Study on the Pathogenesis of Foreign Body Granulomatous Inflammation in the Livers of Sprague-Dawley Rats

Focal granulomatous inflammation developed in the livers of five 10-week-old male Sprague-Dawley rats. The characteristic features of this lesion were the presence of foreign body multinucleated giant cells engulfing calcium deposits and site-specific development in a fissure formed in a sub-lobation in the left lobe or interlobar fissure of the medial lobe of the liver. To clarify the pathogenesis of this lesion, rat livers showing abnormal sub-lobation or lobar atrophy, rat livers in an acute dermal toxicity study and guinea pig livers in a skin sensitization test were also examined histologically. Consequently, the present lesion was considered to be a reactive change against calcium that was dystrophically deposited in the area of hepatocellular necrosis due to delayed circulatory disturbance caused by external pressure or extension force. Granulomatous lesions like in the present cases should be differentiated from those caused by evident exogenous pathogens such as chemicals or microorganisms

Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray

BACKGROUND: Mycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity. RESULTS: Citrinin inhibited growth of yeast cells at a concentration higher than 100 ppm. We monitored the citrinin-induced mRNA expression profiles in yeast using the ORF DNA microarray and Oligo DNA microarray, and the expression profiles were compared with those of the other stress-inducing agents. Results obtained from both microarray experiments clustered together, but were different from those of the mycotoxin patulin. The oxidative stress response genes – AADs, FLR1, OYE3, GRE2, and MET17 – were significantly induced. In the functional category, expression of genes involved in "metabolism", "cell rescue, defense and virulence", and "energy" were significantly activated. In the category of "metabolism", genes involved in the glutathione synthesis pathway were activated, and in the category of "cell rescue, defense and virulence", the ABC transporter genes were induced. To alleviate the induced stress, these cells might pump out the citrinin after modification with glutathione. While, the citrinin treatment did not induce the genes involved in the DNA repair. CONCLUSION: Results from both microarray studies suggest that citrinin treatment induced oxidative stress in yeast cells. The genotoxicity was less severe than the patulin, suggesting that citrinin is less toxic than patulin. The reproducibility of the expression profiles was much better with the Oligo DNA microarray. However, the Oligo DNA microarray did not completely overcome cross hybridization

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

Reversible formation and cleavage of Pt→Ag dative bonds in a pre-organized cavity of a luminescent heteropolynuclear platinum(ii) complex

A U-shaped Pt2Ag2 complex [Pt2Ag2(ppy)2(Ph2pz)4] with a pre-organized cavity (ppy = 2-phenylpridinate and Ph2pz = 3,5-diphenylpyrazolate) and related complexes have been prepared. The Pt2Ag2 complexes react with Ag(I) ions to give the corresponding Pt2Ag3 complexes containing Pt→Ag dative bonds. It became obvious that the existence of the C(ipso) atom in the chelate ligand is important as the driving force for forming Pt→Ag dative bonds. However, once the Pt2Ag3 complex is formed, the trapped Ag(I) ion is mainly stabilized by the Pt→Ag dative bonds, which are stronger than the Ag?C(ipso) bond. The trapped Ag(I) ion can be abstracted from the cavity selectively by adding an equivalent amount of chloride ion into the solution of Pt2Ag3 complexes to reproduce the original Pt2Ag2 complexes.Related illustration appears in this issue\u27s inside back cover. The URL is as follows: http://dx.doi.org/10.1039/C7CC90232

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

Suv39h-Mediated Histone H3 Lysine 9 Methylation Directs DNA Methylation to Major Satellite Repeats at Pericentric Heterochromatin

AbstractBackground: Histone H3 lysine 9 (H3-K9) methylation and DNA methylation are characteristic hallmarks of mammalian heterochromatin. H3-K9 methylation was recently shown to be a prerequisite for DNA methylation in Neurospora crassa and Arabidopsis thaliana. Currently, it is unknown whether a similar dependence exists in mammalian organisms.Results: Here, we demonstrate a physical and functional link between the Suv39h-HP1 histone methylation system and DNA methyltransferase 3b (Dnmt3b) in mammals. Whereas in wild-type cells Dnmt3b interacts with HP1α and is concentrated at heterochromatic foci, it fails to localize to these regions in Suv39h double null (dn) mouse embryonic stem (ES) cells. Consistently, the Suv39h dn ES cells display an altered DNA methylation profile at pericentric satellite repeats, but not at other repeat sequences. In contrast, H3-K9 trimethylation at pericentric heterochromatin is not impaired in Dnmt1 single- or Dnmt3a/Dnmt3b double-deficient ES cells. We also show that pericentric heterochromatin is not transcriptionally inert and can give rise to transcripts spanning the major satellite repeats.Conclusions: These data demonstrate an evolutionarily conserved pathway between histone H3-K9 methylation and DNA methylation in mammals. While the Suv39h HMTases are required to direct H3-K9 trimethylation and Dnmt3b-dependent DNA methylation at pericentric repeats, DNA methylation at centromeric repeats occurs independent of Suv39h function. Thus, our data also indicate a more complex interrelatedness between histone and DNA methylation systems in mammals. Both methylation systems are likely to be important in reinforcing the stability of heterochromatic subdomains and thereby in protecting genome integrity

Pretransplant serum hepatitis C virus RNA levels predict response to antiviral treatment after living donor liver transplantation.

[Background]Given the limited efficacy and high adverse event rate associated with treatment of recurrent hepatitis C after liver transplantation, an individualized treatment strategy should be considered. The aim of this study was to identify predictors of response to antiviral therapy for hepatitis C after living donor liver transplantation (LDLT) and to study the associated adverse events. [Methods]A retrospective chart review was performed on 125 hepatitis C virus (HCV)-positive LDLT recipients who received interferon plus ribavirin and/or peginterferon plus ribavirin therapy at Kyoto University between January 2001 and June 2011. [Results]Serum HCV RNA reached undetectable levels within 48 weeks in 77 (62%) of 125 patients, and these patients were defined as showing virological response (VR). Of 117 patients, 50 (43%) achieved sustained VR (SVR). Predictive factors associated with both VR and SVR by univariate analysis included low pretransplant serum HCV RNA levels, a non-1 HCV genotype, and low pretreatment serum HCV RNA levels. In addition, LDLT from ABO-mismatched donors was significantly associated with VR, and white cell and neutrophil counts before interferon therapy were associated with SVR. Multivariate analysis showed that 2 variables–pretransplant serum HCV RNA level less than 500 kIU/mL and a non-1 HCV genotype–remained in models of both VR and SVR and that an ABO mismatch was associated with VR. No variables with a significant effect on treatment withdrawal were found. [Conclusions]Virological response to antiviral therapy in patients with hepatitis C recurring after LDLT can be predicted prior to transplant, based on pretransplant serum HCV-RNA levels and HCV genotype. LDLT from ABO-mismatched donors may contribute to more efficacious interferon therapy

Development of healthy lifestyle consciousness index for gynecological cancer patients

PURPOSE: Healthy lifestyle is related to quality of life (QOL) after cancer diagnosis and prognosis. However, there are few reports on patients conscious of healthy lifestyle and patients requiring medical providers' attention regarding healthy lifestyle. We aimed to develop a healthy lifestyle consciousness index (HLCI) for cancer patients and evaluated its validity in gynecological cancer patients. METHODS: The HLCI was designed to assess degree of healthy lifestyle consciousness, including items regarding "diet, " "exercise, " "body weight, " and "sleep." Exploratory factor analysis was performed for dimensionality of the scale; Cronbach's alpha was calculated to assess internal-consistency reliability. For criterion-based validity, we calculated proportions of stage III/IV gynecological malignancies in those with categorized HLCI scores based on tertiles. Concurrent validity was evaluated between HLCI and other quality of life (QOL) scales including European Organization for Research and Treatment of Cancer QLQ-C30 in limited patients. RESULTS: HLCI comprised five 10-point items (0-45); higher values implied improved healthy lifestyle consciousness. Data from 108 gynecological malignancy patients at Kyoto University Hospital were analyzed. The mean age of subjects was 55.8 years; 36.1% of them had uterine corpus cancer; 34.3% were at stage III/IV of gynecological malignancy. The factor analysis revealed HLCI was unidimensional; the reliability based on Cronbach's alpha was satisfactory (0.88). The proportions of stage III/IV gynecological malignancies were 25.7%, 33.3%, and 44.4% in those with first (7-24 points), second (25-30 points), and third (31-46 points) tertiles of HLCI score, respectively. For patients with other QOL scales (n = 25), the mean scores of global health status of QLQ-C30 were 33.3, 50.0, and 83.3 for first, second, and third tertiles of HLCI score, respectively. CONCLUSION: HLCI was successfully validated; thus, patients with advanced stages or higher QOL might have strong consciousness regarding healthy lifestyle. HLCI may be useful in precision care for improved lifestyles and QOL

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