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1.
J Proteome Res ; 21(12): 2936-2946, 2022 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-36367990

RESUMO

Untargeted liquid chromatography-mass spectrometry metabolomics studies are typically performed under roughly identical experimental settings. Measurements acquired with different LC-MS protocols or following extended time intervals harbor significant variation in retention times and spectral abundances due to altered chromatographic, spectrometric, and other factors, raising many data analysis challenges. We developed a computational workflow for merging and harmonizing metabolomics data acquired under disparate LC-MS conditions. Plasma metabolite profiles were collected from two sets of maternal subjects three years apart using distinct instruments and LC-MS procedures. Metabolomics features were aligned using metabCombiner to generate lists of compounds detected across all experimental batches. We applied data set-specific normalization methods to remove interbatch and interexperimental variation in spectral intensities, enabling statistical analysis on the assembled data matrix. Bioinformatics analyses revealed large-scale metabolic changes in maternal plasma between the first and third trimesters of pregnancy and between maternal plasma and umbilical cord blood. We observed increases in steroid hormones and free fatty acids from the first trimester to term of gestation, along with decreases in amino acids coupled to increased levels in cord blood. This work demonstrates the viability of integrating nonidentically acquired LC-MS metabolomics data and its utility in unconventional metabolomics study designs.


Assuntos
Aminoácidos , Metabolômica , Gravidez , Feminino , Humanos , Metabolômica/métodos , Cromatografia Líquida , Espectrometria de Massas/métodos , Aminoácidos/metabolismo , Plasma/metabolismo
2.
Anal Chem ; 93(12): 5028-5036, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33724799

RESUMO

LC-HRMS experiments detect thousands of compounds, with only a small fraction of them identified in most studies. Traditional data processing pipelines contain an alignment step to assemble the measurements of overlapping features across samples into a unified table. However, data sets acquired under nonidentical conditions are not amenable to this process, mostly due to significant alterations in chromatographic retention times. Alignment of features between disparately acquired LC-MS metabolomics data could aid collaborative compound identification efforts and enable meta-analyses of expanded data sets. Here, we describe metabCombiner, a new computational pipeline for matching known and unknown features in a pair of untargeted LC-MS data sets and concatenating their abundances into a combined table of intersecting feature measurements. metabCombiner groups features by mass-to-charge (m/z) values to generate a search space of possible feature pair alignments, fits a spline through a set of selected retention time ordered pairs, and ranks alignments by m/z, mapped retention time, and relative abundance similarity. We evaluated this workflow on a pair of plasma metabolomics data sets acquired with different gradient elution methods, achieving a mean absolute retention time prediction error of roughly 0.06 min and a weighted per-compound matching accuracy of approximately 90%. We further demonstrate the utility of this method by comprehensively mapping features in urine and muscle metabolomics data sets acquired from different laboratories. metabCombiner has the potential to bridge the gap between otherwise incompatible metabolomics data sets and is available as an R package at https://github.com/hhabra/metabCombiner and Bioconductor.


Assuntos
Metabolômica , Cromatografia Líquida , Espectrometria de Massas , Fluxo de Trabalho
3.
Bioinformatics ; 36(6): 1801-1806, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31642507

RESUMO

MOTIVATION: When metabolites are analyzed by electrospray ionization (ESI)-mass spectrometry, they are usually detected as multiple ion species due to the presence of isotopes, adducts and in-source fragments. The signals generated by these degenerate features (along with contaminants and other chemical noise) obscure meaningful patterns in MS data, complicating both compound identification and downstream statistical analysis. To address this problem, we developed Binner, a new tool for the discovery and elimination of many degenerate feature signals typically present in untargeted ESI-LC-MS metabolomics data. RESULTS: Binner generates feature annotations and provides tools to help users visualize informative feature relationships that can further elucidate the underlying structure of the data. To demonstrate the utility of Binner and to evaluate its performance, we analyzed data from reversed phase LC-MS and hydrophilic interaction chromatography (HILIC) platforms and demonstrated the accuracy of selected annotations using MS/MS. When we compared Binner annotations of 75 compounds previously identified in human plasma samples with annotations generated by three similar tools, we found that Binner achieves superior performance in the number and accuracy of annotations while simultaneously minimizing the number of incorrectly annotated principal ions. Data reduction and pattern exploration with Binner have allowed us to catalog a number of previously unrecognized complex adducts and neutral losses generated during the ionization of molecules in LC-MS. In summary, Binner allows users to explore patterns in their data and to efficiently and accurately eliminate a significant number of the degenerate features typically found in various LC-MS modalities. AVAILABILITY AND IMPLEMENTATION: Binner is written in Java and is freely available from http://binner.med.umich.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Metabolômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Humanos , Íons , Espectrometria de Massas por Ionização por Electrospray
4.
Int J Mol Sci ; 23(1)2021 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-35008662

RESUMO

Metabolic remodeling plays an important role in the pathophysiology of heart failure (HF). We sought to characterize metabolic remodeling and implicated signaling pathways in two rat models of early systolic dysfunction (MOD), and overt systolic HF (SHF). Tandem mass tag-labeled shotgun proteomics, phospho-(p)-proteomics, and non-targeted metabolomics analyses were performed in left ventricular myocardium tissue from Sham, MOD, and SHF using liquid chromatography-mass spectrometry, n = 3 biological samples per group. Mitochondrial proteins were predominantly down-regulated in MOD (125) and SHF (328) vs. Sham. Of these, 82% (103/125) and 66% (218/328) were involved in metabolism and respiration. Oxidative phosphorylation, mitochondrial fatty acid ß-oxidation, Krebs cycle, branched-chain amino acids, and amino acid (glutamine and tryptophan) degradation were highly enriched metabolic pathways that decreased in SHF > MOD. Glycogen and glucose degradation increased predominantly in MOD, whereas glycolysis and pyruvate metabolism decreased predominantly in SHF. PKA signaling at the endoplasmic reticulum-mt interface was attenuated in MOD, whereas overall PKA and AMPK cellular signaling were attenuated in SHF vs. Sham. In conclusion, metabolic remodeling plays an important role in myocardial remodeling. PKA and AMPK signaling crosstalk governs metabolic remodeling in progression to SHF.


Assuntos
Insuficiência Cardíaca Sistólica/metabolismo , Redes e Vias Metabólicas , Metabolômica , Adenilato Quinase/metabolismo , Animais , Cromatografia Líquida , Ciclo do Ácido Cítrico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glicólise , Espectrometria de Massas , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Ratos , Transdução de Sinais
5.
Metabolomics ; 16(10): 113, 2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33044703

RESUMO

INTRODUCTION: The metabolomics quality assurance and quality control consortium (mQACC) evolved from the recognized need for a community-wide consensus on improving and systematizing quality assurance (QA) and quality control (QC) practices for untargeted metabolomics. OBJECTIVES: In this work, we sought to identify and share the common and divergent QA and QC practices amongst mQACC members and collaborators who use liquid chromatography-mass spectrometry (LC-MS) in untargeted metabolomics. METHODS: All authors voluntarily participated in this collaborative research project by providing the details of and insights into the QA and QC practices used in their laboratories. This sharing was enabled via a six-page questionnaire composed of over 120 questions and comment fields which was developed as part of this work and has proved the basis for ongoing mQACC outreach. RESULTS: For QA, many laboratories reported documenting maintenance, calibration and tuning (82%); having established data storage and archival processes (71%); depositing data in public repositories (55%); having standard operating procedures (SOPs) in place for all laboratory processes (68%) and training staff on laboratory processes (55%). For QC, universal practices included using system suitability procedures (100%) and using a robust system of identification (Metabolomics Standards Initiative level 1 identification standards) for at least some of the detected compounds. Most laboratories used QC samples (>86%); used internal standards (91%); used a designated analytical acquisition template with randomized experimental samples (91%); and manually reviewed peak integration following data acquisition (86%). A minority of laboratories included technical replicates of experimental samples in their workflows (36%). CONCLUSIONS: Although the 23 contributors were researchers with diverse and international backgrounds from academia, industry and government, they are not necessarily representative of the worldwide pool of practitioners due to the recruitment method for participants and its voluntary nature. However, both questionnaire and the findings presented here have already informed and led other data gathering efforts by mQACC at conferences and other outreach activities and will continue to evolve in order to guide discussions for recommendations of best practices within the community and to establish internationally agreed upon reporting standards. We very much welcome further feedback from readers of this article.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Humanos , Laboratórios , Controle de Qualidade , Projetos de Pesquisa , Inquéritos e Questionários
6.
Metabolomics ; 15(1): 4, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30830465

RESUMO

We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.


Assuntos
Metabolômica/métodos , Metabolômica/normas , Humanos , Laboratórios , Controle de Qualidade , Melhoria de Qualidade
7.
Am J Physiol Heart Circ Physiol ; 314(2): H160-H169, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28986357

RESUMO

Maintaining a balance of ω-6 and ω-3 fatty acids is essential for cardiac health. Current ω-6 and ω-3 fatty acids in the American diet have shifted from the ideal ratio of 2:1 to almost 20:1; while there is a body of evidence that suggests the negative impact of such a shift in younger organisms, the underlying age-related metabolic signaling in response to the excess influx of ω-6 fatty acids is incompletely understood. In the present study, young (6 mo old) and aging (≥18 mo old) mice were fed for 2 mo with a ω-6-enriched diet. Excess intake of ω-6 enrichment decreased the total lean mass and increased nighttime carbohydrate utilization, with higher levels of cardiac cytokines indicating low-grade chronic inflammation. Dobutamine-induced stress tests displayed an increase in PR interval, a sign of an atrioventricular defect in ω-6-fed aging mice. Excess ω-6 fatty acid intake in aging mice showed decreased 12-lipoxygenase with a concomitant increase in 15-lipoxygenase levels, resulting in the generation of 15( S)-hydroxyeicosatetraenoic acid, whereas cyclooxygenase-1 and -2 generated prostaglandin E2, leukotriene B4, and thromboxane B2. Furthermore, excessive ω-6 fatty acids led to dysregulated nuclear erythroid 2-related factor 2/antioxidant-responsive element in aging mice. Moreover, ω-6 fatty acid-mediated changes were profound in aging mice with respect to the eicosanoid profile while minimal changes were observed in the size and shape of cardiomyocytes. These findings provide compelling evidence that surplus consumption of ω-6 fatty acids, coupled with insufficient intake of ω-3 fatty acids, is linked to abnormal changes in ECG. These manifestations contribute to functional deficiencies and expansion of the inflammatory mediator milieu during later stages of aging. NEW & NOTEWORTHY Aging has a profound impact on the metabolism of fatty acids to maintain heart function. The excess influx of ω-6 fatty acids in aging perturbed electrocardiography with marked signs of inflammation and a dysregulated oxidative-redox balance. Thus, the quality and quantity of fatty acids determine the cardiac pathology and energy utilization in aging.


Assuntos
Envelhecimento/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Arritmias Cardíacas/induzido quimicamente , Eletrocardiografia , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos Ômega-6/toxicidade , Sistema de Condução Cardíaco/efeitos dos fármacos , Inflamação/induzido quimicamente , Potenciais de Ação/efeitos dos fármacos , Fatores Etários , Ração Animal , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Doença Crônica , Citocinas/genética , Citocinas/metabolismo , Ácidos Graxos Ômega-6/administração & dosagem , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/fisiopatologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Estado Nutricional , Medição de Risco , Fatores de Risco
8.
J Lipid Res ; 58(12): 2275-2288, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28986437

RESUMO

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.


Assuntos
Benchmarking , Ensaio de Proficiência Laboratorial/estatística & dados numéricos , Lipídeos/sangue , Humanos , Cooperação Internacional , Metabolismo dos Lipídeos/fisiologia , Lipídeos/normas , Variações Dependentes do Observador , Padrões de Referência , Reprodutibilidade dos Testes
11.
Cancer Discov ; 13(11): 2370-2393, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37584601

RESUMO

Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. SIGNIFICANCE: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293.


Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Glioma/genética , Glioma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Histonas/genética , Resultado do Tratamento , Epigênese Genética , Mutação
12.
J Biol Chem ; 285(10): 7197-207, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20056612

RESUMO

During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, cargo, and cytoskeletal proteins. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil 84 proteins with significant changes in abundance under the mitotic condition compared with the interphase condition. Among these proteins, several COPI coatomer subunits (alpha, beta, gamma, and delta) are of particular interest. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.


Assuntos
Ciclo Celular/fisiologia , Complexo de Golgi , Membranas Intracelulares , Proteoma/análise , Proteômica/métodos , Animais , Sistema Livre de Células , Análise por Conglomerados , Biologia Computacional , Citosol/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Fígado/citologia , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ratos
13.
Nutrients ; 13(10)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34684365

RESUMO

As the incidence of obesity and type 2 diabetes (T2D) is occurring at a younger age, studying adolescent nutrient metabolism can provide insights on the development of T2D. Metabolic challenges, including an oral glucose tolerance test (OGTT) can assess the effects of perturbations in nutrient metabolism. Here, we present alterations in the global metabolome in response to an OGTT, classifying the influence of obesity and insulin resistance (IR) in adolescents that arrived at the clinic fasted and in a random-fed state. Participants were recruited as lean (n = 55, aged 8-17 years, BMI percentile 5-85%) and overweight and obese (OVOB, n = 228, aged 8-17 years, BMI percentile ≥ 85%). Untargeted metabolomics profiled 246 annotated metabolites in plasma at t0 and t60 min during the OGTT. Our results suggest that obesity and IR influence the switch from fatty acid (FA) to glucose oxidation in response to the OGTT. Obesity was associated with a blunted decline of acylcarnitines and fatty acid oxidation intermediates. In females, metabolites from the Fasted and Random-Fed OGTT were associated with HOMA-IR, including diacylglycerols, leucine/isoleucine, acylcarnitines, and phosphocholines. Our results indicate that at an early age, obesity and IR may influence the metabolome dynamics in response to a glucose challenge.


Assuntos
Jejum/metabolismo , Comportamento Alimentar , Resistência à Insulina , Metaboloma , Obesidade/metabolismo , Caracteres Sexuais , Adolescente , Glicemia/metabolismo , Criança , Feminino , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Cinética , Masculino , Obesidade/sangue
14.
J Trauma Acute Care Surg ; 90(3): 507-514, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33196629

RESUMO

OBJECTIVE: Traumatic brain injury (TBI) is a leading cause of trauma-related morbidity and mortality. Valproic acid (VPA) has been shown to attenuate brain lesion size and swelling within the first few hours following TBI. Because injured neurons are sensitive to metabolic changes, we hypothesized that VPA treatment would alter the metabolic profile in the perilesional brain tissues to create a neuroprotective environment. METHODS: We subjected swine to combined TBI (12-mm cortical impact) and hemorrhagic shock (40% blood volume loss and 2 hours of hypotension) and randomized them to two groups (n = 5/group): (1) normal saline (NS; 3× hemorrhage volume) and (2) NS-VPA (NS, 3× hemorrhage volume; VPA, 150 mg/kg). After 6 hours, brains were harvested, and 100 mg of the perilesional tissue was used for metabolite extraction. Samples were analyzed using reversed-phase liquid chromatography-mass spectrometry in positive and negative ion modes, and data were analyzed using MetaboAnalyst software (McGill University, Quebec, Canada). RESULTS: In untargeted reversed-phase liquid chromatography-mass spectrometry analysis, we detected 3,750 and 1,955 metabolites in positive and negative ion modes, respectively. There were no significantly different metabolites in positive ion mode; however, 167 metabolite features were significantly different (p < 0.05) in the negative ion mode, which included VPA derivates. Pathway analysis showed that several pathways were affected in the treatment group, including the biosynthesis of unsaturated fatty acids (p = 0.001). Targeted amino acid analysis on glycolysis/tricarboxylic acid (TCA) cycle revealed that VPA treatment significantly decreased the levels of the excitotoxic amino acid serine (p = 0.001). CONCLUSION: Valproic acid can be detected in perilesional tissues in its metabolized form. It also induces metabolic changes in the brains within the first few hours following TBI to create a neuroprotective environment.


Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Choque Hemorrágico/tratamento farmacológico , Choque Hemorrágico/metabolismo , Ácido Valproico/uso terapêutico , Animais , Lesões Encefálicas Traumáticas/patologia , Modelos Animais de Doenças , Feminino , Neuroproteção , Choque Hemorrágico/patologia , Suínos
15.
Cancer Discov ; 11(11): 2904-2923, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34039636

RESUMO

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.


Assuntos
Acil-CoA Desidrogenase , Glioblastoma , Peroxidação de Lipídeos , Mitocôndrias , Acil-CoA Desidrogenase/metabolismo , Apoptose , Ácidos Graxos/metabolismo , Glioblastoma/enzimologia , Glioblastoma/genética , Humanos , Mitocôndrias/metabolismo , Estresse Oxidativo
16.
Anaerobe ; 16(4): 454-60, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20338254

RESUMO

Clostridium sordellii is a toxin-producing anaerobic bacillus that causes severe infections in humans and livestock. C. sordellii infections can be accompanied by a highly lethal toxic shock syndrome (TSS). Lethal toxin (TcsL) is an important mediator of TSS. We recently obtained a clinical strain of C. sordellii (DA-108) lacking the TcsL-encoding tcsL gene, which was not fatal in rodent models of infection, in contrast to a tcsL(+) reference strain (ATCC9714). Protein preparations derived from cell-free, stationary phase cultures obtained from ATCC9714 were lethal when injected into mice, while those obtained from DA-108 were not, a difference that was attributed to the unique presence of TcsL in the ATCC9714-derived proteins. We questioned whether there were other major differences between the extracellular proteomes of these two strains, apart from TcsL. Two-dimensional gel electrophoresis was conducted using crude cell-free supernatants from these strains and 14 differentially expressed proteins were subjected to mass spectrometric analysis. Nine of these 14 proteins were more highly expressed by DA-108 and 5 by ATCC9714. Twelve of the 14 proteins isolated from the 2-D gels were putatively identified by mass spectrometry. Several of these proteins were identical, possibly reflecting enzymatic cleavage, degradation, and/or post-translational modifications. Excluding identical sequences, only 5 unique proteins were identified. Four proteins (ferredoxin-nitrite reductase; formate acetyltransferase; Translation Elongation Factor G; and purine nucleoside phosphorylase) were over-expressed by DA-108 and 1 (N-acetylmuramoyl-l-alanine amidase) by ATCC9714. These results support the concept that TcsL is the major determinant of C. sordellii TSS during infection.


Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Clostridium sordellii/química , Proteoma/análise , Fatores de Virulência/análise , Toxinas Bacterianas/genética , Clostridium sordellii/patogenicidade , Eletroforese em Gel Bidimensional , Espectrometria de Massas
17.
J Clin Endocrinol Metab ; 105(7)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32413135

RESUMO

CONTEXT: A person's intrinsic metabolism, reflected in the metabolome, may describe the relationship between nutrient intake and metabolic health. OBJECTIVES: Untargeted metabolomics was used to identify metabolites associated with metabolic health. Path analysis classified how habitual dietary intake influences body mass index z-score (BMIz) and insulin resistance (IR) through changes in the metabolome. DESIGN: Data on anthropometry, fasting metabolites, C-peptide, and dietary intake were collected from 108 girls and 98 boys aged 8 to 14 years. Sex-stratified linear regression identified metabolites associated with BMIz and homeostatic model assessment of IR using C-peptide (HOMA-CP), accounting for puberty, age, and muscle and fat area. Path analysis identified clusters of metabolites that underlie the relationship between energy-adjusted macronutrient intake with BMIz and HOMA-CP. RESULTS: Metabolites associated with BMIz include positive associations with diglycerides among girls and positive associations with branched chain and aromatic amino acids in boys. Intermediates in fatty acid metabolism, including medium-chain acylcarnitines (AC), were inversely associated with HOMA-CP. Carbohydrate intake is positively associated with HOMA-CP through decreases in levels of AC, products of ß-oxidation. Approaching significance, fat intake is positively associated with HOMA-CP through increases in levels of dicarboxylic fatty acids, products of omega-oxidation. CONCLUSIONS: This cross-sectional analysis suggests that IR in children is associated with reduced fatty acid oxidation capacity. When consuming more grams of fat, there is evidence for increased extramitochondrial fatty acid metabolism, while higher carbohydrate intake appears to lead to decreases in intermediates of ß-oxidation. Thus, biomarkers of IR and mitochondrial oxidative capacity may depend on macronutrient intake.


Assuntos
Resistência à Insulina , Mitocôndrias/metabolismo , Estado Nutricional , Adolescente , Índice de Massa Corporal , Criança , Estudos Transversais , Dieta , Humanos , Masculino , Metaboloma , Avaliação Nutricional
18.
Nat Commun ; 11(1): 3811, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732914

RESUMO

Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming therapy resistance. Treatments that are effective independent of genotype are urgently needed. By correlating intracellular metabolite levels with radiation resistance across dozens of genomically-distinct models of GBM, we find that purine metabolites, especially guanylates, strongly correlate with radiation resistance. Inhibiting GTP synthesis radiosensitizes GBM cells and patient-derived neurospheres by impairing DNA repair. Likewise, administration of exogenous purine nucleosides protects sensitive GBM models from radiation by promoting DNA repair. Neither modulating pyrimidine metabolism nor purine salvage has similar effects. An FDA-approved inhibitor of GTP synthesis potentiates the effects of radiation in flank and orthotopic patient-derived xenograft models of GBM. High expression of the rate-limiting enzyme of de novo GTP synthesis is associated with shorter survival in GBM patients. These findings indicate that inhibiting purine synthesis may be a promising strategy to overcome therapy resistance in this genomically heterogeneous disease.


Assuntos
Neoplasias Encefálicas/radioterapia , Reparo do DNA/genética , Glioblastoma/radioterapia , Guanosina Monofosfato/metabolismo , Tolerância a Radiação/genética , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Feminino , Glioblastoma/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Nucleosídeos de Purina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Biochem Biophys Res Commun ; 379(2): 179-85, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19073145

RESUMO

Two new peptide antibiotics were secreted by a Gram-positive bacterial strain isolated from fermented tomato fruit. Based on its 99% 16S rDNA sequence similarity with Paenibacillus alvei, the isolate was designated as P. alvei NP75. Among these two peptides, one is active against Gram-positive pathogens while the other against Gram-negative pathogens; thus these peptides were named as paenibacillin P and paenibacillin N, respectively. After the purification of those peptide antibiotics from the cell free culture supernatant by RP-HPLC, they were analyzed for their temperature sensitivity and susceptibility to proteases. Higher-temperature tolerant paenibacillin N was easily degraded by proteinase K, while the temperature sensitive paenibacillin P was not affected by any of the proteases used in this study other than a specific protease that was secreted by the same NP75 strain. Mass-spectrometry analysis of the above peptide antibiotics further confirmed their distinction among the known peptide antibiotics. We are reporting first of its kind the co-production of two different new peptide antibiotics from a single bacterial isolate of P. alvei strain.


Assuntos
Antibacterianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/biossíntese , Bacillus/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus/genética , Bacillus/isolamento & purificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Solanum lycopersicum/microbiologia , Espectrometria de Massas
20.
Clin Appl Thromb Hemost ; 15(2): 201-8, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19028772

RESUMO

Microparticles are small membrane vesicles released from activated cells and are associated with thrombosis and inflammation. Microparticle contain a unique subset of surface protein derived form the parent cell and may be responsible for their diverse biological functions. To identify these proteins, juvenile baboons (Papio anubis, n = 4) underwent iliac vein thrombosis with 6-hour balloon occlusion. Plasma samples were taken at baselines and at 2 days postthrombosis for microparticle analysis. Microparticles were extracted from platelet-poor plasma, digest separately with trypsin and tagged using isobaric tagging for relative and absolute quantitation reagents. The digests were subjected to 2-dimensional liquid chromatographic separation followed by matrix-assisted laser desorption/ionization tandem mass spectrometry. Peak lists were generated and searched against all primate sequences. For protein identity, a minimum of 2 peptides at 95% confidence interval was required. Later, isobaric tagging for relative and absolute quantitation ratios were generated comparing relative protein level of day 2 to baseline. The proteomic analysis was performed twice for each blood sample, totaling 8 experiments. Proteins were considered elevated of depressed if the isobaris tagging for relative and absolute quantitation ratio deviated by 20% changes from normal and a P value less than .05. Significantly, 7 proteins were differentially expressed on day 2 compared to baseline, and appeared in at least 3 animals and regulated in at least 4 experiment. Among these 7 proteins, upregulated proteins include various forms of fibrinogen and alpha-1-antichymotrypsin and downregulated proteins include immunoglobulins. These proteins influence thrombosis and inflammation through hemostatic plug formation (fibrinogen), inhibiting neutrophil adhesion (alpha-1-antichymoptrypsin), and immunoregulation (immunoglobulins). Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis.


Assuntos
Proteínas/metabolismo , Trombose Venosa/sangue , Animais , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional , Fibrinogênio/metabolismo , Modelos Animais , Selectina-P/metabolismo , Papio , Tamanho da Partícula , Proteômica/métodos , Propriedades de Superfície
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