RESUMEN
Exotoxin A is one of the virulence factors of Pseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosing P. aeruginosa infections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant (Kd ) of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool.
Asunto(s)
ADN de Cadena Simple/sangre , Exotoxinas/sangre , Técnica SELEX de Producción de Aptámeros/métodos , Biomarcadores/sangre , Análisis Químico de la Sangre/métodos , ADN de Cadena Simple/genética , Exotoxinas/genética , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.
Asunto(s)
Toxinas Bacterianas/sangre , ADN de Cadena Simple/metabolismo , Staphylococcus aureus/metabolismo , Secuencia de Bases , ADN de Cadena Simple/química , Ensayo de Inmunoadsorción Enzimática , Humanos , Cinética , Conformación de Ácido Nucleico , Técnica SELEX de Producción de Aptámeros , Resonancia por Plasmón de SuperficieRESUMEN
Mediator complex is essential for transcription by RNA polymerase II in eukaryotes. Although chromatin remodeling is an integral part of transcriptional activation at many promoters, whether Mediator is required for this function has not been determined. Here we have used the yeast CHA1 gene to study the role of Mediator in chromatin remodeling and recruitment of the transcription machinery. We show by chromatin immunoprecipitation that Mediator subunits are recruited to the induced CHA1 promoter. Inactivation of Mediator at 37 degrees C in yeast harboring the srb4-138 (med17) ts mutation severely reduces CHA1 activation and prevents recruitment to the induced CHA1 promoter of Med18/Srb5, from the head module of Mediator, and Med14/Rgr1, which bridges the middle and tail modules. In contrast, recruitment of Med15/Gal11 from the tail module is unaffected in med17 ts yeast at 37 degrees C. Recruitment of TATA-binding protein (TBP) is severely compromised in the absence of functional Mediator, whereas Kin28 and polymerase II recruitment are reduced but to a lesser extent. Induced levels of histone H3K4me3 at the CHA1 promoter are not diminished by inactivation of Mediator, whereas recruitment of Paf1 and of Ser2- and Ser5-phosphorylated forms of Rbp1 are reduced but not eliminated. Loss of histone H3 from the induced CHA1 promoter is seen in wild type yeast but is greatly reduced by loss of intact Mediator. In contrast, Swi/Snf recruitment and nucleosome remodeling are unaffected by loss of Mediator function. Thus, Mediator is required for recruitment of the transcription machinery subsequent to chromatin remodeling during CHA1 induction.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , L-Serina Deshidratasa/biosíntesis , Complejos Multiproteicos/metabolismo , Regiones Promotoras Genéticas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Histonas/genética , Histonas/metabolismo , Calor , L-Serina Deshidratasa/genética , Complejos Multiproteicos/genética , Mutación , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Sir2 is a conserved deacetylase that modulates life span in yeast, worms, and flies and stress response in mammals. In yeast, Sir2 is required for maintaining replicative life span, and increasing Sir2 dosage can delay replicative aging. We address the role of Sir2 in regulating chronological life span in yeast. Lack of Sir2 along with calorie restriction and/or mutations in the yeast AKT homolog, Sch9, or Ras pathways causes a dramatic chronological life-span extension. Inactivation of Sir2 causes uptake and catabolism of ethanol and upregulation of many stress-resistance and sporulation genes. These changes while sufficient to extend chronological life span in wild-type yeast require severe calorie restriction or additional mutations to extend life span of sir2Delta mutants. Our results demonstrate that effects of SIR2 on chronological life span are opposite to replicatve life span and suggest that the relevant activities of Sir2-like deacetylases may also be complex in higher eukaryotes.
Asunto(s)
Histona Desacetilasas/fisiología , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuinas/fisiología , Adenilil Ciclasas/genética , Alcohol Deshidrogenasa/metabolismo , Reparación del ADN , Regulación hacia Abajo/genética , Etanol/metabolismo , Etanol/farmacología , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/genética , Histona Desacetilasas/genética , Calor , Peróxido de Hidrógeno/farmacología , Modelos Biológicos , Mutación/genética , Estrés Oxidativo , Proteínas Quinasas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2 , Sirtuinas/genética , Factores de Tiempo , Regulación hacia Arriba/genética , Vitamina K 3/farmacologíaRESUMEN
Aging is believed to be a nonadaptive process that escapes the force of natural selection. Here, we challenge this dogma by showing that yeast laboratory strains and strains isolated from grapes undergo an age- and pH-dependent death with features of mammalian programmed cell death (apoptosis). After 90-99% of the population dies, a small mutant subpopulation uses the nutrients released by dead cells to grow. This adaptive regrowth is inversely correlated with protection against superoxide toxicity and life span and is associated with elevated age-dependent release of nutrients and increased mutation frequency. Computational simulations confirm that premature aging together with a relatively high mutation frequency can result in a major advantage in adaptation to changing environments. These results suggest that under conditions that model natural environments, yeast organisms undergo an altruistic and premature aging and death program, mediated in part by superoxide. The role of similar pathways in the regulation of longevity in organisms ranging from yeast to mice raises the possibility that mammals may also undergo programmed aging.