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1.
Methods Mol Biol ; 2511: 161-174, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35838959

RESUMO

Testing of large populations for virus infection is now a reality worldwide due to the coronavirus (SARS-CoV-2) pandemic. The demand for SARS-CoV-2 testing using alternatives other than PCR led to the development of mass spectrometry (MS)-based assays. However, MS for SARS-CoV-2 large-scale testing have some downsides, including complex sample preparation and slow data analysis. Here, we describe a high-throughput targeted proteomics method to detect SARS-CoV-2 directly from nasopharyngeal and oropharyngeal swabs. This strategy employs fully automated sample preparation mediated by magnetic particles, followed by detection of SARS-CoV-2 nucleoprotein peptides by turbulent flow chromatography coupled with tandem mass spectrometry.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Pandemias , Espectrometria de Massas em Tandem/métodos
2.
J Chromatogr A ; 1654: 462457, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34404016

RESUMO

Signal variation is a common drawback in untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS), mainly due to the complexity of biological matrices and reduced sample preparation, which results in the accumulation of sample components in the column and the ion source. Here we propose a simple, easy to implement approach to improve data quality in untargeted metabolomics by LC-MS. This approach involves the use of a divert valve to direct the column effluent to waste at the beginning of the chromatographic run and during column cleanup and equilibration, in combination with longer column cleanups in between injections. Our approach was tested using urine samples collected from patients after renal transplantation. Analytical responses were contrasted before and after introducing these modifications by analyzing a batch of untargeted metabolomics data. A significant improvement in peak area repeatability was observed for the quality controls, with relative standard deviations (RSDs) for several metabolites decreasing from ∼60% to ∼10% when our approach was introduced. Similarly, RSDs of peak areas for internal standards improved from ∼40% to ∼10%. Furthermore, calibrant solutions were more consistent after introducing these modifications when comparing peak areas of solutions injected at the beginning and the end of each analytical sequence. Therefore, we recommend the use of a divert valve and extended column cleanup as a powerful strategy to improve data quality in untargeted metabolomics, especially for very complex types of samples where minimum sample preparation is required, such as in this untargeted metabolomics study with urine from renal transplanted patients.


Assuntos
Cromatografia Líquida , Confiabilidade dos Dados , Espectrometria de Massas , Metabolômica , Urinálise , Humanos , Urinálise/métodos , Urinálise/normas , Urina/química
3.
Nat Commun ; 11(1): 6201, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273458

RESUMO

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Here, we develop a high-throughput targeted proteomics assay to detect SARS-CoV-2 nucleoprotein peptides directly from nasopharyngeal and oropharyngeal swabs. A modified magnetic particle-based proteomics approach implemented on a robotic liquid handler enables fully automated preparation of 96 samples within 4 hours. A TFC-MS system allows multiplexed analysis of 4 samples within 10 min, enabling the processing of more than 500 samples per day. We validate this method qualitatively (Tier 3) and quantitatively (Tier 1) using 985 specimens previously analyzed by real-time RT-PCR, and detect up to 84% of the positive cases with up to 97% specificity. The presented strategy has high sample stability and should be considered as an option for SARS-CoV-2 testing in large populations.


Assuntos
Teste para COVID-19/métodos , Técnicas de Laboratório Clínico , Espectrometria de Massas/métodos , Humanos , Nasofaringe/virologia , Orofaringe/virologia , Proteômica , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Proteínas Virais
4.
Am J Physiol Cell Physiol ; 317(2): C326-C338, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31067084

RESUMO

Atherosclerotic plaque development is closely associated with the hemodynamic forces applied to endothelial cells (ECs). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. ECs under low or oscillatory SS (LSS) show upregulation of inflammatory, adhesion, and cellular permeability molecules. On the contrary, cells under high or laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind SS regulation of an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes in endothelial cells (human umbilical vein endothelial cells) under in vitro LSS and HSS that promote an atheroprone or atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism, in special cholesterol metabolism, was downregulated in cells under LSS. The low-density lipoprotein receptor (LDLR) showed significant alterations both at the quantitative expression level as well as regarding posttranslational modifications. Under LSS, LDLR was seen at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of a HSS metabolic phenotype in EC under LSS. Altogether, our data suggest that there is significant modulation of lipid metabolism in endothelial cells under different SS intensities and that this could contribute to the atheroprone phenotype of LSS. Statin treatment was able to partially recover the protective profile of these cells.


Assuntos
Aterosclerose/metabolismo , Hemodinâmica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Metabolismo dos Lipídeos , Lipidômica/métodos , Mecanotransdução Celular , Proteômica/métodos , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Atorvastatina/farmacologia , Células Cultivadas , Colesterol/metabolismo , Glicosilação , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mecanotransdução Celular/efeitos dos fármacos , Fenótipo , Placa Aterosclerótica , Processamento de Proteína Pós-Traducional , Receptores de LDL/metabolismo , Fluxo Sanguíneo Regional , Estresse Mecânico
5.
Sci Rep ; 6: 27882, 2016 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-27292372

RESUMO

It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 µg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early ß-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.


Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Proteoma/efeitos dos fármacos , Tecido Adiposo/metabolismo , Administração Intranasal , Animais , Peso Corporal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol/sangue , Glucose/metabolismo , Insulina/sangue , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Triglicerídeos/sangue , Regulação para Cima/efeitos dos fármacos
6.
Fertil Steril ; 105(3): 617-628, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26621572

RESUMO

OBJECTIVE: To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. DESIGN: Cross-sectional study. SETTING: University andrology and research laboratories. PATIENT(S): A total of 156 normozoospermic men. INTERVENTION(S): Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. MAIN OUTCOME MEASURE(S): Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. RESULT(S): In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. CONCLUSION(S): Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations.


Assuntos
Sêmen/metabolismo , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/metabolismo , Acrossomo/metabolismo , Acrossomo/patologia , Adulto , Biomarcadores/metabolismo , Cromatografia Líquida , Estudos Transversais , Dano ao DNA , DNA Mitocondrial/metabolismo , Análise Discriminante , Fertilidade , Humanos , Análise dos Mínimos Quadrados , Modelos Lineares , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Análise Multivariada , Proteômica/métodos , Espermatozoides/patologia , Espectrometria de Massas em Tandem
7.
BMC Cancer ; 15: 985, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26680993

RESUMO

BACKGROUND: Chronic hepatitis B (CHB) virus infection is a major cause of hepatocellular carcinoma (HCC), as late diagnosis is the main factor for the poor survival of patients. There is an urgent need for accurate biomarkers for early diagnosis of HCC. The aim of the study was to explore the serum lipidome profiles of hepatitis B-related HCC to identify potential diagnostic biomarkers. METHODS: An ultraperformance liquid chromatography mass spectrometry (UPLC-MS) lipidomic method was used to characterize serum profiles from HCC (n = 32), liver cirrhosis (LC) (n = 30), CHB (n = 25), and healthy subjects (n = 34). Patients were diagnosed by clinical laboratory and imaging evidence and all presented with CHB while healthy controls had normal liver function and no infectious diseases. RESULTS: The UPLC-MS-based serum lipidomic profile provided more accurate diagnosis for LC patients than conventional alpha-fetoprotein (AFP) detection. HCC patients were discriminated from LC with 78 % sensitivity and 64 % specificity. In comparison, AFP showed sensitivity and specificity of 38 % and 93 %, respectively. HCC was differentiated from CHB with 100 % sensitivity and specificity using the UPLC-MS approach. Identified lipids comprised glycerophosphocolines, glycerophosphoserines and glycerophosphoinositols. CONCLUSIONS: UPLC-MS lipid profiling proved to be an efficient and convenient tool for diagnosis and screening of HCC in a high-risk population.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/virologia , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hepatite B Crônica/diagnóstico , Lipídeos/sangue , Neoplasias Hepáticas/virologia , Adulto , Idoso , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Diagnóstico Diferencial , Detecção Precoce de Câncer , Feminino , Hepatite B Crônica/sangue , Humanos , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Masculino , Pessoa de Meia-Idade , Adulto Jovem
8.
Fertil Steril ; 104(2): 292-301, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26048152

RESUMO

OBJECTIVE: To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. DESIGN: Cross-sectional study. SETTING: University andrology and research laboratories. PATIENT(S): A total of 156 normozoospermic men. INTERVENTION(S): Seminal lipid peroxidation levels were assessed in individual samples through thiobarbituric acid reactive substances quantification. Subsequently, lipid peroxidation data were used to divide the samples into the experimental groups: low lipid peroxidation levels (control group, bottom 15%, n = 23) and high lipid peroxidation levels (study group, top 15%, n = 23). Seminal plasma proteins from these groups were pooled (four pools per group, with biological variation between the pools) and used for a shotgun proteomic analysis using a liquid chromatography-tandem mass spectrometry approach. Quantitative data were used for univariate (unpaired Student's t test) and multivariate (partial least-squares discriminant analysis, logistic regression, and discriminant analyses) statistical analyses. Significant proteins were also used for functional enrichment analysis. MAIN OUTCOME MEASURE(S): Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. RESULT(S): In total, 629 proteins were quantified in seminal plasma. Of these, 23 proteins were absent or underexpressed and 71 were exclusive or overexpressed in the study group. The main enriched functions in association with seminal lipid peroxidation were unsaturated fatty acids biosynthesis, oxidants and antioxidants activity, cellular response to heat stress, and immune response. Moreover, we suggested mucin-5B as a potential biomarker of semen oxidative stress. CONCLUSION(S): The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.


Assuntos
Estresse Oxidativo/fisiologia , Proteoma/biossíntese , Sêmen/metabolismo , Proteínas de Plasma Seminal/biossíntese , Adulto , Estudos Transversais , Humanos , Peroxidação de Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Proteoma/genética , Análise do Sêmen/métodos , Proteínas de Plasma Seminal/genética , Adulto Jovem
9.
Arthritis Res Ther ; 17: 168, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-26099944

RESUMO

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis. METHODS: Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope. RESULTS: After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases. CONCLUSION: M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.


Assuntos
Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Anidrase Carbônica III/sangue , Técnicas de Visualização da Superfície Celular/métodos , Modelos Animais de Doenças , Mimetismo Molecular/fisiologia , Adulto , Idoso , Sequência de Aminoácidos , Animais , Artrite Reumatoide/genética , Anidrase Carbônica III/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estrutura Secundária de Proteína
10.
Rev. bras. farmacogn ; 21(2): 349-354, mar.-abr. 2011. graf
Artigo em Inglês | LILACS | ID: lil-590184

RESUMO

We present here the effect of heavy metals and of different light intensities on the biosynthesis of fatty acids and pigments in the macroalga Gracilaria tenuistipitata (var. liui Zhang & Xia). In order to verify the fatty acid content, gas chromatography with flame ionization detection (GC-FID) was employed. Pigments (major carotenoids and chlorophyl-a) were monitored by liquid chromatography with diode array detection (HPLC-DAD). Cultures of G. tenuistipitata were exposed to cadmium (Cd2+, 200 ppb) and copper (Cu2+, 200 ppb), as well as to different light conditions (low light: 100 µmol.photons.m-2.s-1, or high light: 1000 µmol.photons.m-2.s-1). Cd2+ and Cu2+ increased the saturated and monounsaturated fatty acid content [14:0, 16:0, 18:0, 18:1 (n-7) and 18:1 (n-9)] and all major pigments (violaxanthin, antheraxanthin, lutein, zeaxanthin, chlorophyll-a and β-carotene). Both heavy metals decreased the levels of polyunsaturated fatty acids (PUFA) [18:2 (n-6), 18:3 (n-6), 18:5 (n-4), 20:4 (n-6), 20:5 (n-3), 22:6 (n-3)]. G. tenuistipitata cultures were exposed to high light intensity for five days and no statistically significant differences were observed in the content of fatty acids. On the other hand, the levels of pigments rose markedly for chlorophyll-a and all of the carotenoids studied.

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