HUBUNGAN ANTARA KEAKTIFAN MENGIKUTI EKSTRAKURIKULER PENDIDIKAN KEPRAMUKAAN DENGAN KEDISIPLINAN SISWA KELAS TINGGI SEKOLAH DASAR NEGERI 69 KOTA BENGKULU

Penelitian ini bertujuan untuk mengetahui hubungan antara keaktifan mengikuti ekstrakurikuler pendidikan kepramukaan dengan kedisiplinan siswa kelas tinggi Sekolah Dasar Negeri 69 Kota Bengkulu. Jenis penelitian ini adalah penelitian kuantitatif. Populasi dalam penelitian ini adalah seluruh siswa kelas tinggi Sekolah Dasar Negeri 69 Kota Bengkulu yang berjumlah 291 siswa. Sampel dalam penelitian ini adalah 25% dari jumlah populasi yaitu 102 siswa. Dengan teknik pengumpulan data menggunakan angket. Instrumen penelitian diuji dengan menggunakan uji validitas dan uji reliabilitas. Sedangkan uji prasyarat analisis dengan menggunakan uji normalitas dan uji homogenitas. Teknik analisis data yang digunakan adalah teknik analisis korelasi Pearson Product Moment. Teknik ini digunakan untuk mengetahui signifikasi hubungan antara variabel bebas dan variabel terikat dengan membandingkan rhitung dengan rtabel dengan taraf signifikan 5%. Berdasarkan hasil analisis data diperoleh nilai rhitung = 0,98 > rtabel = 0,195 yang artinya terdapat hubungan yang berarti antara keaktifan mengikuti ekstrakurikuler pendidikan kepramukaan dengan kedisiplinan siswa dengan interpretasi sangat kuat. Selanjutnya, dari hasil uji signifikansi diperoleh thitung= 49,25 ≥ 1,658 yang artinya terdapat hubungan yang signifikan antar variabel yang berlaku untuk seluruh populasi, dengan konstribusi sebesar 96,04% antara keaktifan mengikuti ekstrakurikuler pendidikan kepramukaan dengan kedisiplinan siswa kelas tinggi Sekolah Dasar Negeri 69 Kota Bengkulu. Berdasarkan hal tersebut, dapat disimpulkan bahwa Ha diterima, dimana terdapat hubungan antara keaktifan mengikuti ekstrakurikuler pendidikan kepramukaan dengan kedisiplinan siswa kelas tinggi Sekolah Dasar Negeri 69 Kota Bengkulu

Intracellular trafficking of O157 OMVs.

<p>(A) CLSM of HBMEC preincubated with OMVs 5791/99 for 30 min at 4°C, and postincubated at 37°C for 30 min to 20 h. The indicated single fluorescence channels are shown in the right panels (enlargements are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s012" target="_blank">S12</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s013" target="_blank">S13</a> Figs) and the merged images in the left panels (green, OMVs; red, compartment-specific marker proteins; blue, nuclei; yellow, colocalized green and red signals). The percentages of OMV colocalizations with compartment-specific marker proteins (white numbers) and with nucleus (blue numbers in panels ER) were calculated with the BioImageXD6 tool. Scale bars are 10 μm. (B) Graphical summary of CLSM data shown in A. (Means of colocalizations from at least five different samples are shown in A and B; for standard deviations and significance analysis see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s008" target="_blank">S8B Fig</a>). (C) Immunoblot detection of OMVs (anti-OmpA antibody) in isolated subcellular fractions of HBMEC which were postincubated for the times indicated with 5791/99 OMVs or for 72 h without OMVs or with TRIS-HCl OMV buffer (negative controls); 5791/99 OMVs without cells (OMV ctrl) were a positive control. (D) Densitometric quantification of OmpA lysosomal signals shown in C. Ee, early endosomes; Le/Lyso, late endosomes/lysosomes; ER, endoplasmic reticulum; Mito, mitochondria; Nucl, nucleus; Cyto, cytosol; DU, densitometric unit.</p

OMV-associated virulence proteins differentially separate from OMVs during intracellular trafficking.

<p>(A) CLSM of HBMEC exposed to 5791/99 OMVs for 30 min at 4°C (OMV binding; panels 0 min) followed by 15 min to 20 h at 37°C (OMV internalization). The indicated single fluorescence channels are shown in the right panels (enlargements are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s009" target="_blank">S9</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s010" target="_blank">S10</a> Figs) and the merged images in the left panels (red, OMVs; green, virulence factors; blue, nuclei; yellow, colocalized red and green signals). The percentages of colocalizations between OMVs and virulence proteins were determined with the BioImageXD6 tool (white numbers). Scale bars are 10 μm. (B) Graphical summary of colocalizations of virulence factors with OMVs during time based on CLSM data shown in A. (Means of colocalizations from at least five different samples are shown in A and B; standard deviations and significance analysis see in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s008" target="_blank">S8A Fig</a>).</p

Retrograde transport of OMV-delivered CdtV-B does not require CdtV-A and CdtV-C.

<p>HBMEC were preincubated (30 min, 4°C) with recombinant CdtV-B-containing OMVs from strain BL21(<i>cdt</i>V-<i>B</i>) or with CdtV-B-lacking OMVs from BL21(pET23) vector control and postincubated at 37°C for 90 min to 20 h. (A) OMV uptake was determined by CLSM after 4 h. Green, OMVs; red, actin; blue, nuclei. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. (B, C) Trafficking of BL21(<i>cdt</i>V-<i>B</i>) OMVs and OMV-delivered CdtV-B, and separation of CdtV-B from the vesicles during the trafficking were determined by CLSM. (B) Colocalization of CdtV-B with OMVs upon OMV cellular binding (time 0 min). (C) Colocalizations of BL21(<i>cdt</i>V-<i>B</i>) OMVs with late endosomes/lysosomes (panels OMV/Le-Lyso), of CdtV-B with OMVs (panels CdtV-B/OMV), and of CdtV-B with the indicated subcellular compartments after postincubation at 37°C for 90 min to 20 h. (D) CLSM of control cells postincubated for 20 h with BL21(pET23) OMVs. The indicated single fluorescence channels in B to D are shown in the right panels (enlargements are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s029" target="_blank">S29</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s030" target="_blank">S30</a> Figs) and the merged images in the left panels (green, OMV or CdtV-B, as indicated; red, OMV or compartment-specific marker proteins, as indicated; blue, nuclei; yellow, colocalized green and red signals). The percentages of colocalizations of the respective signals (white numbers) were calculated with the BioImageXD6 tool; blue numbers in panels ER indicate colocalization of CdtV-B with nucleus (means of colocalizations from three different samples are shown). Scale bars in A to D are 10 μm. (E) Immunoblot detection of CdtV-B in isolated subcellular fractions of HBMEC which were postincubated with the indicated OMVs for 90 min to 20 h. BL21(<i>cdt</i>V-<i>ABC</i>) OMVs were a positive control and BL21(<i>cdt</i>V-<i>ACΔB</i>) OMVs, BL21(pET23) OMVs and untreated cells (no OMV) negative controls; lanes OMV ctrl contain 5791/99 OMVs without cells. (F) Densitometric quantification of CdtV-B signals shown in E. For abbreviations see legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.g004" target="_blank">Fig 4</a>.</p

Intracellular trafficking of OMV O157-delivered Stx2a.

<p>(A) CLSM of HBMEC preincubated with OMVs 5791/99 for 30 min at 4°C, and postincubated at 37°C for 30 min to 20 h. The indicated single fluorescence channels are shown in the right panels (enlargements are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s015" target="_blank">S15</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s016" target="_blank">S16</a> Figs) and the merged images in the left panels (green, Stx2a; red, compartment-specific marker proteins; blue, nuclei; yellow, colocalized green and red signals). The percentages of Stx2a colocalizations with compartment-specific marker proteins (white numbers) and with nucleus (blue numbers in panels ER) were calculated with the BioImageXD6 tool. Scale bars are 10 μm. (B) Graphical summary of Stx2a colocalizations with subcellular compartments based on CLSM data shown in A, and with OMVs (based on data shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.g003" target="_blank">Fig 3A</a>). (Means of colocalizations from at least five different samples are shown in A and B; for standard deviations and significance analysis see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s008" target="_blank">S8C Fig</a>). (C) Immunoblot detection of Stx2a A subunit in isolated subcellular fractions of HBMEC which were postincubated for the times indicated with 5791/99 OMVs or for 72 h without OMVs or with TRIS-HCl OMV buffer (negative controls); 5791/99 OMVs without cells (OMV ctrl) were a positive control. Molecular weights of Stx2a A subunit (32 kDa) and Stx2a A1 fragment (27.5 kDa) are shown on the right side. (D) Densitometric quantification of Stx2a A/A1 signals shown in C. For abbreviations see legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.g004" target="_blank">Fig 4</a>.</p

Sírépítmények tükörábrázolással Pannoniában

<p>(A) CLSM of HBMEC preincubated with OMVs 5791/99 for 30 min at 4°C, and postincubated at 37°C for 30 min to 20 h. The indicated single fluorescence channels are shown in the right panels (enlargements are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s023" target="_blank">S23</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s024" target="_blank">S24</a> Figs) and the merged images in the left panels (green, CdtV-A; red, compartment-specific marker proteins; blue, nuclei; yellow, colocalized green and red signals). The percentages of CdtV-A colocalizations with compartment-specific marker proteins (white numbers) and with nucleus (blue numbers in panels ER) were calculated with the BioImageXD6 tool. Scale bars are 10 μm. (B) Graphical summary of CdtV-A colocalizations with subcellular compartments based on CLSM data shown in A, and with OMVs (based on data shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.g003" target="_blank">Fig 3A</a>). (Means of colocalizations from at least three different samples are shown in A and B; for standard deviations and significance analysis see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s008" target="_blank">S8E Fig</a>). (C) Immunoblot detection of CdtV-A in isolated subcellular fractions of HBMEC which were postincubated for the times indicated with 5791/99 OMVs or for 72 h without OMVs or with TRIS-HCl OMV buffer (negative controls); 5791/99 OMVs without cells (OMV ctrl) were a positive control. (D) Densitometric quantification of CdtV-A signals shown in C. For abbreviations see legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.g004" target="_blank">Fig 4</a>.</p

CdtV-B is responsible for the biological effects caused by OMV-delivered CdtV holotoxin.

<p>HBMEC were incubated with OMVs from the indicated recombinant strains containing the CdtV holotoxin (BL21(<i>cdt</i>V-<i>ABC</i>) OMVs), CdtV-B subunit (BL21(<i>cdt</i>V-<i>B</i>) OMVs), or CdtV-AC subunits (BL21(<i>cdt</i>V-<i>AC</i>Δ<i>B</i>) OMVs). OMVs from BL21(pET23) vector control and untreated cells (no OMV) were negative controls. (A) DNA damage response was determined by immunoblotting of cell lysates with antibodies against phosphorylated forms of H2AX (γ-H2AX) and cdc2 (p-cdc2) after 20 h of exposure. Actin served as a loading control. (B) G2 arrest was measured by flow cytometric detection of cells with 4n DNA content after 48 h of exposure. Positions of the G1 (2n DNA) and G2 (4n DNA) peaks are indicated in the first histogram, and the proportions (%) of cells in the G1 and G2 cell cycle phase, respectively, are shown in all histograms. (C) Cell distension was visualized by light microscopy of Giemsa-stained cells after 72 h of exposure. Scale bars are 20 μm.</p

Retrograde transport of OMV-delivered Stx2a involves the toxin´s interactions with DRM-associated Gb3.

<p>(A, B, C) Colocalization of Stx2a with Gb3 (A), and colocalization of Gb3 with the Golgi marker GM130 (B), and the endoplasmic reticulum marker BiP (C) in cells exposed to 5791/99 OMVs or free Stx2a (control) for 4 h (A, C) or 90 min (B) and processed for CLSM after extraction with a Triton X-100-containing buffer (1 min on ice). Green, Gb3; red, Stx2a (A) or GM130 (B) or BiP (C); blue, nuclei; yellow, colocalized green and red signals. (D) Colocalization of OMV-delivered Stx2a and free Stx2a (control) with the Golgi complex (90 min) and the endoplasmic reticulum (4 h) in HBMEC untreated (no Triton) or pretreated with Triton X-100 (+Triton) before being processed for CLSM. Green, Stx2a; red, GM130 or BiP, as indicated; blue, nuclei; yellow, colocalized green and red signals. In all pictures, the indicated single fluorescence channels are shown in the right panels (enlargements are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s020" target="_blank">S20 Fig</a>) and the merged images in the left panels. The percentages of colocalizations of the respective signals (white numbers) were calculated with the BioImageXD6 tool (means of colocalizations from three different samples are shown). Scale bars are 10 μm.</p

OMVs are taken up by target cells via dynamin-dependent endocytosis.

<p>(A, D, G) Kinetics of R-18 OMV uptake by indicated cell types. Fluorescence of cells incubated with OMVs was normalized to that of OMVs without cells (net fluorescence intensity). (C, F) CLSM visualization of OMV uptake by Caco-2 cells (C) and HBMEC (F) after 30 min and 24 h of incubation. Green, OMVs; red, actin; blue, nuclei. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. Scale bars are 10 μm. (B, E, H) Uptake of R-18 OMVs by Caco-2 cells (B), HBMEC (E), and HRGEC (H) which were pretreated with the indicated endocytosis inhibitors. OMV uptake in the presence of inhibitors was expressed as the percentage of OMV uptake by inhibitor-untreated cells (100%). Data in A, D, G, and B, E, H are means ± standard deviations from three independent experiments. ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001 compared to inhibitor-untreated cells (one-way ANOVA).</p

The role of Gb3 in the uptake and retrograde transport of OMV-delivered Stx2a.

<p>(A, D) FACS analysis of Gb3 content in (A) PPMP-untreated (no PPMP) and PPMP-treated (+PPMP) HBMEC and (D) DLD-1 cells stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD77/Gb3 antibody or unstained (control). One representative experiment is shown for each cell line (geometric mean fluorescence ± standard deviations from three independent experiments are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s018" target="_blank">S18A and S18B Fig</a>). (B, E) CLSM analysis of trafficking of OMV-delivered and free Stx2a (control) into the endoplasmic reticulum (ER) and late endosomes/lysosomes (Le-Lyso) in (B) PPMP-untreated and PPMP-treated HBMEC (20 h) and (E) DLD-1 cells (4 h). The indicated single fluorescence channels are shown in the right panels (enlargements are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006159#ppat.1006159.s019" target="_blank">S19 Fig</a>) and the merged images in the left panels (green, Stx2a or OMVs, as indicated; red, PDI or CD63, as indicated; blue, nuclei; yellow, colocalized green and red signals). The percentages of colocalizations of the respective signals (white numbers) were calculated with the BioImageXD6 tool (means of colocalizations from three different samples are shown). (C) Uptake of 5791/99 OMVs by DLD-1 cells after 4 h of incubation. Untreated cells (no OMV) were a negative control. Green, OMVs; red, actin; blue, nuclei. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. Scale bars in all panels are 10 μm. (F, G) Immunoblot detection of Stx2a in isolated endoplasmic reticulum (ER) and lysosomal (Lyso) fractions of (F) PPMP-treated HBMEC and (G) DLD-1 cells after 20 h and 4 h of incubation, respectively, with the indicated samples or left untreated (no OMV); 5791/99 OMVs without cells (OMV ctrl) were a positive control.</p