Genetic characterization of pancreatic cancer patients and prediction of carrier status of germline pathogenic variants in cancer-predisposing genes.

BACKGROUND: National Comprehensive Cancer Network (NCCN) recently recommended germline genetic testing for all pancreatic cancer patients. However, the genes targeted by genetic testing and the feasibility of selecting patients likely to carry pathogenic variants have not been sufficiently verified. The purpose of this study was to genetically characterize Japanese patients and examine whether the current guideline is applicable in this population. METHODS: Using targeted sequencing, we analyzed the coding regions of 27 cancer-predisposing genes in 1,005 pancreatic cancer patients and 23,705 controls in Japan. We compared the pathogenic variant frequency between cases and controls and documented the demographic and clinical characteristics of carrier patients. We then examined if it was possible to use machine learning to predict carrier status based on those characteristics. FINDINGS: We identified 205 pathogenic variants across the 27 genes. Pathogenic variants in BRCA2, ATM, and BRCA1 were significantly associated with pancreatic cancer. Characteristics associated with carrier status were inconsistent with previous investigations. Machine learning classifiers had a low performance in determining the carrier status of pancreatic cancer patients, while the same classifiers, when applied to breast cancer data as a positive control, had a higher performance that was comparable to that of the NCCN guideline. INTERPRETATION: Our findings support the clinical significance of multigene panel testing for pancreatic cancer and indicate that at least 3.4% of Japanese patients may respond to poly (ADP ribose) polymerase inhibitor treatments. The difficulty in predicting carrier status suggests that offering germline genetic testing for all pancreatic cancer patients is reasonable. FUNDING: AMED under Grant Number JP19kk0305010 and Australian National Health and Medical Research funding (ID177524).

Towards identification of bioactive compounds in cold vacuum extracted double cherry blossom (Gosen-Sakura) leaves.

The present research examines the possibility of finding bio-molecular compounds from the double cherry blossom (termed as 'Gosen-Sakura' of Gosen-city, Niigata-prefecture, Japan) leaves, which have been long used in the preparation of the traditional Japanese sweet (wagashi) - 'sakura-mochi'. Based on its indicated anti-microbial properties historically, our study provides a new low temperature vacuum extraction method for extracting 'near natural form of water soluble leaf (cell) extracts from the Gosen-Sakura, and demonstrates the presence of some 'novel' compound(s) with anti-tumor cell lines proliferation inhibitory affects through the MTT assay. To our knowledge, no reports exist on the sakura tree 'leaf (cell) extracts' inhibiting tumor cell line growth. We further examined and compared the effects of known compounds with anti-tumor activity, coumarin and benzyl alcohol with Gosen-Sakura leaf extract; results lead us to hypothesize that the Gosen-Sakura leaf extract contains substance(s) other than the above two known compounds, with antitumor effect. Additionally, we speculate on the underlying mechanism of action of the Gosen-Sakura leaf extract by targeting cell division at the point of DNA synthesis and causing apoptosis. In conclusion, we present scientific evidence on the presence of certain 'novel' biomolecule(s), with anti-tumor activity, in the Gosen-Sakura leaf which has been long used in Japanese sweet - the 'sakura-mochi'.

Long-term dietary supplementation with saury oil attenuates metabolic abnormalities in mice fed a high-fat diet: combined beneficial effect of omega-3 fatty acids and long-chain monounsaturated fatty acids.

BACKGROUND: Pacific saury is a common dietary component in East Asia. Saury oil contains considerable levels of n-3 unsaturated fatty acids (PUFA) and long-chain monounsaturated fatty acids (LCMUFA) with aliphatic tails longer than 18 carbons. In our previous study, consumption of saury oil for 4 to 6 wk improved insulin sensitivity and the plasma lipid profile in mice. However, the long-term effects of saury oil on metabolic syndrome (MetS) risk factors remain to be demonstrated. In the current study, we examined the long-term effects of saury oil on mice fed a high-fat diet, and compared the effect of n-3 PUFA EPA and LCMUFA on MetS risk factor in diet-induced obese mice. METHODS AND RESULTS: In Experiment 1, male C57BL/6 J mice were fed either a 32% lard diet (control) or a diet containing 22% lard plus 10% saury oil (saury oil group) for 18 weeks. Although no differences were found in body weight and energy expenditure between the control and saury oil groups, the saury oil diet decreased plasma insulin, non-HDL cholesterol, hepatic steatosis, and adipocyte size, and altered levels of mRNA transcribed from genes involved in insulin signaling and inflammation in adipose tissue. Organ and plasma fatty acid profile analysis revealed that consumption of saury oil increased n-3 PUFA and LCMUFA (especially n-11 LCMUFA) levels in multiple organs, and decreased the fatty acid desaturation index (C16:1/C16:0; C18:1/C18:0) in liver and adipose tissue. In Experiment 2, male C57BL/6 J mice were fed a 32% lard diet (control), a diet containing 28% lard plus 4% EPA (EPA group), or a diet containing 20% lard plus 12% LCMUFA concentrate (LCMUFA group) for 8 weeks. EPA or LCMUFA intake increased organ levels of EPA and LCMUFA, respectively. Consumption of EPA reduced plasma lipid levels and hepatic lipid deposition, and decreased the fatty acid desaturation index in liver and adipose tissue. Consumption of LCMUFA decreased plasma non-HDL cholesterol, improved hyperinsulinemia, and decreased the fatty acid desaturation index in adipose tissue. EPA accumulated mainly in liver, and LCMUFA (especially n-11 LCMUFA) accumulated mainly in white adipose tissue, suggesting their possible individual biological effects for improving MetS. CONCLUSION: Our results suggest that saury oil-mediated improvement of metabolic syndrome in diet-induced obese mice may possibly be due to a combined effect of n-3 PUFA and LCMUFA.

Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells.

The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an "epimutagen combination" that is effective at low concentrations as detected in maternal peripheral and cord blood.

Dietary supplementation with long-chain monounsaturated fatty acids attenuates obesity-related metabolic dysfunction and increases expression of PPAR gamma in adipose tissue in type 2 diabetic KK-Ay mice.

The objective of present study was to examine the effect of long-chain monounsaturated fatty acids (LC-MUFAs) with chain lengths longer than 18 (i.e., C20:1 and C22:1 isomers combined) on obesity-related metabolic dysfunction and its molecular mechanisms. Type-2 diabetic KK-Ay mice (n = 20) were randomly assigned to the 7% soybean oil-diet group (control group) and 4% LC-MUFA concentrate-supplemented-diet group (LC-MUFA group). At 8 weeks on the diet, the results showed that plasma, liver and adipose tissue levels of C20:1 and C22:1 isomers increased significantly with LC-MUFA treatment. Supplementation with LC-MUFAs markedly reduced white fat pad weight as well as adipocyte size in the mice. The levels of plasma free fatty acids, insulin, and leptin concentration in the obese diabetic mice of the LC-MUFA group were also decreased as compared with the mice in the soybean oil-diet control group. Dietary LC-MUFAs significantly increased the mRNA expression of peroxisome proliferator-activated receptor gamma (Pparg), lipoprotein lipase (Lpl), fatty acid transport protein (Fatp), fatty acid translocase/CD36 (Cd36), as well as mRNA expression of genes involved in lipid oxidation such as carnitine palmitoyltransferase-1A (Cpt1a) and citrate synthase (Cs), and decreased the mRNA expression of inflammatory marker serum amyloid A 3 (Saa3) in the adipose tissues of diabetic mice. The results suggest that LC-MUFAs may ameliorate obesity-related metabolic dysfunction partly through increased expression of Pparg as well as its target genes, and decreased inflammatory marker expression in white adipose tissue.

Characterization of nitrated phenolic compounds for their anti-oxidant, pro-oxidant, and nitration activities.

Coffee is one of the most widely consumed beverages worldwide. Evidence of the health benefits and the important contribution of coffee brew to the intake of anti-oxidants in the diet has increased coffee consumption. Chlorogenic acid (ChA) and caffeic acid (CaA) are the major phenolic compounds in coffee. However, phenolic compounds, which are generally effective anti-oxidants, can become pro-oxidants in the presence of Cu(2+) to induce DNA damage under certain conditions. On the other hand, sodium nitrite (NaNO(2)) is widely used as a food additive to preserve and tinge color on cured meat and fish. It is possible that phenolic compounds react with NaNO(2) under acidic conditions, such as gastric juice. In this study, we identified compounds produced by the reaction between ChA or CaA in coffee and NaNO(2) in artificial gastric juice. The identified phenolic compounds and nitrated phenolic compounds were assessed for their anti-oxidant, pro-oxidant, and nitration activities by performing an in vitro assay. The nitrated phenolic compounds seemed to show increased anti-oxidant activity and decreased pro-oxidant activity. However, one nitrated CaA compound that has a furoxan ring showed the ability to release NO(2)(-) in the neutral condition.

Development of quantitative analysis of 8-nitroguanine concomitant with 8-hydroxydeoxyguanosine formation by liquid chromatography with mass spectrometry and glyoxal derivatization.

Under inflammatory conditions, both 8-nitroguanine (NO2Gua) and 8-hydroxydeoxyguanosine (8-OHdG) are found in tissues. Measurements of the two types of damaged bases on nucleotides are expected to provide information pointing to the possible correlation between inflammation and carcinogenesis. For the establishment of an in vivo model, in this study, a sensitive and precise method for the determination of NO2Gua, which uses liquid chromatography with mass spectrometry (LC-MS) and 6-methoxy-2-naphthyl glyoxal (MTNG) derivatization, was developed in vitro. The procedure for DNA digestion in this method is identical to that widely used for 8-OHdG measurement, which enables us to detect the two damaged bases in the same DNA sample. In order to validate our method, we measured NO2Gua levels in DNA sample using LC-MS. A mass spectrometer equipped with an electrospray atmospheric pressure ionization source and operated in the negative ion mode (ESI-) was set up with selective ion monitoring at m/z 391 and 394 for NO2Gua-MTNG and [13C, 15N2]-NO2Gua-MTNG as surrogate standard, respectively. The average recoveries from DNA samples spiked with 25, 50 and 250 nM NO2Gua were 99.4, 99.8 and 99.1% with correction using the added surrogate standard, respectively. The limit of quantification was 3.0 nM for NO2Gua. To ascertain the applicability of our method to DNA samples harboring the two damaged bases, we measured NO2Gua and 8-OHdG levels in calf thymus DNA treated with ONOO-. As a result, both NO2Gua and 8-OHdG levels were clearly increased with ONOO- dose dependency, the amount of NO2Gua at the high dose ONOO- being almost the same as those of 8-OHdG. LC-MS was able to determine NO2Gua in a small amount of DNA sample, and is therefore expected to be a very powerful tool for the evaluation of DNA damage induced by reactive nitrogen species.