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1.
Int J Mol Sci ; 22(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920954

RESUMO

Mitocryptides are a novel family of endogenous neutrophil-activating peptides originating from various mitochondrial proteins. Mitocryptide-2 (MCT-2) is one of such neutrophil-activating peptides, and is produced as an N-formylated pentadecapeptide from mitochondrial cytochrome b. Although MCT-2 is a specific endogenous ligand for formyl peptide receptor 2 (FPR2), the chemical structure within MCT-2 that is responsible for FPR2 activation is still obscure. Here, we demonstrate that the N-terminal heptapeptide structure of MCT-2 with an N-formyl group is the minimum structure that specifically activates FPR2. Moreover, the receptor molecule for MCT-2 is suggested to be shifted from FPR2 to its homolog formyl peptide receptor 1 (FPR1) by the physiological cleavages of its C-terminus. Indeed, N-terminal derivatives of MCT-2 with seven amino acid residues or longer caused an increase of intracellular free Ca2+ concentration in HEK-293 cells expressing FPR2, but not in those expressing FPR1. Those MCT-2 derivatives also induced ß-hexosaminidase secretion in neutrophilic/granulocytic differentiated HL-60 cells via FPR2 activation. In contrast, MCT-2(1-4), an N-terminal tetrapeptide of MCT-2, specifically activated FPR1 to promote those functions. Moreover, MCT-2 was degraded in serum to produce MCT-2(1-4) over time. These findings suggest that MCT-2 is a novel critical factor that not only initiates innate immunity via the specific activation of FPR2, but also promotes delayed responses by the activation of FPR1, which may include resolution and tissue regeneration. The present results also strongly support the necessity of considering the exact chemical structures of activating factors for the investigation of innate immune responses.


Assuntos
Peptídeos/química , Peptídeos/metabolismo , Receptores de Formil Peptídeo/química , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/química , Receptores de Lipoxinas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Cálcio/metabolismo , Diferenciação Celular , Dicroísmo Circular , Células HEK293 , Células HL-60 , Humanos , Imunidade Inata , Modelos Biológicos , Simulação de Acoplamento Molecular , Neutrófilos/metabolismo , Peptídeos/sangue , Fatores de Tempo , beta-N-Acetil-Hexosaminidases/metabolismo
2.
Res Vet Sci ; 136: 609-615, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33901785

RESUMO

In this study, we conducted study to explore the association between serum cross-linked N-telopeptide of type I collagen (NTx), a marker of bone resorption, and age, body weight, and blood biochemical parameters as well as the neutered and intact status in male and female dogs. We targeted 145 healthy dogs (aged 0.33-18.33 years); 70 were males (38 intact, 32 castrated), and 75 were females (31 intact, 44 ovariohysterectomized). We found that the NTx levels were significantly higher in dogs aged ≤2 years than in older dogs. NTx concentration tended to decrease with age in dogs aged ≤2 years, but not significantly, and remained constant in dogs aged >2 years. Accordingly, we investigated sex/sterilization status in two age cohorts (juvenile-to-young-adult, ≤2 years of age; adult-to-geriatric, >2 years of age). In the adult-to-geriatric cohort, NTx concentration was highest in intact males, followed by neutered males, neutered females, and intact females. The intact vs. neutered difference was significant in males, but not in females. Our results suggested that estradiol deficiency may not affect bone metabolism in female dogs, but androgen deficiency may affect bone metabolism in male dogs. Furthermore, age did not affect bone metabolism after 2 years. NTx concentrations were significantly higher in the juvenile-to-young-adult cohort than in the adult-to-geriatric cohort and tended to decrease with age, similar to young humans. This study unveils novel sex differences in canine serum NTx concentrations and suggests the effect of neutering on bone metabolism, showing that serum NTx concentrations change with age.


Assuntos
Envelhecimento/sangue , Colágeno Tipo I/sangue , Cães/sangue , Peptídeos/sangue , Animais , Biomarcadores/sangue , Peso Corporal , Reabsorção Óssea/sangue , Reabsorção Óssea/veterinária , Castração , Feminino , Masculino , Caracteres Sexuais
3.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33670003

RESUMO

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359-394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1-0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.


Assuntos
Anticorpos Monoclonais/imunologia , Peptídeos/imunologia , alfa 1-Antitripsina/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos/imunologia , Armadilhas Extracelulares , Humanos , Lipopolissacarídeos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Peptídeos/sangue , Peptídeos/química , Desnaturação Proteica
4.
Anal Chem ; 93(11): 4782-4787, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33656857

RESUMO

The outbreak of coronavirus disease 2019 (COVID-19) caused by SARS CoV-2 is ongoing and a serious threat to global public health. It is essential to detect the disease quickly and immediately to isolate the infected individuals. Nevertheless, the current widely used PCR and immunoassay-based methods suffer from false negative results and delays in diagnosis. Herein, a high-throughput serum peptidome profiling method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed for efficient detection of COVID-19. We analyzed the serum samples from 146 COVID-19 patients and 152 control cases (including 73 non-COVID-19 patients with similar clinical symptoms, 33 tuberculosis patients, and 46 healthy individuals). After MS data processing and feature selection, eight machine learning methods were used to build classification models. A logistic regression machine learning model with 25 feature peaks achieved the highest accuracy (99%), with sensitivity of 98% and specificity of 100%, for the detection of COVID-19. This result demonstrated a great potential of the method for screening, routine surveillance, and diagnosis of COVID-19 in large populations, which is an important part of the pandemic control.


Assuntos
COVID-19/diagnóstico , Peptídeos/sangue , SARS-CoV-2/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Área Sob a Curva , COVID-19/metabolismo , COVID-19/virologia , Estudos de Casos e Controles , Análise Discriminante , Ensaios de Triagem em Larga Escala , Humanos , Análise dos Mínimos Quadrados , Aprendizado de Máquina , Análise de Componente Principal , Curva ROC , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Tuberculose/metabolismo , Tuberculose/patologia
5.
Medicine (Baltimore) ; 100(3): e24259, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33546047

RESUMO

BACKGROUND: Osteoporosis (OP) results in an increased risk of fragility fractures, representing a major public health problem. In preventing OP, complementary and alternative medicine, such as acupuncture, was recommended because of the low efficiency and side effects of medications. Recently, there is insufficient evidence on electroacupuncture as an effective therapy for OP management. Hence, we evaluated the effectiveness of electroacupuncture for OP treatment. METHODS: We conducted a systematic review and meta-analysis of clinical studies on patients with OP. Five databases (PubMed, Embase, Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure, and Wanfang) were searched from the earliest publication date to March 12, 2020. Randomized controlled trials (RCTs) were included if electroacupuncture was applied as the sole treatment or as an adjunct to other treatments compared with medications in patients with OP. The measurement outcomes included serum aminoterminal propeptide of type I procollagen (PINP) and C-telopeptide of type I collagen (CTX) levels, bone mineral density (BMD) of lumbar, and visual analog scale scores for OP-related pain. Acupoints were extracted when available. RESULTS: In total, 11 RCTs involving 731 participants were included for further meta-analysis. The meta-analysis showed that the use of electroacupuncture as a sole treatment or as an adjunct to other treatments could relieve OP-related pain compared with medications [mean difference (MD) =  -0.58, 95% confidence interval (CI); MD =  -0.97 to -0.19, P = .003, I2 = 88%; MD =  -1.47, 95% CI = -2.14 to -0.79, P < .001, I2 = 96%). Meanwhile, the results showed a favorable effect of electroacupuncture on decreasing serum beta-CTX levels. However, there were no significant differences in serum PINP levels and BMD of lumbar. Shenshu (BL23) was the most frequent acupoint stimulation among these studies. CONCLUSIONS: The application of electroacupuncture as an independent therapy or as an adjunct to other treatments might attenuate OP-related pain and serum beta-CTX levels. However, to overcome the methodological shortcomings of the existing evidence, due to a small size of samples and high risk of bias in these included RCTs, further rigorous studies are required.


Assuntos
Eletroacupuntura , Osteoporose/terapia , Dor nas Costas/terapia , Densidade Óssea , Colágeno Tipo I/sangue , Humanos , Osteoporose/sangue , Fragmentos de Peptídeos/sangue , Peptídeos/sangue , Pró-Colágeno/sangue
6.
J Bone Miner Metab ; 39(3): 484-493, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33389132

RESUMO

INTRODUCTION: There have been no reports of the effects of baseline lumbar spine bone mineral density (LS-BMD) and bone turnover marker levels on the therapeutic effect of a 28.2-µg teriparatide formulation for twice-weekly use (2/W-TPTD). MATERIALS AND METHODS: An analysis was performed using data from a double-blind, randomized, non-inferiority trial (TWICE study) conducted with patients who received 2/W-TPTD or a 56.5-µg teriparatide formulation for once-weekly use (1/W-TPTD) for 48 weeks. The patients were divided into tertile groups based on baseline LS-BMD, urinary type I collagen cross-linked N-telopeptide (u-NTX), and serum type I procollagen-N-propeptide (P1NP) levels, respectively. Time profiles of these measurements were analyzed. Furthermore, whether a change in P1NP is a predictor for percentage change in BMD was assessed. RESULTS: Across all tertile groups divided based on baseline LS-BMD and levels of bone turnover markers, the LS-BMD increased significantly. The u-NTX level decreased throughout the study period in the high- and middle-u-NTX-level groups. The P1NP level increased after 4 weeks, but subsequently decreased after 12 weeks and thereafter in the high-P1NP-level group; it increased after 4 weeks and subsequently fluctuated near the baseline level in the middle-P1NP-level group. A cut-off value of 12.0 µg/L for change in P1NP after 4 weeks of 2/W-TPTD as a predictor for percentage change in LS-BMD of 3% or more after 48 weeks gave a positive predictive value of 89.6%. CONCLUSION: 2/W-TPTD, just like 1/W-TPTD, improved LS-BMD significantly, regardless of baseline LS-BMD and bone turnover marker levels.


Assuntos
Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Vértebras Lombares/fisiologia , Teriparatida/farmacologia , Idoso , Conservadores da Densidade Óssea/farmacologia , Conservadores da Densidade Óssea/uso terapêutico , Colágeno Tipo I/sangue , Método Duplo-Cego , Esquema de Medicação , Análise Fatorial , Feminino , Humanos , Vértebras Lombares/efeitos dos fármacos , Masculino , Peptídeos/sangue , Curva ROC , Fatores de Tempo
7.
Chem Commun (Camb) ; 57(7): 895-898, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33367306

RESUMO

Solid-phase synthesis of peptides (SPPS) with release through formation of C-terminal γ-, δ-, or ε-lactams is presented. The natural products ciliatamide A and C were synthesized in up to 90% yield. Peptides carrying C-terminal lactams were shown to possess increased bio-stability and comparable biological activity as compared to the parent non-lactamized peptide amides.


Assuntos
Lactamas/química , Peptídeos/química , Técnicas de Síntese em Fase Sólida , Estabilidade de Medicamentos , Humanos , Lipopeptídeos/síntese química , Lipopeptídeos/química , Peptídeos/sangue , Peptídeos/síntese química
8.
Anal Chem ; 93(3): 1578-1585, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33372771

RESUMO

Fast, robust, and high-throughput mass spectrometry-based serum proteomic pipelines have great potential to yield information for biomarker discovery and daily clinical practice. Here, we developed a simple and rapid sample preparation (RSP) workflow by reducing the classical pretreatment time from overnight to less than 1.5 h in an ordinary system. In HeLa cell lysates and serum samples, the number of proteins and tryptic peptides generated using the RSP was comparable to that generated using conventional methods. For fast scanning of the serum proteome, the RSP-supported pipeline could complete a test in less than 2 h with 30 min of LC-MS/MS analysis. Nearly 390 proteins spanning 8 magnitudes of abundance range were identified with high reproducibility, containing over 90 cancer-associated proteins and over 50 FDA-approved biomarkers. For fast assay development, eight candidate biomarker peptides for cardiovascular disease (CVD) were quantified by MRM with high accuracy (CV% <10). After a simple highly abundant protein removal, a deep serum proteome of over 1400 proteins was reached. By analyzing the depleted serum in DIA acquisition mode, over 700 proteins were quantified. The differentially expressed proteins could help us unambiguously distinguish the serum samples from healthy people and patients with pancreatic cancer (PC). Potential biomarkers for PC were also found. The new RSP method, which is rapid and simple, meets the demands of both deep mining and fast analysis of serum proteins. We believe that it will be widely used in serum protein studies and accelerate the transformation from biomarker discovery to clinical application.


Assuntos
Proteínas Sanguíneas/análise , Doenças Cardiovasculares/sangue , Peptídeos/sangue , Proteômica , Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico , Cromatografia Líquida , Células HeLa , Humanos , Espectrometria de Massas em Tandem
9.
Clin Interv Aging ; 15: 501-518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32308378

RESUMO

Serum biomarkers of osteoarticular diseases have been in the limelight of current clinical research trends. Laboratory validation of defined and candidate biomarkers for both osteoarthritis and osteoporosis is of key importance for future decisional algorithms in the diagnosis, monitoring, and prognosis of these diseases. The current guidelines recommend the use of collagen degradation remnants, eg, CTX-I and CTX-II, in the complementary diagnosis of both osteoporosis and osteoarthritis. Besides the collagen degradation markers, enzymes that regulate bone and articular metabolism are useful in the clinical evaluation of osteoarticular pathologies. Along these, several other recommended and new nominee molecules have been recently studied. Wnts and Wnt-related molecules have a cardinal role in the bone-joint homeostasis, making them a promising target not only for pharmaceutical modulation, but also to be considered as soluble biomarkers. Sclerostin and dickkopf, two inhibitor molecules of the Wnt/ß-catenin signaling, might have a dual role in the assessment of the clinical manifestations of the osteoarticular unit. In osteoarthritis, besides fragments of collagen type II many pathway-related molecules have been studied and proposed for biomarker validation. The most serious limitation is that a significant proportion of studies lack statistical power due to the reduced number of cases enrolled. Serum biomarkers of bone and joint turnover markers represent an encouraging possibility for the diagnosis and prognosis of osteoarticular diseases, although further studies and laboratory validations should be carried out as to solely rely on them.


Assuntos
Osteoartrite/sangue , Osteoporose/sangue , Biomarcadores , Osso e Ossos/metabolismo , Colágeno Tipo I/sangue , Colágeno Tipo II/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/sangue , Via de Sinalização Wnt/fisiologia
10.
Res Vet Sci ; 130: 133-138, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32172002

RESUMO

Ovariohysterectomized (OHE) female dogs do not develop the osteopenia and osteoporosis associated with decreasing estrogen in post-menopausal women, possibly due to post-OHE bone mineral density retention through a mechanism that remains unclear. In this study, we aimed to elucidate this mechanism by investigating estradiol (E2) and bone markers. Samples were collected from 56 OHE and 43 intact bitches (0.33 to 17.58 years old) and analyzed for serum E2, osteoclast-secreted cysteine protease cathepsin K (CTK), and N-telopeptide of type I collagen (NTx) by ELISA. OHE and intact bitches showed no significant difference in serum E2 or NTx, and there was no correlation between serum E2 and NTx and age and time since OHE. Intact bitches showed a very low correlation between E2 and NTx, but OHE bitches showed no correlation, and serum CTK was generally undetectable in both groups. Our findings suggest the influence of gonadal hormones on bone metabolism does not work effectively in dogs; this is consistent with a shorter duration of exposure to E2 in bitches (through the 4-to-8-month anestrus phase) than women.


Assuntos
Catepsina K/sangue , Colágeno Tipo I/sangue , Cães/metabolismo , Estradiol/sangue , Peptídeos/sangue , Animais , Biomarcadores/sangue , Feminino , Histerectomia/veterinária , Ovariectomia/veterinária , Estudos Retrospectivos
11.
Artigo em Inglês | MEDLINE | ID: mdl-32062367

RESUMO

Protein-arginine methyltransferases catalyze the methylation of the guanidine (NG) group of proteinic L-arginine (Arg) to produce monomethyl and dimethylarginine proteins. Their proteolysis releases the free amino acids monomethylarginine (MMA), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), respectively. MMA, SDMA and ADMA are inhibitors of the nitric oxide synthase (NOS) activity. High circulating and low urinary concentrations of ADMA and SDMA are considered risk factors in the cardiovascular and renal systems, mainly due to their inhibitory action on NOS activity. Identity, biological activity and concentration of NG-methylated proteins are largely unknown. The present study addressed these issues by using GC-MS and LC-MS/MS approaches. GC-MS was used to quantify free ADMA released by classical HCl-catalyzed hydrolysis of three synthetic Arg-vasopressin (V) peptides and of unknown endogenous NG-dimethylated proteins. The cyclic (c) disulfide forms of Arg-vasopressin analogs, i.e., Arg-vasopressin (cV-Arg-Gly-NH2), asymmetrically NG-dimethylated vasopressin (cV-ADMA-Gly-NH2) and symmetrically NG-dimethylated vasopressin (cV-SDMA-Gly-NH2) were used as model peptides in quantitative GC-MS analyses of ADMA, SDMA and other expected amino acids from the hydrolyzed Arg-vasopressin analogs. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 were discriminated from cV-Arg-Gly-NH2 by LC-MS and LC-MS/MS, yet they were indistinguishable from each other. The same applies to the respective open (o) reduced and di-S-acetamide forms of oV-ADMA-Gly-NH2, oV-SDMA-Gly-NH2 and oV-Arg-Gly-NH2. Our LC-MS and LC-MS/MS studies suggest that the Arg-vasopressin analogs form [(M-H)]+ and [(M-H)+H]+ in the positive ESI mode and undergo in part conversion of their terminal Gly-NH2 (NH2, 16 Da) group to Gly-OH (OH, 17 Da). The product ion mass spectra of the di-S-acetamide forms are complex and contain several intense mass fragments differing by 1 Da. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 induced platelet aggregation in platelet-rich human plasma with moderately different initial velocity and maximal aggregation rates compared to cV-Arg-Gly-NH2. Previous studies showed that human red blood cells are rich in large (>50 kDa) ADMA-containing proteins of unknown identity. Our LC-MS/MS proteomic study identified several membrane and cytosolic erythrocytic NG-dimethylated proteins, including spectrin-α (280 kDa), spectrin-ß (247 kDa) and protein 4.1 (80 kDa). Being responsible for the stability of the erythrocyte membrane, the newly identified main targets for NG-dimethylation in human erythrocytes should be given a closer look in erythrocytic diseases like hereditary spherocytosis.


Assuntos
Arginina Vasopressina , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Guanidina/química , Espectrometria de Massas em Tandem/métodos , Arginina/análogos & derivados , Arginina/análise , Arginina/sangue , Arginina/química , Arginina Vasopressina/análise , Arginina Vasopressina/sangue , Arginina Vasopressina/química , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Humanos , Modelos Lineares , Masculino , Peptídeos/análise , Peptídeos/sangue , Peptídeos/química , Projetos Piloto , Processamento de Proteína Pós-Traducional
12.
J Cardiovasc Magn Reson ; 22(1): 13, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32036784

RESUMO

BACKGROUND: Using cardiovascular magnetic resonance imaging (CMR), it is possible to detect diffuse fibrosis of the left ventricle (LV) in patients with atrial fibrillation (AF), which may be independently associated with recurrence of AF after ablation. By conducting CMR, clinical, electrophysiology and biomarker assessment we planned to investigate LV myocardial fibrosis in patients undergoing AF ablation. METHODS: LV fibrosis was assessed by T1 mapping in 31 patients undergoing percutaneous ablation for AF. Galectin-3, coronary sinus type I collagen C terminal telopeptide (ICTP), and type III procollagen N terminal peptide were measured with ELISA. Comparison was made between groups above and below the median for LV extracellular volume fraction (ECV), followed by regression analysis. RESULTS: On linear regression analysis LV ECV had significant associations with invasive left atrial pressure (Beta 0.49, P = 0.008) and coronary sinus ICTP (Beta 0.75, P < 0.001), which remained significant on multivariable regression. CONCLUSION: LV fibrosis in patients with AF is associated with left atrial pressure and invasively measured levels of ICTP turnover biomarker.


Assuntos
Fibrilação Atrial/diagnóstico por imagem , Ventrículos do Coração/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética , Função Ventricular Esquerda , Remodelação Ventricular , Adulto , Idoso , Fibrilação Atrial/sangue , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/cirurgia , Função do Átrio Esquerdo , Pressão Atrial , Biomarcadores/sangue , Ablação por Cateter , Colágeno Tipo I/sangue , Técnicas Eletrofisiológicas Cardíacas , Feminino , Fibrose , Galectina 3/sangue , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , Peptídeos/sangue , Valor Preditivo dos Testes , Pró-Colágeno/sangue
13.
Adv Clin Chem ; 94: 1-30, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31952570

RESUMO

Hypertensive disorders of pregnancy (HDP) is the most common and widely known as serious complication of pregnancy. As this syndrome is a major leading cause of maternal, fetal, and neonatal morbidity/mortality worldwide, many studies have sought to identify candidate molecules as potential disease biomarkers (DBMs) for use in clinical examinations. Accumulating evidence over the past 2 decades that the many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. This review provides the potential utility of peptidomic analysis for monitoring for pathophysiological status in HDP, and presents an overview of current status of peptide quantification technology. Especially, the technical limitations of the methods used for DBM discovery in the blood are discussed.


Assuntos
Hipertensão/sangue , Peptídeos/sangue , Complicações Cardiovasculares na Gravidez/sangue , Biomarcadores/sangue , Feminino , Humanos , Hipertensão/complicações , Gravidez , Complicações Cardiovasculares na Gravidez/fisiopatologia
14.
PLoS One ; 15(1): e0228006, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999745

RESUMO

A concerted action on the part of international agencies and national governments has resulted in the near-eradication of poliomyelitis. However, both the oral polio vaccine (OPV) and the inactivated polio vaccine (IPV) have deficiencies which make them suboptimal for use after global eradication. OPV is composed of attenuated Sabin strains and stimulates robust immunity, but may revert to neurovirulent forms in the intestine which can be shed and infect susceptible contacts. The majority of IPV products are manufactured using pathogenic strains inactivated with formalin. Upon eradication, the production of large quantities of pathogenic virus will present an increased biosecurity hazard. A logical ideal endgame vaccine would be an inactivated form of an attenuated strain that could afford protective immunity while safely producing larger numbers of doses per unit of virus stock than current vaccines. We report here the development of an ionizing radiation (IR)-inactivated Sabin-based vaccine using a reconstituted Mn-decapeptide (MDP) antioxidant complex derived from the radioresistant bacterium Deinococcus radiodurans. In bacteria, Mn2+-peptide antioxidants protect proteins from oxidative damage caused by extreme radiation exposure. Here we show for the first time, that MDP can protect immunogenic neutralizing epitopes in picornaviruses. MDP protects epitopes in Polio Virus 1 and 2 Sabin strains (PV1-S and PV2-S, respectively), but viral genomic RNA is not protected during supralethal irradiation. IR-inactivated Sabin viruses stimulated equivalent or improved neutralizing antibody responses in Wistar rats compared to the commercially used IPV products. Our approach reduces the biosecurity risk of the current PV vaccine production method by utilizing the Sabin strains instead of the wild type neurovirulent strains. Additionally, the IR-inactivation approach could provide a simpler, faster and less costly process for producing a more immunogenic IPV. Gamma-irradiation is a well-known method of virus inactivation and this vaccine approach could be adapted to any pathogen of interest.


Assuntos
Raios gama , Vacina Antipólio de Vírus Inativado/imunologia , Vacina Antipólio Oral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Ensaio de Imunoadsorção Enzimática , Genoma Viral , Células HeLa , Humanos , Estresse Oxidativo , Peptídeos/sangue , Poliovirus/genética , Poliovirus/imunologia , Poliovirus/patogenicidade , Poliovirus/ultraestrutura , Ratos Wistar , Proteínas Virais/metabolismo
15.
Mol Cell Proteomics ; 19(3): 540-553, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31896676

RESUMO

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.


Assuntos
Proteínas Sanguíneas/análise , Adulto , Biomarcadores , Teste em Amostras de Sangue Seco , Feminino , Humanos , Masculino , Peptídeos/sangue , Estabilidade Proteica , Proteômica , Reprodutibilidade dos Testes , Adulto Jovem
16.
Nutr Metab Cardiovasc Dis ; 30(1): 49-55, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31757570

RESUMO

AIM: The aim of this study is to evaluate the relationship between OPG and the degree of glycaemic control in a population of elderly subjects. METHODS AND RESULTS: Data presented included 172 elderly subjects, of whom 107 were hospitalized for a hip fracture and 65 were non fractured outpatients. All participants received a multidimensional geriatric evaluation and underwent blood sampling. HbA1c, OPG, CTX and OC were measured and DXA scans were performed. Carotid intima-media thickness (IMT) was measured in all outpatients. Diabetic patients had more comorbidities, higher mean values of lumbar spine and femoral neck BMD and T-score, lower circulating levels of OC and CTX, and higher circulating levels of OPG compared to non-diabetic subjects. OPG was directly correlated with HbA1c. This association was most evident in non-fractured elderly subjects. Moreover, diabetic patients with IMT>1.5 mm had greater mean values of OPG than non-diabetic subjects with high IMT and than elderly subjects with IMT < 1.5 mm, with and without T2DM. CONCLUSIONS: Diabetic patients have reduced circulating levels of OC and CTX, and elevated serum levels of OPG, suggesting a state of low bone turnover. Reduced bone turnover causes an increase of BMD and could lead to a poor bone quality. OPG and HbA1c were directly correlated and OPG mean values were higher in diabetic patients with poor glucose control. Diabetic osteopathy could be considered a late complication of T2DM, directly related with the degree of glucose control and the duration of the disease.


Assuntos
Doenças Ósseas/sangue , Diabetes Mellitus Tipo 2/sangue , Osteoprotegerina/sangue , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Glicemia/análise , Densidade Óssea , Doenças Ósseas/diagnóstico por imagem , Doenças Ósseas/epidemiologia , Colágeno Tipo I/sangue , Diabetes Mellitus Tipo 2/diagnóstico por imagem , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Idoso Fragilizado , Hemoglobina A Glicada/análise , Nível de Saúde , Humanos , Masculino , Osteocalcina/sangue , Peptídeos/sangue , Prevalência , Fatores de Risco , Cidade de Roma/epidemiologia
17.
Mol Pharm ; 17(1): 32-39, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31765157

RESUMO

Obesity and metabolic syndrome are threats to the health of large population worldwide as they are associated with high mortality, mainly linked to cardiovascular diseases. Recently, CPN-116 (CPN), which is an agonist peptide specific to neuromedin-U receptor 2 (NMUR2) that is expressed predominantly in the brain, has been developed as a new therapeutic candidate for the treatment of obesity and metabolic syndrome. However, treatment with CPN poses a challenge due to the limited delivery of CPN to the brain. Recent studies have clarified that the direct anatomical connection of the nasal cavity with brain allows delivery of several drugs to the brain. In this study, we confirm the nasal cavity as a promising CPN delivery route to the brain for the treatment of obesity and metabolic syndrome. According to the pharmacokinetic study, the clearance of CPN from the blood was very rapid with a half-life of 3 min. In vitro study on its stability in the serum and cerebrospinal fluid (CSF) indicates that CPN was more stable in the CSF than in the blood. The concentration of CPN in the brain was higher after nasal administration, despite its lower concentrations in the plasma than that after intravenous administration. The study on its pharmacological potency suggests the effective suppression of increased body weight in mice in a dose-dependent manner due to the direct activation of NMUR2 by CPN. This results from the higher concentration of corticosterone in blood after nasal administration of CPN as compared to nasal application of saline. In conclusion, the above findings indicate that the nasal cavity is a promising CPN delivery route to the brain to treat obesity and metabolic syndrome.


Assuntos
Fármacos Antiobesidade/administração & dosagem , Encéfalo/efeitos dos fármacos , Obesidade/tratamento farmacológico , Peptídeos/administração & dosagem , Receptores de Neurotransmissores/agonistas , Administração Intranasal , Animais , Fármacos Antiobesidade/sangue , Fármacos Antiobesidade/líquido cefalorraquidiano , Fármacos Antiobesidade/farmacocinética , Corticosterona/sangue , Células HEK293 , Humanos , Camundongos , Obesidade/sangue , Obesidade/líquido cefalorraquidiano , Peptídeos/sangue , Peptídeos/líquido cefalorraquidiano , Peptídeos/farmacocinética , Ratos , Ratos Wistar
18.
Maturitas ; 132: 24-29, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31883659

RESUMO

OBJECTIVE: To evaluate trabecular bone score (TBS) in Spanish postmenopausal women from our area. To analyze its relationship with bone mineral density (BMD), bone quantitative ultrasound (QUS) and serum concentrations of 25-hydroxyvitamin D (25(OH)D), intact parathyroid hormone (PTH) and bone turnover markers. STUDY DESIGN: A total of 1450 postmenopausal women aged 44-94 (62 ± 10) participated in this cross-sectional study nested in a population-based cohort. BMD and TBS were assessed by DXA. QUS measurements were performed using a Sahara Clinical Sonometer. Serum 25(OH)D, PTH, P1NP, ß-CTX were determined by electrochemiluminescence. RESULTS: Mean TBS of postmenopausal women in our region was 1.341 ± 0.111. Nearly 50 % of them had normal values. Only 11 % had scores compatible with a clearly degraded microarchitecture. TBS decreased with age, correlated negatively with BMI and was lower in current smokers than in non-smokers. An association was observed between TBS and QUS, although the association was weak and lower than that found between TBS and BMD or QUS and BMD. No association was found between TBS and 25(OH)D, PTH or bone turnover markers. CONCLUSIONS: Half of postmenopausal women in our region have TBS values that indicate a preserved microarchitecture. Only about 10 % have scores compatible with a clearly degraded microarchitecture. A weak association was observed between TBS and QUS, suggesting that the two techniques capture different aspects of bone microarchitecture. The absence of association with 25(OH)D, PTH, and bone turnover markers may be due to the fact that TBS assesses a specific (mostly trabecular) part of the skeleton, whilst the three serum factors are related to the whole skeleton.


Assuntos
Densidade Óssea , Osso Esponjoso/diagnóstico por imagem , Pós-Menopausa , Absorciometria de Fóton , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Colágeno Tipo I/sangue , Estudos Transversais , Feminino , Humanos , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Fragmentos de Peptídeos/sangue , Peptídeos/sangue , Pós-Menopausa/sangue , Pró-Colágeno/sangue , Espanha , Ultrassonografia , Vitamina D/análogos & derivados , Vitamina D/sangue
19.
Mol Med Rep ; 21(1): 51-60, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746355

RESUMO

Matrix­assisted laser desorption/ionization time­of­flight mass spectrometry (MALDI­TOF­MS) was employed to analyze differential serum and urine peptides in patients with small cell lung cancer (SCLC) and healthy individuals, and SCLC diagnostic classification models were constructed. Serum and urine samples from 72 patients with SCLC, age­ and gender­matched with 72 healthy individuals, were divided into training and testing sets in a 3:1 ratio. Serum and urine peptides were extracted using copper ion­chelating nanomagnetic beads, and mass spectra were obtained using MALDI­TOF­MS. Peptide spectra for the training set were analyzed, and the classification model was constructed using ClinProTools (CPT). The testing set was used for blinded model validation. For training­set sera, 122 differential peptide signal peaks with a mass of 0.8­10 kDa were observed, and 19 peptides showed significantly different expression [P<0.0005; area under curve (AUC) ≥0.80]. CPT screened 5 peptide peaks (0.8114, 0.83425, 1.86655, 4.11133 and 5.81192 kDa) to construct the classification model. The testing set was used for the blinded validation, which had 95.0% sensitivity and 90.0% specificity. For the training­set urine, 132 differential peptide signal peaks with m/z ratios of 0.8­10 kDa were observed, and 8 peptides had significantly different expression (P<0.0005; AUC ≥0.80). Then, 5 peaks (1.0724, 2.37692, 2.7554, 4.75475 and 4.7949 kDa) were used for classification model construction. The testing set was used for 36 blinded validation, which had 85.0% sensitivity and 80.0% specificity. Among the differential peptides, 3 had the same significant peaks at 2.3764, 0.8778 and 0.8616 kDa, identified as fibrinogen α, glucose­6­phosphate isomerase and cyclin­dependent kinase­1, respectively. The present study highlighted the differences that exist in serum and urine peptides between patients with SCLC and healthy individuals. Serum and urine peptide diagnostic classification models could be constructed using MALDI­TOF­MS, and showed high sensitivity and specificity.


Assuntos
Neoplasias Pulmonares , Proteínas de Neoplasias , Peptídeos , Carcinoma de Pequenas Células do Pulmão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/urina , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/urina , Peptídeos/sangue , Peptídeos/urina , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/urina
20.
J Bone Miner Metab ; 38(2): 240-247, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31667583

RESUMO

INTRODUCTION: In terms of the balance between benefits and risks of long-term treatment with bisphosphonate, uncertainties remain regarding the optimal treatment duration. We investigated effects of continuous long-term treatment for 10 years with bisphosphonate in postmenopausal osteoporosis patients. MATERIALS AND METHODS: Fifty five patients in the outpatient clinic of our hospital have been continuously treated with alendronate or risedronate for 10 years. All data were retrospectively collected. The age, height, weight, total muscle volume, total fat volume, and BMD at the lumbar spine, total hip and distal 1/3 radius, alkaline phosphatase (ALP), urinary type I collagen cross-linked N-telopeptide (uNTX) and tartrate-resistant acid phosphatase-5b (TRAP5b), calcium (Ca) and phosphate (P) levels were measured pre- and after the start of 10-year continuous treatment. RESULTS: BMD at the lumbar spine increased continuously over the 10-year period, while BMD at the total hip slightly but significantly decreased, and that at the 1/3 radius did not show any significant change over the 10 years. Serum Ca value was significantly decreased after the start of treatment, and became stable within the reference range from the second year. Bone resorption markers such as uNTX and TRAP5b significantly decreased from the second year after the start of treatment and no significant changes were observed thereafter. There were no serious medical adverse events including atypical femoral fractures and osteonecrosis of the jaw. CONCLUSION: We believe that the continuous use of alendronate and risedronate for 10 years could be an option for the treatment of postmenopausal osteoporosis patients.


Assuntos
Grupo com Ancestrais do Continente Asiático , Difosfonatos/uso terapêutico , Osteoporose/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Alendronato/farmacologia , Alendronato/uso terapêutico , Fosfatase Alcalina/sangue , Densidade Óssea/efeitos dos fármacos , Cálcio/sangue , Colágeno Tipo I/sangue , Difosfonatos/farmacologia , Feminino , Humanos , Japão , Osteoporose/sangue , Osteoporose/induzido quimicamente , Osteoporose/fisiopatologia , Osteoporose Pós-Menopausa/tratamento farmacológico , Peptídeos/sangue , Fósforo/sangue , Estudos Retrospectivos , Ácido Risedrônico/uso terapêutico , Fosfatase Ácida Resistente a Tartarato/sangue , Fatores de Tempo , Resultado do Tratamento
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