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Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38−/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).
Citation: Hanoun M, Eisele L, Suzuki M, Greally JM, Hüttmann A, Aydin S, et al. (2012) Epigenetic Silencing of the Circadian Clock Gene CRY1 is Associated with an Indolent Clinical Course in Chronic Lymphocytic Leukemia. PLoS ONE 7(3): e34347. doi:10.1371/journal.pone.0034347
Editor: Osman El-Maarri, University of Bonn, Institut of Experimental Hematology and Transfusion Medicine, Germany
Received: September 30, 2011; Accepted: February 26, 2012; Published: March 28, 2012
Copyright: © 2012 Hanoun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Accumulating epidemiological and genetic evidence indicates that disruption of circadian rhythms may increase the susceptibility for developing cancer including non-Hodgkin lymphoma (NHL) and also adversely affects tumor progression [1]–[5]. At the molecular level, several genes constituting the clock machinery have been found to establish functional interplays with regulators of the cell cycle, and disrupted expression of these genes has been shown to result in aberrant cell proliferation [2], [3], [6], [7]. In a previous study we first described disturbances in the molecular circadian machinery of CLL cells and hypothesized that these alterations may play a role in the molecular pathogenesis of the disease [8]. In particular, we found that the core circadian gene CRY1 is up-regulated in samples from high-risk ZAP-70+/CD38+ CLL patients as compared to their ZAP-70−/CD38− counterparts which are characterized by a more benign clinical course [8], [9]. Therefore, based on these data we and others have proposed that CRY1 may serve as a novel prognostic factor which could be useful for the clinical management of CLL patients [8]–[11]. Functionally, CRY1 has been shown to be essential to the maintenance of circadian rhythm because of its role in the negative arm of the circadian feedback loop [12]. However, independent of its circadian function it may have an additional role as a transcriptional regulator of a number of genes involved in cell metabolism and proliferation [4], [6], [12], [13]. In the current study we further investigated the role of CRY1 by comparing its expression pattern in molecularly defined CLL subgroups to that of B cells obtained from the peripheral blood of normal donors. Furthermore, we aimed to determine the molecular mechanism(s) underlying deregulated CRY1 expression in CLL.
Materials and Methods
Patients and samples
Peripheral blood samples from 76 CLL patients and 35 normal donors (ND) were analyzed after obtaining written consent according to our institutional guidelines, approved by the Ethics Commission of the University of Essen-Duisburg. The diagnosis of CLL required a persistent lymphocytosis of more than 5.0×109/l and a typical CD19+, CD20+, CD5+, CD23+, Ig light chain (κ or λ light chain) restricted immunophenotype as revealed by flow cytometry of peripheral blood cells [14]. Blood samples from CLL patients and ND were drawn in the morning hours and immediately processed. Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep density gradient centrifugation (Invitrogen, Karlsruhe; Germany) and cryopreserved until further analysis. Patient selection for this study was based on the availability of viably frozen DMSO preserved PBMC and/or freshly isolated total RNA stored in our CLL cell bank. Clinical and laboratory data of the study population are shown in Table 1.
Five healthy donors and a subgroup of 57 CLL patients were subjected to DNA methylation analysis. Patient characteristics of this subgroup are shown separately in Table S1. For this set of experiments CD19+ cells were positively selected from PBMCs employing the EasySep Human CD19 Selection Kit (StemCell Technologies, Canada) according to the manufacturer's instruction resulting in a >90% purity of CD19+ B cells. DNA was isolated from the immunomagnetically purified CD19+ cell fraction using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany).
Flow cytometry
Cell surface expression of CD38 was examined by flow cytometry using a previously described panel of fluorochrome-labeled monoclonal antibodies (CD38 PE, clone HB7) in a standard three-color flow cytometry approach using a 20% cut-off to define CD38 positivity [15], [16].
Fluorescence in situ hybridization (FISH)
Prognostically relevant anomalies of chromosomal regions 11q, 13q, and 17p, and of chromosome 12 were assessed by fluorescence in situ hybridization, as described previously [16].
Real-time reverse-transcriptase-PCR (qRT-PCR)
Total RNA from PBMC was extracted with RNeasy Midi Kit (Qiagen, Hilden, Germany) and spectrophotometrically quantified as previously described [8]. First strand DNA was synthesized from 1 µg of RNA using oligo(dT) primers employing a commercially available kit (RT-PCR Amplimers, Becton Dickinson, Heidelberg, Germany) according to the manufacturer's instructions. Real-Time PCR was performed with the ABI Prism 7900HT Sequence Detector (Applied Biosystems, Foster City, CA, USA) using the TaqMan Universal PCR Master Mix protocol (Applied Biosystems). Specific assays for CRY1 (Hs00172734_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), BMAL1 (Hs00154147_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), CLOCK (Hs00231857_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), PER1 (Hs00242988_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), PER2 (Hs00256144_m1, Assays-on-Demand Gene Expression System, Applied Biosystems) and GAPDH (Hs99999905_m1, Assays-on-Demand Gene Expression System, Applied Biosystems) were used. All reactions were carried out in a 10 µl final volume containing 5 µl Master Mix, 0.5 µl of the specific assay and 4.5 µl of 1∶2 diluted cDNA. The amplification was performed under following conditions: 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s and annealing/elongation at 60°C for 1 min. Standard curves for all assays show similar gradients (coefficient of variation 6.8%, data not shown). CRY1 mRNA expression was normalized against the housekeeping gene glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous reference by computing the difference between the respective Ct values (ΔCt = Ct[gene]−Ct[GAPDH]). All PCR reactions were performed in duplicate (mean coefficient of variation for all target genes was below 1%). The mean threshold cycle number (Ct) for each tested mRNA was used to quantify the relative expression of each gene: 2−ΔCt.
DNA methylation analysis of the CRY1 gene by bisulphite genomic sequencing.
Bisulphite treatment of genomic DNA was carried out using the EpiTect Bisulphite Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. The primers for amplifying bisulphite-modified DNA were: CRY1-forward: 5′-TTTGTGAGGGAAGGTTTAGTTT-3′, CRY1-reverse: 5′-AACAATTTCCAAACCCTCC-3′. For possible sequencing of the PCR product we attached a tag (forward 5′-CTTGCTTCCTGGCACGAG-3′, reverse 5′-CAGGAAACAGCTATGAC-3′). The PCR was carried out as follows: denaturation at 95°C for 10 minutes followed by 35 cycles comprising a second denaturation of 30 seconds at 94°C, annealing at 61°C for 30 seconds and extension at 72°C for 45 seconds, followed by 7 minutes elongation at 72°C. The sequence of the PCR product is depicted in Figure S1. PCR products were separated by agarose gel electrophoresis, excised and purified by gel extraction with MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). PCR products were ligated into pGEM®-T Easy Vector (Promega, Madison, USA) and transformed in XL1-Blue Competent Cells (Stratagene, La Jolla, CA, USA). Plasmid DNA isolated from multiple colonies derived from each PCR product were sequenced using the CRY1 reverse primer on an ABI 3130 Genetic Analyzer.
DNA methylation analysis of the CRY1 gene by Bisulphite MassArray assays.
Bisulphite treatment of genomic DNA was carried out using the EpiTect Bisulphite Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. The reverse primer contained a T7-promoter tag for in vitro transcription (5′-cagtaatacgactcactatagggagaaggct-3′) and the forward primer was tagged with a 10 mer (5′-aggaagagag-3′). Bisulphite-treated DNA was PCR amplified (denaturation at 95°C for 10 minutes followed by 40 cycles comprising a second denaturation of 30 seconds at 95°C, annealing at 61°C for 30 seconds and extension at 74°C for 30 seconds, followed by 10 minutes elongation at 74°C), PCR products were purified by gel extraction with MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). 5 µL of the PCR product were used for the assay. Bisulphite MassArray assays were performed by the Genomics Core Facility, Albert Einstein College of Medicine. The data were analyzed using the analytical pipeline previously published [17]. DNA quality and no-template controls, 0%, and 100% methylated DNA were included in all assays.
50 K SNP array analysis of CRY1 copy number at chromosome 12q23.
For SNP array studies, genomic DNA was extracted from CD19 positively selected PBMCs using the QIAamp blood kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Array experiments were performed according to the standard protocol for Affymetrix GeneChip Mapping 50 K arrays (Affymetrix). Briefly, a 250 ng sample of DNA was digested with XbaI, ligated to adaptors, amplified by PCR, fragmented with DNAse I, and biotin-labeled. The labeled samples were hybridized to the Affymetrix 50 K SNP XbaI mapping array (Affymetrix Inc., Santa Clara, CA) followed by washing, staining and scanning.
The acquired signal data was normalized with the dChip [18] program, using model-based expression, perfect match (PM)-only background subtraction and quantiles as probe-selection method. The normalized signal was then used as raw copy number per SNP and further analyzed by the GLAD algorithm [19] included in the GenePattern [20] suite, which segmented the data and assigned aggregated copy numbers to segments. A segment was defined as aberrant if its copy number was below 1.7 (loss) or above 2.3 (gain). The resulting list of aberrant regions was in addition filtered for regions consisting of more than 10 SNPs to exclude regions resulting from random noise in the copy number signal.
Statistical analysis
Comparisons of clinical and biological parameters between subgroups were carried out using the Mann-Whitney-U test for continuous variables and Fisher's exact test for categorical data. Correlation between CRY1 delta CT and percentage of promoter methylation was tested using Spearman correlation. The Wilcoxon test for paired samples was used to compare CRY1 delta CT analysed in B and T cells from ND. Survival analysis was carried out with the Kaplan-Meier method and differences in treatment free survival between risk groups were tested with the log-rank test. All analyses were performed using R statistical software version 2.10.1 (R Development Core Team, Vienna, Austria, http://www.r-project.org) and GraphPad Prism Version 5.04 (GraphPad Software).
Analysis of CRY1 expression in normal donor derived vs. CLL PBMC
We measured the mRNA expression of the circadian gene CRY1 in peripheral blood mononuclear cells (PBMC) containing more than 80% CD19+CD5+ leukemic B cells as determined by multiparameter flow cytometry in a cohort of 76 CLL patients (Table 1). In line with our previously published work [8], [9] we observed significantly higher CRY1 mRNA levels in high-risk(HR) patients defined by the expression of CD38 and/or unmutated IgVH genes as compared to their CD38 negative and/or IgVH mutated low risk (LR) counterparts (Figure 1A, 1B). We could also confirm the prognostic value of CRY1 expression in CLL by comparing the clinical outcome of CLL patients with high vs. low CRY1 expression using the median ΔCt value as a cut-off. Significant differences in treatment free survival (TFS) were observed between the two groups (Figure S2). Of note, comparative analysis with PBMC derived from age matched normal blood donors revealed that these expression differences can be attributed to an under-expression of CRY1 in LR CLL cases rather than over-expression in the HR group (Figure 1A, 1B). As the cellular composition of normal PBMC and PBMC isolated from CLL patients is known to vastly differ with regard to the content of B and T cells we compared mRNA expression levels of CRY1 in immunomagnetically purified CD19+ B cells and CD3+ T cells from normal donors (Figure S3). CRY1 expression was very similar in these normal lymphocyte subsets suggesting that under-expression in LR CLL samples relative to ND controls reflected true down-regulation of the gene in the leukemic cells rather than differences in the cellular composition of the PBMC samples.
CRY1 mRNA expression in normal donors (ND, n = 35) in comparison to CLL samples from prognostic subgroups defined by CD38 expression (A, CD38+ samples, n = 36 vs. CD38− samples, n = 39) and IgVH mutational status (B, IgVH unmutated/V3-21, UM/V3-21, n = 23 vs. IgVH mutated, M, n = 18). mRNA levels are relative to GAPDH. Data are presented in a box-and-whisker format: the difference of the 25th and 75th percentile form the box (interquartile range, IQR), with the median marked as a line; the whiskers go down to the smallest value and up to the largest values. The Mann-Whitney U-test was used to compute p-values for pairwise comparisons.
Dysregulation of CRY1 in CLL cannot be explained by chromosomal aberrations
Aiming at investigating the molecular mechanism(s) underlying disrupted CRY1 expression in CLL cells we then speculated that this phenomenon may be explained by aberration of this gene at chromosome 12q23–q24.1. Exploiting a panel of CLL cases which had been previously investigated for the presence of structural chromosomal abnormalities [21] by high resolution SNP array profiling we could not detect loss of chromosomal material at the CRY1 locus in any of the 55 individual CLL samples analyzed (data not shown). Moreover, an elevated copy number at this position is only detected for samples harbouring a trisomy 12, all other samples exhibit the regular two copies of this gene. The CLL patient cohort of this study (n = 76) comprises 12 cases with a trisomy 12. Among the CD38+ population 10 patients had a trisomy 12 whereas 14 were negative. Among the CD38− patients 2 had a trisomy 12 whereas 28 subjects proved to be negative. No differences in CRY1 mRNA expression were noted neither in the CD38+ nor the CD38− subgroups defined by the presence or absence of trisomy 12 (p = 0.54 and 0.49, respectively).
DNA methylation analysis of the CRY1 gene
As CRY1 has been previously shown to undergo aberrant DNA methylation events in various human cancers [7], [22], we then examined whether epigenetic silencing could explain CRY1 mRNA expression differences in CLL. DNA methylation was studied within the promoter region of the CRY1 gene (Figure S1) employing sequencing of cloned PCR products generated from bisulphite-modified DNA extracted from immunomagnetically purified CD19+ B cells from the peripheral blood of CLL (N = 14) and normal donors (N = 4). We observed that patients with low CRY1 expression show hypermethylation of the analyzed region in comparison to patients with a high CRY1 expression and normal donors (Figure 2A). Using the Sequenom MassArray Compact System for methylation analysis in the same CRY1 promoter region we then confirmed these results in a cohort of 58 CLL samples and 5 normal donors (Figures 3A and S4, Table S3). LR CLL patients either defined by CD38 or IgVH mutational status showed a significantly higher degree of methylation of the CRY1 promoter in comparison to the HR group. In all, there was a statistically highly significant inverse correlation between the percentage of methylated CpGs and CRY1 mRNA expression as detected by qRT-PCR (Figure 2B and 3B). Comparing both methods for methylation analysis a high correlation (r = 0.89, p<0.0001) proves the consistency and robustness of each of the applied methods (Figure 3C).
A Results of bisulphite genomic sequencing performed on 6–12 clones of 4 normal donors (ND) and 12 different CLL samples. Top, schematic depiction of the analyzed area of the CRY1 CpG island around the transcription start site (horizontal black line). Short vertical lines represent CpG dinucleotides. The location of PCR primers are indicated as bold black horizontal arrows. Presence of methylated or unmethylated CpG dinucleotides is indicated by solid or open squares respectively, blue squares indicate not analyzable CpG dinucleotides. The mean standard error for all samples is 1.99 reflecting the low variability between the methylation profile of each analyzed clone. At the right hand side of each graph, first the percentage of methylated CpGs for each individual sample is depicted, below is the percentage of methylated CpGs measured by Bisulphite MassArray and at the bottom each sample's CRY1 mRNA expression relative to GAPDH is shown. B Samples from CLL patients and ND were subjected to both CRY1 mRNA expression and DNA methylation analysis by bisulphite genomic sequencing. mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = −0.62, p = 0.006, Spearman correlation). The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable (open circles indicate ND).
A Results of CRY1 CpG island methylation analysis performed on CLL samples and normal donors (ND) grouped by CD38 expression (CD38− samples, n = 28; CD38+ samples, n = 30; ND, n = 5). Each value represents the average amount of methylated CpGs of all analyzable CpGs within the CpG island promoter from one patient. The values represent the mean of duplicate experiments. The IgVH mutational status of each patient (if available) is highlighted in red; circles and squares indicate unmutated IgVH/V3-21 and mutated IgVH status, respectively. The median is marked as a line, error bars indicate SEM. Unpaired two-tailed t-test was used to compute p-values. B Samples from CLL patients and ND were subjected to both CRY1 mRNA expression and DNA methylation analysis with the bisulphite MassArray assay. mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = −0.63, p<0.0001, Spearman correlation). The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable. C Correlation between the methylation data resulting from bisulphite genomic sequencing and the MassArray method showed high consistency (r = 0.86, p<0.0001, Spearman correlation).
Prognostic value of CRY1 methylation pattern in CLL
To assess the prognostic value of the methylation profile of the CRY1 promoter we defined two groups, i.e. patients with lowly methylated and highly methylated promoter region. The threshold value of 9% was derived from the median value of the CLL samples subjected to MassArray methylation analysis. On survival analysis patients with a highly methylated CRY1 promoter region exhibited a significantly longer treatment-free survival as compared to the hypomethylated patient subgroup (Figure 4).
Treatment-free survival of 53 CLL patients with lowly methylated (n = 26) and highly methylated (n = 27) CRY1 promoter.
Analysis of CRY1 expression over time
Twenty-seven patients were studied at two or more time points using the qRT-PCR assay. As illustrated in Figure S5, CRY1 mRNA expression was relatively stable over time in the majority of patients with stable disease (10/13 (77%), Figure S5, left panel) as well as in patients with progressive disease (12/14 (86%), right panel). The fact that three of the five patients with increasing CRY1 mRNA levels had a comparably benign course of disease may suggest that changes in CRY1 expression over time are not stringently related to disease progression. Moreover, in 6 of the 14 patients (marked by arrows in Figure S5) with a progressive course of the disease first treatment was initiated after collection of the baseline sample in this longitudinal analysis. Although all these patients responded to therapy, no reduction in CRY1 mRNA expression could be documented. It is noteworthy that we did not observe a single case with decreasing CRY1 mRNA levels during follow-up in the entire cohort of patients with samples available for sequential analysis.
Interestingly, one patient with a low baseline CRY1 expression and a correspondingly high degree of CpG island methylation experienced disease progression as evidenced by rising lymphocyte counts and transition from Binet stage A to stage C disease. Increasing CRY1 expression in this particular patient was shown to be associated with loss of CRY1 promoter hypermethylation (Figure S6).
Analysis of the role of CRY1 within the circadian clock's transcription/translation-based feedback loop in CLL
At the molecular level, circadian rhythms are encoded by an autoregulatory loop composed of a set of transcription activators (CLOCK/BMAL1) that induce expression of PER and CRY. Accumulated PER and CRY proteins in turn inhibit BMAL1/CLOCK transcriptional activity and repress their expression. Thus, aberrant silencing of CRY1 expression in LR CLL may be associated with dysregulated expression of other circadian genes. Indeed, we could observe increased PER2 and CLOCK mRNA levels in CLL as compared to ND derived PBMC, whereas mRNA levels of BMAL1 and PER1 were similar in both groups (Figure S7). However, changes in PER2 and CLOCK expression were noted in both LR and HR CLL samples and could not be related to alterations in CRY1 expression (Figure S7) suggesting that these abnormalities may occur independently of each other.
Analysis of CRY1 expression and CpG island hypermethylation in other hematologic malignancies
From a cancer biology standpoint we next wanted to test whether deregulated CRY1 expression may also be observed in other lymphoid malignancies than CLL. To this end we employed the CRY1 qRT-PCR assay to screen RNA samples isolated from a wide range of lymphoproliferative disorders including T prolymphocytic leukemia (T-PLL, n = 10), mantle cell lymphoma (MCL, n = 6), hairy cell leukemia (HCL, n = 3), multiple myeloma (MM, n = 8), plasma cell leukemia (PCL, n = 2) and B and T lineage acute lymphoid leukemia (n = 29 resp. n = 19). In disease entities with more than 5 observations, statistical comparisons with ND derived PBMC did not reveal significant expression differences (Figure 5A). However, this finding needs to be interpreted with caution because of the small numbers of samples tested in this series. The availability of a comparably large number of ALL samples (n = 48) allowed for a more detailed analysis of this disease group. The clinical characteristics of this patient cohort are given in Table S2. Similar to its distribution in CLL, CRY1 expression in ALL was found to be very heterogeneous where one patient with a mature B-ALL exhibited a markedly low CRY1 transcript level (Figure 5A). This particular sample demonstrated a high degree of CRY1 promoter methylation while two other ALL samples with a higher CRY1 expression were nearly completely demethylated (Figure 5B).
qRT-PCR analyses of PBMC samples obtained from patients with T-prolymphocytic leukemia (T-PLL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), hairy cell leukemia (HCL), B and T cell acute lymphoblastic leukemia (B-ALL, T-ALL), CLL and normal donors (ND), A. Red characters indicate samples that were further subjected to DNA methylation analysis of the CRY1 promoter, A. Analysis of CRY1 CpG island promoter methylation status, B. For experimental details and description of the graph in panel B refer to the legend of Figure 2.
Next we investigated the prognostic value of CRY1 expression in ALL. To this end we compared the clinical outcome of ALL patients with high vs. low CRY1 expression using the median ΔCt value as a cut-off. No significant differences in overall survival (OS) were observed between the two groups (Figure S8).
In this study, we analyzed the expression of CRY1 mRNA, which encodes a key component of the central and peripheral circadian oscillator, in the PBMC from normal blood donors and patients with CLL [8], [11], [23], [24]. In line with our previous work [8], [9] and that of others [10], [11] we detected elevated CRY1 transcript levels in patients with high risk disease defined by the expression of CD38 and/or unmutated IgVH (UM) genes as compared to their CD38 negative and/or IgVH mutated (M) low risk (LR) counterparts. Lewintre et al. confirmed these results in a recently published microarray-based gene expression profiling study including 36 patients with early-stage CLL [11]. Therefore, determination of CRY1 may have potential as a novel prognostic marker in CLL and should be tested in comparison to other established molecular risk factors in the setting of prospective randomized trials.
We now uncover that HR CLL cases and ND derived B cells exhibit comparable levels of CRY1 mRNA expression. Thus, disrupted CRY1 expression in CLL can be attributed to down-modulation of CRY1 in LR CLL cases rather than over-expression in the HR group. We then aimed to investigate the molecular mechanisms underlying down-regulation of CRY1 in LR CLL. As CRY1 had been previously shown to undergo aberrant DNA methylation events in various solid human malignancies including breast and ovarian cancers [3], [7], [22], we examined whether epigenetic silencing could also explain the observed CRY1 mRNA expression differences in CLL subgroups. To this end we performed comparative DNA methylation analysis of highly purified CD19+ B cells from the peripheral blood of 57 CLL patients and normal donors. Indeed, our results show that CRY1 is transcriptionally silenced by promoter hypermethylation in LR CLL cases while HR cases and ND derived B cells exhibit hypomethylated CRY1 promoter regions. Importantly, DNA methylation analysis was complemented by qRT-PCR performed on the same samples revealing a statistically highly significant inverse correlation between the percentage of methylated CpGs and CRY1 mRNA expression in individual cases, suggesting a direct regulation of CRY1 expression through methylation of its promoter. We could show that the methylation status of the CRY1 promoter predicts clinical outcome in CLL patients; where aberrant hypermethylation was associated with a more benign course of the disease. To date, only few reports describe an aberrant methylation phenotype as a predictor of outcome in haematological diseases. Recently, Irving et al reported a panel of methylation markers (CD38, HOXA4, BTG4) in which an overall methylation score was significantly associated with time to first treatment in CLL [25]. Olk-Batz et al [26] described that a high methylation profile is associated with an aggressive biological variant of juvenile myelomonocytic leukemia.
As stability of CRY1 expression by the leukemic cell clone over time is an important prerequisite for its reliable use as a prognostic marker [27], we sequentially analyzed CRY1 expression in 27 patients. CRY1 transcript levels were remarkably stable in the majority of patients and no consistent changes were noted in relation to alterations in disease activity and treatment history of individual patients. Significant expression changes during follow-up occurred in 19% of the patients, which could limit the value of this marker under routine clinical conditions. However, these findings are limited by the comparably small number of CLL cases analyzed in this study and thus need to be validated in a larger patient cohort. In one patient with a low baseline CRY1 expression and a correspondingly high degree of CpG island methylation disease progression was linked to increasing CRY1 expression and hypomethylation of the CRY1 promoter region. In aggregate, these findings raise the possibility that epigenetic silencing of CRY1 occurs early in the disease and may be lost during disease progression at least in rare cases. Conversely, it appears unlikely that the leukemic cells acquire epigenetic silencing of CRY1 during the course of the disease as none of the 27 patients in the longitudinal analysis showed a decrease of CRY1 expression. It would be interesting to further investigate this hypothesis by systematically comparing the prevalence of CRY1 methylation events in individuals with monoclonal B cell lymphocytosis with Binet stage A, B and C CLL patients.
While differential CRY1 expression in HR vs. LR CLL subgroups is now well established [8]–[11], [28], the functional consequences of CRY1 down-modulation in the leukemic cells are currently unknown. It is tempting to speculate that epigenetic silencing of CRY1 may contribute to the benign clinical behaviour of LR CLL cases. At first sight this notion appears counterintuitive to the general conception that the core components of the molecular clock machinery may function as tumor suppressor genes [29]–[32]. This view is mainly based on epidemiological and genetic evidence indicating that disruption of circadian rhythms might be directly linked to cancer development including non-Hodgkin-lymphoma [2], [5], [33]–[35]. While the circadian genes PER1 and PER2 have been clearly shown to function as tumor suppressors in the mouse model [30], a recent study showed that epidermal deletion of BMAL1 in a transgenic mouse model which spontaneously develops squamous tumours leads to significantly fewer neoplastic lesions [36]. Along the same line transgenic CRY deficient mice do not show a predisposition to cancer [6]. Furthermore and somewhat unexpectedly, ablation in the mouse of both CRY genes in a TP53−/− background delays the onset of cancer [13]. This latter observation supports our hypothesis that epigenetic silencing of CRY1 may functionally contribute to the indolent clinical behaviour of LR CLL. It is currently unclear whether abrogation of one CRY gene, i.e. CRY1 or CRY2 suffices to fully block the circadian rhythmicity of an individual cell. Studies comparing the biologic characteristics of transgenic mice lacking either one or both CRY genes indicate a certain degree of functional redundancy [37].
Another matter of controversy is whether circadian rhythmicity per se or only certain core clock components are involved in tumorigenesis [4]. To address this issue we correlated CRY1 mRNA expression with other components of the circadian clock's transcription/translation-based feedback loop in individual CLL samples. Recently, a number of in vitro and animal studies have further elucidated the important functional role of CRY proteins for the regulation of the molecular clock. For example, work by Ye et al [38] demonstrated that CRY directly interacts with the BMAL1:CLOCK:E-box complex independent of PER resulting in inhibition of the transactivator function of CLOCK:BMAL1. Furthermore, Busino et al. [39] showed in an in vitro model that Cry1−/− Cry2−/− mouse embryonic fibroblasts are characterized by a loss of oscillation in Per1 and Per2 and exhibit increased Per2 but not Per1 mRNA transcript levels in comparison to control fibroblasts isolated from CRY wild type animals.
Here, we found that the expression of PER2 and CLOCK but not BMAL1 and PER1 are also disrupted in CLL as compared to ND derived control samples. However, contrary to what could be expected from above described findings [38], [39] these abnormalities could neither be correlated with changes in CRY1 expression nor the clinical course of the disease in individual CLL cases. In aggregate, these latter results clearly suggest that defects in the circadian molecular machinery may be a common phenomenon in CLL cells and are not restricted to CRY1. Ongoing work in our laboratory is aimed at determining the molecular and cellular consequences of silencing different circadian genes in CLL cells in vitro using siRNA oligonucleotide technology.
Finally, we wanted to determine whether CRY1 expression may also be disturbed in other lymphoid malignancies than CLL. To this end we measured CRY1 transcript levels in PBMC samples from a range of different disease entities including T-PLL, MCL, HCL, MM, PCL and B and T cell ALL. In a subgroup analysis focussing on ALL we found a heterogeneous expression pattern comparable to that observed in CLL. One patient with a mature B-ALL exhibited a particularly low CRY1 expression which correlated with a high degree of promoter methylation, suggesting that aberrant epigenetic silencing of this gene may also occur in other lymphoid disease entities. However, contrary to CLL survival analysis did not show a significant difference between ALL patients with high and low expression of CRY1, respectively.
In conclusion, our data indicate that the previously reported CRY1 gene expression differences in LR vs. HR CLL patients [8], [11] are caused by aberrant methylation of the CRY1 promoter in the LR patient subgroup. To our knowledge this is the first report in CLL research linking epigenetic silencing of a specific gene to an indolent clinical course of the disease.
Supporting Information
Figure S1.
Map of CRY1 located on chromosome 12q23.3. The chromosomal location and nucleotide sequence of CRY1 on chromosome 12q23.3 (indicated by the red vertical line) extending from 105,909,272–106,011,728. CRY1 has one CpG island consisting of 130 CpGs depicted as the green box (106,010,743–106,011,941). Methylation analyses were performed within this CpG island from 106,011,737–106,011,970. Red letters indicate the primer sequences used for DNA methylation studies.
Figure S2.
Kaplan-Meier curves for treatment-free survival in CLL according to high and low expression of CRY1. The median CRY1 ΔCT value was used as a cut-off to define patient subgroups with high and low CRY1 mRNA transcript levels. Figure S2 illustrates the treatment-free survival in 73 CLL patients with high/low CRY1 mRNA levels. Statistical differences were analyzed by log-rank test.
Figure S3.
Comparative analysis of CRY1 expression in normal B and T cells. qRT-PCR analysis of immunomagnetically purified CD19+ B vs. CD3+ T cells obtained from five normal donors. Statistical comparisons were performed using the Wilcoxon test for paired samples.
Figure S4.
Detailed results of CRY1 CpG island promoter methylation measured with Bisulphite MassArray assays. Results of CRY1 CpG island methylation analysis performed on further 58 CLL samples subdivided by CD38 expression (CD38− samples, n = 28, CD38+ samples, n = 30, ND, n = 5). Each row represents the methylation profile of one individual patient. Putative fragmentation patterns are shown for T -cleavage reaction on the plus strand of an amplicon of the human genome. CG dinucleotides (filled circles) are numbered and color-coded according to their ability to be assayed, where gray indicates that the CG is located on a fragment whose molecular weight is outside the usable mass window, red indicates a molecular weight overlap with another fragment and blue indicates a uniquely assayable site. Fragmentation patterns are shown in corresponding colors, yellow highlights primer sequences. Methylation data are shown as an average from duplicates. Bar height denotes percent methylation on a scale from 0% (low) to 100% (high), error bars indicate median absolute deviation. CG sites that are putatively outside the usable mass window are indicated as boxes with gray background.
Figure S5.
Analysis of CRY1 expression over time. In all, 27 patients were analyzed for CRY1 expression using qRT-PCR at two or more time points. Each line represents an individual patient. The left graph depicts 13 patients with a stable course of disease, on the right 14 patients with progressive disease are shown. Three patients indicated with dashed lines had a progressive disease but were not treated (right graph). Those patients receiving first line chemotherapy after collection of the first sample (baseline) are highlighted by individual colors whereas the time point of first therapy is indicated by vertical arrows. mRNA expression values are normalized to each patient's baseline, which is set to 1.
Figure S6.
Time course analysis of CRY1 mRNA expression and CpG island promoter methylation in patient #13 showing disease progression during follow-up. Methylation analyses in panel B correspond to samples obtained at baseline (a) and after 24 months of follow-up (b). For experimental details and description of the graph in panel B refer to the legend of Figure 2.
Figure S7.
Analysis of circadian gene transcript levels in CLL cells by qRT-PCR. Figure S7 shows PER1, PER2, CLOCK and BMAL1 expression in normal donors (ND, n = 24) in comparison to CLL samples from prognostic subgroups defined by CD38 expression (CD38+samples, n = 24 vs. CD38− samples, n = 27). mRNA expression is relative to GAPDH. Data are presented in a box-and-whisker format: the difference of the 25th and 75th percentile form the box (interquartile range, IQR), with the median marked as a line; the whiskers go down to the smallest value and up to the largest values. The Mann-Whitney U-test was used to compute p-values for pairwise comparisons.
Figure S8.
Kaplan-Meier curves for overall survival in ALL according to high and low expression of CRY1. The median CRY1 ΔCT value was used as a cut-off to define patient subgroups with high and low CRY1 mRNA transcript levels. Figure S8 illustrates the overall survival for 48 patients with ALL according to high and low expression of CRY1 mRNA levels. Statistical differences were analyzed by log-rank test.
Table S1.
Characteristics of CLL patients tested for CRY1 promoter hypermethylation.
Table S2.
Clinical characteristics of ALL patients.
Table S3.
Individual methylation profile (MassArray assay) and CRY1 expression values.
We thank Sima Vagaie, Michael Möllmann, Sana Mohamad and Olga Rempel for excellent technical assistance, Anja Führer and Sabrina Kieruzel for maintaining the CLL biobank and Ute Schmücker for FACS analyses.
Author Contributions
Conceived and designed the experiments: MH LKH JD. Performed the experiments: MH. Analyzed the data: MH LE MS RS LKH JD. Contributed reagents/materials/analysis tools: JG AH SA UD. Wrote the paper: MH JD.
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Feeder Road, Shyamnagar
20+ Yrs experience
Classes: Music Hindustani Music
Sane Mihar ghorana.Disciple of pt.Tarun Bhattacharjee and pt.Shyamal Chatterjee.Trenied as vocal music and instrument like SANTOOR, harmonium, tabla.teach SANTOOR...
Mulajore, Shyamnagar
5 Yrs experience
Classes: Music, Class XI-XII Tuition (PUC) and more.
I have started teaching students since I was in class 8.After completing my ICSE I joined a kindergarten school as a part time teacher along with which i continued...
Feeder Road, Shyamnagar
3 Yrs experience
Classes: Music, Class IX-X Tuition and more.
I am a good teacher in English and Maths and also a guitarist. I have passed H.S. exam from science and taking admission to polytechnic in Computer Science.
Feeder Road, Shyamnagar
Classes: Music Vocal Music
I have no experience because i am a fresher, but i will prove my self when an opportunity was come.
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Join Date: Aug 2006
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i made a couple of aio dipes for my babe with some thin vinyl (used for tablecloths)from wal mart $1.47 a yard,and it works just as good as her pul aio.it is kind of fuzzy on the underside of the vinyl,just sandwich it between the diaper shells.works great.
found this 'ripstop' fab. the wee weka pattern refers to it for a waterproof layer. i havent seen it but i think its maybe like kite material. says waterproof.....tent repair and such.
have you ever woken up with your face against the wet inside of a tent?? or wet sleeping bad near the walls of the tent? i wonder if it wicks with pressure?
anyone ever tried this?? pretty inexpensive too
I'm surprised! I have used vinyl tablecloth fabric (to make tablecloths) and had problems with it tearing and separating from the fuzzy part. Maybe it was different stuff from what you used.
It was the Wee Weka website that made me think that maybe I could find some. Did they actually have a link to some that I missed??? My nylon covers are not the same texture as tent fabric, and don't seem to wick at all through the actual fabric. They do leak at the legs or waist if the child is very very soaked and/or the legs are gaping a bit. I think I will try other types of waterproof layers, too, but those nylon pull-ons just work so well for me.
p.s. I had wondered about the Indisposables covers but never tried them. Glad to hear that they work well!!
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Garlic Basil Steak Salad is a filling meal or elegant appetizer that's bursting with summer flavor! Have a question about one of my recipes? The best place to get answers is the Overtime Cook Recipes Facebook Group. Am I the only one who gets...panicky when the summer is almost over? Where did the time go? What happened to my dreams of days in the sun and relaxing at the beach? Summer BBQ's, farmers markets...all the dreams of wonderful ways to spend the summer, and I find myself sitting inside in my air conditioned office all over again. Luckily, the summer isn't over yet, so consider this your official wake up call. Get out there. Do the things you wanted to do. Spend some time in … [Read more...]
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Mac and Cheese Latkes and a Chanukah Blog Party and Double Giveaway!
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Desktop Table Tennis
Guide price: £11
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Desktop Table Tennis
Work can be boring and stressful at times so why not brighten up your day with a game of Desktop Table Tennis. As soon as your boss goes into a meeting set up your game on a flat surface challenge someone to a quick game and get a third person to keep their eyes open for the boss returning. Desktop Table Tennis folds neatly back into its box when not being used making it perfect for any office environment.
(Jemma Stockton) Contents: 1 x Desktop table 2 x Bats 1 x Net 1 x Ball Features: Desktop Table Tennis Great to brighten your day up Comes with everything you need for a good game Pack neatly back onto its box when not being used Perfect for any flat surface Dimensions: 40 x 54 x 5.5 cm
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Chocolaty Cake - New Year Gift Wishes for 2016
Send The Happiness And Prosperity To Your Loved Ones On This New Year With This Gift Hamper Which Includes : 1/2 kg Chocolate Truffle Round Shape Cake along with New Year Greeting card.


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Ladies! BIKE SHORTS! Please!
When it gets hot in the city, finding a speedy way to get around it a must, and riding a bike is an excellent and environmentally friendly way to cover as much ground as possible as quickly as possible. But hopping on and off of your bike can make you highly susceptible to wardrobe malfunctions, and though the breeze up blowing your skirt may feel nice, the cat calls you get can become excessive. The Solution: BIKE SHORTS! Find a cute pair to wear under your skirt and worry no more! Check out our faves, after the jump…Bike shorts are cute on their own or add flare to a mini skirt blowing in the wind. Don't be caught flashing!
Runway looks like Alexander Wang


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Take a tour through twenty-five modern log homes reborn in the Arts & Crafts tradition, and witness how Craftsman style combines with the latest in log hybrid architecture to create a totally new and unique genre of building in The Arts & Crafts Cabin. In this new title from log building expert Robbin Obomsawin, the rich Arts & Crafts heritage of the late 1800s ...more
Hardcover, 160 pages
Published April 5th 2004 by Gibbs Smith
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This is my all-time favorite design style. I love the craftsmanship involved. The photos in this book are stunning. Check out the stained-glass door on the front cover! Beautiful!
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To support their highly anticipated and widely received debut album, Zeds Dead has taken their signature bass heavy sound on the road for the Fall 2016 Northern Lights Tour. After bouncing down under to Australia from the United States, the duo are coming back ...[more]
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In the 90's, Julien Engelhard developed a passion for electronic music, emerging at that time. Self-taught, he participates in unauthorized events development, wild parties...
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Caribbean Pineapple
Total Time
Prep 1 min
Cook 0 mins
A sweet treat to sip, this simple adult drink recipe comes from the internet.
Ingredients Nutrition
- 1 part Malibu rum
- 3 parts pineapple juice
- Stir and serve over ice. The drink is pretty garnished with a fresh pineapple wedge and maraschino cherry.
Most Helpful
One for my "sip your way slim" diet.
This was a nice cocktail with a definite tropical touch to it. I thought the pineapple juice overpowered it a bit though because I didn't taste any of the rum at all. But it was a lovely drink that would be perfect for Sunday brunch or a light lunch when you don't want a strong alcohol kick.


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Eleven Madison Park sommelier Dustin Wilson, one of four featured in a new documentary, sees his calling as a lifestyle rather than merely a job. In this wide-ranging interview, he charts the path that took him from San Francisco to Colorado to New York, talks about the importance of sommelier certification and describes life in the world of wine. "Everyone I hang out with is a wine person. Most of my time off is doing wine related things like going to tastings and visiting wine regions."
Full Story:Eater


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Think Brick Awards: Entries Open
March 22, 2011
Win a trip to the 2012 Venice Architecture Biennale and enter the Think Brick Awards. Entries close 31 May.
Win a trip to the 2012 Venice Architecture Biennale and enter the Think Brick Awards. Entries close 31 May.
Update: Last chance to enter Think Brick Australia's 2011 awards program: Entries close 7 June. Enter online at www.thinkbrick.com.au
Think Brick has launched its annual awards program, which promotes the design possibilities of building with brick. For 2011, the program encompasses four separate architecture awards:
Open Face Award: A new award for 2011, the Open Face Award is a conceptual design competition challenging architects to design a sustainable, two-bedroom suburban home. The brief calls for "architectural concepts that engage with the suburban by envisioning a future for the Australian suburb that embraces increased densities while accepting our established desire to engage with suburban life."
The competition is open to Australian architects, with the winner receiving two business class airfares to Milan and five nights' accommodation to be used for the period of the Venice Biennale 2012.
About Face Award: The About Face Award is an invitation-only award, with six Australian firms selected to compete for the award – with the same brief as the Open Face Award. For 2011, the participating architects are Ian McDougall of ARM, Olivia Hyde of BVN Architecture, LAVA's Chris Bosse, Trent Woods and Jennie Officer from Officer Woods, Adrian Spence from Richards & Spence, and Thomas Bailey, Nathan Crump, Aaron Roberts and James Wilson of Room11.
About Face Student Award: Students are challenged to design a suburban house using half the number of bricks used in the average suburban bungalow on a typical quarter acre lot. The winner receives a return economy class flights and 3 nights' accommodation to attend the Venice Biennale 2012. Three Highly Commended prizes of $500 cash will also be awarded.
Horbury Hunt Award: The Horbury Hunt Award rewards innovation and craftsmanship in brickwork, in completed projects. The Horbury Hunt rewards commercial and residential built projects.
Entries to each award are now open.
Entries close 31 May.
Deadline extended: entries now close 7 June.
For full details on conditions of entry and to submit your work into any of the Think Brick Awards, go to www.thinkbrick.com.au
Winners will be announced at a Gala Dinner on 11 August, at the Myer Mural Hall on Bourke Street in Melbourne.
Image Durbach Block's Infinity House, the winning entry to the 2010 About Face Award.


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By combining a sleek, cat-eye silhouette with statement-making proportions, this pair of sunglasses is designed to flatter most face shapes. Featuring a powder blue crystal frame for just the right amount of flair. Image Shown: Chelsea Powder Blue Crystal, 000000000010009585 Light Blue Mirror (889556166954)
- 100% UVA/UVB protection
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- Case included
With every pair of eyewear you purchase, TOMS will help give sight to a person in need. One for One®.
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Fly in a vintage aircraft, view world-class art, cast a fly for brown trout, sample Hokonui moonshine or enjoy walks in native bush. You're sure to find a unique experience in Gore.
Gore's central location makes it an ideal base to explore the best of Southland, as well as coastal and Central Otago.
It is readily accessible by main highways from Dunedin, Invercargill, Te Anau, Queenstown and Alexandra and is proud to be New Zealand's southernmost motorhome friendly town.
Famous for Hokonui moonshine, fly fishing and country music, Gore has plenty more to entice visitors to stay a while.
Walkers and mountain bikers will enjoy the tracks and trails in and surrounding Dolamore Park, art lovers will be amazed by the collections at the Eastern Southland Gallery and it's a garden lover's delight.
Major events include the Hokonui Moonshiners' Festival, the New Zealand Gold Guitar Awards, the NZ International Film Festival and the Hokonui Fashion Design Awards.


text: Hi all, I see I've been negligent about posting when My Esri adds new functionality. We generally release something every two weeks. We've been working on changes for the 10.5 and Developer Subscription releases. We're also working on a major overhaul of the My Esri user interface plus a bunch of new features for managing users. Here's what… Show more


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Event: Riteish Deshmukh with wife Genelia D'Souza Deshmukh, Sohail Khan, Sunil Shetty, cricketer Srisanth and many other B-town celebs thronged at the Celebrity Cricket League's party at Vizag.
Guest List: Sreesanth, Vatsal Seth, Sohail Khan, Sunil Shetty, Ritesh Deshmukh, Genelia D'Souza, Ashish Chowdhry, Bobby Deol
Date: 08-Feb-2012
Bollywood badshah Shah Rukh Khan was snapped at lightbox. He's all set for the trailer screening of his forthcoming film RAEES.
Bollywood actress Shraddha Kapoor was snapped at Bandra.
Bollywood actress Dia Mirza was snapped at the inauguration of Dr.Batra's photo exhibition.
Bollywood actor Akshay Kumar was snapped on the sets of Yaaron Ki Baraat.
Bollywood actress Sushmita Sen was snapped at the inauguration of art show Masterstrokes 35.


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This mainly NHS Dental practice are looking to recruit for a part time Dental nurse to start Around April/May 2015.
This is a part time role , where candidates must be able to work between 2-3 days per week and the days of work are open to flexibility.
The practice provide both NHS and cosmetic treatment and are equipped with Kodak R4 software.
Candidates must be fully qualified and GDC registered and the practice will pay £9.50ph
Practice Opening hours:
Monday : 9.00am - 6.30pm
Tuesday : 9.00am - 4.00pm
Wednesday : 8.30am - 5.00pm
Thursday : 8.30am - 5.00pm
Friday : 8.30am - 1.00pm
For more information contact Ann-Marie and quote reference DEN-31781
Call us on 01332 609318
Email us at [email protected]
View more information at http://www.medicruit.co.uk/positiondetails.aspx?id=31781
Dental Nurse – Northumberland
Posted by Medicruit on 10 March 2015
Reference: 31781Apply Back


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Creative Crafting all Year Round: Crafts to Celebrate the Holidays
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Latest Comments
"I saw this pattern and thought it would be great to go in my gift tins for my homemade spa sets for Christmas. I started the pattern but kept having trouble with the puff stitch. When I try to pull through the loop the hook gets stuck and pull a strand or two out making it look un-neat. Is there a way to prevent this? Could it be the yarn I am using?"
"I love this pattern for making dishcloths! It is so simple and it's so easy to remember. It makes a great,durable dishcloth. Does anyone tried this pattern with that new yarn that is made for making dishcloths? I think it might be a rayon blend. I'm going to try it next time I need dishcloths."
"This is a great beginner project, it was easy to follow and gave great instruction. I was able to work up several of these for co-workers who thought it was so cute. I am going to make several more for my home for company to use when they come over and have coffee."
"These spiral beaded trees are so pretty! For extra sparkle, I am going to place a battery tea light candle in the center of the spiral. Several of these trees would look great placed within a strand of twinkle or small rice lights on a mantle or table center piece!"
"That's a great idea!"
Purim Time: Jewish Traditions of Purim, Purim Masks, History and More
By: The Editors of FaveCrafts.com
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Click here to view your Craft Projects.
Purim, a Jewish celebration that occurs in March, is a great holiday for crafters! Kids love to dress up in costume for carnivals and the megillah reading. You can also try some great Purim recipes for the traditional holiday cookie, Hamantashen.
Table of Contents
What is the festival of Purim?
Jewish Traditions of Purim
Purim Masks
Our Favorite Purim Craft Project
More Jewish Holiday Craft Project Ideas
What is the festival of Purim? What does Purim celebrate?
Purim is celebrated every year during the Hebrew month of Adar (which typically coincides with our month of March) and commemorates the deliverance of the Jews from Ancient Persia from Haman's plot to destroy the Jewish people. During the Purim celebration, Jews read the Purim story of how Queen Esther managed to trick Haman and ultimately had him hanged and saved the Jews. To this day, Jews use Purim as a fun, festive celebration that represents the salvation of their people.
Jewish Traditions of Purim
Purim is not considered a religious holiday by most Jews. In celebrating Purim, there are many common Jewish traditions. These include reading the megillah (or the Purim story), dressing up in costumes and Purim masks, eating Hamantashen (fruit-filled triangle cookies) and singing songs. Some religious schools or synagogues host Purim carnivals for their students or younger members where kids can dress up, eat and play games. In honor of the Jewish traditions of Purim, we have included some of our favorite mask projects. While these were not designed specifically for Purim, they are fun projects to do with kids and make a great addition to any Purim costume.
Purim Masks - Fun and Easy Craft Projects
Beaded Celebration Mask
Turn this Mardi Gras-themed mask into a the perfect glittering addition to your Purim costume. Use beads, tape and thread on a platstic mask and have this grand look in no time. You can customize the mask for any costume or ensemble.
Gold Celebration Mask
This dark painting technique looks great on this Purim mask. Design your own or follow this tutorial for a fun costume mask that would make a great add-on to any villain costume, like Haman.
Fun and Easy Full-Face Purim Masks
This project is easy and great for kids! Just take a store-bought plastic mask, grab some threads, beads, sequins and feathers and customize your mask to fit with your costume.
Our Favorite Purim Craft Project: Celebrate Purim Card
It's tradition to give gifts of food on Purim, so why not give a card too? This beautiful handmade Purim card features a group dancing and is crystal-embellished. It's makes a great touch to an already sweet holiday gift.
- Ruth's Jewish Stamps
- Vertical Purim – 779
- Group Dancing – 445
Other Materials
- 3mm Crystalina® Peridot Crystals
- 3mm Turquoise Round Holographic
- 6mm Gold Heart Nailheads
- Kandi's Professional Touch® HotFix Applicator
- Green, Blue and Stripe Cardstock
- Black and Gold ink pad
- Black and Gold embossing powder
- Adhesive
1. Use green cardstock for the card.
2. Cut a strip of stripe paper as a left border.
3. Cut blue cardstock 3"x 3".
4. Using Black dye ink, stamp Purim stamp onto stripe paper.
5. Using Black embossing powder, emboss Purim stamp.
6. Using Gold dye ink, stamp Group Dancing stamp onto blue cardstock.
7. Using Gold embossing powder, emboss Group Dancing stamp.
8. Using adhesive, place the stripe paper and blue paper on the green card.
9. Using Kandi's Professional Touch hotfix Applicator Wand, apply embellishments.
More Jewish Holiday Craft Project Ideas
Dreidel Quilt Block
Hanukkah Candle Quilt Block
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Tate's Bake Shop Passover Brownies
Total Time
Prep 10 mins
Cook 30 mins
Kathleen King, owner of Tate's Bake Shop, developed this recipe to bring with her to a seder in 2004. They are simple, fudgy and dense. They keep well in the refrigerator. *For lighter brownies, substitute 1/2 cup of applesauce for 1/2 cup of the butter.
Ingredients Nutrition
- 1⁄2 teaspoon espresso powder
- 1 tablespoon hot water
- 1 cup white sugar
- 3⁄4 cup dark brown sugar or 3⁄4 cup light brown sugar, firmly packed
- 1 cup unsalted butter, melted
- 3 large eggs
- 3⁄4 cup Dutch-processed cocoa powder, unsweetened, sifted
- 1⁄4 teaspoon salt
- 1 cup matzo meal, minus 2 tbsp
- Preheat oven to 350 degrees.
- Prepare 9" square baking pan with nonstick spray (kosher for Passover) and waxed paper on the bottom. The waxed paper allows easy removal of the bownies and they can be cut more evenly.
- Mix the espresso powder and water and set it aside.
- In a medium bowl, mix the sugars into the melted butter until they are combined.
- Add the eggs, coffee mixture, cocoa powder, salt, and matzo meal. (Mix to blend.).
- Pour the mixture into the prepared pan and smooth the batter evenly.
- Bake for 30 minutes. DO NOT OVERBAKE. The center should still be a bit soft.
- Cool the brownies in the pan.
- Loosen the edges and remove them from the pan and cut into squares.


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items cannot be shipped via air due to being flammable, magnetic, contains batteries,
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Product Information:
Freud's Safety Dado Sets provide the highest quality results while maintaining a superior level of safety. This stacked dado set with its razor sharp outside blades will produce grooves or rabbets which are flat-bottomed and virtually splinter-free in solid woods. Stacked dado sets are more precise than other styles and with the use of Freud's Shim Set the kerf width can be adjusted by as little as .004" for the correct fit.
Use on all table saws, and radial arm saws for cutting slots, grooves, dadoes, and rabbets in softwood or hardwood.
Includes 2 blades, shim set and 5 chippers (4 - 1/8" chippers and 1 - 1/16" chipper).
Shipping / Billing Info:
Returns accepted only for defective goods. If you have a return issue, contact Woodcraft Supply customer service @ 1-800-535-4482 for return details.
When returning product, please include the invoice number or customer number and refer to the back of the invoice for shipping instructions.
If purchased at a Woodcraft Store, please return the item directly to the store where your purchase was made.
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Cutting Tools: Lifetime warranty against defects in workmanship & material
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The infinity maxi dress in infinity print by talulah is sold out. We did have infinity print / s, infinity print / m


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This item requires a vet's prescription
Select from the following options:
In stock. See our delivery options
Activyl Spot On Small Cat (up to 4kg)
Activyl Spot-On Solution for Small Cats is a highly effective innovation for the treatment and prevention of flea infestations in small cats weighing less than or equal to 4kg.
The solution contains the active substance Indoxacarb which works using a process called bio-activation. This mode of action uses enzymes inside the flea to activate and produce an active compound that interferes with the flea's nervous system.
Once activated, Activyl will not only kill adult fleas but also break the flea life cycle and prevent eggs developing into larvae.
Activyl Spot-On Solution for Small Cats can also be used as part of a treatment strategy for the control of Flea Allergy Dermatitis (FAD).
Each 0.51ml pipette contains 99.45mg Indoxacarb.
It is supplied in packs of 4 single use pipettes.
To check the most up to date information about this product, please read its Summary of Product Characteristics (SPC) on the Veterinary Medical Directorate's (VMD) Product Information Database:
We offer a range of delivery options:
Our standard delivery is free by Royal Mail 2nd Class - there are some exceptions and you can see these on our Delivery page. These include refridgerated items and aerosols which have to be sent via our Next Day Courier service.
We also offer delivery by Royal Mail 1st Class or a Next Day Courier service for a small fee (for items in stock ordered before 12pm Monday to Friday - excludes Saturday delivery).
Orders under £20 have a £2.50 handling fee automatically added.
Food deliveries weighing 2kg and over ordered before 12pm (Monday to Thursday) will be delivered next working day by courier - Monday to Friday - excludes Saturday delivery. Orders placed on a Friday, Saturday and Sunday will be delivered on a Tuesday. Orders weighing over 6kg are sent free.
For more information, view our Delivery and Returns page.
Product reviews for prescription products are not allowed by the RCVS. But here is what our customers say about our service:
Service reviews have been compiled from 22461 customer reviews
This is the first time I have bought from Pet Drugs Online. The prices are good and the service excellent so I will use again.
Had the goods, the price and the 'free prescription'service. The latter was hassle free (after the receptionist was helped by a vet!)
First class service. I use Pet Drugs on Line for prescription medicine for my dog. Always receive confirmation of dispatch either same day or next working day from order. Great customer service who resolved issue quickly; they even contacted me to check quantity of one tablet on order, as I had order 3 rather than 30.
Having only just started my cat on the royal canin she is still getting used to it, she seems to like it, I would have liked to have had more flavours.
On 03 Dec 2016 Pet Drugs Online responded:
Dear Rosemary
Many thanks for your feedback, glad to hear that your cat is getting used to the renal diet. Most cats do seem to like the renal food but some prefer certain flavours chicken being one of the favourites. Prescription diets aren't always the most palatable as they do a specific job and have not got any bad things which can make your pet eat them more readily, sometimes if you warm the food slightly that can make the food more palatable and this will also give off more aroma
Kind regards
Site very easy to use and delivery very prompt.Big savings on prescription drugs compared to vet prices.
The required medicine was easily found and ordered. Arrived promptly and in good order. Cheapest I could find and free shipping too.


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- M10 of the greatest places in the world to retire if you're not richmarketwatch.com
- M10 of the best places in the world to retiremarketwatch.com
- MThe coming train wreck for older workersmarketwatch.com
- Columbus is among the top cities for new college gradsWBNS-10TV
- MRetirement is even more fun than you've heardmarketwatch.com
- STop jobs for college dropoutssbstatesman.com
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- Whales in Captivityhuffingtonpost.comIceland
- MShould you rent or buy in retirement?marketwatch.com
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Haweli Of Clapham - Clapham
Address: 3 Cavendish Parade, Clapham Common South Side, Clapham, Greater London, SW4 9DW
Telephone: 02086734900
Cuisine Type: Indian
Tell this Venue You Saw Them On Sugarvine
Reader Reviews
Haweli of Clapham reopening under new management by the award winning restaurant haweli of Belmont in Sutton surrey. Anonymous By Request
Review Haweli Of Clapham, Clapham
Have you recently visited Haweli Of Clapham? Send us your comments about Haweli Of Clapham, Clapham and get £50 off a case of wine *
Our Policy on Reader Reviews
* While offer lasts. Terms and conditions apply. You will receive your promotional code for your £50 discount with Virgin Wines by return email once the review has been approved for upload by our Site Administrator (this can take up up 72 hours).
Restaurants Near Haweli Of Clapham
350 Kings Road, Chelsea and West Brompton, Greater London, SW3 5UU
617 King's Road, Chelsea, Chelsea and West Brompton, Greater London, SW6 1ER
020 7371 4105
1 Denbigh Street, Pimlico, Westminster, Greater London, SW1V 2HF
020 7834 1001
261 Old Brompton Road, Kensington, Greater London, SW5 9JA
020 7483 2927
2 Caxton Street, Westminster, SW1H 0QW
0800 652 1498
View all Restaurants in Clapham
Latest London Deals
Special Price Set Menu: 2 Courses for £12.95.
A 10oz ribeye steak or a whole lobster, both served with garlic and chive butter, French fries and a side salad. Just £20 for either dish.
Tapas Club: As a Tapas Club member, you will be informed of all special events and offers which the group is running throughout the year,giving you the opportunity to be first in line to receive an invitation. Benefits include: Live Music Events, Special Celebratory Menus, Discounts on special event evenings and exclusive Wine Tasting Evenings. Click here to join.
Landmark Special Offers: Click here to view Landmark Special Offers.


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Charity Rakestraw is the author of Ministers and Masters: Methodism, Manhood, and Honor in the Old South (published as Charity R. Carney, LSU Press, 2011). She has taught or served in leadership positions in higher education for over eight years and currently holds the position of Project Manager for Program Development at Western Governors University. WGU is a non-profit, competency-based online university largely focused on serving non-traditional students. Rakestraw has written as a regular columnist for the Daily Sentinel, in Nacogdoches, Texas, and is a contributor to the Religion in American History blog (usreligion.blogspot.com). Currently, her research interests include the making of the modern megachurch and its impact on American religious culture. She is working on a monograph on the historical underpinnings of the megachurch phenomenon in the southern United States.
Please contact Charity at firstname.lastname@example.com for inquiries regarding her research or curriculum development.


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Mister Master and Little Miss
Avg. Saving £9.00*
* average saving based on two adults and two children
More About This Offer
Mister Master & Little Miss was created by a mother and daughter to provide parents with beautiful designer clothing options from across Europe for their children from 0 to 12 years old. We pride ourselves on presentation as well as providing good quality products which can't be found on the high street. On our website we will have a section called "Pandora's Box" which will encompass a variety of mums and kids related offers and items of interest: from children's party planners, to charity events organised by children, and activity camps.
Contact Information
- The Boatyard, Dell Quay, Chichester, West Sussex, PO20 7EE
- Opening Times: Please call for details
- 07787 554311


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Burlesque, Beyoncé, and Healing from Heartbreak
"You gonna wear heels?" she shouted from beside the stereo in the back of the darkened studio. Her brown eyes locked into mine in the mirror. Pausing to think, I pursed my lips together. "No, I think I'll be barefoot. In stockings," I called back, nodding my head to myself as I envisioned the fishnets, the garter belt.


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The California coast, from its majestic redwoods and rocky shores to its palm trees and sandy beaches, is an area of unsurpassed beauty. The new fifth edition of the California Coastal Access Guide is an essential handbook for anyone exploring the 1, 100 miles of one of the world's most diverse and spectacular shorelines. With up-to-date maps and information, this is an indispensable guide for all beachgoers -- hikers, campers, swimmers, divers, wheelchair users, joggers, and boaters. Details on where to go, how to get there, and what facilities and type of environment to expect are readily accessible. The handbook also contains articles on a broad range of topics, including natural history, marine and coastal wildlife, environmental issues, and sports and recreation.cPicnic tables, trailer sanitation facility. ... 31 spaces for trailer camping with electric and water hookups; $10/night. 1500 Bay ... Intended mainly for commercial fishing boats, the marina will also have slips for recreational boats, boat repair yards, anbsp;...
|California Coastal Access Guide
|California Coastal Commission
|Univ of California Press - 1991


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Finding the go-to-shoes for all occasions? Something smart-looking and versatile so that you can wear with any outfit, but also super comfy so you can be on your feet all day! Meet the Skechers GoWalk 3 because innovation seriously never felt this good! Featuring a GoGa Mat for responsive feedback and high rebound energy in your every step; and a FitKnit upper for breathability so you can even wear these slip-ons without socks!
Add to Wishlist Email to a Friend
1300 818 849
8am - 4:00pm WST
|This product has no reviews yet.


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A friend of mine recently restored a house in upstate Ohio. This photo exhibits just how nice of a job they did. Though focusing more on the financing and overall redesign than on concrete remodeling examples, projects like this continue to be a great resource to get ideas of what you might want your space to be like. You can also get a magazine for remodeling tips and designs. Most of these magazines are a treasure trove of lovely ideas, useful tips and tricks from all around the world, before-and-after examples of redesign projects and decor inspiration. With an emphasis on bedroom and kitchen remodels, in addition to living rooms, you never run out of fascinating suggestions. Many of these are accessible online as well. We think that you are able to fulfill your inspirational needs with them. If you want extra ideas to design your interior, you may want to keep checking this website as well. For example with my upcoming posts about Kitchen Remodeling, Bathroom Remodeling, Bedroom Ideas, Living Room Ideas and more. I am planning on building a nice collection of design and decoration concepts on the Internet. With lots of inspiring pictures and ideas from around the world. Including remodeling ideas and inspiration for designing your kitchen, tub, patio. The little rectangular table on this rustic log cabin from the picture below had completely no presence, making the eating area feel virtually nonexistent. A vintage-type kitchen cupboard was created with old barn wood. One other thing I would like to share: I myself got a very nice pressure washer recently. It makes cleaning so much more fun! And my house looks absolutely beautiful.
The best 5 trees for smaller gardens
What are the most suitable trees for your smaller sized garden?
Gardening is great for everyone who wants to be surrounded with a nice peaceful and natural environment. Depending on the space available in your home, you can make a large, medium, or small size garden to plant a number of useful trees and plants. Planting plants and trees in your private garden will not only be useful to get flowers, fruits, or something like that but also useful to have positive thoughts and a peaceful mind.
Planting a tree is an art and one should be very careful planting and maintaining your trees with enough care. When you have less space for gardening, you might be confused about which trees to plant or what is the right number. Here are a few suggestions from the gardening experts to plant trees even in the smallest of gardens.
Topiary shapes tree:
Topiary box, bay, and yew are flexible trees, ideal for small gardens. This is because these types of trees will add instant style and a gorgeous look to your garden. You can buy them ready-to-plant. You can select the lollipop shaped standards, tail upright spirals, pyramids, and cubes to have entrance features and wonderful focusing points.
Apricot tree:
Next to the topiary trees, you can also plant apricot trees in your small gardens. These are really attractive trees with impressive blossom and a heavy harvest of fruits in the July and August months. The production from the apricot trees is huge compared to nectarines and peaches. This is why it is recommended to plant these apricot trees in your garden.
Crab apple tree:
Crab apple trees are also the best choices for the house which has a smaller size garden. These are nice all rounder trees to provide very good spring blossom, wildlife value, and also colourful tasty fruits. The gardeners can raise different varieties of crab apple trees in your garden to get increased production every year. It will also be a smaller business to the gardeners to make income from selling crab apples in the market.
Acer palmatum (Dissectum) tree:
It is another amazing choice of tree for the small gardens. When you are planting this Acer palmatum trees in the garden, they will really provide you impressive look, lacy foliage, and great shape along with the colourful autumn bonus. The gardeners have to keep this tree from the direct sun light because it is very slow growing tree requires only the shady place for the growth.
Espalier apple tree:
Apple is the most favorite fruit to many women and it is also healthy to eat. So, you have to plant the espalier apple tree even you have smaller sized garden. It is the most familiar tree to have different types of apple fruits. It can able to grow even in the limited size of space with the nice feature. It can look like the garden divider or warm sunny wall to screen the direct light from the sun.