A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.     

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background

Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.

Methodology

DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.

Results

A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.

Conclusions

This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.     

Biological Activities of Triterpenoids and Phenolic Compounds from Myrica cerifera Bark

Seven triterpenoids, 1  –  7 , two diarylheptanoids, 8 and 9 , four phenolic compounds, 10  –  13 , and three other compounds, 14  –  16 , were isolated from the hexane and MeOH extracts of the bark of Myrica cerifera L. (Myricaceae). Among these compounds, betulin ( 1 ), ursolic acid ( 3 ), and myricanol ( 8 ) exhibited cytotoxic activities against HL60 (leukemia), A549 (lung), and SK‐BR‐3 (breast) human cancer cell lines (IC₅₀ 3.1 – 24.2 μm ). Compound 8 induced apoptotic cell death in HL60 cells (IC₅₀ 5.3 μm ) upon evaluation of the apoptosis‐inducing activity by flow cytometric analysis and by Hoechst 33342 staining method. Western blot analysis on HL60 cells revealed that 8 activated caspases‐3, ‐8, and ‐9 suggesting that 8 induced apoptosis via both mitochondrial and death receptor pathways in HL60. Upon evaluation of the melanogenesis‐inhibitory activity in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), erythrodiol ( 7 ), 4‐hydroxy‐2‐methoxyphenyl β‐d ‐glucopyranoside ( 13 ), and butyl quinate ( 15 ) exhibited inhibitory effects (65.4 – 86.0% melanin content) with no, or almost no, toxicity to the cells (85.9 – 107.4% cell viability) at 100 μm concentration. In addition, 8 , myricanone ( 9 ), myricitrin ( 10 ), protocatechuic acid ( 11 ), and gallic acid ( 12 ) revealed potent DPPH radical‐scavenging activities (IC₅₀ 6.9 – 20.5 μm ).     

Network Analysis of a Comprehensive Knowledge Repository Reveals a Dual Role for Ceramide in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of senile dementia. Many inflammatory factors such as amyloid-β and pro-inflammatory cytokines are known to contribute to the inflammatory response in the AD brain. Sphingolipids are widely known to have roles in the pathogenesis of inflammatory diseases, where the precise roles for sphingolipids in inflammation-associated pathogenesis of AD are not well understood. Here we performed a network analysis to clarify the importance of sphingolipids and to model relationships among inflammatory factors and sphingolipids in AD. In this study, we have updated sphingolipid signaling and metabolic cascades in a map of AD signaling networks that we named “AlzPathway,” a comprehensive knowledge repository of signaling pathways in AD. Our network analysis of the updated AlzPathway indicates that the pathways related to ceramide are one of the primary pathways and that ceramide is one of the important players in the pathogenesis of AD. The results of our analysis suggest the following two prospects about inflammation in AD: (1) ceramide could play important roles in both inflammatory and anti-inflammatory pathways of AD, and (2) several factors such as Sphingomyelinase and Siglec-11 may be associated with ceramide related inflammation and anti-inflammation pathways in AD. In this study, network analysis of comprehensive knowledge repository reveals a dual role for ceramide in AD. This result provides a clue to clarify sphingolipids related inflammatory and anti-inflammatory pathways in AD.     

Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54^high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54^high or B2^high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.     

Association between Smoking Status and Obesity in a Nationwide Survey of Japanese Adults

Objective

A positive association between the number of cigarettes smoked per day and obesity has been reported, whereas how other smoking-related indices, such as pack-years and duration of smoking, are related with obesity has been less investigated. We analyzed the age-adjusted cross-sectional association between smoking and obesity in a general Japanese population.

Methods

We used data from a nationwide epidemiological study of Japanese adults (N = 23,106). We compared the prevalence of obesity (defined as body mass index ≥ 25kg/m²) among groups classified by smoking behavior, pack-years, number of cigarettes per day, duration of smoking, and duration and time of smoking cessation.

Results

In men, current smokers had a lower odds ratio (OR) for obesity of 0.80 (95% confidence interval (CI): 0.72–0.88) compared to non-smokers, whereas past smokers had a higher OR of 1.23 (95% CI: 1.09–1.37) compared to current smokers. In women, there were no differences in obesity between the three groups classified by smoking behavior. However, in both sexes, the prevalence of obesity tended to increase with pack-years and the number of cigarettes per day, but not with duration of smoking in current and past smokers. Further, in male smokers, the risks for obesity were markedly higher in short-term heavy smokers compared with long-term light smokers, even with the same number of pack-years. Regarding the impact of smoking cessation, female past smokers who quit smoking at an age > 55-years had an elevated OR of 1.60 (95% CI:1.05–2.38) for obesity.

Conclusions

In a general Japanese population, obesity is progressively associated with pack-years and number of cigarettes per day, but not with the duration of smoking. When investigating the association between obesity and cigarette smoking, the daily smoking burden and the duration of smoking require to be independently considered.     

Agatharesinol biosynthesis-related changes of ray parenchyma in sapwood sticks of Cryptomeria japonica during cell death

Planta - The work demonstrates a relationship between the biosynthesis of the secondary metabolite, agatharesinol, and cytological changes that occur in ray parenchyma during cell death in sapwood...     

Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.