Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.295
Filtrar
1.
Diagn Microbiol Infect Dis ; 98(4): 115148, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32920452

RESUMO

Infection with the virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) stimulates an immune response which can serve as a marker for current or past exposure to this pathogen, and possibly for resistance to re-infection. This response to COVID-19 can be monitored based on the production of antibodies, and thus, serologic tests have become available for diagnostic purposes. Despite progress in this area, concerns have been raised that too many of the commercially available serologic detection systems are not completely reliable. To address this issue, Western blots should be considered for confirming a positive or borderline-positive result from a screening test, such as an ELISA. An additional benefit of Western blots would be to identify antigens that could form the basis for developing a vaccine. Little is known about the cell-mediated immune response against COVID-19. One way to address this would be to use skin testing to measure the delayed-type hypersensitivity response in patients recovering from COVID-19.


Assuntos
Anticorpos Antivirais/sangue , Western Blotting/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/métodos , Pneumonia Viral/diagnóstico , Testes Cutâneos/métodos , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Humanos , Hipersensibilidade Tardia/diagnóstico , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Testes Sorológicos/métodos
2.
PLoS One ; 15(7): e0235563, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645092

RESUMO

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.


Assuntos
Western Blotting/métodos , Proteínas de Transporte de Cátions/análise , Fracionamento Celular/métodos , Células A549 , Anticorpos/imunologia , Células CACO-2 , Proteínas de Transporte de Cátions/imunologia , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Limite de Detecção , Células MCF-7
3.
Am J Respir Crit Care Med ; 202(8): 1133-1145, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569477

RESUMO

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems.Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection.Methods: Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models.Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to P. aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection.Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration.


Assuntos
Ceramidase Ácida/metabolismo , Ceramidase Ácida/farmacologia , Fibrose Cística/tratamento farmacológico , Pneumonia/diagnóstico , Infecções por Pseudomonas/diagnóstico , Esfingolipídeos/metabolismo , Adolescente , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Western Blotting/métodos , Células Cultivadas , Criança , Fibrose Cística/diagnóstico , Humanos , Inflamação/diagnóstico , Inflamação/tratamento farmacológico , Espectrometria de Massas/métodos , Camundongos , Pneumonia/tratamento farmacológico , Reação em Cadeia da Polimerase/métodos , Infecções por Pseudomonas/tratamento farmacológico , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Adulto Jovem
4.
PLoS One ; 15(6): e0234645, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555693

RESUMO

Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.


Assuntos
Quinase do Linfoma Anaplásico/química , Western Blotting/métodos , Medições Luminescentes/normas , Fosfotirosina/química , Linhagem Celular Tumoral , Receptores ErbB/análise , Humanos , Luminescência , Proteínas Recombinantes/química , Padrões de Referência
5.
Invest Ophthalmol Vis Sci ; 61(4): 32, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32334435

RESUMO

Purpose: Oxidative stress in retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD). Resveratrol exerts a range of protective biologic effects, but its mechanism(s) are not well understood. The aim of this study was to investigate how resveratrol could affect biologic pathways in oxidatively stressed RPE cells. Methods: Cultured human RPE cells were treated with hydroquinone (HQ) in the presence or absence of resveratrol. Cell viability was determined with WST-1 reagent and trypan blue exclusion. Mitochondrial function was measured with the XFe24 Extracellular Flux Analyzer. Expression of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit was evaluated by qPCR. Endoplasmic reticulum stress protein expression was measured by Western blot. Potential reactions between HQ and resveratrol were investigated using high-performance liquid chromatography mass spectrometry with resveratrol and additional oxidants for comparison. Results: RPE cells treated with the combination of resveratrol and HQ had significantly increased cell viability and improved mitochondrial function when compared with HQ-treated cells alone. Resveratrol in combination with HQ significantly upregulated HO-1 mRNA expression above that of HQ-treated cells alone. Resveratrol in combination with HQ upregulated C/EBP homologous protein and spliced X-box binding protein 1. Additionally, new compounds were formed from resveratrol and HQ coincubation. Conclusions: Resveratrol can ameliorate HQ-induced toxicity in RPE cells through improved mitochondrial bioenergetics, upregulated antioxidant genes, stimulated unfolded protein response, and direct oxidant interaction. This study provides insight into pathways through which resveratrol can protect RPE cells from oxidative damage, a factor thought to contribute to AMD pathogenesis.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Heme Oxigenase-1/genética , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Resveratrol/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting/métodos , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroquinonas/farmacologia , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real/métodos , Epitélio Pigmentado da Retina/citologia
6.
Invest Ophthalmol Vis Sci ; 61(4): 40, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32340032

RESUMO

Purpose: To determine whether high glucose (HG) compromises internalization of lysyl oxidase (LOX) through excess binding of LOX with extracellular matrix (ECM) proteins. Methods: To determine whether HG promotes binding of LOX with ECM proteins, fibronectin (FN) and collagen IV (Coll IV), total or ECM-only proteins from rat retinal endothelial cells grown in normal (N; 5 mM) or HG (30 mM) medium were analyzed by coimmunoprecipitation and Western blot (WB). In parallel, coimmunostaining was performed to determine changes in LOX binding to FN or Coll IV. To determine the effect of HG on extracellular LOX levels, medium in which cells were grown for 1, 3, 5, and 7 days were assessed for LOX levels. Results: WB analysis using total protein showed LOX overexpression and elevated levels of LOX bound to Coll IV or FN in HG condition. Similarly, a significant increase in LOX bound to FN or Coll IV was observed in ECM-only protein. These data were supported by Z-stack confocal microscopy images from coimmunostaining. Furthermore, immunostaining performed on ECM layer revealed increased presence of LOX bound to Coll IV or FN. Additionally, when media from cells grown in HG was monitored, a maximal increase in LOX level was observed by day 3, which declined by day 7. Conclusions: Findings indicate that HG promotes binding of LOX to FN and Coll IV extracellularly that results in reduced LOX internalization, attenuation of negative feedback, and upregulation of LOX expression associated with diabetic retinopathy.


Assuntos
Retinopatia Diabética/metabolismo , Matriz Extracelular/genética , Hiperglicemia/metabolismo , Proteína-Lisina 6-Oxidase/genética , Regulação para Cima/genética , Animais , Western Blotting/métodos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Endoteliais , Fibronectinas/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Hiperglicemia/fisiopatologia , Ligação Proteica/genética , Ratos
7.
Mutat Res ; 852: 503165, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265046

RESUMO

Human risk assessment of genotoxic chemicals is an important area of research. However, the specificity of in vitro mammalian genotoxicity assays is sometime low, as they yield to misleading positive results that are not observe in in vivo studies. Apoptosis can be a confounding factor in the interpretation of the results. Recently, a new strategy for genotoxicity screening, based on the combined analysis of phosphorylated histones H2AX (γH2AX) and H3 (pH3), was proposed to discriminate efficiently aneugenic from clastogenic compounds. However, γH2AX biomarker could also be induce by apoptosis. The aim of the present study was to investigate the specificity of this genotoxic biomarker. For this purpose, we analyzed 26 compounds inducing apoptosis by different mechanism of action, with the γH2AX assay in three human cell lines after 24 h treatment. Most of the tested chemicals were negative in the assay, whatever the cell line tested. The few compounds that generated positive data have also been report positive in other genotoxicity assays. The data presented here demonstrate that the γH2AX assay is not vulnerable to the generation of misleading positive results by apoptosis inducers. Currently, no formal guidelines have been approve for the γH2AX assay for regular genotoxicity studies, but we suggest that this biomarker could be used as a new standard genotoxicity assay.


Assuntos
Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Western Blotting/métodos , Histonas/genética , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Mutagênicos/toxicidade , Apoptose/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Células Hep G2 , Histonas/metabolismo , Humanos , Testes para Micronúcleos , Testes de Mutagenicidade , Mutagênicos/classificação , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
8.
Mil Med ; 185(Suppl 1): 383-389, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-32074315

RESUMO

INTRODUCTION: Military and civil aviation have documented physiological episodes among aircrews. Therefore, continued efforts are being made to improve the internal environment. Studies have shown that exposures to many organic compounds present in emissions are known to cause a variety of physiological symptoms. We hypothesize that these compounds may reversibly inhibit acetylcholinesterase, which may disrupt synaptic signaling. As a result, neural proteins leak through the damaged blood-brain barrier into the blood and in some, elicit an autoimmune response. MATERIALS AND METHODS: Neural-specific autoantibodies of immunoglobulin-G (IgG) class were estimated by the Western blotting technique in the sera of 26 aircrew members and compared with the sera of 19 normal healthy nonaircrew members, used as controls. RESULTS: We found significantly elevated levels of circulating IgG-class autoantibodies to neurofilament triplet proteins, tubulin, microtubule-associated tau proteins (Tau), microtubule-associated protein-2, myelin basic protein, and glial fibrillary acidic protein, but not S100 calcium-binding protein B compared to healthy controls. CONCLUSION: Repetitive physiological episodes may initiate cellular injury, leading to neuronal degeneration in selected individuals. Diagnosis and intervention should occur at early postinjury periods. Use of blood-based biomarkers to assess subclinical brain injury would help in both diagnosis and treatment.


Assuntos
Militares/estatística & dados numéricos , Fenômenos Fisiológicos/fisiologia , Medicina Aeroespacial/métodos , Medicina Aeroespacial/estatística & dados numéricos , Aeronaves , Autoanticorpos/análise , Autoanticorpos/sangue , Biomarcadores/análise , Biomarcadores/sangue , Western Blotting/métodos , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/sangue , Humanos , Imunoglobulina G/análise , Imunoglobulina G/sangue , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/sangue , Proteína Básica da Mielina/análise , Proteína Básica da Mielina/sangue , Proteínas de Neurofilamentos/análise , Proteínas de Neurofilamentos/sangue , Proteínas S100/análise , Proteínas S100/sangue , Tubulina (Proteína)/análise , Tubulina (Proteína)/sangue
9.
J Vis Exp ; (156)2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32090995

RESUMO

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%-20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/sangue , Esclerose Amiotrófica Lateral/diagnóstico , Plaquetas/metabolismo , Técnicas de Química Analítica/métodos , Proteínas de Ligação a DNA/sangue , Anticorpos/sangue , Anticorpos/imunologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Western Blotting/métodos , Proteínas de Ligação a DNA/metabolismo , Eletroforese Capilar/métodos , Humanos , Imunoensaio/métodos , Reprodutibilidade dos Testes
10.
J Immunol Methods ; 479: 112749, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31972214

RESUMO

Traditional immunoprobing techniques like Western blot continue to play a crucial role in the discovery and validation of biomarkers. This technique suffers from several limitations that affect reproducibility and feasibility for large-scale studies. Modern immunoprobing techniques have addressed several of these limitations. Here we contrast the use of Western blot and an automated capillary nano-immunoassay (CNIA), Wes™. We provide evidence highlighting the methodological advantages of Wes™ over Western blot in the validation of a novel biomarker, Calcium-binding protein and spermatid-associated 1 (hCABS1). While Wes™ offers a faster, more consistent approach with lower requirements for sample and antibody volumes, variations in expected molecular weights and computational algorithms used to analyze the data must receive careful consideration and assessment. Our data suggests that CNIA approaches are likely to positively impact biomarker discovery and validation.


Assuntos
Western Blotting/métodos , Imunoensaio/métodos , Nanotecnologia/métodos , Adolescente , Adulto , Automação Laboratorial , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Saliva/metabolismo , Adulto Jovem
11.
Sci Rep ; 10(1): 1039, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974468

RESUMO

Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible EVs; however, the separation of EVs from serum proteins and non-EV lipid particles represents a considerable challenge. In this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-EV protein and lipid particles present in serum. The size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of EVs from soluble protein was determined by calculating the ratio of EV particle count to protein concentration. Finally, lipoprotein particles co-isolated with EVs was determined by Western blot analysis of lipoprotein markers APOB and APOE. Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Vesículas Extracelulares/metabolismo , Lipoproteínas/isolamento & purificação , Adulto , Biomarcadores/metabolismo , Proteínas Sanguíneas/metabolismo , Western Blotting/métodos , Centrifugação com Gradiente de Concentração/métodos , Cromatografia em Gel/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo
12.
Cancer Biomark ; 27(3): 377-387, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31958077

RESUMO

BACKGROUND: METCAM/MUC18 expression was increased with the malignant progression of prostate cancer and also a bona fide metastatic gene, capable of initiating and driving the metastasis of a non-metastatic human prostate cancer cell line to multiple organs. OBJECTIVE: We explored if METCAM/MUC18 was detectable in human serum and a novel biomarker to predict malignant propensity of prostate cancer. MATERIALS AND METHODS: Two antibodies were identified by Western blot analysis having the highest sensitivity and specificity to establish calibration curves from the recombinant METCAM/MUC18 proteins. They were used in ELISA and LFIA to determine the METCAM/MUC18 concentrations in serum samples from 8 normal individuals, 4 BPH patients, 1 with PIN, 6 with high-grade prostate cancer, and 2 treated cancer patients. RESULTS: Serum METCAM/MUC18 concentrations were statistically significantly higher in the patients with PIN and prostate cancer than those with BPH, the treated patients and normal individuals. The LFIA results were statistically better than ELISA and Western blot methods. Serum METCAM/MUC18 concentrations were in direct proportional to most of serum PSA concentrations.


Assuntos
Neoplasias da Próstata/sangue , Adulto , Idoso , Biomarcadores Tumorais/sangue , Western Blotting/métodos , Antígeno CD146/sangue , Progressão da Doença , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoensaio/métodos , Calicreínas/sangue , Masculino , Pessoa de Meia-Idade , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia
13.
Protein Expr Purif ; 169: 105572, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31972264

RESUMO

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.


Assuntos
Tripsina/biossíntese , Tripsinogênio/biossíntese , Antígenos/biossíntese , Western Blotting/métodos , Clonagem Molecular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoensaio/métodos , Mutação/genética , Pichia/genética , Pichia/metabolismo , Isoformas de Proteínas/genética , Proteínas Recombinantes/imunologia , Tripsina/genética , Tripsina/imunologia , Tripsinogênio/genética , Tripsinogênio/imunologia
14.
Parasitol Int ; 75: 102022, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31711975

RESUMO

Chickens are considered to act as paratenic hosts for agents, Toxocara canis, T. cati and Ascaris suum; which cause ascarid larva migrans syndrome (ascarid LMS) in humans. In addition, they are the definitive host for Ascaridia galli, considered not to be infective for humans. All ascarid parasites can have a high homology of antigenicity, leading to cross-reactivity in serodiagnostic assays. This study was conducted to establish a procedure for the serological detection of those roundworm infections in chickens. Twenty-five male Julia chickens were divided into five groups (n = 5); T. canis-, T. cati-, Ascaris suum- and Ascaridia galli-infected, and an uninfected control group. In Ascaris suum-soluble worm antigen preparation (As-SWAP) ELISA, all infected groups showed an elevation of anti-ascarid antibodies, indicating the usefulness of As-SWAP as a screening antigen for the detection of ascarid infections. For infecting species identification, T. canis-excretory/secretory (Tc-ES) and Ascaris suum-ES (As-ES) antigen ELISA were conducted by serial dilution sera. Toxocara spp.-infected sera showed stronger binding to Tc-ES than As-ES, while Ascaris suum and Ascaridia galli-infected sera bound to As-ES more strongly than Tc-ES. To discriminate between Ascaris suum and Ascaridia galli infection, sera were pre-incubated with Ascaridia galli-SWAP antigen and applied to Tc-ES and As-ES ELISAs. In this pre-adsorbed ES antigen ELISAs, only the Ascaris suum infected group showed positive binding to As-ES, resulting from the adsorption of cross-reactive antibodies in Ascaridia galli-infected sera. Finally, anti-Toxocara specific antibodies were confirmed by Tc-ES western blot (WB). Toxocara spp.-infected sera showed toxocariasis-specific band pattern in Tc-ES WB, while no specific band appeared on any strip incubated with Ascaris suum, Ascaridia galli-infected and uninfected sera. In conclusion, the serodiagnostic assays evaluated in this study are useful for the detection of ascarid infections in chickens.


Assuntos
Ascaríase/veterinária , Ascaris suum/isolamento & purificação , Galinhas , Doenças das Aves Domésticas/diagnóstico , Testes Sorológicos/veterinária , Toxocara/isolamento & purificação , Toxocaríase/diagnóstico , Animais , Ascaríase/diagnóstico , Western Blotting/métodos , Western Blotting/veterinária , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Masculino , Testes Sorológicos/métodos
15.
Rheumatology (Oxford) ; 59(5): 1026-1030, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31728542

RESUMO

OBJECTIVES: To describe the prevalence and clinical associations of autoantibodies to a novel autoantigen, eukaryotic initiation factor 3 (eIF3), detected in idiopathic inflammatory myositis. METHODS: Sera or plasma from 678 PM patients were analysed for autoantigen specificity by radio-labelled protein immunoprecipitation (IPP). Samples immunoprecipitating the same novel autoantigens were further analysed by indirect immunofluorescence and IPP using pre-depleted cell extracts. The autoantigen was identified through a combination of IPP and MALDI-TOF mass spectrometry, and confirmed using commercial antibodies and IPP-western blots. Additional samples from patients with DM (668), DM-overlap (80), PM-overlap (191), systemic sclerosis (150), systemic lupus erythematosus (200), Sjogren's syndrome (40), rheumatoid arthritis (50) and healthy controls (150) were serotyped by IPP as disease or healthy controls. RESULTS: IPP revealed a novel pattern in three PM patients (0.44%) that was not found in disease-specific or healthy control sera. Indirect immunofluorescence demonstrated a fine cytoplasmic speckled pattern for all positive patients. Mass spectrometry analysis of the protein complex identified the target autoantigen as eIF3, a cytoplasmic complex with a role in the initiation of translation. Findings were confirmed by IPP-Western blotting. The three anti-eIF3-positive patients had no history of malignancy or interstitial lung disease, and had a favourable response to treatment. CONCLUSION: We report a novel autoantibody in 0.44% of PM patients directed against a cytoplasmic complex of proteins identified as eIF3. Although our findings need further confirmation, anti-eIF3 appears to correlate with a good prognosis and a favourable response to treatment.


Assuntos
Autoantígenos/imunologia , Progressão da Doença , Fator de Iniciação 3 em Eucariotos/sangue , Polimiosite/imunologia , Adulto , Autoanticorpos/sangue , Biomarcadores/sangue , Western Blotting/métodos , Estudos de Casos e Controles , Fator de Iniciação 3 em Eucariotos/imunologia , Feminino , Humanos , Imunoprecipitação/métodos , Imunossupressores/administração & dosagem , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Polimiosite/tratamento farmacológico , Polimiosite/fisiopatologia , Valores de Referência , Estudos Retrospectivos , Febre Reumática/imunologia , Febre Reumática/fisiopatologia , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/fisiopatologia
16.
Drug Test Anal ; 12(1): 109-118, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31668004

RESUMO

Erythropoietins (EPOs) are substances listed in S2 of the World Anti-Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR-PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two-step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Eritropoetina/sangue , Eritropoetina/urina , Western Blotting/economia , Western Blotting/métodos , Doping nos Esportes , Eletroforese em Gel de Poliacrilamida/economia , Humanos , Reprodutibilidade dos Testes , Detecção do Abuso de Substâncias/economia , Detecção do Abuso de Substâncias/métodos , Fatores de Tempo
17.
Methods Mol Biol ; 2052: 205-218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31452164

RESUMO

MicroRNAs (miRNAs) represent a subclass of endogenous small noncoding RNAs that have been identified in both mammalian and nonmammalian cells. miRNAs are an essential part of the complex regulatory networks that control numerous biological processes and may play an important role in host defense and/or microbial offense during host-parasite interactions. Here, several methodologies to explore the role for miRNAs in host-parasite interactions are briefly summarized, including the detection, quantification, and intracellular localization of miRNAs, identification and validation of miRNA targets, and functional manipulation of specific miRNAs.


Assuntos
Cryptosporidium/genética , Interações Hospedeiro-Parasita/genética , MicroRNAs/genética , Northern Blotting/métodos , Western Blotting/métodos , Linhagem Celular , Cryptosporidium/patogenicidade , Bases de Dados Genéticas , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Genes Reporter , Humanos , Hibridização In Situ/métodos , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fluxo de Trabalho
18.
BMC Urol ; 19(1): 133, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842847

RESUMO

BACKGROUND: Human sperm cryopreservation is a simple and effective approach for male fertility preservation. METHODS: To identify potential proteomic changes in this process, data-independent acquisition (DIA), a technology with high quantitative accuracy and highly reproducible proteomics, was used to quantitatively characterize the proteomics of human sperm cryopreservation. RESULTS: A total of 174 significantly differential proteins were identified between fresh and cryoperservated sperm: 98 proteins decreased and 76 proteins increased in the cryopreservation group. Bioinformatic analysis revealed that metabolic pathways play an important role in cryopreservation, including: propanoate metabolism, glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism. Four different proteins involved in glycolysis were identified by Western blotting: GPI, LDHB, ADH5, and PGAM1. CONCLUSIONS: Our work will provide valuable information for future investigations and pathological studies involving sperm cryopreservation.


Assuntos
Criopreservação , Preservação da Fertilidade/métodos , Espectrometria de Massas/métodos , Proteoma/análise , Espermatozoides/química , Western Blotting/métodos , Glicólise , Humanos , Masculino , Mapas de Interação de Proteínas , Proteínas/análise , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Espermatozoides/metabolismo
19.
Vet Res ; 50(1): 97, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767033

RESUMO

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.


Assuntos
Western Blotting/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças das Cabras/classificação , Scrapie/classificação , Animais , Western Blotting/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Europa (Continente) , Doenças das Cabras/diagnóstico , Cabras , Scrapie/diagnóstico
20.
Adipocyte ; 8(1): 357-361, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31755337

RESUMO

Beige adipocytes, which consume energy mainly in an uncoupling protein 1 (UCP1)-dependent manner, are risen in white adipose tissue (WAT) depots. Since beige adipocyte development is gaining attention as a potential strategy for conquering obesity, worldwide researchers are making efforts to study its biological aspects. However, assessing UCP1 protein levels in beige adipocytes is challenging because of the high level of lipid contaminants in WAT. This study showed that an acetone precipitation method had advantages over conventional methods for eliminating lipid contaminants, achieving clear Western blot bands for WAT proteins, especially UCP1. Our results suggest that the acetone precipitation cleaning method could be useful for the clear analysis and precise evaluation of WAT proteins.


Assuntos
Tecido Adiposo Bege/metabolismo , Western Blotting/métodos , Proteína Desacopladora 1/metabolismo , Acetona/química , Animais , Precipitação Química , Camundongos , Camundongos Endogâmicos C57BL
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA