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1.
Nat Commun ; 11(1): 4994, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020485

RESUMO

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Criança , Pré-Escolar , Mapeamento de Epitopos , Humanos , Lactente , Infecções Meningocócicas/prevenção & controle , Biblioteca de Peptídeos , Análise Serial de Proteínas , Ensaios de Anticorpos Bactericidas Séricos , Adulto Jovem
2.
Biosens Bioelectron ; 169: 112643, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007615

RESUMO

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/sangue , Coronavirus/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Influenza Humana/sangue , Pneumonia Viral/sangue , Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Células HEK293 , Humanos , Influenza Humana/diagnóstico , Influenza Humana/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Vírus da SARS/isolamento & purificação , Sensibilidade e Especificidade
3.
PLoS One ; 15(9): e0238503, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925968

RESUMO

Clinacanthus nutans (CN) (Acanthaceae) is well-known for its anti-inflammatory properties among Asian communities; however, there are currently no data specifically focused on the anti-inflammatory effects of CN on the brain tissue. Neuroinflammation is a common consequence of toxin intrusion to any part of the central nervous system (CNS). As an innate immune response, the CNS may react through both protective and/or toxic actions due to the activation of neuron cells producing pro- and/or anti-inflammatory cytokines in the brain. The unresolved activation of the inflammatory cytokines' response is associated with the pathogenesis of neurological disorders. The present study aimed to decipher the metabolic mechanism on the effects of 14 days oral treatment with CN aqueous extract in induced-lipopolysaccharides (LPS) rats through 1H NMR spectroscopic biomarker profiling of the brain tissue and the related cytokines. Based on the principal component analysis (PCA) of the nuclear magnetic resonance (NMR) spectral data, twenty-one metabolites in the brain tissue were profiled as biomarkers for the LPS (10 µL)-induced neuroinflammation following intracerebroventricular injection. Among the twenty-one biomarkers in the neuroinflammed rats, CN treatment of 1000 and 500 mg/kg BW successfully altered lactate, pyruvate, phosphorylcholine, glutamine, and α-ketoglutarate when compared to the negative control. Likewise, statistical isolinear multiple component analysis (SIMCA) showed that treatments by CN and the positive control drug, dextromethorphan (DXM, 5 mg/kg BW), have anti-neuroinflammatory potential. A moderate correlation, in the orthogonal partial least squares (OPLS) regression model, was found between the spectral metabolite profile and the cytokine levels. The current study revealed the existence of high levels of pro-inflammatory cytokines, namely IL-1α, IL-1ß, and TNF-α in LPS-induced rats. Both CN dose treatments lowered IL-1ß significantly better than DXM Interestingly, DXM and CN treatments both exhibited the upregulation of the anti-inflammatory cytokines IL-2 and 4. However, DXM has an advantage over CN in that the former also increased the expression of IL-10 of anti-inflammatory cytokines. In this study, a metabolomics approach was successfully applied to discover the mechanistic role of CN in controlling the neuroinflammatory conditions through the modulation of complex metabolite interactions in the rat brain.


Assuntos
Acanthaceae , Anti-Inflamatórios/uso terapêutico , Encéfalo/efeitos dos fármacos , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Acanthaceae/química , Animais , Anti-Inflamatórios/química , Encéfalo/metabolismo , Inflamação/metabolismo , Masculino , Metabolômica , Extratos Vegetais/química , Análise de Componente Principal , Análise Serial de Proteínas , Espectroscopia de Prótons por Ressonância Magnética , Ratos Sprague-Dawley
4.
BMC Bioinformatics ; 21(1): 411, 2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32942983

RESUMO

BACKGROUND: Protein microarray is a well-established approach for characterizing activity levels of thousands of proteins in a parallel manner. Analysis of protein microarray data is complex and time-consuming, while existing solutions are either outdated or challenging to use without programming skills. The typical data analysis pipeline consists of a data preprocessing step, followed by differential expression analysis, which is then put into context via functional enrichment. Normally, biologists would need to assemble their own workflow by combining a set of unrelated tools to analyze experimental data. Provided that most of these tools are developed independently by various bioinformatics groups, making them work together could be a real challenge. RESULTS: Here we present PAWER, the online web tool dedicated solely to protein microarray analysis. PAWER enables biologists to carry out all the necessary analysis steps in one go. PAWER provides access to state-of-the-art computational methods through the user-friendly interface, resulting in publication-ready illustrations. We also provide an R package for more advanced use cases, such as bespoke analysis workflows. CONCLUSIONS: PAWER is freely available at https://biit.cs.ut.ee/pawer .


Assuntos
Biologia Computacional/métodos , Análise Serial de Proteínas/métodos , Humanos
5.
PLoS One ; 15(9): e0238089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903266

RESUMO

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.


Assuntos
Infecções por Coronavirus/patologia , Epitopos/química , Pneumonia Viral/patologia , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Mapeamento de Epitopos , Epitopos/sangue , Epitopos/imunologia , Furina/metabolismo , Humanos , Pandemias , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Pneumonia Viral/virologia , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
6.
Medicine (Baltimore) ; 99(37): e22199, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32925795

RESUMO

Colorectal cancer (CRC) is the most common malignant gastrointestinal tumor worldwide. Serum exosomal microRNAs (miRNAs) play a critical role in tumor progression and metastasis. However, the underlying molecular mechanisms are poorly understood.The miRNAs expression profile (GSE39833) was downloaded from Gene Expression Omnibus (GEO) database. GEO2R was applied to screen the differentially expressed miRNAs (DEmiRNAs) between healthy and CRC serum exosome samples. The target genes of DEmiRNAs were predicted by starBase v3.0 online tool. The gene ontology (GO) and Kyoto Encyclopedia of Genomes pathway (KEGG) enrichment analysis were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool. The protein-protein interaction (PPI) network was established by the Search Tool for the Retrieval of Interacting Genes (STRING) visualized using Cytoscape software. Molecular Complex Detection (MCODE) and cytohubba plug-in were used to screen hub genes and gene modules.In total, 102 DEmiRNAs were identified including 67 upregulated and 35 downregulated DEmiRNAs, and 1437 target genes were predicted. GO analysis showed target genes of upregulated DEmiRNAs were significantly enriched in transcription regulation, protein binding, and ubiquitin protein ligase activity. While the target genes of downregulated DEmiRNAs were mainly involved in transcription from RNA polymerase II promoter, SMAD binding, and DNA binding. The KEGG pathway enrichment analyses showed target genes of upregulated DEmiRNAs were significantly enriched in proteoglycans in cancer, microRNAs in cancer, and phosphatidylinositol-3 kinases/Akt (PI3K-Akt) signaling pathway, while target genes of downregulated DEmiRNAs were mainly enriched in transforming growth factor-beta (TGF-beta) signaling pathway and proteoglycans in cancer. The genes of the top 3 modules were mainly enriched in ubiquitin mediated proteolysis, spliceosome, and mRNA surveillance pathway. According to the cytohubba plugin, 37 hub genes were selected, and 4 hub genes including phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), SRC, cell division cycle 42 (CDC42), E1A binding protein p300 (EP300) were identified by combining 8 ranked methods of cytohubba.The study provides a comprehensive analysis of exosomal DEmiRNAs and target genes regulatory network in CRC, which can better understand the roles of exosomal miRNAs in the development of CRC. However, these findings require further experimental validation in future studies.


Assuntos
Neoplasias Colorretais/genética , Biologia Computacional/métodos , MicroRNAs/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Genes Neoplásicos , Humanos , Análise Serial de Proteínas , Mapas de Interação de Proteínas
7.
Cell Mol Immunol ; 17(10): 1095-1097, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32895485
8.
Mol Cell Proteomics ; 19(11): 1749-1759, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32788344

RESUMO

Coronavirus disease 2019 (COVID-19) is a highly contagious infection and threating the human lives in the world. The elevation of cytokines in blood is crucial to induce cytokine storm and immunosuppression in the transition of severity in COVID-19 patients. However, the comprehensive changes of serum proteins in COVID-19 patients throughout the SARS-CoV-2 infection is unknown. In this work, we developed a high-density antibody microarray and performed an in-depth proteomics analysis of serum samples collected from early COVID-19 (n = 15) and influenza (n = 13) patients. We identified a large set of differentially expressed proteins (n = 132) that participate in a landscape of inflammation and immune signaling related to the SARS-CoV-2 infection. Furthermore, the significant correlations of neutrophil and lymphocyte with the CCL2 and CXCL10 mediated cytokine signaling pathways was identified. These information are valuable for the understanding of COVID-19 pathogenesis, identification of biomarkers and development of the optimal anti-inflammation therapy.


Assuntos
Proteínas Sanguíneas/imunologia , Infecções por Coronavirus/imunologia , Tosse/imunologia , Síndrome da Liberação de Citocina/imunologia , Febre/imunologia , Cefaleia/imunologia , Influenza Humana/imunologia , Mialgia/imunologia , Pneumonia Viral/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/patogenicidade , Proteínas Sanguíneas/genética , Criança , Infecções por Coronavirus/genética , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Tosse/genética , Tosse/fisiopatologia , Tosse/virologia , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/fisiopatologia , Síndrome da Liberação de Citocina/virologia , Citocinas/genética , Citocinas/imunologia , Feminino , Febre/genética , Febre/fisiopatologia , Febre/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Cefaleia/genética , Cefaleia/fisiopatologia , Cefaleia/virologia , Humanos , Influenza Humana/genética , Influenza Humana/fisiopatologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Mialgia/genética , Mialgia/fisiopatologia , Mialgia/virologia , Orthomyxoviridae/patogenicidade , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/fisiopatologia , Pneumonia Viral/virologia , Análise Serial de Proteínas , Proteoma/genética , Proteoma/imunologia , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia , Transdução de Sinais/imunologia
9.
Emerg Microbes Infect ; 9(1): 1965-1973, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32819220

RESUMO

Serology is a crucial part of the public health response to the ongoing SARS-CoV-2 pandemic. Here, we describe the development, validation and clinical evaluation of a protein micro-array as a quantitative multiplex immunoassay that can identify S and N-directed SARS-CoV-2 IgG antibodies with high specificity and sensitivity and distinguish them from all currently circulating human coronaviruses. The method specificity was 100% for SARS-CoV-2 S1 and 96% for N antigen based on extensive syndromic (n=230 cases) and population panel (n=94) testing that also confirmed the high prevalence of seasonal human coronaviruses. To assess its potential role for both SARS-CoV-2 patient diagnostics and population studies, we evaluated a large heterogeneous COVID-19 cohort (n=330) and found an overall sensitivity of 89% (≥ 21 days post onset symptoms (dps)), ranging from 86% to 96% depending on severity of disease. For a subset of these patients longitudinal samples were provided up to 56 dps. Mild cases showed absent or delayed, and lower SARS-CoV-2 antibody responses. Overall, we present the development and extensive clinical validation of a multiplex coronavirus serological assay for syndromic testing, to answer research questions regarding to antibody responses, to support SARS-CoV-2 diagnostics and to evaluate epidemiological developments efficiently and with high-throughput.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Proteínas do Nucleocapsídeo/sangue , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/sangue , Idoso , Antígenos Virais/sangue , Antígenos Virais/imunologia , Betacoronavirus/patogenicidade , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Testes de Neutralização , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Análise Serial de Proteínas , Vírus da SARS/imunologia , Vírus da SARS/patogenicidade , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia
10.
APMIS ; 128(11): 573-582, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32860265

RESUMO

Human epidermal growth factor receptor 2 (HER2) gene status and overexpression, occurring in ~ 13.6% of primary breast cancers, is essential for identifying patients likely to benefit from biological treatment. In this method of evaluation study, we tested and compared the HER2 gene-protein assay (GPA) with routine HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The GPA was evaluated using 67 formalin-fixed paraffin-embedded (FFPE) HER2 equivoval IHC (2+) breast cancer tissue samples. Overall, agreement between GPA silver in situ hybridization (SISH) and FISH was 91.9% (57/62). Regression analysis revealed slightly higher, but non-significant difference in HER2/chromosome enumeration probe 17 (CEP17) ratio for GPA as compared to FISH (p = 0.074). Intraclass correlation coefficients (ICCs) of 0.94 and Spearman´s rank correlation coefficients of 0.93 (p < 0.0001) for FISH and GPA SISH suggested strong inter-observer association for methods with one observer counting on average 0.23 significant higher for GPA SISH (p = 0.014). Intra-observer IHC method reproducibility was 52.6% (κ = 0.3122, p = 0.004) and 79.7% (κ = 0.6428, p = 0.9197), suggesting fair significant and substantial non-significant difference between tests for reviewers. Inter-observer reproducibility for IHC methods was 53%. While inter-observer reproducibility for experienced IHC interpretation suggested significant differences (κ = 0.3636, p = 0.0332), unexperienced interpretation of IHC GPA suggested fair non-significant difference between reviewers (κ = 0.3101, p = 0.0747). Using FISH as reference, the diagnostic indices for GPA SISH were as follows: sensitivity 100%, specificity 95% and accuracy 92%. Inaccuracy between tests was in 80% of cases due to ISH categorization as equivocal by one of the methods. IHC results highlight that it may be beneficial with a method for simultaneously visualization of HER2 gene and protein status.


Assuntos
Neoplasias da Mama/diagnóstico , Carcinoma/diagnóstico , Receptor ErbB-2/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carcinoma/genética , Carcinoma/patologia , Carcinoma/cirurgia , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Metástase Linfática , Variações Dependentes do Observador , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
11.
Biochem Biophys Res Commun ; 530(1): 4-9, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828312

RESUMO

COVID-19 has become one of the worst epidemic in the world, currently already more than four million people have been infected, which probably co-exist with human beings, and has a significant impact on the global economy and political order. In the process of fighting against the epidemic in China, the clinical value of a variety of herbal medicines has been recognized and written into the clinical application guide. However, their effective molecular mechanism and potential targets are still not clear. Pathology and pharmacology research will gradually attract attention in the post-epidemic outbreak term. Here, we constructed a COVID-19 protein microarray of potential therapy targets, which contains the main drug targets to the SARS-CoV-2 virus and the anti-virus, anti-inflammatory cellar targets of the host. Series of quality controls test has been carried out, which showed that it could be applied for drug target screening of bio-active natural products. The establishment of this microarray will provide a useful tool for the study of the molecular pharmacology of natural products.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Pneumonia Viral/tratamento farmacológico , Proteínas/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/metabolismo , Produtos Biológicos/farmacologia , Ácido Clorogênico/farmacologia , Infecções por Coronavirus/metabolismo , Diterpenos/farmacologia , Descoberta de Drogas , Glucosídeos/farmacologia , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/metabolismo , Análise Serial de Proteínas , Estilbenos/farmacologia
12.
Nat Commun ; 11(1): 4015, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782246

RESUMO

Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum. We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention.


Assuntos
Antimaláricos/farmacologia , Eritrócitos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Concentração Inibidora 50 , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Fosforilação/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Análise Serial de Proteínas , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
J Biosci Bioeng ; 130(4): 374-381, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32713812

RESUMO

With increased awareness among consumers regarding food safety and security, food allergen control has become an indispensable requirement in the food industry. Although several methods for detecting allergens in food products are available, highly sensitive techniques are required. In this study, we developed a technique named as peptide array-based inhibition enzyme-linked immunosorbent assay (ELISA), Pep-iEIA, for evaluating antigenicity and detecting cow's milk antigen in infant formula products, using a peptide array consisting of a series of overlapping peptides found in allergenic milk proteins. Pep-iEIA was used to examine five cow's milk-based infant formulas with different degrees of hydrolyzation, and the assay offered both more sensitive detection and detailed analysis of remaining antigenic peptides in allergen compared to conventional ELISA. The antigenicity level of the allergenic peptides identified using Pep-iEIA was confirmed by surface plasmon resonance assay. We believe that Pep-iEIA will be highly useful for antigenicity evaluation of dairy products consumed by infants and patients with cow's milk allergy.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Fórmulas Infantis/efeitos adversos , Análise Serial de Proteínas , Alérgenos/imunologia , Animais , Bovinos , Feminino , Hipersensibilidade Alimentar/imunologia , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas do Leite/imunologia , Peptídeos/imunologia
14.
Ann Rheum Dis ; 79(10): 1349-1361, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651195

RESUMO

OBJECTIVE: The goal of these studies is to discover novel urinary biomarkers of lupus nephritis (LN). METHODS: Urine from systemic lupus erythematosus (SLE) patients was interrogated for 1000 proteins using a novel, quantitative planar protein microarray. Hits were validated in an independent SLE cohort with inactive, active non-renal (ANR) and active renal (AR) patients, in a cohort with concurrent renal biopsies, and in a longitudinal cohort. Single-cell renal RNA sequencing data from LN kidneys were examined to deduce the cellular origin of each biomarker. RESULTS: Screening of 1000 proteins revealed 64 proteins to be significantly elevated in SLE urine, of which 17 were ELISA validated in independent cohorts. Urine Angptl4 (area under the curve (AUC)=0.96), L-selectin (AUC=0.86), TPP1 (AUC=0.84), transforming growth factor-ß1 (TGFß1) (AUC=0.78), thrombospondin-1 (AUC=0.73), FOLR2 (AUC=0.72), platelet-derived growth factor receptor-ß (AUC=0.67) and PRX2 (AUC=0.65) distinguished AR from ANR SLE, outperforming anti-dsDNA, C3 and C4, in terms of specificity, sensitivity and positive predictive value. In multivariate regression analysis, urine Angptl4, L-selectin, TPP1 and TGFß1 were highly associated with disease activity, even after correction for demographic variables. In SLE patients with serial follow-up, urine L-selectin (followed by urine Angptl4 and TGFß1) were best at tracking concurrent or pending disease flares. Importantly, several proteins elevated in LN urine were also expressed within the kidneys in LN, either within resident renal cells or infiltrating immune cells, based on single-cell RNA sequencing analysis. CONCLUSION: Unbiased planar array screening of 1000 proteins has led to the discovery of urine Angptl4, L-selectin and TGFß1 as potential biomarker candidates for tracking disease activity in LN.


Assuntos
Proteína 4 Semelhante a Angiopoietina/urina , Biomarcadores/urina , Nefrite Lúpica/diagnóstico , Análise Serial de Proteínas , Fator de Crescimento Transformador beta1/urina , Humanos , Nefrite Lúpica/urina
15.
Nat Commun ; 11(1): 3581, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665645

RESUMO

We still know very little about how the human immune system responds to SARS-CoV-2. Here we construct a SARS-CoV-2 proteome microarray containing 18 out of the 28 predicted proteins and apply it to the characterization of the IgG and IgM antibodies responses in the sera from 29 convalescent patients. We find that all these patients had IgG and IgM antibodies that specifically bind SARS-CoV-2 proteins, particularly the N protein and S1 protein. Besides these proteins, significant antibody responses to ORF9b and NSP5 are also identified. We show that the S1 specific IgG signal positively correlates with age and the level of lactate dehydrogenase (LDH) and negatively correlates with lymphocyte percentage. Overall, this study presents a systemic view of the SARS-CoV-2 specific IgG and IgM responses and provides insights to aid the development of effective diagnostic, therapeutic and vaccination strategies.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Análise Serial de Proteínas/métodos , Adulto , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , L-Lactato Desidrogenase/sangue , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/imunologia , Proteoma/análise , Proteoma/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas não Estruturais Virais/imunologia
16.
Medicine (Baltimore) ; 99(27): e20802, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32629664

RESUMO

It is imperative to further the understanding of the drug resistance mechanisms of ovarian cancer (OC) and to identify useful biological markers for prognosis prediction.Cormine, cBioportal, and The Cancer Genome Atlas databases were used to search microarray data of gene methylation related to OC, drug resistance in OC, and prognosis, and to analyze methylated genes potentially inducing the drug resistance in OC. Fifty-five DNA-methylated genes significantly associated with drug resistance in OC were screened, and the regulatory mechanisms underlying changes in methylation levels of these genes were systematically integrated.Enrichment and annotation of biological processes indicated that most of the above DNA-methylated genes were significantly associated with cell proliferation and cell cycle. In addition, pathway enrichment demonstrated that the above DNA-methylated genes were significantly associated with PI3K-AKT and P53 signaling pathways. Among the 55 genes, 4 were significantly associated with OC prognostic disease-free survival, namely bromodomain containing 4, PDZ domain containing 1 (PDZK1), phosphatase and tensin homolog, and TNF receptor superfamily member 10c; 5 were significantly related to overall survival, namely bromodomain containing 4, PDZK1, PIK3C2B, Rh associated glycoprotein, and DYRK; among them, the degree of methylation of TNF receptor superfamily member 10c, PDZK1, and Rh associated glycoprotein genes was significantly correlated with mRNA expression. Furthermore, PDZK1, Rh associated glycoprotein, and TNF receptor superfamily member 10c genes showed significant hypomethylation in drug-resistance tissues of OC, and their mRNAs had significantly high expression.The association between the methylation of these 55 genes and OC and drug resistance in OC, in addition to bioinformatics analyses clarify the important mechanisms of gene methylation in the development, progression, and drug resistance of OC.


Assuntos
Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Big Data , Proliferação de Células , Classe II de Fosfatidilinositol 3-Quinases/genética , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Ovarianas/mortalidade , Prognóstico , Análise Serial de Proteínas , Transdução de Sinais
17.
Artigo em Inglês | MEDLINE | ID: mdl-32609256

RESUMO

Coronavirus disease 2019 (COVID-19) is an infectious disease initially reported in China and currently worldwide dispersed caused by a new coronavirus (SARS-CoV-2 or 2019-nCoV) affecting more than seven million people around the world causing more than 400 thousand deaths (on June 8th, 2020). The diagnosis of COVID-19 is based on the clinical and epidemiological history of the patient. However, the gold standard for COVID-19 diagnosis is the viral detection through the amplification of nucleic acids. Although the quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR) has been described as the gold standard for diagnosing COVID-19, there are several difficulties involving its use. Here we comment on RT-PCR and describe alternative tests developed for the diagnosis of COVID-19.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Betacoronavirus/genética , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoensaio/métodos , Técnicas Imunoenzimáticas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Análise Serial de Proteínas/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Avaliação de Sintomas/métodos
18.
PLoS One ; 15(7): e0236950, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730335

RESUMO

The use of natural products as feed additives in the poultry industry is increasing; however, most studies focus on performance and growth with little regard for determining mechanism. Our laboratory designed a chicken (Gallus gallus)-specific immunometabolic kinome peptide array. Using this tool to examine the active enzymes responsible for phosphorylation events (kinases) provides important information on host and cellular functions. The objective of this project was to determine if feeding a microencapsulated product comprised of a blend of organic acids and botanicals (AviPlus®P) impacts the intestinal kinome of broiler chickens (Gallus gallus). Day-of-hatch chicks were provided 0 or 500g/MT of the additive and jejunal and ileal segments collected for kinome analysis to determine the mode-of-action of the additive. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed by uploading the statistically significant peptides to the Search Tool for the Retrieval of Interacting Genes database. As a whole, GO and KEGG analysis showed similar activities in the ileum and jejunum. However, there were a small number of KEGG pathways that were only activated in either the ileum or jejunum, but not both. Analysis of the adipocytokine and PI3K-AKT signaling pathways showed differences between ileal and jejunal activity that were controlled, in part, by AKT3. Additionally, cytokine/chemokine evaluation showed the ileum had higher IL1ß, IL6, IL10, TNFα, IFNγ, CXCL8, and CCL4 mRNA expression levels (P<0.05). As a whole, the data showed the addition of microencapsulated organic acids and botanicals to a broiler diet activated many of the same signaling pathways in the ileum and jejunum; however, distinctions were observed. Taken together, the findings of this study begin to define the mode-of-action that microencapsulated organic acids and botanicals have on two important intestinal segments responsible for nutrient digestion and absorption in chickens.


Assuntos
Ácidos/farmacologia , Dieta/veterinária , Íleo/metabolismo , Jejuno/metabolismo , Compostos Fitoquímicos/farmacologia , Proteínas Quinases/metabolismo , Ração Animal/análise , Animais , Galinhas , Perfilação da Expressão Gênica , Íleo/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Plantas/química , Análise Serial de Proteínas , Proteínas Quinases/genética
19.
PLoS Negl Trop Dis ; 14(6): e0008366, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579606

RESUMO

Snakebite envenoming is a major neglected tropical disease that affects millions of people every year. The only effective treatment against snakebite envenoming consists of unspecified cocktails of polyclonal antibodies purified from the plasma of immunized production animals. Currently, little data exists on the molecular interactions between venom-toxin epitopes and antivenom-antibody paratopes. To address this issue, high-density peptide microarray (hdpm) technology has recently been adapted to the field of toxinology. However, analysis of such valuable datasets requires expert understanding and, thus, complicates its broad application within the field. In the present study, we developed a user-friendly, and high-throughput web application named "Snake Toxin and Antivenom Binding Profiles" (STAB Profiles), to allow straight-forward analysis of hdpm datasets. To test our tool and evaluate its performance with a large dataset, we conducted hdpm assays using all African snake toxin protein sequences available in the UniProt database at the time of study design, together with eight commercial antivenoms in clinical use in Africa, thus representing the largest venom-antivenom dataset to date. Furthermore, we introduced a novel method for evaluating raw signals from a peptide microarray experiment and a data normalization protocol enabling intra-microarray and even inter-microarray chip comparisons. Finally, these data, alongside all the data from previous similar studies by Engmark et al., were preprocessed according to our newly developed protocol and made publicly available for download through the STAB Profiles web application (http://tropicalpharmacology.com/tools/stab-profiles/). With these data and our tool, we were able to gain key insights into toxin-antivenom interactions and were able to differentiate the ability of different antivenoms to interact with certain toxins of interest. The data, as well as the web application, we present in this article should be of significant value to the venom-antivenom research community. Knowledge gained from our current and future analyses of this dataset carry the potential to guide the improvement and optimization of current antivenoms for maximum patient benefit, as well as aid the development of next-generation antivenoms.


Assuntos
Antivenenos/farmacologia , Reações Cruzadas , Gerenciamento de Dados , Peptídeos , Análise Serial de Proteínas/métodos , África , Animais , Sítios de Ligação , Epitopos/química , Humanos , Mordeduras de Serpentes/terapia , Venenos de Serpentes/química , Serpentes/classificação , Serpentes/metabolismo
20.
Medicine (Baltimore) ; 99(19): e20124, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32384491

RESUMO

Recent studies have suggested an increased risk of prostate cancer in men with Lynch syndrome driven by germline mutations in mismatch repair (MMR) genes. However, the incidence and clinical implication of MMR deficiency in sporadic prostate cancers remain poorly understood. We immunohistochemically stained for MLH1, MSH2, MSH6, and PMS2 in a set of tissue microarray consisting of 220 radical prostatectomy specimens and evaluated the relationship between loss of their expression and available clinicopathological features. MLH1, MSH2, MSH6, and PMS2 were lost in 2 (0.9%), 6 (2.7%), 37 (16.8%), and 27 (12.3%) prostate cancers, respectively. Loss of at least 1 MMR protein was identified in 50 (22.7%) cases. There were no statistically significant associations between MMR deficiency and patient age, family history of prostate cancer, Gleason score, or pT/pN stage. Nonetheless, the levels of preoperative prostate-specific antigen (PSA) were significantly (P = .015) higher in patients with MMR deficiency (mean ±â€ŠSD: 9.12 ±â€Š9.01 ng/mL) than in those without abnormal MMR (5.76 ±â€Š3.17 ng/mL). There were 15 (6.8%) cases showing loss of at least 2 MMR proteins, which was not significantly associated with PSA level or tumor grade/stage. Additionally, 5 and 2 cases showed losses of at least 3 MMR proteins and all 4 proteins, respectively. Kaplan-Meier analysis revealed no significant associations between loss of MLH1 (P = .373), MSH2 (P = .348), MSH6 (P = .946), or PMS2 (P = .681), or at least 1 (P = .477), 2 (P = .486), or 3 (P = .352) MMR proteins and biochemical recurrence. Further analyses of the data on programmed death-ligand 1 (PD-L1) expression previously stained in the same set of tissue microarray demonstrated associations between loss of ≥2 MMR proteins and a higher rate of PD-L1 expression in cancer cells (17.2% vs 5.2%; P = .033) as well as between cases showing both loss of ≥1 MMR protein(s) and PD-L1 expression in tumor-infiltrating immune cells vs a higher risk of biochemical recurrence (P = .045). MMR protein loss was seen in a subset of prostate cancers. Interestingly, it was associated with significantly higher levels of PSA. Moreover, immunohistochemical detection of MMR proteins together with other proteins, such as PD-L1, might be helpful in predicting tumor recurrence following radical prostatectomy.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/fisiologia , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/genética , Fatores Etários , Idoso , Antígeno B7-H1/biossíntese , Biomarcadores Tumorais , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Proteínas de Ligação a DNA/biossíntese , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento/biossíntese , Proteína 1 Homóloga a MutL/biossíntese , Proteína 2 Homóloga a MutS/biossíntese , Gradação de Tumores , Recidiva Local de Neoplasia , Antígeno Prostático Específico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Análise Serial de Proteínas
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