Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 98.283
Filtrar
2.
Anticancer Res ; 40(9): 5159-5170, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878804

RESUMO

BACKGROUND/AIM: The aim of this study was to elucidate the possibility of sensitizing colon cancer cells to the chemotherapeutic drug SN38 and investigate its mechanism of action after combined treatment with electroporation (EP). MATERIALS AND METHODS: Cells were treated with SN38, EP and their combination for 24/48 h. The cell viability, actin cytoskeleton integrity, mitochondrial superoxide, hydroperoxides, total glutathione, phosphatidyl serine expression, DNA damages and expression of membrane ABC transporters were analyzed using conventional analytical tests. RESULTS: The combination of EP and SN38 affected cell viability and cytoskeleton integrity. This effect was accompanied by: (i) high production of intracellular superoxide and hydroperoxides and depletion of glutathione; (ii) increased DNA damage and apoptotic/ferroptotic cell death; (iii) changes in the expression of membrane ABC transporters - up-regulation of SLCO1B1 and retention of SN38 in the cells. CONCLUSION: The anticancer effect of the combined treatment of SN38 and EP is related to changes in the redox-homeostasis of cancer cells, leading to cell death via apoptosis and/or ferroptosis. Thus, electroporation has a potential to increase the sensitivity of cancer cells to conventional anticancer therapy with SN38.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Oxirredução , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Imunofluorescência , Glutationa/metabolismo , Humanos , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
3.
Life Sci ; 259: 118374, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891613

RESUMO

OBJECTIVE: Dipeptidyl peptidase IV (DPP4) has been indicated as a possible prognostic biomarker in papillary thyroid cancer (PTC). However, the mechanism of DPP4 during metastasis of PTC remains unclear. In this study, we investigated whether lysine acetyltransferase 5 (KAT5) and FBJ murine osteosarcoma viral oncogene homolog B (FosB) synergistically regulate high DPP4 expression in PTC. METHODS: PTC tissues and matched paracancerous tissues were harvested, followed by the establishment of IHH-4 and TPC-1 cells with downregulation of DPP4. The relevance of DPP4 on the metastasis of PTC cells was assessed. Subsequently, the effect of KAT5 on the transcription of DPP4 was verified. The binding relationship between FosB and DPP4 was predicted by a bioinformatics website. Functional rescue experiments were performed to evaluate cell activities after overexpression of KAT5 or FosB in cells with DPP4 knockdown. RESULTS: DPP4 was overexpressed in PTC tissues and cell lines, which was correlated with higher risks for metastases and poorer survival. DPP4 downregulation curtailed cell growth and metastasis. Moreover, KAT5 acetylated DPP4 promoter histone, which promoted transcription activation of DPP4. Subsequently, FosB recruited KAT5 at the DPP4 promoter, thereby enhancing DPP4 transcriptional activation. Further overexpression of KAT5 or FosB in cells with low expression of DPP4 promoted cell activity. Finally, DPP4 expedited p62 nuclear translocation to elevate Keap1/Nrf2 expression, thus facilitating the growth and metastasis of PTC cells. CONCLUSION: FosB enhanced the growth and metastasis of PTC cells by recruiting histone acetyltransferases KAT5 to increase DPP4 transcription and activate the p62/Keap1/Nrf2 signaling.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Lisina Acetiltransferase 5/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo
4.
Life Sci ; 259: 118390, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896556

RESUMO

AIMS: This study aimed to evaluate the function and pathway of ATP-binding cassette transporter member A1 (ABCA1)-induced anti-inflammatory response in cells at the feto-maternal interface. MAIN METHODS: The primary amniotic mesenchymal cells (AMCs), chorion cells and decidual cells were isolated from placental membranes of women with uncomplicated pregnancies at full-term (not in labor) using enzymatic digestion. Flow cytometry was used to measure the purity of isolated cells. Immunofluorescence assay was performed to detect the location of ABCA1 and toll-like receptor 4 (TLR4). Reverse transcription PCR and western blotting analyses were used to examine ABCA1, TLR4 and inflammatory factor expression in primary cells. ELISA was used to detect cytokine secretions from the primary cells. KEY FINDINGS: ABCA1 and TLR4 were mainly located in the cell nucleus and cytoplasm of feto-maternal interface cells. ABCA1 expression remained the highest in chorion cells, medium in decidual cells, and weakest in AMCs. Upregulated expression of ABCA1 decreased expression of TLR4 and the levels of pro-inflammatory factors, but increased cytoprotective factors in all cell types. In contrast, downregulated expression of ABCA1 increased the expression of TLR4 and pro-inflammatory factors, but decreased the levels of cytoprotective factors. Downregulated ABCA1 expression followed by decreased TLR4 expression using a small interference RNA (siRNA) induced reduction of interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α) in all cell types. SIGNIFICANCE: ABCA1 at feto-maternal interface acts as an anti-inflammatory role by reducing the expression of TLR4 in uncomplicated pregnancies. ABCA1 might be a potential therapeutic target for preventing gestational diseases.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Troca Materno-Fetal , Receptor 4 Toll-Like/metabolismo , Western Blotting , Células Cultivadas , Córion/metabolismo , Decídua/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Confocal , Placenta/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Methods Mol Biol ; 2203: 119-134, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833209

RESUMO

Well-differentiated primary airway epithelial cell (AEC) cultures have been widely used for the characterization of several human respiratory viruses including coronaviruses. In recent years, there has been an increase in interest toward animal AEC cultures and their application to characterize veterinary viruses with zoonotic potential, as well as studying host-pathogen interactions in animal reservoir host species. In this chapter, we provide a revised and improved protocol for the isolation and establishment of well-differentiated AEC cultures from diverse mammalian species and the use of the cultures for the characterization of veterinary coronavirus. We also describe immunohistochemistry protocols with validated antibodies for the visualization and identification of viral cell tropism in well-differentiated AEC cultures from human, swine, bovine, and feline origin.


Assuntos
Brônquios/citologia , Coronavirus/fisiologia , Células Epiteliais/virologia , Cultura Primária de Células/métodos , Traqueia/citologia , Animais , Gatos , Bovinos , Diferenciação Celular , Células Epiteliais/citologia , Imunofluorescência , Humanos , Imuno-Histoquímica/métodos , Cultura Primária de Células/instrumentação , Suínos , Tropismo Viral
6.
Methods Mol Biol ; 2203: 187-204, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833213

RESUMO

Biotin-based proximity labeling circumvents major pitfalls of classical biochemical approaches to identify protein-protein interactions. It consists of enzyme-catalyzed biotin tags ubiquitously apposed on proteins located in close proximity of the labeling enzyme, followed by affinity purification and identification of biotinylated proteins by mass spectrometry. Here we outline the methods by which the molecular microenvironment of the coronavirus replicase/transcriptase complex (RTC), i.e., proteins located within a close perimeter of the RTC, can be determined by different proximity labeling approaches using BirAR118G (BioID), TurboID, and APEX2. These factors represent a molecular signature of coronavirus RTCs and likely contribute to the viral life cycle, thereby constituting attractive targets for the development of antiviral intervention strategies.


Assuntos
Coronavirus/patogenicidade , Enzimas/genética , Interações Hospedeiro-Patógeno/fisiologia , Proteômica/métodos , Proteínas Virais/metabolismo , Animais , Ascorbato Peroxidases/genética , Biotinilação , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Coronavirus/genética , Enzimas/metabolismo , Proteínas de Escherichia coli/genética , Imunofluorescência , Microrganismos Geneticamente Modificados , Proteínas Repressoras/genética , Proteínas Virais/química , Proteínas Virais/genética
7.
Methods Mol Biol ; 2203: 231-238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833216

RESUMO

Ribopuromycylation enables the visualization and quantitation of translation on a cellular level by immunofluorescence or in total using standard western blotting. This technique uses ribosome catalyzed puromycylation of nascent chains followed by immobilization on the ribosome by antibiotic chain elongation inhibitor emetine. Detection of puromycylated ribosome-bound nascent chains can then be achieved using a puromycin-specific antibody.


Assuntos
Coronavirus/genética , Puromicina/farmacologia , Infecções por Coronavirus , Imunofluorescência , Interações Hospedeiro-Patógeno , Humanos , Biossíntese de Proteínas , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
8.
Tidsskr Nor Laegeforen ; 140(11)2020 08 18.
Artigo em Inglês, Norueguês | MEDLINE | ID: mdl-32815342

RESUMO

BACKGROUND: Robust serological assays for SARS-CoV-2 are essential for determining prior infection and the suitability of donated convalescent plasma for plasma therapy. We compared two in-house and three commercial serological assays in a family cohort with SARS-CoV-2-infected members. CASE PRESENTATION: Three individuals in a family of five developed COVID-19 confirmed by PCR, following a trip abroad. Three to four weeks after the onset of symptoms, three commercial ELISAs and an in-house immunofluorescence test demonstrated antibodies to SARS-CoV-2. An in-house neutralisation test also demonstrated neutralising antibodies. INTERPRETATION: The in-house assays and one commercial assay gave congruent results, which were also consistent with the initial PCR and/or clinical evaluation, indicating prior infection in three of the five family members. The other commercial assays indicated possible infection in a fourth family member, but this result was likely due to cross-reactivity. The neutralising antibodies suggest complete or partial immunity against reinfection.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus , Ensaio de Imunoadsorção Enzimática , Saúde da Família , Imunofluorescência , Humanos , Testes de Neutralização , Pandemias , Testes Sorológicos
9.
Nat Commun ; 11(1): 4060, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792512

RESUMO

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid-binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain-hindbrain lineages and self-renewal-differentiation choices in NPCs.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Drosophila , Epigênese Genética/genética , Citometria de Fluxo , Imunofluorescência , Histona-Lisina N-Metiltransferase/genética , Imunoprecipitação , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética
10.
Nat Commun ; 11(1): 4061, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792541

RESUMO

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.


Assuntos
Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Animais , Benzofenonas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Doxiciclina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia , Humanos , Imuno-Histoquímica , Isoxazóis/farmacologia , Lipossomos/metabolismo , Camundongos , Células NIH 3T3 , Parabiose , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia
11.
Nat Commun ; 11(1): 4102, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796823

RESUMO

Emerging evidence suggests that intestinal stromal cells (IntSCs) play essential roles in maintaining intestinal homeostasis. However, the extent of heterogeneity within the villi stromal compartment and how IntSCs regulate the structure and function of specialized intestinal lymphatic capillary called lacteal remain elusive. Here we show that selective hyperactivation or depletion of YAP/TAZ in PDGFRß+ IntSCs leads to lacteal sprouting or regression with junctional disintegration and impaired dietary fat uptake. Indeed, mechanical or osmotic stress regulates IntSC secretion of VEGF-C mediated by YAP/TAZ. Single-cell RNA sequencing delineated novel subtypes of villi fibroblasts that upregulate Vegfc upon YAP/TAZ activation. These populations of fibroblasts were distributed in proximity to lacteal, suggesting that they constitute a peri-lacteal microenvironment. Our findings demonstrate the heterogeneity of IntSCs and reveal that distinct subsets of villi fibroblasts regulate lacteal integrity through YAP/TAZ-induced VEGF-C secretion, providing new insights into the dynamic regulatory mechanisms behind lymphangiogenesis and lymphatic remodeling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fibroblastos/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Transcrição/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Fibroblastos/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Hibridização in Situ Fluorescente , Mucosa Intestinal/ultraestrutura , Linfangiogênese/genética , Linfangiogênese/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fator C de Crescimento do Endotélio Vascular/genética
12.
Biomed Environ Sci ; 33(7): 493-501, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32807268

RESUMO

Objective: The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP Sc in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification. Methods: We prepared a PrP-specific polyclonal antibody (pAb P54) in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays. Results: Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP C from healthy rodents and humans, and pathological PrP Sc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP C and PrP Sc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei. Conclusion: The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.


Assuntos
Anticorpos/imunologia , Imunização , Proteínas PrPC/imunologia , Proteínas PrPSc/imunologia , Proteínas Priônicas/imunologia , Animais , Western Blotting , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Proteínas Recombinantes/imunologia
13.
Nat Commun ; 11(1): 4118, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807789

RESUMO

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Imunofluorescência , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Proteômica
14.
Nat Commun ; 11(1): 4116, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807793

RESUMO

Glioblastoma contains a rare population of self-renewing brain tumor stem cells (BTSCs) which are endowed with properties to proliferate, spur the growth of new tumors, and at the same time, evade ionizing radiation (IR) and chemotherapy. However, the drivers of BTSC resistance to therapy remain unknown. The cytokine receptor for oncostatin M (OSMR) regulates BTSC proliferation and glioblastoma tumorigenesis. Here, we report our discovery of a mitochondrial OSMR that confers resistance to IR via regulation of oxidative phosphorylation, independent of its role in cell proliferation. Mechanistically, OSMR is targeted to the mitochondrial matrix via the presequence translocase-associated motor complex components, mtHSP70 and TIM44. OSMR interacts with NADH ubiquinone oxidoreductase 1/2 (NDUFS1/2) of complex I and promotes mitochondrial respiration. Deletion of OSMR impairs spare respiratory capacity, increases reactive oxygen species, and sensitizes BTSCs to IR-induced cell death. Importantly, suppression of OSMR improves glioblastoma response to IR and prolongs lifespan.


Assuntos
Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Radiação Ionizante , Receptores de Oncostatina M/metabolismo , Animais , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos SCID , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Oncostatina M/metabolismo , Estresse Oxidativo/efeitos da radiação , Receptores de Oncostatina M/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos da radiação
15.
Nat Commun ; 11(1): 4115, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807795

RESUMO

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.


Assuntos
Anticorpos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células A549 , Anticorpos/genética , Western Blotting , Calorimetria , Cristalografia por Raios X , Citometria de Fluxo , Polarização de Fluorescência , Imunofluorescência , Humanos , Espectrometria de Massas , Ligação Proteica , Domínios Proteicos/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Biologia Sintética
16.
Exp Parasitol ; 217: 107962, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763249

RESUMO

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.


Assuntos
FMN Redutase/metabolismo , Ferro/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Membrana Celular/enzimologia , Doença de Chagas/parasitologia , Colorimetria , FMN Redutase/análise , FMN Redutase/química , Imunofluorescência , Humanos , Filogenia , Distribuição de Poisson , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Trypanosoma cruzi/classificação , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Regulação para Cima
17.
Placenta ; 100: 69-74, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32862058

RESUMO

INTRODUCTION: While the COVID-19 pandemic continues to have a significant global health impact, rates of maternal to infant vertical transmission remain low (<5%). Parenchymal changes of placentas from COVID-19 infected mothers have been reported by several groups, but the localization and relative abundance of SARS-CoV-2 viral proteins and cellular entry machinery has not been fully characterized within larger placental tissue cohorts. METHODS: An extended placental tissue cohort including samples from 15 COVID-19 positive maternal-fetal dyads (with n = 5 cases with evidence of fetal transmission) in comparison with 10 contemporary COVID-19 negative controls. Using comparative immunofluorescence, we examined the localization and relative tissue abundance of SARS-CoV2 spike glycoprotein (CoV2 SP) along with the co-localization of two SARS-CoV2 viral entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). RESULTS/CONCLUSIONS: CoV2 SP was present within the villous placenta in COVID-19 positive pregnancies with and without evidence of fetal transmission. We further identified the predominance of ACE2 expression in comparison with TMPRSS2. Importantly, both CoV2 SP and ACE2 expression consistently localized primarily within the outer syncytiotrophoblast layer placental villi, a key physiologic interface between mother and fetus. Overall this study provides an important basis for the ongoing evaluation of SARS-CoV-2 physiology in pregnancy and highlights the importance of the placenta as a key source of primary human tissue for ongoing diagnostic and therapeutic research efforts to reduce the global burden of COVID-19.


Assuntos
Betacoronavirus/química , Vilosidades Coriônicas/química , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/análise , Pneumonia Viral , Serina Endopeptidases/análise , Glicoproteína da Espícula de Coronavírus/análise , Feminino , Feto , Imunofluorescência/métodos , Humanos , Transmissão Vertical de Doença Infecciosa , Placenta/química , Gravidez , Complicações Infecciosas na Gravidez/virologia , Receptores Virais/análise , Trofoblastos/química
18.
Nat Commun ; 11(1): 3457, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651364

RESUMO

Glioblastoma is a deadly cancer, with no effective therapies. Better understanding and identification of selective targets are urgently needed. We found that advillin (AVIL) is overexpressed in all the glioblastomas we tested including glioblastoma stem/initiating cells, but hardly detectable in non-neoplastic astrocytes, neural stem cells or normal brain. Glioma patients with increased AVIL expression have a worse prognosis. Silencing AVIL nearly eradicated glioblastoma cells in culture, and dramatically inhibited in vivo xenografts in mice, but had no effect on normal control cells. Conversely, overexpressing AVIL promoted cell proliferation and migration, enabled fibroblasts to escape contact inhibition, and transformed immortalized astrocytes, supporting AVIL being a bona fide oncogene. We provide evidence that the tumorigenic effect of AVIL is partly mediated by FOXM1, which regulates LIN28B, whose expression also correlates with clinical prognosis. AVIL regulates the cytoskeleton through modulating F-actin, while mutants disrupting F-actin binding are defective in its tumorigenic capabilities.


Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas dos Microfilamentos/metabolismo , Animais , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citoesqueleto/metabolismo , Imunofluorescência , Glioblastoma/genética , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real
19.
Nat Commun ; 11(1): 3503, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665547

RESUMO

DNA replication timing is tightly regulated during S-phase. S-phase length is determined by DNA synthesis rate, which depends on the number of active replication forks and their velocity. Here, we show that E2F-dependent transcription, through E2F6, determines the replication capacity of a cell, defined as the maximal amount of DNA a cell can synthesise per unit time during S-phase. Increasing or decreasing E2F-dependent transcription during S-phase increases or decreases replication capacity, and thereby replication rates, thus shortening or lengthening S-phase, respectively. The changes in replication rate occur mainly through changes in fork speed without affecting the number of active forks. An increase in fork speed does not induce replication stress directly, but increases DNA damage over time causing cell cycle arrest. Thus, E2F-dependent transcription determines the DNA replication capacity of a cell, which affects the replication rate, controlling the time it takes to duplicate the genome and complete S-phase.


Assuntos
Cromatina/metabolismo , Replicação do DNA/fisiologia , Western Blotting , Cromatina/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Replicação do DNA/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Fase S/genética , Fase S/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Nat Commun ; 11(1): 3515, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665557

RESUMO

An unmet clinical need in solid tumor cancers is the ability to harness the intrinsic spatial information in primary tumors that can be exploited to optimize prognostics, diagnostics and therapeutic strategies for precision medicine. Here, we develop a transformational spatial analytics computational and systems biology platform (SpAn) that predicts clinical outcomes and captures emergent spatial biology that can potentially inform therapeutic strategies. We apply SpAn to primary tumor tissue samples from a cohort of 432 chemo-naïve colorectal cancer (CRC) patients iteratively labeled with a highly multiplexed (hyperplexed) panel of 55 fluorescently tagged antibodies. We show that SpAn predicts the 5-year risk of CRC recurrence with a mean AUROC of 88.5% (SE of 0.1%), significantly better than current state-of-the-art methods. Additionally, SpAn infers the emergent network biology of tumor microenvironment spatial domains revealing a spatially-mediated role of CRC consensus molecular subtype features with the potential to inform precision medicine.


Assuntos
Neoplasias Colorretais/genética , Recidiva Local de Neoplasia/genética , Biomarcadores/metabolismo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Medicina de Precisão , Biologia de Sistemas , Microambiente Tumoral/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA