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1.
Methods Mol Biol ; 2596: 429-443, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36378455

RESUMO

Proteins can be separated according to their size by gel electrophoresis and further analyzed by Western blotting. The proteins can be transferred to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF), which results in a replica of the protein's separation patterns. The proteins on the membrane can be detected by specific antibodies followed by visualization either on the membrane itself, on film, or by CCD cameras. Western blotting is a sensitive technique to verify data obtained from fluorescence two-dimensional difference gel electrophoresis (2D-DIGE)-based proteomics.


Assuntos
Proteínas , Proteômica , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Immunoblotting , Western Blotting , Proteínas/análise , Eletroforese em Gel de Poliacrilamida , Eletroforese em Gel Bidimensional/métodos
2.
Methods Mol Biol ; 2587: 183-196, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36401031

RESUMO

Dysferlinopathies are a group of disabling muscular dystrophies  that includes limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT) as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for many patients with dysferlinopathies. It was reported that exons 28-29 of DYSF are dispensable for dysferlin functions. Here, we present a method for multiexon skipping of DYSF exons 28-29 using a cocktail of two phosphorodiamidate morpholino oligomers (PMOs) on cells derived from a dystrophinopathy patient. Also, we describe assays to characterize the multiexon skipped dysferlin at several levels by using one-step RT-PCR, immunoblotting, and a membrane wounding assay.


Assuntos
Miopatias Distais , Proteínas Musculares , Humanos , Disferlina/genética , Morfolinos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Musculares/genética , Proteínas de Membrana/genética , Mutação , Éxons/genética , Miopatias Distais/genética , Immunoblotting
3.
Commun Biol ; 5(1): 1147, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36307570

RESUMO

Protein degradation mediated by the ubiquitin-proteasome pathway regulates signaling events in many physiological and pathological conditions. In vitro degradation assays have been instrumental in the understanding of how cell proliferation and other fundamental cellular processes are regulated. These assays are direct, time-specific and highly informative but also laborious, typically relying on low-throughput polyacrylamide gel-electrophoresis followed by autoradiography or immunoblotting. We present protein degradation on chip (pDOC), a MITOMI-based integrated microfluidic technology for discovery and analysis of proteins degradation in cell-free extracts. The platform accommodates hundreds of microchambers on which protein degradation is assayed quickly, simultaneously and using minute amounts of reagents in one or many physiochemical environments. Essentially, pDOC provides a sensitive multiplex alternative to the conventional degradation assay, with relevance to biomedical and translational research associated with regulated proteolysis.


Assuntos
Microfluídica , Microfluídica/métodos , Proteólise , Extratos Celulares , Eletroforese em Gel de Poliacrilamida , Immunoblotting
4.
Curr Protoc ; 2(9): e546, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36094175

RESUMO

Expressing recombinant proteins in heterologous host cells is a prerequisite for purification and other downstream processes. Cell cultures require a protein expression test to optimize incubation time, temperature, and additives (like chemical inducers) to identify the best growth conditions with maximum recombinant protein yield. However, running SDS-PAGE followed by western blotting is cumbersome and results are not quick. Here, I describe a simple protocol to quickly check the presence of recombinant protein in cell cultures using a dot-blot experiment. The cells can be rapidly lysed and directly spotted on the nitrocellulose membrane. Then, the membrane is incubated with a horseradish peroxidase (HRP) conjugated antibody raised against the affinity tag present on the recombinant protein to confirm the protein expression by chemiluminescence. It takes less than an hour to get results. This method rapidly investigates recombinant protein expression in different cell lines and tests other variables. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protein expression analysis for eukaryotic systems Basic Protocol 2: Protein expression analysis for bacterial systems.


Assuntos
Proteínas Recombinantes , Western Blotting , Colódio , Peroxidase do Rábano Silvestre , Immunoblotting
5.
Front Immunol ; 13: 963401, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36003369

RESUMO

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease. Although the pathomechanism of BP onset has yet to be elucidated in detail, BP autoantibodies targeting two hemidesmosomal components, BP180 and BP230, are known to play a pivotal role in BP pathogenesis. Thus, the detection and measurement of BP autoantibodies are necessary for diagnosing BP and monitoring the disease activity. Immune assays such as immunofluorescence microscopy, immunoblotting, and ELISAs using BP180 and BP230 detect BP autoantibodies in most BP cases with high specificity; however, BP autoantibodies are sometimes detected in BP patients before the onset of this disease. BP autoantibodies that are detected in patients without typical tense blisters are defined as "preclinical BP autoantibodies". These preclinical BP autoantibodies are detected even in a low percentage of normal healthy individuals. Although the importance of preclinical BP autoantibodies remains elusive, these autoantibodies might be a potential risk factor for subsequent BP development. Therefore, previous comparative epidemiological studies have focused on the prevalence of preclinical BP autoantibodies in populations susceptible to BP (e.g., the elderly) or in diseases with a higher risk of comorbid BP. This mini-review summarizes the literature on the prevalence of preclinical BP autoantibodies in patients with various conditions and diseases, and we discuss the significance of preclinical BP autoantibody detection.


Assuntos
Colágenos não Fibrilares , Penfigoide Bolhoso , Idoso , Autoanticorpos , Autoantígenos , Humanos , Immunoblotting
6.
Front Immunol ; 13: 948108, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36032160

RESUMO

Pemphigoid diseases (PD) are autoimmune skin blistering diseases characterized by autoantibodies directed against proteins of the cutaneous basement membrane zone (BMZ). One of the major antigens is type XVII collagen (BP180), a transmembrane glycoprotein, which is targeted in four PDs: bullous pemphigoid, mucous membrane pemphigoid, linear IgA dermatosis, and pemphigoid gestationis. To date, different epitopes on BP180 have been described to be recognized by PD disease patients' autoantibodies. Different BP180 epitopes were associated with distinct clinical phenotypes while the underlying mechanisms are not yet fully understood. So far, the main effects of anti-BP180 reactivity are mediated by Fcγ-receptors on immune cells. More precisely, the autoantibody-antigen interaction leads to activation of complement at the BMZ and infiltration of immune cells into the upper dermis and, by the release of specific enzymes and reactive oxygen species, to the degradation of BP180 and other BMZ components, finally manifesting as blisters and erosions. On the other hand, inflammatory responses independent of Fcγ-receptors have also been reported, including the release of proinflammatory cytokines and internalization and depletion of BP180. Autoantibodies against BP180 can also be found in patients with neurological diseases. The assumption that the clinical expression of PD depends on epitope specificity in addition to target antigens, autoantibody isotypes, and antibody glycosylation is supported by the observation that epitopes of PD patients differ from those of PD patients. The aim of the present review is to describe the fine specificities of anti-BP180 autoantibodies in different PDs and highlight the associated clinical differences. Furthermore, the direct effects after binding of the autoantibodies to their target are summarized.


Assuntos
Doenças Autoimunes , Doenças do Sistema Nervoso , Penfigoide Bolhoso , Autoanticorpos , Autoantígenos , Proteínas de Transporte , Proteínas do Sistema Complemento , Epitopos , Humanos , Immunoblotting , Colágenos não Fibrilares , Receptores de IgG
7.
Pathology ; 54(7): 900-903, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35931495

RESUMO

Serum anti-Ro60 is one the most frequently encountered autoantibodies in the diagnostic immunopathology laboratory and in clinical practice. A large variety of assays are available to detect this including the popular multiplex line immunoblot (IB) assay. We evaluated the analytical performance of the IB for anti-Ro60 detection, using the counterimmunoelectrophoresis (CIEP) method as the 'gold standard'. We also undertook a survey of international laboratories, who use the IB, about their reporting practices for anti-Ro60. Using the manufacturer's reported cut-off of 15 units, the IB has an analytical sensitivity of 90.9% and specificity of 99.3% for anti-Ro60 detection. The optimal cut-off to balance sensitivity and specificity was determined to be 5 units with a sensitivity of 100% and specificity of 97.4%. Most laboratories use the manufacturer's specified cut-off (15 units) when determining a positive anti-Ro60 result. Whilst the commercial IB generally performs well, laboratorians need to be mindful of the limitations of IB in detecting antibodies that recognise conformational epitopes and what cut-offs they use. A vast majority of laboratories could potentially miss detection of this clinically important autoantibody.


Assuntos
Anticorpos Antinucleares , Autoanticorpos , Humanos , Immunoblotting , Sensibilidade e Especificidade
8.
Methods Cell Biol ; 171: 81-109, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35953207

RESUMO

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.


Assuntos
Transtornos Mieloproliferativos , Policitemia Vera , Mielofibrose Primária , Calreticulina/genética , Humanos , Immunoblotting , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Fosfatidilinositol 3-Quinases/genética , Policitemia Vera/genética , Mielofibrose Primária/genética , Proteínas Proto-Oncogênicas c-akt/genética , Células-Tronco , Serina-Treonina Quinases TOR/genética
9.
Transbound Emerg Dis ; 69(5): e2994-e3006, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35801561

RESUMO

Toxocariasis is an infection caused by the round worms Toxocara canis and Toxocara cati. It occurs worldwide though it is more prevalent in developing countries. For the diagnosis of toxocariasis, the most used method is the indirect enzyme-linked immunosorbent assay (indirect ELISA), based on the detection of specific antibodies using the excreted/secreted products from T. canis larvae (TES) as antigens, but it cross-reacts with several helminth infections. For this reason, there is a need to investigate species-specific immunoreactive proteins, which can be used for the development of a more sensitive and specific diagnosis. This study aims to investigate immunoreactive protein candidates to be used for the development of a more sensitive and specific diagnosis of Toxocara spp. infection in humans. We have used immunoblotting and mass spectrometry to select four Toxocara canis immunoreactive proteins that were recombinantly expressed in bacteria and evaluated as potential new diagnostic antigens (rMUC3, rTES 26, rTES32 and rCTL4). The recognition of these recombinant proteins by total serum IgG and IgG4 was assayed using the purified proteins in an isolated manner or in combination. The IgG ELISAs performed with individual recombinant antigens reached values of sensitivity and specificity that ranged from 91.7% to 97.3% and 94.0% to 97.9%, respectively. Among the analyses, the IgG4 immunoassay was proven to be more effective, revealing a sensitivity that ranged from 88.8% to 98.3% and a specificity of 97.8%-97.9%. The IgG4 ELISA was shown to be more effective and presented no cross-reactivity when using combinations of the rTES 26 and rCTL4 recombinant proteins. The combination of these two molecules achieved 100% sensitivity and specificity. The use of only two recombinant proteins can contribute to improve the current panorama of toxocariasis immunodiagnosis for, with a better optimization and reduced cost.


Assuntos
Toxocara canis , Toxocaríase , Animais , Antígenos de Helmintos , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Humanos , Immunoblotting/veterinária , Imunoglobulina G , Testes Imunológicos/veterinária , Proteômica , Proteínas Recombinantes , Toxocara , Toxocaríase/diagnóstico
10.
Methods Mol Biol ; 2523: 265-279, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35759203

RESUMO

Detection of microbes relies on the expression of germline-encoded pattern recognition receptors (PRRs). While PRRs can directly sense conserved pattern expressed by various microbes, they can also induce effector-triggered immunity (ETI) by sensing pathogenic alterations of cellular homeostasis. One consequence of ETI is the death of the infected cell through the induction of inflammasome-dependent cell death, namely, pyroptosis. Such process can be easily studied in macrophages and epithelial cells, yet neutrophils encode an arsenal of proteolytic enzymes that imped easy and reliable study of ETI-triggered inflammasome response. Here, we describe an immunoblotting methodology to study both ETI- and PRR-driven inflammasome responses in neutrophils upon bacterial infections. This method is also transposable to other microbial pathogen- and toxin-induced inflammasome response in neutrophils.


Assuntos
Inflamassomos , Neutrófilos , Bactérias/metabolismo , Immunoblotting , Inflamassomos/metabolismo , Neutrófilos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo
11.
Exp Gerontol ; 164: 111821, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35504483

RESUMO

Adiponectin (APN), a major adipokine secreted from white adipose tissue, prevents inflammation and improves insulin sensitivity. APN exists as distinct multimeric complexes with different physiological activities, including low, middle and high molecular weight complexes (LMW, MMW and HMW, respectively) in peripheral blood. Caloric restriction (CR), an intervention that suppresses aging-related pathophysiological changes and extends lifespan, reportedly elevates the expression levels of Adipoq (encoding APN) and total circulating APN. Circulating APN levels have generally been measured using ELISA, but ELISA fails to directly and separately detect APN multimeric complexes other than HMW. Here, we aimed to evaluate the association of aging and CR with oligomerization of APN in rodent models, using immunoblotting to distinguish multimeric complexes based on molecular sizes. In mice, aging elevated plasma levels of HMW and MMW, while CR only elevated HMW. In contrast, LMW and monomeric APN levels were unchanged, suggesting that aging and CR can induce the assembly of APN oligomers in adipocytes. In rats, plasma levels of all multimeric complexes and monomeric APN were not significantly changed by aging or CR. Collectively, levels of circulating APN in mice were consistent with previous findings, whereas those of rats were partially inconsistent, probably because of experimental differences. Moreover, aging reduced Adipoq mRNA levels in mice and rats, while CR prevented this reduction only in rats. Such a discrepancy between Adipoq expression and circulating APN levels may be attributed to proteasomal regulation in adipocytes or tissue accumulation of APN. In conclusion, this study provides new findings of aging- and CR-related changes of each APN multimeric complex and underscores the importance of qualitative approaches for a greater understanding of physiological changes in APN.


Assuntos
Adiponectina , Resistência à Insulina , Envelhecimento , Animais , Restrição Calórica , Immunoblotting , Camundongos , Sobrepeso , Ratos
12.
Clin Lab ; 68(4)2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35443596

RESUMO

BACKGROUND: Countries use national policies to screen and diagnose people infected with the hepatitis C virus to prevent transmission and eliminate the disease. In May 2016, the World Health Organization set a target of 90% diagnosis and elimination of the disease by 2030. The aim of this study was to evaluate the screening and diagnostic algorithm of hepatitis C by serological methods. METHODS: The blood samples of people referring to blood transfusion centers in Kerman province in southeastern Iran from January 2014 to January 2020 were examined with the defined algorithm for the presence of antibodies against hepatitis C virus by ELISA and confirmation test (RIBA). RESULTS: Based on the algorithm used, little/no correlations were found between the effect of age on OD in ELISA and RIBA test results, respectively (r = 0.07, p = 0.03) and (r = 0.19, p = 0.001). The correlation between the amount of OD in the ELISA test and the results of RIBA test was (r = 0.24, p = 0.01) and no significant correlation was observed between OD in ELISA and the indeterminate immunoblot test results (r = -0.04 and p = 0.2). CONCLUSIONS: The results of this study show, in low-risk populations, all samples that have reactive ELISA results should be confirmed without considering the amount of ELISA OD and the signal-to-cut-off (S/Co) ratios. The existing algorithm should be modified as soon as possible and newer technologies should be used to perform the test.


Assuntos
Doadores de Sangue , Hepatite C , Algoritmos , Ensaio de Imunoadsorção Enzimática , Hepacivirus , Anticorpos Anti-Hepatite/análise , Anticorpos Anti-Hepatite C , Humanos , Immunoblotting
13.
Comp Immunol Microbiol Infect Dis ; 86: 101816, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35472655

RESUMO

Cystic echinococcosis (CE) is a disease caused by Echinococcus granulosus sensu lato (s.l.), an ubiquitous worldwide zoonotic agent affecting humans and animals. Diagnosis of CE in humans is usually performed by imagine techniques along with immunoassays. The aim of our study was to evaluate and compare four commercial diagnostic kits, based on the detection of IgG antibodies against E. granulosus and E. multilocularis. The study was performed on a total of 259 sera: the positive (n = 74) and the negative (n = 185) group. The following analytic and diagnostic performances of the four kits were evaluated: operator skills, specificity, sensitivity, repeatability, reproducibility, accuracy, positive and negative predictive values. Based on the parameters evaluated, all four tests demonstrated excellent quality and proved to be reliable diagnostic tools to support the clinical evaluation of human patients suspected of having CE. The four commercial assays, in our hands, presented altogether, a range of performances from good to excellent, being immunoblotting (IB) the most reliable, used as gold standard, followed by the immunochromatographic test (ICT) and finally the two enzyme linked immunosorbent assay (ELISAs).


Assuntos
Equinococose , Echinococcus granulosus , Animais , Equinococose/diagnóstico , Equinococose/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Humanos , Immunoblotting/veterinária , Reprodutibilidade dos Testes
14.
STAR Protoc ; 3(2): 101265, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35391936

RESUMO

Pharmacologic inhibition of the protein-protein interaction (PPI) interface of the Keap1:Nrf2 complex, which leads to Nrf2 activation and cytoprotective gene expression, offers a promising strategy for disease prevention and treatment. To facilitate identification and validation of small-molecule Keap1:Nrf2 PPI inhibitors in the cellular environment in a low- and medium-throughput manner, we detail two adapted cellular thermal shift assay (CETSA) protocols, Keap1-CETSA, an immunoblotting-based methodology for detecting endogenous Keap1, and Keap1-Glow CETSA, a microtiter plate assay of overexpressed fluorescently-tagged Keap1. For an example of the use and execution of this protocol, please refer to Dayalan Naidu et al. (2021).


Assuntos
Bioensaio , Fator 2 Relacionado a NF-E2 , Immunoblotting , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Ligação Proteica
15.
PLoS Biol ; 20(3): e3001548, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35239649

RESUMO

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions.


Assuntos
Fase G1/genética , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Divisão Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Immunoblotting , Ligação Proteica , RNA-Seq/métodos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Methods Mol Biol ; 2488: 1-12, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35347678

RESUMO

Cell signaling governs the basic functions of cells by molecular interactions that involve of many proteins. The abundance of signaling proteins can directly influence cellular responses to external signal, contributing to cellular heterogeneity. Absolute quantification of proteins is important for modeling and understanding the complex signaling network. Here, we introduce how to measure the amount of TGF-ß signaling proteins using quantitative immunoblotting. In addition, we discuss how to convert the measurements of protein abundance to the quantities of absolute molecules per cell. This method is generally applicable to the absolute quantification of other proteins.


Assuntos
Proteínas Smad , Fator de Crescimento Transformador beta , Western Blotting , Immunoblotting , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo
17.
STAR Protoc ; 3(1): 101018, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35243365

RESUMO

Following lysosomal damage, activation and nuclear translocation of transcription factor EB (TFEB) is the key event to maintain lysosomal homeostasis. Here, we describe steps to induce lysosomal damage in HeLa cells. This can be followed by monitoring the changes in TFEB localization using widefield fluorescence microscopy. As a complementary approach, we describe the use of immunoblotting to follow the activation and localization of TFEB in cell lysates. These protocols enable quantitative analysis of TFEB. For complete details on the use and execution of this protocol, please refer to Nakamura et al. (2020).


Assuntos
Autofagia , Microscopia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Núcleo Celular , Células HeLa , Humanos , Immunoblotting , Lisossomos
18.
PLoS One ; 17(3): e0265453, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35333910

RESUMO

Several SARS-CoV-2 variants emerged that harbor mutations in the surface unit of the viral spike (S) protein that enhance infectivity and transmissibility. Here, we analyzed whether ten naturally-occurring mutations found within the extended loop harboring the S1/S2 cleavage site of the S protein, a determinant of SARS-CoV-2 cell tropism and pathogenicity, impact S protein processing and function. None of the mutations increased but several decreased S protein cleavage at the S1/S2 site, including S686G and P681H, the latter of which is found in variants of concern B.1.1.7 (Alpha variant) and B.1.1.529 (Omicron variant). None of the mutations reduced ACE2 binding and cell-cell fusion although several modulated the efficiency of host cell entry. The effects of mutation S686G on viral entry were cell-type dependent and could be linked to the availability of cathepsin L for S protein activation. These results show that polymorphisms at the S1/S2 site can modulate S protein processing and host cell entry.


Assuntos
Polimorfismo Genético/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Animais , Chlorocebus aethiops , Células HEK293/virologia , Humanos , Immunoblotting , Células Vero/virologia
19.
Sci Rep ; 12(1): 1687, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35105907

RESUMO

The aim of this study is to evaluate the relationship between antinuclear antibody (ANA) titer and specificity, as well as the relationship between the number of positive-autoantibodies (AAbs) in antinuclear antibodies (ANAs) and specificity for systemic lupus erythematosus (SLE), so as to explore their significance in the diagnosis of SLE. A total of 1297 patients with ANA results was enrolled in this study, including 148 patients with SLE patients. The sensitivity, specificity, sensitive likelihood ratio and specific likelihood ratio of indicators in SLE were determined by receiver-operator characteristic (ROC) curve after measurement of ANA and ANAs by indirect immunofluorescence (IIF) and immunoblotting, respectively. ROC analysis showed that the specificity of ANA titer ≥ 1 +, ≥ 2 + and ≥ 3 + for SLE was estimated to be 81.29%, 90.69% and 96.52% respectively, with a increased titer-specific likelihood ratio (5.16, 9.29 and 19.60, respectively). The specificity of the number of positive-AAbs ≥ 1, ≥ 2 and ≥ 3 in ANAs for SLE was estimated to be 80.42%, 94.95% and 99.3% respectively, with a increased number-specific likelihood ratio (4.8, 15.26 and 72.48, respectively). The estimated sensitivity of the number of positive-AAbs ≥ 3, AnuA and anti-rRNP was higher than that of anti-Sm (p < 0.01) (50.68%, 41.89% and 31.76% vs. 16.89%, respectively), while there was no significant difference in their specificity (99.3%, 99.74% and 99.56% vs. 99.74%, respectively) (p > 0.05). High titers of ANA and the presence of multiple AAbs in ANAs are highly specific for SLE and highly suggestive of SLE. The likelihood of SLE can be assessed by ANA titer and the number of positive-AAbs in ANAs.


Assuntos
Anticorpos Antinucleares/imunologia , Doenças Hematológicas/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Insuficiência Renal/imunologia , Doenças Reumáticas/imunologia , Transtornos Urinários/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Immunoblotting/métodos , Masculino , Pessoa de Meia-Idade , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
20.
J Orthop Surg Res ; 17(1): 70, 2022 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-35120538

RESUMO

BACKGROUND: Osteoarthritis (OA) is the most prevalent degenerative joint disease. In vitro experiments are an intuitive method used to investigate its early pathogenesis. Chondrocyte inflammation models in rats and mice are often used as in vitro models of OA. However, similarities and differences between them in the early stages of inflammation have not been reported. OBJECTIVE: This paper seeks to compare the chondrocyte phenotype of rats and mice in the early inflammatory state and identify chondrocytes suitable for the study of early OA. METHODS: Under similar conditions, chondrocytes from rats and mice were stimulated using the same IL-1ß concentration for a short period of time. The phenotypic changes of chondrocytes were observed under a microscope. The treated chondrocytes were subjected to RNA-seq to identify similarities and differences in gene expression. Chondrocytes were labelled with EdU for proliferation analysis. Cell proliferation-associated proteins, including minichromosome maintenance 2 (MCM2), minichromosome maintenance 5 (MCM5), Lamin B1, proliferating cell nuclear antigen (PCNA), and Cyclin D1, were analysed by immunocytochemical staining, cell immunofluorescence, and Western blots to verify the RNA-seq results. RESULTS: RNA-seq revealed that the expression patterns of cytokines, chemokines, matrix metalloproteinases, and collagen were similar between the rat and mouse chondrocyte inflammation models. Nonetheless, the expression of proliferation-related genes showed the opposite pattern. The RNA-seq results were further verified by subsequent experiments. The expression levels of MCM2, MCM5, Lamin B1, PCNA, and Cyclin D1 were significantly upregulated in rat chondrocytes (P < 0.05) and mouse chondrocytes (P < 0.05). CONCLUSIONS: Based on the findings, the rat chondrocyte inflammation model may help in the study of the early pathological mechanism of OA.


Assuntos
Proliferação de Células/genética , Condrócitos/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Osteoartrite/metabolismo , Animais , Ciclina D1 , Modelos Animais de Doenças , Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Interleucina-1beta/genética , Camundongos , Osteoartrite/genética , Antígeno Nuclear de Célula em Proliferação , RNA-Seq , Ratos
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