RESUMEN
EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.
Asunto(s)
Ciclo Celular/genética , Linaje de la Célula/genética , Células Asesinas Naturales/inmunología , Proteínas de Dominio T Box/genética , Animales , Secuencia de Bases , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Diferenciación Celular , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/inmunología , Epigénesis Genética/inmunología , Interleucina-12/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Unión Proteica , Bazo/citología , Bazo/inmunología , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/inmunología , Transcripción Genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
An ultrafast microfluidic PCR module (30 PCR cycles in 6 min) based on the oscillating fluid plug concept was developed. A robust amplification of native genomic DNA from whole blood samples could be achieved at operational conditions established from systematic investigations of key parameters including heat transfer and in particular flow velocities. Experimental data were augmented with results from computational fluid dynamics simulations. The reproducibility of the current system was substantially improved compared to previous concepts by integration of a closed reservoir instead of utilizing a vented channel end at ambient pressure rendering the devised module suitable for integration into complex sample-to-answer analysis platforms such as point-of-care applications.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Actinas/genética , Simulación por Computador , ADN/sangre , ADN/química , Diseño de Equipo , Humanos , Masculino , Sistemas de Atención de Punto , Reproducibilidad de los Resultados , TemperaturaRESUMEN
Ultraflat nanostructured substrates have been used as a template to create patterned solid-supported bilayer membranes with polymerizable tethered lipids acting as diffusion barriers. Patterns in the size range of 100 nm were successfully produced and characterized. The diffusion barriers were embedded directly into the phospholipid bilayer and could be used to control the fluidity of the membrane as well as to construct isolated membrane corrals. By using nanosphere lithography to structure the templates it was possible to systematically adjust the lipid diffusion coefficients in a range comparable to those observed in cellular membranes. Single colloids applied as mask in the patterning process yielded substrates for creation of isolated fluid membrane patches corralled by diffusion barriers. Numerous potential applications for this new model system can be envisioned, ranging from the study of cellular interactions or of molecular diffusion in confined geometries to biosensor arrays.
Asunto(s)
Membrana Dobles de Lípidos/química , Membranas Artificiales , Nanoestructuras/química , Nanotecnología/métodos , Aire , Difusión , Polimerizacion , Agua/químicaRESUMEN
Nanoparticle arrays created by nanosphere lithography are widely used in sensing applications since their localized surface plasmon resonances are extremely sensitive to changes in the local dielectric environment. A major drawback for any biologically oriented sensing application of conventionally produced particle arrays is the lack of stability of the nanoparticles in aqueous media and buffer solutions. Here, a robust and reusable nanoscale sensing platform based on localized surface plasmon resonances of gold nanoparticles embedded in a silicon dioxide matrix is presented. The architecture exhibits extremely high stability in aqueous environments and can be regenerated several times by simple mechanical cleaning of the surface. The platforms surface is ultraflat by design, thus making it an ideal substrate for any bio-oriented sensing application.