At present, FLD, which is typically diagnosed by imaging, is highly prevalent (∼27% urban population) in China and is mainly related to obesity and metabolic syndrome (MetS). However, the percentage of alcoholic liver disease (ALD) among patients with chronic liver diseases in clinic is increasing as well, and a synergetic effect exists between heavy alcohol drinking and obesity in ALD. Prevalence figures reveal regional variations, with a median prevalence of ALD and nonalcoholic

FLD (NAFLD) of 4.5% and 15.0%, respectively. The prevalence of NAFLD in children is 2.1%, although the prevalence increases to 68.2% among obese children. With the increasing pandemic of obesity and MetS in the general population, China is likely to harbor an increasing reservoir of patients with FLD. The risk factors for FLD resemble to those of Caucasian counterparts, BMS907351 but the ethnic-specific definitions of obesity and MetS are more useful in assessment of Chinese people. Therefore, FLD/NAFLD has become a most common chronic liver disease in China. Public health interventions are needed to halt the worldwide trend of obesity and alcohol abuse to ameliorate liver injury and to improve metabolic health. Although viral hepatitis, especially chronic hepatitis B (CHB), remains a major cause of liver-related morbidity and mortality in China, the prevalence of CHB infection in mainland China has decreased from 10% to 7% between 1992 and 2006.[1] On

the other hand, fatty liver disease (FLD) is emerging as a leading cause of chronic ZVADFMK liver disease in China as a result of the aging population, the improved control of viral hepatitis, and the obesity and alcoholism epidemics.[2-4] FLD refers to a wide clinical and histological spectrum from simple hepatic steatosis to steatohepatitis to cirrhosis, and FLD has been classified as nonalcoholic FLD (NAFLD) and alcoholic liver disease Mannose-binding protein-associated serine protease (ALD) according to etiology.[5,

6] In addition, steatosis can occur in other chronic liver diseases with deleterious effects on the treatment and prognosis.[2, 3, 5] Beyond damage to the liver, steatosis can also worsen and/or induce insulin resistance, and is correlated with the incident of the metabolic syndrome (MetS), type 2 diabetes (T2D), and atherosclerosis.[2, 3, 5] Therefore, the Chinese Fatty Liver and ALD Study Group established in 2001 have issued a series of consensus guidelines for the diagnosis and management of NAFLD and ALD (Table 1).[7-12] Increased epidemiological studies have revealed that FLD is highly prevalent and more often linked to obesity than to alcoholism in China.[3, 13] Imaging surveys for FLD, typically based on ultrasound, have been underway in China since the 1990s.[3, 13] The reported point prevalence of FLD varies widely (1% to more than 50%) mainly based on the information available in a given population (age, gender, occupation, geographic locality), the diagnostic criteria used, and the time of the study.

At present, FLD, which is typically diagnosed by imaging, is highly prevalent (∼27% urban population) in China and is mainly related to obesity and metabolic syndrome (MetS). However, the percentage of alcoholic liver disease (ALD) among patients with chronic liver diseases in clinic is increasing as well, and a synergetic effect exists between heavy alcohol drinking and obesity in ALD. Prevalence figures reveal regional variations, with a median prevalence of ALD and nonalcoholic

FLD (NAFLD) of 4.5% and 15.0%, respectively. The prevalence of NAFLD in children is 2.1%, although the prevalence increases to 68.2% among obese children. With the increasing pandemic of obesity and MetS in the general population, China is likely to harbor an increasing reservoir of patients with FLD. The risk factors for FLD resemble to those of Caucasian counterparts, find more but the ethnic-specific definitions of obesity and MetS are more useful in assessment of Chinese people. Therefore, FLD/NAFLD has become a most common chronic liver disease in China. Public health interventions are needed to halt the worldwide trend of obesity and alcohol abuse to ameliorate liver injury and to improve metabolic health. Although viral hepatitis, especially chronic hepatitis B (CHB), remains a major cause of liver-related morbidity and mortality in China, the prevalence of CHB infection in mainland China has decreased from 10% to 7% between 1992 and 2006.[1] On

the other hand, fatty liver disease (FLD) is emerging as a leading cause of chronic MLN0128 in vitro liver disease in China as a result of the aging population, the improved control of viral hepatitis, and the obesity and alcoholism epidemics.[2-4] FLD refers to a wide clinical and histological spectrum from simple hepatic steatosis to steatohepatitis to cirrhosis, and FLD has been classified as nonalcoholic FLD (NAFLD) and alcoholic liver disease only (ALD) according to etiology.[5,

6] In addition, steatosis can occur in other chronic liver diseases with deleterious effects on the treatment and prognosis.[2, 3, 5] Beyond damage to the liver, steatosis can also worsen and/or induce insulin resistance, and is correlated with the incident of the metabolic syndrome (MetS), type 2 diabetes (T2D), and atherosclerosis.[2, 3, 5] Therefore, the Chinese Fatty Liver and ALD Study Group established in 2001 have issued a series of consensus guidelines for the diagnosis and management of NAFLD and ALD (Table 1).[7-12] Increased epidemiological studies have revealed that FLD is highly prevalent and more often linked to obesity than to alcoholism in China.[3, 13] Imaging surveys for FLD, typically based on ultrasound, have been underway in China since the 1990s.[3, 13] The reported point prevalence of FLD varies widely (1% to more than 50%) mainly based on the information available in a given population (age, gender, occupation, geographic locality), the diagnostic criteria used, and the time of the study.

12 Because the minor allele frequencies of rs12980275, rs11881222, and rs7248668 are all less than 1% in Taiwanese,19 rs8105790, rs8099917, rs4803219, and rs10853728 were selected as candidate SNPs in the present study. The genotypes of the patients were determined with the ABI TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA) and with predesigned commercial genotyping assays (ABI assay C__11710096_10). Briefly, PCR primers and two allelic-specific probes were designed to detect a specific SNP target. The PCR

reactions were performed in 96-well microplates with ABI 7500 real-time PCR (Applied Biosystems). Allele discrimination was achieved by the detection of fluorescence with System SDS 1.2.3. In the initial analysis, rs8105790, rs8099917, and rs4803219 were noted to be in very strong linkage disequilibrium with one another selleck kinase inhibitor (r2 = 0.94-0.96). BIBW2992 Therefore, rs8099917 and rs10853728 were selected for the final analysis with respect

to the other variables in the current study (Fig. 1 and Supporting Information Table 2). The Hardy-Weinberg disequilibrium test was performed for each SNP. The linkage disequilibrium index (Lewontin’s D′ and r2) was calculated with Haploview 4.2.20 The frequencies were compared between groups with the χ2 test with the Yates correction or Fisher’s exact test. Group means, presented as means and standard deviations (SDs), were compared with analysis of variance and the Student t test. Serum HCV RNA levels were expressed after the logarithmic transformation of the original values. Creatinine clearance was estimated with the Cockcroft-Gault equation, which includes the sex, age, body weight, and serum creatinine level as values. The frequencies of the rare alleles of rs8099917 and rs10853728 genotypes were too low, and we combined the rare

homozygote and heterozygote together when Selleckchem Rucaparib we analyzed these two SNPs. To assess the relative contributions of predictors of RVR and SVR, we applied stepwise logistic regression analysis and used age, sex, baseline HCV RNA levels, the degree of liver fibrosis, IL-28B genotypes, and pretreatment aminotransferase levels as covariants. The statistical analyses were performed with the SPSS 12.0 statistical package (SPSS, Chicago, IL). All statistical analyses were based on two-sided hypothesis tests with a significance level of P < 0.05. The basic demographic, virological, and clinical features of the patients are shown in Table 1. The rates of RVR, EVR, EOTVR, SVR, and relapse were 83.8%, 96.7%, 96.9%, 89.0%, and 8.1%, respectively. In the univariate analysis, the genotypes of rs10853728 were not associated with RVR or SVR (Table 2). The TT genotype of rs8099917, low baseline HCV RNA levels (<400,000 IU/mL), low pretreatment levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and less advanced liver fibrosis were significantly associated with a higher RVR rate.

2). Immunostains learn more were analyzed by a liver histopathologist (A. Q.) who was blinded to the clinical data. A cell count was performed using an eyepiece graticule (Datasights limited, Middlesex, UK) as described by Going30 (Supporting Information, section 1.3). Transmission electron

microscopy was performed on liver tissue from three AALF explants as described in the Supporting Information (section 1.4). Areas of necrotic and viable parenchyma were obtained from snap-frozen liver tissue samples using laser capture microdissection (Supporting Information, section 1.5). Tissue lysate was prepared using protein lysate buffer according to the protocol developed by Mustafa et al.31 (supplementary section 1.6). Protein array of tissue lysate HSP inhibitor was performed by Aushon Biosystems (Billerica, Boston, MA;USA) as described in supplementary section 1.7. Results are expressed as pg/mL. To identify differences between groups, nonparametric analysis was used (Mann-Whitney U test, Kruskal-Wallis test, Wilcoxon rank test). Correlations were analyzed using Spearman’s rank test. Results are expressed as the median and interquartile range (IQR). Changes in white blood cell counts were analyzed using one-way analysis of variance. There was no significant difference in median ages of AALF patients

(34 years [IQR, 27-43]) when compared with healthy controls (33.5 years [IQR, 29-40]; Baf-A1 chemical structure P = 0.8), whereas CLD patients were significantly older (50.0 years [IQR, 44.61]; P < 0.05). The mean number of circulating monocytes was significantly reduced in all AALF patients when compared with CLD patients (0.42 × 109/L [0.53] versus 0.63 × 109/L [0.29]; P = 0.002). Table 1 shows the clinical and biochemical indices and circulating inflammatory cytokine levels in the AALF patients categorized

according to clinical outcome. AALF patients were divided into those who survived with conservative medical management (AALF-S), underwent emergency OLT (AALF-O), and died without undergoing OLT (AALF-D). Compared with the AALF-S group, AALF-O and AALF-D patients had significantly lower arterial pH and significantly greater derangement of physiology as evidenced by higher INR, arterial blood lactate, level of encephalopathy, vasopressor and hemofiltration requirements, MELD score, and circulating levels of proinflammatory (TNF-α, IL-6) and anti-inflammatory (IL-10) cytokines. As has been described, serum levels of TNF-α, IL-6, and IL-10 were significantly higher in AALF patients compared with CLD patients and healthy controls (data not shown).5 The number of circulating monocytes was significantly reduced in AALF-D (median, 0.04 × 109/L [range, 0.01-0.22]) and AALF-O (median, 0.145 × 109/L [range, 0.0-1.07]) compared with AALF-S (median, 0.54 × 109/L [range, 0.1-1.05]; both P = 0.0004) at 24 hours following admission.

“
“Background and Aim:  The etiology of autoimmune hepatitis (AIH) is unknown, and limited epidemiological data are available. Our aim was to perform a population based epidemiological study of AIH in Canterbury, New Zealand.

Methods:  To calculate point prevalence, all adult and pediatric outpatient clinics and hospital discharge summaries were searched to identify all cases of AIH in the Canterbury region. Incident cases were recruited prospectively in 2008. Demographic and clinical data were extracted from case notes. Both the original revised AIH criteria and the simplified criteria were applied and cases were included in the study if they had definite or probable AIH. Results:  When the original revised criteria were used, 138 cases (123 definite Decitabine mouse and 14 probable AIH), were identified. Prospective incidence in 2008 was 2.0/100 000 (95% confidence interval [CI] 0.8–3.3/100 000). Point prevalence on 31 December 2008 was 24.5/100 000 (95% CI 20.1–28.9). Age-standardized (World Health Organization standard population) incidence and prevalence were 1.7 and 18.9 per 100 000, respectively. Gender-specific prevalence confirmed a female predominance, while ethnicity-specific prevalence showed higher prevalence in Caucasians. 72% of cases presented after 40 years selleck chemicals of age and

the peak age of presentation was in the sixth decade of life. Conclusions:  This is the first and largest population-based epidemiology study of AIH in a geographically defined region using standardized inclusion criteria. The observed incidence and

prevalence rates are among the highest reported. The present study confirms that AIH presents predominantly in older women, with a peak in the sixth decade, contrary mafosfamide to the classical description of the disease. “
“The p53 family of proteins regulates the expression of target genes that promote cell cycle arrest and apoptosis, which may be linked to cellular growth control as well as tumor suppression. Within the p53 family, p53 and the transactivating p73 isoform (TA-p73) have hepatic-specific functions in development and tumor suppression. Here, we determined TA-p73 interactions with chromatin in the adult mouse liver and found forkhead box O3 (Foxo3) to be one of 158 gene targets. Global profiling of hepatic gene expression in the regenerating liver versus the quiescent liver revealed specific, functional categories of genes regulated over the time of regeneration. Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative cellular proliferation. p53 and TA-p73 bind a Foxo3 p53 response element (p53RE) and maintain active expression in the quiescent liver. During regeneration of the liver, the binding of p53 and TA-p73, the recruitment of acetyltransferase p300, and the active chromatin structure of Foxo3 are disrupted along with a loss of Foxo3 expression.

We identified glides as segments where the absolute value of the Hilbert transform of the pitch rate signal was <0.05 (Woodward

et al. 2006a), and visually checked these sequences. Based on previously described gliding behaviors in right whales (Nowacek et al. 2001, Woodward et al. 2006a), we defined the minimum glide duration as 5 s. Following Wilson et al. (2006) Protease Inhibitor Library manufacturer and Fahlman et al. (2008), we calculated Overall Dynamic Body Acceleration (ODBA, g) by smoothing accelerometer measurements in three separate axes, with a window size of 3 s. We then subtracted these smoothed data (static acceleration) from the unsmoothed data to estimate the dynamic acceleration in each axis. Finally, we then calculated ODBA as the sum of the absolute value of dynamic acceleration in each axis. We observed peaks and identified outliers in ODBA at each surfacing event, and therefore

calculated mean ODBA values within dives, between dives, and during descent and ascent periods of each dive. We defined three phases of the sedation and disentanglement of Eg 3911 (Table 2) hereafter referred to as (1) Sedation/Entangled: animal towing gear and attached buoys, and sedative injection; (2) Disentangled: following removal of most of trailing gear and buoys, administration of antibiotics, and attachment of the satellite LIMPET tag (Andrews et al. 2008); and (3) Recovery: retrieval of injection darts, PARP signaling dart tethers and floats (Moore et al. 2010), and the end of active boat approaches. To determine the behavioral effects of sedation on an entangled whale, we used Wilcoxon rank sum

tests to compare dive parameters and respiration rates within the Sedation/Entangled phase, between the 21 min prior to and the 50 min following sedative injection, but prior to removal of the gear and buoys. We used Three-sample Kruskal-Wallis single factor analysis of variance tests with tied ranks and post hoc Bonferroni-corrected (α =  0.05/3 = 0.0167) Wilcoxon rank sum tests to compare the distributions of various dive Abiraterone order parameters between Sedation/Entangled, Disentangled and Recovery phases. To compare the observed vs. expected ratio of time spent above and below the wave drag limit between phases, we used Chi-square contingency tables. We compared fluke stroke rate, RMS, and the frequency and duration of glides across phases within the single tag deployment to infer changes in thrust intensity and power requirements. As propulsive (thrusting) forces should equal resistive forces (net buoyancy and drag), we expect thrusting intensity (stroke rate and RMS) to be greater and for fewer and shorter glides to occur in entangled vs. nonentangled conditions.

We identified glides as segments where the absolute value of the Hilbert transform of the pitch rate signal was <0.05 (Woodward

et al. 2006a), and visually checked these sequences. Based on previously described gliding behaviors in right whales (Nowacek et al. 2001, Woodward et al. 2006a), we defined the minimum glide duration as 5 s. Following Wilson et al. (2006) selleckchem and Fahlman et al. (2008), we calculated Overall Dynamic Body Acceleration (ODBA, g) by smoothing accelerometer measurements in three separate axes, with a window size of 3 s. We then subtracted these smoothed data (static acceleration) from the unsmoothed data to estimate the dynamic acceleration in each axis. Finally, we then calculated ODBA as the sum of the absolute value of dynamic acceleration in each axis. We observed peaks and identified outliers in ODBA at each surfacing event, and therefore

calculated mean ODBA values within dives, between dives, and during descent and ascent periods of each dive. We defined three phases of the sedation and disentanglement of Eg 3911 (Table 2) hereafter referred to as (1) Sedation/Entangled: animal towing gear and attached buoys, and sedative injection; (2) Disentangled: following removal of most of trailing gear and buoys, administration of antibiotics, and attachment of the satellite LIMPET tag (Andrews et al. 2008); and (3) Recovery: retrieval of injection darts, http://www.selleckchem.com/products/obeticholic-acid.html dart tethers and floats (Moore et al. 2010), and the end of active boat approaches. To determine the behavioral effects of sedation on an entangled whale, we used Wilcoxon rank sum

tests to compare dive parameters and respiration rates within the Sedation/Entangled phase, between the 21 min prior to and the 50 min following sedative injection, but prior to removal of the gear and buoys. We used Three-sample Kruskal-Wallis single factor analysis of variance tests with tied ranks and post hoc Bonferroni-corrected (α =  0.05/3 = 0.0167) Wilcoxon rank sum tests to compare the distributions of various dive Adenosine parameters between Sedation/Entangled, Disentangled and Recovery phases. To compare the observed vs. expected ratio of time spent above and below the wave drag limit between phases, we used Chi-square contingency tables. We compared fluke stroke rate, RMS, and the frequency and duration of glides across phases within the single tag deployment to infer changes in thrust intensity and power requirements. As propulsive (thrusting) forces should equal resistive forces (net buoyancy and drag), we expect thrusting intensity (stroke rate and RMS) to be greater and for fewer and shorter glides to occur in entangled vs. nonentangled conditions.

STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN-α-based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK Everolimus mw cytotoxicity

(tumor necrosis factor–apoptosis-inducing ligand [TRAIL] expression and degranulation) and decreased IFN-γ production. NK cells from patients with a greater than 2 log10 first-phase HCV RNA decline to IFN-α-based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with Torin 1 solubility dmso a less than 2 log10 first-phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN-α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN-α during the second-phase virological response. Conclusion: These data show that IFN-α-induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN-γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN-α-based therapy. (Hepatology 2012) Natural killer (NK) cells are innate immune cells best known for their immediate effector functions

Celecoxib against virus-infected cells and tumor cells.1

These effector functions include the destruction of target cells via perforin/granzyme-mediated lysis or tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the production of cytokines, such as tumor necrosis factor alpha (TNF-α), macrophage-inflammatory protein 1 beta, and interferon gamma (IFN-γ).1 IFN-γ, in particular, has elicited great interest because it is abundantly produced, has direct antiviral activity, and provides a link between innate and adaptive immunity by contributing to the priming of cluster of differentiation (CD)4+ and CD8+ T cells and via the induction of chemokines to T-cell recruitment to the target organ.2 Different effector functions have traditionally been associated with specific NK cell subsets, which can be distinguished based on CD56 expression. Approximately 90% of NK cells in the peripheral blood express low levels of CD56 on their cell surface. These CD56dim NK cells respond quickly to viral infection, exert cytotoxicity, and produce chemokines and cytokines within hours.3-5 The remaining 10% of NK cells with high levels of CD56 expression (CD56bright) respond slower and produce large amounts of IFN-γ and TRAIL with little perforin/granzyme-mediated cytotoxicity. We and others have recently shown that patients with chronic hepatitis C virus (HCV) infection display a polarized NK cell phenotype with increased cytotoxicity and TRAIL production and decreased IFN-γ production.

Both were labeled with α-32P-dCTP using a random primed DNA labeling kit (DECAprime II, Ambion Inc., Austin, TX) as described.12 Results of northern blot

analysis were normalized to Gapdh. See Supporting Methods for details of the microarray analysis, which examined differential mRNA expression profiles and quantitative real-time PCR for confirmation. Protein expression was examined using western blot analysis performed as described16 using anti-PHB1, PHB2, and β-actin antibodies (Abcam, Cambridge, MA). Please see Supporting Methods for details. Histologic examination was done in blinded fashion Apoptosis inhibitor as to the genotype or age of the animal. Please see Supporting Methods for details of these procedures. Plasma bilirubin, ALT, and ALP were determined by total bilirubin kit (Thermo Electron Corp., Waltham, MA),

ALT reagent (Raichem; Cliniqa Corp., San Marcos, CA), and ALP assay kit (Biovision Inc., Mountain View, CA), respectively, following the manufacturer’s protocols. The lipid portion of the liver was extracted by the method of Folch et al.17 using chloroform/methanol (2/1, vol/vol) as solvent matrix. Evaporated and reconstituted lipid from liver and frozen and thawed plasma were subjected to the assays for cholesterol and triglyceride using commercial kits (Thermo DMA, Louisville, CO) following the manufacturer’s manuals. Cell proliferation in PHB1 silenced cells for 24 hours or 48 hours was measured by the incorporation rate of bromodeoxyuridine (BrDU) PI3K inhibitor into DNA using a BrDU assay kit (CalBiochem, San Diego, CA) as described18 with 3000 cells per well in 96-well plates and 4 hours or 1 hour of BrDU incorporation time for AML12

or Huh-7 cells, respectively. Apoptosis was measured by Hoechst staining as described18 in Huh-7 cells treated with sorafenib (10 μM, last 24 hours of knockdown or overexpression). Data are given as mean ± standard error. Statistical analysis for the microarray data is described separately, in that section. For the rest of the results, statistical analysis was performed using unpaired Student t test. For changes in mRNA and protein levels, the ratios of various genes and proteins to the housekeeping gene or protein densitometric values were compared. Atezolizumab datasheet Significance was defined by P < 0.05. Following the scheme shown in Supporting Fig. 1, liver-specific Phb1 KO was generated. Of the 120 mice genotyped from 18 litters of heterozygous mating (Phb1loxP/+; Alb-Cre+/−), 10% were liver-specific KOs (Phb1loxP/loxP; Alb-Cre+/+ or Alb-Cre+/−), 73% were heterozygotes (Phb1loxP/+), and 17% were wild-type (WT) (Phb1+/+). The deletion of Phb1 occurred only in the liver of 3-week-old KO mice (Fig. 1A). However, deletion was not complete at this age, as a faint band remains that corresponds to the WT gene in both liver and isolated hepatocytes. Consistent with this, northern blot analysis using Phb1 exon 2 as the complementary DNA (cDNA) probe shows an 80% reduction in Phb1 mRNA level (Fig.

Nucleotide sequences of the DNA probes used in this study, CP35, CLCK1, MLTF, and SP70, are listed in Table 1. For the electrophoretic mobility shift assay (EMSA) reaction, 2 μL of rKLF15 (100 ng/μL) were mixed with 25 fmol of labeled probe and 4 μL of 5× gelshift buffer (Promega), in a total volume of 20 μL, and incubated at 37°C for 30 minutes. The reaction mixtures were loaded on a 6% polyacrylamide gel and subjected to electrophoresis in 0.5× Tris/Borate/EDTA (TBE) buffer

at 200 V for 2∼3 hours. The gel was dried and analyzed by a Typhoon phosphorimager (GE Healthcare, Waukesha, WI). For selleck screening library the supershift assay using the anti-KLF15 antibody, rKLF15 was incubated with a labeled probe at 37°C for 30 minutes, followed by incubation with either anti-KLF15 or control antibody at room temperature for 40 minutes. The chromatin

immunoprecipitation (ChIP) assay was conducted using the EZ-Magna ChIP G Chromatin Immunoprecipitation Kit (Millipore). Briefly, HepG2 cells in 10-cm dishes were cotransfected with 4.8 μg of pKLF15 and 1 μg of the reporter construct, pCP, or pS1-Luc or the mutated constructs, pCP-2m, pS1Z1/Z2mut-Luc, and pS1M2mut-Luc. Forty-eight hours after transfection, cells were crosslinked with formaldehyde and harvested for immunoprecipitation. An aliquot of the cell lysates was saved to serve as the input DNA control. After the reversal of crosslinking with 5 M of NaCl, ChIP samples were subjected to PCR using the primer pair, HBV1644F/HBV1805R find more (for the core promoter), and primer pair RV3/HBV22R (for the surface promoter). Antibodies used in ChIP assays included KLF15 (Abcam), NF-Y (Thermo Fisher Scientific, Wilmington, MA), Sp1 (Abcam), rabbit control IgG, and goat control IgG (Abcam) antibodies. HepG2 cells cotransfected with pHBV1.3D and pKLF15 or its control vector pcDNA3.1 were harvested in 600 μL of lysis buffer (50 mM Tris-HCl, pH 7.0, and 0.5% Nonidet P-40) 96 hours after transfection.

Ninety microliters of cell lysates or culture medium were mixed with 1 μL of TURBO DNase (Ambion, Austin, TX) and 10× DNase buffer and incubated at 37°C for 30 minutes. After, DNase was inactivated by heating at 75°C for 10 minutes. The mixtures were subsequently processed with the virus extraction column (QIAamp MinElute Virus Spin Kit; Qiagen, Germantown, Niclosamide MD), following the manufacturer’s instruction. Viral genome thus purified was quantified by RT-PCR, using the SYBR green master mix and the HBV DNA-F/R primer pair (Table 1). To extract the encapsidated viral DNA from the mouse serum, 25 or 100 μL of mice serum was used. Experiments involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the San Francisco VA Medical Center. Male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and used at 6-7 weeks of age. To study the effects of KLF15 on HBV viral protein expression and DNA replication, 5 μg of pAAV-HBV1.