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1.
Nature ; 580(7803): 402-408, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296183

RESUMO

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.


Assuntos
Proteoma/metabolismo , Espaço Extracelular/metabolismo , Humanos , Especificidade de Órgãos , Mapeamento de Interação de Proteínas
2.
J Clin Microbiol ; 60(9): e0055122, 2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36040150

RESUMO

The diagnosis of latent tuberculosis (TB) infection (LTBI) is critical to improve TB treatment and control, and the T-SPOT.TB test is a commercial enzyme-linked immunosorbent spot assay used for this purpose. The objective of the study was to increase automation and extend the time between blood collection and processing for the T-SPOT.TB test from 0 to 8 h to 0 to 54 h. The previous maximum time between blood collection and processing for the T-SPOT.TB test is 32 h using T-Cell Xtend. For this, we compared the T-SPOT.TB test using manual peripheral blood mononuclear cell (PBMC) isolation by density gradient separation at 0 to 8 h (reference method, control arm) to an automated PBMC isolation method using magnetic beads (T-Cell Select kit) at 0 to 55 h postcollection. A total of 620 subjects were enrolled from 4 study sites, and blood samples were collected from each volunteer, comprising 1,850 paired samples in total. Overall agreement between both methods was 96.8% (confidence interval [CI], 95.9 to 97.6%), with 95.8% (CI, 93.5 to 97.5%) positive and 97.1% negative agreement (CI, 96.1 to 97.9%). In summary, there was a strong overall agreement between the automated and manual T-SPOT.TB test processing methods. The results suggest that the T-SPOT.TB test can be processed using automated positive selection with magnetic beads using T-Cell Select to decrease hands-on time. Also, this cell isolation method allowed for the time between blood collection and processing to range from 0 to 55 h. Additional studies in larger and diverse patient populations including immunocompromised and pediatric patients are needed.


Assuntos
Tuberculose Latente , Leucócitos Mononucleares , Automação , Separação Celular , Criança , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoadsorventes , Testes de Liberação de Interferon-gama , Tuberculose Latente/diagnóstico , Linfócitos T , Teste Tuberculínico
3.
J Immunol ; 199(8): 2652-2667, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28904128

RESUMO

TCR signaling pathways cooperate to activate the inducible transcription factors NF-κB, NFAT, and AP-1. In this study, using the calcium ionophore ionomycin and/or PMA on Jurkat T cells, we show that the gene expression program associated with activation of TCR signaling is closely related to specific chromatin landscapes. We find that calcium and kinase signaling cooperate to induce chromatin remodeling at ∼2100 chromatin regions, which demonstrate enriched binding motifs for inducible factors and correlate with target gene expression. We found that these regions typically function as inducible enhancers. Many of these elements contain composite NFAT/AP-1 sites, which typically support cooperative binding, thus further reinforcing the need for cooperation between calcium and kinase signaling in the activation of genes in T cells. In contrast, treatment with PMA or ionomycin alone induces chromatin remodeling at far fewer regions (∼600 and ∼350, respectively), which mostly represent a subset of those induced by costimulation. This suggests that the integration of TCR signaling largely occurs at the level of chromatin, which we propose plays a crucial role in regulating T cell activation.


Assuntos
Cálcio/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Ionóforos de Cálcio/imunologia , Humanos , Células Jurkat , Ativação Linfocitária , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fosfotransferases/metabolismo , Receptor Cross-Talk , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo
4.
Front Immunol ; 10: 609, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984185

RESUMO

The NF-κB transcription regulation system governs a diverse set of responses to various cytokine stimuli. With tools from in vitro biochemical characterizations, to omics-based whole genome investigations, great strides have been made in understanding how NF-κB transcription factors control the expression of specific sets of genes. Nonetheless, these efforts have also revealed a very large number of potential binding sites for NF-κB in the human genome, and a puzzle emerges when trying to explain how NF-κB selects from these many binding sites to direct cell-type- and stimulus-specific gene expression patterns. In this review, we surmise that target gene transcription can broadly be thought of as a function of the nuclear abundance of the various NF-κB dimers, the affinity of NF-κB dimers for the regulatory sequence and the availability of this regulatory site. We use this framework to place quantitative information that has been gathered about the NF-κB transcription regulation system into context and thus consider questions it answers, and questions it raises. We end with a brief discussion of some of the future prospects that new approaches could bring to our understanding of how NF-κB transcription factors orchestrate diverse responses in different biological contexts.


Assuntos
Regulação da Expressão Gênica/imunologia , NF-kappa B/imunologia , Elementos de Resposta/imunologia , Transcrição Gênica/imunologia , Animais , Humanos
5.
Sci Signal ; 11(540)2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042130

RESUMO

Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor κB (NF-κB) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-κB signaling in the cells and their size. We compared the rates of TNF-α production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF-α signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF-α response and cellular NF-κB signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF-α propagation was constrained by competitive uptake of TNF-α from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues while avoiding out-of-context responses at longer distances.


Assuntos
Inflamação/imunologia , Ativação de Macrófagos , Macrófagos/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Inflamação/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Células RAW 264.7 , Análise de Célula Única , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia
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