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).
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.
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.
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.
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.
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.
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.
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.
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.
This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on January 21, 2020.
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.
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 ℃.
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.
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.
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.
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.
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 β-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.
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