Деякі проблеми використання тимчасово зайнятих земель

<div><p>Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype <em>Yersinia enterocolitica</em> which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different <em>Yersinia</em> mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, <em>Clostridium difficile</em> toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal <em>GILZ</em> promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the <em>GILZ</em> promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of <em>GILZ</em> promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.</p> </div

Human and mouse BSCs express markers of nonhomologous end joining.

<p>(A) Immunohistochemistry for RAD51, an early marker of homologous recombination, on WT mouse trachea and lung 1 h post γ-irradiation (6 Gy). The insert is a positive control, a mammary tumour from a MMTV-cre;Brca1^fl/flp53^+/- mouse. Black arrows indicate RAD51-positive nuclei. Representative images from <i>n</i> = 3 mice at each time point. Scale bar = 100 μm. (B) Expression of key genes in the NHEJ repair pathway in human BSCs and AT2 cells. <i>n</i> = 3 patients (a 64-y-old male exsmoker, an 83-y-old male exsmoker, and a 53-y-old male current smoker). RPKM, reads per kilobase per million mapped reads. Paired <i>t</i> test. (C) Immunofluorescence staining of phospho-DNA-PKcs and T1α in human airways and alveoli of three patients. Patient 1, a 56-y-old male smoker; patient 2, a 69-y-old female exsmoker; patient 3, a 70-y-old male smoker. Inset, isotype control. Scale bar = 20 μm. (D) Immunofluorescence staining of phospho-DNA-PKcs and T1α in trachea and lung of WT mice following IR (6 Gy). Representative images of one of <i>n</i> = 3 mice at each time point. Inset, isotype control. Scale bar = 20 μm. The underlying data for panel B can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

Human lung BSCs are the putative cells of origin of lung squamous cell carcinoma.

<p>(A) Representative histogram of intracellular DAPI staining of BSCs and AT2 cells isolated from a 56-y-old male exsmoker patient. Gates indicate 2N, 4N, and polyploid cells. (B) Proportion of polyploidy cells in the BSC and AT2 subsets. <i>n</i> = 3 patients (a 69-y-old female exsmoker, a 56-y-old male exsmoker, and a 70-y-old female exsmoker). Paired <i>t</i> test. (C) Boxplots of human lung BSC expression scores by lung tumour subtypes (ADC, adenocarcinoma; SCLC, small cell lung cancer; SqCC, squamous cell carcinoma). The width of each box indicates the sample size. (D) Barcode plot showing strong correlation of the human lung BSC expression signature with that of SqCCs (ROAST <i>p</i> = 0.0001). Genes are sorted left to right from most up- to most down-regulated in SqCC relative to all other cancer subtypes. Positive BSC signature genes are marked with vertical red bars, and negative signatures genes are marked in blue. Variable-height bars show log-fold-change strength for each signature gene. (E) Fold changes in the expression of genes frequently altered in lung SqCC between human BSCs and other human lung epithelial cell types. <i>n</i> = 3 patients (a 64-y-old male exsmoker, an 83-y-old male exsmoker, and a 53-y-old male current smoker). (F) Violin plots showing expression levels of <i>PRKDC</i> and <i>XRCC6</i> in normal lung tissue (<i>n</i> = 54), lung ADCs (<i>n</i> = 125), and lung SqCCs (<i>n</i> = 224) from The Cancer Genome Atlas (TCGA). Violin bodies show log2 counts per million (log CPM) expression values as smoothed densities. All pairwise <i>p</i>-values are <10⁻⁶ by moderated <i>t</i> tests. (G) Proportion of genome altered versus <i>PRKDC</i> and <i>XRCC6</i> expression levels in TCGA lung SqCC data (<i>n</i> = 179). Expression levels are split into quartiles. Significance was determined by Student’s <i>t</i> tests. The underlying data for panels A, B, C, D, E, and G can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

Mouse tracheal BSCs are less sensitive than alveolar cells to DNA damage-induced cell death.

<p>(A) Barcode plots showing strong correlation of mouse and human expression signatures. The upper panel correlates the mouse tracheal BSC signature with that of human lung BSCs; the lower panel correlates the mouse alveolar cell signature with that of human AT2 cells. For each plot, genes are ordered left to right from most up-regulated to most down-regulated in human BSCs or AT2 cells, relative to all other cell populations. Positive mouse signature genes are marked with red vertical bars, and negative mouse signature genes with blue. Variable-height vertical bars show log-fold changes for the mouse signature genes. Worms show relative enrichment of mouse genes in the human ranked list. Rotation gene set tests give <i>p</i> = 0.0001 for each plot. (B) Immunofluorescence staining of γH2AX and T1α in alveoli or trachea of wild-type (WT) mice that are either nonirradiated or 1, 4, 8, 24, or 96 h post irradiation (6 Gy, top panel) or saline injected or 1, 4, 8, 24 or 96 h post bleomycin injection (40 mg/kg intravenously, bottom panel). T1α marks alveolar type 1 cells in the lung and BSCs in the trachea. Representative images of <i>n</i> = 3 mice at each time point. Scale bar = 20 μm. (C) FACS detection of dead cells in lung alveolar (EpCAM⁺) or tracheal BSCs (T1α⁺) isolated from WT mice 24 h post irradiation (6 Gy). <i>n</i> = 4–6 mice per group. Student’s <i>t</i> test. (D) Immunohistochemistry staining of Ki67 in alveoli or trachea of WT mice control or 4 h, 8 h, or 24 h post irradiation (6 Gy). Representative images for one of <i>n</i> = 3 mice at each time point. Scale bar = 20 μm. The underlying data for panel C can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

BSCs use error-prone nonhomologous end joining to repair DNA double-strand breaks.

<p>(A) Immunofluorescence staining of γH2AX and T1α in the lungs and tracheas of WT and SCID^<i>prkdc</i> mice that are nonirradiated or 1, 4 or 8 h post irradiation (6 Gy). Representative images of <i>n</i> = 3 mice at each time point. Arrows indicate γH2AX⁺ T1α⁺ BSCs. Scale bar = 20 μm. (B) Representative FACS plots showing the expression of γH2AX in EpCAM⁺ lung epithelial cells and T1α⁺ tracheal BSCs in WT and SCID^<i>prkdc</i> mice 0, 4, and 7 h following IR (6 Gy). The timing corresponds to the number of hours between time of irradiation and generation of single-cell suspension for FACS analysis. (C) Percentage of γH2AX-positive cells in WT and SCID^<i>prkdc</i> mice in EpCAM⁺ lung epithelial cells and T1α⁺ tracheal BSCs 0, 4, and 7 h following irradiation. <i>n</i> = 6 animals per group. Student’s <i>t</i> test. The timing corresponds to the number of hours between time of irradiation and generation of single-cell suspension for FACS analysis. (D) Immunofluorescence staining of cleaved caspase 3 (CC3), T1α, and 4′,6-diamidino-2-phenylindole (DAPI) in WT and SCID^<i>prkdc</i> tracheas that are nonirradiated or 4, 24, or 96 h post irradiation (6 Gy). Representative images of <i>n</i> = 3 mice at each time point. Arrows indicate CC3⁺ T1α⁺ BSCs. Scale bar = 20 μm. (E) FACS detection of cells in subG1 in tracheal BSCs (T1α⁺) cells isolated from WT or SCID^<i>prkdc</i> mice 24 h post irradiation (6 Gy). <i>n</i> = 7 mice for WT mice and <i>n</i> = 12 for SCID^<i>prkdc</i> mice. Student’s <i>t</i> test. The underlying data for panels C and E can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

Human lung BSC and AT2 populations have progenitor activity.

<p>(A) Representative images of colonies grown from lung epithelial subsets in a 3-D in vitro assay; a 64-y-old male current smoker. Black scale bar = 500 μm; white scale bar = 100 μm. (B) Representative immunofluorescence staining of keratin 5 (KRT5) (a 57-y-old male exsmoker) and pro-surfactant protein C (proSFTPC) (a 54-y-old female exsmoker) in BSC and AT2 colonies. <i>n</i> = 13 patients; 39–78 y old; current, ex-, and never smokers. Scale bar = 100 μm. (C) Colony-forming capacity of sorted lung epithelial cells; <i>n</i> = 24 patients for proximal lung samples and <i>n</i> = 27 patients for distal lung samples; 21–85 y old; male and female; never, ex-, and current smokers. Student’s <i>t</i> test. (D) Representative images of human BSC and AT2 cell colonies from an exsmoker patient (a 57-y-old male) compared to a never-smoker patient (a 71-y-old female). Scale bar = 500 μm. (E) Linear regression analysis of the number of human BSCs (r² = 0.2) or AT2 colonies (r² = 0.3) versus number of years of patient tobacco smoking. <i>n</i> = 21 patients for basal colonies and <i>n</i> = 23 patients for AT2 colonies, 21–83 y old, male and female. The underlying data for panels C and E can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

Human lung BSCs sustain less DNA damage than alveolar progenitor cells.

<p>(A) Multidimensional scaling plot of expression profiles of human lung epithelial subsets from three patients (a 64-y-old male exsmoker, an 83-y-old male exsmoker, and a 53-y-old male current smoker). Distances represent the leading log2-fold change. (B) Gene ontology (GO) terms associated with DNA repair or the cell cycle are significantly up-regulated in BSCs compared to AT2 cells by rotation gene set tests (ROAST) (<i>p</i> < 0.02). Each pair of bars corresponds to a relevant (GO) term. Bars show the proportion of genes associated with the GO term that are more highly expressed in BSCs (orange) or in AT2 cells (blue), as determined by limma’s roast function. (C) Expression of key genes in the DNA repair pathway in BSCs relative to AT2 cells. RPKM, reads per kilobase per million mapped reads. <i>n</i> = 3 patients; a 64-y-old male exsmoker, an 83-y-old male exsmoker, and a 53-y-old male current smoker. Paired <i>t</i> test. (D) Immunofluorescence staining of γH2AX (green) and T1α (purple) in whole human lung fragments that are nonirradiated (control) or 1 or 24 h post irradiation (6 Gy). <i>n</i> = 2 patients (a 60-y-old female never smoker and a 78-y-old female never smoker). Scale bar = 20 μm. The underlying data for panels A, B, and C can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

Ganetespib does not suppress basal circulating blood neutrophils but inhibits LPS induced mobilization without causing neutrophil apoptosis.

<p>Balb/c mice were treated with ganetespib (100 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse tn). <b>(A)</b> 24 h post-LPS peripheral blood counts and blood smears were obtained. Smears were manually differentiated into neutrophils, monocytes and lymphocytes. Total leukocytes were quantified and differentiated using standard morphological criteria of fixed and stained blood smears. Data are mean ± SEM. n = 6–8 mice per group (n = 3 for ganetespib alone). Note that ganetespib did not significantly reduce basal neutrophil numbers but suppressed mobilization post-LPS. <b>(B)</b> Kinetic profile of the effect of ganetespib on neutrophil survival as assayed by live cell imaging. Neutrophils were treated with ganetespib, (concentrations as shown) or 10 µM SN-38 (positive control), for 15mins before addition of 10ng/mL G-CSF or GM-CSF. Data points represent means +/- SEM of three independent samples. p<0.001 for SN-38 vs DMSO from 8h in G-CSF and GM-CSF-treated cells. p<0.05 for 250nM GIB vs DMSO at 24h in G-CSF-treated cells. p< 0.05 for 62.5, 250nM, 1000nM GIB vs DMSO from 19h in GM-CSF-treated cells.</p

Ganetespib inhibits net gelatinase and MMP-9 gelatinase activity in BALF.

<p>Balb/c mice were treated with Ganetespib (50 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse, tn). <b>(A)</b> 102–105 kDa (size markers not shown) MMP9 gelatinase activity of pooled BALF samples determined by gelatin zymography. Left panel, original lucent bands revealed on post-stained gel; right panel corresponding band densitometry in arbitrary units (image pixels). Pooled samples, no statistics. <b>(B)</b> Net gelatinase activity in unfractionated whole BALF collected 3 and 24 h post LPS was determined in individual mice by fluorogenic substrate assay. Data are mean ± SEM. n = 8 mice per group. (naïve; n = 4). *** p<0.001 compared to LPS/vehicle treated mice.</p

Effect of Ganetespib on cellular inflammation and mediator levels and transcripts.

<p><b>(A)</b> Balb/c mice were treated with Ganetespib (50 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse tn). BALF was collected at 3 and 24 h post-LPS and total cells/mL were counted and cellular composition was determined by manual counting microscopy using standard morphological criteria. Data are mean ± SEM. n = 8 mice per group for treatments, n = 4 naïve control. ** p < 0.01, *** p < 0.001 compared to LPS/vehicle treated mice. <b>(B)</b> Balb/c mice were treated with Ganetespib (30 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse, tn). ELISA data for TNFα, MIP-2, IL-6 and KC in BALF obtained at 3 and 24 h (LOD 15.6 pg/ml). Data are mean ± SEM. n = 8 mice per group and n = 4 naïve control. * p<0.05, *** p<0.001 compared to LPS/vehicle treated mice. <b>(C)</b> Relative mRNA transcript abundance for a panel of inflammatory mediators measured in pooled lung tissue (n = 2–3 mice per group) collected 3 h post LPS. Expression is normalized to naïve control levels. Pooled samples, no statistics.</p