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20 Jul 2013 18:57 GMT | Politics, Humanitarian crises, Africa, Congo, Rwanda
A week of renewed fighting in the Democratic Republic of Congo's troubled east has driven more than 4,000 people to seek refuge in the provincial capital Goma, aid workers said.
The new offensive by the M23 rebel group in mineral-rich but unstable North Kivu province has caused 4,200 people to flee their homes and take shelter in schools and churches in Goma, the provincial seat and largest city in the area, the United Nations said on Saturday, citing figures from humanitarian group Premiere Urgence.
Fighting between the Congolese military and the M23, a group launched by Tutsi ex-soldiers who mutinied from the army in April last year, broke out again on July 14 after months of relative calm.Displaced people
The UN's Office for the Co-ordination of Humanitarian Affairs (OCHA) said it had also received reports of hundreds of refugees flooding over the border into neighbouring Rwanda, though an exact figure could not be established.
OCHA says there were 967,000 displaced people in North Kivu province at the end of June, 90 percent of whom had fled their homes because of the fighting or fears that it would spread to their areas.
Humanitarian organisations have voiced concern that those forced from their homes, many of whom are farmers, will not be able to return in time to sow their fields for the next planting season in mid-August raising the risk the conflict could also turn into a food crisis.
The latest hostilities have been focused about 15km north of Goma.
The M23 occupied Goma for 10 days in November before withdrawing from the city under international pressure.
The Congolese government and the UN have accused Rwanda and Uganda of backing the rebel group, a charge both countries deny.
Source: Agencies
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Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model.
We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire−/− mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire−/− mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire+/+ mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire+/+ and Aire−/− mTECs but distinct from that of athymic cells and tissues.
These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.
Keywords:Medullary thymic epithelial cells; Aire; T-DMR
Medullary thymic epithelial cells (mTECs) are highly involved in the establishment of central tolerance by ectopically expressing a variety of tissue-restricted antigens (TRAs) [1]. Deficiency of the autoimmune regulator, Aire—which regulates a large pool of genes in mTECs—results in autoimmune polyglandular syndrome in humans and autoimmunity in mice [2]. There are estimated to be hundreds to over one thousand TRAs that are activated by Aire in mTECs, based on microarray data from the transcriptomes of wild-type and Aire-deficient mTECs [3,4]. However, numerous TRAs are expressed in mature mTECs in an Aire-independent pattern [3,4]. Moreover, single-cell PCR analysis with sorted mTECs demonstrated that approximately 70% [5], or even fewer [6], of mature mTECs express Aire, and TRAs are expressed in Aire-negative mTECs [5]. These findings indicate that Aire is necessary, but not sufficient, for the expression of TRAs in mTECs.
The role of Aire in the maturation of mTECs has been indicated by using Aire-deficient mice, which exhibit contracted compartments in the thymic medulla and an increased number of medullary cells positive for p63, whose expression is diminished in Aire-positive mTECs [6-9]. The role of Aire in the differentiation of mTECs has been reviewed by Matsumoto [10]. However, the existence of the medullary compartment and the increased number of a small subset of K5−K8+ cells—considered to represent end-stage and terminally differentiated mTECs—in the Aire-deficient thymus indicate a limited role of Aire in the lineage determination of mTECs, in which the RANK signaling pathway is involved [11,12]. Thus, Aire-deficient mTECs may represent a suitable model to study not only Aire-independent TRA expression but also the fundamental regulation system that governs cell lineage determination of mTECs.
Genome-wide analysis of DNA methylation has indicated that a specific DNA methylation profile exists for every cell or tissue type. This profile consists of numerous tissue-dependent and differentially methylated regions (T-DMRs) at cytosine residues in CpG dinucleotides (CpGs), which are differentially methylated among distinct cells and tissues [13]. DNA methylation profiles change during cell differentiation, reflect developmental similarity among cell lineages [14,15], and illuminate cell- or tissue-specific gene expression profiles [16-18]. These findings suggest that DNA methylation profiles are involved in the establishment and maintenance of the regulatory system for cell- or tissue-type-specific genome use, and could therefore be used to identify types of cells and tissues.
The DNA methylation profile responsible for the phenotype of mTECs is still largely unknown, although a limited number of genes, including Aire and 2 Aire-independent TRA genes, Csn2 (Csnb) and Gad1 (Gad67), are unmethylated in their promoter regions [19-21]. To explore the genome-wide DNA methylation of mTECs, we applied a microarray-based screening system, T-
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Results and discussion
mTEC-T-DMRs identified in Aire−/− mTECs are associated with mTEC-specific genes
Thymic stromal cells from Aire−/− mice and their Aire+/+ littermates were fractioned into 2 populations: UEA1+CD45− (mTECs) and UEA1−CD45− (control thymic stromal cells). In this study, we use the term "stromal" to refer to UEA1−CD45− cells, which are a mixture of keratin-expressing epithelial cells, including cortical TECs (cTECs), and keratin-negative mesenchymal cells such as fibroblasts, connective tissue cells, and endothelial cells [23]. Predominant expression of Aire, Cd80, Krt5, Krt8, Krt14, and Foxn1 in the UEA1+CD45− population (detected by RT-PCR) indicated enrichment of mTECs in this fraction (Additional file 1: Figure S1). To elucidate the characteristics of genome-wide DNA methylation in mTECs, we compared Aire−/− mTECs with the liver, brain, thymus, and embryonic stem (ES) cells by D-REAM. mTEC-T-DMRs, which were differentially methylated as compared with the other cells and tissue types, were screened as genomic fragments that exhibited significantly different microarray scores after digestion by the methylation-sensitive restriction enzyme, HpyCH4IV (Additional file 2: Figure S2). Among the mTEChypo-T-DMRs, which were hypomethylated in Aire−/−mTECs, we designated those that exhibited scores higher than all or any 2 of the reference cells and tissues as mTEC-unique T-DMRs (mTECu-T-DMRs) or mTECany2-T-DMRs, respectively (Figure 1A, Additional file 3: Table S1).
Additional file 1. Figure S1. Isolation and characterization of CD45-UEA1+ mTECs and CD45-UEA1- thymic stromal cells. Isolation and characterization of CD45-UEA1+ mTECs and CD45-UEA1- thymic stromal cells. A. Isolation of CD45-UEA1+ mTECs (red) and CD45-UEA- thymic stromal cells (yellow) by flow cytometry. Values are representatives of the percentage of each population in repeated isolations. B. Gene expression in mTECs, thymic stromal cells, thymus, liver and ES cells by RT-PCR. Compared with the stromal cells, higher expressions of Aire, Cd80, Krt5, Krt14, Krt8, Foxn1 in both the Aire+/+ and Aire−/− mTECs were detected. The Aire−/− mice strain used in this research lacks the exon2 of Aire, but the transcription of Aire could be detected [1]. E2, exon2; E3, exon3; E5, exon5. C. Real-time RT-PCR quantification of transcript levels of Aire in mTECs isolated from Aire+/+ mice, as well as mTEC, thymus from C57BL/6 strain, and ES cells. The Exon2 to Exon3 of Aire mRNA was amplified. Standard curve method calculation was used to compute transcript levels relative to Actb mRNA. The y axis indicates arbitrary unit normalized to Actb, with the expression level in thymus as 1. The expression level of Aire in mTECs was over 50 times higher than in the thymus, which showed a similar pattern as human AIRE in mTECs and the thymus [2]. The expression level of Aire in ES was about half of the thymus. Expression of Aire in liver was not detected.
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Additional file 2. Figure S2. Illustration of the D-REAM method for identification of T-DMRs. D-REAM was developed via integration of a methylation-sensitive restriction enzyme (HpyCH4IV), ligation-mediated PCR (LM-PCR), and DNA tiling microarrays [3,4]. Briefly, for a certain genomic locus that contains a HpyCH4IV recognizing site (ACGT), in Sample A, the ACGT containing 5′-methylcytosine cannot be digested by HpyCH4IV and the corresponding fragment is not amplified by LM-PCR, thus does not detected by microarray. In contrast, in Sample B, the ACGT with unmethylated cytocine is digested and the corresponding fragment is efficiently amplified, leading to microarray signal, reflected by D-REAM score. Comparing the two samples using the D-REAM score, the locus is defined as a T-DMR and hypomethylated in Sample B. (PDF 242 kb)
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Additional file 3. Table S1. Summary of mTEC-T-DMRs. (XLSX 12 kb)
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Figure 1. mTECany2-T-DMRs correlate with gene expression in mTECs and associate with marker genes of mTECs. A. Hierarchical clustering of D-REAM scores corresponding to mTECany2-T-DMRs identified by comparison of Aire−/− mTECs with the brain, thymus, liver, and ES cells by Pearson's correlations. Among mTECany2-T-DMRs, those hypomethylated in Aire−/− mTECs in comparison with all of the reference cells and tissues were defined as mTECu-T-DMRs (indicated by asterisk). Br, brain; Thy, thymus; Lv, liver. B. Relative expression levels of genes associated with mTECu-T-DMRs in proximal regions (−6 to +2.5 kb) from TSS. The horizontal axis of the boxplot represents the log ratios of gene expression levels relative to Aire−/− mTECs. The numbers in round brackets indicate independent gene expression profiles for each type of cell or tissue (Additional file 5: Table S2). The p-values of t-tests showing significant differences in relative expression levels are shown. C. Bisulfite sequencing of regions around the TSS of Aire. The arrowhead indicates the position of the mTECu-T-DMR. The regions upstream (−674 to −191 bp) and downstream (+1672 to +2082 bp) of the TSS were analyzed independently using bisulfite sequencing. Open and closed circles represent unmethylated and methylated CpGs, respectively. Positions of CpGs are shown as vertical bars above the genomic structure (shown at the bottom of the panel), and the boxes indicate exons. D. COBRA analysis of mTECu-T-DMRs associated with Cd80 and Trp63. Positions of HpyCH4IV sites and mTECu-T-DMRs are shown above the genomic sequences within −1 to +2.5 kb of the TSS. Electropherograms represent the bisulfite PCR products after digestion with HpyCH4IV. Hypomethylated fragments were resistant to HpyCH4IV digestion (+). The levels of DNA methylation (%) are shown under each lane of the electropherograms.
The mTECany2-T-DMRs mapped to within 8.5-kb regions around the transcription start sites (TSSs) of 3081 ENSTs (Ensembl transcripts; mm9) (Additional file 3: Table S1). The distribution of mTECany2-T-DMRs exhibited a similar pattern to that observed in previous analyses of somatic tissues and ES cells [16,17], displaying a peak of T-DMRs at the 3'-downstream regions of TSSs (Additional file 4: Figure S3). Hypomethylated signals in these regions are often associated with cell- and tissue-specific highly expressed genes [16,17].
Additional file 4. Figure S3. Distributions of positions relative to TSS of mTECany2-T-DMRs and mTECu-T-DMRs. The highest frequencies of T-DMRs are in the 3′ downstream to TSSs in both mTECany2-T-DMRs and mTECu-T-DMRs. For the histogram, on unit represents 250 bp. (PDF 87 kb)
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We analyzed the expression of ENSTs associated with mTECany2-T-DMRs in mTECs using the gene expression profile of CDR1intB7-1hi CD45− mTECs from the Gene Expression Omnibus (GEO) database, and compared with those of mouse liver, and CD4+CD8+ thymocytes as a representative of the thymic cells (Additional file 5: Table S2). The differences in expression levels of genes associated with mTECany2-T-DMRs were relatively small between Aire−/− and Aire+/+ mTECs as compared with those between mTECs and the liver or thymocytes, indicating that the expression patterns of genes associated with mTECany2-T-DMRs in mTECs are not affected by functional Aire (Additional file 6: Figure S4). High gene expression in mTECs was more prominent for genes associated with mTECany2-T-DMRs in the −1.5 kb upstream and +2.5 kb downstream (proximal) regions of TSSs. Among these genes, those associated with proximal mTECu-T-DMRs showed significantly higher expression in mTECs compared with both the liver and thymocytes (Figure 1B). These data indicate that mTEC-specific genes are likely to be associated with mTECu-T-DMRs, and methylation may be responsible for gene silencing in Aire non-expressing tissues and cells. Indeed, we found that among the mTECu-T-DMR–associated genes, some had been previously reported as mTEC-specific genes, of which the majority showed Aire-independent expression in mTECs (Additional file 7: Table S3). Moreover, marker genes of mTECs, including Aire, Cd80, and Trp63, were found to be associated with proximal mTECu-T-DMRs.
Additional file 5. Table S2.List of microarray data of transcriptomes used in this study.
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Additional file 6. Figure S4. Log ratio of the expression levels of genes associated the mTECu-T-DMRs and mTECany2-T-DMRs. Relative expression levels of genes associated with mTECu-T-DMRs (upper) and mTECany2-T-DMRs (lower) in Aire−/− mTECs, Aire+/+ mTECs, liver and CD4+CD8+ thymocytes to Aire−/− mTECs. From left to right, the data sets represented the all probed genes and the T-DMRs, classified according to their relative distance to TSSs as −6 to +2.5 kb (whole window), -1 to +2.5 kb (proximal) and −6 to −1 kb (distal) regions. The vertical axis of the box-plot represents the values of gene expression levels of duplicated data sets relative to one microarray profile of Aire−/− mTECs. The numbers in the round brackets indicate independent gene expression profiles of each type of cell or tissues listed in Table S2. * indicates significant differences in relative expression levels where the p values of t-tests are less than −0.1. (PDF 128 kb)
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Additional file 7. Table S3. List of mTEC specific genes that associated with mTECu-T-DMRs. (XLSX 12 kb)
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We analyzed the DNA methylation status around the mTECu-T-DMR of Aire which is located downstream of its TSS, and the upstream region by using bisulfite sequencing (Figure 1C). In the −624 to −218 bp upstream region, the liver, which represents an Aire non-expressing tissue, showed a methylated status comparable with Aire+/+ and Aire−/− mTECs as well as with ES cells, which express Aire [24]. The promoter regions of human AIRE (−295 to +65 bp) and mouse Aire (−287 to +133 bp) are both associated with a CpG island (>50% CpG content) and are unmethylated in mTECs [19,20]. Since Aire-negative cTECs and several extrathymic tissues were also found to be unmethylated at this region [20], hypomethylation of the promoter is considered to be necessary, but not sufficient, for expression. In contrast, Aire+/+ and Aire−/− mTECs, but not ES cells, exhibited a hypomethylated status at the 9 CpGs in the region between +2082 and +1672 bp from the TSS, including the CpG corresponding to the mTECu-T-DMR. Among the tissue-specific genes carrying CpG islands within 1 kb of their TSSs, hypomethylated T-DMRs tend to be located downstream of TSSs [16,17]. Thus, the mTEC-TDMR for Aire located 3'-downstream of the Aire TSS could serve as an epigenetic marker to distinguish mTECs from Aire-expressing ES cells, in which the expression level of Aire was significantly less than that in the thymus and mTECs (Additional file 1: Figure S1).
The mTECu-T-DMRs associated with CD80 and p63 (Trp63) were analyzed with combined bisulfite restriction analysis (COBRA). Both Aire+/+ and Aire−/− mTECs exhibited a hypomethylated status compared with the liver, thymus, and ES cells (Figure 1D). Notably, the mTECu-T-DMR of p63 was found to be associated with ΔNp63, the short isoform of p63, which is essential for maintenance of the progenitor status of thymic epithelial cells [25] and not expressed in Aire-positive mTECs [7-9]. Hypomethylation at this T-DMR in Aire+/+ mTECs suggests that DNA methylation does not determine the repression of p63 in Aire+/+ mTEC cells.
mTECany2-T-DMRs are specific to mTECs, as compared with the other thymic stromal cells
We compared the DNA methylation levels of 15 selected genes—which were shown to be associated with mTECany2-T-DMRs—between the mTECs and stromal cells from Aire+/+ and Aire−/− mice by COBRA (Figure 2). In contrast to the hypomethylated status in mTECs, Aire, Cd80, and Trp63 were hypermethylated in stromal cells. Some genes that associated with mTECany2-T-DMRs were found to be involved in the RANK/RANKL signaling pathway, which stimulates the downstream NF-κB pathway and is required for the development of mTECs [11,12,26] (Additional file 8: Figure S5). For example, Tnfrsf11a (RANK) was hypomethylated in mTECs but hypermethylated in stromal cells. Traf2, the gene downstream of RANK, was hypomethylated not only in mTECs but also in the stroma, while its downstream genes, Tank, Birc2, and Edaradd demonstrated similar methylation patterns to Tnfrsf11a. We also found that H2-Dma (H2-Dm), which exhibited 2 mTECu-T-DMRs surrounding its second exon—a region essential for antigen presentation by MHCII [27]—was hypomethylated in mTECs and hypermethylated in the remaining stromal cells. In addition to these genes, similar methylation patterns were observed at mTECany2-T-DMRs associated with genes exhibiting tissue-specific expression patterns, such as Aplp2 (adipose tissue and brain), Arg1 (liver), Ctla4 (Treg), Ehhadh (kidney and liver), Mtap7 (lens), and Slc36a3 (testis). Importantly, these methylation patterns were observed in both Aire−/− and Aire+/+ mTECs, demonstrating that a DNA methylation pattern distinct from other thymic stromal cells is maintained in Aire+/+ mTECs.
Additional file 8. Figure S5. Genes involved in the development of mTECs with mTEC-T-DMRs. White, mTECu-T-DMR; gray, mTECany2-T-DMR; black, no mTEC-T-DMR. The pathway was modified from Genome Network Platform (http://genomenetwork.nig.ac.jp webcite). Tnfrsf11a (RANK) and its down-stream genes Traf2 and Lyn were associated with mTECany2-T-DMRs and Traf5 was associated with mTECu-T-DMR. TANK (Tank), cIAP2 (Birc2) and Crinkled (Edaradd), which were recruited by TRAF2 to activate the downstream NF-κB [9-11]. Stimulation by RANK leads to activation of STAT1 which in turn up-regulates IFN-stimulated gene expression in mTECs [12]. Stat1 and Jak1 were found to be associated with mTECu-T-DMR and mTECany2-T-DMR, respectively. (PDF 82 kb)
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Figure 2. UEA1+CD45−mTECs and UEA1−CD45−thymic stromal cells display distinct DNA methylation levels at mTECany2-T-DMRs. DNA methylation levels at the mTECany2-T-DMRs in mTECs and stromal cells isolated from Aire+/+ and Aire−/− littermate mice, as detected by COBRA. The levels of DNA methylation at CpGs within the HpyCH4IV site are indicated by the vertical axis. For Aire and CD80 genes—containing 2 HpyCH4IV sites inside the investigated region—the DNA methylation rate was calculated when any 1 of the CpGs was methylated. For the remaining genes, the levels represent DNA methylation levels at a single CpG. Bar graphs indicate means ± standard errors of the data from at least 3 independent bisulfite PCRs using independently isolated cells from 3 Aire+/+ mice and 3 Aire−/− mice.
Aire+/+ and Aire−/− mTECs show similar DNA methylation levels at mTEC-T-DMRs
To further compare the DNA methylation status between Aire+/+ and Aire−/− mTECs, we used COBRA to analyze 55 loci, including mTEC-T-DMRs and HpyCH4IV sites in the vicinity and some previously identified T-DMRs of other cell and tissue types [16,17] (Figure 3, Additional file 9: Table S4). Hierarchical clustering of DNA methylation levels at these mTEC-T-DMRs showed that Aire+/+ and Aire−/− mTECs display the most similar pattern (less than 20% difference in DNA methylation) compared with the liver, thymus, and ES cells. DNA methylation levels at mTEChypo-T-DMRs in both mTECs showed a unique or common hypomethylated status compared with the reference cell and tissues (Figure 3, upper panel), as revealed by D-REAM analysis (Figure 1A). Genes associated with T-DMRs characteristic of other cell and tissues were hypermethylated in mTECs (Figure 3. lower panel). These data indicate that the presence of Aire does not greatly change DNA methylation levels in mTECs.
Additional file 9. Table S4. T-DMRs analyzed by COBRA.
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Figure 3. Aire+/+andAire−/−mTECs show similar DNA methylation profiles. Hierarchical clustering with Euclidean distance of DNA methylation levels estimated by COBRA at 39 mTECany2-T-DMRs and adjacent T-DMRs (upper panel) and 16 previously identified tissue T-DMRs [16,17], which overlap with mTEChyper-T-DMRs (lower panel, Additional file 3: Table S1). DNA methylation levels from 0% to 100% are represented as the continuous gradation from white to black. Each row represents a different gene associated with T-DMRs.
Among the analyzed genes, Fgg (fibrinogen gamma chain), which is a liver-specific gene that is expressed in mTECs in an Aire-dependent pattern [3,4], was hypermethylated in both Aire+/+ and Aire−/− mTECs, similar to its status in ES cells and the thymus. The gene expression of TRAs in mTECs occurs in specific subpopulations and in a stochastic manner [1,2]; therefore, DNA methylation changes may occur at specific loci in an Aire-dependent manner in subpopulations of mTECs that express a specific TRA.
During thymic organogenesis, Aire is expressed at E16.5 [28], while the stromal cells of the medulla and cortex are separated at embryonic day 13 (E13) [29]. In the absence of Aire, mTECs survive Aire-induced apoptosis, reach a terminally differentiated stage [10], and maintain the ability to express TRAs [3,4]. Thus, the similar DNA methylation status at T-DMRs in Aire+/+ and Aire−/− mTECs suggests the existence of an Aire-independent DNA methylation profile that serves as an intrinsic system to determine the phenotype of mTECs.
In this study, we demonstrated the DNA methylation profile for Aire−/− mTECs by genome-wide identification of T-DMRs. The DNA methylation status at dozens of mTEC-T-DMRs in Aire+/+ mTECs was similar to that in Aire−/− mTECs, while distinct from athymic cells and tissues as well as the other thymic stromal cells, indicating that the Aire-independent DNA methylation profile is involved in differentiation of the mTEC lineage and other thymic stroma cells that form the thymic microenvironment.
Aire+/− mice (B6.129S2-Airetm1.1Doi/J) were obtained from the Jackson Laboratory [22] and backcrossed onto C57BL/6J (B6) mice (Oriental Yeast, Japan). Aire−/− mice and their Aire+/+ littermates were used for isolation of mTECs. The mice were housed and bred under specific pathogen-free conditions.
All experiments using mice were carried out according to the institutional guidelines for the care and use of laboratory animals (Graduate School of Agricultural and Life Sciences, the University of Tokyo).
Isolation of mTECs
The isolation of mTECs was conducted by modifying previously described methods [16,30]. Briefly, thymi were collected from 6- to 7-week-old male mice, finely minced, washed in RPMI 1640 medium, and sequentially digested twice with 0.2 mg/mL collagenase D for 15 min at 37°C. The pellets were incubated with 0.2 mg/mL collagenase D and 0.2 mg/mL Dispase I (Roche Applied Science) with 25 μg/mL DNase I (Takara), and the reaction was stopped with 5 mM EDTA. Subsequently, after centrifugation at 1350 × g on a Percoll gradient, supernatants between ρ = 1.06 g/mL and ρ = 1.0 g/mL were incubated with Mouse BD Fc Block (2.4G2), PE Rat Anti-Mouse CD45 (30-F11) (BD Pharmingen), and FITC-conjugated UEA-1 (Sigma) and sorted using an EPICS Altra flow cytometer (Beckman-Coulter). The fractionation of cells was monitored by RT-PCR (Additional file 10).
Additional file 10. Methods and references to additional files.
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D-REAM analysis
In D-REAM analysis, differential methylation status at HpyCH4IV loci is indicated by differential scores corresponding to fragments generated by this methylation-sensitive restriction enzyme between 2 samples [16; Additional file 2: Figure S2]. In the present study, we performed microarray experiments using mTECs from Aire−/− mice and the thymus from C57BL/6J mice. Briefly, after digestion of genomic DNA with HpyCH4IV (New England Biolabs), 50 ng of DNA was ligated with an adaptor, followed by TaqI digestion and ligation with a second adaptor. The fragments were amplified by ligation-mediated PCR to allow selective amplification of unmethylated fragments generated by HpyCH4IV digestion and then hybridized using the GeneChip Mouse Promoter 1.0R Array (Affymetrix), which comprises probes that map to the regions (−6 to +2.5 kb) around the TSSs of approximately 50,000 transcripts (ENST IDs; mm9) obtained from the UCSC genome browser. Hybridization signals were processed with MAT [31] and can be found at Array Express (http://www.ebi.ac.uk/arrayexpress/ webcite; accession number: E-MTAB-1113). The data from Aire−/− mTECs and the thymus were compared with D-REAM data from the liver, brain, and ES cells [17] to identify fragments that exhibited a difference in microarray scores, thereby indicating the DNA methylation status at corresponding HpyCH4IV sites. We set the cut-off value at 0.5% of FDR (false detecting rates) for T-DMRs. The Galaxy genome browser (https://main.g2.bx.psu.edu/ webcite) [32] and "R" (http://www.R-project.org webcite) [33] were used for genomic fragment analysis.
To analyze the gene expression of Aire+/+ and Aire−/− mTECs, we obtained their gene expression profiles from the GEO database (http://www.ncbi.nlm.nih.gov/geo/ webcite) [34] and compared with those of the liver and CD4+CD8+ thymocytes on the same microarray platform (Additional file 5: Table S2). The array data were normalized using the gcrma package [35]. The ENST IDs were converted into Affymetrix IDs by using BioMart software (http://www.biomart.org webcite) [36].
DNA methylation analysis with bisulfite-converted genomic DNA
mTEC cells and gDNAs from the thymus, liver, and ES cells were subjected to bisulfite treatment using the EZ DNA Methylation-Direct Kit (Zymo Research Cooperation) according to the manufacturer's instructions. Unmethylated cytosine residues are converted into thymine residues by the sodium bisulfite reaction, while methylated cytosine residues remain unchanged. PCR was carried out using the bisulfite-converted genomic DNA with the primers listed in Additional file 11: Table S5. To evaluate the DNA methylation status at specific CpGs, COBRA was performed. The PCR products were evenly divided and incubated with or without HpyCH4IV for 5 h at 37°C and then analyzed with a microchip electrophoresis system (MultiNA, Shimadzu Biotech). DNA methylation levels were calculated as the percentage of digested fragment compared with the sum of digested and undigested fragments. To evaluate the DNA methylation status at CpGs within hundreds of base pairs, the PCR fragments were cloned into the pGEM-T Easy Vector (Promega), and more than 10 clones were sequenced using an ABI 3130 sequencer with a BigDye Terminator kit (Applied Biosystems).
Additional file 11. Table S5.List of primers for bisulfite PCR.
Format: XLSX Size: 16KB Download file
mTEC: Medullary thymic epithelial cells; cTEC: Cortical thymic epithelial cells; TRA: Tissue-restricted antigen; Aire: Autoimmune regulator; T-DMR: Tissue-dependent and differentially methylated region; D-REAM: T-DMR profiling with restriction tag-mediated amplification; UEA1: Ulex europaeus agglutinin I.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
GW conceived the study, carried out experiments, analyzed the data, prepared the figures, and drafted the manuscript. KH performed the microarray experiment. SS assisted in bioinformatical analysis of D-REAM data. NA and TA assisted in isolating mTECs and control stromal cells. KS participated in the design of experiments. SY participated in the design of the experiments, performed bioinformatical analysis of D-REAM data, prepared the figures, and drafted the manuscript. All authors read and approved the final manuscript.
This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (no. 21221008 to K.S.); by a grant from the Japanese Society for the Promotion of Science (to TA); and by CREST from the Japan Science and Technology Agency (JST) (to SY).
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subset: commoncrawl


text:
Roleplay Discussion Thread
View Single Post
March 6th, 2013 (1:34 PM).
Xlugon Pyro
Dragon Tamer
Join Date: May 2007
Location: Wisconsin
Gender: Male
Nature: Bold
Posts: 307
Originally Posted by
What do I have waiting to be made?
A medieval fantasy roleplay - debating whether I should keep the Pokemon aspect to it
A Pokemon journey roleplay, in a new "region" of eighteen islands. Revive of my first roleplay.
A superhero roleplay
And possible a to-the-death kind of roleplay
Which would y'all like to see first?
I'm working on a fantasy Pokemon RP myself but I'm taking my time with it. We might end up competing for sign-ups but I have no idea when I'll have it finished.
I do like the second one. I do like journey RPs.
My posts look too short, so I'm going to misguide the general public and artificially enhance the length of my posts with this garbage of a signature!
In a world without substance, a nonsense signature is only appropriate!
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text: Discipline What veterinary associates today want and need in a great job VETERINARY ECONOMICS: Sep 30, 2015 By Matthew Kenwright, Assistant Content Specialist Our fictional Dr. Alex Doe exemplifies real-life survey results on what they're looking for in the workplace. Whistleblowing becomes insubordination when tech questions the boss DVM360 MAGAZINE: Sep 01, 2015 By Marc Rosenberg, VMD A concerned veterinary team member suspects impropriety and pays the price for questioning authority. 3 tales to test interest in practice ownership DVM360 MAGAZINE: Aug 26, 2015 By Christopher J. Allen, DVM, JD Lawsuits and nuisance employees should give aspiring veterinary owners perspective. 8 steps to bark back at bullies before they bite your team FIRSTLINE: Aug 05, 2015 By Cheryl Sanchez, MBA A bully on your veterinary team may start with a little growling and nipping, but you need to bark back before they ravage your team's culture and ruin more than just the workday. Veterinary employees across the nation head to court to face theft allegations DVM360 MAGAZINE: Jul 24, 2015 By dvm360.com staff Accusations range from embezzlement to third-degree burglary. What do you do when veterinary staff members break equipment? VETERINARY ECONOMICS: Jul 16, 2015 By Jeff Rothstein, DVM, MBA Training and communication are your best bets when it comes to preventing accidents. Everyone in veterinary practice should be using this time management tactic VETERINARY ECONOMICS: Mar 25, 2015 By Candiss Schneider, MBA Organize your life with a legal pad, a portfolio and efficient time use. Letter: Mark Opperman's story on sexual harassment falls short VETERINARY ECONOMICS: Mar 09, 2015 By dvm360.com staff A letter writer didn't think veterinary consultant Mark Opperman took a claim of sexual harassment seriously enough, but Opperman says different. Relief veterinarians: Rules to live by DVM360 MAGAZINE: Feb 19, 2015 By Christopher J. Allen, DVM, JD A greater level of flexibility in your veterinary career comes with increased responsibility. Ask Shawn: We're sick of chronic absenteeism FIRSTLINE: Feb 04, 2015 By Shawn G. McVey, MA, MSW Shawn McVey counsels a manager who's struggling with a veterinary technician and receptionist who frequently call in sick. Team tool: Choose your attitude cards VETERINARY ECONOMICS: Nov 01, 2014 By dvm360.com staff Use these cards to gently remind veterinary team members that everyone chooses their attitude at work. Addressing a veterinary employee about a visible tattoo VETERINARY ECONOMICS: Sep 19, 2014 By dvm360.com staff In this type of scenario, showing respect for the offending team member is key. Address the dress: Male veterinary managers discussing dress code policy with female employees VETERINARY ECONOMICS: Sep 19, 2014 By dvm360.com staff Get a tip from an expert to help you navigate this sticky situation in practice. How our veterinary hospital survived an employee mismatch VETERINARY ECONOMICS: Jul 14, 2014 By Erika Valente Why one DVM's style didn't fit—and what we did about it. BizQuiz: Are you the bully at work? FIRSTLINE: Jun 05, 2014 By Oriana D. Scislowicz, BS, LVT Are you the bully at work? Take this quiz to find out if you need to make a change in your professional behaviors at your veterinary practice. 1 2 3 4 5 ... 10 Next » Last » Hot topics on dvm360Feline posture: A visual dictionary Nov 04, 2015The F-word: A dvm360 Leadership Challenge Nov 01, 2015DIY: Build a better cat trap Oct 27, 2015Veterinarians could lose access to ketamine if drug is reclassified Oct 27, 2015Texas board seeks to revoke Dr. Kristen Lindsey's license Oct 05, 2015|More|


text:
Nov 17, 2012 EbenGregory News 7
Frank Ocean, who came out of his closet earlier this year, kinda ended all speculation that he was dating Willy Cartier when he took him to the GQ magazine party earlier this month.
Reports Global Grind:
Frank recently posted some Instagram pictures of the two hanging out together doing some meditation. He also added a photo of him sitting across the table from a guy while grabbing a bite to eat.
Although Frank doesn't try to make it too obvious that Willy is the man on the other end of the table, it's a good chance that it's him, since Frank also took Willy to the GQ Magazine red carpet recently.
And now…meet Frank's alleged boyfriend Willy Cartier.
This is EbenGregory.com…telling you no matter how far in or out of the closet you are, you still have a next step.
He looks like a woman that's dressed as a man for halloween…
classic lol
thats why eben need to do so the other day stories
gay fish
hold up i got another one:
1 fish, 2 fish, gay fish, NE-YO
D*ck n' da booty a$$ ninjas!
i used to know a nigga named mr malik from philly


text:
2003 Toyota Celica • 140,000 miles
When the car is running and I turn on anything electrical the dashboard goes out. Its not the alternator and checking the wires hasn'tt produced any results
March 18, 2013.
March 18, 2013.
Check out the related content below while we wait for the question to be answered by a professional mechanic.
How do i properly remove the alternator? Step by step please, I'm kind of slow.
1 answer • 2000 Jeep Wrangler • 137,000 miles
Do you have a walkthrough for replacing an alternator on a 97' Dodge Caravan with a 2.4L frontwheel drive engine? If you can help me Please include all the tools necessary for doing ...
1 answer • 1997 Dodge Caravan


text:
Kodak Ektonol was a favorite of mine to use for warm-toned printing for years. After Kodak stopped making it I stocked up with a full case but soon after I switched to mixing my own from established formulas. Last year I needed to do a quick printing session and was short/low on a few standard chems from Photographers Formulary, and I spied the case of Ektonol in my darkroom, and mixed up a gallon. It worked just as I remembered; a standard warm-toned developer with good capacity and keeping qualities, and the finished fiber prints were quite lovely. I mixed one more gallon size and have used that up, and now I would like to reverse engineer this formula but have not found a formula that is close enough as a starting point. I do have a few more packets to use as a control, and I have a good idea of the components they are pretty standard based on the package: Sodium Sulfite, Metol, Hydroquinone, Sodium Tetraborate (as per the package info), Potassium Bromide and here's the kicker, Sodium Hydroxide separately packed in Part B. Part A package states total weight as 227g, no weight given for Part B but a quick weigh with packaging is 31.5g ( I will be weighing the packaging of both A and B after I mix up this next batch to see what the weight of the chemicals is.)
SO, how can I go about devising either the formula or a close approximate? Anyone know of a warm-toned paper developer that used Sodium Hydroxide and no carbonate with lots of Potassium Bromide that I can use as a starting point?
I'd be willing to part with one of my treasured 1 gallon package to someone who wants to help figure this out, many thanks in advance.


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Managing local practices in global contexts
It is hard to think of a part of the world that has not been touched by globalisation....
It is hard to think of a part of the world that has not been touched by globalisation. From 'Big Macs' in Moscow to Blockbuster video in Beijing the world seems less distant and twenty-four-hour-a-day news makes foreign places more familiar. This unit examines the dimensions of globalisation and the processes that connect people together.
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- Duration 20 hours
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- 1 The management of local knowledge-generating practices
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Managing local practices in global contexts
This unit looks at the management of local knowledge-generating practices. You will explore the processes that link practices to global contexts and learn to identify the key dimensions of globalisation and explore the implications for knowing how to 'do things' in a variety of contexts. You will go on to compare the approaches to managing and organising, based on universally applicable principles, with context-specific rationalities and look at how viable interpretations of reality might be contructed from a variety of different perspectives.
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text: 'Regulation' No Longer a Dirty Word in DC Wall St. woes, toy and food scares have pols of all stripes on board By Sam Gale Rosen, Newser Staff Posted Mar 24, 2008 11:38 AM CDT STORY COMMENTS (0) (Newser) – "Regulation" is becoming less of a dirty word in Washington in the wake of the mortgage meltdown, woes on Wall Street, and scares over tainted food and toys. Many Democrats and even some Republicans want a shift from voluntary industry standards in vogue since the Reagan administration. "We're in for a potentially significant regulatory response," one economist tells the Wall Street Journal. When Congress gets back from its recess, it is expected to start work on an overhaul of the financial regulatory system. And a House committee wants to give food and pharmaceutical regulating agencies new power after a series of huge recalls of contaminated food. Meanwhile, all three presidential candidates have shown willingness to strengthen regulation. Ronald Reagan helped to inaugurate the idea that any regulation of business is bad for the economy. (PRNewsFoto/Ronald Reagan Presidential Foundation) (AP Photo) House Speaker Nancy Pelosi of Calif. holds up a recalled toy during a news conference on recalled toys and toy safety, Tuesday, Oct. 30, 2007, on Capitol Hill in Washington. (AP Photos/Susan Walsh) (AP Photo) House Speaker Nancy Pelosi, accompanied by Rep. Diana DeGette, D-Colo., left, and Rep. Bobby Rush, D-Ill., points out a recalled toy during a news conference on toy safety. (AP Photos/Susan Walsh) (AP Photo) ADVERTISEMENT(Click Prev or Next to continue viewing images.) « Prev« Prev | Next »Next » Slideshow Wall Street Journal (Source Grid ») Not everyone is on the same page New York Times Money • Entertainment Bush administration • Barack Obama • Hillary Clinton • John McCain • pet food recall • food contamination • SEC • Christopher Cox • credit crisis • regulation • toy recalls • lobbyists • House Energy and Commerce Committee • subprime crisis • Glenn Hubbard To report an error on this story, notify our editors.


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Etudes (12) For Piano, Op. 25, Ct. 26-37: XI. Etude No. 23 in A Minor, Op 25, CT. 36
Features of This Tracka well-known composer
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Train Your Mind: Seeing Confusion as the four kayas
Atisha's 59 Lojong Slogans with commentary by Acharya Judy Lief
The Mind-Training Slogans, Slogan #14
Each Friday, Acharya Judy Lief, teacher in the Shambhala tradition of Chogyam Trungpa Rinpoche, comments on one of Atisha's 59 mind-training (Tib. lojong) slogans, which serve as the basis for a complete practice.
Atisha (980-1052 CE) was an Indian adept who brought to Tibet a systematized approach to bodhicitta (the desire to awaken for the sake of all sentient beings) and loving-kindness, through working with these slogans. Judy edited Chogyam Trungpa's Training the Mind (Shambhala, 1993), which contains Trungpa Rinpoche's commentaries on the lojong ("mind-training") teachings.
Each entry includes a practice.
Read all the lojong slogans here.
14. Seeing confusion as the four kayas
Is unsurpassable shunyata protection.
With this slogan, once again we are joining what we usually consider as undesirable with practice. In this case it is confusion. At first glimpse, this slogan seems rather obscure and even esoteric. What kind of confusion? What are the four kayas? What is shunyata, anyway, and what form of protection can it provide? Protection from what?
In everyday experience, it is often hard to pin down what exactly is happening and why. Whenever we begin to figure things out, there is always some kind of slippage. Things begin to make sense, but just almost and not quite. We keep trying to chip away at our confusion, to straighten it out, to get rid of it, imagining ourselves somehow coming out on the other side, into a nonconfused state where everything is workable. But according to this slogan, rather than getting rid of our confusion, what we really need to do is to examine it and in doing so transform our view of it. We need to look below the surface to how we perceive reality altogether.
Basically, the point here is that if we really look closely at the way our mind works, even in the midst of confusion, we alway find the same process: one of continual awakening. This process is described in terms of what are called the four kayas or "bodies." Through careful attention and meditative practice we begin to see how every perception begins with uncertainty and openness (dharmakaya); then starts to come into focus (nirmanakaya); then develops energy and begins to come together (sambhogakaya), and finally clicks, synthesized as immediate present-moment experience (svabhavikakaya). It is as though confusion is awakening in disguise.
This pattern of continual awakening (seeing confusion as the four kayas) is paired with one of continual letting go (supreme shunyata protection). So in this slogan, not only do we transform how we view confusion, but we also see that although it may seem solid and intractable, fundamentally it is empty (shunyata). Combining all this, when we see everything as empty and awake, we have no ground to defend and nothing to protect—which is the most excellent protection of all.
Today's practice
In your sitting practice, pay attention to the arising and dissolving of perceptions. Notice how your sense of self seems to arise simultaneously with each perception, ready to respond to any threat; notice the subtle undertone of fear. What are you actually protecting?
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I found these teachings (Atisha's mind training techniques as interpreted by Chogyum Trungpa) to be quite confusing the longer I engaged with them. (Many years ago) It was precisely this practice that had me begin to question that which seemed to bring confusion and remain mindful of that which developed release from confusion.
As a westerner, I found that trying to develop an understanding of the Buddha's teachings through foreign cultural influences to be nearly impossible. It may be unclear to some readers that Atisha's (Or Mr. Trungpa's) teachings in mind training are not a practice that the Buddha taught. This may be why some later schools teach that awakening will take "countless eons."
The Buddha taught that any human could awaken in the present lifetime if they would engage the Dhamma whole-heartedly and free of distraction. It was not until I put my mindfulness on the original teachings of the Buddha, free from cultural or patriarchal influences, was I able to understand the Four Noble Truths and the Eightfold Path.
Confusion may almost be awakening in disguise for some, as Ms. Lief states. For me when something is presented as the Dhamma, or at least what one Buddhist teacher is presenting as the Dhamma, and develops confusion rather than release, I return to the framework of the Eightfold Path.
The Buddha's teaching of putting aside all views arising from ignorance by incorporating mind-training within the framework of the Eightfold Path became clear and accessible and effective in delivering the Buddha's stated purpose of his Dhamma: To recognize stress and suffering and to put aside all causes of stress and suffering.
I have great respect for Judy Lief and I have read much of her material. Lojong is a much honored and useful practice for many. Atisha preserved Tibetan Buddhism at a time (11th century) that was critical for Tibetan Buddhism and has led many in the Mahayana and Vajrayana teachings as developed in Tibet.
Many westerners may find the original teachings of the Buddha to be more effective and less confusing in recognizing and putting aside all causes of stress and suffering. Peace.
John Haspel


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Opinion of State Governments Drops With the Economy, Budget Gaps
The falloff in favorable views has been greater in states with the largest budget gaps. Also, the new administration has shifted partisan views of the federal government dramatically.
Obama's Ratings Slide Across the Board
Support for Obama's job performance — as well as his handling of health care, the economy and deficit–has fallen, but most remain confident his policies will be positive in the long term. The public supports many of his health care goals but opposes many proposals being debated in Congress.
Recession Dot Net
More than two-thirds of Americans have logged on to the internet looking for financial information. Of these "online economic users" most are looking for good deals and job opportunities. More said that what they learned on the internet made them more anxious than said they were made more confident.
Public More Optimistic About the Economy, But Still Reluctant to Spend
Increasingly, Americans express the view that the nation's economy will improve in the next year, and a growing number also expect their personal finances to get better. But this has not caused people to open their wallets.
Recession Pounds States' Budgets
Stateline.org's legislative review finds state budgets in such dire straits that they are closing prisons in Colorado and Kansas, raising taxes on the rich in New York and even taxing bourbon in Kentucky — and the worst is yet to come.
Gen Next Squeezed By Recession, But Most See Better Times Ahead
While the economic downturn is falling quite heavily on younger Americans, their overall outlook remains optimistic. A new survey also finds Generation Next expressing more liberal views when compared with older age cohorts as well as evidence of increased political engagement.
Independents Take Center Stage in the Obama Era
Centrism has emerged as a dominant factor in public opinion as the Obama administration begins. Republicans and Democrats are even more divided than in the past, while the growing political middle is steadfastly mixed in its beliefs about government, the free market and other values that underlie views on contemporary issues and policies. Both political parties have lost adherents since the election and an increasing number of Americans identify as independents.
Support for Free Trade Recovers Despite Recession
Despite the economic recession, support for free trade agreements is up by nine percentage points — from 35% to 44% — putting positive opinions of trade back in line with long-term trends. People in low-income families and Democrats are much more supportive of trade now than they were a year ago.
Public Knows Basic Facts About Financial Crisis
High percentages of Americans know that the government assistance to banks is aimed at getting them to lend more money, not less money and that China is the foreign country holding the most U.S. government debt. Notably, more Americans know the current unemployment rate than the current level of the Dow Jones Industrial Average.
The Phantom Recovery
The eight-year period from 1999 through 2007 is the longest in modern U.S. economic history in which inflation-adjusted median household income failed to surpass an earlier peak.


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Seven Valencia High School seniors received the John Tynes Scholarship this year.
They are: Aleksandra Banas, Biola University; Jessica Cervantes, Fullerton College; Gabriela Sanchez, Cal Poly Pomona; Sean Steinke, Orange Coast College; Jocelyn Pineda, Cal State Fullerton; Eduardo Waldo, Chapman University; and Sergio Gutierrez, Cal State Fullerton.
John Tynes' wife, Clairee, and their son, Jake, continue the scholarship program on behalf of the late John Tynes, whose life's work was dedicated to helping students. Tynes was a former athletic director at Valencia High School and served as our school district's superintendent.
STUDENT HONORSEl Camino Real High School has announced its third quarter top scholars. Students named to the High Honor Roll must earn 20 or more credits, maintain a 3.5 GPA.
Those students were: Graciela Amparan, Aylin Armenta, Christopher Boyd, Natalia Castillo, Courtney Cole, Jaedin Davasligil, Wendy Esparza, Rene Fernandez, Victor Gomez, Vivian Guerrero, Mariel Jimenez, Kimberly Lopez, Matthew Parra, Kevin Peralta, Jacob Richards, Jaclyn Rubio, Jacinto Sanudo, Sarah Sorensen, Jessica Tronco and Irene Valencia.
Honor Roll students must exceed 15 credits of work while maintaining a 3.0 GPA.
Those students were: Pedro Adan, Jayson Alvi, Alberto Aparicio, Alex Arivizo, Garrett Austin, Walter Ascencio, Oscar Baltazar, Stacy Barnes, Roberta Basua, Urbano Belmontes, Jeremy Biggs, Jamie Brown, Julio Castro, Alexis Cedeno, Steven Cervantes, James Coffman, Mckenna Douglas, David Espejo, Samantha Estrada, Andrew Lopez-Garcia, Deyanira Garcia, Tania Garcia-Munoz, Alexandra Gasmena, Nate Gediman, Everardo Hernandez, Gustavo Hernandez, Jr., Junior Hernandez, Angel Hwang and Jesus Jimenez.
More are Dave Kulasingam, Kirsten Larsen, Marilu Limon, Victor M. Lopez, Yazmin Lopez, Jazmin Luna, Laura Martinez, Mariela McBenttes, Greg Miller, Rickey Monsivais, Stephany Nunez, Alfredo Ortiz, Sam Phan, Kacie Phillips, Vivian Ponce, Hayley Prindle, Charlie Roberson, Rigo Rodriguez, Monica Sanchez, Aaron Sandoval, P.J. Singh, Sean Suskie, Jessica Tapia, Julie Tapia, Moises Vargas, Mario Velasco, Alejandro Velazquez, Dafne Villarruel and Cesar Zacarias.
KEY HELPFor the third consecutive year, George Key School put on its carnival. Parents donated their time assisting with set-up and manning booths, as well as donating pizza, drinks and prizes.
Community members also pitched in and a big thanks goes to the Orange County Fire Authority for bringing two fire engines and crews to give students the opportunity to sit in the driver's seat, touch equipment used for fighting fires and pose for pictures. Even neighboring Golden Elementary School gave a hand by assisting Key students play the games.
As a result of so many thoughtful contributions of time, effort, products and services, there was no cost for admission or food and beverages.
The day began with their annual jog-a-thon where they earned money for the purchase and installation of an outdoor wheelchair adapted swing.
FOUNDATION FOR STUDENTSA new organization has been formed with goals to strengthen student performance in our district. The foundation is called R.E.A.C.H., which stands for Resources for the Enrichment of Academics, Art and Athletics for our Children to reach their ultimate Heights.
This new foundation has great plans to fund field trips, materials and activities for the classrooms and teachers. They also hope to provide afterschool and summer programs that will include tutoring, sports, music, art and technology.
The foundation has been established by a group of concerned parents who have volunteered their time and expertise. This nonprofit's board of directors consists of Julie Nibali, president; Chrystie Adams, vice president; Rose Mergelmeyer, treasurer; and Carrie Johnson, secretary.
They are currently conducting a Founding Partners Giving Campaign through July 31. The following sponsorship programs are available: Diamond Founding Partner $25,000, Gold Founding Partner $15,000 and Silver Founding Partner $10,000. For those wishing to donate a smaller amount, the Community Partners Sponsorship Campaign is going on now through the fall. They will also be holding their first inaugural 5K/2K Run event in March.
For more information, email to email@example.com .
HONORING SERVICEYorba Linda High School PTSA President Melinda Shank and Executive Vice President Sondia Buscaino presented this year's PTA Honorary Service awards to the following people: teachers Sarah Shay, Beth Mazurier and Jeff Platt received recognition for yearbook, newspaper and men's athletics, respectively. Parent Jim Hom was honored for being a library volunteer, Janis Heckel for PTAS student advisor and Leticia Rice received the PTSA Very Special Award for her help with the band.
Corinne Griffiths, a retired sixth-grade teacher from the Placentia-Yorba Linda Unified School District, covers our students' accomplishments. E-mail items to email@example.com.


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Above all else, I love dirty fucking a sloppy cunt (and it's always a cunt, never a pussy). Maybe ass is a close second. I like freaks, geeks, nerds, hippies, crazy weird shit, and the occasional artsy shot. One thing I can't stand is a million and one 19 year old bimbolinas, shaved smooth, all the same size and shape, sitting around doing nothing. Boooooring! Enjoy yourself some sloppy cunt today! - Sloppy Cunt (@sloppycunt)


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Track # 13
Description: Hikaru no Go OP1
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Online shopping has some major advantages. Hitting up multiple stores at once, price comparison…and let's not forget 24/7 round-the-clock access (who has not bought a pair of shoes at 3 am?) But e-commerce can get a little lonely. That's where our latest obsession, ThisNext.com comes in. Their addictively easy social shopping platform combines the convenience of buying online with the fun of group browsing. Plus, it's free! Join now, build your guide, and start sharing your styling smarts. Plus, see what we're buying/loving/wanting, starting with Lucky executive fashion director Alexis Bryan Morgan and Lucky beauty director Jean Godfrey-June plus Lucky Style Collective members such as Mary Tomer of the Mrs. O blog. We can't wait to see what you come up with!
What's with All the Spam in the Comments Section?
We're sure that you've noticed the flood of spam in the comments section lately. Spammers beware–we are completely on top of it. As we figure out a way to put a stop to this madness, we ask you for your patience and that you don't stop leaving your own comments, because we love hearing from you!


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Education - Post Primary Teaching
Dublin City University
- Dublin
- Degree - Masters (Level 9 NFQ)
- Graduate Diploma
- Higher Diploma (Level 8 NFQ)
Attendance options
- Evening
- Full time
- Part time
Apply to...
Dublin City University
PAC Application Information
For Application dates, please click on the 'Application Weblink' or 'Course Web Page' links below.
Postgraduate Applications Centre (PAC)
The Postgraduate Applications Centre (PAC) is based in Galway. It is an application centre that provides the facility for applicants to apply online for Postgraduate taught programmes at DCU. There are currently 89 DCU courses available through PAC.
Applicants apply for DCU postgraduate courses online at www.pac.ie. Applicants can apply for up to three programmes when making an application to DCU on PAC. Programmes should be selected in order of preference as sequential processing and assessment of programmes mean that if an applicant is offered their first choice no further assessment of the other programmes is made. The cost of applying is €50 and is a non-refundable fee payable to PAC.
All documentation relevant to the application should be posted to PAC at the following address:-
Postgraduate Application Centre
1 Courthouse Square
On receipt of the application fee PAC scans all documentation onto the system for review at DCU. Applicants are notified by email as each step of the review route is progressed. It is therefore, imperative that applicants have a secure email account and review it regularly for updates, as no other means of communication between DCU and prospective applicants will be used.
To assist in each stage of the application process web links are provided both on the PAC system and here on our webpage. These links, listed opposite, can be reviewed prior to making an application or dipped into during the process. They provide relevant information from the initial stage of making an application to accepting an offer online. Applicants also have the facility to pay the initial deposit online on accepting and subsequent fees.
Application Weblink
2 years full-time (evenings).
Link to Course Fee
Entry Requirements
Entry Requirements:
Applicants will normally hold at least a primary degree that is recognised for teaching at least one subject from the second-level curriculum. In addition you must have all of the following:
- Two references (including current or most recent employer)
- Guaranteed placement of at least 200 hours with 12-18 years olds in a recognised school/college
- Be available to attend for interview prior to the start of the programme
- Relevant experience (desirable)
Applicants MUST read the Programme Regulations for the PDE, complete and return four supplementary forms with your PAC number directly to Postgraduate Applications Centre, 1 Courthouse Square, Galway within 10 days of making your application online.
Why DCU?
- DCU is the largest centre for education and training in Ireland.
- We take a modern and creative approach to teacher education.
- We link initial teacher education programmes to professional development.
- DCU graduates in teacher education are highly regarded.
International Applications
International candidates are expected to have educational qualifications of a standard equivalent to those outlined above. In addition, where such candidates are non-native speakers of the English language they must satisfy the university of their competency in the English language.
Course Content
Programme Overview:
Degree Information:
Do you believe that your efforts and commitments can inspire a young person to achieve great things? As a teacher in a second-level school, it is the quality of the relationships that you form with your students that will define their experience of school and encourage, enlighten and challenge them to achieve the highest standards possible for themselves. Without doubt schools in Ireland are now defined by a diversity of races, faiths, cultures, social and economic backgrounds. If you think you have the courage to work in this way then you should consider becoming a teacher.
By contributing to the development of a young person's values and character you influence not just the quality of one person's life but the quality of the whole of society.
The Professional Master of Education addresses the need for a flexible evening teacher education qualification for those who want to work in second-level schools but who are not in a position to engage in a full-time day programme of study.
Aims and Objectives:
- To provide student teachers with the knowledge, understanding and skills required to adapt to the demands of their professional lives.
- To combine the varied experiences and opinions of the students to bring a synergy to the lectures, shared tutorials and group work, which are key features of the programme.
It is our aim to recruit students for this programme from a variety of backgrounds, including those working in second-level schools as well as those who have recently completed undergraduate study, those who are currently working in Further Education, industry and business, TEFL, Special Needs Education, Youthreach, VTOS and other professional settings.
Programme Structure:
The programme starts in September, with lectures held on Tuesday and Wednesday from 5pm to 8pm. This includes an induction to teaching practice which happens each year prior to students starting their placement. You will participate in lectures, shared seminars, workshops and study in groups to develop your abilities in two subject pedagogies. Please note that it is your responsibility to ensure that upon completion of the programme you meet the General and Special Requirements of the Teaching Council, in order to be able to register as a teacher of your chosen subject in a second-level school in Ireland.
In Year 1, you take five modules, attracting a total of 30 credits (ECTS). In Year 2, seven modules are offered across both semesters, also attracting 30 credits. Each module is continuously assessed, with terminal examination as appropriate. Continuous assessment is by projects, case studies, papers, presentations, reports and computer-based assignments, where relevant.
School Placement
This programme includes a supervised school placement during which you will practice classroom teaching and participate in the general life of the school. You must obtain a minimum of 100 hours of teaching in a classroom, as well as a further 200 hours of systematic observation and participation in school life.
Subjects Taught
Please click on "Course Web Page" below for subject information.
Careers or Further Progression
Career Prospects:
While most of our graduates will work as teachers in voluntary secondary, vocational, community and comprehensive schools, others will pursue further studies in education and related fields, such as guidance, pastoral care, school leadership and management. Others again will work in, Further Education, Higher Education, government departments and other education and training settings.
As this course is fully recognised by the Teaching Council, graduates will receive appropriate salary allowances for permanent positions in second-level schools.
Further Enquiries
Dr James O'Higgins Norman,
email: firstname.lastname@example.com
Tel: +353 1 700 7417
Room: CA107
Course Web Page


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Finally, divergent paths have resulted in uneven development in Shandong and in China. Uneven development can give rise to polarization, and polarization can, in turn, create social tensions and conflicts. Polarization in China would not only shake the very base of the CCP but also upset China's social and political stability, since the ideal of equity and equality of the previous decades is still ingrained in the minds of many Chinese despite the last nineteen years of economic reform. The village conglomerate has set a good example of "getting rich together as a community." In addition, many village conglomerates are helping their neighboring villages and less-developed villages in their regions through financial aid and investment and by sponsoring training programs and providing information.
As China is preparing to enter the new millennium, the village conglomerate may very well be a prototype of political economy. Its significance to China's success in its modernization drive cannot be overestimated.


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Earthship Global Network
". . . the Earthship is the epitome of sustainable design and construction. No part of sustainable living has been ignored in this ingenious building."
Los Técnicos Chixot Education Center
The purpose of the Los Técnicos Chixot Education Center is to provide Comalapan youth with marketable skills that will enable them to be responsible citizens and entrepreneurs. Educational opportunities for teenagers and young adults in rural Guatemala are severely lacking, and this project will give them the tools they need to be competitive in the job market. The school will offer relevant educational opportunities, create jobs, and combat environmental issues in Guatemala.
Not In Your Guidebook: Earthship Lands Besides Lubaantun Maya Site in Southern Belize
from sanpedroscoop.com
Last weekend, on the very last day of the weekend long Belize Chocolate Festival, we took a shuttle bus down the mile long path to the Lubaantun Maya Site in the Toledo District of Belize. Huge trees and greenery was pretty much all we were seeing until just before the ruins – this sign.
Ummm. Earthship Belize. And the view of an odd adobe-like structure behind some brush. What the heck is that? There is a sign…it must be open to the public. Maybe. Surely there could not be attack dogs. There is a sign!
We kept going (we were on a shuttle bus) but I made note to check this out on our exit… […]
Building Belize's First Earthship Alongside the Mayan Ruins
Phil Moore | Monday, 15th October 2012 | from permaculture.co.uk
Belize's first Earthship lies within sight of the Maya ruins of Lubaantun in the south of the country, creating a wonderful juxtaposition between ruined civilization and new ecological design
Renegade architect Mike Reynolds and his crew began construction in March 2012. Commissioned by an ex-pat British family, the building is situated on a huge piece of land with a cacao farm, an important and popular crop in this part of the world.
Richard and Alisa arrived in Belize two years ago having lived in Scotland, Spain, and Mexico with their three children. Now settled with three dogs, three cats, and an army of chickens most of the building work is being done by locals employed from the nearby village of San Pedro Colombia. The Maya crew headed by Cessaerio were taught the ropes by Mike and his team and there are plans to use the tyres and bottle brick approach to create a rain water catchment system for the village school. […]
Final days for this round in Comalapa, Guatemala
The Earthship crew has completed the 3-week build that donated a home to a family in Comalapa, Guatemala for the second time around. Working with Long Way Home, a non-profit organization in Comalapa working to build sustainable schools, we were able to raise the appropriate amount of money as well as gather volunteers and organize to allow this project to take place. We are thankful for all who participated and donated to this project.
Guatemala Tues Wed – Week 3 Update – Enclosure Systems
The house is on its way to full enclosure and completion as the Earthship crew will be leaving Guatemala early Saturday. With two workdays left, we are in a good place to leave Long Way Home foreman, Romeo Apen, to do the finishes to his liking on his new home.
Earthship Guatemala Week 3
It is the final week of construction in Comalapa and the building is moving toward finishes. Lou and his group are working on the black water cells. The front of the house is trenched appropriately and the septic has been piped into the botanical cells. Today, the trenches were lined with plastic and then filled with big rock, gravel – to be filled with dirt.
Earthship Guatemala – Update – End of Week 2
Over the course of 2 weeks, Earthship Biotecture and Long Way Home have been working on the 3-U Survival Pod Earthship for Long Way Home foreman, Romeo. The progress has been impressive with all of the volunteers working and the crew managing its team accordingly. Groups were broken up into ten in order to keep the process structured and efficient. Much to report from the last update earlier this week.
The vaults have been plastered and the parapets have been built and leveled with plastic trash bottles. With a line of people, dirt was moved to bury around the vaults in order to create the thermal mass. Pummus was them bucketed up the line to insulate the thermal mass.
Update: Earthship Guatemala Day 6
Building has been framed for the greenhouse and cement work is continuing. Third coat was done on the vaults and the trash bottle parapets are being raised. The planters were dug and septic lid has been put onto the flush toilet on the east side of the building. Plumbing for the shower and toilet and grey and black water cells were done by Lou and his team.
Update: Earthship Guatemala Day 5
Continue with cementing the vault and creating the parapet behind the house. Can walls are going up and trash and glass bottle walls are being formed . The framing of the south facing windows is about half way completed. The structure is beginning to be enclosed and looking more and more like a home every day.
Update: Earthship Guatemala Day 4
Vaults/Assembly Lines/'Outlaw' Septic
Business as usual today for the Earthship/Long Way Home crew with 60+ volunteers. The third vault is on, bond beams are poured and cement is being hauled up the hill by an assembly line of people throughout the whole workday. Plaster work has begun on the east vault. The grey water planters have been dug out as well as the 'outlaw' septic. The cisterns will be moved to another location on the site because they cannot be buried behind the building and their new space has not yet been determined.


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Illinois Climate Action Plan's goals set back by University solar farm construction postponement
Construction of the University's solar farm has been postponed, causing setbacks in the Illinois Climate Action Plan's goals.
The solar farm is a major component of the plan, which was approved by the Board of Trustees in November 2012 and set for construction this fall. It was originally supposed to contribute 2 percent of University energy by 2015. Morgan Johnston, sustainability coordinator for Facilities and Services, said that goal would no longer be met.
The delay began when State Purchasing Officer Steve Rotello raised questions about the financial standing of Phoenix Solar, the contractor the University picked to build the farm.
"The University explained that the solar market had gone through a decline, and Phoenix is well positioned to survive that decline," Johnston said. "At this point now we're already seeing improvements in their financial situation, but we still do need to address the potential risk."
The University is developing a risk management system for the project as a method of insurance in case anything did go wrong with the company, she added.
With the risk management plan, Johnston is hopeful the solar farms will be approved by Rotello this year, and construction will begin in Spring.
"This project will help us get to about 2 percent renewable electricity for the campus; the initial goal for the Climate Action Plan was 5 percent by 2015," Johnston said. "We're hopeful to at least have the 2 percent up and running by 2015 with the solar farm."


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Football Groom's Cake
- Uploaded By Bythebullseye
- 2012-09-22 10:03:01
A sports theme groom's cake. The client wanted me to feature both his college and highschool colors/mascot. The black and white is from his high school where he played both sports. The baseball is pretty much made like a cake pop (crumbled cake pieces and buttercream balled together, covered in fondant). The football is a layered cake with 4 thick layers of buttercream and is completely hand/knife sculpted.
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©2015 General Mills, Inc. All Rights Reserved. This information will only be used to send an email to your friend(s) and will not be saved. Please read our Privacy Policy
Bake and serve this cheesy sausage and vegetable strata that can be assembled ahead to save time!
"I served this at brunch the day after my daughter Amy's wedding, and it was a real hit! It was convenient for me because the assembly is done the day before; all I had to do the day of serving was bake it."
Green leafy vegetables such as spinach are a good source of vitamin K, essential for normal blood clotting. If taking a blood thinner such as Coumadin®, however, avoid excessive intake of dietary vitamin K because it may interfere with the medication.
High in calcium, folic acid and vitamins A and C
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Cowboys Photos
Photos: Cowboys all smiles as they beat Dolphins in Hall of Fame Game
Photo: Tom Fox/Staff Photographer
Dallas Cowboys quarterback Alex Tanney (7) throws a fourth quarter pass against the Miami Dolphins in the Pro Football Hall of Fame Game, the opening preseason game of the season, at Fawcett Stadium in Canton, Ohio, Sunday, August 4, 2013.
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Speaker Hany Hassan Awadalla and Fei Xia
Affiliation MSR Machine Translation, Linguistics Department at UW
Host Michael Gamon
Duration 01:30:41
Date recorded 16 May 2014
Domain adaptation via effective feature engineering across domains Yan Song and Fei Xia
Domain adaptation aims at bridging the performance gap when training and test data come from different domains. In the field of natural language processing, domain adaptation techniques have been applied to tasks such as POS tagging, parsing, machine translation, and sentiment analysis, and there have been extensive studies in this area in the past decade.
In this talk, we present two novel approaches to domain adaptation. In the first approach, we address the limitations of two existing domain adaptation methods, training data selection and feature augmentation, by combining the two methods; that is, we propose to use training data selection to divide the source domain training data into two parts, pseudo target data (the selected part) and source data (the unselected part), and then apply feature augmentation on the two parts of the training data. In the second approach, we improve system performance by introducing new features that represent shared properties among two domains. Our experiments show that these approaches can boost system performance not only when the training and test data come from different domains, but also when they are from two closely related languages.
Yan Song was a visiting student at UW in 2011-2012. After receiving his PhD from City University of Hong Kong in 2014, he joined Microsoft Search Technology Center Asia in Beijing, China as a speech scientist. Fei Xia is an Associate Professor at the Linguistics Department at UW. She is one of the organizers of the UW/MS NLP Symposium.
Graph-based Semi-Supervised Learning of Translation Models from Monolingual Data Hany Hassan Awadallah, Senior Research Scientist, MSR Machine Translation
Statistical phrase-based translation learns translation rules from bilingual corpora, and has traditionally only used monolingual evidence to construct features that rescore existing translation candidates.
In this work, we present a semi-supervised graph-based approach for generating new translation rules that leverages bilingual and monolingual data. We report results on a large Arabic-English system and a medium-sized Urdu-English system. Our proposed approach significantly improves the performance of competitive phrase- based systems, leading to consistent improvements between 1 and 4 BLEU points on standard evaluation sets.
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