Pradefovir Treatment in Patients With Chronic Hepatitis B: Week 24 Results From a Multicenter, Double-Blind, Randomized, Noninferiority, Phase 2 Trial.

BACKGROUND: Pradefovir is a liver-targeted prodrug of adefovir, a nucleoside/nucleotide analogue with antiviral activity against hepatitis B virus (HBV) DNA polymerase. This phase 2 study compared the efficacy and safety of oral pradefovir (30, 45, 60, or 75 mg) versus tenofovir disoproxil fumarate (TDF; 300 mg) and aimed to identify the most appropriate dose of pradefovir for the forthcoming phase 3 study. METHODS: Treatment-naive and experienced (not on treatment >6 months) patients with chronic hepatitis B were eligible. RESULTS: A total of 240 participants were randomized and treated in the study (48 per group). Approximately 80% were hepatitis B e antigen (HBeAg) positive, and 10% had liver cirrhosis. The reductions from baseline in HBV DNA levels achieved at week 24 were 5.40, 5.34, 5.33, and 5.40 log10 IU/mL, with pradefovir doses of 30-, 45-, 60-, and 75-mg, respectively, compared with 5.12 log10 IU/mL with TDF. However, HBeAg loss was attained by more participants who received 45-, 60-, or 75-mg pradefovir than by those receiving TDF (12%, 6%, and 9% vs 3%). The TDF group exhibited a more significant increase in serum creatinine than the pradefovir 30- and 45-mg groups, and serum phosphate levels were comparable among all groups. Most adverse events (AEs) were mild (grade 1). No treatment-related severe AEs were reported. Overall, AEs and laboratory abnormalities were comparable to those in the TDF group. CONCLUSIONS: Pradefovir and TDF exhibited comparable reductions in HBV DNA levels. All treatments were safe and well tolerated. CLINICAL TRIALS REGISTRATION: NCT00230503 and China Drug Trials CTR2018042.

Overexpression of long non-coding RNA cancer susceptibility 11 is involved in the development of chemoresistance to carboplatin in hepatocellular carcinoma.

The long non-coding (lnc)RNA cancer susceptibility 11 (CASC11) promotes gastric cancer, however its role in other diseases is unknown. The present study demonstrated upregulation of lncRNA CASC11 and microRNA (miR)-21 in hepatocellular carcinoma (HCC). Furthermore, the expression of CASC11 was positively correlated with that of miR-21 in HCC tumors. Moreover, overexpression of lncRNA CASC11 led to upregulation of miR-21 in HCC cells, whereas overexpression of miR-21 had no effect on CASC11 levels. The levels of lncRNA CASC11 and miR-21 were found to be upregulated in the plasma of patients with HCC during chemotherapy. In vitro cell experiments demonstrated upregulation of lncRNA CASC11 in HCC cells treated with carboplatin. Additionally, overexpression of lncRNA CASC11 promoted, whereas its knockdown inhibited the viability of HCC cells following carboplatin treatment. Finally, overexpression of miR-21 ameliorated the effects of lncRNA CASC11 knockdown on cell viability. Thus, these findings suggest that upregulation of lncRNA CASC11 is involved in the development of chemoresistance to carboplatin in patients with HCC, via the upregulation of miR-21.

Subunit interactions as mediated by "non-interface" residues in living cells for multiple homo-oligomeric proteins.

Protein-protein interaction, including protein homo-oligomerization, is commonly believed to occur through a specific interface made of a limited number of amino acid residues. Here our systematic in vivo photo-crosslinking analysis via genetically incorporated unnatural amino acids unexpectedly shows that the dimerization of HdeA, an acid stress chaperone, is mediated by the residues along its whole polypeptide. These include those "forbidden" residues that are far away from the dimerization interface as judged according to the reported 3-D structure. We demonstrate that such dimerization, though intriguing, is neither a result of protein over-expression nor of any structural disturbance caused by the residue replacement. Similar unexpected dimerization also occurs for two other oligomeric proteins, IbpB (a molecular chaperone existing as polydispersed oligomers in vitro) and DegP (a protease existing as hexamers in vitro). In contrast to these three proteins, dimerization of a few other oligomeric proteins (e.g., OmpF, LamB, SurA, FtsZ and FkpA) that we similarly examined in living cells seems to be mediated only by specific residues. Together, our unexpected observations suggest that, for some oligomeric proteins such as HdeA, IbpB and DegP, their subunit interactions in living cells can also be mediated by residues other than those located at the interfaces as revealed by in vitro structure determination. Our observations might be partially explained by the formation of "encounter complex" or by protein conformational dynamics. Our findings provide new insights on understanding protein-protein interactions and encounter complex formation in living cells.

GC-MS-based metabolomics approach to diagnose depression in hepatitis B virus-infected patients with middle or old age.

Depression is concomitantly presented in hepatitis B virus (HBV)-infected patients (HB). However, there is still no objective method to diagnose HBV-infected patients with depression (dHB). Therefore, in this study, a gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach was employed to profile urine samples from 59 dHB and 52 HB (the training set) in order to identify urinary metabolite biomarkers for dHB. Then, 41 dHB and 35 HB (the testing set) were used to independently validate the diagnostic generalizability of these biomarkers. In total, 13 differential metabolites responsible for the discrimination between dHB and HB were identified. These differential urinary metabolites belonged mainly to Lipid metabolism and Amino acid metabolism. A panel consisting of six urinary metabolite biomarkers (ethanolamine, azelaic acid, histidine, threitol, 2,4-dihydroxypyrimidine and levulinic acid) was identified. This panel was capable of distinguishing dHB from HB with an area under the receiver operating characteristic curve (AUC) of 0.986 in the training set. Moreover, this panel could classify blinded samples from the testing set with an AUC of 0.933. These findings indicated that the GC-MS-based metabolomics approach could be a useful tool in the clinical diagnosis of dHB, and the identified biomarkers were helpful for future developing an objective diagnostic method for dHB.

DegP functions as a critical protease for bacterial acid resistance.

To counteract the lethal acid stress, bacteria explore such strategies as cytoplasmic decarboxylase-catalyzed proton consumption and periplasmic chaperone-assisted protein refolding. Here, we report a periplasmic protease-mediated acid resistance mechanism in Escherichia coli. Deletion of the protease gene degP dramatically decreases the viability of late log or early stationary phase cells against the extreme acid stress (pH 2.3), which can only be minimally rescued by complementary expression of the protease-deficient DegP(S210A) mutant protein. Similarly, DegQ, a homolog of DegP, also contributes to the bacterial acid resistance, but SurA as an important periplasmic chaperone hardly exhibits protection effect. In vitro studies reveal that DegP completely loses its protease activity under acidic condition but is able to partially reactivate upon neutralization. Importantly, we demonstrate the interaction of DegP with typical cellular substrate proteins in cells during acid stress and/or recovery stages by using unnatural amino acid-mediated in vivo photo-crosslinking, as well as the degradation of periplasmic proteins by DegP during recovery after acidic denaturation. These data illustrate the role of DegP in bacterial acid resistance conceivably via degrading those acid-induced misfolded proteins. Our findings, together with earlier reports, suggest a comprehensive acid resistance strategy adopted by bacteria such that in the ATP-deficient extra-cytoplasm, the inevitable misfolded proteins induced by acid stress are refolded by a chaperone (e.g., HdeA/HdeB) and/or cleaved by a protease (e.g., DegP/DegQ) while in the cytoplasm excessive protons are directly consumed or exported.

[Effect and mechanism of danshensu on hepatitis B virus reverse transcriptase and antigen expression].

MTT assay was used in this study to investigate the inhibitory effect of danshensu on the activity of 2.2.15 cells among human hepatoma cell line (HepG2); indirect fluorescence labeling method was used to measure the changes of reactive oxygen levels in the cells; ELISA method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in cellular supernatants; HBV DNA level was measured with fluorogenic quantitative PCR method. The inhibitory effect of danshensu on HBV RT(hepatitis B virus reverse transcriptase) was studied by using enzyme inhibition dynamics, and the effect of danshensu on secondary structure of HBV reverse transcriptase was monitored by using circular dichroism. The results showed that danshensu had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC50) of (15.35±2.43) µmol•L⁻¹; danshensu could significantly inhibit HBsAg and HBeAg expressions, and showed an inhibitory effect on HBV DNA replication. In addition, danshensu was an effective inhibitor for HBV reverse transcriptase [IC50 (21.32±2.43) µmol•L⁻¹]. The fluorescence labeling results showed that the reactive oxygen levels in the cells were increased with the increase of danshensu concentration. Circular dichroism analysis showed that danshensu could induce partial change of conformation of HBV reverse transcriptase and gradually increased α-helical content. These results indicated that danshensu could make the structure of the enzyme become closer by binding to HBV reverse transcriptase, which was not conducive to the formation of the active center, so it could finally decrease the activity of HBV reverse transcriptase. Such decrease in enzyme activity would directly affect the HBV DNA replication, and combined with the decrease of the antigen levels, the effect of danshensu on HBV was increased.

Inhibition of microRNA-199a-5p reduces the replication of HCV via regulating the pro-survival pathway.

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that post-transcriptionally regulate the pathological processes of various liver diseases including hepatitis C virus (HCV) infection. In the present study, we demonstrated that HCV infection enhanced the expression of miR-199a-5p in HCV infected human hepatocytes and Huh7.5.1cells, as well as liver biopsy specimens. Inhibition of miR-199a-5p decreased HCV replication not only in terms of HCV RNA, but also the protein levels of NS3 and NS5A. Furthermore, we discovered that miR-199a-5p knockdown in Huh7.5.1 cells infected with genotype 2a (JFH1) or genotype 1b (SN1a) resulted in the remarkable inhibition of pro-survival pathways, as observed by the down-regulation of p-Akt, p-ERK and ß-catenin protein levels. Moreover, pre-treatment with the pro-survival pathway specific activator prominently ablated the inhibition of HCV replication induced by miR-199a-5p knockdown. Collectively, our results highlight the up-regulation of miR-199a-5p expression with HCV infection and the promotion of HCV replication by miR-199a-5p. Moreover, miR-199a-5p may facilitate HCV replication by regulating pro-survival pathways through PI3K/Akt, Ras/ERK and Wnt/ß-catenin. miR-199a-5p might be a potential drug target for developing a novel strategy to combat HCV infection.

Urinary metabonomics for diagnosis of depression in hepatitis B virus-infected patients.

BACKGROUND: Depression is concomitantly presented in Hepatitis B Virus (HBV)-infected patients and decreases these patients' quality of life. However, there are no laboratory-based methods to objectively diagnose this disorder. OBJECTIVES: The aim of this study was to investigate the alteration of urinary metabolites in depressed HBV-infected patients. PATIENTS AND METHODS: In this study, 81 depressed HBV-infected patients, 68 non-depressed HBV-infected patients and 64 Healthy Controls (HC) were recruited. A nuclear magnetic resonance (NMR)-based urinary metabonomic method was used to characterize the urinary metabolic profiling of depressed and non-depressed subjects. RESULTS: Seventeen differential urinary metabolites responsible for discriminating depressed HBV-infected patients from non-depressed HBV-infected patients and HC were identified. Among these metabolites, pyruvate, isobutyrate, N-methylnicotinamide, α-hydroxybutyrate, acetoacetate and malonate were identified as potential biomarkers for diagnosing depression in HBV-infected patients. A combined panel of these potential biomarkers could effectively discriminate depressed HBV-infected patients from non-depressed HBV-infected patients and HC, with an average accuracy of 89.6% in the training set and a predictive accuracy of 86.4% in the test set. CONCLUSIONS: These findings suggest that NMR-based urinary metabonomics approach might be a useful tool for the clinical diagnosis of depression in HBV-infected patients and the six potential biomarkers could be helpful for developing an objective diagnostic method. Limited by the number of recruited subjects, future studies are required to validate our conclusions.

Expression of serum exosomal microRNA-21 in human hepatocellular carcinoma.

New strategies for the diagnosis of hepatocellular carcinoma (HCC) are urgently needed. There is an increasing interest in using microRNAs (miRNAs) as biomarkers in diseases. In this study, we examined the expression of miR-21 in serum exosomes from patients with HCC or chronic hepatitis B (CHB) and investigated the potential clinical significance of miR-21. Quantitative RT-PCR indicated that the concentration of miR-21 was significantly higher in exosomes than in exosome-depleted supernatants or the whole serum. Further, the expression level of serum exosomal miR-21 was significantly higher in patients with HCC than those with CHB or healthy volunteers (P < 0.0001, P < 0.0001, resp.). High level of miR-21 expression correlated with cirrhosis (P = 0.024) and advanced tumor stage (P = 0.001). Although serum level of miR-21 was higher in patients with HCC than in patients with CHB and healthy volunteers, the sensitivity of detection is much lower than using exosomal miR-21. These findings indicate that miR-21 is enriched in serum exosomes which provides increased sensitivity of detection than whole serum. Exosomal miR-21 may serve as a potential biomarker for HCC diagnosis.

The association between CCL2 polymorphisms and drug-resistant epilepsy in Chinese children.

The treatment of drug-resistant epilepsy remains a major challenge, affecting approximately 30% of epilepsy patients. More recently, immunity and inflammation are considered to be key elements of epilepsy. Targeting brain inflammation may represent a novel therapeutic strategy for epilepsy and refractory epilepsy. In this study, we investigated the association of a tag SNP of the CCL2 gene, rs1024611 (originally designated as -2578G>A or -2518G>A) with drug-resistant epilepsy in Chinese children with epilepsy. We enrolled 484 epilepsy patients, including 98 drug-resistant patients and 386 drug-responsive patients. The rs1024611 was genotyped by PCR-RPLP. The rs1024611 AA genotype was associated with a greater susceptibility to drug-resistant epilepsy (p=0.008; OR=2.51, 95% CI: 1.33-4.72), adjusted for age, sex, and seizure type, and the association remained significant after Bonferroni correction for multiple testing (p<0.05). Our results demonstrate that the CCL2 genetic polymorphism is associated with drug-resistant epilepsy in Chinese paediatric patients.

Elevated levels of adiponectin and other cytokines in drug naïve, first episode schizophrenia patients with normal weight.

OBJECTIVE: The study was to examine the levels of adiponectin (APN) and other cytokines, and body metabolism in drug naïve, first episode schizophrenia patients with normal weight. METHODS: Ninety-six drug naïve, first episode schizophrenia patients with normal weight (SZ group), 60 healthy individuals with normal weight (control group), and 60 overweight or obese but otherwise healthy individuals (obesity group) were enrolled in the study. Serum levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and APN were measured using enzyme linked immunosorbent assay (ELISA). Glucose oxidase was used to measure plasma glucose level. Lipid levels were measured using the enzymatic colorimetric method. RESULTS: Serum levels of IL-1ß, IL-6 and TNF-α in both the SZ group and the obesity group were significantly higher than those in the control group (p's<0.001). There were no significant differences between the SZ group and the obesity group on those cytokines (p's>0.05). In addition, the levels of APN were significantly higher in the SZ group (p<0.001), and significantly lower in the obesity group (p<0.01) compared with the control group. Further, there were significant positive relationships between levels of APN and levels of other cytokines within the SZ group (p's<0.05); in contrast, there were significant negative relationships between levels of APN and levels of other cytokines within the obesity group (p's<0.05). Fasting serum levels of glucose, LDL, triglycerides and total cholesterol were significantly higher, and fasting serum levels of HDL were significantly lower in the obesity group compared with the other two groups (p's<0.01). There were no significant differences in any of the metabolic parameters between the control group and the SZ group (p's>0.05). CONCLUSIONS: Drug naïve, first episode schizophrenia patients with normal weight seem to present an up-regulated inflammatory status as reflected by elevated levels of IL-1ß, IL-6, and TNF-α. APN may play a unique pro-inflammatory role in this patient population. Implications of the findings in relation to the pathogenesis of schizophrenia and the vulnerability for metabolic problems were discussed.

[Correlation of the expression of NF-κB p65 and hepatic fibrosis in hepatitis patients].

OBJECTIVE: To explore the relationship between the expression of NF-κB p65 and hepatic fibrosis in chronic hepatitis B (CHB) patients. METHODS: Sixty CHB patients with hepatic fibrosis underwent liver biopsy to determine the stages of liver fibrosis (S0-S4). The distribution and expression of collagens I, III and NF-κB p65 in different stages of fibrosis in liver tissue were observed by immunohistochemistry and the results analyzed statistically. RESULTS: The expression of NF-κB p65 was positively correlated with the stage of hepatic fibrosis. That was S4 > S3 > S2 > S1 (S0) (P < 0.01). And it was also positively correlated with the expression of collagens I and III (P < 0.01). CONCLUSION: The elevated expression of NF-κB p65 is closely associated with the occurrence and development of hepatic fibrosis. Its mechanism is probably related with the increased secretion of collagens I and III after the activation of hepatic stellate cell.

A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance.

Acid chaperones are essential factors in preserving the protein homeostasis for enteric pathogens to survive in the extremely acidic mammalian stomach (pH 1-3). The client proteins of these chaperones remain largely unknown, primarily because of the exceeding difficulty of determining protein-protein interactions under low-pH conditions. We developed a genetically encoded, highly efficient protein photocrosslinking probe, which enabled us to profile the in vivo substrates of a major acid-protection chaperone, HdeA, in Escherichia coli periplasm. Among the identified HdeA client proteins, the periplasmic chaperones DegP and SurA were initially found to be protected by HdeA at a low pH, but they subsequently facilitated the HdeA-mediated acid recovery of other client proteins. This unique, ATP-independent chaperone cooperation in the ATP-deprived E. coli periplasm may support the acid resistance of enteric bacteria. The crosslinker would be valuable in unveiling the physiological interaction partners of any given protein and thus their functions under normal and stress conditions.

[Effects of oxymatrine on serum cytokines and hepatic fibrotic indexes in patients with chronic hepatitis B].

OBJECTIVE: To study the effect of oxymatrine on serum cytokines and hepatic fibrotic indexes in patients with chronic hepatitis B (CHB). METHODS: Eighty-two CHB patients were randomly assigned to the control group treated with Western routine therapy and the treated group treated by Western routine therapy plus oxymatrine. The treatment course was 24 weeks. Another group with 20 healthy subjects was set as the normal control group. The serum levels of transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6), hyaline acid (HA), collagen type IV (C-IV) and laminin (LN) were measured by ELISA and RIA before and after treatment. RESULTS: The serum levels of TGF-beta1, IL-6, HA, C-IV and LN in CHB patients were significantly higher than those in the normal control group (P < 0.01). After 24-week treatment with oxymatrine, these abnormal indexes in the treated group were significantly lowered, as compared with those before treatment and in the control group, the differences were significant (all P < 0.01). CONCLUSION: Oxymatrine could lower the levels of cytokines, including TGF-beta1, IL-6, etc. in patients with CHB, which might be one of the mechanisms of its action in reversing liver fibrosis.

Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells.

AIM: To investigate the effect of the serum of patients with chronic hepatitis B (CHB) on apoptosis of renal tubular epithelial cells in vitro and to study the role of hepatitis B virus (HBV) and transforming growth factor-beta (1) (TGF-beta (1)) in the pathogenesis of hepatitis B virus associated glomerulonephritis (HBV-GN). METHODS: The levels of serum TGF-beta(1) were measured by specific enzyme linked immunosorbent assay (ELISA) and HBV DNA was tested by polymerase chain reaction (PCR) in 44 patients with CHB ,and 20 healthy persons as the control. The normal human kidney proximal tubular cell (HK-2) was cultured together with the sera of healthy persons, CHB patients with HBV-DNA negative (20 cases) and HBV-DNA positive (24 cases) for up to 72 h. Apoptosis and Fas expression of the HK-2 were detected by flow cytometer. RESULTS: The apoptosis rate and Fas expression of HK-2 cells were significantly higher in HBV DNA positive serum group 19.01%+/-5.85% and 17.58%+/-8.35%, HBV DNA negative serum group 8.12%+/-2.80% and 6.96%+/-2.76% than those in control group 4.25%+/-0.65% and 2.33%+/-1.09%, respectively (P<0.01). The apoptosis rate and Fas expression of HK-2 in HBV DNA positive serum group was significantly higher than those in HBV DNA negative serum (P<0.01). Apoptosis rate of HK-2 cells in HBV DNA positive serum group was positively correlated with the level of HBV-DNA (r = 0.657). The level of serum TGF-beta (1) in CHB group was 163.05+/-91.35 microg/L, significantly higher as compared with 81.40+/-40.75 microg/L in the control group (P<0.01). CONCLUSION: The serum of patients with chronic hepatitis B promotes apoptotic damage in human renal tubular cells by triggering a pathway of Fas up-regulation. HBV and TGF-beta (1) may play important roles in the mechanism of hepatitis B virus associated glomerulonephritis.