RESUMO
Cholangiocarcinoma is a fatal disease with limited therapeutic options. We screened genes required for cholangiocarcinoma tumorigenicity and identified FADS2, a delta-6 desaturase. FADS2 depletion reduced in vivo tumorigenicity and cell proliferation. In clinical samples, FADS2 was expressed in cancer cells but not in stromal cells. FADS2 inhibition also reduced the migration and sphere-forming ability of cells and increased apoptotic cell death and ferroptosis markers. Lipidome assay revealed that triglyceride and cholesterol ester levels were decreased in FADS2-knockdown cells. The oxygen consumption ratio was also decreased in FADS2-depleted cells. These data indicate that FADS2 depletion causes a reduction in lipid levels, resulting in decrease of energy production and attenuation of cancer cell malignancy.
RESUMO
The accuracy of the structural annotation of unidentified peaks obtained in metabolomic analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be enhanced using retention time (RT) information as well as precursor and product ions. Unified-hydrophilic-interaction/anion-exchange liquid chromatography high-resolution tandem mass spectrometry (unified-HILIC/AEX/HRMS/MS) has been recently developed as an innovative method ideal for nontargeted polar metabolomics. However, the RT prediction for unified-HILIC/AEX has not been developed because of the complex separation mechanism characterized by the continuous transition of the separation modes from HILIC to AEX. In this study, we propose an RT prediction model of unified-HILIC/AEX/HRMS/MS, which enables the comprehensive structural annotation of polar metabolites. With training data for 203 polar metabolites, we ranked the feature importance using a random forest among 12,420 molecular descriptors (MDs) and constructed an RT prediction model with 26 selected MDs. The accuracy of the RT model was evaluated using test data for 51 polar metabolites, and 86.3% of the ΔRTs (difference between measured and predicted RTs) were within ±1.50 min, with a mean absolute error of 0.80 min, indicating high RT prediction accuracy. Nontargeted metabolomic data from the NIST SRM 1950-Metabolites in frozen human plasma were analyzed using the developed RT model and in silico MS/MS prediction, resulting in a successful structural estimation of 216 polar metabolites, in addition to the 62 identified based on standards. The proposed model can help accelerate the structural annotation of unknown hydrophilic metabolites, which is a key issue in metabolomic research.
Assuntos
Metaboloma , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Metabolômica/métodos , Ânions , Interações Hidrofóbicas e HidrofílicasRESUMO
Recently, various metabolites derived from host microbes have been reported to modulate the immune system, with potential involvement in health or diseases. Archaea, prokaryotic organisms, are present in the human body, but their connection with the host is largely unknown when compared to other microorganisms such as bacteria. This study focused on unique glycerolipids from symbiotic methanogenic archaea and evaluated their activities toward an innate immune receptor. The results revealed that archaeal lipids were recognized by the C-type lectin receptor Mincle and induced immune responses. A concurrent structure-activity relationship study identified the key structural features of archaeal lipids required for recognition by Mincle. Subsequent gene expression profiling suggested qualitative differences between the symbiotic archaeal lipid and the pathogenic bacteria-derived lipid. These findings have broad implications for understanding the function of symbiotic archaea in host health and diseases.
Assuntos
Archaea , Lectinas Tipo C , Humanos , Archaea/metabolismo , Lectinas Tipo C/metabolismo , Receptores Imunológicos/metabolismo , Relação Estrutura-Atividade , LipídeosRESUMO
Free radical-mediated lipid peroxidation (LPO) induces the formation of numerous lipid radicals, which contribute to the development of several oxidative diseases. To understand the mechanism of LPO in biological systems and the significance of these radicals, identifying the structures of individual lipid radicals is imperative. In this study, we developed an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and a profluorescent nitroxide probe, N-(1-oxyl-2,2,6-trimethyl-6-pentylpiperidin-4-yl)-3-(5,5-difluoro-1,3-dimethyl-3H,5H-5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-7-yl)propanamide (BDP-Pen), for the detailed structural analysis of lipid radicals. The MS/MS spectra of BDP-Pen-lipid radical adducts showed product ions and thus allow the prediction of the lipid radical structures and individual detection of isomeric adducts. Using the developed technology, we separately detected the isomers of arachidonic acid (AA)-derived radicals generated in AA-treated HT1080 cells. This analytical system is a powerful tool for elucidating the mechanism of LPO in biological systems.
Assuntos
Espectrometria de Massas em Tandem , Cromatografia Líquida , Radicais Livres/química , Peroxidação de Lipídeos , Ácido AraquidônicoRESUMO
C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors.
RESUMO
There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.
Assuntos
Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/biossíntese , Adenilato Quinase/genética , Tecido Adiposo Branco/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Canagliflozina/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Glicólise , Força da Mão , Fígado/efeitos dos fármacos , Masculino , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transportador 2 de Glucose-Sódio/fisiologia , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Serina-Treonina Quinases TOR/biossíntese , Serina-Treonina Quinases TOR/genéticaRESUMO
One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.
Assuntos
Metaboloma , Espectrometria de Massas em Tandem , Humanos , Células HeLa , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Metabolômica/métodos , Ânions , Aminas , PolímerosRESUMO
Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we propose a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis.
Assuntos
Lipídeos/análise , Neoplasias Hepáticas/diagnóstico , Animais , Dietilnitrosamina/administração & dosagem , Modelos Animais de Doenças , Radicais Livres/análise , Injeções Intraperitoneais , Neoplasias Hepáticas/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura MolecularRESUMO
Quantitatively and rapidly analyzing lipids is necessary to elucidate their biological functions. Herein, we developed a quantitative method for various lipid classes using supercritical fluid chromatography (SFC) coupled with a charged aerosol detector (CAD), providing high-throughput data analysis to detect a large number of molecules in each lipid class as one peak. Applying the CAD was useful for analyzing lipid molecules in the same lipid class with a constant response under the same mobile phase composition. First, we optimized the washing method for the diethylamine column, achieving baseline separation of lipid classes while maintaining good peak shapes. In addition, the CAD conditions (organic solvent evaporation and numerical correction of the CAD data) were optimized to improve the signal-to-noise ratio. We used an internal standard (ceramide phosphoethanolamine d17:1-12:0), which did not coelute with the lipid classes and showed high extraction efficiency. Based on a quantitative analysis of HepG2 cells, the concentration of lipid classes detected by CAD was adequate compared with that obtained by triple-quadrupole MS (QqQMS) in a previous study because the deviations of the concentrations were 0.6- to 2.3-fold. These results also supported the quantitative performance of SFC-QqQMS developed in our previous report.
Assuntos
Cromatografia com Fluido Supercrítico , Lipídeos/análise , Espectrometria de Massas , Aerossóis , Células Hep G2 , HumanosRESUMO
Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.-Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba.
Assuntos
Cromatografia com Fluido Supercrítico , Metabolismo dos Lipídeos , Espectrometria de Massas , Metabolômica/métodos , Animais , Isomerismo , Lipídeos/sangue , Lipídeos/química , Lipídeos/isolamento & purificação , Infarto do Miocárdio/metabolismo , Coelhos , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
A generally applicable method to discover xenobiotic metabolites is important to safely and effectively develop xenobiotics. We propose an advanced method to detect and identify comprehensive xenobiotic metabolites using stable isotope labeling, liquid chromatography coupled with benchtop quadrupole Orbitrap high-resolution tandem mass spectrometry (LC/HRMS/MS), data mining techniques (alignment, peak picking, and paired-peaks filtering), in silico metabolism prediction, and time-dependent profiling. The LC/HRMS analysis was carried out using Arabidopsis T87 cultured cells treated with unlabeled or with 13C- or 2H-labeled 2,4-dichlorophenoxyacetic acid (2,4-D). Paired-peak filtering enabled the accurate detection of 83 candidates for 2,4-D metabolites without any false positive peaks derived from solvents or the biological matrix. We confirmed 10 previously reported 2,4-D metabolites and identified 16 novel 2,4-D metabolites. Our method provides accurate detection and identification of comprehensive xenobiotic metabolites and represents a potentially useful tool for elucidating xenobiotic metabolism.
Assuntos
Ácido 2,4-Diclorofenoxiacético/metabolismo , Arabidopsis/metabolismo , Herbicidas/metabolismo , Espectrometria de Massas em Tandem/métodos , Xenobióticos/metabolismo , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Mineração de Dados/métodos , Marcação por Isótopo/métodosRESUMO
Expression of CD56 has recently been introduced as one of the adverse prognostic factors in acute promyelocytic leukemia (APL). However, the clinical significance of CD56 antigen in APL has not been well elucidated. We assessed the clinical significance of CD56 antigen in 239 APL patients prospectively treated with all-trans retinoic acid and chemotherapy according to the Japan Adult Leukemia Study Group APL97 protocol. All patients were prospectively treated by the Japan Adult Leukemia Study Group APL97 protocol. The median follow-up period was 8.5 years. Positive CD56 expression was found in 23 APL patients (9.6%). Expression of CD56 was significantly associated with lower platelet count (P = 0.04), severe disseminated intravascular coagulation (P = 0.04), and coexpression of CD2 (P = 0.03), CD7 (P = 0.04), CD34 (P < 0.01) and/or human leukocyte antigen-DR (P < 0.01). Complete remission rate and overall survival were not different between the two groups. However, cumulative incidence of relapse and event-free survival (EFS) showed an inferior trend in CD56(+) APL (P = 0.08 and P = 0.08, respectively). Among patients with initial white blood cell counts of 3.0 × 10(9)/L or more, EFS and cumulative incidence of relapse in CD56(+) APL were significantly worse (30.8% vs 63.6%, P = 0.008, and 53.8% vs 28.9%, P = 0.03, respectively), and in multivariate analysis, CD56 expression was an unfavorable prognostic factor for EFS (P = 0.04). In conclusion, for APL with higher initial white blood cell counts, CD56 expression should be regarded as an unfavorable prognostic factor.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno CD56/biossíntese , Leucemia Promielocítica Aguda/sangue , Leucemia Promielocítica Aguda/tratamento farmacológico , Adolescente , Adulto , Idoso , Antígeno CD56/genética , Citarabina/administração & dosagem , Intervalo Livre de Doença , Feminino , Humanos , Idarubicina/administração & dosagem , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Contagem de Plaquetas , Prognóstico , Estudos Prospectivos , Indução de Remissão , Resultado do Tratamento , Tretinoína/administração & dosagem , Adulto JovemRESUMO
Recently, the presence of CEBPA mutation was identified as an important prognostic factor for normal karyotype (NK) acute myeloid leukemia (AML). Because AML with CEBPA mutation is closely associated with CD7, CD15, CD34, and HLA-DR expression, we investigated the prognostic implications of CD7+ CD15+ CD34+ HLA-DR + immunophenotype in NK-AML. We analyzed the immunophenotype of 329 patients with NK-AML from the Japan Adult Leukemia Study Group (JALSG) AML97 population. NK-AML with the CD7+ CD15+ CD34+ HLA-DR + immunophenotype was classified as the CEBPA type, and NK-AML that did not meet this criterion was considered as the non-CEBPA type. The influence of the CEBPA status on event-free survival (EFS) and overall survival (OS) was assessed using log-rank test and a multivariate Cox proportional hazard regression model. Furthermore, the surface antigen expression profile in AML according to the CEBPA mutation status (monoallelic or biallelic) was also investigated. Of the 329 NK-AML patients that were studied, 39 and 243 were classified as having CEBPA and non-CEBPA type NK-AML, respectively. Patients with CEBPA type NK-AML had significantly better EFS and OS than those with non-CEBPA type NK-AML. Multivariate analysis showed that the CEBPA type and white blood cell (WBC) counts of >20 × 10(9)/L were independent prognostic factors for EFS and OS. Moreover, NK-AML with the biallelic CEBPA mutation was more closely associated with CD34 positivity than that with the monoallelic CEBPA mutation. NK-AML with the CD7+ CD15+ CD34+ HLA-DR + immunophenotype is a clinically discrete entity, and this may have a possible role in risk stratification.
Assuntos
Cariótipo , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Idoso , Antígenos CD34/análise , Antígenos CD7/análise , Antimetabólitos Antineoplásicos/uso terapêutico , Proteínas Estimuladoras de Ligação a CCAAT/genética , Citarabina/uso terapêutico , Feminino , Fucosiltransferases/análise , Antígenos HLA-DR/análise , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , Cariotipagem , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/tratamento farmacológico , Contagem de Leucócitos , Antígenos CD15/análise , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto/estatística & dados numéricos , Modelos de Riscos Proporcionais , Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Estudos Retrospectivos , Medição de Risco , Resultado do Tratamento , Adulto JovemRESUMO
Twice-daily administration of cyclosporine A (CyA) has often been used for prophylaxis of acute graft versus host disease in allogeneic hematopoietic stem cell transplantation (allo-HSCT). But there have not been any reports that calculated the conversion ratio of the switch from twice-daily intravenous infusion to oral administration of CyA in adult patients. To establish the conversion ratio and the best strategy of twice-daily administration of CyA, the authors investigated the serial changes in the blood CyA concentration during the switch from twice-daily intravenous infusion to oral administration while maintaining high-peak concentration (over 1000 ng/mL) and calculated the bioavailability of Neoral, a micro emulsion cyclosporine, in 11 patients. All the patients underwent allo-HSCT with graft versus host disease prophylaxis consisting of CyA at a high-peak concentration of twice-daily infusion with short-term methotrexate and oral administration. Neoral was started at an oral dose, 2 times daily, at twice the latest dose of intravenous dose according to the bioavailability of healthy volunteers. Micafungin, a mild inhibitor of CYP3A4, was administered for prophylaxis against fungal infection. The total area under the concentration-time curve during oral administration (AUCpo) was nearly the same as AUC during intravenous infusion (AUCiv) (mean ± SD, 7206 ± 1557 ng·h·mL and 7783 ± 897.7 ng·h·mL, respectively). The bioavailability of Neoral, defined as F = AUCpo × DOSEiv/AUCiv × DOSEpo was 0.58 ± 0.15 (mean ± SD, range: 0.41-0.94). When patients were switched from twice-daily infusion to oral administration, the dose conversion ratio of 1:2 seemed to be appropriate. At that time, the target trough level of Neoral was nearly the same as that of the infusion.
Assuntos
Ciclosporina/farmacocinética , Transplante de Células-Tronco Hematopoéticas , Imunossupressores/farmacocinética , Administração Oral , Adulto , Área Sob a Curva , Ciclosporina/administração & dosagem , Feminino , Doença Enxerto-Hospedeiro/etiologia , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
In metabolomic analysis, one of the most commonly used techniques to support the detection sensitivity and quantitation of mass spectrometry is combining it with liquid chromatography. Recently, we developed a method that enables comprehensive single-run measurement of hydrophilic metabolites using unified-hydrophilic interaction/anion exchange liquid chromatography/high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) with a polymer-based mixed amines column (Gelpack GL-HilicAex). However, the importance of stationary phase functional groups and mobile phase conditions for the separation mechanisms and sensitive detection in unified-HILIC/AEX/HRMS is not yet fully understood. This study aimed to understand the importance of the mobile and stationary phases in unified-HILIC/AEX/HRMS. Two different alkali-resistant polymer-based amines-modified columns (Gelpack GL-HilicAex, primary, secondary, tertiary, and quaternary amine-modified polyglycerol dimethacrylate gel; Asahipak NH2P-50 2D, secondary amine-modified polyvinyl alcohol gel) and two eluents (acetonitrile and ammonium bicarbonate solution, pH 9.8) were used for comparative validation. A comparison of mobile phase conditions using both columns confirmed that the two-step separation from HILIC to AEX characteristic of unified-HILIC/AEX requires a linear gradient condition from acetonitrile to nearly 50% water and AEX with up to 40 mM bicarbonate ions. We found that when alkali-resistant hydrophilic polymer packing materials are modified with amines, unified-HILIC/AEX separation can be reproduced if at least one secondary amine associated with the amine series is present in the stationary phase. Furthermore, the difference in sensitivity in the HILIC and AEX modes owing to the different columns indicates the need for further improvements in the mobile phase composition and stationary phase.
RESUMO
Upregulation of nuclear factor κB (NFκB) signaling is a hallmark of aging and a major cause of age-related chronic inflammation. However, its effect on cellular senescence remains unclear. Here, we show that alteration of NFκB nuclear dynamics from oscillatory to sustained by depleting a negative feedback regulator of NFκB pathway, NFκB inhibitor alpha (IκBα), in the presence of tumor necrosis factor α (TNFα) promotes cellular senescence. Sustained NFκB activity enhanced inflammatory gene expression through increased NFκB-DNA binding and slowed the cell cycle. IκBα protein was decreased under replicative or oxidative stress in vitro. Furthermore, a decrease in IκBα protein and an increase in DNA-NFκB binding at the transcription start sites of age-associated genes in aged mouse hearts suggested that nuclear NFκB dynamics may play a critical role in the progression of aging. Our study suggests that nuclear NFκB dynamics-dependent epigenetic changes regulated over time in a living system, possibly through a decrease in IκBα, enhance the expression of inflammatory genes to advance the cells to a senescent state.
RESUMO
Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin-based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue-derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function.
Assuntos
Células T Invariantes Associadas à Mucosa , Animais , Camundongos , Ácidos e Sais Biliares , Ligantes , Sulfatos , Antígenos de Histocompatibilidade Menor/metabolismo , AntígenosRESUMO
Dysregulation of liver metabolism associated with obesity during feeding and fasting leads to the breakdown of metabolic homeostasis. However, the underlying mechanism remains unknown. Here, we measured multi-omics data in the liver of wild-type and leptin-deficient obese (ob/ob) mice at ad libitum feeding and constructed a differential regulatory trans-omic network of metabolic reactions. We compared the trans-omic network at feeding with that at 16 h fasting constructed in our previous study. Intermediate metabolites in glycolytic and nucleotide metabolism decreased in ob/ob mice at feeding but increased at fasting. Allosteric regulation reversely shifted between feeding and fasting, generally showing activation at feeding while inhibition at fasting in ob/ob mice. Transcriptional regulation was similar between feeding and fasting, generally showing inhibiting transcription factor regulations and activating enzyme protein regulations in ob/ob mice. The opposite metabolic dysregulation between feeding and fasting characterizes breakdown of metabolic homeostasis associated with obesity.
RESUMO
Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen-sensing systems inducing acute hypoxic responses, including the hypoxia-inducible factor pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here we show that pyridoxine 5'-phosphate oxidase (PNPO), which catalyses bioactivation of vitamin B6, serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decreased PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and inhibited lysosomal acidification, which in macrophages led to iron dysregulation, TET2 protein loss and delayed resolution of the inflammatory response. Among PLP-dependent metabolism, supersulfide synthesis was suppressed in prolonged hypoxia, resulting in the lysosomal inhibition and consequent proinflammatory phenotypes of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing that gradually shuts down PLP-dependent metabolism in response to prolonged oxygen deprivation.
Assuntos
Lisossomos , Macrófagos , Fosfato de Piridoxal , Lisossomos/metabolismo , Macrófagos/metabolismo , Animais , Camundongos , Fosfato de Piridoxal/metabolismo , Hipóxia/metabolismo , Hipóxia Celular , Vitamina B 6/metabolismo , Oxigênio/metabolismo , Inflamação/metabolismoRESUMO
BACKGROUND: Contemporary treatment protocols for adult acute myeloid leukemia (AML) are age-specific, and older patients are generally treated less intensively than younger patients. However, it remains uncertain whether older but fit patients with AML really need to have their treatment attenuated. METHODS: To evaluate the contribution of age to outcome for patients with AML receiving intensive chemotherapy, data were analyzed for 2276 patients aged less than 65 years who were treated uniformly, regardless of age, in 3 consecutive prospective studies conducted by the Japan Adult Leukemia Study Group. RESULTS: A substantial drop in overall survival (OS) between patients aged 40 to 49 years and 50 to 64 years led to a focus on 2 comparisons: 1) age < 50 versus ≥ 50 years; and 2) age 50 to 54 versus 55 to 59 versus 60 to 64 years. OS was significantly better for patients aged < 50 years than that for those aged ≥ 50 years (49.6% and 37.0% at 5 years; P < .001); older patients were more susceptible to relapse, but not to early death or nonrelapse mortality. The significant differences in OS between these 2 age groups were equally seen for patients with favorable, intermediate, and adverse cytogenetics (P < .001 each). Outcomes for those aged 50 to 54, 55 to 59, and 60 to 64 years were similar, with 5-year OS rates of 38.2%, 35.1%, and 38.0%, respectively (P = .934), and no differences in early death or nonrelapse mortality were observed among these age groups. CONCLUSIONS: These findings justify the use of intensive chemotherapy without dose attenuation toward older but fit patients with AML, at least up to the age of 64 years.