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1.
Sci Adv ; 6(4): eaay5952, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32010788

RESUMO

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.


Assuntos
MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Técnicas de Amplificação de Ácido Nucleico/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
2.
Nat Commun ; 10(1): 674, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787297

RESUMO

Direct cardiac reprogramming from fibroblasts can be a promising approach for disease modeling, drug screening, and cardiac regeneration in pediatric and adult patients. However, postnatal and adult fibroblasts are less efficient for reprogramming compared with embryonic fibroblasts, and barriers to cardiac reprogramming associated with aging remain undetermined. In this study, we screened 8400 chemical compounds and found that diclofenac sodium (diclofenac), a non-steroidal anti-inflammatory drug, greatly enhanced cardiac reprogramming in combination with Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2. Intriguingly, diclofenac promoted cardiac reprogramming in mouse postnatal and adult tail-tip fibroblasts (TTFs), but not in mouse embryonic fibroblasts (MEFs). Mechanistically, diclofenac enhanced cardiac reprogramming by inhibiting cyclooxygenase-2, prostaglandin E2/prostaglandin E receptor 4, cyclic AMP/protein kinase A, and interleukin 1ß signaling and by silencing inflammatory and fibroblast programs, which were activated in postnatal and adult TTFs. Thus, anti-inflammation represents a new target for cardiac reprogramming associated with aging.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/efeitos dos fármacos , Diclofenaco/farmacologia , Dinoprostona , Fibroblastos , Fator de Transcrição GATA4/metabolismo , Humanos , Inflamação , Interleucina-1beta , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas com Domínio T/metabolismo
3.
Biochem Biophys Res Commun ; 495(1): 884-891, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29158084

RESUMO

The coronary vascular system is critical for myocardial growth and cardiomyocyte survival. However, the molecular mechanism regulating coronary angiogenesis remains elusive. Vascular endothelial growth factor (VEGF) regulates angiogenesis by binding to the specific receptors Flk1 and Flt1, which results in different functions. Despite the importance of Flk1 and Flt1, their expression in the coronary vasculature remains largely unknown due to the lack of appropriate antibodies for immunostaining. Here, we analyzed multiple reporter mice including Flk1-GFP BAC transgenic (Tg), Flk1-LacZ knock-in, Flt1-DsRed BAC Tg, and Flk1-GFP/Flt1-DsRed double Tg animals to determine expression patterns in mouse hearts during cardiac growth and after myocardial infarction (MI). We found that Flk1 was expressed in endothelial cells (ECs) with a pattern of epicardial-to-endocardial transmural gradients in the neonatal mouse ventricle, which was downregulated in adult coronary vessels with development. In contrast, Flt1 was homogeneously expressed in the ECs of neonatal mouse hearts and expression was maintained until adulthood. After MI, expression of both Flk1 and Flt1 was induced in the regenerating coronary vessels at day 7. Intriguingly, Flk1 expression was downregulated thereafter, whereas Flt1 expression was maintained in the newly formed coronary vessels until 30 days post-MI, recapitulating their expression kinetics during development. This is the first report demonstrating the spatiotemporal expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after MI; thus, this study suggests that these factors have distinct and important functions in coronary angiogenesis.


Assuntos
Envelhecimento/metabolismo , Vasos Coronários/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Vasos Coronários/crescimento & desenvolvimento , Progressão da Doença , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Neovascularização Fisiológica/fisiologia
4.
Int J Mol Sci ; 18(8)2017 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-28825623

RESUMO

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Doxiciclina/farmacologia , Miócitos Cardíacos/metabolismo , Tetraciclina/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/efeitos dos fármacos , Doxiciclina/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Humanos , Fatores de Transcrição MEF2/genética , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Medicina Regenerativa/tendências , Proteínas com Domínio T/genética , Transativadores/genética , Transdução Genética , Transgenes/efeitos dos fármacos
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