DNA methyltransferase 3B plays a protective role against hepatocarcinogenesis caused by chronic inflammation via maintaining mitochondrial homeostasis

Most hepatocellular carcinomas (HCCs) develop on the basis of chronic hepatitis, but the mechanism of epigenetic regulation in inflammatory hepatocarcinogenesis has yet to be elucidated. Among de novo DNA methyltransferases (DNMTs), DNMT3B has lately been reported to act specifically on actively transcribed genes, suggesting the possibility that it plays a role in the pathogenesis of cancer. We confirmed that DNMT3B isoforms lacking its catalytic domain were highly expressed in HCCs compared with non-tumorous liver tissue. To elucidate the role of DNMT3B in hepatocarcinogenesis, we generated a genetically engineered mouse model with hepatocyte-specific Dnmt3b deletion. The liver of the Dnmt3b-deficient mice exhibited an exacerbation of thioacetamide-induced hepatitis, progression of liver fibrosis and a higher incidence of HCC compared with the liver of the control mice. Whole-genome bisulfite sequencing verified a lower CG methylation level in the Dnmt3b-deficient liver, demonstrating differentially methylated regions throughout the genome. Transcriptome analysis revealed decreased expression of genes related to oxidative phosphorylation in the Dnmt3b-deficient liver. Moreover, primary hepatocytes isolated from the Dnmt3b-deficient mice showed reduced mitochondrial respiratory capacity, leading to the enhancement of oxidative stress in the liver tissue. Our findings suggest the protective role of DNMT3B against chronic inflammation and HCC development via maintaining mitochondrial homeostasis

Single-molecular real-time deep sequencing reveals the dynamics of multi-drug resistant haplotypes and structural variations in the hepatitis C virus genome

While direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have dramatically progressed, patients still suffer from treatment failures. For the radical eradication of HCV, a deeper understanding of multiple resistance-associated substitutions (RASs) at the single-clone level is essential. To understand HCV quasispecies and their dynamics during DAA treatment, we applied single-molecule real-time (SMRT) deep sequencing on sera from 12 patients with genotype-1b HCV infections with DAA treatment failures, both pre- and post-treatment. We identified >3.2 kbp sequences between NS3 and NS5A genes of 187, 539 clones in total, classifying into haplotype codes based on the linkage of seven RAS loci. The number of haplotype codes during the treatment, per sample, significantly decreased from 14.67 ± 9.12 to 6.58 ± 7.1, while the number of nonsynonymous codons on the seven RAS loci, per clone, significantly increased from 1.50 ± 0.92 to 3.64 ± 0.75. In five cases, the minority multi-drug resistant haplotypes at pre-treatment were identical to the major haplotypes at relapse. Moreover, various structural variations (SVs) were detected and their dynamics analysed. These results suggest that SMRT deep sequencing is useful for detecting minority haplotypes and SVs, and to evaluate the dynamics of viral genomes at the single-clone level

Evolutional transition of HBV genome during the persistent infection determined by single-molecule real-time sequencing

BACKGROUND: Although HBV infection is a serious health issue worldwide, the landscape of HBV genome dynamics in the host has not yet been clarified. This study aimed to determine the continuous genome sequence of each HBV clone using a single-molecule real-time sequencing platform, and clarify the dynamics of structural abnormalities during persistent HBV infection without antiviral therapy. PATIENTS AND METHODS: Twenty-five serum specimens were collected from 10 untreated HBV-infected patients. Continuous whole-genome sequencing of each clone was performed using a PacBio Sequel sequencer; the relationship between genomic variations and clinical information was analyzed. The diversity and phylogeny of the viral clones with structural variations were also analyzed. RESULTS: The whole-genome sequences of 797, 352 HBV clones were determined. The deletion was the most common structural abnormality and concentrated in the preS/S and C regions. Hepatitis B e antibody (anti-HBe)-negative samples or samples with high alanine aminotransferase levels have significantly diverse deletions than anti-HBe-positive samples or samples with low alanine aminotransferase levels. Phylogenetic analysis demonstrated that various defective and full-length clones evolve independently and form diverse viral populations. CONCLUSIONS: Single-molecule real-time long-read sequencing revealed the dynamics of genomic quasispecies during the natural course of chronic HBV infections. Defective viral clones are prone to emerge under the condition of active hepatitis, and several types of defective variants can evolve independently of the viral clones with the full-length genome

Mutational spectrum of hepatitis C virus in patients with chronic hepatitis C determined by single molecule real-time sequencing

The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure

Deep-vein thrombosis detection rates and consideration of the living environment in a tsunami disaster area during the disaster reconstruction phase: A cross-sectional study

Introduction: Tsunami victims of the Great East Japan Earthquake were screened for deep-vein thrombosis(DVT) in order to compare the DVT incidence rates between temporary and non-temporary housing residentgroups. Material and methods: Lower extremity venous ultrasonography was performed on 290 subjects (64 menand 226 women; mean age = 71.9 ± 7.9 years) at 44 months after the disaster. All subjects completedquestionnaires to gather information about their background factors which included the Kessler PsychologicalDistress Scale: K6. Results: The DVT detection rate was 10.7% in the temporary group. In the non-temporary group, it was 11.3%among the subjects who previously lived in temporary housing. For the subjects who were living in their own homes it was 9.2%. Psychological distress levels measured by K6 were significantly higher in the temporary housinggroup than in the non-temporary housing group. The multivariate analysis showed that the background factorassociated with DVT risk was SV (soleal vein) dilatation in all subjects as well as in the non-temporary housinggroup, while hypertension and use of sleeping pills were found to be the factors in the temporary housing group. Conclusions: DVT detection rates were similar between the temporary and non-temporary housing groups,and were higher than that in the Japanese general population. The psychological distress level of the tsunamivictims measured by K6 was also higher in the temporary housing group than in the non-temporary housinggroup. It is necessary to establish a long-term and awareness-raising disaster victim support system

Markedly improved outcomes and acceptable toxicity in adolescents and young adults with acute lymphoblastic leukemia following treatment with a pediatric protocol: a phase II study by the Japan Adult Leukemia Study Group

The superiority of the pediatric protocol for adolescents with acute lymphoblastic leukemia (ALL) has already been demonstrated, however, its efficacy in young adults remains unclear. The ALL202-U protocol was conducted to examine the efficacy and feasibility of a pediatric protocol in adolescents and young adults (AYAs) with BCR\u27ABL-negative ALL. Patients aged 15\u2724 years (n = 139) were treated with the same protocol used for pediatric B-ALL. The primary objective of this study was to assess the disease-free survival (DFS) rate and its secondary aims were to assess toxicity, the complete remission (CR) rate and the overall survival (OS) rate. The CR rate was 94%. The 5-year DFS and OS rates were 67% (95% confidence interval (CI) 58\u2775%) and 73% (95% CI 64\u2780%), respectively. Severe adverse events were observed at a frequency that was similar to or lower than that in children treated with the same protocol. Only insufficient maintenance therapy significantly worsened the DFS (hazard ratio 5.60, Po0.001).These results indicate that this protocol may be a feasible and highly effective treatment for AYA with BCR\u27ABL-negative ALL