RNA-seq heat map revealing endogenous gene expression patterns during cardiomyogenesis induced with CHIR alone.

<p>Pluripotent H1 ESCs expanded on Matrigel in mTeSR1 medium were induced to differentiate by changing the medium to RPMI/B27 (without insulin) including the small MW inhibitors CHIR (12 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5; insulin (4,000 ng/ml) was included after Day 7. RNA was purified from duplicate cultures on the indicated days, converted to cDNA, and processed to RNA-seq libraries as described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118670#pone.0118670.s011" target="_blank">S1 Methods</a></b>. Colors indicate the range of each gene’s expression during the 14 day period, with least expression shown in green and highest expression shown in red (see inset). The number in each panel indicates the expression of each gene in transcripts per million (TPM) within each culture dish.</p

Wnt-modulated cardiomyogenesis is enhanced by Matrigel overlay.

<p>Pluripotent H1 ESCs expanded on Matrigel in mTeSR1 medium were treated with Matrigel overlays on Day -3 or -1. Differentiation was induced by changing medium to RPMI/B27 (no insulin) containing the small MW inhibitors CHIR (12 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5; insulin (4,000 ng/ml) was included after Day 7. <b>A</b>, scheme of cardiomyogenic induction using Matrigel overlay and small MW inhibitors; arrows denote days when medium was changed, before (green) and after (red) induction. <b>B</b>, typical flow cytometry results showing percentages of cardiac troponin-T (cTnT)-positive cells at Day 14. <b>C</b>, αMHC (MF20) immunostaining at Day 14. <b>D</b>, bar graph showing averaged flow cytometry results obtained in three independent determinations. Vertical lines depict ± SEM. Size bars in C = 200 μm. The p-value in D was calculated using Student’s t-test.</p

Activin-A levels during Day 0–1 modulate CM vs. DE differentiation.

<p>Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. <b>Panel A</b>, a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); <b>e</b> is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. <b>Panel A f-i</b> shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. <b>Panel B</b> depicts the effect of Activin-A levels during Day 0–1 on cardiomyocyte differentiation at Day 14, determined by flow cytometry using anti-cTnT. Cultures treated with 10 ng/ml Activin-A began to rhythmically contract at Day 6. Cells treated with 50 or 100 ng/ml Activin-A did not beat at any time. Bars indicate the average values combined from multiple experiments. Vertical lines = ±SEM. P-values were calculated by Student’s t-test. The p-value over the bar denoting 10 ng/ml Activin-A is relative to cells treated with CHIR only (0 ng/ml Activin-A), whereas the p-values over the bars denoting 50 and 100 ng/ml Activin-A are relative to cells treated with 10 ng/ml Activin-A. The size bar in Aa, which pertains to panels a-i, = 200 μm.</p

Spontaneous Development of Written Literacy

Poměrně delší dobu sleduji, jak děti zacházejí s písmem a jak se mu věnují, ať už se jedná o děti předškolní nebo školou povinné, ať už je dítě ve škole nebo doma a na školu se připravuje, nebo je mimo ni, v družině, v zájmovém kroužku, na táboře, na škole v přírodě, u prarodičů nebo s kamarády. Ve všech těchto a mnoha dalších situacích se často děti v tomto věku věnují psaní záměrně i nezáměrně, hrou nebo "prací", tedy činností, která je podmíněna svým dokončením (školní učební látka). Středem mého zájmu je vztah dítěte ve vývojovém období školní zralosti1 a jeho spontánního psaní jako komunikativní dovednosti ve vztahu k výchovněvzdělávacímu působení. Komunikativní psaní je součástí vzdělávací oblasti Český jazyk a literatura2 a je obsaženo ve všech jejich částech: Komunikační a slohové výchově, Jazykové výchově i Literární výchově

Tip60-Haploinsufficiency Augments 4-OHT-Induced Induction of Cell-Cycle Activity in MycER Transgenic Hearts.

<p>Eight week old MycER transgenic Tip60 wild-type (WT) and heterozygous (Het) mice were induced with 4-OHT for seven days and assessed for cell-cycle activity by immunostaining phosphorylated histone H3 (H3P; arrows in <b>A</b>,<b>B</b>). This was verified by immunostaining BrdU-incorporated nuclei (arrows in <b>D</b>,<b>E</b>). Percentages of labeled cells were determined by evaluating at least 5,000 (<b>C</b>) or 2,500 (<b>F</b>) hematoxylin-stained nuclei for H3P or BrdU antigen, respectively. (N)  =  number of hearts; vertical bars  =  ±SEM; p-values were calculated using Student's t-Test (two-tailed, unpaired). The scale bar in all images  =  10 µm. (Note: A control utilizing WT-MycER mice demonstrated that 4-OHT had no effect on these parameters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031569#pone.0031569-Xiao1" target="_blank">[26]</a>).</p

Increased Cell-Cycle Activity in Tip60^+/− Neonatal Cardiomyocytes.

<p>Cardiomyocytes were isolated from 2 day-old neonatal hearts and cultured on gelatin-coated dishes. When the cells were ∼60% confluent (72 hrs later) they were double-immunostained for phosphorylated histone-H3 (H3P) to detect M-phase cells (arrows in <b>A</b> & <b>C</b>) and for sarcomeric α-actin (not shown) to verify cardiomyocyte identity. Nuclei were stained with DAPI (<b>B</b>,<b>D</b>); H3P-labeled nuclei are encircled because DAPI is obscured DAB-stained nuclei. E shows percentages of H3P-positive neonatal cardiomyocytes, based on enumerating 1,000-2,000 cells in each dish; error bars  =  +/−SEM. Scale bars in <b>A</b>–<b>D</b> = 10 µm.</p

Over-Expression of Tip60β, but not Tip60α, Inhibits Cell Proliferation in NIH/3T3 Cells.

<p>Cultured NIH/3T3 cells were transfected with plasmid p3xFLAG-CMV-7.1 (empty vector), or with the same vector containing cDNA encoding either Tip60α or Tip60β. <b>A</b> shows the average cell number in each well of a 12-well plate, five days after transfecting the cells with plasmid. Error bars  =  ±SEM; p-values were calculated using Student's t-test (two-tailed, unpaired). <b>B</b> shows western blots confirming exogenous expression of Tip60α and Tip60β isoproteins.</p

Most H3P-Positive Nuclei are in Cardiomyocytes.

<p>Sections from hearts processed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031569#pone-0031569-g005" target="_blank">Figure 5</a> were double-immunostained for phosphorylated H3P and Nkx2.5 to determine cardiomyocyte identity. <b>A</b> & <b>D</b> show Nkx2.5 staining (nucleus-specific brown DAB reaction product) in cardiomyocyte nuclei, as distinct from smaller non-myocyte nuclei in which only (blue) hematoxylin counter-stain is seen. <b>B</b> & <b>E</b> show H3P fluorescent green signal in the same nuclei. <b>C</b> and <b>F</b> are merged images of A–B and D–E, respectively. In <b>C</b> & <b>F</b>, dark arrows denote nuclei double-stained for Nkx2.5 and H3P; white arrows denote nuclei expressing only Nkx2.5. <b>G</b> summarizes results from enumerating a minimum of 150 H3P-positive nuclei per heart for co-localization of Nkx2.5. Error bars  =  ±SEM; statistical significance was determined by Student's t-Test (two-tailed, unpaired). Scale bars  = 20 µM.</p

Tip60-Haploinsufficiency Increases 4-OHT-Induced Expression of Cyclin D2.

<p>Eight week old MycER transgenic Tip60 wild-type (WT) or heterozygous (Het) mice were induced with 4-OHT for seven days and assessed for cyclin D2 by western blotting. <b>A</b> is a western blot showing cyclin D2 and GAPDH levels in non-stressed (no 4-OHT) and stressed (+4-OHT) WT and Het myocardium. Each lane contained 10 µg total protein from separate hearts. <b>B</b> shows densitometry of the bands in <b>A</b>, with cyclin D2 normalized to GAPDH. Numbers (N) in parentheses indicate numbers of hearts evaluated. Vertical bars  =  ±SEM; p-values were calculated using Student's t-Test (two-tailed, unpaired).</p

Tip60 Expression in Adult Organs.

<p>Tissues from 14 week-old adult Tip60^+/+ and Tip60^+/− mice were processed for (<b>A</b>) semi-quantitative RT/PCR to assess transcript levels (of bulk Tip60 isoforms) in WT and Het tissues and (<b>B</b>) Western blotting to determine Tip60 protein levels. The primer pair used for RT/PCR (<b>A</b>) detected transcripts encoding both Tip60α and Tip60β; GAPDH was assessed as loading control. The bar graph in the lower part of <b>B</b> shows densitometric analysis of protein bands assessed from duplicate animals, normalized to GAPDH and expressed as a percentage of the most abundant band (in WT heart); vertical bars  =  range. +/+  =  WT; +/−  =  Het.</p