RESUMEN
The human microbiome has begun to emerge as a potential forensic tool, with varied applications ranging from unique identification to investigative leads that link individuals and/or locations. The relative abundance of the combined DNA of the microbiome, compared to human nuclear DNA, may expand potential sources of biological evidence, especially in cases with transfer or low-copy number DNA samples. This work sought to determine the optimal swab type for the collection and analysis of microorganisms. A bacterium (Proteus mirabilis) was deposited by pipette onto four swab types (cotton, flocked, dental applicators, and dissolvable), and extraction and real-time PCR quantitation of the bacterial DNA were performed, which allowed for absolute microbial DNA recovery and comparison of yields across the four sampling substrates. Flocked swabs had the highest yield (~1240 ng) compared to the cotton swabs (~184 ng), dental applicators (~533 ng), and dissolvable swabs (~430 ng). The collection efficiency was further evaluated for cotton and flocked swabs using dried microbial samples spotted onto non-porous surfaces (treated wood, glass, plastic, and tile). Flocked swabs performed consistently better across wood, glass, and tile, but showed decreased recovery from plastic. The cotton swabs failed in the recovery of P. mirabilis DNA across all surfaces. Knowing the appropriate sampling substrate will be useful as others continue to investigate the use of the microbiome as a forensics tool.
Asunto(s)
Técnicas Bacteriológicas/instrumentación , ADN Bacteriano/análisis , Microbiota , Proteus mirabilis/aislamiento & purificación , Manejo de Especímenes/instrumentación , ADN Bacteriano/aislamiento & purificación , Humanos , Proteus mirabilis/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Retroalimentación Fisiológica , Proteínas de Homeodominio/metabolismo , Células Madre Multipotentes/citología , Miocardio/citología , Miocitos Cardíacos/citología , Proteína Smad1/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proteína Morfogenética Ósea 2 , Proliferación Celular , ADN Complementario , Embrión de Mamíferos , Corazón/embriología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Humanos , Proteínas con Homeodominio LIM , Ratones , Células Madre Multipotentes/metabolismo , Miocitos Cardíacos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Factores de Transcripción/genéticaRESUMEN
Cited2 is a transcriptional co-factor that is widely expressed in both embryonic and extraembryonic cells during early development. It is essential for embryonic development with Cited2 null embryos showing abnormal development of organs including heart, neural tube, adrenal glands, and placenta (both in trophoblast derivatives and invading fetal vasculature), as well as having defects in the establishment of the left-right body axis. We report the generation of two conditional null alleles allowing Cre-recombinase-mediated somatic cell gene inactivation. Mice heterozygous or homozygous for these alleles are viable and fertile. Crossing conditional mutants with CMV-Cre transgenic mice produces an embryonic-lethal phenotype in the offspring indistinguishable from germline null mutants. We also demonstrate that conditional deletion results in lacZ expression under the control of the Cited2 promoter. These alleles are therefore useful genetic tools for dissecting the functions of Cited2 in the formation of different organs and patterning of the developing embryo. genesis
Asunto(s)
Alelos , Proteínas de Unión al ADN/genética , Desarrollo Embrionario/genética , Proteínas Represoras/genética , Transactivadores/genética , Animales , Cartilla de ADN , Componentes del Gen , Silenciador del Gen , Ingeniería Genética/métodos , Vectores Genéticos , Integrasas , Ratones , Mutagénesis , Regiones Promotoras Genéticas/genética , beta-Galactosidasa/metabolismoRESUMEN
Homeodomain factor Nkx2-5 is a central component of the transcription factor network that guides cardiac development; in humans, mutations in NKX2.5 lead to congenital heart disease (CHD). We have genetically defined a novel conserved tyrosine-rich domain (YRD) within Nkx2-5 that has co-evolved with its homeodomain. Mutation of the YRD did not affect DNA binding and only slightly diminished transcriptional activity of Nkx2-5 in a context-specific manner in vitro. However, the YRD was absolutely essential for the function of Nkx2-5 in cardiogenesis during ES cell differentiation and in the developing embryo. Furthermore, heterozygous mutation of all nine tyrosines to alanine created an allele with a strong dominant-negative-like activity in vivo: ES cell<-->embryo chimaeras bearing the heterozygous mutation died before term with cardiac malformations similar to the more severe anomalies seen in NKX2.5 mutant families. These studies suggest a functional interdependence between the NK2 class homeodomain and YRD in cardiac development and evolution, and establish a new model for analysis of Nkx2-5 function in CHD.