Pengembangan Kurikulum Pendidikan Lingkungan di Kota Tangerang Selatan: Bagaimana Mengintegrasikan Deklarasi Tbilisi dalam Kurikulum

This study aims to develop environmental education curriculum based on Tbilisi Declaration. There were five environmental education goals of Tbilisi Declaration namely knowledge, awareness, attitudes, skills, and participation. Curriculum was developed for supporting education policy in South Tangerang, Banten Province, Indonesia. Letter of Education Division South Tangerang City nomor 800/KEP 1222-dikdas/2014 stated environmental education as local curriculum in South Tangerang Elementary School. Curriculum was developed by developmental research. There was four steps to develop curriculum, every steps had three activities, i.e. making, reviewing, and fixing. This study result some standard and basic competences. Research developed environmental education was integrated with thematic subject matter in 1st and 2nd grade, but as monolithic subject matter in 3rd – 6rd grade. Using exiting competence standard from 2014 curriculum and format standard competence 2006 made this curriculum have flexibility to implement for the school that used 2006 or 2013 curriculum

Model of events during early KSHV latency establishment.

<p>(<b>A</b>) Immediately after infection, MLL/SET containing complexes are recruited to viral episomes by as of yet unknown cellular and/or viral factors, resulting in the establishment of the early activation marks and transcription of a subset of KSHV genes that includes ORF73/LANA. Recruitment or activity of PRC2 to KSHV episomes is inhibited by the presence of soluble Sp100. (<b>B</b>) After accumulation of <i>de novo</i> expressed LANA protein, LANA induces SUMOylation and relocalization of Sp100 into insoluble fractions which may be ND10s, but could also represent another matrix-associated fraction. Depletion of soluble nucleoplasmic or chromatin-associated Sp100 allows widespread recruitment of PRC2 complexes via molecular mechanisms that remain to be determined. PRC2 recruitment establishes H3K27me3 patterns, allowing repression of lytic genes after extinction of activation marks (e.g., on the K2 promoter) or establishment of bivalent chromatin (e.g. at the ORF50/Rta promoter). The major latency promoter upstream of ORF73/LANA is protected from H3K27me3 acquisition and remains active throughout latency.</p

Proteasomal degradation and transcriptional silencing are not responsible for loss of soluble Sp100.

<p>(<b>A</b>) KSHV negative SLK cells or long-term infected SLK_P cells were treated with either the proteasome inhibitor MG-132 or DMSO for 8 h. Subsequently, soluble RIPA extracts were prepared and analyzed for Sp100 and LANA protein levels by immunoblotting. (<b>B</b>) Transcript levels of genes encoding the ND10 core components (PML, SP100, DAXX) and the three housekeeping genes GAPDH, ADH5 and VPS29. Transcript levels were analyzed by RNAseq (see complete dataset in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274.s013" target="_blank">Dataset S1</a>) in mock infected (0 h value) or KSHV infected SLK cells after 2, 4, 8, 12, 16, 24, 48 or 96 hours of infection and are given as RPKM (reads per kilobase and million mapped reads) values. Baseline expression levels as observed in mock infected cells are marked across plots by a dashed gray line. The fold range (FR) of maximum up- or down-regulation across the entire time course is indicated in each panel.</p

LANA foci do not co-localize with ND10/PML.

<p>SLK cells were infected with KSHV, fixed at the indicated time point, and analyzed for LANA and PML using standard IF staining and confocal microscopy procedures. DAPI images represent one confocal plane, whereas LANA and PML are depicted as maximum intensity projections to demonstrate separate localization of both proteins in all three dimensions.</p

Histone modification profiles acquired by transfected KSHV bacmids and de novo infecting episomes.

<p>(<b>A</b>) SLK cells were transfected with KSHV BAC16 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274-Brulois1" target="_blank">[143]</a> DNA and selected with hygromycin for 24 days to select for bacmid-carrying cells. Histone modifications were analyzed by high resolution ChIP on microarray analysis with antibodies directed against H3K4me3 (upper panel) or H3K27me3 (lower panel). (<b>B</b>) SLK cells were infected with KSHV and chromatin was prepared at indicated time points, and histone modification profiles were investigated by high resolution ChIP on microarray analysis with antibodies directed against H3K4me3 (upper panel) or H3K27me3 (lower panel). Arrows above H3K4me3 profiles denote peaks that are prominent at 24 h post infection, but were diminished upon acquisition of repressive H3K27me3 marks at later time points. Normalized signal intensity values from the profiles shown in A and B as well as from previously investigated SLK cells at 5 d post infection (5 dpi) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274-Gunther1" target="_blank">[14]</a> were used to generate the heat maps shown in (<b>C</b>). The heat maps indicate the chromatin status as being either naïve (black), dominated by H3K4me3 (green) or H3K27me3 (red), or characterized by the presence of both modifications (yellow). The latter state is designated as ‘putative bivalent’ since co-occurrence of both modifications on the same nucleosome has formally been proven only for the ORF50/Rta promoter <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274-Gunther1" target="_blank">[14]</a>.</p

Depletion of PML or Sp100 does not interfere with latency establishment in HUVEC cells.

<p>(<b>A</b>) Western blot analysis of HUVEC cells transduced with shRNA-expressing lentiviruses directed against PML, Daxx, Sp100 or GFP. shDaxx transduced cells did not show a reduction of Daxx protein levels and were thus not included in further analyses. Although residual levels of PML and Sp100 are still visible in bulk protein extracts, immunofluorescence analysis suggests that a considerable number of cells is devoid of PML or Sp100 positive nuclear bodies (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274.s009" target="_blank">Figure S9</a>). (<b>B</b>) FACS analysis of stably shRNA-expressing HUVEC cultures analysed 48 h ater infection with KSHV. The rightmost columns show cells which were treated with sodium butyrate (5 mM) immediately after infection. Mock infected cells were used to correct for background staining levels. Error bars represent SEM of three biological replicates. (<b>C</b>) Transcript levels of ORF50, ORF59, ORF64 and ORF73 in EA.hy cells at 48 hours post infection. Expression was calculated by normalization to GAPDH and is shown relative to shGFP controls (set to 1). Error bars represent SEM of at least three data sets.</p

LANA does not co-localize with Sp100, and KSHV infection does not disperse Sp100 from ND10s.

<p>(<b>A</b>) SLK cells were infected with KSHV and analyzed for LANA and Sp100 using standard IF staining procedures. LANA and Sp100 are depicted as maximum intensity projections from z-stacks to demonstrate separate localization of both proteins in all three dimensions. (<b>B</b>) The number of Sp100 containing dots per nucleus (left panel) and total volume of Sp100 dots (sum of volume of all dots within each individual nucleus; right panel) were determined in >60 cells in mock infected or KSHV infected cells at 72 h post infection using the Volocity software (see material and methods for details). Each dot represents a single cell. Bars represent mean and SEM values.</p

Soluble Sp100 levels are reduced upon KSHV infection of SLK cells.

<p>(<b>A</b>) Analysis of low-salt soluble RIPA extracts prepared from mock infected or KSHV infected SLK cells after 48 hours. (<b>B</b>) Analysis of soluble low-salt RIPA extracts prepared from long-term KSHV infected SLK cells (SLKp, lane 6), mock infected SLK cells (lane 1), or SLK cells that had been infected with KSHV for the indicated time points (lanes 2–5). (<b>C</b>) Analysis of total protein extracts prepared from mock infected SLK cells (lane 1) or SLK cells infected with KSHV for the indicated time points. (<b>D</b>) Analysis of soluble low-salt extractable (left panel) or total (right panel) protein fractions in uninfected (SLK) or long-term KSHV infected SLK cells (SLKp). β-actin served as a loading control in all panels.</p

Sp100 accumulates in the insoluble matrix upon <i>de novo</i> infection and in PEL-derived B-cell lines.

<p>Western-blot analysis of sub-fractionated cellular extracts (<i>cyto</i>: cytoplasmic; <i>memb</i>: membrane-associated; <i>nucpl</i>: nucleoplasmic; <i>chrom</i>: chromatin-associated; <i>matrix</i>: associated with the insoluble matrix) from: (<b>A</b>) EA.hy cells <i>de novo</i> infected with KSHV and harvested 72 h after infection or (<b>B</b>) PEL-derived (BCBL1, HBL6, AP2) or BL-derived (BJAB, Jijoye, Raji) B cell lines. For each B cell line, infection status for KSHV and EBV is indicated to the right of the panel. Successful fractionation was confirmed by detection of specific marker proteins (Hsp70, Sp1, HDAC2 and Vimentin).</p

Depletion of Sp100 but not Daxx or PML accelerates acquisition of H3K27me3.

<p>(A+B) SLK-shSp100, SLK-shDaxx, SLK-shPML and SLK-shGFP cells were <i>de novo</i> infected with KSHV and chromatin was prepared at 36 h post infection. Deposition of activating H3K4me3 (<b>A</b>) or repressive H3K27me3 (<b>B</b>) marks was evaluated by ChIP-qPCR using specific primers as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274.s012" target="_blank">Table S1</a>. (C+D) ChIP-qPCR analysis of selected loci in <i>de novo</i> KSHV infected shSp100 (red squares) or shGFP (control; blue diamonds) SLK (<b>C</b>) or EA.hy (<b>D</b>) cells was performed to analyze deposition of H3K27me3 marks at the indicated time points.</p