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- COMMUNITY OUTREACH SPECIALIST - Position Announcement Community Outreach Specialist/Latino Ombudsman The City of West Chicago, Illinois is seeking a bilingual Community Outreach Specialist who will play a key role in promoting and coordinating services and opportunities offered by the City. This individual will develop and implement programs to increase community awareness of programs and services, targeting the Spanish speaking population and also conduct assessments to identify conditions, barriers and factors that are limiting the Latino population from understanding and/or accessing City services and developing detailed strategies to address them. This position is part of the Administration Department and reports directly to the City Administrator. Position will also be responsible for developing and translating materials in Spanish to ensure effective communication with the Latino Community. Position requires: Equivalent to a Bachelor's degree from an accredited college or university with major coursework in Human Service Discipline (e.g. social work, sociology, psychology, public policy, etc.) or a related field. Four years of increasingly responsible experience in community based work such as advocacy and/or serving as a liaison/ombudsman with the Spanish speaking population. Bilingual -English/Spanish required. Salary range: $63,674 -$89,144. Send resume, salary history, letter of interest and three professional references to: Ms. Carol LeBeau, Human Resources Coordinator, City of West Chicago, 475 Main Street, West Chicago, IL 60185. Position open until filled. EOE (id:4461466) PLANT MAINTENANCE TECHNICIAN - PLANT MAINTENANCE TECHNICIAN Elgin -General plant maint, hvac, plumbing, electrical firstname.lastname@example.com (id:4461830) MARKET RESEARCH ANALYST - Huron Consulting Services, LLC is seeking an Oracle Consultant Manager in Chicago, IL with the following requirements: Bachelor's degree in Business Administration, Finance, Accounting or related field or foreign academic equivalent plus 9 years of related experience OR Three years of undergraduate study in Business Administration, Finance, Accounting or related field or foreign academic equivalent plus 9 years of related experience. Required skills: design and develop planning, budgeting and forecasting solutions using Oracle Hyperion Essbase, Hyperion Planning, and Oracle Planning and Budgeting Cloud Service (PBCS) (5 yrs); develop interfaces between legacy systems and Hyperion to load metadata and data using Oracle Data Integrator and Oracle Data Management (5 yrs); develop web based financial reports using Hyperion Financial Reports and excel based reports using Smart-View (5 yrs); manage projects through all project stages, from the design stage through to completion, including onsite and offshore team co-ordination (5 yrs). Periodic travel may be required to various unanticipated worksites in the US; individuals may reside anywhere in the US. Apply on-line at www.huronconsultinggroup.com, Careers, and search for Keyword: 2305. (id:4461018)
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Jul 24, 2013, 08.08 AM | Source: PTI
Aditya Birla Group's Kumar Mangalam Birla resigned from the board of the Reserve Bank of India; weeks after his group firm applied for a banking license from the RBI.
Also read: Aditya Birla Nuvo enters race for banking licence
Birla was a nominated as a member of the Directors of the Central Board of the RBI in 2006. His group firm Aditya Birla Nuvo is among 26 entities that have applied for a bank licence. The last date for applying for a bank licence expired on July 1.
With Birla continued to be on the Board of RBI even after his group firm applied for a licence. CPI had recently raised objections, saying there is conflict of interest as he is a member of the RBI board.
RBI Governor D Subbarao had responded by saying the central bank would look into the issue. The Reserve Bank is expected to grant new licences by March next year.
India has 26 public sector banks, 22 private sector banks and 56 regional rural banks.
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Early aberrant DNA methylation events in a mouse model of acute myeloid leukemia
- Miriam Sonnet1,
- Rainer Claus1, 6,
- Natalia Becker2,
- Manuela Zucknick2,
- Jana Petersen1,
- Daniel B Lipka1,
- Christopher C Oakes1,
- Mindaugas Andrulis3,
- Amelie Lier4,
- Michael D Milsom4,
- Tania Witte1,
- Lei Gu1, 10,
- Soo-Zin Kim-Wanner1,
- Peter Schirmacher3,
- Michael Wulfert5,
- Norbert Gattermann5,
- Michael Lübbert6,
- Frank Rosenbauer7,
- Michael Rehli8,
- Lars Bullinger9,
- Dieter Weichenhan†1 and
- Christoph Plass†1Email author
DOI: 10.1186/gm551
© Sonnet et al.; licensee BioMed Central Ltd. 2014
Received: 25 March 2014
Accepted: 25 April 2014
Published: 30 April 2014
Aberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown.
We screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors.
We identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML.
Our study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.
Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with severe morbidity and poor prognosis. It comprises a highly heterogeneous group of blastic myeloid malignancies and constitutes the most frequent type of acute leukemia in adults [1]. AML can arise de novo but also secondarily from preceding myelodysplastic syndrome (MDS), or after cytotoxic treatment or radiotherapy. It is characterized by an aggressive clonal proliferation of immature hematopoietic progenitor cells (myeloblasts) and impaired differentiation [2]. Recurrent chromosomal aberrations and rearrangements occur in more than 50% of cases and represent important predictive factors for response to therapy and outcome of the disease [3]. Altered gene function in AML is often a consequence of distinct cytogenetic aberrations [4], but also results from mutations in genes like CEBPA (CCAAT/enhancer-binding protein, alpha), FLT3 (fms-like tyrosine kinase receptor-3), or NPM1 (nucleophosmin 1) [3, 4]. Although novel high-resolution genome-wide technologies have enabled the detection of numerous gene mutations, the multistep process of leukemogenesis is still poorly understood. In recent years, many reports have proposed that additional pathogenetic mechanisms, such as aberrant loss or gain of gene function due to epigenetic dysregulation, are of similar relevance for AML pathogenesis [5–8].
Methylation of cytosines in the context of CpG dinucleotides is a stable and common epigenetic modification in the mammalian genome. Most human gene promoters overlap with CpG-rich regions, designated CpG islands (CGIs), which are usually excluded from DNA methylation and, consequently, keep genes transcriptionally active. Conversely, promoter methylation is commonly linked with transcriptional silencing.
Hypermethylation and subsequent inactivation of genes are hallmarks of AML pathogenesis [9, 10]. Prominent examples include the epigenetically silenced tumor suppressor genes CDH1 or p15/CDKN2B[11, 12]. In addition, gene hypomethylation is frequently found in myeloid malignancies. The mechanistic link, however, between promoter hypomethylation and tumorigenesis is incompletely understood. Global hypomethylation is common in many cancers, including AML, and is suspected to destabilize genome integrity by re-activating retrotransposons [6, 13, 14]. Alterations in DNA methylation contribute to initiation, expansion, and evolution of the leukemic clone and promoter hypermethylation is a frequent observation in specimens of patients with MDS and AML [15–17]. The mechanisms underlying the establishment of aberrant DNA methylation patterns are still largely unknown. Aberrant DNA methylation might be explained by the aberrant binding of transcription factors to their genomic target sequences. Transcription factor binding may prevent DNA methylation at these sequences, while diminished binding may result in de novo DNA methylation [18].
To gain a better insight into the molecular mechanisms and pathways underlying AML onset and progression, different mouse models recapitulating human AML have been generated, many based on the prevalent fusion genes AML1/ETO, PML/RARA or MLL/ENL (for review see [19]). These models mimic various human leukemogenic processes in the context of distinct disease genotypes and phenotypes associated with the different forms of AML [20].
Targeted deletion of an upstream regulatory element of the mouse gene Sfpi1 (common human name SPI1) encoding the transcription factor PU.1, a key hematopoietic regulator for myeloid differentiation, results in homozygous PU.1 hypomorphs that develop AML (or much less frequently T-cell lymphomas) after a latency of 3 to 8 months [21, 22]. As a consequence of reduced PU.1 expression in homozygous animals, epigenetic alterations of tumor suppressor genes are suspected to be involved in leukemogenesis. A previous screen of those animals developing a lymphoma but not an AML phenotype revealed promoter hypermethylation of the tumor-suppressor gene Id4[21].
Here, we provide a first comprehensive characterization of the methylome at CGIs in bone marrow (BM) cells from PU.1 hypomorphic animals during onset and progression of AML. Genome-wide DNA methylation screening during the process of leukemogenesis reveals the extent as well as the spatial and temporal distribution of altered DNA methylation. We aimed at identifying early differentially methylated genes preceding the fully established AML phenotype. Early differentially methylated genes may contribute to onset and progression of the disease, while the far more abundant and diverse differentially methylated genes at the late leukemic disease stage may reflect clonal diversification of AML and bystander events. We propose that hypomorphic PU.1 expression contributes to the initiation of aberrant DNA methylation of PU.1 target genes. Our study uncovers known and novel targets of aberrant epigenetic regulation occurring at onset and during progression of AML and, hence, may help to develop novel therapeutic strategies by revealing new pharmacologic targets at different stages of the disease.
Animals, sample collection and histopathology
Transcription factor PU.1 hypomorphic Balb/c mice with a homozygous deletion of an upstream regulatory element of gene Sfpi1 encoding PU.1 were described previously [21, 22]. Whole BM of age- and gender-matched homozygous wild-type (PU.1-wt) and PU.1 knockdown (PU.1-kd) mice were collected at three different disease stages. BM blast counts of PU.1-kd animals were used to define disease stages as preleukemic stage (BM blasts <20%, age 4 to 18 weeks, n = 7), early leukemic stage with residual non-malignant hematopoiesis (BM blasts between 20% and 50%, 4 to 12 weeks, n = 5) and late leukemic stage with full blown AML (>50%, 12 to 27 weeks, n = 7). From two of the seven animals of the late leukemic stage, the blast count could not be determined because the two mice died at the age of 22 to 24.5 weeks shortly before sampling. Both mice were considered late leukemic since they displayed phenotypic characteristics of their stage, such as enlarged spleens. BM cells were freshly collected from mouse femurs flushed with cold phosphate-buffered saline. Bone sections from forelegs were stained with hematoxylin and eosin and subjected to histopathological inspection for assessment of the disease stages.
Mouse BM cells were collected from the femorae, tibiae and iliae of PU.1-wt and preleukemic PU.1-kd mice by gentle crushing in Iscove's modified Dulbecco's medium. To confirm that the selected PU.1-kd animals were preleukemic, a May-Grünwald/Giemsa staining was performed on BM cytospins. The blast count was below 20% in each animal. Five PU.1-wt animals were pooled to obtain enough cells for sorting. For PU.1-kd animals, two groups with four preleukemic animals per group were collected. Murine lineage-depleted BM cells were isolated essentially as described in [23]. Low density mononuclear cells (LDMNCs) were purified by density gradient centrifugation using Histopaque 1083 (Sigma-Aldrich, Taufkirchen, Germany). LDMNCs were stained with the following rat anti-mouse biotin-conjugated lineage markers (all from BD Biosciences, Franklin Lake, NJ, USA): anti-CD5 (53-7.3), anti-CD8a (53-6.7), anti-CD11b (M1/70), anti-CD45R/B220 (RA3-6B2), anti-Ly-6G/Ly-6C (RB6-8C5) and anti-TER-119 (TER-119). The labeled LDMNCs were subsequently incubated with Biotin Binder Dynabeads (Life Technologies, Darmstadt, Germany) and the lineage-positive cells were depleted using a Dynamag-15 magnet, resulting in lineage-depleted cells. The lineage-depleted cells were stained with the following panel of antibodies: FITC-conjugated rat anti-mouse CD34 (RAM34; eBioscience, Frankfurt, Germany); eFlour®450-conjugated rat anti-mouse CD16/32 (93, eBioscience); APC-conjugated rat anti-mouse CD127 (A7R34, eBioscience); PE-conjugated rat anti-mouse CD117/c-Kit (2B8, eBioscience); APC-Cy7-conjugated rat anti-mouse Ly-6A/E/Sca-1 (D7; BD Biosciences, Heidelberg, Germany); and PE-Cy7-conjugated Streptavidin (eBioscience). Lineage-negative, CD127-, c-Kit+, Sca-1- cell fractions corresponding to granulocyte-macrophage progenitor cells (GMPs; CD16/32+, CD34+), common myeloid progenitor cells (CMPs; CD16/32-, CD34+), and megakaryocyte-erythroid progenitor cells (MEPs; CD16/32-, CD34-) as well as the lineage-negative, CD127-, c-Kit+, Sca-1+ cell (LSK) fraction were then prospectively isolated using a BD FACSAria I, II or III flow cytometer (BD Biosciences). All animal experiments were performed in accordance with the institutional guidelines of the German Cancer Research Center and were approved by the Regierungspräsidium Karlsruhe, Germany.
MDS patient samples from whole BM (n = 149) and from peripheral blood (n = 1) reflecting the entire disease spectrum as displayed by the World Health Organization (WHO) classification were obtained from the Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine University, Düsseldorf, Germany with patient informed consent and the University Clinic Düsseldorf review board approval in accordance with the Declaration of Helsinki. The sample set contained the following MDS subgroups: MDS with del(5q) (5q-, n = 5), refractory anemia (RA, n = 6), refractory anemia with ringed sideroblasts (RARS, n = 8), refractory cytopenia with multilineage dysplasia (RCMD, n = 43), refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS, n = 30), refractory anemia with excess of blasts type I and II (RAEBI, n = 18; RAEBII, n = 28), chronic myelo-monocytic leukemia type I and II (CMMLI, n = 10; CMMLII, n = 2). Normal karyotype AML patient samples from whole BM (n = 46) and from peripheral blood (n = 5) were obtained from the Department of Hematology/Oncology, University Medical Center, Freiburg, Germany with patient informed consent and the University Clinic Freiburg review board approval in accordance with the Declaration of Helsinki. Healthy granulocytes were isolated from blood of 14 healthy donors using Leukosep (Greiner bio-one, Frickenhausen, Germany) according to the manufacturer's instructions. CD34+ cells were from two healthy female and one healthy male donor (median age 36 years) and purchased from Lonza (Verviers, Belgium).
DNA and RNA extraction
DNA and RNA were extracted from mouse BM using the Allprep Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol and stored at 4°C (DNA) or -80°C (RNA).
DNA of human MDS and AML samples was isolated with the QIAmp DNA Mini Kit (QIAGEN) according to the manufacturer's instructions.
Methyl-CpG immunoprecipitation
Methyl-CpG immunoprecipitation (MCIp) was performed as described previously [24]. In brief, a total of 2.5 μg DNA was sonicated with the Bioruptor NextGen (Diagenode, Liege, Belgium) to fragments of 100 to 600 bp as monitored on a 1.5% agarose gel. MCIp enrichment of highly methylated DNA was performed, as described, with minor modifications using SX-8G IP-Star robot (Diagenode). Sonicated DNA was enriched with 90 μg purified methyl-CpG-binding domain-Fc protein coupled to 60 μl protein A-coated magnetic beads (Diagenode). DNA was eluted by incubation with increasing NaCl concentrations (fraction A, 300 mM; B, 400 mM; C, 500 mM; D, 550 mM; E, 1,000 mM). Desalted eluates were controlled for enrichment of methylated DNA by real-time PCR analyzing the imprinted gene Mest. The non-methylated allele elutes at low-salt while the methylated allele elutes at high-salt concentration.
Methylome profiling by microarray analysis
Highly methylated DNA, corresponding to fraction E of the enrichment procedure, from age- and gender-matched PU.1-kd and PU.1-wt animals was labeled with Alexa 3 (PU.1-wt) or Alexa 5 (PU.1-kd) and co-hybridized to a mouse CGI array (Agilent, Böblingen, Germany) covering the about 16,000 CGIs, represented by 88,358 probe sequences with a length of 45 to 60 bp per probe sequence, of the mouse genome (approximately 0.4%; NCBI36/mm8). Henceforth, probe sequences are designated 'probes' throughout this study and 'differentially methylated probes' (DMPs), if they were differentially methylated between PU.1-kd and PU.1-wt animals. Agilent's annotation, according to NCBI36/mm8, assigned the probes to 5,285 gene promoters, 7,872 gene bodies, 480 locations downstream of genes and 1,869 locations with unknown genomic feature. Here, promoter probes were defined as those being located 2,000 bp upstream to 500 bp downstream of a gene's transcription start site. In all other cases, we followed the annotation of Agilent. Microarrays were analyzed using a DNA microarray scanner (Agilent) and Feature Extraction Software 10.5 (Agilent) with the ChIP protocol setting. Data processing and statistical analyses were done within the R statistical environment, v. 2.13.1 [25]. Background correction and log 2-ratio transformation were performed according to the NormExp method with offset = 50; any intensity that is less than 0.5 after background subtraction is reset to be equal to 0.5 [26]. Variation between co-hybridized samples was reduced by intensity-based LOESS normalization on rank-invariant probes and negative controls [27].
The CGI array data of this study has been deposited at the NCBI Gene Expression Omnibus [28] under accession number GSE37315.
Quantitative DNA methylation analysis
The degree of DNA methylation was determined by MALDI-TOF mass spectrometry (MassARRAY, Sequenom, San Diego, USA) as previously described [29]. Amplicon primers (Additional file 1) flanked genomic stretches that covered at least one of the hypermethylated probes represented on the CGI array. In vitro methylated standard DNA served as control. Unmethylated DNA was produced using the REPLI-g Mini Kit (QIAGEN) and purified with the QIAmp DNA Mini Kit (QIAGEN). Half of the unmethylated DNA was methylated using M.SSSI enzyme and purified with the QIAquick gel extraction kit (QIAGEN). Unmethylated and methylated DNA was mixed to obtain different ratios for the standard DNA (0%, 20%, 40%, 60%, 80% and 100% DNA methylation).
Overlap of publicly available chromatin immunoprecipitation sequencing data with methylome data
Genomic coordinates from publicly available chromatin immunoprecipitation sequencing (ChIP-Seq) data of transcription factor PU.1 (NCBI37/mm9) [30] were converted to the NCBI36/mm8 (2006) genome by using the lift over tool of the UCSC Genome Browser [31]. In total, 22,625 out of 22,720 peak regions could be successfully converted. The PU.1 ChIP coordinates were overlapped with genomic coordinates of the preleukemic hypermethylated genes, and Fisher's exact test was used to test for significant enrichment of the PU.1 ChIP peaks. We randomly permutated the PU.1 ChIP-Seq peaks 1,000 times over the genomic coordinates of the preleukemic hypermethylated CGIs and compared the randomly permutated numbers with the actual numbers of PU.1 ChIP-Seq peak/preleukemic hypermethylated CGI overlap.
Statistical analysis
Following CGI array normalization, one class significance analysis of microarrays (SAM; package samr, version 2.0 [32]), was performed for each disease stage separately to find significantly hyper- and hypomethylated probes between PU.1-kd and PU.1-wt mice with a false discovery rate of 5%. Principal component analysis (PCA; package pcaMethods, version 1.36.0) was performed using the array methylation patterns to analyze homogeneity between individual animals at the molecular level in the course of the disease.
Unsupervised clustering with Euclidian distance and the average agglomeration method was used for clustering MassARRAY data. Unsupervised clustering was performed by multiscale bootstrap resampling [33] (package pvclust, version 1.2.2) to calculate approximately unbiased (AU) P-values for each cluster in the result of hierarchical clustering. The number of permutations was set to 1,000.
Mann-Whitney U test was performed to test for methylation differences derived from MassARRAY data between PU.1-kd and PU.1-wt animals and between AML/MDS samples and healthy granulocytes/CD34+ cells.
The computational analysis was performed by custom Perl scripts and the motif discovery was conducted with the software suite HOMER (Hypergeometric Optimization of Motif EnRichment) [34].
Pathway analysis
Pathway analysis was performed using genes that were represented by at least two DMPs of the preleukemic stage through the use of Qiagen's Ingenuity® Pathway Analysis [35].
Disease progression is associated with alterations in global DNA methylation
Early aberrant DNA methylation is followed by the diversification of hypermethylation in the late leukemic disease stage
We used the array-derived M-values to characterize the three disease stages by SAM. Of the 88,358 probes, 20,787 (23.5%) were aberrantly methylated in at least one stage of the disease. Aberrant DNA methylation was equally distributed across the genome, and no chromosome was preferentially hypermethylated (Figure 1C) or hypomethylated (Additional file 2). In the preleukemic stage, 1,184 (1.34%) hyper- and 802 (0.91%) hypomethylated probes were found, covering 762 and 504 genes or other genomic locations, respectively (Additional file 3). Both numbers slightly decreased in the early leukemic stage, suggesting either partial reversion of initial methylation changes or concomitant molecular processes other than aberrant DNA methylation (for example, genetic alterations) driving malignant clone selection (Figure 1D). The number of hypermethylated, but not that of hypomethylated probes abruptly increased in the late leukemic stage to 19,316 (21.9%), covering 5,465 genes or other genomic locations (Figure 1D). Within the respective stages, several unique hyper- and hypomethylated probes were observed. For example, 162 probes were uniquely hypermethylated in the preleukemic stage, but were unchanged or even hypomethylated in the other stages. Throughout all stages, 297 (0.3%) probes were commonly hypermethylated and 47 probes were commonly hypomethylated (Figure 1E).
We observed a significant gradual increase of methylation levels with disease progression in the majority of analyzed genes as exemplified by Fzd5 and Fzd8 (Figure 2B,C). Fzd5 methylation was 12.1% (range 6.8 to 21.3%) in PU.1-wt animals and 33.4% (range 18.2 to 44.9%), 40.9% (range 34 to 47.8%) and 50.4% (range 39.8 to 92.8%) in preleukemic, early leukemic and late leukemic stage animals, respectively. The stage-dependent increase of Fzd8 methylation was similar to that of Fzd5, starting with 22.8% (range 15.9 to 37.3%) in PU.1-wt animals and increasing to 30.6% (range 24.3 to 44.7%), 35.4% (range 28.1 to 41.9%) and 44.8% (range 36.1 to 67.2%) in the preleukemic, early leukemic and late leukemic stage animals, respectively.
Acute myeloid leukemia-specific methylation changes
To ensure that the observed changes in DNA methylation are not the result of tissue-specific methylation patterns and, thus, reflect differences in cell type composition, we analyzed Fzd5, Fzd8 and eight additional differentially methylated regions in four hematopoietic cell types, LSK (lineage-negative, c-Kit+, Sca-1+ cells), CMPs, GMPs and MEPs, which were enriched from both PU.1-wt and PU.1-kd BM, respectively, and which represent different stages of hematopoietic commitment. We found both genes to be similarly hypermethylated in all four cell types in PU.1-kd BM (Figure 2D,E; Additional file 5). This indicates that hypermethylation at these loci is an early event that is specific for PU.1-kd and does not just reflect the expansion of specific hematopoietic compartments. In summary, our quantitative methylation data indicate dynamic changes from onset to the late leukemic stage of the disease. The correlation between DNA methylation level and myeloblast infiltration suggests that aberrant DNA methylation is a feature of the malignant clone. Early aberrant DNA methylation at specific loci, in turn, can ubiquitously be found in the myeloid compartment and may characterize the (pre)-malignant clone in its early stage.
Early targets of aberrant DNA methylation in the PU.1 mouse model are relevant for the pathogenesis of human myeloid malignancies
To identify genes potentially involved in the onset of AML, we looked for overlaps between the list of 1,229 genes or other genomic locations indicated by aberrantly methylated probes in the preleukemic stage (Additional file 3) and gene lists from previously published genome-wide DNA methylation data derived from the HELP (Hpa II tiny fragment enrichment by ligation-mediated PCR) assay in human MDS and AML [17]. We detected 291 common genes with MDS-associated DNA methylation targets (5,390 in total) and 30 common genes with the AML gene list (475 de novo targets in total) (Additional file 6). These overlaps corroborate the relevance of the animal model for acute human myeloid malignancies. Well-known examples of human leukemogenesis, such as RUNX1, CEBPA, and ABL1, were aberrantly methylated in both the murine preleukemic stage and in human MDS. Remarkably, the ratios between overlapping genes and the aberrantly methylated human disease genes (291/5,390 ~ 0.05 for MDS and 30/475 ~ 0.06 for AML) were rather similar, suggesting that the mouse model was not confined to only a distinct disease type of either MDS or AML.
We tested by gene ontology analysis (Ingenuity Pathways Analysis) whether the set of aberrantly methylated, preleukemic genes represented functional groups of genes or pathways relevant for AML pathogenesis. Among several partially overlapping signaling pathways, Wnt/β-catenin and embryonic stem cell signaling were prominently overrepresented (Additional file 7). These pathways are known to be involved in onset and progression of human malignancies, including AML [36, 37]. Moreover, Wnt signaling has already been linked to the inappropriate regulation of the PU.1 transcription factor associated with T-cell lymphoma in mice [21].
We selected three additional genes, PRDM16, ROBO3, and CXCL14, that displayed promoter hypermethylation already in the preleukemic or early leukemic stage (Additional file 4) for validation in human AML samples. Thus far, these genes have not been shown to be aberrantly methylated in human AML; however, PRDM16 and ROBO3 are differentially methylated in MDS [17]. PRDM16 is a fusion partner of RPN1, RUNX1, and other genes in hematopoietic malignancies [38, 39], and rearrangement of PRDM16 was associated with poor prognosis [38]. ROBO3 is hypermethylated in cervical cancer [40]. CXCL14 is important in the progression of many malignancies, including colorectal cancer [41], and is epigenetically silenced in lung and prostate cancer [42, 43]. All three genes showed promoter hypermethylation in the AML samples, and that of ROBO3 and CXCL14 reached statistical significance (P < 0.05; Additional file 9).
Loss of PU.1 binding contributes to aberrant DNA methylation
To identify PU.1 target genes associated with the preleukemic hypermethylated CGIs, we searched for overlaps between the genomic coordinates of PU.1 binding sites obtained from publicly available PU.1 ChIP-Seq data [30] and the coordinates of the preleukemic hypermethylated CGIs. We found an overlap of 109 genes or other genomic locations (Figure 4B). In a more stringent search, we selected only the preleukemic CGIs that were covered by at least two hypermethylated probes. Here, 17 from 214 preleukemic hypermethylated CGIs overlapped with a PU.1 ChIP peak (Figure 4B; Additional file 11). Enrichment of PU.1 targets among the preleukemic hypermethylated genes was significant for both search stringencies (P < -2.2e-16 for 109/787 genes and P = 0.002049 for 17/214 genes). From the 17 genes identified under higher stringency, we selected three gene promoters, Bcor, Itpka, and Hes6, for validation by quantitative methylation analysis in PU.1-kd and -wt animals. Bcor mutations have been found in AML [46], Itpka contributes to differentiation of human embryonic stem cells [47] and is downregulated in oral squamous cell carcinoma [48], and Hes6 is overexpressed in glioma and breast cancer [49, 50]. From those overlapped regions only covered by a single hypermethylated probe (109; Figure 4B), we selected a fourth gene, Tal1, a known PU.1 target, because of its function in normal hematopoiesis and leukemogenesis [51, 52]. Hypermethylation of all four PU.1 target genes was confirmed by MassARRAY in the PU.1-kd samples (Figure 4C; Additional file 12). We also analyzed the genes in four hematopoietic cell types, LSK, CMP, GMP and MEP. We found all genes similarly methylated in all cell types (Additional file 5).
Taken together, our results suggest that loss of PU.1 binding contributes to the establishment of aberrant DNA methylation patterns. Similarities between DNA methylation patterns of the PU.1-kd mice and human MDS and AML patients underscore the benefit of studying early epigenetic changes in the mouse model for the identification of genes potentially involved in onset and progression of the disease. We present various novel genes associated with AML as potential early targets for aberrant DNA methylation during leukemogenesis.
AML pathogenesis is a complex multistep process that involves an interplay of genetic and epigenetic aberrations. The time from disease onset to its full-blown clinical picture requires detailed knowledge about the timing of disease-driving molecular mechanisms to successfully interfere with these processes by targeted therapy. Here, we addressed the question whether epigenetic aberrations already contribute to the early events and processes in AML pathogenesis by utilizing a murine AML progression model with a stable down-regulation of the hematopoietic transcription factor PU.1 [22]. We characterized DNA methylation dynamics over three stages of disease development and demonstrated that distinct DNA methylation changes occur early and subsequently expand during leukemogenesis. The reliability and relevance of methylation assessment by our genome-wide, array-based approach was corroborated by independently confirming 34 of 40 selected genes/chromosomal locations using quantitative, high-resolution mass spectrometry.
PCA with the DNA methylation values of all CGI array probes distinguished late leukemic stage from preleukemic and early leukemic stage animals. Preleukemic animals with low or absent myeloblasts already exhibited a high number of hypermethylated sequences, indicating extensive involvement of epigenetic mechanisms at this stage. These sequences represented 762 genes or other genomic locations. Approximately one-fourth of the preleukemic hypermethylated sequences were consistently hypermethylated throughout all leukemogenic stages, underscoring the pathogenic relevance of the affected genes for disease initiation and progression. Compared to hypermethylation, hypomethylation was considerably less abundant, probably due to its preferential occurrence outside of CGIs. Thus, our findings highlight that CGI hypermethylation accompanies AML onset and, therefore, may contribute to AML development.
From the preleukemic to the early leukemic stage, the number of hypermethylated probes appeared largely stable. At the late leukemic stage, however, the number of hypermethylated probes strikingly increased approximately 20-fold, accompanied by genomic diversification of DNA methylation. Cluster analysis of quantitative methylation values clearly discriminated between PU.1-wt and PU.1-kd animals.
The increase of aberrant DNA methylation abundance in the late leukemic stage cannot simply be explained by the mere increase in blast counts, but might rather be the consequence of a vigorous 'epigenetic' clonal evolution or of severe disturbance of the epigenetic machinery. When we examined the methylation levels in diverse hematopoietic progenitors at the preleukemic stage, all cell types displayed hypermethylation, similar to that of the bulk of myelogenic cells at this stage. Accordingly, we could so far neither dissect the AML cell of origin nor attribute leukemic expansion to the expansion of a distinct hematopoietic lineage.
Our study supports a model of an epigenetic outburst targeting distinct regions early in disease progression. This could be a consequence of genetic alterations in enzymes regulating epigenetic patterns, such as gene mutations found in human myeloid malignancies, including DNMT3a, TET2, IDH1, IDH2, EZH2, or ASXL1[53]. With respect to the dramatic outburst of aberrant DNA methylation in the late stage, our AML-like mouse model differs from a recently reported chronic lymphocytic leukemia-like mouse model, where early DNA methylation events are followed by a gradual increase of aberrantly hypermethylated genomic regions over time [54].
We identified a wealth of known and novel AML-associated genes, epigenetically altered already at the preleukemic disease stage, and provide a repository of 762 early hypermethylated and 504 hypomethylated genes, together constituting a valuable resource for investigating potential key pathogenic factors in AML. Since methylation of cytosine is a reversible epigenetic modification, and demethylating drugs are already used in the clinical setting for treatment of both MDS and AML patients [55, 56], the novel early candidates identified in this study may point towards druggable mechanisms and pathways for targeted therapy. In line with observations by others [36, 37], a prominent role at disease onset can be ascribed to the Wnt signaling pathway, since members of this pathway, Fzd5, Fzd8, Fzd10, and Wnt3 (Additional file 7), were overrepresented among the early aberrantly methylated targets. The link between Wnt signaling and the PU.1-kd-driven AML mouse model is corroborated by earlier observations that PU.1 is targeted by Wnt pathway members [21].
We detected a considerable overlap between early aberrantly methylated genes and genes involved in human myeloid malignancies (MDS and AML) [17], indicating the relevance of the observed epigenetic changes in the mouse model for human disease. Hypermethylated genes in the preleukemic stage such as Cebpa and Hic1 have already been described as being hypermethylated as well in AML [57, 58]. Moreover, normal karyotype AML and MDS patients (of different WHO subtypes) displayed hypermethylation of the Wnt pathway members FZD5 and FZD8, as observed in the mouse model. We confirmed three additional candidates, PRDM16, ROBO3 and CXCL14, to be hypermethylated in the AML patient cohort. So far, none of these five genes has been validated as being aberrantly methylated in AML by a quantitative high resolution method, albeit FZD5, FZD8, ROBO3 and PRDM16 have been found in other genome-wide methylation screens of MDS samples [17]. The concordant presence of aberrant methylation in these candidate genes already in early stages of our mouse model as well as in both MDS and AML suggests a disease driving potential of these aberrations.
It has been shown previously that binding of transcription factors to target DNA sequences may prevent their methylation [18]. In line with this, knockdown of transcription factor PU.1 was associated with preleukemic hypermethylation at a considerable number of PU.1 target sequences derived from publicly available ChIP-Seq data [30]. Looking closer at four selected PU.1 target genes by quantitative methylation analysis, we confirmed hypermethylation in both PU.1-kd animals and human AML samples. However, a correlation between PU.1 mRNA expression and methylation levels of the selected target genes BCOR, HES6, ITPKA and TAL1 could not be demonstrated in AML patients, suggesting other mechanisms than mere PU.1 down-regulation to be required for hypermethylation of these genes in human AML.
Taken together, our results suggest that the PU.1-kd mouse is a valuable model to study epigenetic changes during AML progression. The newly identified early hypermethylated genes are potential determinants for aberrant DNA methylation patterns in the disease course and, consequently, may contribute to disease development in humans. Early epigenetic changes are suspected drivers of malignancies and, hence, may offer the chance to identify suitable drug targets for early therapeutic intervention. As shown here, epigenetic profiling of tumor progression models is a promising strategy to highlight the role of epigenetics in disease initiation and progression.
In the present study, we utilized a mouse model of leukemogenesis to identify epigenetically altered genomic loci on a global scale and to determine the timing of altered epigenetic reprogramming. DNA methylation profiling of the PU.1 mouse model of leukemogenesis enabled detailed insight into the extent and dynamics of aberrant epigenetic mechanisms and created a valuable resource of early aberrantly methylated genes. We demonstrate that DNA methylation changes occur along AML pathogenesis in mice, and that these specific alterations recapitulate the alterations seen in human myeloid malignancies. Thus, this mouse model represents a suitable tool to investigate the molecular mechanisms leading to epigenetic reprogramming. We found high numbers of genes affected by epigenetic changes. The wealth of early affected loci strongly argues for a prominent role of epigenetic mechanisms in the pathogenesis and progression of MDS and AML.
Authors' information
MS and TW hold a stipend of the Helmholtz International Graduate School.
acute myeloid leukemia
bone marrow
- bp:
base pair
CpG island
- ChIP:
chromatin immunoprecipitation
chronic myelo-monocytic leukemia
common myeloid progenitor cell
differentially methylated probe
granulocyte-macrophage progenitor cell
- kd:
low density mononuclear cell
- MCIp:
methyl-CpG immunoprecipitation
myelodysplastic syndrome
megakaryocyte-erythroid progenitor cell
principal component analysis
refractory anemia
refractory anemia with excess of blasts
refractory anemia with ringed sideroblasts
refractory cytopenia with multilineage dysplasia
refractory cytopenia with multilineage dysplasia and ringed sideroblasts
significance analysis of microarrays
World Health Organization
- wt:
wild type.
We are grateful to Oliver Mücke for technical support with MassARRAY-based DNA methylation analyses. We thank Olga Bogatyrova for support with gene ontology analyses. We thank Sarah Messnard for preparation of the bone histological sections and Anders Lindroth for providing DNA of healthy granulocytes. We thank Nadja Blagitko-Dorfs and Mahmoud Abdelkarim for preparing the AML specimens. The study was supported by fellowships from the German Research Council (DFG) to RC (CL 427/2-1), LB (Heisenberg-Stipendium BU 1339/3-1), and the DFG priority program SPP1463 to ML (LU 429/7-1) and CP (PL 202/3-1).
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- Chen SS, Raval A, Johnson AJ, Hertlein E, Liu TH, Jin VX, Sherman MH, Liu SJ, Dawson DW, Williams KE, Lanasa M, Liyanarachchi S, Lin TS, Marcucci G, Pekarsky Y, Davuluri R, Croce CM, Guttridge DC, Teitell MA, Byrd JC, Plass C: Epigenetic changes during disease progression in a murine model of human chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2009, 106: 13433-13438. 10.1073/pnas.0906455106View ArticlePubMedPubMed CentralGoogle Scholar
- Lubbert M, Suciu S, Baila L, Ruter BH, Platzbecker U, Giagounidis A, Selleslag D, Labar B, Germing U, Salih HR, Beeldens F, Muus P, Pfluger KH, Coens C, Hagemeijer A, Eckart Schaefer H, Ganser A, Aul C, de Witte T, Wijermans PW: Low-dose decitabine versus best supportive care in elderly patients with intermediate- or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy: final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. J Clin Oncol. 2011, 29: 1987-1996. 10.1200/JCO.2010.30.9245View ArticlePubMedGoogle Scholar
- Lubbert M, Ruter BH, Claus R, Schmoor C, Schmid M, Germing U, Kuendgen A, Rethwisch V, Ganser A, Platzbecker U, Galm O, Brugger W, Heil G, Hackanson B, Deschler B, Dohner K, Hagemeijer A, Wijermans PW, Dohner H: A multicenter phase II trial of decitabine as first-line treatment for older patients with acute myeloid leukemia judged unfit for induction chemotherapy. Haematologica. 2012, 97: 393-401. 10.3324/haematol.2011.048231View ArticlePubMedPubMed CentralGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


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Images of Brian Zartman(0 from 0 )
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Brian Zartman (Age: 41)
46580 Warsaw IN
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07724 Eatontown NJ
Brian K Zartman (Age: 52)
45880 Payne OH
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1999-2000 Football | Cardinals Hall of Champions
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Twitter Program (Automatic Read & Monitor Twitter Handle and Respond based on specific Tweet Format) - repost
- Status Closed
- Budget N/A
- Total Bids 3
Project Description
Looking to get this done fast - 3-8 days.
Program specification: (Could be client or mobile based)
1. Monitor a specific group of twitter handles (i.e. nikeboston, nikeroosvelt, etc) for structured messages
2. Upon a specific tweet from targeted handle, send a FORMATED direct message back to the handle
A. The DM's sent must be able to attach the specific hashtag sourced from the tweet posted and formatted as #specifichastag.
B. Must attach and direct message source twitter handle with hashtag and pre-entered/formated data containing 3 dimensions. Example #hastag, FirstName, LastName, size
App Features
App should be able to include multiple names and shoe sizess and once a target twitter account post's an RSVP tweet, the software will immediately send direct messages, each from multiple twitter account, in the specific format, with the random hashtag.
Speed is a priority, direct messages should be sent within 0-5 seconds from when the tweet is posted.
Program to work on ms WINDOWS, MAC OS LION 10.7.5
Use Case:
Example of the situation: A Nike Account User will post a tweet similar to this "Twitter #RSVP is now open for the Air Jordan 8 in sizes 7-12, 13, 14. #AJ8; Once the user posts this tweet, I want to construct an app that will automatically send a Direct Message to that same user containing some pre written text as well as the random hashtag, which in the example is #AJ8. An entire example of the complete direct message I need to send back in response to the user is " #AJ8, Jon Doe, Size 10,;
For fast delivery willing to pay moreGet free quotes for a project like this
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What do Kate Winslet, Ricky Gervais and Tony Blair have in common? Aside from a propensity for making embarrassing speeches, that is. The answer, unexpectedly, lies with butterflies.
They are among 80 artists, designers, writers and politicians who have contributed to The Butterfly Book, a charity art book in aid of Leukaemia Research.
Winslet drew a "butterfly person", inspired by her daughter Mia, while Gervais, drawing on his Flanimals cartoons, provided a bigger, hairier version. Blair sent in a silk butterfly plucked from a display at an official dinner. Among the artists, Grayson Perry has dreamed up a typically outrageous killer insect with swastikas on its wings and nipple piercings (yes, his butterflies have nipples) while Cornelia Parker created a Rorschach blot of a butterfly using snake venom for ink. Other contributors, from a wide field, include Gavin Turk, Manolo Blahnik, Ewan McGregor, Maggi Hambling, Matthew Williamson, Alan Ayckbourn, and Quentin Blake.
Alongside the £25 hardback book there are also 70 limited-edition books for sale which come with one of the original artworks inside, costing £100. The artworks are pre-sealed and there is no way of choosing: like the popular Royal Academy postcard exhibition, it's a case of lucky dip, meaning you could end up with a £100 Peter Blake bargain or, less luckily, a doodle from Ann Widdecombe.
Among the non-visual contributions, Lembit Opik has written a verse comparing the flight of the butterfly to the unpredictability of life, Jeffrey Archer sent in an ode written by somebody else (plus ça change) and Jeanette Winterson summed up the transient appeal of the most beautiful – and rapidly disappearing – of insects with a poem: "I love them because they are so brief and so beautiful. A line of poetry. A run of colour. A bar of music. Gone."
- More about:
- Butterflies
- Grayson Perry
- Jeffrey Archer
- Lembit Opik


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Welcome. Happy. Safe.
Loudon, Tennessee Pet Friendly Hotels & Lodging
Looking for a hotel in Loudon, TN that you can bring your pet to? We made sure that each one of the hotels below are pet friendly, so you can enjoy your trip with your pet by your side! Once you have found a pet friendly hotel, you can book your stay instantly from our website or call 866-212-1803 to speak with a pet travel agent.
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Rates from
1500 Highway 72 - Loudon, TN (Map)
Americas Best Value Inn - Loudon/Lenoir City is pet friendly. The pet fee is $7 per pet, per day.
Overview | Pet Policy | Map
Rates from
12452 Hwy 72 North - Loudon, TN (Map)
Pet guests are welcome at pet friendly Inn of Loudon. Each room can have no more than 2 pets which can be up to 165 pounds. The pet fee is $10 per pet, per night.
Overview | Pet Policy | Map


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Jessica's Day With Tina, JT, and Thom Yorke
Jessica's Day With Tina, JT, and Thom Yorke
Just like Mary-Kate Olsen, Justin Timberlake and Jessica Biel hit up the Hollywood Bowl last night to check out Radiohead. While it was hopefully less of a logistical nightmare than the band's show in SF on Friday night, it was certainly more star-studded. Before tending to their rock concert needs, Jessica took her four-legged best friend Tina for her usual walk. Seems like a relaxing Monday for Biel after an exciting weekend showing off her short film at the Palm Springs International Film Festival's Short Fest. Justin didn't join her for that red carpet, but he did come out to see the screening, support his girl, and celebrate, aww.
Source and X17 Online


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|Greg Stein <gst...@lyra.org>
|linking libraries (was: Re: cvs commit: httpd-2.0 configure.in)
|Thu, 21 Dec 2000 21:59:48 GMT
On Thu, Dec 21, 2000 at 07:26:17PM +0100, Sascha Schumann wrote: > > That might work for libmain, if I could figure out our build system to > > implement it. But, for apr-util, which must be usable outside of Apache > > as either a shared library or a static one, a convenience library isn't an > > option. > > Why cannot we create a two step process for apr-util then? > > 1. Create a convenience library. > > 2. Create a normal libtool library, pulling in the > convenience library. > > Apache could then use the lib produced in step 1, while the > result from step 2 would be installed (or whatever you are > doing with it). This would be good, in general. I can easily see other libtool-based apps wanting a convenience library. In other words, APR(UTIL) would build three things: 1) classic .a object archive 2) .so shared library 3) libtool convenience library Sascha: what is the right magic for doing this? [ I'd also like to know because I think it might be useful within SVN ] Cheers, -g -- Greg Stein, http://www.lyra.org/
|View raw message


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They can be soooooooooooo goooooood at guilt trips, can't they?
----- Original Message -----
From: <firstname.lastname@example.com>
To: <email@example.com>
Sent: Monday, July 12, 2004 2:03 PM
Subject: Re: [CHAT] OT: food bowl
> In a message dated 07/12/2004 8:48:52 AM Eastern Daylight Time,
> email@example.com writes:
> My cat, Hecate, only likes to drink running water in the bathroom sink. I
> cannot tell you how many times I have spit on her head! She jumps in the
> sink
> every time I turn on the faucet.
> One of my young cats, Lottie, wants to drink from the kitchen faucet. She
> tries to get
> to the water every time it is turned on, and begs for me to turn it on
when I
> am doing
> any kind of food preparation. The problem is that if I let her drink more
> than a few
> sips, she will throw it up on the floor shortly afterward. I've tried to
> explain to her why
> I don't let her have all the water she wants to drink that way - there is
> always a good
> supply of water in her dish - but she still pretends to think I am not
> kind to her.
> Auralie
> ---------------------------------------------------------------------
> Support hort.net -- join the hort.net fund drive!
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Hotel Andreina
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TripAdvisor Reviews Hotel Andreina Rome
Travel Blogs from Rome
A trip to the Vatican City – a most grand pl
... the square was
being shut down for tours at about 1:00 PM. We did not have time to go to the
Vatican Museum and the Sistine Chapel but I would certainly like to do that
another day.
It was well worth the effort to
stay in line and St Peters Basilicas was a truly amazing place to enter. I
could take pictures but they cannot in any way do it Justice We were not kicked
out after a length of time and could stay as long as we ...
Roman Forum
The Roman Forum is the center of ancient Rome where many ruins lay. Many of these ancient ruins were government buildings that helped make Rome what it is today. It is thought to be the most celebrated area of Rome,as well. The Roman Forum is located a valley between the Palatine Hill, which basically was ancient Rome's home to the elite and the ...
... buy our Roma Passes after only a few detours and
Wrong turns
We tour the Forum following the voice of Rick Steves
On my IPAD Mini
He's corny but there with the facts
At every turn
Next is the Coliseum
Again, old Rick to the rescue
By this time - it's almost 2
So, we forego the bus and grab a taxi
To the Pantheon
An amazingly beautiful, perfectly formed open church
To ooh and aah at ...
When it rains, it pours
... toilets seem pretty scarce in Rome!
The building is designed in an art nouveau style.
We passed another church, Santa Maria Maddalena. But we'd had enough churches for the day, and we wanted to get to the Trevi Fountain before the rain got heavier, so we didn't ...
Rome Day 2
... of fresh basil, WOW, 3 pasta dishes with a few glasses of very nice Chianti, arancinni balls and the best of all .... Gelato .... Everyone needs to come here and eat this Gelato. Of course it wasn't all food. There was a bit of history included. A pyramid....yes I know you didn't know Rome had a Pyramid right. Well there is one smack bang in the middle of Testaccio, been there since about 18 BC. We ate in ...


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By John
on December 12, 2013
Arts at its finest quality together with the ambience of nature opened it's doors to the young and master artists to showcase their masterpiece to the public.
Artists and their crew began to put up their booths and soulful arts to the public as critiques, enthusiast and visitors marvel at their genuine work of art.
Rising to the peak of excitement, the event finally freed its viewers with the ribbon cutting ceremony and followed by a media interview of Greenfield District's Executive VP Atty. Duanne A.X. Santos.
Blending modern culture and lifestyle to contemporary and passé of creativity and uniqueness of arts, Greenfield District opened its doors to a fresh and vast horizon, giving visitors the unforgettable experience of nature inside urban living.
Artists across the country gathered to present and sell their masterpiece to collectors and art enthusiasts showcasing everything from paintings to crafts that an artist can think of...
A lot of booths can also be visited and arts to be viewed made by prominent local artist of the country and their art gallery booths as follows: Art Nove, Big Gallery, Gallery Joaquin, Galeria de las Islas, Craft MNL, EDI+ION, Coast Thru Life, Chae'z Kuro, Cevio Art House, Art Verité, une ART hed, Prometheus, OMG (One Manila Gallery) NOW Gallery, Metro Gallery and Liongoren Gallery.
As they etched their names in history using their skills and artworks, some of the masterpiece were made beyond your wildest imagination as it will tease your creativity and appreciation for art.
While others are solely for entertainment... Like these... But still a piece of art is still a piece of art right?
Selling some of their artworks not to grab the opportunity of being rich and famous but a portion of the proceeds will go to a charitable institution that will greatly help the people affected by the super typhoon #yolandaPH.
A flicker of curiosity was entertained by Atty. Santos on our lunch interview at Kanin Club, at The Hub as the social media's turn to throw questions at the organizing team and to Atty. Santos.
Film viewing was also made available featuring the winners of Gawad Urian and unfortunately, we weren't able to watch it due to time constraints and the weather condition.
Together with Jaja Ramirez of www.TheRogueWriter.net and Me of ManilaZine (also a writer / contributor to www.Azraelsmerryland.blogspot.com) pinched a question "what inspired you to produce such an art event like this?" as Atty. Santos answered "This is to showcase Filipino contemporary and traditional arts to be appreciated by the future customers and artist young and masters alike outside the central parks of urban living." a good cause indeed.
Heightened with the excitement, local and famous foods were served as we dine together for lunch like a family as we have shared and enjoyed each and every serving.
For more information about the Art for Generations event, like them on facebook at GreenfieldDistrictArtFair and for information on Greenfield Development Corp. visit www.greenfield.com.ph, and get in touch with them thru e-mail at email@example.com lastly, you can reach them at (02) 72-GREEN, (02)53-GREEN and 0917-53-GREEN.
This is one terrific and valuable event to be treasured by young and old, future cutomers and enthusiast and the best of all for the world to appreciate the wonder and beauty of ART. - John Bautista of ManilaZine
This is my VLOG channel, pls watch and SUBSCRIBE https://www.youtube.com/user/azraelcoladilla/
Tuesday, December 17, 2013
2013 Art for Generations - A 2-day art exhibit at it's finest in Greenfield District, Mandaluyong City
By John


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