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1.
Nat Commun ; 11(1): 4928, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004791

RESUMO

High-altitude adaptation of Tibetans represents a remarkable case of natural selection during recent human evolution. Previous genome-wide scans found many non-coding variants under selection, suggesting a pressing need to understand the functional role of non-coding regulatory elements (REs). Here, we generate time courses of paired ATAC-seq and RNA-seq data on cultured HUVECs under hypoxic and normoxic conditions. We further develop a variant interpretation methodology (vPECA) to identify active selected REs (ASREs) and associated regulatory network. We discover three causal SNPs of EPAS1, the key adaptive gene for Tibetans. These SNPs decrease the accessibility of ASREs with weakened binding strength of relevant TFs, and cooperatively down-regulate EPAS1 expression. We further construct the downstream network of EPAS1, elucidating its roles in hypoxic response and angiogenesis. Collectively, we provide a systematic approach to interpret phenotype-associated noncoding variants in proper cell types and relevant dynamic conditions, to model their impact on gene regulation.


Assuntos
Aclimatação/genética , Cromatina/metabolismo , Grupos Étnicos/genética , Redes Reguladoras de Genes , Modelos Genéticos , Altitude , Doença da Altitude/etnologia , Doença da Altitude/genética , Doença da Altitude/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/genética , Células Cultivadas , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Resistência à Doença/genética , Feminino , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Oxigênio/metabolismo , Polimorfismo de Nucleotídeo Único , Gravidez , Cultura Primária de Células , RNA-Seq , Elementos Reguladores de Transcrição/genética , Seleção Genética , Tibet/etnologia , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Genoma
2.
Sci Total Environ ; 747: 142095, 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33076209

RESUMO

Despite the detection of a wide range of contaminants in the blood of green turtle populations foraging in three locations of northern Queensland - Upstart Bay, Cleveland Bay and the Howick Group of Reefs, little is known about the effects of these contaminants on turtle health. Newly developed cell-based bioassays using green turtle primary cell cultures provide an ethical, reproducible, and high-throughput method for assessing the risk of chemical exposure sea turtles. In this project, the toxicity of six priority metals (Mn, Co, Mo, As, Sb, Cu) and blood extracts from foraging turtles were tested in two bioassays adapted to green turtle primary skin and liver cells. Cytotoxicity of metals and blood extracts was measured in primary skin fibroblast cells using a resazurin assay. Glutathione-S-transferase (GST) activity was measured in primary skin fibroblasts and primary liver epithelial cells following exposure to metals and blood extracts. Arsenic, molybdenum, cobalt and copper were found to be cytotoxic to green turtle skin cells. Only manganese, cobalt and copper were found to alter GST activity, predominantly in skin cells, indicating a higher sensitivity of green turtle skin cells compared to liver cells. Effect concentrations of metals in both bioassays were above concentrations found in turtle blood. Turtle blood extracts from the three foraging grounds showed differences in cytotoxicity and GST activity. In both assays, blood extracts of turtles from Upstart Bay were the most toxic, followed by those from Cleveland Bay, then the Howick Reefs, suggesting turtles from Upstart Bay and Cleveland Bay may be at risk from current concentrations of organic contaminants. This study demonstrates that species-specific cell-based bioassays can be used effectively to assess chemical risk in sea turtles and their foraging grounds, and could be applied to assess chemical risk in other marine wildlife.


Assuntos
Tartarugas , Poluentes Químicos da Água , Animais , Bioensaio , Cultura Primária de Células , Queensland , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidade
3.
Nat Commun ; 11(1): 4917, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004802

RESUMO

Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3'-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Animais , Técnicas de Cultura Embrionária , Feminino , Fertilização In Vitro/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Meiose/genética , Camundongos , Mutação , Oócitos/metabolismo , Cultura Primária de Células , RNA-Seq , Tubulina (Proteína)/genética , Zigoto/metabolismo
4.
Nat Commun ; 11(1): 4402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879318

RESUMO

Genome-wide association studies have identified genetic variation contributing to complex disease risk. However, assigning causal genes and mechanisms has been more challenging because disease-associated variants are often found in distal regulatory regions with cell-type specific behaviours. Here, we collect ATAC-seq, Hi-C, Capture Hi-C and nuclear RNA-seq data in stimulated CD4+ T cells over 24 h, to identify functional enhancers regulating gene expression. We characterise changes in DNA interaction and activity dynamics that correlate with changes in gene expression, and find that the strongest correlations are observed within 200 kb of promoters. Using rheumatoid arthritis as an example of T cell mediated disease, we demonstrate interactions of expression quantitative trait loci with target genes, and confirm assigned genes or show complex interactions for 20% of disease associated loci, including FOXO1, which we confirm using CRISPR/Cas9.


Assuntos
Artrite Reumatoide/genética , Linfócitos T CD4-Positivos/metabolismo , Cromatina , Proteína Forkhead Box O1/genética , Doenças Autoimunes/genética , Linfócitos T CD4-Positivos/citologia , Cromatina/química , Cromatina/genética , Elementos Facilitadores Genéticos , Proteína Forkhead Box O1/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Cultura Primária de Células , Regiões Promotoras Genéticas , Locos de Características Quantitativas
5.
Nat Commun ; 11(1): 4527, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913197

RESUMO

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Evolução Clonal , Variações do Número de Cópias de DNA , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Cultura Primária de Células , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA-Seq , Estudos Retrospectivos , Esferoides Celulares , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Microtomografia por Raio-X
6.
Nat Commun ; 11(1): 4387, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873795

RESUMO

The role of neutrophils in solid tumor metastasis remains largely controversial. In preclinical models of solid tumors, both pro-metastatic and anti-metastatic effects of neutrophils have been reported. In this study, using mouse models of breast cancer, we demonstrate that the metastasis-modulating effects of neutrophils are dictated by the status of host natural killer (NK) cells. In NK cell-deficient mice, granulocyte colony-stimulating factor-expanded neutrophils show an inhibitory effect on the metastatic colonization of breast tumor cells in the lung. In contrast, in NK cell-competent mice, neutrophils facilitate metastatic colonization in the same tumor models. In an ex vivo neutrophil-NK cell-tumor cell tri-cell co-culture system, neutrophils are shown to potentially suppress the tumoricidal activity of NK cells, while neutrophils themselves are tumoricidal. Intriguingly, these two modulatory effects by neutrophils are both mediated by reactive oxygen species. Collectively, the absence or presence of NK cells, governs the net tumor-modulatory effects of neutrophils.


Assuntos
Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Mamárias Animais/imunologia , Neutropenia/prevenção & controle , Neutrófilos/imunologia , Animais , Linhagem Celular Tumoral/transplante , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Humanos , Injeções Intravenosas , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/secundário , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/sangue , Neoplasias Mamárias Animais/complicações , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos NOD , Neutropenia/sangue , Neutropenia/etiologia , Neutropenia/imunologia , Neutrófilos/efeitos dos fármacos , Cultura Primária de Células
7.
Signal Transduct Target Ther ; 5(1): 186, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883951

RESUMO

Sterol regulatory element binding protein-2 (SREBP-2) is activated by cytokines or pathogen, such as virus or bacteria, but its association with diminished cholesterol levels in COVID-19 patients is unknown. Here, we evaluated SREBP-2 activation in peripheral blood mononuclear cells of COVID-19 patients and verified the function of SREBP-2 in COVID-19. Intriguingly, we report the first observation of SREBP-2 C-terminal fragment in COVID-19 patients' blood and propose SREBP-2 C-terminal fragment as an indicator for determining severity. We confirmed that SREBP-2-induced cholesterol biosynthesis was suppressed by Sestrin-1 and PCSK9 expression, while the SREBP-2-induced inflammatory responses was upregulated in COVID-19 ICU patients. Using an infectious disease mouse model, inhibitors of SREBP-2 and NF-κB suppressed cytokine storms caused by viral infection and prevented pulmonary damages. These results collectively suggest that SREBP-2 can serve as an indicator for severity diagnosis and therapeutic target for preventing cytokine storm and lung damage in severe COVID-19 patients.


Assuntos
Betacoronavirus/patogenicidade , Colesterol/biossíntese , Infecções por Coronavirus/genética , Síndrome da Liberação de Citocina/genética , Interações Hospedeiro-Patógeno/genética , Leucócitos Mononucleares/imunologia , Pneumonia Viral/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Betacoronavirus/imunologia , Estudos de Casos e Controles , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/mortalidade , Síndrome da Liberação de Citocina/virologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Unidades de Terapia Intensiva , Interleucina-1beta/genética , Interleucina-1beta/imunologia , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/virologia , NF-kappa B/genética , NF-kappa B/imunologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Cultura Primária de Células , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/imunologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
8.
Sci Adv ; 6(31)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32937590

RESUMO

The outbreak of the highly contagious and deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease 2019 (COVID-19), has posed a serious threat to public health across the globe, calling for the development of effective diagnostic markers and therapeutics. Here, we report a highly reliable severity diagnostic biomarker, acetylated 676th lysine transforming growth factor-beta-induced protein (TGFBIp K676Ac). TGFBIp K676Ac was consistently elevated in the blood of patients with SARS-CoV-2 pneumonia (n = 113), especially in patients in the intensive care unit (ICU) compared to non-ICU patients. Patients' blood samples showed increased cytokines and lymphopenia, which are exemplary indicators of SARS-CoV-2 pneumonia. Treatment with TGFBIp neutralizing antibodies suppressed the cytokine storm. The increased level of TGFBIp K676Ac in ICU patients suggests the promise of this protein as a reliable severity diagnostic biomarker for severe SARS-CoV-2 disease.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Síndrome da Liberação de Citocina/diagnóstico , Proteínas da Matriz Extracelular/imunologia , Leucócitos Mononucleares/imunologia , Pneumonia Viral/diagnóstico , Processamento de Proteína Pós-Traducional , Insuficiência Respiratória/diagnóstico , Fator de Crescimento Transformador beta/imunologia , Acetilação , Anticorpos Neutralizantes/farmacologia , Betacoronavirus/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Humanos , Unidades de Terapia Intensiva , Contagem de Leucócitos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Lisina/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Cultura Primária de Células , Prognóstico , Insuficiência Respiratória/sangue , Insuficiência Respiratória/imunologia , Insuficiência Respiratória/patologia , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética
9.
PLoS One ; 15(9): e0237809, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915792

RESUMO

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.


Assuntos
Canalículos Biliares/efeitos dos fármacos , Desenvolvimento de Medicamentos/métodos , Hepatócitos/efeitos dos fármacos , Cultura Primária de Células/métodos , Quimeras de Transplante , Animais , Canalículos Biliares/citologia , Canalículos Biliares/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Ciclosporina/farmacologia , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Eur J Immunol ; 50(9): 1283-1294, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32910469

RESUMO

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and naïve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM- plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies.


Assuntos
Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Isotipos de Imunoglobulinas/sangue , Pulmão/imunologia , Pneumonia Viral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/classificação , Linfócitos B/virologia , Betacoronavirus/imunologia , Proteína C-Reativa/imunologia , Estudos de Casos e Controles , Proliferação de Células , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Estudos Transversais , Citocinas/genética , Citocinas/imunologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/imunologia , Humanos , Imunidade Humoral , Memória Imunológica , Pulmão/patologia , Pulmão/virologia , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Escores de Disfunção Orgânica , Pandemias , Pneumonia Viral/mortalidade , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Cultura Primária de Células , Índice de Gravidade de Doença , Análise de Sobrevida
11.
Aquat Toxicol ; 227: 105586, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32882451

RESUMO

Estrogenic effects triggered by androgens have been previously shown in a few studies. Aromatization and direct binding to estrogen receptors (ERs) are the most proposed mechanisms. For example, previously, a modulation of vitellogenin A (VtgA) by testosterone (T), an aromatizable androgen, was reported in brown trout primary hepatocytes. The effect was reversed by an ER antagonist. In this study, using the same model the disruption caused by T and by the non-aromatizable androgen - dihydrotestosterone (DHT), was assessed in selected estrogenic targets. Hepatocytes were exposed (96 h) to six concentrations of each androgen. The estrogenic targets were VtgA, ERα, ERß1 and two zona pellucida genes, ZP2.5 and ZP3a.2. The aromatase CYP19a1 gene and the androgen receptor (AR) were also included. Modulation of estrogenic targets was studied by quantitative real-time PCR and immunohistochemistry, using an HScore system. VtgA and ERα were up-regulated by DHT (1, 10, 100 µM) and T (10, 100 µM). In contrast, ERß1 was down-regulated by DHT (10, 100 µM), and T (100 µM). ZP2.5 mRNA levels were increased by DHT and T (1, 10, 100 µM), while ZP3a.2 was up-regulated by DHT (100 µM) and T (10, 100 µM). Positive correlations were found between VtgA and ERα mRNA levels and ZPs and ERα, after exposure to both androgens. The mRNA levels of CYP19a1 were not changed, while AR expression tended to increase after micromolar DHT exposures. HScores for Vtg and ZPs corroborated the molecular findings. Both androgens triggered estrogen signaling through direct binding to ERs, most probably ERα.


Assuntos
Androgênios/toxicidade , Di-Hidrotestosterona/toxicidade , Estrogênios/metabolismo , Hepatócitos/efeitos dos fármacos , Testosterona/toxicidade , Truta/metabolismo , Poluentes Químicos da Água/toxicidade , Androgênios/metabolismo , Animais , Células Cultivadas , Di-Hidrotestosterona/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Estrogênios/genética , Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Cultura Primária de Células , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Testosterona/metabolismo , Vitelogeninas/genética , Vitelogeninas/metabolismo , Poluentes Químicos da Água/metabolismo
12.
Mol Pharmacol ; 98(3): 267-279, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32817462

RESUMO

Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.


Assuntos
Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Doadores de Óxido Nítrico/farmacologia , Tirosina/química , Linhagem Celular , Citocromo P-450 CYP2B6/genética , Regulação para Baixo , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cultura Primária de Células , Proteólise
13.
PLoS One ; 15(8): e0236164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760085

RESUMO

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.


Assuntos
Ácido Hialurônico/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/terapia , Medicina Regenerativa/métodos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/metabolismo , Géis , Humanos , Ácido Hialurônico/química , Peróxido de Hidrogênio/toxicidade , Microscopia Intravital , Peso Molecular , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/patologia , Miogenina/análise , Miogenina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Ratos , Superóxido Dismutase/análise , Superóxido Dismutase/metabolismo , Imagem com Lapso de Tempo , Fator de Necrose Tumoral alfa/metabolismo
14.
PLoS One ; 15(8): e0237746, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810144

RESUMO

In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and ß2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and ß2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4ß1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in ß2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4ß1 and ß2 was significantly reduced 70 and 67%, respectively), but α4ß1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.


Assuntos
Oxigenação Hiperbárica , Integrina alfa4beta1/antagonistas & inibidores , Neutrófilos/imunologia , Peptidomiméticos/uso terapêutico , Úlcera Cutânea/terapia , Idoso , Idoso de 80 Anos ou mais , Antígenos CD18/análise , Antígenos CD18/metabolismo , Adesão Celular/imunologia , Doença Crônica/terapia , Terapia Combinada/métodos , Feminino , Seguimentos , Humanos , Integrina alfa4beta1/análise , Integrina alfa4beta1/metabolismo , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Peptidomiméticos/farmacologia , Cultura Primária de Células , Úlcera Cutânea/sangue , Úlcera Cutânea/imunologia , Úlcera Cutânea/patologia , Resultado do Tratamento , Cicatrização/efeitos dos fármacos , Cicatrização/imunologia
15.
Nat Commun ; 11(1): 3945, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770028

RESUMO

TP53 missense mutations leading to the expression of mutant p53 oncoproteins are frequent driver events during tumorigenesis. p53 mutants promote tumor growth, metastasis and chemoresistance by affecting fundamental cellular pathways and functions. Here, we demonstrate that p53 mutants modify structure and function of the Golgi apparatus, culminating in the increased release of a pro-malignant secretome by tumor cells and primary fibroblasts from patients with Li-Fraumeni cancer predisposition syndrome. Mechanistically, interacting with the hypoxia responsive factor HIF1α, mutant p53 induces the expression of miR-30d, which in turn causes tubulo-vesiculation of the Golgi apparatus, leading to enhanced vesicular trafficking and secretion. The mut-p53/HIF1α/miR-30d axis potentiates the release of soluble factors and the deposition and remodeling of the ECM, affecting mechano-signaling and stromal cells activation within the tumor microenvironment, thereby enhancing tumor growth and metastatic colonization.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Complexo de Golgi/patologia , Síndrome de Li-Fraumeni/genética , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Biópsia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Feminino , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Síndrome de Li-Fraumeni/patologia , Camundongos , Microtúbulos/metabolismo , Microtúbulos/patologia , Mutação , Cultura Primária de Células , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Transdução de Sinais/genética , Pele/citologia , Pele/patologia , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Nat Commun ; 11(1): 4311, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855420

RESUMO

Pulmonary disease increases the risk of developing abdominal aortic aneurysms (AAA). However, the mechanism underlying the pathological dialogue between the lungs and aorta is undefined. Here, we find that inflicting acute lung injury (ALI) to mice doubles their incidence of AAA and accelerates macrophage-driven proteolytic damage of the aortic wall. ALI-induced HMGB1 leaks and is captured by arterial macrophages thereby altering their mitochondrial metabolism through RIPK3. RIPK3 promotes mitochondrial fission leading to elevated oxidative stress via DRP1. This triggers MMP12 to lyse arterial matrix, thereby stimulating AAA. Administration of recombinant HMGB1 to WT, but not Ripk3-/- mice, recapitulates ALI-induced proteolytic collapse of arterial architecture. Deletion of RIPK3 in myeloid cells, DRP1 or MMP12 suppression in ALI-inflicted mice repress arterial stress and brake MMP12 release by transmural macrophages thereby maintaining a strengthened arterial framework refractory to AAA. Our results establish an inter-organ circuitry that alerts arterial macrophages to regulate vascular remodeling.


Assuntos
Lesão Pulmonar Aguda/complicações , Aneurisma da Aorta Abdominal/patologia , Proteína HMGB1/metabolismo , Macrófagos/metabolismo , Remodelação Vascular , Lesão Pulmonar Aguda/patologia , Animais , Aorta Abdominal/citologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Dinaminas/antagonistas & inibidores , Dinaminas/metabolismo , Humanos , Macrófagos/citologia , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Estudos Retrospectivos , Regulação para Cima
17.
Plast Reconstr Surg ; 146(2): 309-320, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32740581

RESUMO

BACKGROUND: Adipose-derived stem cells are considered as candidate cells for regenerative plastic surgery. Measures to influence cellular properties and thereby direct their regenerative potential remain elusive. Hyperbaric oxygen therapy-the exposure to 100% oxygen at an increased atmospheric pressure-has been propagated as a noninvasive treatment for a multitude of indications and presents a potential option to condition cells for tissue-engineering purposes. The present study evaluates the effect of hyperbaric oxygen therapy on human adipose-derived stem cells. METHODS: Human adipose-derived stem cells from healthy donors were treated with hyperbaric oxygen therapy at 2 and 3 atm. Viability before and after each hyperbaric oxygen therapy, proliferation, expression of surface markers and protein contents of transforming growth factor (TGF)-ß, tumor necrosis factor-α, hepatocyte growth factor, and epithelial growth factor in the supernatants of treated adipose-derived stem cells were measured. Lastly, adipogenic, osteogenic, and chondrogenic differentiation with and without use of differentiation-inducing media (i.e., autodifferentiation) was examined. RESULTS: Hyperbaric oxygen therapy with 3 atm increased viability, proliferation, and CD34 expression and reduced the CD31/CD34/CD45 adipose-derived stem cell subset and endothelial progenitor cell population. TGF-ß levels were significantly decreased after two hyperbaric oxygen therapy sessions in the 2-atm group and decreased after three hyperbaric oxygen therapy sessions in the 3-atm group. Hepatocyte growth factor secretion remained unaltered in all groups. Although the osteogenic and chondrogenic differentiation were not influenced, adipogenic differentiation and autodifferentiation were significantly enhanced, with osteogenic autodifferentiation significantly alleviated by hyperbaric oxygen therapy with 3 atm. CONCLUSION: Hyperbaric oxygen therapy with 3 atm increases viability and proliferation of adipose-derived stem cells, alters marker expression and subpopulations, decreases TGF-ß secretion, and skews adipose-derived stem cells toward adipogenic differentiation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.


Assuntos
Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Engenharia Celular/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxigênio/administração & dosagem , Tecido Adiposo/citologia , Adulto , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Pressão , Cultura Primária de Células/métodos
18.
PLoS Biol ; 18(8): e3000826, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776935

RESUMO

Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and ßCaMKII-deficient (αßCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αßCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αßCaMKII DKO neurons was restored by active ßCaMKII but not inactive ßCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to ßCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.


Assuntos
Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/genética , Animais , Astrócitos/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Fosforilação , Cultura Primária de Células , Subunidades Proteicas/deficiência , Sinapses/fisiologia , Transmissão Sináptica , Imagem com Lapso de Tempo
19.
PLoS Biol ; 18(8): e3000808, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817651

RESUMO

Although dysregulation of mitochondrial dynamics has been linked to cellular senescence, which contributes to advanced age-related disorders, it is unclear how Krüppel-like factor 5 (Klf5), an essential transcriptional factor of cardiovascular remodeling, mediates the link between mitochondrial dynamics and vascular smooth muscle cell (VSMC) senescence. Here, we show that Klf5 down-regulation in VSMCs is correlated with rupture of abdominal aortic aneurysm (AAA), an age-related vascular disease. Mice lacking Klf5 in VSMCs exacerbate vascular senescence and progression of angiotensin II (Ang II)-induced AAA by facilitating reactive oxygen species (ROS) formation. Klf5 knockdown enhances, while Klf5 overexpression suppresses mitochondrial fission. Mechanistically, Klf5 activates eukaryotic translation initiation factor 5a (eIF5a) transcription through binding to the promoter of eIF5a, which in turn preserves mitochondrial integrity by interacting with mitofusin 1 (Mfn1). Accordingly, decreased expression of eIF5a elicited by Klf5 down-regulation leads to mitochondrial fission and excessive ROS production. Inhibition of mitochondrial fission decreases ROS production and VSMC senescence. Our studies provide a potential therapeutic target for age-related vascular disorders.


Assuntos
Aneurisma da Aorta Abdominal/genética , Células Endoteliais/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Mitocôndrias/metabolismo , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Idoso , Angiotensina II/genética , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Aorta/diagnóstico por imagem , Aorta/metabolismo , Aorta/patologia , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Senescência Celular/efeitos dos fármacos , Ecocardiografia , Células Endoteliais/patologia , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/deficiência , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo
20.
Ecotoxicol Environ Saf ; 205: 111154, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810643

RESUMO

The study focused on the toxicological effect of Di-n-butyl phthalate (DBP) on the expression of Phosphorylated signal transducer and activator of transcription 1 (pSTAT1) -regulated Forkhead box protein M1 (FoxM1), which might provide a new understanding of gestational diabetes mellitus (GDM) development and a potential target for treatment. Streptozotocin (STZ) (40 mg/kg) was introduced in maternal rats by intraperitoneal injection on gestation day 0 (GD 0) in the STZ and STZ + DBP groups. DBP was introduced in maternal rats by oral feeding in the STZ + DBP group over the following 3 days (750 mg/kg/day). The changes in fasting blood glucose level in rats were detected on GD 1 and GD 5. The insulin levels in maternal rats and PIBCs were measured on GD 18. The Oral Glucose Tolerance Test (OGTT) test was performed on GD 18 to check the stability of the GDM model. The primary islet ß cells (PIBCs) were established for in vitro experiments. We examined the FoxM1 and pSTAT1 expression in pancreas by immunohistochemistry. Real-time PCR and Western blot were used to detect the pSTAR1 and FoxM1 protein and mRNA gene expression levels in PIBCs. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis was used to test the viability and apoptosis of cells. The results showed that the STZ + DBP group had higher glucose and lower insulin secretion levels than the other groups by both fasting test and OGTT. FoxM1 was significantly suppressed while pSTAT1 was highly expressed after DBP exposure. FoxM1 could be regulated by pSTAT1. DBP can influence the progression of GDM through its toxicological effect, which significantly increases the expression of pSTAT1 and suppresses FoxM1, causing a decline in ß cell viability.


Assuntos
Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Gestacional/induzido quimicamente , Dibutilftalato/toxicidade , Disruptores Endócrinos/toxicidade , Proteína Forkhead Box M1/metabolismo , Exposição Materna/efeitos adversos , Fator de Transcrição STAT1/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Gestacional/metabolismo , Feminino , Proteína Forkhead Box M1/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Fosforilação , Gravidez , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1/genética , Transdução de Sinais
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