Quantification of risperidone and paliperidone by liquid chromatography-tandem mass spectrometry in biological fluids and solid tissues obtained from two deceased using the standard addition method.

Risperidone (RIS) is an atypical antipsychotic agent and its 9-hydroxylated metabolite named paliperidone (PAL) also has pharmacological properties similar to that of RIS. Quantifications of RIS and PAL in authentic human biological fluids and solid tissues by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) have not been reported yet although those in plasma (and blood) were reported abundantly. In the present work, a quantification method for RIS and PAL based on the standard addition method was devised and validated for the human fluid and solid tissue specimens. RIS and PAL in biological fluids were quantified only after their dilution and deproteinization. The concentrations of RIS and PAL in the heart whole blood, pericardial fluid, stomach contents, bile, urine, liver, kidney and cerebrum were determined for a deceased who had been treated with RIS therapeutically, and also a deceased who had ingested RIS with other drugs intentionally. To our knowledge, this is the first report on the quantification of RIS and PAL by LC-MS/MS in the authentic human tissues and biological fluids.

Lung adenocarcinoma and squamous cell carcinoma difficult for immunohistochemical diagnosis can be distinguished by lipid profile.

In patients with unresectable non-small cell lung cancer, histological diagnosis is frequently based on small biopsy specimens unsuitable for histological diagnosis when they are severely crushed and do not retain their morphology. Therefore, establishing a novel diagnostic method independent of tissue morphology or conventional immunohistochemistry (IHC) markers is required. We analyzed the lipid profiles of resected primary lung adenocarcinoma (ADC) and squamous cell carcinoma (SQCC) specimens using liquid chromatography-tandem mass spectrometry. The specimens of 26 ADC and 18 SQCC cases were evenly assigned to the discovery and validation cohorts. Non-target screening on the discovery cohort identified 96 and 13 lipid peaks abundant in ADC and SQCC, respectively. Among these 109 lipid peaks, six and six lipid peaks in ADC and SQCC showed reproducibility in target screening on the validation cohort. Finally, we selected three and four positive lipid markers for ADC and SQCC, demonstrating high discrimination abilities. In cases difficult to diagnose by IHC staining, [cardiolipin(18:2_18:2_18:2_18:2)-H]- and [triglyceride(18:1_17:1_18:1) + NH4]+ showed the excellent diagnostic ability for ADC (sensitivity: 1.00, specificity: 0.89, accuracy: 0.93) and SQCC (sensitivity: 0.89, specificity: 0.83, accuracy: 0.87), respectively. These novel candidate lipid markers may contribute to a more accurate diagnosis and subsequent treatment strategy for unresectable NSCLC.

Uric acid is a major chemical constituent for the whitish coloration in the medaka leucophores.

In whitish parts of teleost skin, the coloration is attributed to a light scattering phenomenon within light-reflecting chromatophores, namely leucophores and iridophores, which contain high refractive index materials in their cytoplasmic organelles, leucosomes and light-reflecting platelets, respectively. Previous chemical examinations revealed that guanine is a major constituent of the materials in the platelets of the iridophores, while, in leucophores, the detailed chemical nature of the materials contained in the leucosomes has not been reported. Here, using liquid chromatography-tandem mass spectroscopy, we investigated the chemical features of materials eluted from scales, larvae, and single chromatophores of the medaka. Results of the liquid chromatography-tandem mass spectroscopy suggested that uric acid is a major constituent of the high refractive index materials in medaka leucophores and is a unique marker to investigate the presence of leucophores in the fish. The whitish appearance of the medaka leucophores may be attributed to the light-scattering phenomenon in leucosomes, which contain highly concentrated uric acid.

Updating "Data on changes in lipid profiles during the differentiation and maturation of human subcutaneous white adipocytes analyzed using chromatographic and bioinformatic tools" with data on the changes in protein profiles.

Previously, we reported changes in the lipid profile of cultured human subcutaneous white preadipocytes during their differentiation and maturation. Here, using the same cells, we report changes in the protein profiles during differentiation and maturation as multi-omics data. The three cell lines of Caucasian-derived subcutaneous preadipocytes were divided into five stages: stage-1, subcutaneous preadipocytes; stage-2, following induction of differentiation into adipocytes; stages-3 to -5, from the initiation of lipid droplet formation to mature subcutaneous adipocytes (depending on the lipid droplet amount and formation). In each stage, proteins were extracted from the cells, proteolytically cleaved, and analyzed using untargeted liquid chromatography and mass spectrometry. The proteins were then identified and statistically analyzed. A total of 1,871 proteins were identified with high confidence, of which, 381 were statistically significant (P-value < 0.05) between any two stages. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the proteins significantly altered during the differentiation and maturation of preadipocytes were enriched in various pathways, including "ribosome," "Coronavirus disease-COVID-19," and "extracellular matrix (ECM)-receptor interaction" (FDR < 0.05).

Quantification of olanzapine and its three metabolites by liquid chromatography-tandem mass spectrometry in human body fluids obtained from four deceased, and confirmation of the reduction from olanzapine N-oxide to olanzapine in whole blood in vitro.

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 â„ƒ 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.

Lipid biomarkers that reflect postoperative recurrence risk in lung cancer patients who smoke: a case-control study.

BACKGROUND: The risk of postoperative recurrence is higher in lung cancer patients who smoke than non-smokers. However, objective evaluation of the postoperative recurrence risk is difficult using conventional pathological prognostic factors because of their lack of reproducibility. Consequently, novel objective biomarkers that reflect postoperative risk in lung cancer patients who smoke must be identified. Because cigarette smoking and oncogenesis alter lipid metabolism in lung tissue, we hypothesized that the lipid profiles in lung cancer tissues are influenced by cigarette smoking and can reflect the postoperative recurrence risk in smoking lung cancer patients. This study aimed to identify lipid biomarkers that reflect the smoking status and the postoperative recurrence risk. METHODS: Primary tumor tissues of lung adenocarcinoma (ADC) (n = 26) and squamous cell carcinoma (SQCC) (n = 18) obtained from surgery were assigned to subgroups according to the patient's smoking status. The ADC cohort was divided into never smoker and smoker groups, while the SQCC cohort was divided into moderate smoker and heavy smoker groups. Extracted lipids from the tumor tissues were subjected to liquid chromatography-tandem mass spectrometry analysis. Lipids that were influenced by smoking status and reflected postoperative recurrence and pathological prognostic factors were screened. RESULTS: Two and 12 lipid peaks in the ADC and SQCC cohorts showed a significant positive correlation with the Brinkman index, respectively. Among them, in the ADC cohort, a higher lipid level consisted of three phosphatidylcholine (PC) isomers, PC (14:0_18:2), PC (16:1_16:1), and PC (16:0_16:2), was associated with a shorter recurrence free period (RFP) and a greater likelihoods of progressed T-factor (≥ pT2) and pleural invasion. In the SQCC cohort, a lower m/z 736.5276 level was associated with shorter RFP and greater likelihood of recurrence. CONCLUSIONS: From our data, we propose three PC isomers, PC (14:0_18:2), PC (16:1_16:1), and PC (16:0_16:2), and a lipid peak of m/z 736.5276 as novel candidate biomarkers for postoperative recurrence risk in lung ADC and SQCC patients who are smokers.

Long-term stability of 24 synthetic cannabinoid metabolites spiked into whole blood and urine for up to 168 days, and the comparable study for the 6 metabolites in non-spiked real case specimens stored for 1-5 years.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 ℃.

A fatal case involved in pyrethroid insecticide ingestion: quantification of tetramethrin and resmethrin in body fluids of a deceased by LC-MS/MS.

PURPOSE: 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). METHODS: 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. RESULTS: 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 â„ƒ. CONCLUSIONS: To our knowledge, this is the first report for quantification of unchanged tetramethrin and resmethrin in human specimens obtained in a fatal case.

Data on changes in lipid profiles during the differentiation and maturation of human subcutaneous white adipocytes analyzed using chromatographic and bioinformatic tools.

In this dataset, we have described the changes in the lipid profile occurring during the differentiation and maturation of cultured human subcutaneous white preadipocytes into mature adipocytes. We divided three cell lines of Caucasian-derived subcutaneous preadipocytes into five stages (stage-1 to stage-5), from subcutaneous preadipocytes to mature subcutaneous adipocytes filled with many lipid droplets. Lipids were extracted from the cells at each stage by employing the Bligh and Dyer method and processed using untargeted liquid chromatography coupled with Q-Exactive Orbitrap tandem mass spectrometry. The lipids were identified using LipidSearch 4.2.13, and statistical analysis was performed using MetaboAnalyst 5.0. Dendrogram and principal component analysis clearly separated different stages of cells such as subcutaneous preadipocytes (stage-1), after the induction of differentiation into adipocytes (stage-2), and after the start of fat accumulation (stage-3 to stage-5). Of the 309 lipid species detected in LipidSearch 4.2.13, a total of 145 were statistically significant (false discovery rate < 0.05). The data are available at Metabolomics Workbench, Study ID ST001958: [https://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Project&ProjectID=PR001245].

Decreased sphingomyelin (t34:1) is a candidate predictor for lung squamous cell carcinoma recurrence after radical surgery: a case-control study.

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 < 0.05) and one was increased (≥ 2 fold change; P < 0.05) in the recurrent group. Among the six candidates, the top three final candidates (selected by AUC assessment) were all decreased SM(t34:1) species, showing strong performance in recurrence prediction that is equivalent to that of histopathological prognostic factors. Meta-analysis indicated that decreases in a limited number of SM species were observed in the SQCC cohort as a lipidomic characteristic associated with recurrence, in contrast, significant increases in a broad range of lipids (including SM species) were observed in the ADC cohort. 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.

In Vivo Metabolites of AB-PINACA in Solid Tissues Obtained from Its Abuser: Comparison with In Vitro Experiment.

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 ß-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.

Quantification of Major Metabolites of AB-FUBINACA in Solid Tissues Obtained from an Abuser.

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.

Sensitive quantification of BB-22 and its metabolite BB-22 3-carboxyindole, and characterization of new metabolites in authentic urine and/or serum specimens obtained from three individuals by LC-QTRAP-MS/MS and high-resolution LC-Orbitrap-MS/MS.

PURPOSE: A synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole have not yet been quantified in human urine. The aim of this study is to establish a sensitive analytical method for the quantification of BB-22 and its 3-carboxyindole in human serum and urine specimens, and the characterization of the unreported metabolites of BB-22 in authentic urine specimens from three individuals. METHODS: These compounds were extracted from ß-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum 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 new metabolites was made by high-resolution LC-MS/MS. RESULTS: The limits of detection of BB-22 and BB-22 3-carboxyindole were 3 and 30 pg/mL in urine, respectively. The devised method was applied to quantify these compounds in authentic serum and urine obtained from two drug abusers and in urine from one drug abuser. The serum levels of BB-22 were 149 and 6680 pg/mL, and those of BB-22 3-carboxyindole were 0.755 and 38.0 ng/mL in cases 1 and 2, respectively. The urine levels of BB-22 were 5.64, 5.52 and 6.92 pg/mL and those of BB-22 3-carboxyindole were 0.131, 21.4 and 5.15 ng/mL in cases 1, 2 and 3, respectively. New monohydroxyl metabolites retaining the structure of BB-22 were found in the urine specimens. CONCLUSIONS: The synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole were identified and quantified in authentic human serum and urine specimens for the first time, and new metabolites of BB-22 were tentatively identified in authentic urine specimens obtained from three drug users in this study.

A case of intoxication with a mixture of synthetic cannabinoids EAM-2201, AB-PINACA and AB-FUBINACA, and a synthetic cathinone α-PVP.

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 ±â€¯4.2 and 12.6 ±â€¯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 ±â€¯34, 355 ±â€¯30, 124 ±â€¯12 and 59.0 ±â€¯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.

Identification of differentially methylated region (DMR) networks associated with progression of nonalcoholic fatty liver disease.

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.

Identification of the genomic region under epigenetic regulation during non-alcoholic fatty liver disease progression.

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.

Viral Population Changes during Murine Norovirus Propagation in RAW 264.7 Cells.

Laboratory adaptation of viruses is an essential technique for basic virology research, including the generation of attenuated vaccine strains, although the principles of cell adaptation remain largely unknown. Deep sequencing of murine norovirus (MuNoV) S7 during serial passages in RAW264.7 cells showed that the frequencies of viral variants were altered more dynamically than previously reported. Serial passages of the virus following two different multiplicity of infections gave rise to distinct haplotypes, implying that multiple cell-adaptable sequences were present in the founder population. Nucleotide variants lost during passage were assembled into a viral genome representative of that prior to cell adaptation, which was unable to generate viral particles upon infection in cultured cells. In addition, presence of the reconstructed genome interfered with production of infectious particles from viruses that were fully adapted to in vitro culture. Although the key nucleotide changes dictating cell adaptation of MuNoV S7 viral infection are yet to be elucidated, our results revealed the elaborate interplay among selected sequences of viral variants better adapted to propagation in cell culture. Such knowledge will be instrumental in understanding the processes necessary for the laboratory adaptation of viruses, especially to those without relevant cell culture systems.

Identification of core gene networks and hub genes associated with progression of non-alcoholic fatty liver disease by RNA sequencing.

AIM: 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. METHODS: 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. RESULTS: A total of 1777 genes differentially expressed between mild and advanced NAFLD (q-value <0.05) 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-value <0.05). Among clinical parameters, serum type IV collagen 7 s was most strongly associated with the epigenetic status in NAFLD. CONCLUSIONS: 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.

CDH13 Polymorphisms are Associated with Adiponectin Levels and Metabolic Syndrome Traits Independently of Visceral Fat Mass.

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.

Development of a simple genotyping method for the HLA-A*31:01-tagging SNP in Japanese.

AIM: To construct a simple, low-cost typing method for the surrogate marker of HLA-A*31:01, a risk factor for carbamazepine (CBZ) related Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). MATERIALS & METHODS: DNAs from Japanese SJS/TEN patients were used for genotyping and developing the assay. RESULTS: HLA-A*31:01 was confirmed to be significantly associated with definite/probable cases of CBZ-related SJS/TEN (p = 0.0040). Three single nucleotide polymorphisms, rs1150738, rs3869066 and rs259945, were in absolute linkage disequilibrium with HLA-A*31:01 in 210 Japanese SJS/TEN patients. Robust genotyping of rs3869066 in ZNRD1-AS1 was developed using polymerase chain reaction-restriction fragment length polymorphism assays. CONCLUSION: Single nucleotide polymorphism genotyping is less time consuming and cheaper than conventional HLA typing, and would be useful for identifying Japanese patients at risk of CBZ-related SJS/TEN.