Project description:We tested the hypothesis on the mechanisms responsible for the early control of monocytes function by identifying a discrete set of genes in circulating monocytes that were altered by exercise. Exercise leads to a rapid change in the profile of gene expression in monocytes. Twelve healthy men (22-30 yr old) performed 10 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to about 82% of VO2max. A baseline blood sample was taken before the and immediately after the exercise. Monocytes were isolated from PBMC using a negative magnetic cell separation methods (Miltenyi Biotec Monocyte Isolation Kit II human #130-091-153 and the autoMACS® Pro Separator). Total RNA was extracted using TRIzol. For this study we used Affymetrix plus 2 (total of 24 chips).
Project description:Exercise leads to a rapid change in the profile of gene expression in monocytes. We hypothesized that microRNA expression in circulating monocytes would also be affected by brief exercise. Twelve healthy men (22-30yr old) performed 10 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to about82% of VO2max. A baseline blood sample was taken before the and immediately after the exercise. Monocytes were isolated from PBMC using a negative magnetic cell separation methods (Miltenyi Biotec Monocyte Isolation Kit II, human #130-091-153 and the autoMACSM-BM-. Pro Separator). Total RNA was extracted using TRIzolM-BM-.. For this study we used Agilent Human miRNA microarrays V2 (total of 24 arrays).
Project description:We tested the hypothesis on the mechanisms responsible for the early control of NK cell function by identifying a discrete set of genes in circulating NK cells that were altered by exercise. Exercise leads to a rapid change in the profile of gene expression in NK cells. Twelve healthy men (20-29 yr old) performed 10 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to about 77% of VO2max. A baseline blood sample was taken before the and immediately after the exercise. NK cells were isolated from PBMC using a negative magnetic cell separation methods (Miltenyi Biotec Kit #130-092-657 and the autoMACSM-BM-. Pro Separator). Total RNA was extracted using TRIzolM-BM-.. For this study we used Affymetrix plus 2 (total of 24 chips).
Project description:The Ptf1a gene has essential functions during several stages of pancreas development. It is expressed in the naM-CM-/ve endoderm and required pancreas cell fate specification; it is also required later in the differentiation and maintenance of acinar cells. To identify the regulatory genetic program downstream of Ptf1a required for early pancreatic fate acquisition, we used microarrays to perform a comprehensive gene expression analysis of Ptf1a overexpressing endodermal tissue at NF32 and NF36. The results revealed an up-regulation on 1142 probe sets over 2-fold. Additional analyses, by in situ hybridizations, identified 9 genes that were endodermally expressed after the onset of endogenous Ptf1a; STXBP1, putative transmembrane protein TA-2, C25H, IGFBP1, IRF1, HALPN3, Hey1, sestrin 1, syndecan-4. These results provide insight into the regulatory network activated by Ptf1a during early pancreas development. In order to identify downstream targets of Ptf1a, two microarrays were performed at different time-points. The two microarrays compared control pancreatic tissue (GFP) and pancreatic tissue over-expressing Ptf1a (Ptf1a+GFP). The first microarray was performed at NF32, hence renamed MA32, 8 hours after the initial expression of endogenous Ptf1a. A second microarray was performed at NF36 (MA36), 8 hours after the first one. Ptf1a+gfp mRNA or gfp mRNA alone was injected into the two dorso-vegetal blastomeres of eight-cell embryos, targeting the anterior endoderm, and 40/48 hours later the anterior endoderm was dissected out. Approximated 15 endoderm explants were pooled for each RNA preparation, and both control and experimental samples were collected from the same batch of embryos; this was done in triplicate at NF32 and in quadruplicate at NF36 (Fig. 1A). For the NF32 microarray, we used the Affymetrix 3M-bM-^@M-^Y Xenopus laevis Genome 2.0 GeneChip, whereas for the NF36 microarray we used the Affymetrix 3M-bM-^@M-^Y Xenopus laevis Genome 1.0 GeneChip; the NF36 microarray was performed prior to the release of the 2.0 GeneChip.
Project description:Exercise leads to a rapid change in the profile of gene expression in NK cells. We hypothesized that microRNA expression in circulating NK cells would also be affected by brief exercise. Eleven healthy men (20-29 yr old) performed 10 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to about 77% of VO2max. A baseline blood sample was taken before the and immediately after the exercise. NK cells were isolated from PBMC using a negative magnetic cell separation methods (Miltenyi Biotec Kit #130-092-657 and the autoMACSM-BM-. Pro Separator). Total RNA was extracted using TRIzolM-BM-.. For this study we used Agilent Human miRNA microarrays V2 (total of 22 chips).
Project description:Dendritic cells (DCs) are major antigen-presenting cells that play a key role in initiating and regulating innate and adaptive immune responses. DCs are critical mediators of tolerance and immunity. The functional properties of DCs changes with age. The purpose of this study was to define the age-associated molecular changes in DCs by gene array analysis using Affymatrix GeneChips. We identified up and down-regulated gene expression changes in DC from aged donors compared to young donors. Total 9 MoDC RNA samples from four young donors (20-27 years) and five aged donors (77-84 years) were analyzed using Affymetrix HG-U133A_2 Gene Arrays to compare differential gene expression changes in MoDC between aged and young groups
Project description:Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition or EMT). Recent studies suggest that EMT endows differentiated epithelial cells with stem cell traits, posing the interesting question of how epithelial plasticity is properly restricted to ensure epithelial differentiation during tissue morphogenesis. Here we identify zinc-finger transcription factor Ovol2 as a key suppressor of EMT of mammary epithelial cells. Epithelia-specific deletion of Ovol2 completely arrests mammary ductal morphogenesis, and depletes epithelial stem/progenitor cell reservoirs. Further, Ovol2-deficient epithelial cells undergo EMT in vivo to become non-epithelial cell types, and that Ovol2 directly represses key EMT inducers such as Zeb1 and regulates stem/progenitor cell responsiveness to TGF-beta. We also provide evidence for a suppressive role of Ovol2 in breast cancer progression. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity to balance stemness with epithelial differentiation in development and cancer. TEBs from control and conditional Ovol2-knockout mammary glands were physically isolated for RNA extraction and hybridization on Affymetrix microarrays. In order to identify primary changes, we analyzed TEBs from 24-25-day-old mice, when morphological differences between control and Ovol2 SSKO were still minimal.
Project description:During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/2-deficient epidermal cells fail to undertake alpha-catenin–driven actin cytoskeletal reorganization and adhesive maturation, and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations as well as defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-a-catenin sequential repression and highlight novel functions of Ovol as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity. Skin from control and Ovol2 overexpression (Ovol2 BT) were physically isolated for RNA extraction and hybridization on Affymetrix microarrays. In order to identify primary changes, we analyzed skin from E16.5 mice, when morphological differences between control and Ovol2 overexpression were still minimal.
Project description:During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/2-deficient epidermal cells fail to undertake alpha-catenin–driven actin cytoskeletal reorganization and adhesive maturation, and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations as well as defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-a-catenin sequential repression and highlight novel functions of Ovol as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity. Isolated keratinocytes from control, Ovol1 knockout and Ovol1/2 double knockout were physically isolated for RNA extraction and hybridization on Affymetrix microarrays. In order to identify primary changes, we isolated the keratinocytes from mouse skin and allowed them to grow in culture for 2-5 days.