RESUMO
The rapid spread of SARS-CoV-2 infection throughout the world led to a global public health and economic crisis triggering an urgent need for the development of low-cost vaccines, therapies and high-throughput detection assays. In this work, we used a combination of Ideal-Filter Capillary Electrophoresis SELEX (IFCE-SELEX), Next Generation Sequencing (NGS) and binding assays to isolate and validate single-stranded DNA aptamers that can specifically recognize the SARS-CoV-2 Spike glycoprotein. Two selected non-competing DNA aptamers, C7 and C9 were successfully used as sensitive and specific biological recognition elements for the development of electrochemical and fluorescent aptasensors for the SARS-CoV-2 Spike glycoprotein with detection limits of 0.07 fM and 41.87 nM, respectively.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , Aptâmeros de Nucleotídeos/genética , COVID-19/diagnóstico , Humanos , SARS-CoV-2/genética , Técnica de Seleção de Aptâmeros , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Prior to and during the process of human labor, maternal circulating leukocytes infiltrate the maternal-fetal interface (choriodecidua) and become activated resembling choriodecidual leukocytes. Since, there is no evidence comparing maternal circulating and choriodecidual leukocytes, herein, we characterized their transcriptome and explored the biological processes enriched in choriodecidual leukocytes. From women undergoing spontaneous term labor we isolated circulating and choriodecidual leukocytes, performed microarray analysis (n = 5) and qRT-PCR validation (n = 9) and interaction network analysis with up-regulated genes. We found 270 genes up-regulated and only 17 genes down-regulated in choriodecidual leukocytes compared to maternal circulating leukocytes. The most up-regulated genes were CCL18, GPNMB, SEPP1, FN1, RNASE1, SPP1, C1QC, and PLTP. The biological processes enriched in choriodecidual leukocytes were cell migration and regulation of immune response, chemotaxis, and humoral immune responses. Our results show striking differences between the transcriptome of choriodecidual and maternal circulating leukocytes. Choriodecidual leukocytes are enriched in immune mediators implicated in the spontaneous process of labor at term.
Assuntos
Decídua/metabolismo , Trabalho de Parto/genética , Leucócitos/metabolismo , Transcriptoma , Adulto , Decídua/citologia , Feminino , Humanos , Trabalho de Parto/sangue , Trabalho de Parto/metabolismo , GravidezRESUMO
BACKGROUND AND AIMS: Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. METHODS: Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. RESULTS: After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. CONCLUSIONS: The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer.
Assuntos
Aptâmeros de Nucleotídeos/isolamento & purificação , Papillomavirus Humano 16 , Proteínas E7 de Papillomavirus/química , Neoplasias do Colo do Útero/virologia , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Extratos Celulares/química , Células Cultivadas , Feminino , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Mapeamento de Nucleotídeos , Ligação Proteica , Técnica de Seleção de Aptâmeros , Neoplasias do Colo do Útero/diagnósticoRESUMO
To determine the role of Dp71 in neuronal cells, we generated PC12 cell lines in which Dp71 protein levels were controlled by stable transfection with either antisense or sense constructs. Cells expressing the antisense Dp71 RNA (antisense-Dp71 cells) contained reduced amounts of the two endogenous Dp71 isoforms. Antisense-Dp71 cells exhibited a marked suppression of neurite outgrowth upon the induction with NGF or dibutyryl cyclic AMP. Early responses to NGF-induced neuronal differentiation, such as the cessation of cell division and the activation of ERK1/2 proteins, were normal in the antisense-Dp71 cells. On contrary, the induction of MAP2, a late differentiation marker, was disturbed in these cells. Additionally, the deficiency of Dp71 correlated with an altered expression of the dystrophin-associated protein complex (DAPC) members alpha and beta dystrobrevins. Our results indicate that normal expression of Dp71 is essential for neurite outgrowth in PC12 cells and constitute the first direct evidence implicating Dp71 in a neuronal function.
Assuntos
Distrofina/análogos & derivados , Distrofina/fisiologia , Neuritos/ultraestrutura , Células PC12/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , DNA Antissenso/farmacologia , Distrofina/genética , Humanos , Cinética , Proteínas Associadas aos Microtúbulos/análise , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/química , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/ultraestrutura , Neuropeptídeos/análise , Isoformas de Proteínas/análise , Ratos , TransfecçãoRESUMO
El cáncer es un proceso multifactorial y con múltiples etapas. Los protooncogenes, antioncogenes y virus oncogénicos están involucrados en el desarrollo de diversas neoplasias. La expresión alterada de los protooncogenes (por mutaciones, rearreglos o amplificaciones), así como la cooperación entre ellos puede llevar a la célula a una estado transformado. Los genes p53 y RB codifican para dos proteínas antioncogénicas que regulan las decisiones celulares de proliferación o diferenciación. La ausencia de RB (pérdida de ambos alelos) lleva a la liberación de factores de transcripción. p53 en su forma mutada, favorece el crecimiento celular. El cáncer cervicouterino ejemplifica claramente la intervención de este tipo de factores en su desarrollo. Los papilomavirus humanos genitales (PVH) estan implicados en su etiología como iniciadores de la proliferación celular. La inactivación de las proteínas antioncogénicas p53 y p105 RB porparte de los oncogenes virales E6 y E7, respectivamente, mantiene el estado de divición celular continua. Adicionalmente, la participación de ooncogenes expresados en forma alterada (c-myc) y otros cofactores contribuyen a modificar los periodos de latencia y la gravedad de la enfermedad
Assuntos
Fatores de Transcrição/genética , Genes Supressores de Tumor/genética , Oncogenes/genética , Papillomaviridae/genética , Papillomaviridae/ultraestrutura , Proto-Oncogenes/genética , Transformação Celular Viral/genética , Neoplasias do Colo do Útero/genética , Análise Mutacional de DNA , DNA Viral/ultraestrutura , Genes myc , Genes rasRESUMO
El cáncer cérvico uterino (CaCU) ocupa el 30 por ciento de todas las neoplasias de la mujer en México, resultando una de las principales causas de muerte por cáncer. En un estudio anterior en la ciudad de México, nuestro grupo reportó que sólo cinco de 16 muestras analizadas por transferencia Southern contenían secuencias virales integradas del papilomavirus humano tipo 16 (HPV16). En el presente trabajo, hemos extendido esta observación en un estudio comparativo de las ciudades de México, D!F!, y Monterrey, Nuevo León, incluyendo además otro tipo viral, el papilomavirus humano tipo 18 (HPV18), para determinar la frecuencia con la que se presentan en población mexicana los dos tipos de papilomavirus humanos más comunes en CaCU. Se determinó en primera instancia que la prevalencia de secuencias del HPV16 era similar en ambas poblaciones (4 de 14 para la ciudad de México y 6 de 23 para Monterrey); sin embargo, la presencia de secuencias de HPV18 fue aún más baja (una de 14 y una de 10, para las ciudades de México y Monterrey, respectivamente). En todos los tumores, las secuencias virales se encontraron integradas al genoma celular. Nuestros resultados muestran que existen una relativamente baja proporción de HPV16 Y 18 en tumores cervicales de población mexicana, sugiriendo que otros tipos de papilomavirus humanos o la presencia de un nuevo factor de riesgo (p.ej., activación de oncogenes) están involucrados en el desarrollo del CaCU en México.