RESUMEN
The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell-derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.
RESUMEN
We performed next generation sequencing- and microarray-based gene expression profiling of CD44(+)/CD24(-)/CD45(-) breast CSCs (cancer stem cells) isolated from primary ERα-positive breast cancer. By combining semi-automated dissociation of human tumor tissue, magnetic cell sorting and cDNA amplification less than 500 CSCs were required for transcriptome analyses. Besides overexpressing genes involved in maintenance of stemness, the CSCs showed higher levels of genes that drive the PI3K pathway, including EGFR, HB-EGF, PDGFRA/B, PDGF, MET, PIK3CA, PIK3R1 and PIK3R2. This suggests that, in CSCs of ERα-positive breast cancer, the PI3K pathway which is involved in endocrine resistance is hyperactive.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Estrógenos , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/fisiología , Neoplasias Hormono-Dependientes/patología , Técnicas de Amplificación de Ácido Nucleico/métodos , Fosfatidilinositol 3-Quinasas/fisiología , Neoplasias de la Mama/enzimología , Antígeno CD24/análisis , Carcinoma Ductal de Mama/enzimología , Receptor alfa de Estrógeno/análisis , Femenino , Humanos , Receptores de Hialuranos/análisis , Separación Inmunomagnética , Inmunofenotipificación , Isoenzimas/fisiología , Proteínas de Neoplasias/genética , Neoplasias Hormono-Dependientes/enzimología , Células Madre Neoplásicas/química , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , ARN Mensajero/genética , ARN Neoplásico/genética , Sensibilidad y Especificidad , TranscriptomaRESUMEN
The differentiation of CD4(+) or CD8(+) T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4(+) or CD8(+) T cell differentiation. Surprisingly, our data revealed that while CD4(+) and CD8(+) T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4(+) and CD8(+) T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Perfilación de la Expresión Génica , Subgrupos de Linfocitos T/inmunología , Timo/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Línea Celular , Linaje de la Célula/inmunología , ADN Complementario/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Timo/citologíaRESUMEN
Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls expression of genes involved in nitrogen metabolism including urease and nitrogen fixation genes. The ntrY-ntrX genes, which are located immediately downstream of the nifR3-ntrB-ntrC operon, code for a two-component system of unknown function. Transcription of ntrY starts within the ntrC-ntrY intergenic region as shown by primer extension analysis, but maximal transcription requires, in addition, the promoter of the nifR3-ntrB-ntrC operon. While ntrB and ntrY single mutant strains were able to grow with either urea or N2 as sole nitrogen source, a ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use urea or N2. These findings suggest that the histidine kinases NtrB and NtrY can substitute for each other as phosphodonors towards the response regulator NtrC.