Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90° and forward scatter measurements.

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxyuridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M-and G2-phase cells was obtained after 30–50 min heat treatment at 95°C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90° scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulselabeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrdunlabeled S-phase cells

Stromerzeugung aus Windenergie

Im Gegensatz zu der direkten Sonnenenergie ist die Windenergie eine indirekte Art der Sonnenenergie. Die Einstrahlung der Sonne erwärmt die Erdoberfläche und die darüber liegenden Luftschichten unterschiedlich – d. h., wegen ihrer niedrigen Wärmekapazität werden im Sommerhalbjahr die Kontinentalflächen bei Tag stärker erwärmt als die Ozeane. Dies bewirkt auf verschiedenen Gebieten der Erdoberfläche Dichte- und Druckunterschiede, die in fluktuierenden Luftströmungen ihren Ausgleich finden. Diese fluktuierenden Luftströmungen bzw.Winde können technisch durchWindenergieanlagen (WEA) genutzt werden, die in den strömenden Luftmassen enthaltene kinetische Energie in elektrische Energie umwandeln. Dabei wird die Energie des Windes über die Rotorblätter zunächst in mechanische Rotationsenergie und dann über einen Generator in elektrische Energie umgewandelt (Abb. 7.1)

Decreased expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4⁺ T cells and peripheral blood from tuberculosis patients.

The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4(+) T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4(+) T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease

Four miRNAs are differentially expressed in CD4⁺ T cells from tuberculosis patients and LTBI.

<p>Expression of miR-21 (triangles), miR-26a (squares), miR-29a (circles), and miR-142-3p (diamonds) in CD4⁺ T cells from tuberculosis patients (TB) (black symbols, n = 6), LTBI (grey symbols, n = 7), and PPD negative healthy controls (PPDneg) (open symbols, n = 3). Median expression of candidate miRNAs relative to the housekeeping gene RNU48 is shown. Significant differences are indicated as asterisks (* for p<0.05; ** for p<0.01, Mann-Whitney U-test).</p

Expression of candidate miRNAs in T_H1 and T_H17 clones.

<p>Expression of miR-21 (triangles), miR-26a (squares), miR-29a (circles), miR-142-3p (diamonds), and miR-155 (hexagons) was compared between IFNγ-expressing T_H1 (open symbols, n = 11) and IL-17-expressing T_H17 clones (black symbols, n = 9) of healthy donors prior to (<b>A</b>) and after <i>in vitro</i> activation (<b>B</b>). Expression of candidate miRNAs was normalized to the housekeeping gene RNU48. Median expression and range is shown. Significant differences are indicated as asterisks (* for P<0.05, Mann-Whitney-U test). (<b>B</b>) MiRNA expression after 12 h <i>in vitro</i> re-stimulation with PMA/Ionomycin. Relative expression to the respective non-activated control is shown (dotted line).</p

Target genes and overlaps for miR-21, miR-26a, miR-29a and miR-142-3p.

<p>(<b>A</b>) A Venn diagram indicates the overlap of target genes for miR-21, miR-26a, miR-29a and miR-142-3p. Common targets of at least three candidate miRNAs are listed by name. (<b>B</b>) Comparison between expression miR-21 (upper left graph), miR-26a (upper right graph), miR-29a (lower graph) and protein expression of the common target PTEN in lymphocytes from children with tuberculosis (n = 19) and LTBI (n = 3) is shown. We indicate relative miRNA expression to house keeping gene RNU48 by quantitative PCR and compare this to PTEN protein expression determined by flow cytometry using median fluorescence intensity (MFI) analysis. Each symbol represents data from an individual donor.</p

IFNγ and miR-29a expression of infected children and ectopic miR-29a suppression in T cells.

<p>(<b>A</b>) IFNγ expression after short-term <i>in vitro</i> re-stimulation of PBMC from children with tuberculosis (upper left graph) prior to chemotherapy (black symbols), three months under chemotherapy (grey symbols), after recovery (open symbols), as well as children with LTBI (upper right graph) with <i>M. tuberculosis</i>-specific PPD is shown. Proportions of IFNγ expressing CD4⁺ T cells (y-axis) for different time points after treatment onset of patients (x-axis) are depicted. Each symbol represents data from an individual patient with tuberculosis (hexagon) or LTBI (filled trigon). <i>Ex vivo</i> whole blood miR-29a expression (x-axis) in comparison to the proportions of IFNγ expressing CD4⁺ T cells (x-axis) for individual children with TB under therapy (circles) is shown in the lower graph. (<b>B</b>) Modulation of miR-29a expression (upper left graph) and control miR-16 (upper right graph) of CD4⁺ T cells by specific antagomirs (Homo sapiens (Hsa)-29a, Hsa-16, and Caenorhabditis elegans (Cel)-67) and effects on IFNγ expression (lower graphs) are depicted. IFNγ expression of activated CD4⁺ T cells after miR-29a or miR-16 suppression is shown as proportions of IFNγ⁺ cells (lower left graph) or as the IFNγ expression level per cell (indicated by median fluorescence intensities (MFI)) (lower right graph).</p

MiRNA candidate expression of peripheral blood cells from children with tuberculosis and LTBI.

<p>Expression of miR-21 (triangles), miR-26a (squares), miR-29a (circles), and miR-142-3p (diamonds) in whole blood is shown for children with tuberculosis (TB, black symbols), healthy latently <i>M. tuberculosis</i> children (LTBI, grey symbols), and PPD negative contacts (PPD_neg, open symbols) (<b>A</b>) as well as for children with tuberculosis under therapy and recovery (<b>B</b>). Each symbol indicates miRNA candidate expression for an individual donor relative to ‘housekeeping’ control RNU48. Significant differences are indicated as asterisks (* for P<0.05; ** for P<0.01, Mann-Whitney U-test). Exact P-values (Mann-Whitney U-test) are indicated for tendencies.</p