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The aim of this study is to investigate the stabilities of the 24 synthetic cannabinoid metabolites (SCMs) in blood and urine at various temperatures from - 30 to 37 ℃ stored for 1-168 days. In addition, experiments of stabilities at lower temperatures and for much longer duration have been performed as described below.The quantification was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The blank blood and urine spiked with SCMs and non-spiked real case (authentic) specimens were incubated at 37 ℃ up to 56 days and at 22, 4 or - 30 ℃ up to 168 days. The non-spiked authentic blood and urine specimens were also stored at - 30 or - 80 ℃ for 1, 3 or 5 years to investigate stabilities during very long time frames.All the 24 SCMs were much more stable in urine than in blood at 37, 22 or 4 ℃. All 24 SCMs spiked into blood or urine were stable at - 30 ℃ for up to 168 days. The 6 SCMs in the authentic specimens exhibited long stabilities at - 30 or - 80 ℃ for 3-5 years. Some tendencies were observed according to the relation between the structures of SCMs and their stabilities.The long-term stabilities of 24 SCMs in spiked samples and those of 6 SCMs in the authentic specimens were examined using LC-MS/MS. SCMs were largely very stable and usable several years after storage at - 30 or - 80 ℃.

Purpose Quantification of olanzapine (OLZ) and its metabolites such as N-desmethylolanzapine (DM-O), 2-hydroxymethylolanzapine (2H-O) and olanzapine N-oxide (NO-O) in five kinds of human body fluids including whole blood by liquid chromatography (LC)–tandem mass spectrometry (MS/MS) has been presented; the quantification methods were carefully devised and validated using the matrix-matched calibration and standard addition methods. Methods OLZ and its three metabolites were extracted from 40 μL each of body fluids by two-step liquid–liquid separations. The samples and reagents were pre-cooled in a container filled with ice for the extraction because of the thermal instability of OLZ and its three metabolites especially in whole blood. Results The limits of quantification (LOQs) of OLZ and 2H-O were 0.05 ng/mL and those of DM-O and NO-O were 0.15 ng/mL in whole blood and urine, respectively. The concentrations of OLZ and its metabolites in heart whole blood, pericardial fluid, stomach contents, bile and urine were determined for two cadavers and those in whole blood and urine for the other two cadavers. The reduction from NO-O to OLZ was observed at 25 ℃ in whole blood in vitro. Conclusions To our knowledge, this is the first report on the quantification of metabolites of olanzapine in the authentic human body fluids by LC–MS/MS as well as on the confirmation of in vitro reduction from NO-O to OLZ in whole blood that seems to have induced the quick decrease of NO-O.

The quantification of parent molecules of pyrethroids tetramethrin and resmethrin in human specimens by a mass spectrometry (MS) technique has not been reported yet. A woman in her 60s was found dead in a wasteland. At the scene, an empty beer can and a spray for insecticides containing tetramethrin and resmethrin were found. Therefore, the concentrations of tetramethrin and resmethrin in postmortem specimens and the methanol solution used for rinsing the inside of the beer can were determined using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). The quantification method by LC–MS/MS for intact parent molecules of tetramethrin and resmethrin in whole blood and urine has been devised and validated in this work. The method was applied to the quantification of tetramethrin and resmethrin in whole blood, urine and stomach contents obtained from a cadaver at autopsy. The limits of detection of tetramethrin and resmethrin were 0.06 and 0.03 ng/mL; limits of quantification were 0.2 and 0.1 ng/mL in blood and urine, respectively. The concentrations of tetramethrin of the deceased were 11.1 ± 1.2 and 0.425 ± 0.017 ng/mL for stomach contents and urine, respectively; the concentration of resmethrin in stomach contents was 1.77 ± 0.18 ng/mL. The tetramethrin and resmethrin were unstable in blood and urine at room temperature; they should be kept at not higher than 4 ℃. To our knowledge, this is the first report for quantification of unchanged tetramethrin and resmethrin in human specimens obtained in a fatal case.

A decrease in nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of aging in multiple species, including humans. Our previous clinical trial showed that supplementation of 200 mg nicotinamide, a form of vitamin B3, increased blood NAD+ levels. Here we report a clinical trial to assess the efficacy of a lower and a higher dose nicotinamide on increasing NAD+ levels. Blood NAD+ levels were evaluated before and after treatment of 100 mg nicotinamide, 500 mg nicotinamide, or water only. The three treatment arms were tested in the same cohort of 5 healthy adults. Oral supplementation of 500 mg nicotinamide led to a significant increase in blood NAD+ after 12 h and showed a trend of increase after 48 h (P = 0.056), whereas 100 mg nicotinamide or water intake only did not change the NAD+ levels in the same subjects. Blood lipidome analysis showed that the levels of 277 lipid species significantly changed after 12 h among 1463 species detected, while no changes were detected at 0 h among the 3 groups. These lipid changes largely reflected the difference between the nicotinamide supplemented groups and the water control group. Together with our previous results, this study shows that oral supplementation of nicotinamide within the daily tolerable upper level is an effective way to transiently increase NAD+ levels and affect the composition of blood lipids.

In this study, solid tissues such as the lung, liver, kidney and urine were highlighted to profile the AB-PINACA in vivo metabolites in a fatal abuse case, although such metabolite analysis is usually made with urine specimens. We compared the relative peak intensities of in vivo metabolites of AB-PINACA in lung, liver, kidney and urine specimens collected at the autopsy of its abuser with its in vitro metabolites in human hepatocytes. The metabolites of AB-PINACA in tissues were extracted after homogenization. The urine specimen and portions of the extracted metabolites from tissues were firstly hydrolyzed with β-glucuronidase, and the metabolites were extracted. For in vitro experiment, AB-PINACA was incubated with human hepatocytes for 3 h to produce its metabolites. The identification of the in vivo and in vitro metabolites was performed using liquid chromatography (LC)–high-resolution Orbitrap-tandem mass spectrometry (MS-MS), and the relative intensities of these metabolites were measured using low resolution LC–quadrupole-ion trap-MS-MS. Thirteen metabolites of AB-PINACA were characterized in vivo in several human specimens and in in vitro human hepatocytes. They were produced by the terminal amide hydrolysis to carboxylic acid, hydroxylation, carbonyl formation and/or glucuronidation. The most detectable metabolite in the hepatocytes, lung or liver was the one produced by the terminal amide hydrolysis, whereas the top metabolite in the kidney or urine was the one produced by hydroxylation or carbonyl formation on the pentyl side chain after the terminal amide hydrolysis, respectively. At least 12 metabolites of AB-PINACA were detected in authentic human lung, liver or kidney specimen from a cadaver. It is concluded that the postmortem metabolite profiling of AB-PINACA can be fulfilled with solid tissues, and the lung and kidney were most recommendable especially when urine specimen is not available.

A decrease in nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of aging in multiple organisms, including humans. We report a human clinical trial designed to assess the efficacy of single-dose supplementation of nicotinamide, a form of vitamin B3, on increasing NAD+ levels. Nicotinamide intake within the daily tolerable upper level (200 mg) led to the maximal whole blood concentration by a 30-fold increase at 0.5 h with a consistent decrease until 6 h in all 6 healthy male volunteers. The increase in blood nicotinamide amounted to 17%, whereas renal clearance accounted for 1.8% of the ingested dose. NAD+ levels reached a maximum concentration at 12 h, with all time points showing a trend of increase. Blood metabolome assay indicates that the metabolomic changes with NAM supplementation lasted 24 h and then returned to baseline by 48 h. The basal levels of whole blood NAD+ varied among individuals with a negative correlation with body height as well as nicotinamide mononucleotide (NMN) levels. These data warrant consideration for further clinical evaluation with repeated doses of nicotinamide within the safety dose range for the potency to increase NAD+ levels and potential accompanying benefits for healthy longevity.

The aim of this study is to characterize the metabolites of EAM-2201 in human hepatocytes obtained in vitro and those in liver and urine specimens obtained in vivo from the autopsy of an EAM-2201 abuser. EAM-2201 was incubated with human hepatocytes for 3 h in a CO2 incubator and the metabolites of EAM-2201 were produced. The human liver specimen was homogenized and the metabolites were extracted. The urine specimen was hydrolyzed first with β-glucuronidase and the metabolites were extracted. The tentative detection and identification of those metabolites were performed using liquid chromatography (LC)–quadrupole-ion trap tandem mass spectrometry (MS/MS) and high-resolution LC–Orbitrap-MS/MS, respectively. Twelve metabolites of EAM-2201 could be characterized in hepatocytes, liver and urine. They were produced by defluorination, hydroxylation, carboxylation, dehydrogenation, N-dealkylation and/or glucuronidation of EAM-2201. The most detectable metabolite obtained from the in vitro hepatocyte incubation was that monohydroxylated at the 4-ethylnaphthalene moiety of EAM-2201 after dehydrogenation. However, only a trace amount of the same metabolite was detected in the liver tissue obtained postmortem from an EAM-2201 abuser. In the liver tissue, the highest metabolite was 5-carboxypentyl derivative of EAM-2201, which was monohydroxylated at the 4-ethylnaphthalene moiety, followed by monohydroxylpentyl and monohydroxy-4-ethylnaphthalenyl derivative of EAM-2201 without dehydrogenation. Twelve metabolites of a synthetic cannabinoid, EAM-2201, were tentatively identified in human hepatocytes in vitro, and in human liver and urine specimens in vivo for the first time. There was a distinct difference in metabolism profile between the in vitro and in vivo results.

Background To reduce disease recurrence after radical surgery for lung squamous cell carcinomas (SQCCs), accurate prediction of recurrent high-risk patients is required for efficient patient selection for adjuvant chemotherapy. Because treatment modalities for recurrent lung SQCCs are scarce compared to lung adenocarcinomas (ADCs), accurately selecting lung SQCC patients for adjuvant chemotherapy after radical surgery is highly important. Predicting lung cancer recurrence with high objectivity is difficult with conventional histopathological prognostic factors; therefore, identification of a novel predictor is expected to be highly beneficial. Lipid metabolism alterations in cancers are known to contribute to cancer progression. Previously, we found that increased sphingomyelin (SM)(d35:1) in lung ADCs is a candidate for an objective recurrence predictor. However, no lipid predictors for lung SQCC recurrence have been identified to date. This study aims to identify candidate lipid predictors for lung SQCC recurrence after radical surgery. Methods Recurrent (n = 5) and non-recurrent (n = 6) cases of lung SQCC patients who underwent radical surgery were assigned to recurrent and non-recurrent groups, respectively. Extracted lipids from frozen tissue samples of primary lung SQCC were analyzed by liquid chromatography-tandem mass spectrometry. Candidate lipid predictors were screened by comparing the relative expression levels between the recurrent and non-recurrent groups. To compare lipidomic characteristics associated with recurrent SQCCs and ADCs, a meta-analysis combining SQCC (n = 11) and ADC (n = 20) cohorts was conducted. Results Among 1745 screened lipid species, five species were decreased (≤ 0.5 fold change; P P Conclusion We identified decreased SM(t34:1) as a novel candidate predictor for lung SQCC recurrence. Lung SQCCs and ADCs have opposite lipidomic characteristics concerning for recurrence risk. Trial registration This retrospective study was registered at the UMIN Clinical Trial Registry (UMIN000039202) on January 21, 2020.

AB-FUBINACA M3 was reported to be a major metabolite of the drug, but its in vivo concentration in authentic human solid tissues has not been quantified yet. Another metabolite AB-FUBINACA M4 did not receive much attention previously and also has not been quantified yet in any authentic human specimens. The aims of this study are to establish a sensitive method for quantification of M3 and M4 in solid tissues and to compare the metabolite profile of AB-FUBINACA in authentic human specimens in vivo with that produced by human hepatocytes in vitro. The quantification was performed by liquid chromatography (LC)–quadrupole-ion trap-tandem mass spectrometry (MS-MS), and the characterization by LC–quadrupole Orbitrap MS-MS The limits of quantification of M3 were 10 pg/mL and 60 pg/g, and those of M4 were 100 pg/mL and 600 pg/g in urine and tissues, respectively. In the present work, M3 and M4 were identified and quantified in human lung, liver and kidney obtained from a cadaver for the first time; the concentrations of M3 were 226, 255, 202 and 155 pg/mL or g, and those of M4 14,400, 768, 637 and 1,390 pg/mL or g in urine, lung, liver and kidney, respectively. The peak intensity profiles of seven metabolites in these specimens were compared with that produced by human hepatocytes; the top three metabolites in urine specimen were completely different from those of hepatocytes. M3 was reported as the predominant metabolite in several previous works and M4 was listed as a minor metabolite in only one work, but, in this work, M4 has been found to be the major metabolite in all of the authentic urine, lung, liver and kidney specimens. The M3 plus M4 metabolites in lung or kidney were found most recommendable to prove AB-FUBINACA consumption, when urine specimen is lacking.

The progression of nonalcoholic fatty liver disease (NAFLD) is affected by epigenetics. We performed differentially methylated region (DMR) and co-methylation analyses to identify DMR networks associated with the progression of NAFLD. DMRs displaying differences in multiple consecutive differentially methylated CpGs between mild and advanced NAFLD were extracted. The average values of topological overlap measures for the CpG matrix combining two different DMRs were calculated and two DMR networks that strongly correlated with the stages of fibrosis were identified. The annotated genes of one network included genes involved in transcriptional regulation, cytoskeleton organization, and cellular proliferation. The annotated genes of the second network were primarily associated with metabolic pathways. The CpG methylation levels in these networks were strongly affected by age and fasting plasma glucose levels, which may be important co-regulatory factors. The methylation status of five DMRs in the second network was reversible following weight loss. Our results suggest that CpG methylation in DMR networks is regulated concomitantly via aging and hyperglycemia and plays important roles in hepatic metabolic dysfunction, fibrosis, and potential tumorigenesis, which occur during the progression of NAFLD. By controlling weight and blood glucose levels, the methylation of DMRs in the second network may be reduced.

A synthetic cannabinoid 5F-NNEI and its metabolites in authentic human specimens have not been reported yet. The aim of this study is, firstly, to establish a sensitive quantification method of 5F-NNEI in human serum and urine specimens, and, secondly, the characterization of its metabolites in authentic urine specimens obtained from three individuals. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum specimens via liquid-liquid extraction. The identification and quantification were performed using liquid chromatography (LC)–QTRAP tandem mass spectrometry (MS/MS), and the characterization of the metabolites was done using LC–high-resolution-MS/MS. The limits of detection for unchanged 5F-NNEI were 6 pg/mL with the quantification range of 10–2000 pg/mL in serum, and 3 pg/mL with the quantification range of 5–1000 pg/mL in urine. The devised method was applied to quantify 5F-NNEI in authentic serum and/or urine specimens from three individuals. The levels of 5F-NNEI were 11.6 and 3680 pg/mL in serum specimens of cases 1 and 2, respectively, and were 5.07 and 7.90 pg/mL in urine specimens of cases 2 and 3, respectively. Several metabolites of 5F-NNEI produced by amide hydrolysis, defluorination, hydroxylation, carboxylation and/or glucuronization were tentatively identified in the urine specimens by the LC–high-resolution-MS/MS. A synthetic cannabinoid, 5F-NNEI, was identified and quantified in its unchanged form, and its several metabolites were tentatively identified in authentic specimens obtained from three individuals, for the first time to our knowledge.

Urine is the most suitable specimen to collect from individuals because of noninvasiveness and relatively large volumes obtainable. In authentic urine specimens, however, synthetic cannabinoids having the structures of quinolinyl ester indoles, such as 5F-PB-22, PB-22 and BB-22, in unchanged forms as well as their metabolites, have not been quantified yet. Therefore, the aim of this study was to establish a sensitive analytical method for the quantification of 5F-PB-22 and its three metabolites 5F-PB-22 3-carboxyindole, PB-22 N-5-hydroxypentyl and PB-22 N-pentanoic acid in authentic urine samples in four cases. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine via liquid-liquid extraction. The identification and quantification were performed using the QTRAP type of a liquid chromatography–tandem mass spectrometer. The limits of detection were 3–30 pg/mL and their summed quantitation range was 10–10,000 pg/mL. The devised method was applied to quantify these compounds in authentic urine specimens obtained from four individuals. The levels of 5F-PB-22 were 5.1, 13.6, 94.7 and 470 pg/mL; those of 5F-PB-22 3-carboxyindole were 8.25, 3.39, 23.2 and 880 ng/mL; and those of PB-22 N-pentanoic acid were 12.0, 57.4, 959 and 2090 pg/mL, respectively, in four unhydrolyzed urine samples, and the levels of PB-22 N-5-hydroxypentyl could be quantified as 29.9 and 131 pg/mL in two unhydrolyzed urine samples. The 5F-PB-22 and its metabolites PB-22 N-5-hydroxypentyl have been detected from authentic human urine samples for the first time. Also, this is the first report dealing with the quantification of the three metabolites in human urine samples.

Aim Nonalcoholic fatty liver disease (NAFLD) progresses because of the interaction between numerous genes. Thus, we performed weighted gene co-expression network analysis to identify core gene networks and key genes associated with NAFLD progression. Methods We enrolled 39 patients with mild NAFLD (fibrosis stages 0 to 2) and 21 with advanced NAFLD (fibrosis stages 3 or 4). Total RNA was extracted from frozen liver biopsies, and sequenced to capture a large dynamic range of expression levels. Results A total of 1,777 genes differentially expressed between mild and advanced NAFLD (q-value

We report a case of intoxication with a mixture of three synthetic cannabinoids and a synthetic cathinone, which have been disclosed by a highly sensitive progressing technology. A man was found dead, and his forensic autopsy was performed at our department. After further examinations of his specimens, EAM-2201 and α-PVP have been newly found in his lung. The concentrations of EAM-2201 have not been reported yet in any authentic human specimens although its existence (not quantified) in blood was reported in 2015. Therefore, a sensitive quantitation method of these compounds in blood and solid tissues has been devised using the sensitive instrument. The limits of detection of these compounds were in the range of 3–10 pg/ml with their quantification range of 10–1000 pg/ml in blood. The femoral vein blood levels of EAM-2201 and AB-PINACA were 56.6 ± 4.2 and 12.6 ± 0.1 pg/ml, respectively, and AB-FUBINACA could be detected but not quantifiable in the blood specimens; α-PVP could not be detected. The standard addition method was employed for the quantification of these compounds in the lung, liver and kidney specimens. The lung levels of EAM-2201, AB-PINACA, AB-FUBINACA and α-PVP were 348 ± 34, 355 ± 30, 124 ± 12 and 59.0 ± 7.4 pg/g, respectively. In conclusion, in this study, the concentrations of EAM-2201 in authentic human specimens including blood and solid tissues and those of AB-PINACA and AB-FUBINACA in solid tissue specimens were quantified for the first time to our knowledge.

AIM Visceral fat accumulation contributes to the development of metabolic syndrome. As visceral fat accumulation increases, adiponectin levels decrease; therefore, adiponectin provides a link between visceral fat accumulation and metabolic disorders. Genome-wide association studies (GWASs) have identified genetic variations in the cadherin 13 (CDH13) gene that are associated with adiponectin levels. METHODS We investigated whether single nucleotide polymorphisms (SNPs) in CDH13 was associated with adiponectin levels and metabolic syndrome traits independent of the visceral fat area (VFA), as measured using computed tomography (CT) in 945 Japanese individuals. RESULTS We found that three CDH13 SNPs reported by recent GWASs (i.e., rs3865188, rs4783244, and rs12051272) were significantly associated with higher adiponectin levels (P ＜ 1 × 10 (-14)), even after adjustment for VFA. However, these adiponectin-inducing alleles of CDH13 SNPs were significantly associated with traits consistent with deteriorating metabolic symptoms, such as higher fasting insulin, homeostasis model assessment-insulin resistance (HOMA-IR) scores, and triglycerides and lower high-density lipoprotein (HDL)-cholesterol levels, similar to increasing VFA and decreasing adiponectin levels. CONCLUSION These results suggested that CDH13 SNPs cause an adiponectin-resistant status to compensate for increasing adiponectin levels and could result in the deterioration of metabolic syndrome traits.

Visceral fat accumulation contributes to the development of insulin resistance, leading to metabolic syndrome. Adiponectin provides a link between visceral fat accumulation and insulin resistance. In addition to environmental factors, genetic factors play important roles in visceral fat accumulation and circulating adiponectin levels. Genome-wide association studies (GWASs) have identified genetic variations in the adiponectin, C1Q and collagen domain containing (ADIPOQ) gene that are associated with adiponectin levels. In this study, we investigated whether ADIPOQ single nucleotide polymorphisms (SNPs) were associated with visceral fat accumulation and insulin resistance. We measured the visceral fat area (VFA) by computed tomography (CT) and examined the presence of the insulin resistance-related phenotype (fasting plasma glucose, fasting insulin, and homeostasis model assessment-insulin resistance [HOMA-IR]) in a set of Japanese individuals (731 men and 864 women) who were genotyped for seven ADIPOQ SNPs reported by recent GWASs (namely, rs6810075, rs10937273, rs1648707, rs864265, rs182052, rs17366568, and rs6773957). SNPs associated with the phenotype (P < 0.05) were then evaluated by association analysis using a second set of the study participants (383 men and 510 women). None of the SNPs was associated with body mass index (BMI) or VFA in men or women. However, the adiponectin-decreasing alleles of rs10937273 and rs1648707 were significantly associated with HOMA-IR (P = 0.0030 and P = 0.00074, respectively) in women, independently of BMI. These SNPs were significantly associated with decreased adiponectin levels in women. Our results suggested that rs10937273 and rs1648707 may affect insulin sensitivity by regulating adiponectin production by adipose tissue in women.

Aim The progression of non-alcoholic fatty liver disease (NAFLD) is affected by epigenetics. We undertook co-methylation and differentially methylated region (DMR) analyses to identify the genomic region that is under epigenetic regulation during NAFLD progression. Methods We collected liver biopsy specimens from 60 Japanese patients with NAFLD and classified these into mild (fibrosis stages 0-2) or advanced (fibrosis stages 3-4) NAFLD. We carried out a genome-wide DNA methylation analysis and identified the differentially methylated CpGs between mild and advanced NAFLD. Differentially methylated regions with multiple consecutive differentially methylated CpGs between mild and advanced NAFLD were extracted. Results Co-methylation analysis showed that individual differentially methylated CpG sites were clustered into three modules. The CpG sites clustered in one module were hypomethylated in advanced NAFLD and their annotated genes were enriched for "immune system" function. The CpG sites in another module were hypermethylated and their annotated genes were enriched for "mitochondria" or "lipid particle", and "lipid metabolism" or "oxidoreductase activity". Hypomethylated DMRs included tumorigenesis-related genes (FGFR2, PTGFRN, and ZBTB38), the expressions of which are upregulated in advanced NAFLD. Tumor suppressor MGMT had two DMRs and was downregulated. Conversely, FBLIM1 and CYR61, encoding proteins that reduce cell proliferation, showed hypomethylated DMRs and were upregulated. Expression of the antioxidant gene NQO1 was upregulated, with a hypomethylated DMR. The DMR containing cancer-related MIR21 was hypomethylated in advanced NAFLD. Conclusions Co-methylation and DMR analyses suggest that the NAFLD liver undergoes mitochondrial dysfunction, decreased lipid metabolism, and impaired oxidoreductase activity, and acquires tumorigenic potential at the epigenetic level.

The genomic regions containing PNPLA3, SAMM50 and PARVB are susceptibility loci for the development and progression of nonalcoholic fatty liver disease (NAFLD). In order to search for all common variations in this region, we amplified the genomic DNA of 28 NAFLD patients by long-range PCR, covering the entire susceptibility region and sequenced the DNA using indexed multiplex next-generation sequencing. We found 329 variations, including four novel variations. Fine mapping of variations including insertion/deletions was performed for 540 NAFLD patients (488 with nonalcoholic steatohepatitis (NASH) and 52 with simple steatosis) and 1012 control subjects. HaploView analysis showed that linkage disequilibrium (LD) block 1 and 2 occurred in PNPLA3, block 3 in SAMM50 and block 4 in PARVB. Variations in LD blocks 1-4 were significantly associated with NAFLD as compared with control subjects (P, Published online 13 March 2014

In several genome-wide association studies, nonalcoholic fatty liver disease and alanine aminotransferase susceptibility variants have been identified in several genes, including LYPLAL1, ZP4, GCKR, HSD17B13, PALLD, PPP1R3B, FDFT1, TRIB1, COL13A1, CPN1, ERLIN1, CWF19L1, EFCAB4B, PZP, and NCAN. To investigate the relationship between these genes and nonalcoholic fatty liver disease in the Japanese population, we genotyped 540 patients and 1012 control subjects for 18 variations. We performed logistic regression analyses to characterize the association between the tested variations and nonalcoholic fatty liver disease. Metabolic syndrome and histological traits were also analyzed by linear regression. We also examined GCKR rs780094, TRIB1 rs2954021, and PNPLA3 rs738409 for epistatic effects. The A-allele of rs780094 in GCKR (P = 0.0024) and the A-allele of rs2954021 TRIB1 (P = 4.5×10-5) were significantly associated with nonalcoholic fatty liver disease. GCKR rs780094 was also associated with decreased plasma glucose, and increased triglycerides in the patient and control groups. GCKR rs780094 was also associated with an increased ratio of visceral to subcutaneous fat area in the patients with nonalcoholic fatty liver disease. Variations in GCKR, TRIB1, and PNPLA3 independently influenced nonalcoholic fatty liver disease and had no epistatic effects. Our data suggest variations in GCKR and TRIB1 are involved in the development of nonalcoholic fatty liver disease.

The predominant risk factor of metabolic syndrome is intra-abdominal fat accumulation, which is determined by waist circumference, waist-hip ratio measurements and visceral fat area (VFA); the latter can be accurately measured by performing computed tomography (CT). In addition to environmental factors, genetic factors play an important role in obesity and fat distribution. New genetic loci associated with body mass index (BMI) and adiposity have been identified by genome-wide association studies (GWASs). This study utilized CT to investigate whether single nucleotide polymorphisms (SNPs) that confer susceptibility to higher BMI are associated with VFA, subcutaneous fat area (SFA), and the ratio of VFA to SFA (V/S ratio). We measured the VFA and SFA of 1424 obese Japanese subjects (BMI ≥ 25 kg/m(2), 635 men and 789 women) who were genotyped for 13 single nucleotide polymorphisms (SNPs) reported by recent GWASs, namely, TNNI3K rs1514175, PTBP2 rs1555543, ADCY3 rs713586, IRS1 rs2943650, POC5 rs2112347, NUDT3 rs206936, LINGO2 rs10968576, STK33 rs4929949, MTIF3 rs4771122, SPRY2 rs534870, MAP2K5 rs2241423, QPCTL rs2287019, and ZC3H4 rs3810291. The G-allele of NUDT3 rs206936 was significantly associated with increased BMI (P = 5.3 × 10(-5)) and SFA (P = 0.00039) in the obese Japanese women. After adjustment with BMI, the association between rs206936 and SFA was not observed. This significant association was not observed in the men. The other SNPs analyzed were not significantly associated with BMI, VFA, SFA, or V/S ratio. Our results suggest that NUDT3 rs206936 is associated with BMI in Japanese women.

AIM Visceral fat accumulation plays an integral role in morbidity and mortality rates by increasing the risk of developing metabolic disorders such as type 2 diabetes, dyslipidemia, and hypertension. New genetic loci associated with fat distribution, measured by waist-hip ratios and computed tomography (CT), have recently been identified by genome-wide association studies in European-descent populations. This study used CT to investigate whether single nucleotide polymorphisms (SNPs) that confer susceptibility to fat distribution are associated with visceral fat area (VFA) and subcutaneous fat area (SFA) in the Japanese population. METHODS We measured the VFAs and SFAs of 1424 obese Japanese subjects (BMI≥25 kg/m(2), 635 men and 789 women) that were genotyped at 15 SNPs, namely, TBX15 rs984222, DNM3 rs1011731, LYPLAL1 rs4846567, GRB14 rs10195252, NISCH rs6784615, ADAMTS9 rs6795735, CPEB4 rs6861681, LY86 rs1294421, VEGFA rs6905288, RSPO3 rs9491696, NFE2L3 rs1055144, ITPR2 rs718314, HOXC13 rs1443512, ZNRF3 rs4823006 and THNSL2 rs1659258. RESULTS The G-allele of LYPLAL1 rs4846567 was borderline associated with an increased ratio of VFA to SFA (V/S ratio; p= 0.0020). LYPLAL1 rs4846567 had a stronger effect on the V/S ratio in women (p= 0.0078) than in men (p= 0.12); however, neither result was significant after Bonferroni correction for multiple comparisons. NISCH rs6784615 was nominally associated with increased VFA (p=0.040) and V/S ratio (p= 0.020). The other SNPs analyzed were not significantly associated with body mass index (BMI), VFA, or SFA. CONCLUSION Our results suggest that LYPLAL1 rs4846567 and NISCH rs6784615 may influence fat distribution in the Japanese population.

The predominant risk factor of metabolic syndrome is intra-abdominal fat accumulation, which is determined by waist circumference, waist-hip ratio measurements and visceral fat area (VFA); the latter can be accurately measured by performing computed tomography (CT). In addition to environmental factors, genetic factors play an important role in obesity and fat distribution. New genetic loci associated with body mass index (BMI) and adiposity have been identified by genome-wide association studies (GWASs). This study utilized CT to investigate whether single nucleotide polymorphisms (SNPs) that confer susceptibility to higher BMI are associated with VFA, subcutaneous fat area (SFA), and the ratio of VFA to SFA (V/S ratio). We measured the VFA and SFA of 1424 obese Japanese subjects (BMI ≥ 25 kg/m(2), 635 men and 789 women) who were genotyped for 13 single nucleotide polymorphisms (SNPs) reported by recent GWASs, namely, TNNI3K rs1514175, PTBP2 rs1555543, ADCY3 rs713586, IRS1 rs2943650, POC5 rs2112347, NUDT3 rs206936, LINGO2 rs10968576, STK33 rs4929949, MTIF3 rs4771122, SPRY2 rs534870, MAP2K5 rs2241423, QPCTL rs2287019, and ZC3H4 rs3810291. The G-allele of NUDT3 rs206936 was significantly associated with increased BMI (P = 5.3 × 10(-5)) and SFA (P = 0.00039) in the obese Japanese women. After adjustment with BMI, the association between rs206936 and SFA was not observed. This significant association was not observed in the men. The other SNPs analyzed were not significantly associated with BMI, VFA, SFA, or V/S ratio. Our results suggest that NUDT3 rs206936 is associated with BMI in Japanese women.

Non-alcoholic fatty liver disease (NAFLD) progresses because of the interaction between numerous genes. Thus, we carried out a weighted gene coexpression network analysis to identify core gene networks and key genes associated with NAFLD progression.We enrolled 39 patients with mild NAFLD (fibrosis stages 0-2) and 21 with advanced NAFLD (fibrosis stages 3-4). Total RNA was extracted from frozen liver biopsies, and sequenced to capture a large dynamic range of expression levels.A total of 1777 genes differentially expressed between mild and advanced NAFLD (q-value0.05) clustered into four modules. One module was enriched for genes that encode cell surface or extracellular matrix proteins, and are involved in cell adhesion, proliferation, and signaling. This module formed a scale-free network containing four hub genes (PAPLN, LBH, DPYSL3, and JAG1) overexpressed in advanced NAFLD. PAPLN is a component of the extracellular matrix, LBH and DPYSL3 are reported to be tumor suppressors, and JAG1 is tumorigenic. Another module formed a random network, and was enriched for genes that accumulate in the mitochondria. These genes were downregulated in advanced NAFLD, reflecting impaired mitochondrial function. However, the other two modules did not form unambiguous networks. KEGG analysis indicated that 71 differentially expressed genes were involved in "pathways in cancer". Strikingly, expression of half of all differentially expressed genes was inversely correlated with methylation of CpG sites (q-value0.05). Among clinical parameters, serum type IV collagen 7 s was most strongly associated with the epigenetic status in NAFLD.Newly identified core gene networks suggest that the NAFLD liver undergoes mitochondrial dysfunction and fibrosis, and acquires tumorigenic potential epigenetically. Our data provide novel insights into the pathology and etiology of NAFLD progression, and identify potential targets for diagnosis and treatment.

Visceral fat accumulation has an important role in increasing the morbidity and mortality rates, by increasing the risk of developing several metabolic disorders, such as type 2 diabetes, dyslipidemia and hypertension. New genetic loci that are associated with increased systolic and diastolic blood pressures have been identified by genome-wide association studies in Caucasian populations. This study investigates whether single nucleotide polymorphisms (SNPs) that confer susceptibility to high blood pressure are also associated with visceral fat obesity. We genotyped 1279 Japanese subjects (556 men and 723 women) who underwent computed tomography for measuring the visceral fat area (VFA) and subcutaneous fat area (SFA) at the following SNPs: FGF5 rs16998073, CACNB2 rs11014166, C10orf107 rs1530440, CYP17A1 rs1004467, NT5C2 rs11191548, PLEKHA7 rs381815, ATP2B1 rs2681472 and rs2681492, ARID3B rs6495112, CSK rs1378942, PLCD3 rs12946454, and ZNF652 rs16948048. In an additive model, risk alleles of the CYP17A1 rs1004467 and NT5C2 rs11191548 were found to be significantly associated with reduced SFA (P=0.00011 and 0.0016, respectively). When the analysis was performed separately in men and women, significant associations of rs1004467 (additive model) and rs11191548 (recessive model) with reduced VFA (P=0.0018 and 0.0022, respectively) and SFA (P=0.00039 and 0.00059, respectively) were observed in women, but not in men. Our results suggest that polymorphisms in the CYP17A1 and NT5C2 genes influence a reduction in both visceral and subcutaneous fat mass in Japanese women.

The revised building standard law established in 2007 confused building confirmation application process in building work places. This cause of the confusion is related to the strict examination duty ruled by the revised law. In this paper, the contents of the strict examination were analyzed, the work rate was measured, and the working time was measured. Furthermore two techniques to make the examination process more efficient were proposed. One is an effective technique in strict examination, and another is an outline examination. In the outline examination the work rate is small, however a building confirmation application can’t be judged exactly. In contrast, in strict examination work rate is heavy, however a building confirmation application can be judged more exactly. Both examination methods have different concept, however using both methods in coordination can make the examination process more effectively. In the previous paper, the strict examination process was shown, and in this paper outline one is mainly discussed.

Given a bivariate polynomial $f(x,y)$, let $\phi(y)$ be a power series root of $f(x,y)=0$ with respect to $x$, i.e., $\phi(y)$ is a function of $y$ such that $f(\phi(y),y)=0$. If $\phi(y)$ is analytic at $y=0$, then we have its power series expansion \begin{equation} \phi(y)=\alpha_{0}+\alpha_{1}y+\alpha_{2}y^{2}+\cdots+\alpha_{r}y^{r}+\cdots. \end{equation} Let $\phi^{(k)}(y)$ denote $\phi(y)$ truncated at $y^{k}$, i.e., \begin{equation} \phi^{(k)}(y)=\alpha_{0}+\alpha_{1}y+\alpha_{2}y^{2}+\cdots+\alpha_{k}y^{k}. \end{equation} Then, it is well known that, given initial value $\phi^{(0)}(y)=\alpha_{0}\in\mathbf{C}$, the symbolic Newton's method with the formula \begin{equation} \phi^{(2^{m}-1)}(y)\gets\phi^{(2^{m-1}-1)}(y) -\frac{f(\phi^{(2^{m-1}-1)}(y),y)}{\frac{\partial f}{\partial x}(\phi^{(2^{m-1}-1)}(y),y)} \quad (\mod y^{2^{m}}) \end{equation} computes $\phi^{(2^{m}-1)}(y)$ ($1\le m$) in (2) with quadratic convergence (the roots are computed in the order $\phi^{(0)}(y) \to \phi^{(2^{1}-1)}(y) \to \phi^{(2^{2}-1)}(y) \to \cdots \to \phi^{(2^{m}-1)}(y)$). References [1] and [3] indicate that the symbolic Newton's method can be generalized so that its convergence degree is an arbitrary integer $p$ where its roots are computed in the order $\phi^{(0)}(y) \to \phi^{(p-1)}(y) \to \phi^{(p^{2}-1)}(y) \to \cdots \to \phi^{(p^{m}-1)}(y)$. Although the high degree convergent formula in [1] and [3] requires fewer iterations than the symbolic Newton's method, it may not be efficient as expected, since one iteration of the formula requires more computations than one in the symbolic Newton's method. ¶ In this paper, we combine the polynomial evaluation method in [9] with the formula of arbitrary degree convergence and propose an algorithm that computes the above power series root $\phi^{(k)}(y)$. We analyze the complexity of the algorithm and give the number of multiplications/divisions required to compute a power series root in an explicit form. It is shown that when the degree of polynomial $f(x,y)$ is high, high degree convergent formula is advantageous over the symbolic Newton's method.

Let M(y) be a matrix whose entries are polynomial in y, λ(y) and v(y) be a set of eigenvalue and eigenvector of M(y). Then, λ(y) and v(y) are algebraic functions of y, and λ(y) and v(y) have their power series expansions λ(y) = β0 + β1y + ₀ + βkyk + ₀ (βj e C),            (1) v(y) = γ0 + γ1y + ₀ + γkyk + ₀ (γj e Cn),            (1) provided that y = 0 is not a singular point of λ(y) or v(y). Several algorithms are already proposed to compute the above power series expansions using Newton's method (the algorithm in [4]) or the Hensel construction (the algorithm in [5],[12]). The algorithms proposed so far compute high degree coefficients βk and γk, using lower degree coefficients βj and γj (j = 0,1,₀,k-1). Thus with floating point arithmetic, the numerical errors in the coefficients can accumulate as index k increases. This can cause serious deterioration of the numerical accuracy of high degree coefficients βk and γk, and we need to check the accuracy. In this paper, we assume that given matrix M(y) does not have multiple eigenvalues at y = 0 (this implies that y = 0 is not singular point of λ(y) or v(y)), and presents an algorithm to estimate the accuracy of the computed power series βi,γj in (1) and (2). The estimation process employs the idea in [9] which computes a coefficient of a power series with Cauchy's integral formula and numerical integrations. We present an efficient implementation of the algorithm that utilizes Newton's method. We also present a modification of Newton's method to speed up the procedure, introducing tuning parameter p. Numerical experiments of the paper indicates that we can enhance the performance of the algorithm by 12 ~ 16%, choosing the optimal tuning parameter p.

This study relates to examination technology of a structure check. Since a structure check camouflage case in 2005, a building administration system was argued, and the building-code was amended. In this paper, examination technology was systematized and developed. This examination technology with check lists and calculation tools realizes detection of a camouflaged structure check. And it corresponds to rigid examination by Japanese amended building-code.

In recent years, algorithms based on Computer Algebra ([1]-[3]) have been introduced into a range of control design problems because of the capacity to handle unknown parameters as indeterminates. This feature of algorithms in Computer Algebra reduces the costs of computer simulation and the trial and error process involved, enabling us to design and analyze systems more theoretically with the behavior of given parameters. In this paper, we apply Computer Algebra algorithms to H∞ control theory, representing one of the most successful achievements in post-modern control theory. More specifically, we consider the H∞ norm minimization problem using a state feedback controller. This problem can be formulated as follows: Suppose that we are given a plant described by the linear differential equation dx/dt = Ax + B1w + B2u, z = Cx + Du, where A, B1, B2, C, D are matrices whose entries are polynomial in an unknown parameter k. We apply a state feedback controller u = -Fx to the plant, where F is a design parameter, and obtain the system dx/dt = (A-B2F)x + B1w, z = (C-DF)x. Our task is to compute the minimum H∞ norm of the transfer function G(s)(=(C-DF)(sI-A + B2F)-1B1) from w to z achieved using a static feedback controller u = -Fx, where F is a constant matrix. In the H∞ control theory, it is only possible to check if there is a controller such that ||G(s)||∞ < γ is satisfied for a given number γ, where ||G(s)||∞ denotes the H∞ norm of the transfer function G(s). Thus, a typical procedure to solve the H∞ optimal problem would involve a bisection method, which cannot be applied to plants with parameters. In this paper, we present a new method of solving the H∞ norm minimization problem that can be applied to plants with parameters. This method utilizes QE (Quantifier Elimination) and a variable elimination technique in Computer Algebra, and expresses the minimum of the H∞ norm as a root of a bivariate polynomial. We also present a numerical example to illustrate each step of the algorithm.