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Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5hmC and 5mC.
Methodology/Principal Findings
We have developed specific MethylScreen restriction enzyme digestion and qPCR-based protocols to more rapidly quantify DNA methylation at four CpG sites in the FXN upstream GAA region. Increased DNA methylation was confirmed at all four CpG sites in both FRDA cerebellum and heart tissues. We have also analysed the DNA methylation status in FRDA cerebellum and heart tissues using an approach that enables distinction between 5hmC and 5mC. Our analysis reveals that the majority of DNA methylation in both FRDA and unaffected tissues actually comprises 5hmC rather than 5mC. We have also identified decreased occupancy of the chromatin insulator protein CTCF (CCCTC-binding factor) at the FXN 5' UTR region in the same FRDA cerebellum tissues.
Increased DNA methylation at the FXN upstream GAA region, primarily 5hmC rather than 5mC, and decreased CTCF occupancy at the FXN 5' UTR are associated with FRDA disease-relevant human tissues. The role of such molecular mechanisms in FRDA pathogenesis has now to be determined.
Citation: Al-Mahdawi S, Sandi C, Mouro Pinto R, Pook MA (2013) Friedreich Ataxia Patient Tissues Exhibit Increased 5-Hydroxymethylcytosine Modification and Decreased CTCF Binding at the FXN Locus. PLoS ONE 8(9): e74956. https://doi.org/10.1371/journal.pone.0074956
Editor: Annalisa Pastore, National Institute for Medical Research, Medical Research Council, London, United Kingdom
Received: July 11, 2013; Accepted: August 1, 2013; Published: September 4, 2013
Copyright: © 2013 Al-Mahdawi 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: The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS (CS), the Wellcome Trust [089757] (SA) and Ataxia UK (RMP) to MAP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have no conflicts of interest to declare.
FRDA is an autosomal recessive neurodegenerative mitochondrial disorder caused primarily by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene [1]. Unaffected individuals have up to 43 GAA repeats, while affected individuals have 44 to 1700 GAA repeats, most commonly between 600–900 GAA repeats [2,3]. The length of the smaller GAA repeat correlates with FRDA disease severity and inversely correlates with the age of onset [4,5]. The effect of the GAA repeat expansion is to decrease expression of the essential and ubiquitously expressed mitochondrial frataxin protein, with levels in FRDA patients ranging from 4% to 29% that of normal [6]. Reduced levels of frataxin in FRDA patients are associated with defects of iron–sulphur (Fe–S) cluster biosynthesis [7], mitochondrial iron accumulation in the heart, spinal cord and dentate nucleus of the cerebellum [8] and increased susceptibility to oxidative stress [9].
Two main hypotheses have been proposed to link FXN GAA repeat expansions with decreased frataxin expression. Firstly, evidence from in vitro and cell transfection studies suggests that GAA repeat expansions may adopt abnormal non-B DNA structures (triplexes or "sticky DNA") or DNA•RNA hybrid structures (R-loops), which impede the process of RNA polymerase II and thus reduce FXN gene transcription [10,11]. Secondly, there is evidence that GAA repeat expansions can induce heterochromatin-mediated gene silencing effects [12]. Consistent with the latter hypothesis, several FRDA-related epigenetic changes have been identified in the immediate vicinity of the expanded GAA repeats of the FXN gene [13]. An initial investigation of DNA methylation of the FXN gene by bisulfite sequencing revealed hypermethylation of the cytosine residue of specific CpG sites upstream of the GAA repeat sequence in FRDA patient lymphoblastoid cells compared to cells derived from unaffected individuals [14]. We have subsequently used bisulfite sequencing to identify increased DNA methylation at the upstream GAA repeat region in FRDA patient cerebellum and heart autopsy tissues, which are clinically relevant tissues in FRDA [15]. Interestingly, we also identified reduced levels of DNA methylation in the downstream GAA repeat region in FRDA patient tissues compared with controls. These findings have now been confirmed by bisulfite-based EpiTYPER MassARRAY analysis of blood and buccal cell samples from a large cohort of FRDA patients, where a significant inverse correlation was also detected between the level of DNA methylation in the upstream GAA region and the level of FXN expression [16]. Yet another study has shown that the degree of DNA methylation in the upstream GAA repeat region in FRDA patients correlates with the length of the GAA repeats and inversely correlates with the age of disease onset [17]. Therefore, there is good evidence that DNA methylation may have some role to play in the molecular mechanism of GAA repeat induced FRDA disease. However, bisulfite sequencing is a somewhat time-consuming procedure. Therefore, we now report a more rapid MethylScreen restriction enzyme digestion and qPCR procedure, which we have validated by confirming increased DNA methylation at the upstream GAA repeat region in FRDA patient cerebellum and heart autopsy tissues.
Methylation of mammalian DNA primarily occurs as 5mC modification of CpG dinucleotides. However, recent studies have revealed the existence of an alternative modification, 5hmC, which is formed by oxidation of 5mC by ten-eleven translocation (TET) enzymes [18,19]. The overall levels of 5hmC in the mammalian genome are approximately 10% of 5mC levels [20], although higher levels have been detected in tissues of the central nervous system [21]. For example, 5hmC is approximately 40% as abundant as 5mC in the DNA of Purkinje cells of the cerebellum [18]. There are two main theories regarding the function of 5hmC. Firstly, 5hmC may be an intermediate in the removal of 5mC by an active demethylation process that involves the base excision repair pathway [22]. Secondly, 5hmC may be an epigenetic modification in its own right, regulating chromatin or transcriptional factors involved in processes such as neurodevelopment [23]. Therefore, it is possible that 5hmC may also be involved in epigenetic-based disease mechanisms, such as those proposed for FRDA. However, useful information cannot be obtained from previous bisulfite sequence DNA methylation data, because this method does not distinguish between 5mC and 5hmC [24]. Therefore, we have analysed the DNA methylation status of one of the FXN upstream GAA CpG sites in FRDA cerebellum and heart tissues using a procedure that does allow distinction between 5hmC and 5mC and we report that the majority of the methylated DNA at this CpG residue comprises 5hmC rather than 5mC.
In addition, another factor that has been proposed to be involved in silencing of the FXN gene in FRDA is the chromatin insulator protein CTCF, because decreased occupancy of CTCF has been identified at the FXN 5' UTR region of FRDA fibroblast cells [25]. Therefore, we have decided to further investigate CTCF occupancy in FRDA cerebellum tissue and we report decreased occupancy of CTCF at the FXN 5' UTR region. Overall we show that increased 5hmC at the FXN upstream GAA region and decreased CTCF occupancy at the FXN 5' UTR are both associated with FRDA disease-relevant human tissues.
Materials and Methods
DNA preparation from human tissues
Human cerebellum and heart tissue samples were obtained from autopsies of four FRDA patients (aged 17, 24, 36 and 47, each homozygous for GAA repeat expansions within the range of approximately 550-750 units) and four unaffected individuals (aged 69-82, obtained from NDRI). Research ethical approval was obtained from the SHSSC Research Ethics Committee, Brunel University, and samples were obtained with written consent and were stored in accordance with UK Human Tissue Authority ethical guidelines. For the FRDA patient aged 17, written informed consent was obtained from the next of kin under the organisation of the Friedreich's Ataxia Group tissue donor scheme. Genomic DNA was isolated from frozen tissues by standard phenol/chloroform extraction and ethanol precipitation.
MethylScreen analysis
Four CpG sites in the FXN upstream GAA repeat region, designated 3, 6, 11 and 13 (numbered according to [14], Figure 1), were investigated using adaptations of the previously described MethylScreen assay [26]. 1µg of genomic DNA was digested with: (1) a methylation-sensitive restriction enzyme (MSRE), (2) a methylation-dependent restriction enzyme (MDRE), (3) both MSRE and MDRE (double digest, DD), and (4) neither MSRE or MDRE (mock control). The MSREs used for CpGs 3, 6, 11 and 13 were AciI (Fermentas), Hpy188III (New England Biolabs), AjiI (Fermentas) and Eco72I (Fermentas), respectively. The MDRE used for all four CpGs was McrBc (Fermentas). A 50ng aliquot of digested DNA was then amplified by quantitative PCR using SYBR® Green (Applied Biosystems) and an ABI7900HT Fast Real-Time PCR System with the following primers: CpG3 F 5'-GAGACGTGGCTTTGTTTTCTG-3' and R 5'-GTTTCCTCCTTTCAAGCCGTG-3'; CpG6 F 5'-GAAGATGCCAAGGAAGTGGTAG-3' and R 5'-GAGCAACACAAATATGGCTTGG-3'; CpG11 and CpG13 F-met 5'-GAACCGTCTGGGCAAAGGCCAG-3' and R-met 5'-ATCCCAAAGTTTCTTCAAACACAATG-3'. PCR quantification was carried out using the ΔCt method (values were calculated as 2 ΔCt (mock – digest) with the mock value set at 100%) and RQ Manager software (Applied Biosystems). Each qPCR reaction for each sample was performed in triplicate in a 96-well plate. MethylScreen DNA methylation values were then calculated as follows: Densely methylated (DM) = (MSRE-DD)/(100-DD) x 100; unmethylated (UM) = (MDRE-DD)/(100-DD) x 100; intermediately methylated (IM) = 100 - (DM+UM).
Schematic diagram of 2.5kb of the FXN exon 1 and intron 1 region showing the GAA repeat within an Alu sequence (light grey box), together with the regions of 5mC/5hmC MethylScreen (dark grey box) and 5hmC EpiMarkTM (black box) analysis. The numbers represent the CpG sites, which are numbered according to [13]. Asterisks indicate the positions of the three potential CTCF binding sites that were analysed by EMSA.
5hmC analysis
Levels of 5mC and 5hmC were determined at CpG site 12 (numbered according to [14]) of the FXN upstream GAA repeat region (Figure 1) using the EpiMarkTM Analysis Kit (New England Biolabs). 1µg genomic DNA was glucosylated with T4 B-glucosyltransferase (T4-BGT), and then both glucosylated (G) and non-glucosylated (NG) DNA samples were digested with the methylation sensitive restriction enzyme, HpaII, or the methylation insensitive restriction enzyme, MspI. Digested DNA, together with non-digested (mock control) DNA samples were then amplified by quantitative PCR using SYBR® Green (Applied Biosystems) on an ABI7900HT Fast Real-Time PCR System with F-met and R-met PCR primers (the same as used for MethylScreen analysis). PCR quantification was carried out using the ΔCt method (values were calculated as 2 ΔCt (mock+G – digest) with the mock+G value set at 100%) and RQ Manager software (Applied Biosystems). Each qPCR reaction for each sample was performed in duplicate in a 96-well plate.
CTCF electrophoretic mobility shift assay (EMSA)
EMSA was performed with recombinant CTCF protein and three γ-32P end-labelled FXN probes, together with CTCF-positive (DM1 (1)) and CTCF-negative (DM1 (3)) probes [27]. The probes were prepared by PCR amplification of human genomic DNA using the following primer pairs: FXN 5' UTR, F 5'-AAGCAGGCTCTCCATTTTTG-3', R 5'-CGAGAGTCCACATGCTGCT-3'; FXN GAA Upstream, F 5'-GAAACCCAAAGAATGGCTGTG-3', R 5'-TTCCCTCCTCGTGAAACACC-3'; FXN GAA Downstream, F 5'-TGGGTTGTCAGCAGAGTTGT-3', R 5'-CCGATAATCCCAGCTACTCG-3'; DM1 (1), F 5'-GCCTGCCAGTTCACAACC-3', R 5'-AGCAGCATTCCCGGCTAC-3'; DM1 (3), F 5'-AGCTTTCTTGTGCATGACG-3', R 5'-GGTTGTTGGGGGTCCTGTAG-3'. PCR products were gel purified using Geneclean III (BIO 101) and end-labelled with γ-32P ATP using T4 polynucleotide kinase. Labelled PCR products were put through a Microspin S-200 HR column (GE HealthCare) and then incubated with 0.2µg of CTCF full-length recombinant protein (Abnova) in PBS buffer containing 5mM MgCl2, 0.1mM ZnSO4, 1mM DTT, 0.1% Nonidet P-40, 10% glycerol and 1µg of double-strand competitor DNA poly(dI-dC) (Sigma). After incubation for 30 minutes at room temperature, samples were run on a 5% nondenaturing polyacrylamide gel in 0.5X TBE buffer at 100V for 1-2 hours. Gels were then vacuum dried and exposed to ECL Hyperfilm (GE HealthCare), followed by film development.
CTCF chromatin immunoprecipitation (ChIP)
Human cerebellum tissue samples (30mg from each of three FRDA and three unaffected controls, the same as used for DNA preparation) were homogenised, followed by cross-linking of DNA and protein by formaldehyde treatment. Cell nuclei were isolated by centrifugation and then lysed in the presence of protease inhibitors [15]. DNA was sheared by sonication, followed by immunoprecipitation with anti-CTCF antibody (Upstate 07-729). After reversal of cross-linking, qPCR amplification of the resultant co-immunoprecipitated DNA was carried out with SYBR® Green in an ABI7400 sequencer (Applied Biosystems) using FXN 5' UTR primers and GAPDH (negative for CTCF) control primers as previously described [25]. PCR quantification was carried out using the ΔΔCt method and RQ Manager software (Applied Biosystems). Each value of immunoprecipitated DNA was normalised to both input and GAPDH values. Each tissue sample was subjected to four independent ChIP procedures, followed by triplicate qPCR analysis.
MethylScreen analysis is a rapid method to quantify DNA methylation in the FXN upstream GAA repeat region
We, and others, have previously used bisulfite sequencing to quantify DNA methylation changes at the FXN upstream GAA repeat region in FRDA cells and tissues [14–17]. However, bisulfite sequencing is a somewhat time-consuming procedure, which can be subject to errors due to incomplete bisulfite treatment. Therefore, we have now adapted the previously described MethylScreen assay [26] to rapidly quantify the DNA methylation status at four CpG sites at the FXN upstream GAA repeat region, designated 3, 6, 11 and 13 according to [14] (Figure 1). The MethylScreen method does not require bisulfite treatment of DNA, but involves digestion of genomic DNA with a MSRE, a MDRE, and both a MSRE and a MDRE, followed by qPCR amplification of the residual non-digested DNA using an amplicon that spans the CpG site of interrogation. The output data is expressed as three levels of DNA methylation status: unmethylated (UM), intermediately methylated (IM) and densely methylated (DM). We validated the MethylScreen assay by investigating the levels of DNA methylation at CpG11 and CpG 13 in cerebellum and heart tissue from FRDA patients and unaffected controls, which allowed a direct comparison with our previously reported bisulfite sequence analysis [15]. In addition, we investigated CpG3 and CpG6 in cerebellum and heart tissue, since these sites together with CpG13 have previously been identified as the three sites of most significant DNA methylation increase in FRDA versus control lymphoblastoid cells [14]. Our results for cerebellum tissue reveal an increase in DNA methylation at all four CpG sites in FRDA tissue compared with unaffected control tissue, in agreement with all previous bisulfite sequencing data (Figure 2). However, the magnitude of this DNA methylation increase was found to vary from a non-statistically significant small increase in DM of 6% to 20% at CpG3, through an intermediate level of increase in DM of 4% to 68% at CpG6 (P<0.001), to high levels of increase in DM of 14% to 96% at both CpG11 and CpG13 (both P<0.001). The results for heart tissue also reveal significant increases in DNA methylation at all four CpG sites in FRDA tissue compared with unaffected control tissue, with DM values increasing from 0% to 93% at CpG3 (P<0.001), 15% to 92% at CpG6 (P<0.001), 32% to 83% at CpG11 (P<0.01) and 21% to 97% at CpG13 (P<0.001), again in agreement with all previous bisulfite sequencing data (Figure 3). Therefore, the specific MethylScreen protocols that we have developed can be considered as both rapid and robust procedures to investigate DNA methylation status at the FXN locus.
MethylScreen analysis of four CpG sites in the FXN upstream GAA repeat region of DNA from FRDA and control cerebellum tissues. UM = unmethylated, IM = intermediately methylated, DM = densely methylated. Error bars = s.e.m. n=4.
MethylScreen analysis of four CpG sites in the FXN upstream GAA repeat region of DNA from FRDA and control heart tissues. UM = unmethylated, IM = intermediately methylated, DM = densely methylated. Error bars = s.e.m. n=4.
5hmC is the predominant constituent of DNA methylation in the FXN upstream GAA repeat region and is increased in FRDA cerebellum and heart tissues
Levels of 5mC and 5hmC were determined at CpG site 12 of the FXN upstream GAA repeat region (Figure 1) using the EpiMarkTM Analysis Kit (New England Biolabs). This CpG site was chosen because it resides within a CCGG motif, which is required by the EpiMarkTM procedure for differential restriction enzyme digestion by HpaII and MspI. The principle of this technique depends on the addition of glucose to the hydroxyl group of 5hmC via an enzymatic reaction utilising T4-BGT. Genomic DNA is glucosylated, then digested with a methylation sensitive restriction enzyme, HpaII, and a methylation insensitive restriction enzyme, MspI. If 5hmC is present in the context of CCGG, this modification converts a normally cleavable MspI site to a non-cleavable one.
Initial determination of DNA methylation levels (combined 5mC and 5hmC) by observing HpaII + G and HpaII qPCR values at CpG12 in cerebellum tissue reveals a significant increase in DNA methylation in FRDA tissue compared with unaffected control tissue (Figure 4A), at levels similar to those previously identified by bisulfite sequencing. Thus, DNA methylation levels increase from either 36% to 77% (HpaII + G, P<0.01) or 30% to 87% (HpaII, P<0.05) compared with previously reported bisulfite sequence values of 40% to 95% [15]. However, more detailed analysis reveals that there is no difference in the MspI + G and HpaII + G qPCR results from unaffected cerebellum tissues, and similarly there is no difference in the MspI + G and HpaII + G qPCR results from FRDA cerebellum tissues (Figure 4A). This indicates that all methylated DNA in both the unaffected and the FRDA cerebellum tissue comprises 5hmC rather than 5mC. Therefore, we can now conclude that a characteristic feature of FRDA cerebellum tissue is an increase in 5hmC-modified DNA in the FXN upstream GAA repeat region, at least at CpG site 12. Determination of DNA methylation levels (combined 5mC and 5hmC) in heart tissue also reveals a significant increase in DNA methylation in FRDA tissue compared with unaffected control tissue (Figure 4B), at levels similar to those previously identified by bisulfite sequencing. Thus, DNA methylation levels increase from either 44% to 100% (HpaII + G, P<0.01) or 37% to 89% (HpaII, P<0.001) compared with previously reported bisulfite sequence values of 65% to 95% [15]. However, more detailed analysis of the MspI + G compared with HpaII + G qPCR results indicates that the methylated DNA comprises approximately 64% 5hmC in the unaffected heart tissues and only 37% 5hmC in the FRDA heart tissues, although there is still an overall increase in 5hmC levels (MspI + G values) in FRDA compared with controls (Figure 4B). This suggests that, while there is increased DNA methylation in both cerebellum and heart tissues, there are also tissue specific differences: DNA methylation is entirely due to 5hmC in the cerebellum, but only partially due to 5hmC in the heart.
EpiMarkTM 5hmC and 5mC analysis of CpG site 12 in the FXN upstream GAA repeat region of DNA from FRDA and control cerebellum (A) and heart (B) tissues. G = glucosylated DNA. MspI values represent complete digestion control levels of DNA; MspI + G represents 5hmC levels, HpaII and HpaII + G represent combined 5mC and 5hmC levels; Mock and Mock +G represent undigested control levels of DNA. Error bars = s.e.m. n=4.
CTCF occupancy at the FXN 5' UTR region is decreased in FRDA cerebellum tissue
We firstly performed in silico screening of a 40kb region spanning the FXN gene for potential CTCF binding sites using the bioinformatics web tool designed by Klenova and colleagues (http://www.essex.ac.uk/bs/molonc/spa.htm) and we identified three regions of interest at the start of the FXN gene. We then investigated the three potential CTCF binding regions, which were located within the 5' UTR, GAA upstream and GAA downstream regions of the FXN locus (Fig 1), by EMSA using recombinant full length CTCF protein. Only the 5' UTR probe proved capable of binding CTCF protein to cause a gel shift (Figure 5A). This result is in agreement with a previous report that identified CTCF binding in the FXN 5' UTR region using fibroblast cell lysates [25]. We then performed ChIP analysis to assess the level of CTCF binding at the 5' UTR site in three FRDA cerebellum tissue lysates compared with three unaffected controls, using the same qPCR amplicons as previously described [25]. A statistically significant decrease (P<0.05) in CTCF binding was detected in FRDA cerebellum, with levels falling to only 20% of the levels detected in unaffected cerebellum controls (Figure 5B).
(A) EMSA showing a mobility shift for the FXN 5' UTR probe and CTCF-positive DM1 (1) probe [35], while there is no such shift detected for the FXN upstream GAA (Up) probe, FXN downstream GAA (Down) probe or CTCF-negative DM1 (3) probe [35]. (B) ChIP analysis showing the relative CTCF occupancy in the FXN 5' UTR of DNA from FRDA and control cerebellum tissues. Error bars = s.e.m. n=3. * = P<0.05.
We have investigated two molecular mechanisms that may be involved in the disease process in FRDA patient cerebellum and heart tissues. Firstly, we have developed a rapid and robust MethylScreen method to determine the general DNA methylation status (5mC and 5hmC) at the FXN upstream GAA repeat region. The level of DNA methylation in FRDA cerebellum tissue showed a gradation across the four CpG sites investigated, progressively increasing as you move closer to the GAA repeat locus. This suggests that the GAA repeat expansion mutation may act as focal point to induce only a localised effect of DNA methylation, spreading upstream from this point. However, this gradation effect was not seen in FRDA heart tissue, which demonstrated high levels of DNA methylation at all 4 CpG sites, indicating a degree of variability in the DNA methylation process in different FRDA tissues. Such variability may have some bearing on the previously identified differences in GAA repeat somatic instability, with cerebellum showing high levels of instability, while heart is comparatively stable [28].
Subsequently, we have shown that the methylated DNA at one CpG site in the FXN upstream GAA region comprises entirely 5hmC in FRDA cerebellum, but only partially 5hmC in FRDA heart. Our results are in agreement with the recent report that 5hmC is particularly associated with human cerebellum throughout development [29]. However, we can discount the possibility that the increased 5hmC that we have observed in FRDA tissues is a non-specific age-related effect in neuronal tissues, as previously reported for mouse hippocampus and cerebellum [23,30], because our FRDA patients are actually younger than the unaffected controls. Further studies will be required to determine if our finding is a general increased 5hmC effect for CpG sites within the FXN upstream GAA region, or indeed within other regions of the genome. The high levels of 5hmC that we have detected at the FXN upstream GAA region could be explained by either a passive or an active process. Thus, 5hmC could merely be a non-functioning intermediate in a demethylation process that is attempting to reverse the GAA repeat expansion-induced 5mC DNA methylation that is associated with FXN gene silencing [17]. In this case, the differences that we have detected between 5hmC levels in cerebellum and heart tissues could perhaps be due to tissue-selective differences in the efficiency of the TET enzymes that convert 5mC to 5hmC [18,19] or the DNA repair systems (e.g. base excision repair) that are required to convert 5hmC to cytosine [22]. Alternatively, 5hmC may have an as yet unidentified role in regulation of gene expression. For example, it has shown in vitro that 5hmC at the promoter but not in the gene body negatively regulates gene expression [31]. Furthermore, it has been proposed that 5hmC may be involved in the regulation of genes that are silent but transcriptionally poised for activation, where there is loss of 5mC, recruitment of Polycomb repressive complex 2 (PRC2) and, therefore, methylation of histone 3 at lysine 27 (H3K27me3) [20]. Indeed, 5hmC and H3K27me3 have been shown to have high correlation across a variety of somatic tissues [32]. Therefore, it is interesting to note that the FXN gene demonstrates RNA polymerase II pausing and FRDA lymphoblastoid cells have shown increased levels of H3K27me3 in the FXN upstream GAA repeat region [33,34]. In addition, it has been proposed that 5hmC may inhibit DNMT1 binding and may therefore function as a negative regulator of 5mC levels at protein-DNA interaction sites, allowing the binding of enhancer proteins or transcription factors [35]. Furthermore, 5hmC has been reported to bind MECP2, particularly in the CNS, although the functional consequences of such an interaction are not yet known [36]. Although the close positioning of the GAA repeat expansion mutation and the increased 5hmC modification of DNA in the FXN upstream GAA repeat region in FRDA would suggest a likely causal relationship, there may also be other more general factors involved in the formation of 5hmC. For example, the TET enzymes that produce 5hmC are known to be dependent on Fe(II) and α-ketoglutarate. Therefore, disruptions of iron metabolism, oxidative stress and the TCA cycle, processes which are highly relevant to FRDA, may all potentially affect 5hmC levels [37].
The second molecular change that we have identified in FRDA cerebellum is reduced CTCF occupancy at the FXN 5' UTR region, confirming previous findings in FRDA fibroblast cells [25]. It has been hypothesized that decreased binding of CTCF in FRDA cells may be an epigenetic switch that allows increased frataxin antisense (FAST-1) transcription, spread of heterochromatin and resultant FXN gene silencing. DNA methylation has not been thought to play an active role in this process, because increased DNA methylation has not been detected at the CTCF binding site in the FXN 5' UTR region of FRDA cells or tissues [15,16,25], and the increased DNA methylation at the FXN upstream GAA repeat region has been considered to be an unconnected marker of the heterochromatin state. However, if there is a demethylation process occurring at the FXN upstream GAA repeat region, as indicated by the high levels of 5hmC that we have now identified, then this may indeed be involved in increasing FAST-1 expression, CTCF depletion and hence FXN gene silencing. In conclusion, we can propose a model for FRDA that combines both the increased 5hmC modification of DNA and decreased CTCF binding at the FXN locus. Further studies will now be required to determine the exact relationship between these molecular mechanisms and FRDA pathogenesis.
The unaffected human tissues used in this study were provided by the National Disease Research Interchange (NDRI).
Author Contributions
Conceived and designed the experiments: SA RMP MAP. Performed the experiments: SA CS RMP MAP. Analyzed the data: SA CS RMP MAP. Wrote the manuscript: SA CS RMP MAP.
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- 19. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H et al. (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324: 930-935. doi:10.1126/science.1170116. PubMed: 19372391.
- 20. Branco MR, Ficz G, Reik W (2011) Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat Rev Genet 13: 7-13. doi:10.1038/ni.2192. PubMed: 22083101.
- 21. Globisch D, Münzel M, Müller M, Michalakis S, Wagner M et al. (2010) Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLOS ONE 5: e15367. doi:10.1371/journal.pone.0015367. PubMed: 21203455.
- 22. Guo JU, Su Y, Zhong C, Ming GL, Song H (2011) Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145: 423-434. doi:10.1016/j.cell.2011.03.022. PubMed: 21496894.
- 23. Szulwach KE, Li X, Li Y, Song CX, Wu H et al. (2011) 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nat Neurosci 14: 1607-1616. doi:10.1038/nn.2959. PubMed: 22037496.
- 24. Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR et al. (2010) The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLOS ONE 5: e8888. doi:10.1371/journal.pone.0008888. PubMed: 20126651.
- 25. De Biase I, Chutake YK, Rindler PM, Bidichandani SI (2009) Epigenetic silencing in Friedreich ataxia is associated with depletion of CTCF (CCCTC-binding factor) and antisense transcription. PLOS ONE 4: e7914. doi:10.1371/journal.pone.0007914. PubMed: 19956589.
- 26. Holemon H, Korshunova Y, Ordway JM, Bedell JA, Citek RW et al. (2007) MethylScreen: DNA methylation density monitoring using quantitative PCR. BioTechniques 43: 683-693. doi:10.2144/000112597. PubMed: 18072598.
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Filippova GN, Thienes CP, Penn BH, Cho DH, Hu YJ et al. (2001) CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus. Nat Genet 28: 335-343. PubMed: 11479593.
- 28. De Biase I, Rasmussen A, Endres D, Al-Mahdawi S, Monticelli A et al. (2007) Progressive GAA expansions in dorsal root ganglia of Friedreich's ataxia patients. Ann Neurol 61: 55-60. doi:10.1002/ana.21052. PubMed: 17262846.
- 29. Wang T, Pan Q, Lin L, Szulwach KE, Song CX et al. (2012) Genome-wide DNA hydroxymethylation changes are associated with neurodevelopmental genes in the developing human cerebellum. Hum Mol Genet 21: 5500-5510. doi:10.1093/hmg/dds394. PubMed: 23042784.
- 30. Chen H, Dzitoyeva S, Manev H (2012) Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus. Restor Neurol Neurosci 30: 237-245. PubMed: 22426040.
- 31. Robertson J, Robertson AB, Klungland A (2011) The presence of 5-hydroxymethylcytosine at the gene promoter and not in the gene body negatively regulates gene expression. Biochem Biophys Res Commun 411: 40-43. doi:10.1016/j.bbrc.2011.06.077. PubMed: 21703242.
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- 33. Chan PK, Torres R, Yandim C, Law PP, Khadayate S et al. (2013) Heterochromatinization induced by GAA-repeat hyperexpansion in Friedreich's ataxia can be reduced upon HDAC inhibition by vitamin B3. Hum Mol Genet 22: 2662-2675. doi:10.1093/hmg/ddt115. PubMed: 23474817.
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- 37. Chia N, Wang L, Lu X, Senut MC, Brenner C et al. (2011) Hypothesis: environmental regulation of 5-hydroxymethylcytosine by oxidative stress. Epigenetics 6: 853-856. doi:10.4161/epi.6.7.16461. PubMed: 21617369.


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Author Tags: Outdoors, Women
Daisy Callison once accompanied her father's prospecting party through the Cassiar Mountain Range, seven people in total, travelling through dangerous, uncharted territory with sixty head of horses. She and her sister were the first white women to pass that way, in what is now part of the Muskwa Kechika park. Her memoir/novel Mountain Trails is based on the diary of a sixteen-year-old girl for a four-month period in 1935.
DATE OF BIRTH: July 22, 1919
PLACE OF BIRTH: Pouce Coupe, BC
ANCESTRAL BACKGROUND: Irish-American
EMPLOYMENT OTHER THAN WRITING: artist
Mountain Trails (Victoria: Desktop Publishers, 2004)
[BCBW 2005] "Women" "Outdoors"

subset: commoncrawl


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Michael Rocque
Michael Rocque received his PhD from Northeastern University's School of Criminology and Criminal Justice. Professor Rocque is from Augusta, Maine and attended the University of Maryland for his Master's degree. His research interests are criminological theory, racial disparities in the criminal justice system, and desistance from crime.
Professional Affiliations
- Senior Research Advisor, Maine Department of Corrections
Selected Publications
- Jennings, WG, Rocque, M, Fox, BH, Piquero, AR, & Farrington, DP. (Forthcoming). Can they recover? An assessment of adult adjustment problems among males in the abstainer, recovery, life-course persistent, and adolescent-limited pathways followed up to age 56 in the Cambridge Study in Delinquent Development. Development and Psychopathology.
- Rocque, M. (Forthcoming). The lost concept: The (re)emerging link between maturation and desistance from crime. Criminology & Criminal Justice. DOI: 0.1177/1748895814547710
- Rocque, M, Posick, C, & Felix, S. (2015). The role of the brain in urban violent offending: Integrating biology with structural theories of "the streets." Criminal Justice Studies. 28(1): 84-103.
- Rocque, M, Posick, C, Barkan, SE, & Paternoster, R. (2015). The relationship between marriage and county-level crime rates: A research note. Journal of Research in Crime & Delinquency. 52(1): 130-145.
- Rocque, M., & Plummer-Beale, J. (2014). In the eye of the beholder? An examination of the inter-rater reliability of the LSI-R and YLS/CMI in a correctional agency. Journal of Criminal Justice. 42(6): 568-578.
- Welsh, BC, & Rocque, M. (2014). When crime prevention harms: A review of systematic reviews.Journal of Experimental Criminology. 10(3): 245-266.
Professor Michael Rocque's CV – August 2017


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Fresh Park Venlo was established as an auction site in the 1960s at the initiative of the Coöperatieve Venlose Veiling (CVV). The CVV bought a plot of land measuring 60 hectares between the Nijmegen-Venlo railway line and the A67motorway that was then still under construction. 1965 saw the official opening of what was then still called the 'auction site'. The site grew tremendously from the very beginning. By the 1980s this explosive growth induced the CVV to join forces with its rival auction company VGV. After the merger, on 1 January 1990, the two auctions' products were sold jointly. In 1992 the two parties joined the Coöperatieve Veiling Zuidoost-Nederland (ZON; Cooperative Auction South East Netherlands).
In 2001 ZON opted for a divisional structure with a separate branch for the management and expansion of the auction site. Introduced in 2000, the Fresh Park concept focuses on promoting chain cooperation in the fresh produce sector both within and outside the ZON site. Since then, dozens of initiatives have been launched.
As a result of a municipal reorganisation, Fresh Park Venlo has formed part of the municipality of Venlo since 1 January 2001. Strong international ambitions urged the organisation to change its name in 2008. Since 6 June 2008 the business park's official name has been Fresh Park Venlo. A name that paved the way for attracting fresh products other than fruit & vegetables and ornamental plants, such as meat, fish and dairy products.


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Prism layer to capture the Boundary Layer
3 Attachment(s)
Hi, I've been following a tutorial of a wingbody that shows how to add a layer of prism elements around a body.
I have followed the tutorial but it seems to be adding the layer of elements on the inside of my wing rather than the outside. Is there any way to change the side of the surface that the elements are added to?
I have attached some pictures of what is happening and what it should look like.
Edit: Should have said I'm using ICEM.
Have you planned to study the flow around the wingbody?
If yes, I'm not sure you need cells inside the wingbody. So you could delete them, and the prisms would be in the good part.
Anyway, when you create prisms, you can choose in which part the prisms will be (Mesh -> Compute Mesh -> Prism Mesh : select part for prism mesh).
Good luck ;)
|All times are GMT -4. The time now is 11:42.


text:
Question: A small metal crucible is taken from a 200 C
A small metal crucible is taken from a 200°C oven and immersed in a tub full of water at room temperature (this process is often referred to as quenching). What is the approximate final equilibrium temperature?
View Solution:
View Solution:
Answer to relevant QuestionsWhat is a major problem that arises in measuring specific heats if a sample with a temperature above 100°C is placed in water?Concrete has a higher specific heat than soil. Use this fact to explain (partially) why cities have a higher average nighttime temperature than the surrounding countryside. If a city is hotter than the surrounding ...Why can potatoes be baked more quickly when a metal skewer has been inserted through them?Updrafts of air are familiar to all pilots and are used to keep non-motorized gliders aloft. What causes these currents?A shaft is loaded in bending and torsion such that Ma = 600 lbf • in, Ta = 400 lbf • in, Mm = 500 lbf • in, and Tm = 300 lbf • in. For the shaft, Su = 100 kpsi and Sy = 80 kpsi, and a fully corrected endurance limit ...
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text:
Mercedes-Benz automobile enthusiasts have grown accustomed to a certain level of a reputation for strength when driving their S55 AMG around town. When your Mercedes-Benz S55 AMG was born, it was destined for a person with discerning taste who wants German engineering in their automobile. It's not all the time that you find yourself in need of a replacement Tie Rod to fix your automobile. While it's not hard to buy replacement parts for Mercedes-Benz vehicles, these days the necessity for nothing but the absolute best are required to repair your car or truck.
If we only drove in straight lines, then none of our vehicles would need any steering components. That would be very dull for Mercedes-Benz S55 AMG owners, and it would also mean that tie rods would likely never have been invented. The tie rod ends on your vehicle connect the center link or the steering rack to the spindles, allowing the wheels to pivot and turn. A broken Mercedes-Benz S55 AMG tie rod means you will have trouble turning, parking, or dodging obstacles in the road.
Given all the hassles associated with repairing damage to your car or truck, let www.carpartsdiscount.com be the most authoritative website you go to make buying it easy. Don't let a broken Tie Rod spell the end of your car, truck, or SUV, and don't gamble with its condition by using anything but the highest quality replacement parts. Trust in the value of the parts you get from carpartsdiscount.com and realize incredible savings on all your upcoming repairs. All the Mercedes-Benz S55 AMG Tie Rod parts on this page will fit vehicles made in 2006, 2005, 2004, 2003, 2002, 2001. Choose your year below to refine your search.


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Oct 2015 Update: Finally lost money in Vegas :(
Movies: La Vita e Bella, The Usual Suspects, The Game, Memento, The Dark Knight, Gladiator, Inception, Braveheart, The Green Mile. In essence, most movies that either toy with your emotions, or mess with you head.
Music: I don't listen to nearly as much music as in my high school/college days. Just about anything other than country or jazz will do though.
Food: You really can't go wrong with Italian, albeit I'm up for just about anything.
If you are, or know of said princess or dragon, then you should probably message me.


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Main season: 1st of April to 31st of October, 2017
* = Parents with children up to 19 years, only with family card.
By arrival per ship approximadetly 5 % reduction (only in the summer saison). Coupons are offered for reduced Pfänder cable car tickets exclusively in advance at the shipping ports and on all ships to Bregenz.
Information: Internationale Bodensee Tourismus GmbH
Tel. +49 7531 90 94 90 - www.bodensee.eu
with family card, e.g. family pass Vorarlberg
Rights are reserved for changes in the timetable and price


text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Form redirects to a closed Adobe Form Central form when submittedAsked by lakurnik on February 24, 2015 at 01:23 PM
Migrated form from Adobe Forms Central has a message that it is closed but it's not - how do I fix this?
This form was migrated from Adobe FC. After I filled it out in preview mode, I get the confirmation by email but on my screen I get the message that the form is closed and it still says Adobe Forms Central on it. Cannot figure out how to change this.
JotForm Support
It's the thank you message/page settings in the form. You have the Adobe FC form as the redirect URL.
Here's our guide for setting up the thank you message/page: http://www.jotform.com/help/211-Setting-Up-the-Thank-You-Page.
Thank you!


text:
At $17.2 million, the largest research grant won by the LSU Agricultural Center is striking at one key question about renewable energy: Can you make fuel from plants pay its freight?
"Paying its freight" is not just an expression. Part of the cost of "biomass" fuels is the expense of harvesting and transporting the plants to processing plants.
The grant over five years is part of a larger initiative by the U.S. Department of Agriculture to come to grips with costs of biomass energy of different kinds, as well as making more fuels from different crops.
LSU's scientists and their partners in the grant will focus on sweet sorghum and sugar cane — particularly the "energy cane" that can be grown for this purpose.
While there are more questions than answers today about biomass fuels, LSU's work will include the costs of farming the plants as well as innovative ways to increase production on the farm and in the conversion facilities. It is a very practical initiative.
What can be definitively said is this grant represents a striking success for LSU scientists, as well as the extension agents who will work closely with farmers on the practical side of the research. A pilot plant for processing the cane or other products is to be built with part of the money.
"We built this grant through many collaborations," noted Vadim Kochergin, head of the Louisiana Institute for Biofuels and Bioprocessing.
It is also built through state investment in a knowledge economy. We don't know yet whether Louisiana will be, as John Russin of the LSU Agricultural Center says, as big in the biomass energy economy as our state has been in the traditional energy sector. But having cutting-edge research here might be more valuable someday than we can readily calculate today.


text:
Buy these useful purses at a great price.
Diamond Plate™ 4pc Ladies' Belt Loop Purse Set with Genuine Leather Accents.
All purses feature adjustable/removable shoulder straps.
4pce Set includes:
7-1/2" x 1-7/8" x 6-1/2" purse with zippered main and front pockets and flap with hook and loop closure;
11-1/2" x 7-7/8" purse with zippered main pocket, zippered privacy pocket and flap with metal skull emblem and snap closure;
7-1/8" x 2-7/8" x 5-3/4" purse with 2 zippered main pockets, 1 zippered back pocket, cell phone holder with flap closure, and front pocket with metal American flag emblem on flap with hook and loop closure;
8-3/4" x 3" x 6-1/4" purse with zippered main compartment with zippered privacy pocket and front pocket with metal rose emblem on flap with snap closure.
These cannot be ordered individually, we only sell these in complete 4pc sets.
We accept Visa, MasterCard, or PayPal.
This will ship via USPS Ground.


text:
Entry requirements
Postgraduate Certificate in Counselling Skills (Integrative pathway) or equivalent (approximately 150 hours training to include counselling skills, integrative counselling. Enhanced DBS Check. Two references uploaded with your application (One from your previous/current Counselling tutor). A degree or a qualification at level 6 or above*. A confirmed suitable placement for counselling practice. Please note that this is essential and you will be asked to provide details of your placement before the course begins. The criteria for a suitable placement will be discussed at interview and will be posted on the Counselling Landing Point along with a list of placements that our students have used before
Months of entry
Course content
The Postgraduate Diploma/MA in Integrative Counselling and Psychotherapy counselling and psychotherapy course, accredited by the British Association for Counselling and Psychotherapy (BACP), emphasises the integration of theory, research, practice, and a commitment to self-awareness to help you develop into a competent, creative and ethically-sound counsellor. The programme has a long tradition of preparing students to be versatile and adaptable in their therapeutic practice to meet the individual needs of different client groups and work settings.
This a professional qualification designed for students seeking a career as a qualified counsellor or psychotherapist working in the statutory and voluntary sectors, in business or private practice.
The course facilitates students in developing a critical understanding of the strengths and weaknesses of integrative counselling practice, with a view to them developing their own coherent, ethical and effective approach to counselling practice, which can be adapted for use in a wide range of settings for short and long term work. On successful completion, you will be ready to work as a trained Integrative therapist.
The PG Diploma stage equips you with the training for professional BACP accreditation. Successful completion of a third year leads to an MA in Integrative Counselling and Psychotherapy.
Visit University of South Wales website for more information.
Fees and funding
Visit www.southwales.ac.uk/money
Qualification and course duration
Course contact details
- Name
- Enquiries & Admissions
- firstname.lastname@example.com
- Phone
- 03455 760 599


text:
"I said," Thom drawled, "that you should tell Alanna to cease healing the queen."
"Are you insane?" she hissed, glancing around the dark corridor Thom had pulled her into. "Lianne could very well die if Alanna does that!"
"And Alanna might die if she doesn't!" Thom snapped back. Cythera blinked, taken aback by his words and astonished by the fear in his eyes.
"What do you mean?" she demanded, arms akimbo.
"I mean," Thom said slowly, "that it's very likely that whoever is behind these assassinations is hoping for Lianne's death. If Alanna wants to stay alive, she needs to stop all her extra activities, and soon."
Cythera frowned. "You're an idiot if you think Alanna will stop what she's doing, just because you're worried," she informed Thom coldly. "She's as stubborn as a mule, and determined to do what she wants to do."
"Which is why," Thom said with exaggerated patience, "I'm asking you to help convince my idiot sister to keep a low profile!"
"No," Cythera said shortly. "Not unless you tell me who is behind the attempt."
Thom froze. "I can't do that," he said after what seemed like forever.
"Then no deal," Cythera replied. She turned on her heel and walked away, head held high.
He knew, Cythera was certain of it. Thom knew who had tried to kill his sister. Now she just had to figure out how to get the information from him.
"So what?" Gary returned, frowning at his friends.
Jon rolled his eyes. "So, you saw her fight. How did she do?"
"You saw the duel we had," Gary pointed out sharply. Jon and Alex were watching him carefully, while Raoul hummed to himself.
"Yes, but she wasn't even really trying," Alex said, frowning. "So how did she do?"
Raoul rolled his eyes, finally shutting up. Gary sighed in relief- that damned humming had been grating on his nerves.
"She held her own," Raoul said, amused by Alex and Jon's persistence. "Against professional assassins, which should tell you something."
"Any idea who was behind the attempts?" Jon queried, head tilted slightly.
Gary sighed. "None. They were freelancers, that much we do know."
"Why, then?" Jon asked, looking honestly stumped.
"Because my father told the Council who had given him the information about the Tusaine incursion," Gary said acidly. "He hadn't thought the spy could be in the Council, but it seems he was wrong. Now it's common knowledge that Alanna was the one to get the information, and someone wants her out of the way."
Jon scowled. "I hate this, not knowing who might be a traitor," he said darkly. "And who in their right mind would try to kill a girl? She's only fifteen!"
"She's stepped on a lot of toes by giving Duke Gareth that information," Alex said mildly. "She's a new player, and no one knows quite what to make of her. Therefore, our spy wants her out of the way. I doubt he cares about her age."
"What did her brother say about the attacks?" Raoul asked, frowning. Gary shot his friend a sharp glance- there was something in Raoul's tone that alarmed the future Duke. Raoul, easy-going Raoul, sounded almost suspicious.
Jon was the one who answered. "Thom didn't say much, actually. But he looked furious. He was terrified that Lady Alanna had been hurt- the first time I've seen him so emotional, really. Though he laughed when he heard Lady Alanna's been assigned a guard."
Gary snorted. "Hasn't done much good," he said acerbically. "She still managed to sneak out to the city."
"I heard about that," Jon said, curious. "What for?"
"She isn't saying," Gary said, unable to hide his smirk. "Father was about ready to start screaming in frustration."
"That'd be a sight," Raoul murmured mischievously.
Duke Gareth rarely lost his temper. But he was getting very, very close to it now.
He was furious with himself, first and foremost. True, he hadn't suspected that the spy might be a member of the King's Council...but he still should have kept Alanna's name quiet. Now there were assassins after her blood.
He was also furious with Alanna herself. What had she been thinking, going to the city like that? And she wouldn't even tell him why!
It was stupidity, sheer idiocy, for her to pull those kinds of tricks. Especially when she was so needed: without Alanna, the queen might very well relapse, the tentative new peace between Naxen and Trebond would fail, and Tortall would lose a fine citizen. She had to learn to be careful, or she would get herself killed before she was twenty.
Alanna growled, glaring at her door. She'd been pretty much confined to her quarters for the next couple of days, by Duke Gareth's order.
He hadn't been at all pleased by her "little stunt", as he'd put it. Especially as she hadn't told him exactly why she'd gone to the city.
Cythera hadn't been at all sympathetic, either. "Act like a child, and you'll be treated like one."
Alanna had barely managed to keep from choking her dear friend.
Coram had been completely furious with her. He'd spent a good hour scolding her, before going off to write her father. They both knew it was a pointless activity, but Coram didn't seem to care.
Thom, oddly enough, had seemed relieved by her internment, muttering about how it was safe.
Truth be told, Thom was really starting to worry her. He looked worried and sickly, and she was willing to bet he hadn't been getting near enough sleep.
Gary's reaction had been the strangest. He'd snapped at her for worrying them- he had an extremely sharp tongue when he was annoyed- then told her to buck up, and that he'd smuggle her some cake that night.
True to his word, Gary had appeared an hour before with some cake and a few books for her to peruse. They'd eated the cake together, discussing the attack and what Alanna had learned from George. She hadn't yet told him who her informant was, and he didn't press. He merely took her at her word, and agreed that keeping Faithful nearby was a good idea.
For some reason, Alanna felt inclined to trust Gary with that information, when she hadn't even told Cythera or Thom. Strangely enough, she rather suspected that the two people she currently trusted the most were George and Gary, despite the fact that she barely knew them.
Now, going on midnight, Alanna was sprawled in her desk chair, glowering at her closed door.
'Frowning isn't going to do any good,' Faithful informed her calmly from where he was curled on her bed. 'Just accept it with good grace.'
Alanna snorted. She wasn't exactly known for her positive attitude.
Then came a tap on her door. Startled, Alanna hesitated, wondering if she'd imagined it.
But no, Faithful had looked up as well, amethyst eyes suspicious.
The tap came again. This time, Alanna slid out of her chair and walked to the door, slowly opening it. She silently thanked Gary for leaving it unlocked.
To her surprise, a small page waited outside. He looked vaguely familiar- the page who'd been in the courtyard when she'd arrived in Corus, she thought. Harry or Harris or something like that.
"Yes?" she demanded, instincively keeping her voice quiet.
Harris- yes, that was the name, she was sure of it- looked nervous.
"Lady, can...can I come in?" the page asked, fidgeting. Alanna's eyebrows shot up- pages weren't allowed in the ladies' rooms.
Alanna, however, was in a rebellious mood. "Very well," she said, standing aside. Harris scampered in, and Alanna closed the door behind.
"Sit down," Alanna ordered, pointing to one of the two armchairs by the fire. Harris obeyed, looking rather shaky.
Alanna sat across from the lad, quite curious. "Now, what brings you here?"
"Edan told me I should come to you," Harris said, flushing. "He said you might understand."
"Understand what?" Alanna demanded, doing her best to be patient.
For a long moment, Harris remained silent. Then, quietly, he said, "I'm a girl."


text: CST Brands, Inc. (NYSE: CST), one of the largest independent retailers of motor fuels and convenience merchandise in North America, today reported financial results for the full year and fourth quarter ended December 31, 2013. Three Months Results For the three month period ended December 31, 2013, the Company reported net income of $34 million, or $0.44 per diluted share. Included in net income are asset impairments of $4 million. Excluding these asset impairments, net income would have been $36 million, or $0.48 per diluted share. Net income was $60 million, or $0.78 per diluted share, for the comparable period in 2012. Revenues totaled $3.1 billion for the fourth quarter of 2013 compared to $3.2 billion for the same period of 2012. Motor fuel revenues in the U.S. segment declined $110 million, driven by a 1 percent decline in motor fuel gallons sold and a 6 percent decline in the Company's per gallon average motor fuel selling price. Motor fuel revenues declined $36 million in our Canada segment, primarily due to weakness of the Canadian dollar relative to the U.S. dollar. Overall revenues in the Canadian segment declined approximately $71 million due to foreign currency effects of the Canadian dollar relative to the U.S. dollar. For the fourth quarter 2013, the U.S. motor fuel gross margin (cents per gallon), after deducting credit card fees, was $0.15 compared to $0.21 in the fourth quarter of 2012. The Company experienced historically strong U.S. motor fuel gross margins in the fourth quarter of 2012, due primarily to the volatility of wholesale gasoline prices during that period. U.S. merchandise gross margin, net of credit card fees, increased from 28.9 percent for the fourth quarter of 2012 to 30.5 percent for the same period in 2013 due primarily to an increase in food services and packaged beverages.


text:
The ultimate 3D Blu-ray starter pack!
47 Ronin 3D
Keanu Reeves makes an explosive return to action-adventure in 47 Ronin. After a treacherous warlord kills their master and banishes their kind, 47 leaderless samurai vow to seek vengeance and restore honour to their people. Driven from their homes and dispersed across the land, this band of Ronin must seek the help of Kai (Reeves)- a half-breed they once rejected-as they fight their way across a savage world of mythic beasts, shape-shifting witchcraft and wondrous terrors. As this exiled, enslaved outcast becomes their most deadly weapon, he will transform into the hero who inspires this band of outnumbered rebels to seize eternity.
Jeff Bridges and Ryan Reynolds headline the supernatural action-adventure R.I.P.D. as two undead cops dispatched by the otherworldly Rest In Peace Department to protect our world from an increasingly destructive array of creatures who refuse to move peacefully to the other side.
Immortals 3D
From the producers of '300', Immortals is a visually stunning and bloody retelling of the epic Greek legend of Theseus. The ruthless King Hyperion (Mickey Rourke) leads his bloodthirsty army on a murderous rampage across Greece to find a deadly weapon that can kill the Gods themselves. Only Theseus (Henry Cavill), a mortal chosen by Zeus, King of the Gods, can lead the fight against Hyperion and his evil army with the fate of mankind and the Gods at stake.
Special Features:
|Sound Information:
|Number of Discs:
|Main Language:
|Dubbing Languages:
|Subtitle Languages:
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If ordered before 9pm, delivered by courier next working day. Available on orders placed before 9pm (2pm weekends and Bank Holidays) for delivery next business day. Applies to most delivery addresses within mainland UK.
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Just wondering - will I need a self-inflating air-matress for a sandy beach? I'm thinking the sand will still be warm from the day, but you know what they say about deserts - though this will only be southwest France in a few days time. Thanks.
Just wondering - will I need a self-inflating air-matress for a sandy beach? I'm thinking the sand will still be warm from the day, but you know what they say about deserts - though this will only be southwest France in a few days time. Thanks.
I'll admit it is a bit of an odd question, so perhaps I should add that I'm traveling really light with just a carry-on bag and want to leave as much food space as I can for when I get there. I don't want to be taking things I won't need. I'm only going for four nights and will stay permanently on the beach, maybe moving along a bit if I get bored or if it gets noisy. Not sure about the legalities of it all, but I don't really care. I don't make a mess. I'm quiet, I'm polite and I'm doing it.
Along with some simple cooking stuff and the usual suspects, all I'm taking is a one-season sleeping bag that will go into my ex-army 'Goretex' bivi bag. The bivi bag will take the rain, but I'll also take a lightweight tarpaulin which will allow me to 'live' in case of poor weather. Some rain is actually forecast, but that's the luck of the draw I suppose and I'll take my chances.
Any response welcome. Thanks.
Not at all practical-beaches become quite cold overnight.
Plus the Frogs will be sure to take offence (it's part of their national character) and call the gendarmes, what with all the trouble they have with immigrants from North Africa you could find yourslef in a spot of bother quite quickly.
And there was me thinking they would just shrug their shoulders in a French sort of way.
The forecast has changed from 'some rain' to 'rain plus thunderstorms' along the coast so I'm giving it a miss.
Thanks for the tip about 'cold beaches'. Seems a sleep mat will be in order if I get to Sardinia next month.
Beaches near hotels and resorts have a tendency to be rat-infested, have you thought about that?
If I was you I would go for a light aluminium mat, the sort of stuff that people put on their car windshields in winter.
Here is an example:
Thanks. Hadn't thought about rats in my planning as I'm more concerned about being away from people than anything else - pretty much the same result? Though, if I didn't have that choice, there's not a lot I could do about it other than keep my own food well secured and away from where I'm sleeping. Seems rats have better taste and live far more comfortably than I do.
edit: Liked the ali mat. If I can figure out the language and postage, I might go for one.
[ Edit: Edited on 17-Sep-2009, at 19:24 by fabyomama ]
Their English ordering gude:
IIRC shipping to the UK was somewhere between 10-12 EUR.
But you should be able to find a mat like that on ebay.co.uk or similar. I have a similar mat and found it in a One-Euro-Store.
So check out your local One-Pound-Store.
Thanks for going to the trouble. I'd have found my way round but it was late and I was tired from getting up early. I'm still at home btw after checking the forecast - thunderstorms Friday and Saturday - so decided to get on with some work. No point in being huddled up in a sandy hole somewhere when all I wanted was a few days swimming.
I normally book ahead even if I'm not sure if I can go. At £1 a pop it's no biggie and I'd actually forgotten about this one until the 'check-in' email last week! On average, I make about half of them. Sardinia in a few weeks - weather permitting.
I'll have a look around the cheap shops for one of those mats. We have 'Poundland' and another place called 'Snips'. Thanks.


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Warren, Elizabeth (Betty), 92, passed away May 21, 2017 in Orlando, Florida. She was born in Akron, Ohio June 6, 1924 to William A. and Mary Yurkowsky. She was preceded in death by her parents, two older brothers and her husband, William P. Warren. She is survived by brother, William Yurkowsky, Jr., and niece Melani (James) Menke, of Orlando, nephew Michael (Ashley) Yurkowsky, of St. Augustine, Florida, nephew, Rick Warren of Hilliard, OH, niece, Maryann Vandegrift of Akron, OH as well as great nieces Briana Menke and Victoria Warren and Great Nephews Nicholas Menke and Grey Yurkowsky. Betty was a graduate of Akron East High and Kent State University. She was a 40+ year employee of Ohio Bell in both Akron and Cleveland. She was a member of the Eastern Star and served as Worthy Matron in 1969. She was an active member of Telephone Pioneers and the American Legion Auxiliary.
Funeral Services will be 11:00AM, TUESDAY, JUNE 6, 2017 at JOHNSON-ROMITO FUNERAL HOME, 19 E. MAIN ST., (ON THE SQUARE), HUDSON, OH (330-650-4181) and will be conducted by Father Nicholas Wyslutsky of St. Nicholas Orthodox Church. Friends will be received at the funeral home before the service from 10-11:00AM. Burial will be at Fairview Cemetery in Boston Heights immediately after the service. In lieu of flowers, donations may be made to St. Nicholas Orthodox Church Cemetery Fund, 755 S. Cleveland Ave., Mogadore, OH 44260. www.johnsonromito.com


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Introducing Copán
Copán is the site of a large set of ruins in Honduras, one of the few such Mayan ruins in the country which is located at the edge of the Maya Region. The site is located in western Honduras near the border with Guatemala, about halfway between the two oceans. It is located in the extreme southeast of the Maya Region and was almost surrounded by non-Maya peoples. The city-state had a population of at least 20,000 people in about 100 square miles at its peak in the Classic period from the 5th to 9th centuries AD. The kingdom then collapsed and lost most of its population. The abandoned city suffered from the encroachment of nature including the Copán River whose course has now been changed to prevent further destruction. The ruins were later rediscovered by Europeans, and the site is now an archaeological park run by the national government. The uncovering of buildings and restoration are ongoing.
The site has been described as the most enchanting of all the Mayan archaeological sites and the most artistic since one of its major features is the many large (10 to 15 feet tall) stone stelae with portraits carved on one side and intricate hieroglyphics on the other in high relief. The site contains more hieroglyphic carvings than any other Mayan site. 4,509 structures have been found so far.
The Park
The Copan Ruins and the Museum of Sculpture are open from 8am to 4pm daily. There is a $15 cost to enter the park, and an additional fee for the Museum of Sculpture. And there is an additional cost to enter two tunnels. Guided tours are recommended and can be arranged at the park entrance or by contacting the Copán Guide Association.
The park is located a pleasant 15-minute walk from the town of Copán Ruinas, a lovely and comfortable town of about 3,000. There are numerous hotels, hostels, restaurants, cabs etc. in the town.
Major Attractions
The Sculpture Museum is a 2-story building whose centerpiece is a full scale, and full color, model of the Rosaline Temple, which was covered over when another temple was built on top of it. The museum also contains much of the original stonework from the site.
In addition, the Principal Group contains five major areas of interest The Acropolis, The Tunnels, The Ball Court, The Hieroglyphic Stairways and the Great Plaza.
The Acropolis is divided into two big plazas and two temples. An altar depicts the 16 members of the Copan Dynasty.
The Tunnels, 4km long, were dug by archaeologists under the acropolis to view earlier stages of Copan civilization. The Maya tended to build new temples over their old ones covering them completely. By digging the tunnels, the archaeologists were able to see these earlier buildings. Two of the tunnels are open to the public for an additional fee.
The ball court is the second largest in Central America.
The Hieroglyphic Stairway is the most famous of Copan's monuments. 63 steps and several thousand glyphs tell the history of the royal house of Copan.
The Great Plaza is famous for its stelae and altars that are found scattered around a well groomed lawn.
Las Sepulturas is another archaeological site, less than a mile from the main Copán site, and the admission is included in the Main Park fee. This site provides examples of how the people actually lived, and is the only such residential site open to the public in the Mayan Region.
How to get there
The ruins are located about a mile outside the town of Copan Ruinas, which is located about 100 miles southwest of the city of San Pedro Sula. The San Pedro Sula International Airport is the major airport serving this region, and this airport can be reached from Miami, Houston, New Orleans, New York, Los Angeles, San Francisco, Orlando, Mexico City and Cancun. In addition, there are connections via the Central American Airlines through San Jose, Costa Rica; San Salvador, El Salvador; Panama City, Panama and Guatemala City, Guatemala.
Once in San Pedro Sula, the easiest way to get to the ruins is to purchase a tour package through one of the tour operators in town. It is a three-hour bus ride to get to the ruins on such a tour. Another alternative is to take a regularly scheduled bus to Copan Ruinas and spend the night in one of the numerous hotels there.


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This article is a stub, an article too short to provide more than rudimentary information about a subject. You can help Pro Wrestling Wiki by expanding it.
The Mexican National Heavyweight Championship (called the Campeonato Nacional Completo in Spanish) is a Mexican Lucha Libre (professional wrestling) championship created and sanctioned by "Comisión de Box y Lucha Libre Mexico D.F." (the Mexico City Boxing and Wrestling Commission). While the Commission sanctions the title, it does not promote the events in which the Championship is defended. From 1933 until the mid-1990s, Consejo Mundial de Lucha Libre (CMLL) controlled the Championship, since then Asistencia Asesoría y Administración (AAA) has controlled the championship, after the Commission granted them the right to the title. In 2006 the title inactivated and replaced by the AAA World Heavyweight Championship but in late 2009 it became active again. As the Championship is designated as a heavyweight title, the Championship can only officially be competed for by wrestlers weighing at least 105 kg. However, the regulation is not strictly adhered to.
Being a professional wrestling championship, it is not won legitimately: it is instead won via a scripted ending to a match or awarded to a wrestler because of a storyline. The earliest documented use of the Mexican National Heavyweight Title was in 1926 and as such the Mexican National Heavyweight Championship was the oldest continuously promoted wrestling title in the world at the time of its inactivation. The earliest recorded champion was Francisco Aguayo. When Empressa Mexicana de Lucha Libre (EMLL, later renamed CMLL) was founded in 1933, it was given the full promotional control of the title, with the Commission only being asked to approve the champions.<In this, "control" refers to the every day use of the title, determining which storylines the title is being used it, who gets to challenge for the title, how to use it in a public relations sense. After Pierroth, Jr. won the title in 1995, he left CMLL and signed with AAA, bringing the Mexican National Heavyweight Championship with him. When Máscara Sagrada became the champion in 1996, it was officially acknowledged by the Commission that AAA controlled the booking of the championship from that point forward. El Halcón, also billed as Halcón Ortiz and Super Halcón, has the record for most championship reigns, with five. The Championship was inactive for several years, starting on September 13, 2006 where it was one of the four titles eliminated to make room for the new AAA World Heavyweight Championship. In the fall of 2009 Charly Manson left AAA, bringing the Mexican National Championship belt with him to defend on the independent circuit. The longest reign belongs to Charly Manson with 2088 days, although the title was inactive for several years. The longest active title reign belongs to El Médico Asesino, who held the title for 2074 days. Mr. Águila was champion for the shortest time, 42 days. The current champion is X-Fly, having won the title from Charly Manson on February 21, 2010 at a Perros del Mal Producciones show. X-Fly is the first "Independent" champion crowned in the title's history.
Hector Garza was the most recent Mexican National Heavyweight champion after defeating former champion X-Fly, Perro Aguayo Jr., Toscano, Texano Jr. and El Mesias in a six-way match to win the title at Arena Aficion on February 14, 2012. Garza announced a temporary retirement from the ring in October 2012 due to lung cancer, then passed away in May of 2013. The championship has not been mentioned since. All of the other national championships not promoted by CMLL have gone defunct by this point, and it's possible this one may not be heard from again.
Title history
- Key
|The reign number for the specific set of wrestlers listed
|The event promoted by the respective promotion in which the titles were won
|The specific information is not known
|Used for vacated reigns so as not to count it as an official reign
|Indicates that a title change took place "no later than" the date listed.


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Invest Now
The only gift too small is the one not made
There are eight rungs in charity. The highest is when you help people to help themselves."Maimonides
The Bennington Area Habitat for Humanity affiliate is a 501 (c) 3 non-profit organization. Our tax-exempt status can be confirmed by clicking on these links. Group Exemption and IRS letter.Thanks to the generosity of area individuals, businesses, service organizations and congregations, we now provide housing for over 65 people and are able to serve two or more families each year with new construction or ABWK, a program of minor repairs making homes safer and more affordable. Our fiscal year ends June 30.
Building homes and changing lives....A brighter future begins at home. We consider your gift to be an investment in our community by helping us to build more, safe, sufficient and affordable homes in southwestern, Vermont. Your gift will make a difference today - especially in the lives of the children who will live in the home!
Your gift could help buy:
|$25 = A Box of Nails
|$500 = Kitchen Counter-tops
|$50 = Paint for one bedroom
|$1,000 = Interior Doors
|$100 = Bathroom Vanity
|$2,500 = Kitchen Cabinets
|$250 = Exterior Door
|$5,000 = Family Room & Kitchen
Some frequently asked questions
If I give to Habitat for Humanity, will my contribution be used to build homes in Vermont?
How does building locally help build more homes in other countries?
What is the Cars for Homes Program?
How can I leave a bequest to Habitat?
How can I give appreciated securities to Habitat?
Charity Navigator and BBB Wise Giving Alliance checklist for giving to non-profit organizations.
Want a small percentage of your purchases from Amazon to benefit our affiliate?
If I give to Habitat for Humanity, will my contribution be used to build homes in Vermont? There are two ways to ensure that your gifts will be used to build homes locally. If you contribute to Habitat for Humanity and want your gift to be used in our part of Vermont, you need to designate this when you make your contribution. The simplest way is to make a contribution directly to our affiliate by mailing a check to Bennington Area Habitat for Humanity, P.O. Box 1159, Manchester, Vermont 05254.
How does building locally help build more homes in other countries? Your gift is used as you specify. If you donate directly to our affiliate or through Habitat for Humanity International and specify that you wish your donation to be allocated to our affiliate, your gift will fund construction projects in Bennington County. Un-designated gifts are used where most needed including administrative purposes. Visit Habitat for Humanity International for more information on how your donations are used. Unless specifically requested by the donor, 10% of all cash contributions are tithed to Habitat International for use in another country. In the past we have split our tithe between Uganda and India and most recently Haiti. We have personal contacts with Habitat affiliates in Uganda and India. Both countries have a tremendous need for housing. Progress is slowly being made in Haiti. Since our founding, we have contributed a total of $64,629 to the tithe program building 17.4 homes in other countries.
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What is the Cars for Homes Program?
Your vehicle donation can help a family build a home, and the hope of a better life! Car, truck, boat and RV donations help Habitat for Humanity build houses in partnership with families in need of decent, affordable shelter. Donate a car, truck, boat, RV or other vehicle to Cars for Homes, Habitat for Humanity's official car donation program, by calling 1-877-277-4344 (toll free) or online at www.carsforhomes.org. In 2008, five cars were donated to Habitat with the proceeds helping us build homes in Bennington County.
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One of the easiest ways to make a gift to the Bennington Area Habitat for Humanity affiliate is by designating BAHfH as a beneficiary of a life insurance policy, a savings account, or an IRA.
You can also leave our affiliate a bequest in your Will. There are several ways you can do this. You can (1) give a specific amount of money to BAHfH; (2) give a specific item of property to BAHfH; or (3) give a specified percentage of the residue of your estate to BAHfH (the residue of your estate is that which is left over after all specific gifts have been made, whether charitable or non-charitable).
We recommend that your bequest be for unrestricted purposes or be directed generally to home building. A bequest may be included in the body of a will or as an amendment (codicil). Gifts made through a bequest typically provide an estate tax deduction.
Below we have listed some information that may help you in your estate planning. We always recommend that you work with an attorney, estate, or financial professional when making any estate plans. The language below is intended to only be a guide for estate planning purposes.
"I give and devise to the Bennington Area Habitat for Humanity Affiliate or its successor, the sum of _________ Dollars ($_________) to be used for such charitable purposes as the Board of Directors of the Bennington Area Habitat for Humanity deems appropriate; provided, however, that at the time of such gift, Bennington Area Habitat for Humanity or its successor, must be a charity as described in Sections 170(c) and 2055(a) of the Internal Revenue Code."
I give and devise ________ percent (________%) of my residuary estate to Bennington Area Habitat for Humanity or its successor, to be used for such charitable purposes as the Board of Directors of the Bennington Area Habitat for Humanity deems appropriate; provided, however, that at the time of such gift, Bennington Area Habitat for Humanity or its successor, must be a charity as described in Sections 170(c) and 2055(a) of the Internal Revenue Code."
How can I give appreciated securities to Habitat?
Many people find they can save taxes and make a larger contribution to Habitat by transferring highly appreciated securities to us. This is easy to do. Stock transfer instructions
Charity Navigator and BBB Wise Giving Alliance checklist for giving to non-profit organizations
We are grateful to the many individuals and organizations that support us, through generous contributions of time, materials, and financial contributions, and we are proud of our stewardship record. In their recent e-newsletter, Richard Male Associates published a checklist provided by the Charity Navigator and the BBB Wise Giving Alliance. It is a rigorous checklist for review by donors to all non-profit organizations. We agree that it is of critical importance for all non-profit organizations to be transparent about how they manage their resources, and we strive for a perfect score! Read checklist.


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While the BRATS continue our search for a permanent home, we will hold our August meeting on Monday, 8/22 at 7 pm at the Greene Turtle in Owings Mills, MD. Come discuss out upcoming balloon launch and JOTA activities!
Kevin KB3PLX
While the BRATS continue our search for a permanent home, we will hold our August meeting on Monday, 8/22 at 7 pm at the Greene Turtle in Owings Mills, MD. Come discuss out upcoming balloon launch and JOTA activities!
Kevin KB3PLX
The BRATS will be supporting the Susan G Komen foundation for its inaugural Promise Ride, a Metric Century (66 mi) "woods and water" ride through Anne Arundel county, MD, on Saturday, June 25.
For more details, please contact email@example.com.
The May meeting of the BRATS will be held 5/16 at 7 pm at the Owings Mills Greene Turtle. Hope to see you there!
The 2016 Baltimore Amateur Radio Club hamfest will be held Sunday, June 19, at the Arcadia Volunteer Fire Company from 8 am until 2 pm.
For more information, please visit the BARC Hamfest page.
The BRATS activated Ft. McHenry for National Parks on the air, setting up two stations and making contacts throughout the continent.
Thanks to WA3AER, K0RYX, K9FXY, and KB3SKN for their attendance and equipment to help us bring this station on the air, and a very special thanks to Mark W3MSR for all his efforts securing permission to operate and making this event happen!
Please join us for the April meeting of the BRATS at Liberty Road Volunteer Fire Company as we welcome Stephen Hamilton KJ5HY. The meeting begins at 7 pm.
In 2011, the United States Military Academy began a Balloon Satellite program where cadets would build a payload and attach it to a weather balloon and send it to heights exceeding 100,000 feet. The payload always includes an APRS tracker with the West Point Amateur Radio club callsign W2KGY. Stephen Hamilton (KJ5HY) has advised the cadets and helped with all 6 launches, to include the last one on 17 April, 2016. Stephen will discuss various issues with balloon satellite building,
launching, and recovery.
The BRATS will be setting up a station at Ft. McHenry on Saturday, April 30, for National Parks on the Air. Please email us firstname.lastname@example.com for more info. Hope to see you there!
The BRATS are working hard to maintain and improve the WB3DZO linked repeater system. As a part of this process, we've installed new radios and filters at several of our sites.
One of the features we're looking into is whether a PL tone will help cut down on activations. For now, we've only enabled it on one of our receive sites, so no PL is necessary to reach the repeater from most of Baltimore, but we encourage you to set PL 156.7 to ensure you can hit every receive site.
Although we hope the summer storm season is quiet, we're working hard to make sure our gear is ready!
The April meeting of the BRATS will be Monday, April 25, at 7 pm at the Liberty Road Volunteer Fire Company. We hope to see you there!
The only Monday we could get the Liberty Road VFC was March 28; we hope to see you there!


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When This Little Boy Sees His Sister Hula Hoop He Desperately Wants To Do The Same!
We mostly want to become like our elder brothers and sisters and the little boy in the video also wishes to do the same. He gets excited seeing his sister doing Hula loop and wishes to give it try. You will see how he enjoys doing the same even if he is not able to complete a single spin.


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Keeping outdoor adventure in the classroom!
Activities: The ROC is a multi-season destination with a wide variety of activities for all skills and abilities. During winter months, the ROC offers you and your students a variety of exciting and exhilarating opportunities such as; snow tubing,tobogganing, ice skating, skiing and snowboarding! Bookings are in 3 hour time increments and can include one or all of the activities listed above. All group bookings require a minimum of 30 students and are booked on our operating days: Wednesday, Thursday and Fridays. Helmets: According to OSBIE (Ontario School Boards' Insurance Exchange) "all participants must wear an appropriate snow sport helmet for school excursions to OSRA member facilities." The ROC is an OSRA member facility and all students, staff, and volunteers must wear an appropriate snow sport helmet for skiing, snowboarding, tubing and ice skating. Booking Options Tubing
|Tubing & Lunch
|$12 per person
|$17 per person
Skiing & Snowboarding
|Skiing & Snowboarding
|Skiing, Snowboarding & Lunch
|$10 per person
|$15 per person
Skating & Tobogganing
|Skating & Tobogganing
|Skating, Tobogganing & Lunch
|$9 per person
|$14 per person
Lunch Options
|Lunch Option A
|Individual Cheese Pizza Veggies n dip (Ranch) Apple Sauce Milk (Chocolate or White)
|Lunch Option B
|Chicken Strips (2) w/ Plum sauce & fries Fruit Cup Brownie Milk (Chocolate or White)
|Lunch Option C
|Grilled Chicken Wrap Cheese & Crackers Mini – Go Yogurt Milk (Chocolate or White)
Food & Beverage Facilities: A variety of pre-ordered lunch options are available for $5.00 per person. Brown bag lunches are welcome. The cafeteria will be open for snacks and beverages.


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The OBC210 Mini combines portability, solid construction and a highly versatile range of tones suitable for any number of styles. Housing a pair of 10" Eminence Legends in a neatly proportioned, rear-ported 15mm birch plywood shell, the cab bears all sonic the hallmarks of a classic 2×10" cab.
Midrange frequencies are pronounced yet detailed, whilst the highs have a smooth and satisfying character. More surprising, though, is the low end clout. Despite its extremely compact stature, the OBC210 Mini manages to retain a warm and defined bass response, with a depth and assertiveness that will shock even the fussiest 5-String players.
Other specifications include:
Features: Compact 2×10″ bass cabinet with parallel speakon jacks for 'daisy chaining'
Speakers: 2 x 10″ Eminence Legends
Impedance: 8 Ohms
Power Handling: 400 Watts
Finish Options: Orange or Black basketweave vinyl
Unboxed Dimensions (W x H x D): 62 x 35 x 34cm (24.4 x 13.8 x 13.4″)
Unboxed Weight: 19.25kg (42.43lb)
For more information about the Orange OBC210 Mini Cab, please contact us.
|Brand New
All Major Credit Cards, Money Order, Cashiers Check, Personal Check, Wire Transfer, and Paypal
Call for details.
Call for details.


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I get to choose daily what type of attitude I "clothe" myself with.
I get to choose whether I see my cup as half empty or half full {on the days that my proverbial cup doth not runneth over}.
And get this: I even get to choose the "climate" of my home.
Wow. What a reality check. What a great honor. What a hefty responsibility!
We, as women {especially as wives and mothers}, have a tremendous impact on the "tone" of our home environment. In short…we set it.
It is sweet. Or it is sour.
Unlike the sauce, It cannot be both.
I get to choose how I respond to my children when they behave poorly. I get to rise above and initiate an energizing cycle, in spite of bad behavior, and regardless of how badly I may have slept the night before {thanks to them}…or simply react negatively out of my own selfishness and immaturity.
Wisdom or impulse. It's my choice.
I get to choose how I respond to my husband when he is weary, unknowingly abrupt, and still in "work mode" {which, translated, means treating me in a way I interpret as unloving}. Choosing to love and serve, even when I am feeling unloved and empty.
I get to choose. Every time.
What will I choose to pour over the situations I find myself standing on the verge of on a day-to-day basis? And those I find myself hopelessly stuck in on rare occasion?
My attitude has the potential to change everything…the basic dynamic of my day…of his day…of their day.
I've heard it said, "attitude is everything". And the older I get, the more I grasp the magnitude of that statement. It's undeniable…our attitude has the potential tomake or break us, and those around us.
And the heart-breaking truth is, I've failed miserably in this department over the past few weeks. Specifically in regards to my sweet husband.
I so easily turn my focus inward. Selfishness consumes my thinking, and a mutually uncomfortable season in our marriage, brief as it may be, becomes one where all I see is my own emptiness.
I am instantly the victim, my husband the culprit.
Sucked into the vortex of my own self-centered, little pity party, I fail to recognize where my man is struggling, where he is feeling worn down and empty, and how my superficial attempt to explain my feelings leaves him feeling dejected and insufficient.
Fuel or water?
I had been choosing fuel almost exclusively for a week. Crying myself to sleep, I made agreements about myself and my marriage that were so far from the truth, but were powerful enough to slowly drive a wedge between us. Intimacy, shot. Heart, hardened. Climate, frosty.
My perspective clouded by selfishness, I held him at arm's length and practically demanded my {emotional} needs got met before his {physical} needs had a chance of being met.
How does the knowledge I've acquired regarding the differences between men and women get forgotten so easily. While I was processing legitimate heart-ache, I was going about it the wrong way. I know that isn't how this relationship thing works! I know that isn't the way God intended me to love my husband. I know that isn't the way to woo my husband's heart.
It starts with dying to self. Laying my agenda aside. Loving extravagantly – without expectation – and allowing God to do "His thing".
I forgot the power of pure water.
I've laid my bucket of fuel aside, pursued my husband's heart {in spite of feeling the nagging ache of loneliness…oh, how I despise 'night shift'}, intentionally prioritizing the refreshment of his weary soul…and now find myself reveling in the sweetness of God's upside-down way of doing love.
My cup runneth over!
It's delightful. It's mind-blowing. It's life-giving. Tis' sweet, my friends!
And it has energized the heck out of my marriage.
I get to choose. Initiate an energizing cycle, or perpetuate the destruction of the crazy cycle.
All because of a small {okay, enormous} decision to change the attitude of my heart.
I've written previously about my love for the often quoted saying by Chuck Swindoll… "The longer I live, the more I realize the impact of attitude on life…The remarkable thing is we have a choice everyday regarding the attitude we will embrace for that day. We cannot change our past… we cannot change the fact that people will act in a certain way. We cannot change the inevitable. The only thing we can do is play on the one string we have, and that is our attitude. I am convinced that life is 10% what happens to me and 90% of how I react to it. And so it is with you… we are in charge of our Attitudes."
I've had an attitude adjustment over the past week {and for this, my husband is eternally grateful}.
It was hard and awkward and tear-filled. But oh, so necessary.
{because marriage, God's way, always is}
By Joy McMillan, Simply Bloom


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