proteinase activity. Meanwhile, the expression

of TIMP-1 and TIMP-2 were increased in a dose-dependent fashion. Thus, the anti-metastatic effect of baicalein on MHCC97H cells is correlated to proteinases and their inhibitors. Moreover, ERK pathway is closely correlated with synthesis of proteinases and their inhibitors. In our study, we found that

baicalein treatment decreased the levels of phosphorylation of MEK1 and ERK1/2. Overexpression of MEK1 partially blocked anti-metastatic effects of baicalein. Conclusion: Baicalein preferentially inhibits HCC invasion through inhibition of ERK pathway and by regulating synthesis of proteinases and their inhibitors. Acknowledgements: Venetoclax order Supported by a grant from Program for changjiang Scholars and Innovative Research Team in University (PCSIRT: 1171). Key Word(s): 1. Baicalein; 2. HCC; 3. ERK pathway; 4. ECM; Presenting Author: WEILI HUANG Additional Authors: XIAOHUI GUAN Corresponding Author: WEILI HUANG Affiliations: Department of Digestion, Affiliated Hospital of Beihua University Objective: To investigate the relationship between the contents of β-catenin in cell nucleus of gastric cancer tissue and sFas in blood plasma and the degree of hyperplasia and infiltration of gastric cancer cells.

The complement component 2(C2) p.Glu318Asp mutation model was built on the crystal structure reported by Milder et AZD3965 al. (PDB ID: 2I6Q).14 It is not feasible to model the mutation on transmembrane protein 2 (TMEM2) at present, as very little is known of the structure of TMEM2 or its homologous proteins. TMEM2 p.Ser1254Asn was found to be associated with CHB, but with no indications of immunological function of the wildtype protein. We therefore performed expression studies. Immunohistochemistry

was performed on formalin-fixed and paraffin-embedded healthy liver tissues from 12 individuals, with polyclonal rabbit antihuman TMEM2 antibody (Aviva Systems Biology, San Diego, CA). The sections were incubated with the first antibody at 1:40-1:160 dilution at 4°C overnight. The second, peroxidase-labeled goat antirabbit/mouse antibody (Dako K5007, Carpinteria, CA) was applied to the sections for 30 minutes at 37°C and the sections were developed with Diaminobenzidine (DAB) solution. The staining was replicated in healthy liver tissues from another six subjects using rabbit polyclonal

antibody to human TMEM2 from a different company (Jin Tiancheng, Beijing, China). Negative controls were performed with phosphate-buffered saline (PBS) replacing the first antibody preparation. Real-time PCR was performed in liver tissues from three CHB patients and normal GDC-0199 liver tissues from three subjects who underwent surgical ablation of hemangioma Succinyl-CoA in the liver. The latter had normal liver function (normal ALT, aspartate aminotransferase [AST], and total bilirubin) and were negative for HBsAg and

HBeAg. Total RNA was extracted. Real-time PCR for TMEM2 was performed using the primers 5′-GGAGATATGCTCCGTCTGACC-3′ and 5′-CATCTGACTTGCCATACAAGGT-3′ and 5′-CCA TCTTCCAGGAGCGAGA-3′ and 5′-TGGTTCACA CCCATGACGAA-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Real-time PCR was also performed with the same primers in two cell lines (1) HepG2.2.15 containing the complete HBV genome and capable of stable HBV expression and replication in the culture system15 and (2) a non-HBV-containing HepG2 cell line (ATCC, Manassas, VA). The two cell lines were maintained in the exponential growth phase in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Carlsbad, CA), supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1% (w/v) streptomycin. The mean and standard error (SE) were calculated from three independent experiments. Western blotting was also performed on the two cell lines. After lysis of the harvested HepG2 and HepG2.2.15 cells and gel electrophoresis, the rabbit antihuman TMEM2 antibody (Aviva Systems Biology) and mouse antihuman GAPDH monoclonal antibody (Kang Chen Biotech, Shanghai, China) were applied for detection of the proteins. Apart from meeting all the criteria described in the “candidate selection” section above, TMEM2 p.

The abundance of Srx protein was not affected by exposure of any of these cells to 100 mM ethanol for 18 hours, whereas the protein levels of Srx were increased slightly in E47 cells (Supporting Information Fig. 2B). Similar treatment of primary mouse hepatocytes also showed no significant effect of ethanol on Srx and CYP2E1 expression (Supporting Information Fig. 2C). It was shown previously that HepG2 cells resist the adverse effect of

ethanol because the cells contain a very low amount of CYP2E1.39 Chronic ethanol feeding of mice was previously shown to increase Nrf2 expression ≈2-fold in the liver.6 The role of Nrf2 in ethanol-induced Autophagy inhibitor Srx expression in the liver was investigated with the use of Nrf2-deficient mice. The amount of Srx protein in the liver was increased ≈9-fold by ethanol feeding in Nrf2+/+ mice but only ≈2-fold in Nrf2−/− mice (Fig. 2B,C). Ethanol feeding also induced similar changes in the hepatic abundance of Srx mRNA (Fig. 2D). In addition, the basal level of Srx mRNA was reduced by ≈50% in Nrf2−/− mice compared with that in Nrf2+/+ animals (Fig. 2D). These results suggested that the Nrf2-ARE pathway plays a key role in the induction of Srx in the liver of ethanol-fed mice. The observation that ethanol still induced an ≈2-fold increase in Srx expression in the liver of Nrf2−/− mice, however, suggested that the AP-1-ARE pathway might also Fostamatinib contribute to this effect. Srx is responsible for

reduction of the Florfenicol hyperoxidized forms of 2-Cys Prx enzymes (Prx I to IV) generated during elimination of peroxides. Hyperoxidized 2-Cys Prxs can be detected by immunoblot analysis with antibodies generated in response to a sulfonylated peptide modeled on the conserved peroxidatic cysteine residue (CP). Given that the amino acid sequences surrounding CP are identical for 2-Cys Prx enzymes, the antibodies react with all of these hyperoxidized proteins.13 To investigate the role of Srx in ethanol-fed mice, we generated Srx−/− mice (Supporting Information Fig. 3). Srx+/+ and

Srx−/− mice were then subjected to chronic ethanol feeding, after which liver proteins were subjected to immunoblot analysis with antibodies to Srx, to sulfinic forms of 2-Cys Prxs (Prx-SO2), and to Prx I to IV (Fig. 3). Prx I and Prx II, which differ by only one amino acid residue in size, cannot be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the molecular sizes of Prx I/II, III, and IV differ sufficiently to allow such separation. In NIH 3T3 cells that had been exposed to 100 μM H2O2 for 10 minutes the cytosolic enzymes Prx I and II as well as the mitochondrial enzyme Prx III were found to be completely hyperoxidized (see below), whereas hyperoxidation of the ER-localized Prx IV was not detected (not shown). Extracts of the H2O2-treated cells are included as a standard for Prx I/II-SO2 and Prx III-SO2 in Figure 3.

In mouse embryonic liver, Gata4 is expressed in the endodermal hepatic bud and in the adjacent mesenchyme of the septum transversum. Previous studies have shown that Gata4 inactivation impairs liver formation. However,

whether these defects are caused by loss of Gata4 in the hepatic endoderm or in the septum transversum mesenchyme remains MI-503 to be determined. In this study, we have investigated the role of mesenchymal GATA4 activity in liver formation. We have conditionally inactivated Gata4 in the septum transversum mesenchyme and its derivatives by using Cre/loxP technology. We have generated a mouse transgenic Cre line, in which expression of Cre recombinase is controlled by a previously identified distal Gata4 enhancer. Conditional inactivation of Gata4 in hepatic mesenchymal cells led to embryonic lethality around mouse embryonic stage 13.5, likely as a consequence of fetal anemia. Gata4 knockout fetal livers exhibited reduced size, advanced fibrosis, accumulation of extracellular matrix components and hepatic stellate cell (HSC) activation. Haploinsufficiency

of Gata4 accelerated CCl4-induced liver fibrosis in adult mice. Moreover, Gata4 expression was dramatically reduced in advanced hepatic fibrosis and cirrhosis in humans. Conclusions: Our data demonstrate that mesenchymal GATA4 activity regulates HSC activation and inhibits the liver fibrogenic process. (Hepatology 2014;59:2358–2370) “
“Aim:  To investigate learn more XPNPEP1 rs17095355 polymorphism in biliary atresia (BA) patients and to determine whether there is an association between XPNPEP1 gene polymorphism and susceptibility to BA Dimethyl sulfoxide in a Thai population. Methods:  A total of 124 cases of BA and 114 controls were genotyped for XPNPEP1 rs17095355 polymorphism. The XPNPEP1 rs17095355 C/T genotype was determined by polymerase chain reaction (PCR) and direct sequencing. Allele and genotype frequencies were established by directed counting from the sequences. Results:  Genotype distributions for the XPNPEP1

rs17095355 polymorphism tested were in Hardy–Weinberg equilibrium for both control and study groups. There were no significant differences in genotype and allele frequencies of the single nucleotide polymorphism between controls and Thai children with BA. Genotype frequencies of rs17095355 of T/T in BA were higher than those of controls (34.68% and 16.67%, P < 0.002). Also, the T allele frequencies of BA were higher than those of controls (56.85% and 42.98%, P < 0.003). Conclusion:  The association between XPNPEP1 rs17095355 polymorphism and BA has been demonstrated, particularly with the T allele. We hypothesize that the XPNPEP1 rs17095355 polymorphism confers increased susceptibility to the disease. "
“Barrett’s esophagus is an acquired metaplastic abnormality in which the normal stratified squamous epithelium lining of the esophagus is replaced by an intestinal-like columnar epithelium.

With genotypes 4, 5, and 6 representing a substantial proportion of all HCV infections worldwide and with 4a and 6a also being poorly responsive to conventional therapy,2 it is crucial that these genotypes are considered in the future development of PIs and other antiviral therapy. Although not as potent as BILN 2061, telaprevir has been shown to be clinically effective in genotype 1-infected patients.12 In

a phase IIa clinical trial, telaprevir also demonstrated substantial activity in genotype 2-infected patients but only limited efficacy in genotypes 3- and 4-infected individuals, for whom as a result treatment was stopped.13, 14 As Talazoparib datasheet demonstrated by our in vitro studies, telaprevir also shows considerable differences in MK-1775 supplier potency against different genotypes, observations that highlight again the potential value of evaluating PIs on all genotypes

before clinical assessment. We found that genotypes 1b and 6a were the most susceptible to telaprevir, followed by 2a, then 3a, and genotypes 4a and 5a being the most resistant. Based on the in vitro findings, genotype 6a- but not 4a- and 5a-infected patients might therefore be effectively treated by this antiviral in the future. However, in this specific case where relatively smaller genotype-associated differences in IC50 values for telaprevir have been found, it is necessary to investigate the extent of within-subtype variability in susceptibility and whether this might have a significant impact on clinical effectiveness. For example, in previous studies, catalytic efficiencies within a subtype were shown to vary widely (by up to 7-fold), especially within genotypes 1a and 1b.30, 31 We have shown that different isolates of genotype 3a exhibited at least 3-fold variability in IC50 values (130 nM to 310 nM) against BILN 2061, attributable to naturally occurring amino acid changes in the protease domain of NS3.16 These strain-associated differences may indeed account for the discrepancies between genotype 3 and 4 susceptibilities in the in vitro system with clinical susceptibility data.13, 14 However, the chimera model correctly reports

their much poorer response compared to genotype 1 and 2. The rapid selection of drug-resistant genetic variants is a major problem substantially limiting the effectiveness of antiviral therapy for HCV.21 Mathematical modeling Histidine ammonia-lyase has suggested that all possible single- and double-mutant viruses already preexist before treatment32 and can be rapidly selected at the start of antiviral therapy. Identification of potential resistance mutations within the individual genotypes towards the different PIs is crucial to preidentify individuals with preexisting resistant variants and adjust treatment options accordingly. We induced resistance mutations in vitro through passaging the intra- and intergenotypic recombinants under subinhibitory concentrations of PIs. Several new potential resistance loci were identified (Fig. 4; Tables 2, 3).

Gender- and age-matched C57/BL6 wild-type (WT) and ATGL KO littermates were intraperitoneally injected with 1 mg/kg body weight of a 0.1-mg/mL suspension of tunicamycin (TM) in saline. Mice were reinjected after 24 hours. At 48 hours after the starting point, mice were killed by cervical dislocation. The experimental protocols were approved by the local animal care and use committees according to criteria outlined in the Guide for

the Care and Use of Laboratory Animals prepared by the U.S. National Academy of Sciences (National Institutes of Health publication 86-23, revised 1985). The animals were kindly provided by Rudolf Zechner from the Institute of Molecular Biosciences at Karl-Franzens University (Graz, Austria) Ceritinib datasheet and were generated as described previously.26 Animals were fed a standard rodent chow and were housed in a controlled environment with 12-hour light-dark cycles. TM, sodium oleate, and sodium palmitate were from Sigma-Aldrich (Vienna, Austria). Enzymatic assays were used to

measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), Veliparib price alkaline phosphatase (ALP), cholesterol (CHOL), TGs (Roche Diagnostics, Mannheim, Germany), and free FAs (Wako Chemical, Neuss, Germany). Lipoprotein subfractions were determined by quantitative agarose gel electrophoresis (Helena Biosciences, Gateshead, UK). For conventional light microscopy, livers were

fixed in 4% neutral buffered formaldehyde solution for 24 hours, embedded in paraffin, and stained with hematoxylin and eosin (H&E) or Sirius Red. Frozen tissue was embedded in Tissue Tec (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands), and sections of Glutamate dehydrogenase 2 μm were stained with Oil Red O. Double immunofluorescence staining for cleaved caspase-3 and cytokeratin 18 was performed as described previously.30 RNA isolation, complementary DNA synthesis, and real-time polymerase chain reaction (PCR) were performed as described previously.31 All messenger RNA (mRNA) expression data were normalized to 36b4. Oligonucleotide sequences are available upon request. Hepa1.6 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium and knockdown for ATGL was performed by using a lentivirus containing a short hairpin RNA against ATGL, as described in the Supporting Materials and Methods. Hepatic nuclei were extracted with the NE-PER Nuclear and Cytoplasmic Extraction kit from Pierce Biotechnology (Rockford, IL). Srebp1c protein levels were determined using the polyclonal antibodiy Srebp-1(K10), sc-367 (Santa Cruz Biotechnology, Santa Cruz, CA).

PHILIPPE HALFON, PHARM, M.D., PH.D. “
“A 71-year-old woman was referred for a second opinion before hospice with progressive abdominal pain, fullness, diarrhea, and weight loss. A workup revealed ascites and esophageal varices. Imaging selleckchem showed seven liver lesions that were suspicious for hepatocellular carcinoma (HCC) on a computed tomography scan (Fig. 1A), and follow-up magnetic resonance imaging revealed arterial enhancement followed by washout. A tissue sample was compatible with well-differentiated HCC (CD34 and glutamine synthetase positivity, reticulin loss, and isolated vessels); the background liver

revealed hepatoportal sclerosis without cirrhosis (Fig. 1B). A further review of the abdominal scan revealed a dilated inferior mesenteric vein (IMV) due to an arteriovenous malformation (AVM), which was confirmed by angiography (Fig. 1C). There was no evidence of trauma or prior surgery. There was no endoscopic evidence of ischemia or a superficial AVM in the terminal ileum or ascending selleck chemicals llc colon, and biopsies were normal. She underwent transhepatic mesenteric

venous coil embolization, which reduced the IMV flow and the main portal venous pressure from 46 to 26 mm Hg. Shortly after the procedure, there was significant improvement in her diarrhea and abdominal pain. Four months later, the ascites had fully resolved, and she had gained weight. Furthermore, abdominal imaging demonstrated complete resolution of the hepatic lesions (Fig. 1D). AVM arteriovenous malformation HCC hepatocellular carcinoma IMV inferior mesenteric vein. This is the first known case in which an intra-abdominal AVM produced (1) chronic intestinal ischemia and diarrhea from arteriovenous shunting of blood; (2) noncirrhotic, presinusoidal portal hypertension with varices and ascites; and (3) multiple hepatic nodules suspicious

for HCC (all of which completely resolved Reverse transcriptase after venous embolization). Splanchnic AVMs commonly involve the hepatic or splenic artery, but IMV involvement is rare.1 Mesenteric AVMs alter vascular flow, reduce the distal arterial pressure, and increase the proximal venous pressure.2 This bypasses the capillary bed and induces a form of mesenteric steal syndrome, which results in abdominal pain, weight loss, diarrhea, and nonocclusive ischemic colitis. Several reports describe inferior mesenteric arteriovenous fistulas resulting in clinically significant arteriovenous shunting.3–5 The symptoms correlate with the amount of blood shunted and the length of time for which the malformation has been present. Hyperdynamic flow from AVMs can also result in presinusoidal portal hypertension. Ascites, varices, and splenomegaly are well-described complications of mesenteric AVMs,1, 6 and arterialization of the portal venous system can significantly increase hepatic blood inflow.

26 Recently, PBMs have been used to define the DNA-binding specificity of large classes of TFs27, 28 and have been shown to correlate well with gel shift results.29 Whereas as others have pioneered the technology using the DNA-binding domain (DBD) of TFs purified from bacteria, here we adapt the PBM technology to more closely approximate physiological conditions. Because HNF4α has a very strong dimerization domain outside of the DBD and a very low affinity for DNA when expressed in bacteria,14, 30, 31 we ectopically

expressed full-length, native HNF4α in COS-7 cells and prepared minimally processed nuclear extracts (Fig. 1B) that we then applied Selleck Tyrosine Kinase Inhibitor Library directly to a PBM specifically designed for HNF4α (Fig. 1C,D). The PBM was developed with a highly specific antibody to the C-terminus of HNF4α (Supporting Fig. 1), allowing us to examine a completely native TF. The full-length HNF4α protein selleck screening library in the crude extracts yielded an excellent signal with a range of intensities, whereas extracts from mock-transfected cells yielded no reproducible signals (Fig. 1E). We compared two species (rat and human) and two isoforms of HNF4α (HNF4α2 and HNF4α8), as well as antibodies that recognized different regions of HNF4α (Fig. 2A). There was an excellent correlation between replicate arrays in the first-generation

PBM (PBM1) using crude nuclear extracts, regardless of antibody used (R2 = 0.78), and results with affinity-purified protein were very similar to those with crude extracts (R2 = 0.68) (Fig. 2B). In a second generation of the PBM (PBM2), different HNF4α isoforms (HNF4α2 versus HNF4α8) and species (human versus rat) also produced excellent correlations (R2 > 0.9), indicating that these isoform and species differences do not influence the binding of HNF4α to DNA. This is not surprising considering that the DBD is identical in these constructs (Fig. 2A). PBM1 identified ∼500 new HNF4α binding sequences with the DR1-derived sequences exhibiting the best binding affinities relative to negative controls 5-FU mouse (P

< 8.274 × 10−12) (Fig. 3A ). Sequences derived from ChIP-chip analysis bound roughly as well as the DR1 variants. In PBM2, an additional ∼1000 novel sequences that strongly bind HNF4α were identified, including sequences identified by SVM1. The signal-to-noise ratio (literature-derived versus random sites) was also significantly improved in PBM2 due to optimization of the binding conditions (P < 2.6 × 10−11 versus P < 2.6 × 10−16, respectively, using the Student t test) (Fig. 3B). The PBM2 results also correlated very well with gel shift results (Fig. 3C). Additionally, SVM2 derived from PBM2 predicted binding sequences with a high degree of accuracy (R2 = 0.76) (Fig. 3D). Even though position weight matrices (PWMs) do not capture the interdependence between the positions in a motif as do PBMs and SVMs, they are useful for describing motifs.