text:
- Research article
- Open Access
A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing
- Sylvain Meylan†1, 2,
- Anna C Groner†1, 2,
- Giovanna Ambrosini1, 3,
- Nirav Malani4,
- Simon Quenneville1, 2,
- Nadine Zangger1, 2,
- Adamandia Kapopoulou1, 2,
- Annamaria Kauzlaric1, 2,
- Jacques Rougemont1,
- Angela Ciuffi5,
- Frederic D Bushman4,
- Philipp Bucher1, 3 and
- Didier Trono1, 2Email author
© Meylan et al; licensee BioMed Central Ltd. 2011
- Received: 12 April 2011
- Accepted: 26 July 2011
- Published: 26 July 2011
KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.
To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.
We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.
Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.
- KRAB-zinc finger proteins
- transcriptional repression
- chromatin
- heterochromatin
- histone modifications
Gene expression is modulated through the alteration of chromatin states by epigenetic regulators. Krüppel-associated box zinc finger proteins (KRAB-ZFPs), which together constitute the single largest group of transcriptional repressors encoded by the human genome, partake in this process [1–3]. The KRAB-ZFP family is evolutionary recent and has expanded and diverged through multiple rounds of gene and segment duplications, to give rise to more than three hundred and fifty annotated members in humans [4–7]. Despite their abundance, KRAB-ZFPs and their transcriptional targets remain largely uncharacterized except for a few [8–10]. KRAB-ZFPs carry a C-terminal array of two to forty C2H2 zinc finger motifs, each potentially capable of recognizing a triplet of nucleotides in a sequence-specific manner [1], while their N-terminal KRAB domain recruits the KAP1 (KRAB associated protein 1) corepressor [11–14]. KAP1 (also named TIF1β, KRIP-1 or TRIM28) binds KRAB and homotrimerizes through its N-terminal RBCC (Ring finger/B box/Coiled-Coil) domain, while its C-terminus acts as a scaffold for various heterochromatin-inducing factors, such as heterochromatin protein 1 (HP1), the histone methyltransferase ESET (also known as SetDB1), the nucleosome-remodeling and histone deacetylation (NuRD) complex, the nuclear receptor corepressor complex 1 (N-CoR1) and, at least during early embryonic development, de novo DNA methyltransferases [15–22]. This results in local loss of histone acetylation, enrichment in histone 3 lysine 9 trimethylation (H3K9me3) and increased chromatin compaction [23, 24].
Using chromatin immunoprecipitation (ChIP) and a tiling array, KAP1 has been documented to bind more than 7000 sites in a human testicular embryonal carcinoma cell line [25]. A more recent publication additionally revealed that KAP1 chromatin targeting falls into different categories, only a subset of which is dependent on its RBCC domain and consequently on its association with KRAB-ZFPs [26]. KAP1 is dynamically associated with both heterochromatin and euchromatin. It is thought to organize constitutive heterochromatin and to stimulate its propagation, as evidenced by its co-localization with HP1 in pericentromeric heterochromatin domains [16, 27]. Using a combination of gene trapping and a drug-controllable KRAB-containing repressor, we recently demonstrated that KRAB/KAP1 can induce long-range repression through HP1-dependent heterochromatin spreading [28]. However, while some promoters located tens of kilobases (kb) from KAP1 docking sites were silenced by this mechanism, others were resistant. Here, we investigated the basis for this differential behavior by comparing the genomic context and the pre-existing levels of specific chromatin marks at repressed and non-repressed genes. This analysis revealed that genes most susceptible to KRAB/KAP1-induced silencing were in genomic regions of high gene activity. More specifically, repression was most efficient at sites with increased levels of pre-existing repressive histone marks at promoters and gene bodies, embedded within gene-rich regions with high levels of transcription.
Characterization of thousands of KRAB/KAP1-targeted gene traps
Since we were interested in elucidating differences between KRAB/KAP1 repressible and non-repressible promoters and genes, we reasoned that "all or none" phenotypes would facilitate subsequent analyses. Therefore, we selected cells in which trapped promoters were highly active at baseline, and either strongly repressed ("repressed clones" containing a "repressing integrant") or almost completely resistant to this process ("non-repressed clones" containing a "non-repressing integrant") when the trans-repressor was allowed to bind its target (Figure 1B). More specifically, we isolated trapped integrants from a population of cells by puromycin selection in the presence of Dox, which impairs tTRKRAB binding and silencing. Then trapped integrants were subjected to subsequent rounds of cell sorting to isolate cells harboring gene traps with repressible promoters and reporter genes. These rounds first included the isolation of GFP negative cells when tTRKRAB was allowed to bind (Dox-), followed by the sorting out of GFP positive cells when its recruitment was inhibited (Dox+) (Figure 1B). Isolation of non-repressible genes was achieved by a similar approach. However, trapped cell populations were cultured in the presence of tTRKRAB binding (Dox-) and GFP positive cells, which did not silence reporter expression, were directly isolated after TrapSil vector infections (Figure 1B).
After the isolation of cell populations with differential silencing phenotypes, we mapped proviral integration sites, in order to identify the trapped genes. For this, we combined linker-mediated PCR (LM-PCR) of proviral-genomic junctions with massive parallel DNA pyrosequencing [31, 33, 34]. The amplified sites were mapped to the human genome with the FetchGWI software [35], and the UCSC known gene annotation was used to subsequently identify the trapped promoters (Figure 1C). We previously described that about 1 in 15 promoters trapped by MLV-TrapSil vectors were non-repressed by tTRKRAB, compared with approximately 1 in 5 for those captured by LV-based vectors [28]. Therefore, we isolated over 7000 integration sites, with an intentional bias for non-repressed clones to obtain integrant numbers comparable to their repressible counterparts. 69% of the promoter-trapping LV integrants mapped within annotated genes, whereas only 54% of their MLV counterparts did (Figure 1C, Additional File 1). This observation is in agreement with previous data indicating that parental MLV as well as MLV-based gene traps integrate in promoter proximal regions, which are less well annotated than gene bodies, which in turn are the preferential integration sites of LV and LV-based traps [36, 37]. Consistently, we mapped 6135 LV-TrapSil integrants to the genome, 4219 of which were located within genes. In contrast, we only found 787 intragenic MLV-TrapSil integrants.
Prior to further analysis, we validated our experimental approach by deriving clones from each population. All of the 32 clones analyzed exhibited the expected silencing profile in flow cytometry measurements. Moreover, the clones comprised 10 non-repressed (LI I-X) and 8 repressed (LR I-VIII) LV-TrapSil clones, in addition to 8 non-repressed (MI I-VIII) and 6 repressed (MR I-VI) MLV-TrapSil clones, (Additional File 2). We also used ChIP analysis to verify that non-repressed genes properly recruited KAP1 and downstream effectors to their tTRKRAB docking site, in a doxycycline-dependent manner (Additional File 3). After this validation, we continued with the characterization of the genomic context of our KRAB/KAP1 repressible or non-repressible genes to find patterns correlating with silencing efficiency.
Genomic environment of repressing and non-repressing gene trap integrants
We characterized the genomic environment of the integrants segregated according to their phenotype by using ROC (Receiver Operator Characteristic) curve analysis [38]. This type of analysis was previously used to identify the genomic features enriched around retroviral integration sites. This study confirmed that both MLV and LV preferentially integrate within transcriptionally active regions, and that this effect is augmented when integrants enabling reporter expression are selected [38]. In addition, this analysis also revealed that the effects of different genomic features on integration can change depending on the size of genomic segments in question [38]. Therefore, we included genomic intervals ranging from 0.1 kb to 10 Mb in our analyses.
We then compared the ROC values, which are proportional to the levels of genomic features at these sites, between TrapSil groups harboring differential susceptibilities to KRAB/KAP1-silencing. We did this by making relative comparisons between a chosen reference and other gene groups. The reference gene groups are indicated by the symbol "---" within the whole results section. Using this approach we compared the levels of specific genomic features between the respective REP gene group and their corresponding NREP counterpart. When statistical differences were assessed, we found that LV-TrapSil repressing integrants were located within gene-denser genomic regions than non-repressible integrants (Figure 2). Furthermore, the environment of repressing LV integrants was enriched in CpG and DNase I sites, as well as in highly expressed genes (based on publicly available microarray data), compared to that of non-repressing LV integrants. While all the described parameters were statistically significantly different between repressible and non-repressible LV traps, comparisons of their MLV-TrapSil counterparts did not reach significant differences, although it showed similar trends (Figure 2). Therefore, a positive correlation between gene activity in the environment of the targeted transcriptional unit and efficient KRAB/KAP1-mediated silencing is established. The lack of significance between the MLV repressible and non-repressible TrapSil groups could be due to smaller integrant numbers or could reflect the presence of other uncharacterized features affecting KRAB/KAP1 recruitment, including the on average closer proximity of MLV integrants to promoters.
Genomic features of matched repressed and non-repressed transcriptional units
The genomic context of the three gene groups was reminiscent of observations made in the integrant-centered analysis (Figure 2), with genes from group 1 being in gene-richer and transcriptionally more active environments, and surrounded by a higher density of DNase I hypersensitivity sites (Figure 3B). These associations, however, did not reach statistical significance. Importantly, no difference in distance between repressor binding site and the trapped promoter was apparent when comparing the three groups, eliminating concerns about this potential bias for subsequent analyses of these genes (Figure 3B). When we examined the expression levels of the different gene groups, we found that genes supporting long-range repression (group 1) were on average more highly expressed than genes that did not enable KRAB/KAP1-mediated repression (group 3) (Figure 3C). Therefore, KRAB/KAP1-mediated silencing seems to be more effective in regions of high gene activity. To further consolidate this result, we assessed the levels of different chromatin features, correlating with transcriptional activation or repression in our different gene groups.
Chromatin features of matched repressed and non-repressed transcriptional units
We first assessed the levels of putative barrier elements such as CTCF, H3.3/H2Az or chromatin modifiers in the different groups [39–41]. This was achieved by utilizing published datasets, which were used to calculate the relative abundance of these features by ROC curve analysis and by comparing these values between the groups. There was no differential association with either one of the three gene groups for the intervals tested (Additional File 5).
Previous analyses on the mechanisms of KRAB/KAP1-mediated gene regulation have mostly examined the impact of this system on the expression of transfected promoter-reporter units. Here, we investigated KRAB/KAP1-induced changes within the context of endogenous genes. Using a combination of promoter trapping and drug-controllable KRAB/KAP1 recruitment, we previously observed that this complex, when docked to the bodies of transcriptionally active genes, could induce silencing over distances of several tens of kilobases [28]. However, we had also noted that repression was more efficient if the distance between the effector and the promoter was less than 20 kb. Furthermore, a significant fraction of trapped promoters/KRAB docking loci escaped these rules, suggesting other counteracting influences. The present large-scale comparison of the genomic features of KRAB/KAP1-responsive and KRAB/KAP1-resistant transcriptional units identified by our gene trap system reveals a positive correlation between efficient KRAB/KAP1-mediated repression of trapped promoters and i) a gene-richer and transcriptionally more active genomic context, ii) a more euchromatic environment, and iii) the pre-existence of some repressive marks at and around the promoter.
Comparing KRAB/KAP1-repressed and non-repressed genes gave no indication for a role of putative obstacles to the spread of heterochromatin, such as CTCF binding, accumulation of H3.3/H2Az or recruitment of HATs (reviewed in [42, 43]). This is consistent with the observation that CTCF recruitment to the HS4 region of the chicken β-globin locus can be prevented without abrogating the barrier function of this DNA sequence [44]. However, it is at odds with a recent study presenting CTCF as a marker of transition between euchromatic and heterochromatic regions [39]. A model reconciling these findings would be that CTCF acts as an H3K27me3 heterochromatin-specific barrier yet has no effect on H3K9me3-based heterochromatin propagation. However, it should be emphasized that our analysis was limited to the transcribed region of genes owing to our gene trap-based approach, precluding overly general conclusions on the possible role of barrier elements.
Although both repressed and non-repressed genes were situated within euchromatic regions, as expected from the promoter-trapping approach used for their selection, we observed significant differences in both their local and broader chromatin environments. Repressed genes were in regions containing generally higher levels of major euchromatin-associated marks and higher levels of transcription compared with non-repressed genes. Therefore, there is a positive correlation between efficient KRAB/KAP1-silencing and high gene activity. This is suggestive of a model whereby genes situated in more heterochromatic environments can only be highly expressed if endowed with an intrinsic ability to resist repressive influences, while genes located in more euchromatic environments do not need such protective mechanisms [45]. Consistently, in our analysis KRAB/KAP1-resistant units were on average closer to telomeres than their KRAB/KAP1-susceptible counterparts, although this difference did not reach statistical significance (data not shown).
Genes repressed by the TrapSil system also carried higher levels of the repressive marks H4K20me3 and H9K9me3 at baseline at and around their promoters, compared with their repression-resistant counterparts. Noteworthy, these contrasting chromatin configurations were not only observed when comparing a selected set of multiply hit repressed and non-repressed genes (Figure 5), but were also present in the complete pools of repressing and non-repressing integrants (Additional File 6). Interestingly, a recent analysis of the chromatin structure of zinc finger genes found that high levels of both H3K36me3 and H3K9me3 co-localized at the 3' exons of these genes [46]. Since KRAB-ZFP genes, which belong to this gene family, are endogenous targets of KRAB/KAP1-repression [25, 47], we performed the same analysis in our HeLa cell system and reproduced the same result (Additional Files 7 and 8). Therefore, the high levels of both H3K9me3 and H3K36me3 at KRAB-ZFP gene bodies may be necessary for efficient KRAB/KAP1-induced heterochromatin spreading. The finding that the repressive H3K9me3 and the activating H3K36me3 marks are not co-regulated further supports this hypothesis [46], since high levels of H3K36me3, which positively correlate with active transcription, may independently enhance the spread of H3K9me3 at KRAB-ZFP genes. This model is reminiscent of results obtained from the TrapSil analysis, where high levels of both active and repressive histone marks can be seen in genes that accommodate KRAB/KAP1-mediated heterochromatin spreading and silencing.
A difference between genes targeted by our TrapSil system and endogenous KRAB-ZFP genes lays in the finding that the latter do not seem susceptible to KRAB/KAP1-mediated long-range repression [28, 46, 47]. This may be due to the use of our ectopic repressor system. Alternatively, certain endogenous promoters may be resistant to KRAB/KAP1-induced heterochromatin spreading. A possible factor in this process is the H3K9me1/2 demethylase PHF8 [48]. Active H3K9 demethylation may prevent the heterochromatization of KRAB-ZFP promoters and subsequent transcriptional silencing. This idea is consistent with recent PHF8 genome-wide binding data that showed it locating to the promoter regions of zinc finger-encoding genes [49].
Other mechanisms potentially involved in conferring resistance to KRAB/KAP1-mediated silencing are suggested by the analysis of genes that were hotspots of proviral TrapSil targeting and carried both repressible and non-repressible integrants (Additional File 9). In this subgroup, the repressible integrants generally clustered closer to the promoter than their non-repressible counterparts, consistent with the overall observation that silencing is most efficient when KRAB/KAP1 is recruited in the proximity of the affected promoter. In some cases, however, the distributions of repressible and non-repressible integrants overlapped within the same gene. This could reflect the differential susceptibilities of the two alleles of a gene to KRAB/KAP1-mediated repression, somewhat reminiscent of what is observed with imprinting, a process that involves a KRAB-ZFP [9, 50]. Additionally, cells within a population may be heterogeneous for the chromatin status of specific loci, which in turn might impact on the consequences of KRAB/KAP1 recruitment. Such a phenomenon would be comparable to variegation, where particular genes are differentially expressed amongst cells of an otherwise apparently homogeneous population [51].
In summary, the present work indicates that the impact of KRAB-mediated docking of KAP1 on the expression of targeted genes is more variable than previously suspected. It further reveals reciprocal influences between the functional outcome of KRAB/KAP1 recruitment to DNA and the chromatin features of the involved loci. More broadly, the approach described in the present study, which combined an analysis of the functional consequences of exogenously introduced cis-acting KRAB/KAP1-recruiting sequences with an examination of the transcriptional activity, genomic context and chromatin features of targeted loci, could be fruitfully applied to the study other epigenetic regulators.
pLV-tTR-KRAB-Red was previously described [52]. pLtTR-KRAB-NG95 was cloned through ligation of a BamHI/XhoI digested MLV-based pNG95 [53] with a compatible tTR-KRAB amplicon with BamHI/XhoI sites added by PCR (primer sequences see Additional File 10). To construct LV- and MLV-based TrapSil vectors, published gene trap vectors [37] were modified by PCR-based mutagenesis (Stratagene mutagenesis kit). A BlpI restriction site was introduced into the MLV U3 region of 3'LTRs (MLV: BlpI Primers MLV Trap F/R - Additional File 10), whereas a SpeI site was introduced in the LV U3 region of 3'LTR (Primers HIV Trap F/R - Additional File 10), these new sites were then used to insert 7 repeats of TetO. LV- and MLV-based particles were produced and titered as described elsewhere http://tcf.epfl.ch/page-6764-en.html. The WPRE of LV-TRAPSIL, the GAG remnant of MLV-TRAPSIL, and the Albumin gene served for proviral and cellular genome quantification by Taqman.
Cell culture and Fluorescence activated cell sorting (FACS)
HeLa cells were grown under standard conditions. Doxycycline (Sigma-Aldrich) was used at a concentration of 1 μg/mL. Clonal tTRKRAB-expressing HeLa cell lines dsRK4 (pLV-tTR-KRAB-Red, LV-backbone) and KiN1.25 (pLTetR-KRAB-NG95, MLV-backbone) were derived after infection with pLV-tTR-KRAB-Red or pTetR-KRAB-NG95, respectively. The LV based HeLa dsRK4 clone contains approx. 15 vector copies as titrated by Taqman and was used for MLV-TRAPSIL assays while the MLV-based KiN1.25 clone contains 10 vector copies and was used for all LV-TRAPSIL assays. In view of this mapping strategy, 2 × 108 dsRK4 or KiN1.25 HeLa cells were infected with 1.6 × 106 MLV-TrapSil or LV-TrapSil infectious particles, respectively, with a multiplicity of infection of 0.04. Cells were sorted based for GFP expression by using the Beckton Dickinson FACSVantage SE turbo Sorter with Diva Option. Flow Cytometry analyses were performed on BD FACScan flow cytometer.
Quantitative PCR (qPCR)
qPCR reactions were carried out with a standard PCR program in ABI PRISM 7900 HT in duplicates or triplicate using either SYBR green detection 1× Power Sybr or 1× Taqman Universal Mix, No AmpErase (Applied Biosystems). Primers were used at a final concentration of 100 nM. When SYBR analysis was performed, cycling reactions were followed by a dissociation curve analysis to validate specificity of amplified products. The increase in fluorescence was analyzed with the SDS software, version 2.2.2 (Applied Biosystems). For all amplification plots the baseline data were set with the automatic cycle threshold function. Primer sequences for all qPCR reactions are listed in Additional File 10.
Linker-mediated PCR (LM-PCR), 454 pyrosequencing and data processing
LM-PCR was used to map integration sites following a previously described protocol [31, 33, 34]. Briefly, 10 μg of genomic DNA (DNeasy, Qiagen) was digested with MseI. Fragments were ligated to a linker and were digested with DpnI and SacI (LV-TrapSil) or SpeI (MLV-TrapSil) to avoid contaminations with bacterial plasmids and to avoid cloning of internal vector fragments. Nested PCR then served to amplify TrapSil vector-gDNA junctions (Takara Advantage 2 kit). Amplicons ranging between 100 and 400 bp were purified, quantified and sent for pyrosequencing at GATC biotech (Konstanz, Germany). Raw sequences were downloaded from the GATC biotech website and converted to FASTA files. Sequences having exact pyrosequencing reaction primers (F: primer A; R: primer B, Additional File 10) were selected and others discarded. Selected sequences were then categorized according to barcode for TrapSil vector type and integrant type (barcodes: LI: TGAC/AGTC; LR: CTGA; MI: TCGA/AGCT; MR: GTAC). After classification, all primer sequences and viral vector overhangs were trimmed yielding only genomic DNA sequence. The 20 bases adjacent to primer B before trimming were used as tags for mapping the inserts to the human genome assembly hg18. The mapping was done using FetchGWI tolerating at most 2 mismatches [35].
Integration site mapping in genes: Integrant orientation was annotated as determined during sequence processing. UCSC known gene [54] were downloaded from UCSC tables with transcript start (Tsx), transcript end (Tsend) and gene orientation. Only integrants mapping with correct orientation within a gene were mapped relative to it. In a second step, a non-redundant gene list was generated (from the original UCSC Gene list) using an aggressive clustering strategy, which groups all transcripts that directly or indirectly (through other transcripts) overlap on the same strand of the same chromosome. In the non-redundant gene list we recorded the 5'most Tsx position and the 3'most Tsend position for each cluster. For analysis of the integrant distance to gene promoters, we considered only integrants falling within the transcribed region of the same gene. Files containing integration sites (sequence-mapping from Insipid, LV_LUI (LV irrepressible), LV_LUR (LV repressible), MLV_MUI (MLV irrepressible), MLV_MUR (MLV_repressible)) and gene groups (20 KB promoter classes with at least 3 integration sites: group 1: "long-range repressible), group 2: "short-range repressible", group 3: "long-range irrepressible") can be found under http://ccg.vital-it.ch/KAP1/.
Receiver Operator Characteristic (ROC) curve analysis
Data analysis was based on a "nested case control" strategy using a collection of TSS characterized by a given behavior with respect to repression along with control sites sampled from the genome to make inferences about the probability of a TSS to display a given response to repression based on genomic/epigenetic features characterizing its environment. More detailed description of statistical basis for this analysis can be found in [38]. Data were analyzed using the R language and environment for statistical computing/graphics version 2.3.0 and several contributed packages. Empirical ROC curve areas were calculated for datasets that used random genomic controls, in which case each TSS of a cluster was compared only with its matched controls to determine the proportions of controls whose values equaled or exceeded that of TSS [55]. Annotations of genomic features were obtained as described previously [38]; the chromatin features analyzed came from ChIP-seq data generated in this and other studies [39–41].
Chromatin immunoprecipitation (ChIP) and ChIP followed by sequencing (ChIP-Seq)
ChIP reactions were performed according to published protocols with minor modifications (http://www.millipore.com/userguides/tech1/mcproto407 and http://cshprotocols.cshlp.org/cgi/content/full/2009/6/pdb.prot5237), using antibodies listed in Additional File 11, either native or pre-bound to beads. For Histone modifications, 2 × 107 HeLa cells were trypsinized and resuspended in MNase buffer. 1 U MNase (Roche) was added for 10 min and adding EDTA to a final of 10 mM arrested the nuclease reaction. Chromatin was sonicated with a Branson digital sonicator (model 250) on ice three times for 20 s and then dialyzed against RIPA with AEBSF protease inhibitor 0.2 mM for 1 h. The chromatin was pelleted after dialysis; glycerol was added to the supernatant to a final 5% concentration and the chromatin was stored at -80°C. 500 ul was incubated with AB-specific pre-coated beads over night (IP). Complexes were washed, eluted, purified, precipitated and resuspended in 50 ul H2O. For KAP1 ChIPs, approximately 2 × 107 cells were cross-linked with 1% formaldehyde for 8 min at RT, quenched by adding glycine and rinsed with PBS, before shearing by sonication with a Branson digital sonicator (model 250) on ice four times for 20 s at 30% intensity. 100 μl of sonicated chromatin was directly de-crosslinked and used as the total input (TI) reference in qPCR analysis at a dilution of 1:100. 100 μl of sonicated chromatin was used for each ChIP reaction and was diluted in 900 μl dilution buffer and precleared with 80 μl salmon-sperm DNA protein A agarose beads (Upstate). Chromatin-antibody complexes were captured washed and eluted with 100 mM NaHCO3, 1% SDS. Cross-links between DNA and proteins were reversed by addition of NaCl and incubation at 65°C. DNA was precipitated after incubation with RNase A (Sigma) and Proteinase K (Roche) and resuspended in 50 μl H2O and subjected to qPCR analysis. qPCR is described above and primers are listed in Additional File 10. Negative control reactions without antibody were run for each sample and in all cases gave negligible results. To validate the relative enrichment of proteins or specific histone modifications at a given sequence a ratio between the relative quantities of IP and TI was established.
The sequencing libraries from all ChIP products were prepared using the ChIP-seq Sample Preparation Kit (Illumina; San Diego, California, USA; Cat. No. IP-102-1001) according to the protocol supplied with the reagents and using 10 ng of ChIP sample quantified using the Qubit fluorometer (Invitrogen; Carlsbad, California, USA). One lane of each library was sequenced on the Illumina Genome Analyzer IIx using the Single-Read Cluster Generation Kit v2 (Cat. No. FC-103-2001) and 36 Cycle Sequencing Kits v3 and v4 (Cat. Nos. FC-104-3002 and FC-104-4002). Data were processed using the Illumina Pipeline Software v1.5.1. Illumina GAII data were mapped to the genome with Bowtie http://bowtie-bio.sourceforge.net/index.shtml. All output files were converted to processable file formats (SGA) for subsequent bioinformatics analysis described below. Enrichment of genomic and chromatin features was assessed with the ChIPcor web-based tool http://ccg.vital-it.ch/chipseq/chip_cor.html. All files were converted into SGA format, settings included: sort input: on, strand option: oriented for references files (gene clusters TSS and poly-A sites) and any for all target features, range was set at -40 kb to +40 kb, window size was at 500/50 (for graphic or statistical analysis) and cut-off value was 1. Raw and processed sga files can also be found under http://ccg.vital-it.ch/KAP1/ and were used as follows: "SGA files for Zhao-produced CTCF and H3K27me3 genome wide" include HeLa-CTCF.sga.gz, HeLa-H3K27me3.sga.gz data; "H3.3-H2A.Z double ChIP (Zhao et al)" contain GSM335958.sga.gz files; "SGA files from genome-wide mapping of HATs and HDACs in human CD4+ T cells" [41]: contain CD4-Tip60.sga.gz; "SGA files for histone modification profiles (ChIP-Seq data)": contains H3K27ac: H3K27acpf.sga, H2BK5me1: H2BK5me1pf.sga, H3K4me1: H3K4me1pf.sga, H3K4me3: H3K4me3p.sga, H3K36me3: H3K36me3b.sga, H4K20me1: H4K20me1b.sga, H3K9me2: H3K9me2p.sga, H3K9me3: H3K9me3pf.sga, H4K20me3: H4K20me3b.sga.
Microarray data
HU133a arrays for HeLa cells were downloaded from GEO/NCBI [56, 57] and data were extracted and normalized using RNA Robust Multichip Average (Quantile normalization) [58]. Specific gene expression scores were extracted and normalized average values for 4 different arrays were calculated. Statistical comparisons were done with the non-parametric Wilcoxon test.
We thank Joanna Roberts and Miguel Garcia for the FACS analyses, and Keith Harshman and colleagues for help with the DNA sequencing. This work was supported by grants from the Swiss National Science Foundation, the Strauss Foundation, the European Union (PERSIST program) and the Infectigen Association to DT, by an MD-PhD scholarship from the Swiss National Science Foundation to SM, and by NIH grants AI52845 and AI082020, by the University of Pennsylvania Center for AIDS Research and by the Penn Genome Frontiers Institute through a grant from the Pennsylvania Department of Health to F.D.B.
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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/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Triax TD88 88cm Satellite Dish with Pole Mount Fittings
Almost any dish can provide the signals needed to get a clear TV-picture. But will consumer satisfaction last, if signals deteriorate due to corrosion of the reflector or if the dish is damaged by a storm?
TD dishes minimize problems of any kind. Long-term corrosion is prevented by an extremely thorough anti-corrosive process and polyester coating. A solid construction of all parts, including the non-slip mast brackets, ensures that the dish remains in its correct position, when other dishes are torn down or twisted by a heavy storm.
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The elevation bracket is pre-mounted and is easily fixed to the mast with non-slip mast brackets A setting scale on the elevation brackets facilitates precise adjustment to the required satellite The pre-mounted feedarm just needs unfolding, and the LNB holder with the LNB is simply clicked on
Manufactured by Triax Uk ltd the TD series of satellite dish antennas clearly stands out from all the other dishes on the market.
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Reflector Material Galvanised Steel
Available Options:
colour:Dark grey
Copyright © 2019 Satwise TV LimitedWebsite design by Sunnyside Web Design Ltd, Dorset, Somerset

subset: commoncrawl


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by Jack Phillips
Naturalists are by nature an optimistic lot because we have learned to find beauty in unexpected places, that nature is resilient, that wildness is all around us as well as within, and that humans are hard-wired for curiosity. That is why so much nature writing tends toward exaggerated sweetness and if we're not careful, toward painting pretty pictures that leave out murky colors. When our workshop leader asked us to write about an adverse or troubling experience in nature I resisted, but not for a lack of these experiences but for simply not wanting to think about them. But writing helps us look at nature honestly and at ourselves in nature as well.
The memory I conjured made me queasy. On a hot summer day, the woods I had known since childhood stank of rotting corpses. The rough road to the cabin was a narrow track through a shanty town of cobbled-together weekend getaways largely made of salvaged materials and home-made dreams, none fancy but generally tidy and painted. The riparian woods canopied this little neighborhood. Maples, mulberries, walnuts, dogwood, basswood, box-elder, and cottonwoods lined the road and the river. But lately, these woods had become a foul gallery of catfish heads and whole bodies of every manner of fish and game nailed to tree trunks or displayed on fallen logs, the handywork of a killer and collector of death we called The Poacher. He probably ate some of his illegal prey, but no freezer in the county could have held the mounting massacre.
My aunt and uncle's cabin, also shared by their descendants that grew in number throughout a lifetime, and also by wanderers and an ever-expanding circle of friends, sat on a few concrete blocks frugally arranged on the soft and shrinking bank of an unruly river. The hospitality of my kin has sometimes seemed excessive. Especially in the case of The Poacher. My uncle, a lover and defender of all things wild and also a delicate harvester of wild fare, showed him a measure of kindness far beyond my tolerance. But that was his way. He showed The Poacher at least as much tolerance as he afforded the wasps with whom he shared his toolshed.
The Poacher appeared on that hot and humid afternoon distressed and in need of one more never-ending favor. Apparently there was at least one creature the fearless slayer of toads and rabbits and catfish and deer was afraid to approach near enough to murder: a snake caught on a hook. He had pronounced it venomous. He needed a long-handled implement with which to dispatch it, with great bravery, as it writhed on one of his scores of set-lines he had deployed across shallow channels shore to shore. Or a volunteer.
I politely choked back my disdain and asked him to show me. My young son wanted to come along and I reluctantly consented. But The Poacher declined to climb down the bank to the water and instead described where he had dragged up the gigantic monster. I was surprised to find it easily, but was not surprised to find a completely harmless eight inch-long northern water snake (Nerodia sipedon) in a tangle. Alive they are harmless, even more so dead. The limp snakeling was attached to length of twine by a deep hook that had grossly stretched its jaws. Snakes have the ability to swallow prey much larger than their heads, but in this case the talent proved deadly. The bait, a waterlogged toad threaded like a charm, hung to the side. There was nothing to do except deprive The Poacher of another corpse. We cut the snakeling loose and gave it to the river.
The experience confirmed for me, once again, that humans need to learn or relearn how to encounter nature with respect and reverence. The Poacher was but an extreme example of the human impulse to collect, master, own, and exploit the natural world. Even among my friends this impulse is obvious when a herpetologist keeps a snake for a pet, a forager collects every mushroom in reach, or an herbalist sees not plants, but product. There are those who practice responsible killing and taking, but far too often those impulses reveal an attitude of entitlement to the natural world that has proven to be corrosive and depleting.
My uncle, true to his perpetual belief in humanity, thought that I was too critical. He believed that The Poacher wanted to live close to nature but just didn't know how. He may have been right, but I was worried that there wouldn't be enough nature left for the rest of us. We later learned that he was hiding out in an empty cabin; he was arrested soon after not for crimes against nature, but for other tresspasses and atrocities.
Emerson wrote: "To speak truly, few adult persons can see nature." That day on the river proved him true. I should not be surprised when I am healed by children, and my gratitude increases every time. On the way back to the cabin my tender son counted turtle tracks, hunted for bison bones and arrowheads, lost and retrieved shoes in sticky muck, caught and released little toads, and tried to skip the rare stone we happened upon. Barefoot on hot sand and cool mud, we wandered sandbars and mudflats and happened upon a wee water snake with an attitude.
By the age of six my son was a river rat in his own right. He tried to grab it. The feisty little snake, just a few hours old and roughly my son's age in snake years, bit his finger and immediately drew blood. With no hesitation whatsoever and before I could react, he deftly grabbed it with the other hand. He didn't complain about his injury or the blood dripping from his finger, but it took a few minutes for my parental panic to subside.
Though not venomous, the northern water snake is a cranky species right out of the womb. (Nerodia sipedon is viviparous, bearing live young.) But their saliva, like that of many snakes, contains an anticoagulant that bleeds and weakens prey. But not so little boys. My son fondly admired the fresh and shiny serpent, in his hands more squirmy than angry, the innocent kin of the one we could not save.
Jack Phillips is a naturalist, nature writer, and Principal of The Naturalist School: Ecology and Creativity for Conservation.


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السلام عليكم "
جآآآآء وقت مفضلات شهر ديسمبر ^_^
الشهر دا ما جربت أشياء كثيرة يعني الأشياء المفضلة قليلة
it's time for December favorites ^_^
this month i didn's try a lot of things so it's a few things
صوووورة جوية ^_^
Atomic Red Mat ) By ( DEBORAH )
my favorite lipstick ever
dark red and Guess what $_$ it's matt
concealer palette By makeup for ever
body spray By victorias secret
A vibrant, floral fragrance. Steal the spotlight in Such a Flirt, a tease of tempting starfruit and white orchid. Refreshing formula is infused with conditioning aloe vera and calming chamomile.
madagascan vanilla flower By the body shop
purple nail polish By flomar
my favorite candle By IKEA
Smelled wonderful
my favorite winter Scarf
i wear it nearly every day
and thaaaat's it
if u like the bost just subscribe
follow me on TWITTER
follow me on INSTAGRAM
ask me on ASK


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Atlas DS16 Digital Mini Servo FCD (High Torque)
Part Number: HP-DS16-FCD
These are simply the best servos you can buy at any price, and a great value too.
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- Gear Train primary material: CarbonPoly
- Dimension: 32 x 16 x 31.5mm
- Weight: 25.5g
Note: See the Manuals and Support Tab for more details for Full Specifications and Programming Guide


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Discover our
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If you are passionate about film and storytelling through images, and always wondered what all the people in the credits of a film are actually doing, then this course is most certainly for you. This hands-on course, accredited by Creative Skillset and recognised by industry professionals, will enable you to produce films to professional standards, write production proposals and give presentations. Our graduates regularly appear in the credits of major TV and cinema releases, win awards at prestigious film festivals and the Royal Television Society, and have their scripts developed into independent film productions.
Our students and graduates frequently win national industry awards, such as Lauren Clark winning the Undergraduate Open Category prize at the Royal Television Society's Student TV Awards.
UCLan Film Production offers scope to develop both personal creative expression and professional level skills in a supportive and inclusive environment. Accredited by Creative Skillset and recognised by industry professionals, the programme delivers "strong emphasis on practical and vocational development" (Diarmid Scrimshaw, Producer 'Tyrannosaur') and prepares "exceptionally professional" students (Derrin Schlesinger, Producer, 'This is England', 'Four Lions', 'Babylon').
By helping students maximise their learning potential, this progressive programme has seen students and graduates regularly appear in the credits of major TV and cinema releases. UCLan Film Production students have been Oscar nominated in 2014 and won the Royal Television Society awards 5 times, with two awards from 2014.
To learn more about our School and the range of courses within it, go to our homepage. If you'd like to learn more about the successes and awards experienced by our students, staff and alumni, visit our news page.
As a student you will be focusing on professional and creative development in a variety of formats including comedy, the short film and the feature film. Around one third of your study will explore the theoretical aspects of Film language and its influence on modern production techniques, its historical context and philosophical underpinnings.
I have worked with the students of the University of Central Lancashire on two large scale productions and found them to be exceptionally professional, hardworking and mature in their approach to the working environment
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This course consists of lectures and seminars, with extensive tutorial support. In scheduled classroom sessions you will develop and present your ideas, discuss and critique other students work and analyse films. There are short week by week assignments alongside your main projects.
You will be actively encouraged to build a portfolio of work in your chosen area be it directing, sound design, online editing and production management etc. to use these skills to access industry.
The course is focused on employability therefore apprenticeships with regional and national broadcasters, employment through commission competition or creative control over story development for small screen, cinema and perhaps radio, are all within the remit of this course.
The course leader for this course is Peter Wobser.
This September the Faculty of Culture and Creative Industries is offering Foundation students a unique opportunity to 'Futureproof' their ambitions and gain a National qualification free of charge as part of your studies.
Futureproof includes the PEARL qualification, a national GQAL accredited certificate, that will enable you to transfer key skills that can be taken forward into all areas of life, especially employment or further study.
The course will open doors for you with creative businesses and offers you extra coaching and access to high profile industry professionals. You will get additional support to help you recognise your strengths and apply them in creative work environments.
The five areas PEARL focuses on are, self-awareness, social skills, managing feelings, empathy and motivation.
Join us now to be a part of this innovative learning experience.
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Part-time: £1,540* per 20 credits studied (UK/EU)
The University offers a range of scholarships and bursaries to support you through your studies.Discover More
Our typical offer is 104 UCAS Points. We operate a flexible admissions policy and treat everyone as an individual. This means that we will take into consideration your educational achievements and predicted grades (where applicable) together with your application as a whole, including work experience and personal statement. General Studies accepted
BTEC Extended Diploma: Merit, Merit, Merit
BTEC Diploma: Distinction, Distinction
Pass Access Course: 106 UCAS Points
International Baccalaureate:Pass Diploma with 104 UCAS points from Higher Level Subjects
IELTS: 6.0 with no subscore lower than 5.5
GCSE: 5 at grade C/4 including Maths & English or equivalent
Applicants will be required to submit an electronic portfolio. You will sent an email by our Admissions Office with further details on what your portfolio should contain and how to submit it.
If you do not meet the formal entry requirements specified, Foundation Entry offers an alternative route to study for this degreeFoundation Entry Route
Not sure how many points you have? Use our handy calculator and find out.Points calculator
Plus 20 credits from:
Plus any 60 credits from:
Plus any 80 credits from:
A 3 year degree qualification typically comprises 360 credits and each 20 credit (a standard module) equates to 200 hours of study, which comprises of a mixture of lectures, seminars, tutorials, practical sessions and independent study. Independent study is an important aspect of your degree course. The exact combination of study time will be detailed within your module descriptors, and will depend on your option choices.
Lectures are delivered by industry practitioners and visiting specialists from different fields of film production and you'll be involved in film screenings, tutorials and technical workshops too.
You will be 'hands-on' focusing on industry practices and conventions to give you the opportunity to develop directly transferable skills in the new and emerging media markets.
Lectures are delivered by industry practitioners and visiting specialists from different fields of film production. These are three hours in length. There are also film screenings, tutorials and technical workshops. Typically, you would be spending 10 hours per week, per module on practical study.
The coursework consists largely of practical assignments. About 25-30 per cent of the assessed work is written work such as journals, production paperwork, evaluations and presentations. There are no examinations.
This course has been awarded the Creative Skillset Tick, the industry kitemark of quality, following a rigorous assessment process by experts working in the Creative Industries. The Creative Skillset Tick is awarded to practice-based courses which best prepare students for a career in the industry.
We have a well-established track record of delivering successful media students, with three recent Royal Television Society (RTS) awards in as many years, and our graduates feature in many credits of major cinema and TV productions. You could pursue a career in broadcasting, producing media management or scriptwriting.
You'll have the opportunity to produce your own full feature film - which is unique for an undergraduate film production course in the UK. You'll also have the opportunity to study abroad in Year 2, either in Europe or America.
UCLan is proud to host The Great Northern Creative Festival – a free four-day media extravaganza which brings together some of the biggest creative talents in the north as well as across the UK, whilst also showcasing the outstanding creative talents of UCLan students and graduates in media, film, photography and journalism. For more information, visit www.thegreatnortherncreativefestival.wordpress.com
Final year undergraduate and postgraduate media students have the opportunity to get involved in our annual cJAM employability event. cJAM shakes-up the traditional career fair format and gives UCLan students face-to-face time with up to 30 successful industry professionals all of whom have come to the event with the offer of valuable work placement opportunities. The cJAM event format consists of four main components:
In 2018, there were four cJAM events and a total of 320 placements were awarded. All of which enabled our students to establish credible links in line with their career aspirations. To find out more about cJAM: Media, see the video and press release.
New 'street' created in Preston
A new 'street' with a difference has been built in Preston city centre to enhance the creative abilities of University students.
The University of Central Lancashire (UCLan) has created an outdoor television set on campus which will allow students to learn more practical media skills in an industry standard environment.
You will have 24 hour access to rooms in our purpose-built Media Factory. You'll spend time in dedicated edit suites running Final Cut Pro and ProTools, with a sound stage, recording studio, Photography Studios and Television Studio.
BA (Hons) Film Production, 2014
UCLan graduate, Anthony Smith, has given his production career a kick-start by setting up his own company, Red Pencil Productions.
Anthony chose to study BA (Hons) Film Production after hearing a lot of great things about the course. He was particularly interested in the feature film module that was taught in the second year. Anthony believed that UCLan was the perfect University to move away to and gain independence.
This course is based in the School of Journalism, Media and Performance
Telephone us for further information +44(0)1772 892400
or email us at: email@example.com | Book a visit
For detailed information about studying this course at UCLan, please see the course handbook for your year of entry:
2018 Entry
| 2019 Entry
For information on possible changes to course information, see our Essential and Important Course Information.
*Tuition Fees are per year unless otherwise stated.
For 2018/19 fees please refer to our fees page.
You can find regulations and policies relating to student life at The University of Central Lancashire on our Student Contract page.
For changes to 2017 UCAS tariff entry requirements please see our Essential and Important Course Information. UCLan requires all undergraduate applicants to have a minimum attainment of five GCSEs at grade C and above, or equivalent, (including Maths and English). In 2017 and beyond we will view the new Grade 4 as being equivalent to a C grade and will therefore require students to achieve GCSE Grade 4 or above. However, if the subject is relevant to our degree programme and requires a higher GCSE grade (e.g. GCSE B grade), and/or includes a Professional body that governs the entry requirements, Grade 5 or above may be required.


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Carnival RideMusic CD - 2007
From the critics
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This big-haired blonde was a one time winner of the abysmal "American Idol". Consequently, I wasn't expecting much. What did we get here? Nothing more than Screamin' Celine DION doing a third-rate country impersonation. Boring "modern" hayseed background with a mighty whack of bathos (Just a Dream) screamed at full volume. (D-)
Contains some unnecessary programming. It should be a priest, not preacher. Tracks 7, 8, 10 and 12 are bad songs, and maybe Track 5 as well. Worth a listen. Featuring Vince Gill.
Carrie Underwood's voice gets better and better with each album that comes out. I wish she would do a Christmas album (two or three Christmas songs are on this CD) but touching songs as well as vengeful love songs are featured.
I love Carrie Underwoods songs they are amazing!!! I haven't heard all of them only just a dream, temporary home, and so small but I like them all. I love her voice so much better than demi lovato, selena gomez, or taylor swift. But I like some of their music to.
Carrie Underwood is a good singer! She actually has country songs that have a bit of pop, so it is good for my style. I didn't actually know she was on Idol before. My favourite songs are: All American Girl, Just a Dream, So Small, You Won't Find This.
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rosepetal16 thinks this title is suitable for 12 years and over
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Carrie Underwood is back with a second CD and is inching away from relying on American Idol. Filled with tracks, such as Twisted, So Small and Just a Dream.
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I was counting on forever now I'll never know
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Hi Jessica & Jonny,
I wanted to tell you how much fun we had on our vacation. Everything was great. It was very kind of you to e-mail me with additional information for our trip.
Your help in our preparation has made this a care free vacation and I can't tell you how much I appreciate that. We all enjoyed ourselves. We all wanted to say thanks for all you did to help us plan our vacation.
Robin Voss
North Carolina — July 2010
These vacations can be further modified according to lodging preferences, price range, number of activities, etc. Click here to customize your vacation.


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Brookstone Aero wine air infuser
Designed to release the full flavor and bouquet of your favorite red wines in mere seconds, the Brookstone Aero infuses air directly into the wine Read More
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The innovative Vinaera electronic aerator is based on some 5 years of research. The aerator offers full and fast aeration though a pressurized air and Read More
Made of the highest quality aluminium, the modular wine rack, Nucleus from Esthys is the best representation of modern minimalist design. The elegant wine rack Read More
This sleek glass front wine bar cabinet from Müller Classic Line is a perfect eye catching expression of contemporary living. Striking and extremely robust, this Read More
If you have forgotten to put the bottle of wine in the fridge and guests are on they way, enlist the help of the Waterfall Read More
Featuring a modern design with clean lines and tough yet elegant stainless steel construction, these wine racks and bottle holders will continue to store your Read More
Bring some mystery and romance in your living room with the incredibly beautiful John Rocha decanter from Waterford Black Cut Collection. The high gloss crystal Read More
[KBIS 2010 Review] Functional and sleek, Vynebar is a new vertical wine rack that offers trendy wine storage solution. The design is the trend for Read More
The easy to use Clatronic BZ3233 is a stylish Bierzapfanlage or beer dispenser with cooling function, designed unfortunately only for European markets. Finished in silver Read More
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Ideal for a smooth decanting process, this sleek hand made Chantilly wine decanter from Umbra is priced at $130. Perfect gift for the holidays.
You can select that favorite wine rack to suite your taste and decorate your modern house. Umbra offers several very interesting and modern designs. Loopsey Read More
Meneghini designs what is claimed to be the first kitchen sink equipped with an air- or water-based refrigeration system that holds your foods and beverages Read More


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Anyone and everyone studies martial arts at USA Karate. USA Karate students come from all walks of life: Moms, Dads, Kids, Grandmas, Grandpas. They come from many professions: Doctors, Lawyers, CPAs, and Engineers. They all benefit from martial arts training. Whether you are in peak physical condition or looking for a unique exercise program, USA Karate has a place for you. The classes are physical in nature because of the kicking drills, forms training and daily exercises and routines. All of these are combined to improve coordination, agility, reflexes and more. Karate also enhances any existing exercise program or sport and is a great release from stress. For parents considering enrolling a child, our report card policy ensures discipline in all aspects of the student's lives, not just karate. Each school age student is required to present a copy of their report card each grading session. The students, grades must be acceptable in order for the student to progress in karate. This helps the students realize the importance of their education and how it reflects on everything they do. And, of course, there is the aspect of self-defense. In today's world, it has become almost necessary to equip oneself with some form of protection. Practical application and continued practice in class help prepare the students for almost any confrontation.
Tae Kwon Do was originated in Korea and was introduced to the west in the late 1950's. It is the most popular of the martial arts practiced in America due largely to its high, flying kicks. Because not everyone has the flexibility and strength to perform such gymnastic-type movements, USA Karate has blended other systems together to provide a comprehensive program of self-defense.
These systems and techniques include:
Judo / Jujitsu -- Japanese arts utilizing leverage and body mechanics.
Hapkido -- Korean art using joint locks and joint manipulation.
Ryukyu Kempo -- Blend of Chinese and Okinawan arts. Forerunner to modern Karate.
Tuite / Kyusho - Jitsu -- Okinawan grappling and striking arts using the detailed science of acupuncture and related pressure points.
Modern Arnis -- Filipino stick fighting art that transposes self defense techniques for weapons and empty hand.
Western Boxing -- Footwork and close quarter fighting.
Q. Is tournament competition required?
A. Tournament competition is not required. If a student wishes to participate in a tournament, we will ensure that the student is well prepared.
Q. Do students have to spar?
A. Sparring is an integral part of Martial Arts, improving speed, self esteem, courage and self confidence. Depending on the readiness, individuals determine their own level of commitment.
Q. Are there injuries in Karate?
A. Safety is our number one priority. In fact, more injuries occur to and from karate than in the actual classes. Because karate is a contact activity, bumps and bruises do happen.
Q. Do children and adults train together?
A. Yes. The interaction between the two promote understanding and acceptance. Children must learn to accept adult discipline while many adults learn patience and understanding. This allows family members to train together.
Q. Am I too old, or is my child to young?
A. Regardless of age, USA Karate is for everyone. Ages five and older train in regular classes, while a special Mighty Mights class is designed for ages 3-5.
Starter Package!


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If You Work In An Office or Factory All Day, You Probably Are Not Getting Enough Sunshine to Make Vitamin D
If you read all the research on vitamin D3 that Dr. Michael Holick and Dr. Hector DeLuca discovered at the University of Wisconsin Madison, you would not go another day without supplementing your diet with vitamin D3. For everyone, it should be a supplement of vitamin D3. Probably there is only one in a thousand who get enough sunshine to make the body's own source of vitamin D3. The further away we live from the equator, the less the sun has the ability to reflect on our bodies to make vitamin D3. If we are overweight or have dark skin, we cannot hold on to vitamin D3. The older we are, the less ability our bodies have to produce vitamin D3. From the research on vitamin D3, it opens up a whole new world of possibilities for good health. It can reduce the risk of 17 different kinds of cancer by 77%. It reduces the risk of type 2 diabetes by 78%. It reduces the risk of depression by 80%. It can reduce influenza (flu) and infectious diseases by 90%. In a recent Japanese study, supplementing with vitamin D3 was almost equally effective as having a flu shot. Vitamin D3 could be your flu shot. According to research at the Vitamin D Council in Omaha Nebraska, adults should get 5,000 IUs of vitamin D3 daily. I take a berry flavored vitamin D3 tablet once daily. I think it makes me more resistant to colds and flu.


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4 is the new 8
9 comments:
- The Dragonfly said...
I'd never thought of having children as serving a mission. But I can totally relate to that idea. This post really resonated with me. I often feel like I'm not accomplishing much, but when I look at those sleeping babies at night, I feel better.
ps. what did you think of Downton?
- February 25, 2011 at 1:41 PM
- Apis Melliflora said...
There's a poem in the middle there somewhere. This was a beautifully written post, so thoughtful. You may not have painted with paint on canvass today, but you did it here with words.
- February 25, 2011 at 7:49 PM
- LisAway said...
Beautiful, MS! I love it. Great writing and message.
I'm not exactly sure that it's so selfish to want both things but I know that it IS unrealistic most days. :)
- February 26, 2011 at 8:10 AM
- Phoenix Peacock said...
I've been doing the same thing with instant netflix! it is an engrossing show that just makes sleep not seem that important - until its time to wake up!
I think it is only natural to feel trapped at certain stages of motherhood. I think it is wonderful that you can remind yourself how much you love it also. I also belive in having a reliable babysitter so that you can go out or have the kids go on a field trip just for a little bit of sanity!
Ps. I'd love it if you took a look at this:
- February 26, 2011 at 11:35 AM
- The Wingnut's said...
Downton Abbey is an amazing series. Cannot wait for the second season. I think I would of sat curled up on the couch watching that all day too!
- February 28, 2011 at 7:08 AM
- ilana said...
An inspiring and beautifully-written post. Just curious, where is the "Count your Blessings" graphic from?
- February 28, 2011 at 11:14 AM
- MelancholySmile said...
Ooops, forgot to give credit. I found the print here: http://svpply.com/item/276191/Blessings_12x18_Gicle_Print_from
- February 28, 2011 at 11:34 AM
- The Queen Vee said...
Beautifully written and well thought out. I think you used all your time wisely on this particular day, especially your run time....we have that to thank for this insightful post.
- March 1, 2011 at 7:55 AM
- hukolb said...
Thanks for this post, too! We must be on the same wavelength with young children at home and a nursing infant! At least you went running today:). My husband will like the new analogy to children, I tend to agree for whatever reason, rearing children is somehow looked at as more complicated and demanding. We had a great talk about "stuffication" which can be anything that eats up your time like things, people, etc...I think its great to slow down and do the important sometimes (I need to take my own advice). And I love your mission analogy, too! Thanks!
- March 1, 2011 at 6:59 PM


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Best barbecue joints in London
National Barbecue Week is 23rd – 30th May, but if you don't fancy lighting up a barbecue yourself or if the great British summer makes such an event impossible, why not treat yourself to a meal out in a top barbecue restaurant, such as these ones in London?
Seven great places to enjoy a barbecue in London:
- One Sixty Smokehouse has branches in the City and in West Hampstead. Enjoy smoked and seasoned meat accompanied by an extensive list of beers from all over the world.
- Pitt Cue Co, just off Carnaby Street, started as a food truck on the South Bank before coming to its current premises. A second restaurant has recently opened in the City.
- The Smokehouse Islington opened in August 2013. It has an extensive beer list and wine sourced from family-owned vineyards. Meat and fish from top UK suppliers are grilled over charcoal and roasted and smoked over English oak.
- Duke's Brew & Que boasts a greet selection of craft beers, which is the perfect accompaniment to this American-style barbecue. Smoking methods are derived from Texas, resulting in tender ribs and pulled pork.
- Berber & Q is a Middle Eastern restaurant in Haggerston. Enjoy the flavours of the Ottoman and North Africa, perhaps accompanied by craft beer, orange wine or a cocktail created from Middle Eastern ingredients.
- The Joint in Brixton started out in 2012 as a pop-up. Treat yourself to fresh free-range meat, including 16-hour slow-cooked pulled pork, accompanied by Brixton Brewery craft beer, St Reatham Lager or a speciality cocktail.
- Smoking Goat is a Thai barbecue in Denmark Street. Meat and day boat fish come direct from small production farmers and fishermen. Other ingredients, such as chillies, fish sauce and palm sugar, come from Bangkok for a truly authentic flavour.


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As Barack Obama traveled to Minneapolis to make his case for tougher gun regulations, the news media back in Washington was working itself into a lather over a photo of the president shooting skeet at Camp David.
In another example of "inside-the-beltway media malpractice," the press is distracting itself with irrelevant stunts rather than covering issues people care about, Nation editor and publisher Katrina vanden Heuvel said Monday on MSNBC's The Ed Show. The photo frenzy comes on the tenth anniversary of the Iraq War, when the Washington media displayed a similar lack of accountability, vanden Heuvel noted.
—Alec Luhn
Need a quick and popular way to raise revenue? Start by closing corporate loopholes, Katrina vanden Heuvel writes.


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Hello everyone!
So it is Tuesday today. I went the whole day thinking it was Monday. We had a long weekend and my brain got screwed up. It wasn't until my sister texted me to tell me she wasn't watching Dance Moms and to please e-mail her a summary of the episode that I realized it was Tuesday. I feel like I have missed a whole day!!!
So I did some baking! Because really, that's what you do when you realize you've been living in the past…kinda…
I started wanting to make banana bread, but I wanted it to be healthy and nutritious. We have gotten a lot of snow and there's just no time to make a full nutritious breakfast and still manage to leave at a decent time and arrive to work on time. So I started emptying out my cupboards…and mixing stuff together…finally I decided it would be more sensible to use my muffin tin because muffins are easier to eat on-the-go, plus there's no cutting involved!
Makes 12 muffins
- 1 cup spelt flour
- 1/4 cup quinoa flour
- 1/2 cup oats
- 1/3 cup brown sugar
- 3 medium ripe bananas (about 1 1/2 cup mashed bananas)
- 2 tsps baking powder
- 1/2 tsp baking soda
- 2 tbsp non-dairy butter
- 2 tbsp peanut butter*
- 2 tbsp vanilla soy milk
- 1 tsp vanilla extract
- handful of pecans (about 1/4 cup chopped)
- pinch of salt
*I used Kraft All Natural Smooth Peanut Butter, I haven't tried it with regular peanut butter, but I think the taste might still be the same.
- Preheat oven to 350F and prepare/grease muffin tin pan.
- In a large bowl, mix the sugar, baking powder, baking soda and salt. Add mashed bananas and mix well.
- Add in milk, peanut butter, and butter a spoonful at a time. Make sure to whisk so everything gets incorporated. Add vanilla extract.
- Add in quinoa flour and mix. Then add spelt flour slowly and mix well. Finally add oats and pecans.
- Scoop out batter into prepared muffin tin.
- Bake at 350F for 18-22 minutes or until toothpick inserted in the middle comes out clean.
I tried one and it was yummy! I will have one for breakfast tomorrow and hopefully it still tastes awesome! My mom will be taking half of them to share with a couple of her co-workers tomorrow. I'm crossing my fingers they'll like them.
Thanks so much for stopping by and have a great day!


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American Legion baseball: Seyler deals Beech Creek past Bellefonte
Bellefonte couldn't solve Beech Creek pitcher Caleb Seyler in a 6-0 loss in American Legion play on Sunday.
Seyler struck out six in a complete game. Beech Creek (3-1) scored the game's first run in the sixth inning on a sacrifice fly by Cole Hanley and put it away with a five runs in the seventh.
Hanley and Ethan McGill each finished with two RBIs for Beech Creek.
Seth Weaver had two singles for Bellefonte (3-3).


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Check out my stamps here~
|Deviant Art
Hello all!
You could call me Aly~ or Ally-Wally if you like
I started out this Deviantart account to post my drawings but then I'm not very good at it. So I skipped around and tried my luck on things like making stamps and fan videos. Currently I'm into cosplaying in which I'm just having so much fun. I'm an architecture student who dreams to build a better homes for all animals (especially) and people of the future generations, but is too lazy to do anything really. I spend most of my time with my pets and on ideas that are usually not realized because of my idleness or so.
You could check out my Architecture work here, my fanarts and my cosplays. Man, this account is old and I haven't really accomplished much but thank you for stopping by!
Feel free to visit my accounts on other sites~!
WorldCosplay worldcosplay.net/member/SabreX…
Cure No. 273954
Ample Site: ample-cosplay.com/?u=000000428…
Instagram: instagram.com/sabrelupe.cos
More of my cosplay here
Credits to the photographers.


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D.E. McNamara and R.P. Buland (2004). Ambient Noise Levels in the Continental United States. Bull. Seism. Soc. Am. 94/4, 1517-1527.
Swiss Data
HHZ channels
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Temporary Deployments
Aftershocks (8D) channels
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MoMaNic (Z9) channels
COSEISMIQ (2C,ON) channels


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It was a bright, summery winters (2010) day here in the middle east so we took the dog out of the city for a much needed hike. We ended up stumbling upon a fantastic huge field of thick, lush overgrown green grass and squatted for a mini picnic of butter croissants and cheesey sandwiches. I ended up tearing off my croissant-flake-covered skinny jeans and thick wool socks mid hike, to sport only an old AA T-shirt dress and pasty white legs. Although winter is and always will be my most beloved season, it did feel nerdily liberating to skinny down along the heated path and bare a little skin. Here's to a odd, warm winter's day (and the coolest dog ever):
Shopping: crop top, wool shark shorts, booties. Check out more FIELD TRIP PICKS.


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Bihar Board 10th Result 2016: The BSEB Matric Result 2016 will accessible on the official site (www.biharboard.ac.in Indiaresults.com). Bihar School Examination Board Patna will annonce Bihar 10th Result in the month of June 2016. The Bihar Board 10th Results 2016 is declared by Bihar Board. BSEB 10th result 2016 Name savvy, School astute, can be check from the online Results Partner of Bihar Board.
Bihar Board 10th Result 2016
The Bihar School Examination Board (BSEB) will declare Bihar Board 10th Results 2016 in the month of June 2016 at the official site of Bihar Board i,e http://www.biharboard.ac.in. The Candidates who are sitting tight for Bihar 10th Results 2016 are encouraged to check it from the official site to know mark sheet of examination. Applicants are asked for to visit this page for most recent reports in regards to all Information about Bihar Board like Bihar Board twelfth Result 2016, Bihar Board 10th Result 2016, BSEB twelfth Result 2016, BSEB Intermediate Result 2016, and BSEB Admit Card and so on or visit fundamental web gateway of Bihar School Examination Board Patna for most recent redesigns. We have likewise given here Bihar Board 10th Results 2016.
BSEB 10th Results 2016
The Bihar School Examination Board is set up for holding and leading an examination toward the end of the Secondary School stage, for recommending course of studies for such examination and for doing such different articles and obligations as might be viewed as important for the reason as expressed in the Act, Rules and Regulations of the Board. Typically consistently the Bihar School Examination Board conducts Annual Secondary School Examination in the month of February/March and Supplementary School Examination in the month of August/September on the premise obviously/syllabus as recommended by the state Government. Aside from the aforementioned Secondary School Examination, the Board additionally directs departmental examinations (not on yearly premise, for example, Diploma in Physical Education, Certificate in Physical Education and Teachers Training Examination on such terms and condition as set around the State Government.
http://www.biharboard.ac.in 10th result 2016
Name of the Board: Bihar School Examination Board
Name of the Exam: BSEB Matric Examination 2016
Exam Date: eleventh March to eighteenth March, 2016
BSEB Matric Result Date: first week of June 2016
Site: http://www.biharboard.nih.nic.in | http://www.biharboard.ac.in
BSEB Class 10th Result 2016
This year around 5, 69,537 (Expected) numbers of understudies was show up in the examination which is somewhat higher than a year ago. Consistently BSEB conducts the exams in the month of March and declares their outcome in the month of June. Scorecard is the last report to advise the contender about their powerless point and solid point. Understudies will have confirmation for advanced education according to their rate in 10th exam as it were.
Bihar Board 10th Result 2016
The board individuals are turning out to be all that much strict towards the tenets and regulations of leading the exam and assessing the answer sheets. This is the primary explanation for why every one of the contenders is so stress over their Result. Their nervousness is should likewise as this is the essential and most imperative stride in the life of every last understudy to get confirmation for further study. After Matric, Students can go for Intermediate and you are additionally allowed to study Diploma in Engineering, Diploma in Polytechnic and ITI course. For your sympathetic data, Students who study Intermediate then they have to choose stream out of Science, Commerce and Arts. There is number of branches are additionally accessible in Diploma, for example, Mechanical, Electrical, Polytechnic, EC, Computer, Civil, Chemical Etc.


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Aromatherapy Starter Kit
Aromatherapy Starter Kit: Either for yourself or as a gift for someone else.....everything you need to get started in the wonderful field of aromatherapy! No fancy labels, no fancy packaging, just lots of good stuff!
The kit consists of twelve 1/2 oz (filled with 1/6 oz product) 5ml amber bottles with orifice reducers filled with essential oils of Aniseseed, Bergamot (bergaptene free), Cedarwood Virginia, Eucalyptus globulus, Geranium, Grapefruit (pink), Lavender 40/42, Lemon, Orange (sweet), Patchouli, Peppermint (Yakima) and Tea Tree, 13 disposable pipettes, 13 ziplock bags to store the pipettes, 2 oz. of jojoba, 2 oz. of almond or sunflower oil, three 1/2 oz. amber glass bottles with orifice reducers, two 1 oz. HDPE plastic bottles and four 1/2 oz. HDPE plastic bottles. (Note: We reserve the right to substitute any item with a similar priced item when out of stock.)
Average Customer Review: Not yet rated
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important information
OUT OF STOCK ISSUES: If a customer orders a large quantity of a product and all of the product is not in stock when the order is entered into our system, once the new stock arrives we will ship separately whatever quantity we may owe the customer. Most of our restocking orders arrive at our warehouse 5-7 business days after we place a restocking order, sometimes sooner. Should the distiller or vendor inform us restocking will take longer than 7 business days, we'll promptly notify the customer.
Please note that we do not allow order cancellations for any reason and we don't allow removal/cancellations of products which become partially or completely out of stock after the customer places an order.
Raw materials/ingredients are dispensed by weight. Many customers ask how many ounces in a pound, there are 16 ounces in one pound. These are not fluid ounces, they are ounces by weight.
EO = essential oil
FO = fragrance oil
Our containers are filled with products to where our commercial digital scales indicate the container should be filled. Some containers will be filled to the top, some will not.
HOW OILS ARE DISPENSED: All our products are dispensed by weight. EO's in 8 oz. through 2 lbs. - amber plastic pharmaceutical-grade bottle. EO's in 5 and 10 lbs. - heavy duty plastic container w/pouring spout, the oil may remain inside the container at least 2 months. EO's in sizes of 25+ lbs. - industry-standard metal or plastic can with a pouring spout, the oil may remain inside the metal can indefinitely and up to 2 months in the plastic pail. It's our decision in which type of pail we dispense the product, metal cans are not readily available. CARRIER OILS - plastic bottles/pails depending on the size ordered, do not need to be transferred to glass unless you wish to do so. FO's - in plastic containers, contents do not need to be transferred to glass unless you wish to do so.
Essential oils in sizes from ½ oz. to 4 oz. are dispensed in amber glass bottles without orifice reducers or glass droppers. If we ever run out of amber glass bottles, we reserve the right to use any other glass bottle available at that time. The only sizes of EO's dispensed with orifice reducers are 1/32, 1/16, 1/8 and 1/4 oz. sizes. If you require bottles/vials with orifice reducers or Eurodroppers, we sell those separately.
HOW TO STORE: Upon receipt, EO's dispensed in plastic bottles MUST be transferred into amber glass bottles. EO's, FO's and BUTTERS should be stored in tightly-sealed containers away from direct light and heat. Many oils could be highly flammable, keep away from open flames. Refrigerating butters could adversely affect the consistency of the butter, please join our Forum if you need to learn how to handle and store products.
S&H QUOTES: Our cart provides all S&H quotes. Please look for the link to enter your zip code and obtain a quote. Shipments weighing over 120 lbs. will not be quoted final S&H by our cart, you'll be quoted a minimum for which you'll pay during checkout. We'll charge the balance due, if any, to the card on file. If you paid with PayPal, we'll ask you to disburse the amount due, if any, before we ship. S&H provided by our cart is always an estimate, please review our Shipping Policies page if you need more information. We do not accept or allow order cancellations because the customer doesn't agree to final S&H, please be sure you understand this before finalizing an order.
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