, 1996, 1996; Spinnler & Tognoni, 1987) were within the normal range (see Table 1). Processes of acquisition of navigational information and re-orientation were assessed with the DDTDB, derived from tests used by Bianchini et al. (2010) in a previous study of DTD, based on theoretical models of normal development and normal navigation stages (Siegel & White, 1975; Wang & Spelke, 2002). The battery included three different categories MAPK Inhibitor Library of tasks. The first category assessed specific

domains such as visual spatial perception, visuospatial memory and visuospatial imagery (see Table 2). The second and third categories of tests assessed specific navigational abilities, respectively, in an experimental and an ecological environment (see Table 2). Dr. WAI’s performance on tests lacking standardization data was compared with that of male volunteers (C) matched

for age and years; the number of C varied from 20 to 5 in different tests. Dr. WAI’s and controls’ performances were compared HM781-36B mw by means of analysis developed by Crawford and Howell (1998; CH), using the computer program SINGLIMS.EXE. This analysis uses a modified t test described by Sokal and Rohlf (1995) and is the more suitable analysis to estimate the abnormality of the individual scores when the normative sample is small (that is less than 50 subjects). Results for each test (as well as size of C group) are described below and shown in Table 2. Assessment of Dr. WAI’s basic visuospatial abilities included tests of visuospatial perception (Visual Object Spatial Perception Battery, Benton’s Facial Recognition Test), visuospatial memory (Corsi Block Tapping Test: Span and Supraspan), and visuospatial imagery (Memory of buildings, Letter Inspection Test, Mental Rotation Test, Rucaparib purchase Generation of imagery from long-term memory as Map drawing of current home) (see Tables 1 and 2). Only tests not commonly used in clinical practice are described below. On the Corsi Block Tapping Test (CBT; Corsi,

1972), Dr. WAI had a normal span, as well as normal Supra-span learning and delayed recall when compared with a group of five controls. His performance in object and space perception (Visual Object Spatial Perception Battery, Warrington & James, 1991) and face recognition (Benton’s Facial Recognition Test, Benton, Van Allen, Hamsher, & Levin, 1975) was well within the normal range (see Table 1). Topographical abilities involve some specific aspects of cognition, such as recognizing landmarks and scenes and describing and drawing a map of a familiar environment, which rely on visual imagery abilities (Farah, 1989; Riddoch & Humphreys, 1989). As in Bianchini et al.’s (2010) study, we referred to Kosslyn’s model (2005) in Dr. WAI’s assessment, to evaluate processes of generation, inspection, and transformation of visual mental images.

While not designed to study such patterns in mothers, early work on human nursing did not detect an isotopic effect in lactating women (Fogel et al. 1997). In contrast, a study of wild horses showed that lactating females had lower δ15N values than other adults (males, nonlactating females) and used mass balance calculations to argue that this 15N-depletion is the expected result of the nitrogen balance perturbations associated with lactation (Koch 1997). Further support for this trend was reported in CSF-1R inhibitor Kurle (2002), where blood δ15N values of a single lactating northern fur seal were approximately 1‰ lower than those for nulliparous females. Fuller

et al. (2004) reported δ15N variations among pregnant human females. They found that δ15N values dropped from conception to birth, and that the magnitude of the drop correlated to the birth weight of the baby as well as the amount of weight gained by the mother. If these phenomena occur in marine mammals, they would reduce Δ15Ntissue-diet values for growing

or pregnant females. Expectations for lactating females are more complex and may Olaparib molecular weight depend on whether animals feed or fast while lactating (i.e., income vs. capital breeders). The δ18O value of a biomineral depends on the temperature at which it forms and the 18O value of the body fluid from which it precipitates (discussion below based on Clementz

and Koch 2001 and Koch 2007). For mammals there is a constant offset between the 18O value of body water and phosphate (∼+18‰), and between the phosphate and carbonate components of bioapatite (∼+8‰), close to values predicted for isotopic equilibrium at typical body temperatures. Physiology affects the 18O value of body water by altering the fluxes of oxygen into and out of the body, as well as fractionations associated with transport and/or transformation of oxygen-bearing compounds. Ingested water is a major flux of oxygen into marine mammals and includes preformed water in food, seawater consumed incidentally when eating, and water taken by active drinking (mariposia). The proportion of water gained from these sources varies widely among marine mammals (Ortiz 2001), yet as these processes 4-Aminobutyrate aminotransferase do not strongly fractionate oxygen, these fluxes should all have 18O values close to that of seawater (0‰ V-SMOW). Metabolic water generated by oxidation of food dry matter may contribute to marine mammal body water. This water may be 18O-enriched relative to ingested water, as atmospheric O2 is much heavier than ingested water (∼+21‰ V-SMOW). Finally, there is evidence in cetaceans for a substantial flux of water across the skin (Hui 1981, Andersen and Nielsen 1983); it is unlikely that this process greatly fractionates oxygen isotopes, though the issue has not been studied.

Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed

sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance. Hepatitis C virus (HCV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma worldwide. Current antiviral treatment consisting of pegylated interferon-alpha (IFN-α) and ribavirin is limited by resistance, adverse effects, and high costs.1 Although the clinical development of novel antiviral compounds that target HCV protein processing KPT-330 price has been shown to markedly improve sustained virological response, toxicity of the individual compounds and development of viral resistance remain major challenges.2 Thus, novel antiviral strategies are urgently needed. Micro-RNAs (miRNAs) are key regulators of gene expression at a posttranscriptional level.3

Their biogenesis is now well characterized and involves the processing of a large primary transcript into a stem-loop pre-miRNA, ultimately leading to the mature single-stranded ∼22-nucleotide miRNA. This functional miRNA is assembled into an RNA-induced silencing complex (RISC) that invariably contains a member of the Argonaute protein family (Fig. 1). Once loaded, the active RISC can be directed toward its messenger RNA target to regulate, predominantly in a negative manner, its translation.4 Besides targeting cellular messenger RNAs, miRNAs R428 were recently shown to interact with transcripts of viral origin.5 The first description of such interactions revealed that miRNAs of cellular origin could negatively regulate viral messenger RNAs. Furthermore, mammalian viruses have been shown to usurp the cellular miRNA repertoire. One remarkable example of such usurpation is provided by HCV, which recruits the liver-specific

miR-122 to enhance its replication.6In vivo, the impact of miRNA for pathogenesis of HCV infection Selleckchem Erastin is more complex: analyzing liver biopsies from subjects with chronic hepatitis C who are undergoing IFN therapy, Sarasin-Filipowicz et al. showed no correlation of miR-122 expression with viral load but markedly decreased pretreatment miR-122 levels in subjects who had no virological response during later IFN therapy.7 To truly assess the importance of miRNAs as a therapeutic target requires the use of chemically modified antisense oligonucleotides complementary to the miRNA to prevent its interaction with the target RNA. This approach was first established in vitro,8 before it was shown that it was also very effective in preventing miRNA action in mouse models.9 The later study was carried out using miR-122 as a model, and it enabled the identification of several cellular targets, most of which are involved in the cholesterol biogenesis pathway, e.g.

Silencing of the SR gene induces a decrease in the basal proliferative capacity of large cholangiocytes compared with large

mock-transfected cholangiocytes. In our evaluation of SR expression, we found a time-dependent increase in the expression of SR in large cholangiocytes during BDL compared with normal large cholangiocytes. This finding was consistent with previous studies showing that: (1) in the rodent liver SR is only expressed by large cholangiocytes,1, 4, 5, 9, 12 (2) SR expression is up-regulated following BDL ligation in large cholangiocytes,14, 17 and (3) the extent of secretin effects on cholangiocyte functions parallel with the duration of BDL.16 This finding parallels recent findings that mouse cholangiocytes share a similar heterogeneous Protease Inhibitor Library profile

as rat cholangiocytes5 and freshly isolated and immortalized large mouse cholangiocytes are the only cell types to express the SR.5, 8, 14 In human, SR expression is present in the biliary tract in click here normal bile ducts and ductules and the majority of cholangiocarcinomas, but is not present in hepatocytes or hepatocellular carcinoma.26, 27 Consistent with animal models of cholestasis, SR expression was up-regulated in ductular reactions in liver cirrhosis.27 In our in vivo model, the level of the reduction of cholangiocyte proliferation is consistent with the paradigm that cholangiocyte proliferation is regulated in autocrine and paracrine mechanisms by a number of stimulatory neurohormonal factors.18, 20, 28 In a knockout mouse model for α-calcitonin gene-related peptide, the lack of circulating α-calcitonin gene-related peptide also reduces biliary proliferation during BDL to a similar degree as the lack of SR,20 which indicates that the regulation of biliary

proliferation during extrahepatic cholestasis is multifactorial and a complex regulatory system.18, 20, 28 The trophic effects of secretin were dependent upon the activation of the cAMP/PKA/ERK1/2 signaling. The second messenger system, cAMP, is a key factor for the function of large cholangiocytes.1, 4, 7, 9, 13 Secretin stimulates bicarbonate secretion of large bile ducts through activation of cAMP-dependent CFTRCl−/HCO3− anion exchanger 2.1, 4, 7, 9, 13 Also, the activation of the cAMP/PKA/ERK1/2 Farnesyltransferase pathway modulates cholangiocyte proliferation.12, 15, 18, 29 In fact, the direct stimulation of adenylyl cyclase activity by the chronic administration of forskolin stimulates normal cholangiocyte proliferation both in vivo and in vitro, which is associated with activation of the PKA/Src/MEK/ERK1/2 pathway.29 Maintenance of cAMP levels by forskolin administration prevents the impairment of cholangiocyte proliferation and enhancement of biliary apoptosis induced by vagotomy.30 Furthermore, Banales et al. have shown31 that cAMP stimulates cholangiocyte proliferation through two downstream effectors (i.e., PKA and Epacs) in an animal model of autosomal recessive polycystic kidney disease.

However, the classification F4 is not included in Japanese clinical trials, and there have been no reports of therapeutic results stratified for the degree of hepatic fibrosis. Taken together, the results of Japanese and overseas clinical trials showed no clear age-related differences in therapeutic effect of SMV + Peg-IFN + RBV Seliciclib ic50 triple therapy. Although IL28B SNPs and the degree of fibrosis may influence therapeutic efficacy, SVR rates of 60–80% were still achieved in patients with IL28B minor alleles and advanced fibrosis ≥ F3. Accordingly, at present we cannot say that age, IL28B SNPs or the degree of fibrosis exerts any great influence on the therapeutic

efficacy of this treatment regimen. Recommendations SMV + Peg-IFN + RBV triple therapy is at present the treatment of first choice in IFN-naïve patients. IL28B polymorphism has little influence on the SVR rate in IFN-naïve patients undergoing SMV + Peg-IFN + RBV triple therapy, with relatively high SVR rates achieved even in patients with Selleckchem PLX3397 the TG/GG minor alleles. In Japanese clinical trials conducted with subjects aged ≤ 70, no clear correlation could be identified between age and SVR rates. Although Japanese data is lacking, the results of overseas clinical trials indicate that advanced hepatic fibrosis may influence SVR rates. From the above, in general, if treatment is likely to be tolerated, SMV-based triple

therapy is indicated in all patients who meet the criteria for antiviral therapy (ALT > 30 U/L or platelet count <150 000/μL), irrespective of IL28B SNP status. In some patients, however, in whom adverse reactions are a concern, and the PRKD3 risk of carcinogenesis is considered low, it may be possible to await the introduction of the new agents with more favorable safety profiles. In this patient group at high risk of hepatocellular carcinogenesis, the best possible antiviral therapy should be promptly commenced. However, the possibility of adverse reactions, and

the possibility that viral eradication may not be achieved, should be thoroughly explained to the patient in advance. Although the introduction of TVR + Peg-IFN + RBV triple therapy improved SVR rates in comparison to Peg-IFN + RBV dual therapy,[1] postmarketing surveys revealed serious adverse reactions in approximately 40% of elderly patients. Accordingly, it is recommended that TVR therapy should be commenced at a reduced dosage of 1500 mg/day,[18] although great caution is still required in its use in this age group. On the other hand, clinical trials of SMV + Peg-IFN + RBV triple therapy for treatment-naïve patients have reported an SVR rate of 86% (19/22) in elderly patients aged ≥ 65 (and ≤70), indicating a therapeutic efficacy similar to that seen in non-elderly patients (Fig. 4). Furthermore, very little difference is seen between SMV-based triple therapy and Peg-IFN + RBV dual therapy in terms of safety.

The design and primary outcomes of the VIRAHEP-C trial have been reported elsewhere.8 Adults who were treatment-naïve, infected with genotype 1, had detectable HCV RNA, and had histological evidence of chronic HCV were eligible to participate. Patients were classified by race as either African American or Caucasian, and by ethnicity as either Hispanic or non-Hispanic, based on self-report. All participants were required to have

been born in the United States. From eight clinical centers across the United States, 401 patients were enrolled and began therapy between July 2002 and December 2003. For the present study, serum samples were acquired from a subset of 272 patients from the total VIRAHEP-C cohort, comprising 157 responders Autophagy activator (104 CA, 53 AA) and 115 nonresponders (34 CA, 81 AA). All specimens analyzed were obtained under Institutional Review Board–approved protocols for which participants

provided written informed consent, including consent for genetic testing. Patients received peginterferon alfa-2a (Pegasys; Roche Pharmaceuticals, Nutley, NJ) 180 μg/week and ribavirin (Copegus; Roche Pharmaceuticals) 1,000-1,200 mg/day for at least 24 weeks. Patients who became HCV RNA–negative by week 24 continued treatment for a total of 48 weeks, whereas patients who remained HCV RNA–positive stopped treatment and were considered nonresponders. The primary endpoint of the trial was SVR, defined as the absence of detectable HCV RNA for at least 24 weeks after stopping therapy. HCV RNA testing was performed at a central laboratory (SeraCare BioServices, Gaithersburg, MD) NVP-BKM120 datasheet using the Cobas Amplicor Assay (sensitivity, 50 IU/mL; Roche Molecular Diagnostics, Alameda, CA). Selected samples were tested for HCV RNA levels using the Cobas Amplicor Monitor Assay and for HCV RNA genotype using Carnitine palmitoyltransferase II the Versant HCV Genotype Assay (Bayer, Tarrytown, NY). All patients had undergone liver biopsy within 18 months of screening, and

the biopsy specimens were assessed by a blinded central pathologist. All biopsies were assessed for severity of hepatitis C by grading the inflammation and staging the fibrosis using Ishak’s modified histological activity index. IP-10 was measured in serum samples collected at baseline, prior to initiation of treatment, using the commercially available Quantikine human CXCL10/IP-10 immunoassay (R&D Systems). All samples were diluted 1:2 and analyzed in duplicate. The linear dynamic range of the IP-10 measurement in this assay was 8-500 pg/mL, with a detection limit at 7.8 pg/mL. Samples with IP-10 concentration above 1,000 pg/mL were diluted 1:5 and reanalyzed. The IL28B polymorphic marker rs12979860 was analyzed using the ABI TaqMan allelic discrimination kit and the ABI7900HT Sequence Detection System (Applied Biosystems) as described by Thomas and colleagues.

They defined the EGJ as the macroscopic junction between brown–red gastric mucosa and gray esophageal mucosa. The authors reported the detection of CG and oxyntocardiac glands in 97% of cases, with a mean length of 5 mm (range: 1–15 mm). They did not identify any case that showed a direct transition of gastric fundic oxyntic glands to the esophageal squamous mucosa. In addition, they also reported the findings

of the intra-esophageal presence of CG and oxyntocardiac glands above the deep esophageal glands and ducts in 25% of cases. However, in as much as 61% of cases in their report, there existed squamous islands among the CG or oxyntocardiac glands, which indicates that their analysis was actually Proteasome inhibitor in the tissues taken from the distal esophagus, not in the proximal stomach, since squamous islands are indicative of the esophagus.25,30 To contribute to the debate on the existence of gastric CG and CM, Marsman et al.22 conducted an endoscopic biopsy study in 63 of 198 unselected patients with biopsies at or immediately below the endoscopically-normal-appearing SCJ that was defined as the EGJ. They Tyrosine Kinase Inhibitor Library reported a uniform presence of the CM in the proximal stomach, including CG in 62% of cases and oxyntocardiac glands in 38%. Therefore,

they concluded that the CG and the CM were congenital, not acquired.22 Their conclusion was confirmed in a similar study of volunteer health-care workers in the USA.31 In summary, all studies showed a consistent presence of CG and Tolmetin the CM on the gastric side of the EGJ in most, but not all, patients, which is slightly different from that shown in fetuses and pediatric populations. The length of the CM in this transitional zone is short; approximately 5 mm on average. The absence of the CM in over 39% of adult Americans, as reported by the Chandrasoma groups, has not been confirmed. It should be noted that most studies used the SCJ as the landmark of the EGJ, which might potentially be the source of errors.25 In

contrast, recent studies on the distribution of CG in the EGJ region in adults from Japan and China show different results from those reported in Europe and North America. In Japan, Misumi et al. studied the relationship between the mucosal EGJ,32 which was defined as the distal end of esophageal longitudinal vessels, and CG in endoscopic biopsy patients without reflux esophagitis, ulcers, hiatal hernia, or tumors in the esophagus and stomach. They also systemically mapped CG in the entire EGJ region in additional, resected specimens for esophageal carcinoma and another cohort of resected specimens with cancers in either the lower esophagus or the proximal stomach. They reported the presence of CG in an area between 7.5 mm proximal and 13 mm distal to the EGJ. Histologically, the CM straddled the EGJ approximately 10 mm proximally and 20 mm distally.

There is considerable evidence Cabozantinib molecular weight that activation

of inflammation targeting the biliary system plays an important role in both extrahepatic and intrahepatic aspects of BA.44 Studies examining the importance of the inflammatory process have strengthened the argument for an infectious pathogenesis to BA, but there is evidence from other diseases that noninfectious etiologies may lead to inflammatory activation, including activation of IFN-γ.45 Here we demonstrate that developmental defects in biliary anatomy and activation of IFN-γ-stimulated genes can be elicited by genetic and pharmacologic inhibition of DNA methylation. IFN-γ activation in biliary cells may lead to cell damage via activation of IFN-γ downstream pathways, or potentially by inhibition of transforming growth factor β (TGF-β). Activation of IFN-γ inhibits TGF-β signaling in several model systems.46 TGF-β exerts a positive effect on the development

of bile duct cells,28 and thus inhibition of TGF-β would be expected to have a negative effect on biliary development. Such a mechanism is attractive in the developing liver, as the differentiation of hepatoblasts into bile duct cells is probably not present in the healthy mature liver. Thus, the specificity of this mechanism would be due to pathways that are developmentally limited. Although there are similarities between our zebrafish with inhibition of DNA methylation and BA, there are also key differences. We did buy MLN0128 not observe extrahepatic biliary defects in dtp, azaC-treated

larvae, or dnmt1 morphants, whereas extrahepatic biliary abnormalities are clearly important in BA. Of note, we have not observed extrahepatic defects in any of our models of abnormal biliary development in zebrafish, including hnf6 morphants, whereas targeted deletion of Hnf6 in mice clearly leads to extrahepatic biliary defects.29 This discrepancy may be due to a lack of evolutionary conservation in development of the extrahepatic biliary tree, or may be due to other factors such as timing of knockdown with respect to development or technical difficulties in observing the extrahepatic biliary tree in developing zebrafish. We also did not observe inflammatory Tideglusib infiltration of the liver or biliary tree in our models, although we did observe activation of inflammatory genes. This activation of IFN-γ-responsive genes in particular was attenuated by prednisone, which also led to rescue of the biliary defects in our fish and has been shown to be of some benefit for patients with BA post-portoenterostomy.47, 48 These results suggest that the gene expression changes elicited by prednisone may be responsible for the rescue of biliary defects, but other possible mechanisms, such as altered expression of non-IFN-γ pathway genes that lead to biliary growth and development, may be functioning as well.

At t = 4 hours, the relative CBF was 228% ± 49% in the triple dosing group and 169% ± 22% in Forskolin molecular weight the intravenous infusion group. Compared with group 1 from experiment B, we found no significant differences (F[2,16] = 0.95, P = 0.41, one-way ANOVA) (Fig. 2B). Based on local clinical recommendations for hypermagnesemia and relevant literature,21 we aimed at achieving a P-Mg > 2 mM after 2 hours. We also aimed at a CSF-Mg level of greater than 20% above baseline after 4 hours. The results of the dose finding study in experiment A demonstrated that groups 2, 3, and 4 achieved a P-Mg greater than 2 mM at t = 2 hours and that group 4 also had a P-Mg

greater than 2 mM at t = 4 hours. Interestingly, we did not find that increased doses of magnesium led to higher CSF-Mg levels at t = 4 hours, even though P-Mg was higher. We concluded that the most appropriate dosing regimen CB-839 was 1.6 mmol/kg MgSO4 intraperitoneally at baseline and 0.8 mmol/kg MgSO4 intraperitoneally after 1 hour, which fulfilled our criteria of acceptable plasma levels and relevant central nervous system bioavailability. Consequently, this dosing regimen was used

in experiment B, in which we, apart from MAP, ICP, and CBF, also studied the potential mechanisms of action of hypermagnesemia. Experiment B showed that induction of hypermagnesemia did not prevent development of intracranial hypertension or cerebral hyperperfusion. We used the well-characterized rat model with PCA and acute hyperammonemia22 and did indeed find cerebral hyperfusion and high ICP. In fact, with the dosing regimen of experiment B, we saw a higher CBF and a tendency toward a higher ICP in rats treated with MgSO4 compared with the corresponding group not receiving MgSO4. Another interesting finding was that

hyperammonemia led to a significant drop in MAP after 1 hour of ammonia infusion and that hypermagnesemia ADAMTS5 in both ammonia and saline infusion animals led to a tendency torward lower MAP; however, it was most pronounced in rats with hyperammonemia. This could indicate that the model itself (PCA + ammonia infusion) induces an initial vasodilatation that is worsened by hypermagnesemia and adds up to the substantial increase in relative CBF that was almost 50% higher in the PCA+NH3+MgSO4 group compared with PCA+NH3+vehicle. To reduce the risk of false-negative results, we also performed experiment C with alternative dosing regimens of MgSO4. The PCA rats appeared to have a lower clearance of MgSO4 than the healthy animals in experiment A, most likely because of the hepatic shunt. We achieved a P-Mg above 2 mM at 2 hours and at the end of the experiment with both a triple-dosing regimen and intravenous infusion. This did not, however, lead to significant positive effects on ICP or CBF. We did observe a tendency toward a slightly lower ICP in the triple dosing group than in the vehicle group.

All miRNAs and siRNAs used in the present study are listed in Supporting Information Table 1. An HBV replication-competent clone INK 128 clinical trial pSM2 harboring a head-to-tail tandem dimer of the HBV genome (GenBank accession number: V01460) was provided by Dr. Hans Will (Heinrich-Pette-Institute, Hamburg, Germany). The expression plasmid encoding full length human HDAC417

was purchased from Addgene (Cambridge, MA). The class I histone deacetylases inhibitor trichostatin A (TSA), FXRA antagonist guggulsterone (GGS), cell cycle synchronization chemicals aphidicolin and nocodazole were purchased from Sigma-Aldrich (Steinheim, Germany). Human hepatoma cell lines

HepG2 and Huh7 were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and maintained at 37°C in a humidified 5% CO2 atmosphere. HepG2.2.15 cells with integrated dimers of the HBV genome (GenBank accession number: U95551) and Con-1 cells with a subgenomic HCV replicon (kindly provided by Prof. AG 14699 Dr. Ralf Bartenschlager, University of Heidelberg, Germany) were cultured with 500 μg/mL of G418 (Sigma-Aldrich). Primary human hepatocytes were isolated from liver transplantation Vitamin B12 donor by perfusion and cultured as described.18 Plasmids, miRNAs, and small interfering RNAs (siRNAs) were transfected into cells at indicated concentrations using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. HBV replicative intermediates (HBV RI) from intracellular core particles and HBV transcripts were extracted from hepatoma cell lines and detected by southern and northern blot, respectively,

according to the published protocols.19 HBV progeny DNA was extracted from cell culture supernatants using QiAamp DNA Blood Mini kit (Qiagen) and quantified by real-time polymerase chain reaction (PCR) as described.20 HBV RNAs in cells were also detected using quantitative real-time reverse transcriptase (RT)-PCR assay (primer sequences are listed in Supporting Information Table 2). A monoclonal antibody (clone 10E11, Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect hepatitis B c-antigen (HBcAg) expression by western blot as described below. The levels of HBsAg and HBeAg in culture supernatants were determined using the Architect system and HBsAg and HBeAg CMIA kits (Abbott Laboratories, Wiesbaden-Delkenheim, Germany) according to the manufacturer’s instructions.