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1.
PLoS One ; 15(8): e0234539, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756554

RESUMO

Diabetes Mellitus (DM) accelerates coronary artery disease (CAD) and atherosclerosis, the causes of most heart attacks. The biomolecules involved in these inter-related disease processes are not well understood. This study analyzes biomolecules in the sera of patients with CAD, with and without type (T) 2DM, who are about to undergo coronary artery bypass graft (CABG) surgery. The goal is to develop methodology to help identify and monitor CAD patients with and without T2DM, in order to better understand these phenotypes and to glean relationships through analysis of serum biomolecules. Aorta, fat, muscle, and vein tissues from CAD T2DM patients display diabetic-related histologic changes (e.g., lipid accumulation, fibrosis, loss of cellularity) when compared to non-diabetic CAD patients. The patient discriminatory methodology utilized is serum biomolecule mass profiling. This mass spectrometry (MS) approach is able to distinguish the sera of a group of CAD patients from controls (p value 10-15), with the CAD group containing both T2DM and non-diabetic patients. This result indicates the T2DM phenotype does not interfere appreciably with the CAD determination versus control individuals. Sera from a group of T2DM CAD patients however are distinguishable from non-T2DM CAD patients (p value 10-8), indicating it may be possible to examine the T2DM phenotype within the CAD disease state with this MS methodology. The same serum samples used in the CAD T2DM versus non-T2DM binary group comparison were subjected to MS/MS peptide structure analysis to help identify potential biochemical and phenotypic changes associated with CAD and T2DM. Such peptide/protein identifications could lead to improved understanding of underlying mechanisms, additional biomarkers for discriminating and monitoring these disease conditions, and potential therapeutic targets. Bioinformatics/systems biology analysis of the peptide/protein changes associated with CAD and T2DM suggested cell pathways/systems affected include atherosclerosis, DM, fibrosis, lipogenesis, loss of cellularity (apoptosis), and inflammation.


Assuntos
Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/complicações , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/sangue , Adulto , Idoso , Biomarcadores/sangue , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , Ponte de Artéria Coronária , Doença da Artéria Coronariana/cirurgia , Estudos Transversais , Angiopatias Diabéticas/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Espectrometria de Massas por Ionização por Electrospray , Biologia de Sistemas , Espectrometria de Massas em Tandem
2.
Cell Syst ; 11(1): 11-24.e4, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32619549

RESUMO

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.


Assuntos
Proteínas Sanguíneas/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Proteômica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Infecções por Coronavirus/classificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/classificação , Pneumonia Viral/classificação , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Adulto Jovem
3.
Nature ; 583(7816): 425-430, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612231

RESUMO

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Barreira Hematoencefálica/metabolismo , Transcitose , Fosfatase Alcalina/metabolismo , Animais , Anticorpos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacocinética , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Saúde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasma/metabolismo , Proteoma/administração & dosagem , Proteoma/metabolismo , Proteoma/farmacocinética , Receptores da Transferrina/imunologia , Transcrição Genética , Transferrina/metabolismo
4.
Eur J Endocrinol ; 183(3): 285-295, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32567559

RESUMO

Objective: Determining the factors associated with new-onset pre-diabetes and type 2 diabetes mellitus (T2D) is important for improving the current prevention strategies and for a better understanding of the disease. Design: To study the factors (clinical, circulating protein and genetic) associated with new onset pre-diabetes and T2D in an initially healthy (without diabetes) populational familial cohort with a long follow-up (STANISLAS cohort). Methods: A total of 1506 participants attended both the visit 1 and visit 4, separated by ≈20 years. Over 400 proteins, GWAS and genetic associations were studied using models adjusted for potential confounders. Both prospective (V1 to V4) and cross-sectional (V4) analyses were performed. Results: People who developed pre-diabetes (n = 555) and/or T2D (n = 73) were older, had higher BMI, blood pressure, glucose, LDL cholesterol, and lower eGFR. After multivariable selection, PAPP-A (pappalysin-1) was the only circulating protein associated with the onset of both pre-diabetes and T2D with associations persisting at visit 4 (i.e. ≈20 years later). FGF-21 (fibroblast growth factor 21) was a strong prognosticator for incident T2D in the longitudinal analysis, but not in the cross-sectional analysis. The heritability of the circulating PAPP-A was estimated at 44%. In GWAS analysis, the SNP rs634737 was associated with PAPP-A both at V1 and V4. External replication also showed lower levels of PAPP-A in patients with T2D. Conclusions: The risk of developing pre-diabetes and T2D increases with age and with features of the metabolic syndrome. Circulating PAPP-A, which has an important genetic component, was associated with both the development and presence of pre-diabetes and T2D.


Assuntos
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Genômica/métodos , Proteômica/métodos , Adulto , Estudos de Coortes , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Masculino , Estado Pré-Diabético/sangue , Estado Pré-Diabético/genética , Estado Pré-Diabético/metabolismo , Estudos Prospectivos , Fatores de Risco , Adulto Jovem
5.
PLoS One ; 15(6): e0234502, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525915

RESUMO

Preservation of blood plasma in the dried state would facilitate long-term storage and transport at ambient temperatures, without the need of to use liquid nitrogen tanks or freezers. The aim of this study was to investigate the feasibility of dry preservation of human plasma, using sugars as lyoprotectants, and evaluate macromolecular stability of plasma components during storage. Blood plasma from healthy donors was freeze dried using 0-10% glucose, sucrose, or trehalose, and stored at various temperatures. Differential scanning calorimetry was used to measure the glass transition temperatures of freeze-dried samples. Protein aggregation, the overall protein secondary structure, and oxidative damage were studied under different storage conditions. Differential scanning calorimetry measurements showed that plasma freeze-dried with glucose, sucrose and trehalose have glass transition temperatures of respectively 72±3.4°C, 46±11°C, 15±2.4°C. It was found that sugars diminish freeze-drying induced protein aggregation in a dose-dependent manner, and that a 10% (w/v) sugar concentration almost entirely prevents protein aggregation. Protein aggregation after rehydration coincided with relatively high contents of ß-sheet structures in the dried state. Trehalose reduced the rate of protein aggregation during storage at elevated temperatures, and plasma that is freeze- dried plasma with trehalose showed a reduced accumulation of reactive oxygen species and protein oxidation products during storage. In conclusion, freeze-drying plasma with trehalose provides an attractive alternative to traditional cryogenic preservation.


Assuntos
Proteínas Sanguíneas/metabolismo , Plasma/química , Preservação Biológica/métodos , Conservantes Farmacêuticos/química , Trealose/química , Proteínas Sanguíneas/química , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Humanos , Agregados Proteicos , Conformação Proteica em Folha beta , Estabilidade Proteica , Temperatura de Transição , Vitrificação
6.
Tumour Biol ; 42(6): 1010428320936410, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32586207

RESUMO

Pancreatic ductal adenocarcinoma is the most common and aggressive type of pancreatic cancer, with a 5-year survival rate that is less than 10%. New biomarkers to aid in predicting the prognosis of pancreatic ductal adenocarcinoma patients are needed. Previous proteomic studies have to a great extent focused on finding proteins of value for the diagnosis of pancreatic ductal adenocarcinoma. There is a lack of studies that have profiled the serum or plasma proteome in order to discover candidates for new prognostic biomarkers. In this study, we have used ultra-performance liquid chromatography-ultra-definition mass spectrometry to analyze the serum samples of 21 pancreatic ductal adenocarcinoma patients with short or long survival. Statistical analysis discovered 31 proteins whose expression differed significantly between pancreatic ductal adenocarcinoma patients with short or long survival. Pathway analysis discovered multiple canonical pathways enriched in this data set, with several pathways having roles in inflammation and lipid metabolism. The serum proteins identified here, which include complement components and several enzymes, could be of value as candidates for new noninvasive prognostic markers.


Assuntos
Adenocarcinoma/mortalidade , Biomarcadores Tumorais/metabolismo , Proteínas Sanguíneas/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Neoplasias Pancreáticas/mortalidade , Proteoma/metabolismo , Proteômica/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/análise , Proteínas Sanguíneas/análise , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Projetos Piloto , Prognóstico , Mapas de Interação de Proteínas , Proteoma/análise , Taxa de Sobrevida
7.
PLoS Genet ; 16(6): e1008863, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559195

RESUMO

Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in Drosophila, we identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). We showed that NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. We showed that NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Acetiltransferase N-Terminal B/metabolismo , Neoplasias/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Alelos , Animais , Animais Geneticamente Modificados , Apoptose/genética , Proteínas Sanguíneas/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Acetiltransferase N-Terminal B/genética , Neoplasias/patologia , Proteína do Retinoblastoma/genética , Mutações Sintéticas Letais , Fatores de Transcrição/genética
8.
Arch Biochem Biophys ; 689: 108476, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585310

RESUMO

BACKGROUND: Proteomics is expected to provide novel insights in the underlying pathophysiology of type 2 diabetes mellitus. In the present study, we aimed to identify and biochemically characterize proteins associated with diabetes mellitus in a Qatari population. METHODS: In a diabetes case-control study (175 cases, 164 controls; Arab, South Asian and Philippine ethnicities), we conducted a discovery study to screen 1141 blood protein levels for associations with diabetes mellitus. Additional analyses were done in controls in relation to Hb1Ac, and biochemical characterization of the main findings was performed with metabolomics (501 metabolites). We performed two-sample Mendelian Randomization to provide evidence of potential causality using data from European descent of the DIAGRAM consortium (74,124 cases of diabetes mellitus and 824,006 controls) for the identified proteins for T2D and Hb1Ac. RESULTS: After accounting for multiple testing, 30 protein levels were different (p-values<8.6e-5) between cases and controls. Of these, a higher Hb1Ac in controls was associated with a lower IGFBP-2 level (p-value = 4.1e-6). IGFBP-2 protein level was found lower among cases compared with controls across all ethnicities. In controls, IGFBP-2 was associated with 21 metabolite levels, but specifically connected to the metabolite citrulline in network analyses. We observed no evidence, however, that the association between IGFBP-2 and diabetes mellitus was causal. CONCLUSIONS: We specifically identified IGFBP-2 to be associated with diabetes mellitus, although with no evidence for causality, which was specifically connected to citrulline metabolism.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Adulto , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , Citrulina/sangue , Citrulina/metabolismo , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Masculino , Metaboloma , Pessoa de Meia-Idade , Proteoma/análise , Proteoma/metabolismo
9.
PLoS One ; 15(5): e0233289, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469899

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by two aggregates, namely, amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein (tau-p), which are released into the blood in a very small amount and cannot be easily detected. An increasing number of recent studies have suggested that S-glutathionylated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is highly correlated with Aß in patients with AD and that S-glutathionylated GAPDH plays a role as a proapoptotic factor in AD. We found that S-glutathionylated GAPDH is abundant in the blood of AD patients, which is unusual because S-glutathionylated GAPDH cannot exist in the blood under normal conditions. The aim of this study was to further explore the correlation between the S-glutathionylated GAPDH levels in blood plasma and AD progression. As controls, we recruited 191 people without AD, which included 111 healthy individuals and 37 patients with depression and insomnia, in the psychosomatic clinic. Moreover, 47 patients with AD (aged 40-89 years) were recruited at the neurology clinic. The blood S-glutathionylated GAPDH levels in the AD patients were significantly (p < 0.001) higher (752.7 ± 301.7 ng/dL) than those in the controls (59.92 ± 122.4 ng/dL), irrespective of gender and age. For AD diagnosis, the criterion blood S-glutathionylated GAPDH level > 251.62 ng/dL exhibited 95.74% sensitivity and 92.67% specificity. In fact, the individuals aged 70-89 years, namely, 37 patients from the psychosomatic clinic and 42 healthy individuals, showed significant blood S-glutathionylated GAPDH levels (230.5 ± 79.3 and 8.05 ± 20.51 ng/dL, respectively). This finding might indicate neurodegenerative AD progression in psychosomatic patients and suggests that the degree of neuronal apoptosis during AD progression might be sensitively evaluated based on the level of S-glutathionylated GAPDH in blood.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/patologia , Proteínas Sanguíneas/metabolismo , Glutationa/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/sangue , Processamento de Proteína Pós-Traducional , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Progressão da Doença , Feminino , Seguimentos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
10.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 32(3): 330-335, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32385998

RESUMO

OBJECTIVE: To investigate the effect of heparin-binding protein (HBP) on the damage of vascular permeability in early burn. METHODS: (1) Clinical research: 12 patients with severe burns admitted to Suzhou Hospital of Nanjing Medical University from January 1st to August 30th in 2019 were enrolled. Eight patients with severe trauma admitted to the same hospital during the same period were also enrolled as controls to explain the specificity of burn injury. Whole blood samples were obtained within 0.5 hour after admission. The white blood cell count (WBC), absolute value and ratio of neutrophils, and serum HBP levels were measured. Serum samples of 12 patients with severe burn were collected within 9 days after admission, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of metabolism products of glycocalyx including syndecan-1 and hyaluronic acid (HA). The correlation between HBP and neutrophils ratio, syndecan-1 and HA were analyzed by linear correlation. (2) Basic research: a 30% total body surface area (TBSA) III degree angle burn model of Sprague-Dawley (SD) rat aged 6-8 weeks was prepared. In low molecular weight heparin (LMWH) intervention group (n = 5), 200 U/kg LMWH was injected subcutaneously immediately and every 2 hours after injury for 4 times in total; the burn group (n = 5) was given the same amount of normal saline. No intervention was given to the normal control group (n = 5). The peripheral venous blood was collected at 0, 2, 4, and 8 hours after injury, and the serum levels of HBP, syndecan-1 and HA were measured; the injury of glycocalyx on pulmonary vascular endothelial cells was observed under transmission electron microscope. RESULTS: (1) Clinical research results: the WBC, neutrophils absolute value and ratio, and HBP levels were increased in 12 patients with severe burn and 8 patients with severe trauma. There was no significant difference in the WBC, absolute value and ratio of neutrophils between severe burn and severe trauma patients [WBC (×109/L): 14.5±6.1 vs. 10.8±3.6, the absolute value of neutrophils (×109/L): 12.0±5.9 vs. 9.0±4.0, the ratio of neutrophils: 0.81±0.10 vs. 0.79±0.14, all P > 0.05], but the HBP levels in the burn patients were significantly higher than those in the trauma patients (µg/L: 192.92±61.73 vs. 51.17±23.05, P < 0.01). Twelve patients with severe burns had a sharp increase in serum syndecan-1 and HA levels after burns, which continued to maintain high levels and peaked at the 9th day [syndecan-1 (µg/L): 16.02±0.39, HA (µg/L): 106.83±4.90]. The analysis showed that HBP was positively correlated with neutrophils ratio, syndecan-1 and HA in severe burn patients at the 1st day after admission (r values were 0.805, 0.732 and 0.900, respectively, all P < 0.01). It indicated that the sharp increase of neutrophils after the burn released a lot of HBP, and the glycocalyx of the vascular endothelium was severely damaged. (2) Basic research results: the levels of serum HBP, syndecan-1 and HA in the burn group were increased sharply as compared with the normal control group, and continued to increase with time, reaching a peak at 8 hours after burn. In the LMWH intervention group, the serum levels of HBP, syndecan-1 and HA were significantly lower than those in the burn group, and the difference was still statistically significant after 8 hours [HBP (µg/L): 6.47±0.25 vs. 12.48±0.08, syndecan-1 (µg/L): 19.06±1.48 vs. 25.92±3.34, HA (µg/L): 35.76±2.10 vs. 54.91±2.64, all P < 0.01]. The results of transmission electron microscopy showed that in the normal control group, the glycocalyx pulmonary vascular endothelial cells was continuous, evenly distributed and dense. The glycocalyx on pulmonary vascular endothelial cells of rats were significantly damaged and shed 2 hours after burn in the burn group, and no glycocalyx was observed at 8 hours. In the LMWH intervention group, the glycocalyx on pulmonary vascular endothelial cells was damaged and the phenomenon of shedding was significantly relieved, and the glycocalyx could be observed 8 hours after the injury. CONCLUSIONS: The massive exudation of body fluids and the significant increase of vascular permeability in patients in early burns may be related to the destruction of the glycocalyx on endothelial cells by HBP released from increased neutrophils.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Permeabilidade Capilar , Heparina de Baixo Peso Molecular , Animais , Células Endoteliais , Humanos , Ratos , Ratos Sprague-Dawley
11.
J Vis Exp ; (158)2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32391810

RESUMO

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as ß-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Proteínas Sanguíneas/metabolismo , Espectrometria de Massas , Proteômica , Automação , Cromatografia Líquida , Humanos , Reprodutibilidade dos Testes , Fluxo de Trabalho
12.
Nat Chem Biol ; 16(6): 667-675, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393901

RESUMO

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.


Assuntos
Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Medo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Receptores de Canabinoides/metabolismo , Transdução de Sinais
13.
J Chromatogr A ; 1621: 461027, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32276854

RESUMO

In the present study, 88 structurally- diverse drugs were investigated by biopartitioning micellar chromatography (BMC) using Brij-35 as surfactant under different chromatographic conditions. It was found that temperature and presence of NaCl have only a minor effect in BMC retention. Correlation of BMC retention factors with octanol-water partitioning required the inclusion of fractions of ionized species as additional parameters, showing that there is a weaker effect of ionization in BMC environment. Compared to Immobilized Artificial Membrane (IAM) Chromatography, BMC retention factors cover a relatively narrow span, two-fold smaller than retention factors on IAM stationary phases as a result of the presence of micelles facilitating elution of lipophilic compounds and the absence of secondary attractive electrostatic interactions in the BMC environment. Similarities/dissimilarities between BMC, octanol-water partitioning and IAM Chromatography were investigated by Linear Free Energy Relationships (LSER). BMC retention factors were used to construct relationships with cell permeability,% Human Oral Absorption (%HOA) and Plasma Protein Binding (%PPB). Linear BMC models were obtained with Caco-2 cell lines and Parallel Artificial Membrane Permeability Assay (PAMPA). For %HOA, a hyperbolic model was established upon incorporation of topological polar surface area (tPSA) as additional parameter. A sigmoidal model was constructed for %PPB and a linear one for the corresponding thermodynamic binding constant logK. In both cases inclusion of the fraction of anionic species with a positive sign was required reflecting the preference of human albumin for acidic drugs.


Assuntos
Cromatografia Líquida , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Absorção Fisiológica , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Humanos , Modelos Lineares , Membranas Artificiais , Micelas , Octanóis/química , Polietilenoglicóis/química , Tensoativos/química , Termodinâmica , Água/química
14.
Int J Nanomedicine ; 15: 1481-1498, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189964

RESUMO

Purpose: It is well known that when exposed to human blood plasma, nanoparticles are predominantly coated by a layer of proteins, forming a corona that will mediate the subsequent cell interactions. Magnetosomes are protein-rich membrane nanoparticles which are synthesized by magnetic bacteria; these have gained a lot of attention owing to their unique magnetic and biochemical characteristics. Nevertheless, whether bacterial magnetosomes have a corona after interacting with the plasma, and how such a corona affects nanoparticle-cell interactions is yet to be elucidated. The aim of this study was to characterize corona formation around a bacterial magnetosome and to assess the functional consequences. Methods: Magnetosomes were isolated from the magnetotactic bacteria, M. gryphiswaldense (MSR-1). Size, morphology, and zeta potential were measured by transmission electron microscopy and dynamic light scattering. A quantitative characterization of plasma corona proteins was performed using LC-MS/MS. Protein absorption was further examined by circular dichroism and the effect of the corona on cellular uptake was investigated by microscopy and spectroscopy. Results: Various serum proteins were found to be selectively adsorbed on the surface of the bacterial magnetosomes following plasma exposure, forming a corona. Compared to the pristine magnetosomes, the acquired corona promoted efficient cellular uptake by human vascular endothelial cells. Using a protein-interaction prediction method, we identified cell surface receptors that could potentially associate with abundant corona components. Of these, one abundant corona protein, ApoE, may be responsible for internalization of the magnetosome-corona complex through LDL receptor-mediated internalization. Conclusion: Our findings provide clues as to the physiological response to magnetosomes and also reveal the corona composition of this membrane-coated nanomaterial after exposure to blood plasma.


Assuntos
Endocitose , Magnetossomos/metabolismo , Magnetospirillum/metabolismo , Coroa de Proteína/metabolismo , Adsorção , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Magnetossomos/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura
15.
Nucleic Acids Res ; 48(8): 4382-4395, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32182359

RESUMO

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1-/-) and FcRn knockout (FcRn-/-).


Assuntos
Oligonucleotídeos Antissenso/farmacocinética , Ácido Palmítico , Albuminas/genética , Albuminas/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Caveolina 1/genética , Feminino , Coração , Células Hep G2 , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Sistema Linfático/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/química , Músculo Quadríceps/metabolismo , Receptores Fc/genética , Distribuição Tecidual
16.
J Photochem Photobiol B ; 204: 111782, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32062389

RESUMO

BACKGROUND: Ultraviolet (UV) fluorescent lamp (FL) was applied in mainstream riboflavin photochemical method (RPM) to inactivate pathogens in blood components. Low UV irradiance emitted by UV-FL resulted in more time to achieve effective inactivation. MATERIALS AND METHODS: A novel light emitting diode (LED) UV illumination with adjustable irradiance was developed by us. Two strains of drug-resistant bacteria (DRB), pan-drug resistant Acinetobacter baumannii (PDRAB) and methicillin-resistant Staphylococcus aureus (MRSA) were cultured and used for evaluating the inactivation effectiveness of RPM using UV-LED or UV-FL against DRB in plasma or platelets. Three plasma factors and four platelet parameters were measured after treatments. RESULTS: There was a linear relationship between UV-LED irradiance and electric current, the minimum UV irradiance was 24 mW/cm2, and the maximum was 258 mW/cm2. At the same UV dose of 15 J/cm2, inactivation effectiveness of UV-LED with 258 mW/cm2 against PDRAB in plasma or platelets were comparable to that of UV-FL with 16 mW/cm2, both above 98%. UV-FL treatment required 10-15 min, but UV-LED only required 1-2 min. However, MRSA showed a resistance to UV-LED (inactivation effectiveness was around 40%) compared with UV-FL (inactivation effectiveness was above 98%). The retention of fibrinogen, factor V, factor VII in plasma and platelet counts in platelets with UV-LED treatment were significantly higher than UV-FL at the same UV dose. CONCLUSION: The treatment of RPM using UV-LED with high UV irradiance was able to dramatically shorten inactivation time against PDRAB in plasma or platelets and improve retention of blood components compared with UV-FL.


Assuntos
Proteínas Sanguíneas/metabolismo , Riboflavina/química , Raios Ultravioleta , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/efeitos da radiação , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , Farmacorresistência Bacteriana/efeitos dos fármacos , Fator V/metabolismo , Fibrinogênio/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos da radiação , Contagem de Plaquetas , Riboflavina/farmacologia
17.
Toxicol Lett ; 323: 41-47, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31982501

RESUMO

Gynura japonica (also named Tusanqi in Chinese) is used as a folk herbal medicine for treating blood stasis or traumatic injury. However, hundreds of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of preparations made from G. japonica because it contains large amounts of hepatotoxic pyrrolizidine alkaloids (PAs). To date, blood pyrrole-protein adducts (PPAs) are suggested as biomarkers for the diagnosis of PA-induced HSOS in clinics. However, the concentration of PPAs in the blood is greatly affected by several factors including the amount of PA exposure, herb intake period, and blood sampling time after the last exposure. In present study, the kinetic characters of PPAs in serum and liver as well as other potential target organs were studied systematically and comprehensively following multiple exposures of PAs in G. japonica extract (GJE). As results, PPAs content reached to a plateau both in serum and liver after the mice were treated with GJE for 2 weeks on daily basis. PPAs cleared significantly slower in liver (T1/2ke∼184.6 h, ∼7.7 days) than in serum (T1/2ke∼95.8 h, ∼4.0 days). Although more than 90 % PPAs were removed 2 weeks after the last dosing, PPAs still persisted in the liver until the end of the experiment, i.e. 8 weeks after the last dosing. The results would be of great help for understanding the importance of PPAs for PA-induced toxicity and its detoxification.


Assuntos
Proteínas Sanguíneas/metabolismo , Medicamentos de Ervas Chinesas/farmacocinética , Hepatopatia Veno-Oclusiva/induzido quimicamente , Pirróis/metabolismo , Alcaloides de Pirrolizidina/farmacocinética , Animais , Medicamentos de Ervas Chinesas/toxicidade , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/análise , Extratos Vegetais/toxicidade , Alcaloides de Pirrolizidina/toxicidade
18.
J Vis Exp ; (155)2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31984952

RESUMO

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-кB/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-кB/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.


Assuntos
Proteínas Sanguíneas/metabolismo , Receptor 1 Toll-Like/isolamento & purificação , Receptor 1 Toll-Like/metabolismo , Adsorção , Células Cultivadas , Humanos , Macrófagos/metabolismo , Transdução de Sinais
19.
Gen Comp Endocrinol ; 290: 113396, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987871

RESUMO

Biological activities in ectothermic vertebrates depend to a great extent on ambient temperature. Adapting their biological systems to annual or short-term alterations in temperature may play an important role in thermal resistance or overwintering survival. Using SDS-PAGE and western blot, we examined plasma proteins in bullfrog (Rana catesbeiana) tadpoles that were seasonally acclimatized (winter vs. summer) or thermally acclimated (4 °C vs. 21 °C) and identified two season-responsive proteins. The first, transthyretin (TTR), is a plasma thyroid hormone distributor protein that was abundant in summer, and the second is a protein containing C-type lectin-like domain (CTLD) that was abundant in winter and cold acclimation of 4 weeks. Sequence analysis revealed that the C-terminal carbohydrate recognition domain of this CTLD protein (termed collectin X) was highly similar to those of the collectin family members, which participate in complement activation of the innate immune system; however, it lacked most of collagen-like domain. Among the hepatic genes involved in the thyroid system, ttr and dio3 were up-regulated, whereas thra and thrb were down-regulated, in summer acclimatization or warm acclimation. In contrast, the collectin X gene (colectx), as well as colect10 and colect11 in the collectin family involved in the innate immune system, were down-regulated during warm acclimation, although fcn2 in the ficolin family was up-regulated during summer acclimatization and warm acclimation. These findings indicate that seasonal acclimatization and thermal acclimation differentially affect some components of the thyroid and innate immune systems at protein and transcript levels.


Assuntos
Aclimatação/fisiologia , Proteínas Sanguíneas/metabolismo , Larva , Rana catesbeiana , Animais , Estações do Ano , Temperatura
20.
Clin Biochem ; 76: 5-10, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31765635

RESUMO

BACKGROUND: Identification and treatment in the early stage can significantly improve the prognosis of gastric cancer (GC). However, to date, there is still no ideal biomarker that can be used for the screening of early stage GC (EGC). The proteomics supported by mass spectrometry offers more possibilities for discovering tumor biomarkers. The aim of this study was to explore candidate protein biomarkers for EGC screening with mass spectrometry and bioinformatics technology. METHODS: Plasma samples were collected from 15 EGC patients and 15 healthy controls. After a selective immune-depletion to remove high abundance proteins, plasma samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with the tandem mass tags (TMT) labeling. RESULTS: A total of 2040 proteins were identified, and 11 proteins were found to be differentially expressed. The results of the logistic regression model and orthogonal signal correction-partial least squares discriminant analysis (OPLS-DA) model showed that the changed proteins identified by plasma proteomics could help distinguish EGC patients from healthy controls. CONCLUSION: The proteins identified by plasma proteomics using LC-MS/MS combined with TMT labeling could help distinguish EGC from healthy controls.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas Sanguíneas/metabolismo , Proteômica , Neoplasias Gástricas/metabolismo , Idoso , Estudos de Casos e Controles , Cromatografia Líquida , Detecção Precoce de Câncer , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/diagnóstico , Espectrometria de Massas em Tandem
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