RESUMEN
This study introduces a thermoplastic microdevice integrated with additive-enhanced allele-specific amplification and hydrazine-induced silver nanoparticle-based detection of single nucleotide polymorphism (SNP) and opportunistic pathogens. For point-of-care testing of SNP, an allele-specific loop-mediated isothermal amplification reaction using nucleotide-mismatched primers and molecular additives was evaluated to discriminate single-nucleotide differences in the samples. The microdevice consists of purification and reaction units that enable DNA purification, amplification, and detection in a sequential manner. The purification unit enables the silica-based preparation of samples using an embedded glass fiber membrane. Hydrazine-induced silver nanoparticle formation was employed for endpoint colorimetric detection of amplicons within three min at room temperature. The versatile applicability of the microdevice was demonstrated by the successful identification of SNPs related to sickle cell anemia, genetically-induced hair loss, and Enterococcus faecium. The microdevice exhibited a detection limit of 103 copies per µL of SNP targets in serum and 102 CFU mL-1 of Enterococcus faecium in tap water within 70 min. The proposed microdevice is a promising and versatile platform for point-of-care nucleic acid testing of different samples in low-resource settings.
Asunto(s)
Escherichia coli O157 , Nanopartículas del Metal , Plata , Colorimetría , Alelos , Escherichia coli O157/genética , Pruebas en el Punto de Atención , Técnicas de Amplificación de Ácido Nucleico , Hidrazinas , NucleótidosRESUMEN
While transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNALeu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNALeu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNALeu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNALeu-binding protein. We suspected that the overexpression of free tRNALeu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNALeu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNALeu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.
Asunto(s)
Neoplasias de la Mama/genética , ARN de Transferencia de Leucina/genética , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Aminoácidos/deficiencia , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación de la Expresión Génica , Células HEK293 , Células HT29 , Humanos , Células MCF-7 , Ratones , Células 3T3 NIH , Fosforilación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN de Transferencia de Leucina/antagonistas & inhibidores , ARN de Transferencia de Leucina/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de SeñalAsunto(s)
Flavobacterium/aislamiento & purificación , Meningitis/diagnóstico , Anciano , Aneurisma/cirugía , Antibacterianos/farmacología , Encefalopatías/cirugía , Craneotomía/efectos adversos , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Femenino , Infecciones por Flavobacteriaceae/etiología , Infecciones por Flavobacteriaceae/microbiología , Flavobacterium/clasificación , Flavobacterium/efectos de los fármacos , Humanos , Meningitis/microbiología , Pruebas de Sensibilidad Microbiana , Filogenia , Complicaciones Posoperatorias , Análisis de Secuencia de ADNRESUMEN
Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein.