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Liver fibrosis is caused by chemicals or viral infection. The progression of liver fibrosis results in hepatocellular carcinogenesis in later stages. Recent studies have revealed the importance of DNA hypermethylation in the progression of liver fibrosis to hepatocellular carcinoma (HCC). However, the importance of DNA methylation in the early-stage liver fibrosis remains unclear.
To address this issue, we used a pathological mouse model of early-stage liver fibrosis that was induced by treatment with carbon tetrachloride (CCl4) for 2 weeks and performed a genome-wide analysis of DNA methylation status. This global analysis of DNA methylation was performed using a combination of methyl-binding protein (MBP)-based high throughput sequencing (MBP-seq) and bioinformatic tools, IPA and Oncomine. To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and in-vitro-methylation assays.
The genome-wide analysis revealed that DNA methylation status was reduced throughout the genome because of CCl4 treatment in the early-stage liver fibrosis. Bioinformatic and biochemical analyses revealed that a gene associated with fibrosis, secreted phosphoprotein 1 (Spp1), which induces inflammation, was hypomethylated and its expression was up-regulated. These results suggest that DNA hypomethylation of the genes responsible for fibrosis may precede the onset of liver fibrosis. Moreover, Spp1 is also known to enhance tumor development. Using the web-based database, we revealed that Spp1 expression is increased in HCC.
Our study suggests that hypomethylation is crucial for the onset of and in the progression of liver fibrosis to HCC. The elucidation of this change in methylation status from the onset of fibrosis and subsequent progression to HCC may lead to a new clinical diagnosis.
Fibrosis is one of the most severe systemic diseases and is characterized by excessive accumulation of fibrous connective tissues, such as collagen, induced by acute or chronic injury [1]. Fibroproliferative diseases occur throughout the body, including in the lungs, kidneys, and liver. The progression of fibrosis leads to the failure of the physiological functions of tissues. Liver fibrosis, in particular, has been extensively investigated because its progression results in hepatocellular carcinoma (HCC), which is the fifth most common cancer worldwide [2,3].
Currently, liver fibrosis is known to be a part of the dynamic process of continuous extracellular matrix (ECM) remodeling in chronic liver injury [4]. In liver fibrosis, a liver injury activates the Kupffer cells--resident macrophages of the liver sinusoids--thereby inducing inflammation [5]. This inflammatory response triggers the activation of hepatic stellate cells (HSCs), which play a key role in fibrogenesis by transdifferentiating into myofibroblasts [1]. The proliferation of myofibroblasts and stimulation of ECM synthesis, ultimately results in liver fibrosis.
Recently, the progression of liver fibrosis has been reported to be associated with hypermethylation of DNA [6]. HSC activation is inhibited by 5'-Azacytidine (5'-Aza), a DNA methylation inhibitor, resulting in the transdifferentiation of HSCs to myofibroblasts [7]. Furthermore, in other tissues such as renal, several studies using heterozygous mice revealed that the DNA methyltransferase Dnmt1 and its inhibitor 5'-aza ameliorate renal fibrosis by inhibiting proliferation of myofibroblasts [8,9]. These results indicate the pivotal role of DNA hypermethylation in the progression of both liver and renal fibrosis. However, the importance of DNA methylation in early-stage liver fibrosis remains unclear.
Here, we performed a global analysis of DNA methylation during the onset of liver fibrosis. A mouse model treated with carbon tetrachloride (CCl4) was used as a model of liver fibrosis. Analysis of the CCl4-treated mouse livers revealed symptoms of early-stage liver fibrosis. To analyze the genome-wide DNA methylation profile of this CCl4-induced early-stage liver fibrosis, we used a combination of methyl-binding protein (MBP)-based precipitation (MBP-IP) and high-throughput DNA sequencing (MBP-seq). This genome-wide analysis can reveal hypo- and hypermethylated sites (125 and 88, respectively) in the genomic DNA. Analyzing the MBP-seq data, we revealed that the DNA methylation status was reduced throughout the genome, and that the enhancer of secreted phosphoprotein 1 (Spp1), also known as osteopontin, was hypomethylated. Two bioinformatics tools, IPA and Oncomine, indicated that Spp1 is related to liver fibrosis and inflammation. Using biochemical methods, such as bisulfite modification and in-vitro-methylation assays, we confirmed that the hypomethylation of Spp1 enhancer up-regulates its mRNA. These results clearly indicate that hypomethylation of the genome may precede the onset of liver fibrosis. Moreover, Spp1 enhances tumor development. This suggests that hypomethylation during the early-stage liver fibrosis may be important in the development of the primary liver cancer HCC, which is an end-stage liver disease.
Five-week-old male C57BL/6 wild-type mice were purchased from CLEA Japan Inc. To induce liver fibrosis, 2 ml/kg CCl4 mixed with olive oil was intraperitoneally administered 3 times per week for 2 weeks. All animal husbandry and animal experiments were performed in accordance with the guidelines of the University of Tsukuba's Regulation of Animal Experiments Committee. Enzymatic activities of the serum proteins, alanine aminotranferease (Alt) and aspartate aminotransferase (Ast), were measured using the Fuji dri-chem 3000 analyzer (Fuji Film).
Mouse liver cell line, Hepa1-6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Buffalo Rat liver cell line, BRL-3 A were maintained in Ham's F12 medium with 10% FBS. 3uM of the 5-Aza deoxy derivative, 5-dAza-C, were treated with BRL-3 A for 36hr, and then harvested for indicated experiments.
After 2 weeks with or without CCl4 treatment, liver tissues were fixed with formalin. Formalin-paraffin livers were cut into 5 μm thick. Hematoxylin and Eosin (H&E) staining, Masson's trichrome (MT) staining and Sirius red staining were performed according to the manufacturer's protocol.
The genomic DNA from each of 4 mouse livers treated with 2 ml/kg CCl4 for 2 weeks were pooled. The genomic DNA from 3 mouse livers treated with olive oil for 2 weeks was pooled and used as control. The genomic DNA purified from the CCl4-treated mouse liver specimens was randomly fragmented into 50-350 bp lengths as described in the SOLiD 5500xl fragment library protocol. The fragmented DNA was then subjected to the MethylMiner methylated DNA enrichment kit according to the manufacturer's protocol. These methylated fragments were eluted with 1000 mM NaCl and used to construct standard fragment libraries using a combination of adaptor ligation and nick translation (SOLiD Fragment Library Construction Kit, Invitrogen). Each DNA library was selected on the basis of size (inserts were approximately 500 bp) by AgencourtAMPure XP (Beckman) before PCR amplification, bead attachment, and emulsion PCR. Libraries were sequenced on a SOLiD 5500xl Analyzer (Applied Biosystems). The resulting tag sequences and quality files were mapped onto the mouse genome (NCBI Build 37, UCSC mm9) using Lifescope version 2.0 (Life Technologies), and peaks were detected using the Genomics Workbench version 4.7.2 (CLC Bio). Parameters for peak mapping and detection are described for details below. Gene annotation of sequence peaks was performed through the BioMart website version 0.7 (http://www.biomart.org). The chromosomal distribution of the MBP-seq peaks was described using R version 1.4 (http://www.r-project.org). UCSC Genome Browser (http://genome.ucsc.edu) [10] was used to obtain data on the epigenetic markers and to display epigenetic features around the target genome locus.
The bioinformatics tool IPA version 1.0 (http://www.ingenuity.com) was used for in silico analysis of the MBP-seq data in the context of known functions and pathways using the Ingenuity Pathways Knowledge Base as a reference set, filtering for molecules and relationships associated with physiological and pathological processes of the liver. For the in silico functional analysis, a right-tailed Fisher's exact test was used to calculate the p-value determining the probability that the hepatotoxic function assigned to that data set was owing to chance alone.
The web-based human cancer microarray database Oncomine (https://www.oncomine.com) was used to analyze the mRNA expression of target genes associated with HCC identified in three studies [11-13]. Details of standardized normalization techniques and statistical calculations can be found on the Oncomine website (https://www.oncomine.com) [14]. In brief, Student's t-test was performed to generate a p-value indicative of the significance of an observation. The lower the p-value, the more confidence in the difference between the groups. The Student's t-test statistic provided for the Oncomine visualizations reflects the magnitude of the difference between groups. Fold change is the magnitude of difference between the primary class and the other control classes, shown on a linear scale. An over-expression fold change is designated with a positive number.
The purified genomic DNA from the mouse livers was randomly fragmented into 50-350 bp lengths as described in the SOLiD 5500xl fragment library protocol. The fragmented DNA was then subjected to the MethylMiner methylated DNA enrichment kit, according to the manufacturer's protocol (Invitrogen). qPCR was then performed to amplify and quantify fragments representative of the methylated genome using the Thermal Cycle Dice TP800 (TaKaRa) and SYBR Premix Ex Taq (TaKaRa). The primer sequences and genome locus used for MBP-IP are summarized in Additional file 1.
qPCR was performed as described previously [15]. Tissues were homogenized in 1 ml of Sepazol and total RNA was extracted according to the manufacturer's instructions (Nacalai Tesque). cDNA was synthesized from total RNA using RevatraAce reverse transcriptase (TOYOBO) and oligodT primer. qPCR was performed to amplify and quantify fragments representative of the indicated mRNA expression using a Thermal Cycle Dice TP800 (TaKaRa) and SYBR Premix Ex Taq (TaKaRa). Cyclophilin and Gapdh was used as the normalization control (Additional file 2). The primer sequences for qPCR are summarized in Additional file 1.
The DNA fragments encoding Spp1 enhancer region (chr5:104846058-104846328) was amplified by PCR. The Spp1 enhancer fragment was methylated by the DNA methylase Sss1 (2U enzyme/10ug DNA), and then cloned into a pGL3-basic reporter plasmid. Each plasmid harboring methylated or unmethylated fragment was directly transfected into Hepa1-6 cells by Lipofectamine 2000 (Invitrogen), according to the manufacturer's instruction. Twenty-four hours after transfection, luciferase assays were performed by Dual-Luciferase Reporter 1000 Assay System (Promega), according to the manufacturer's instruction. phRG-TK (Promega) was used as a reference plasmid to normalize transfection efficiency.
The purified genomic DNA from the mouse livers were denatured in 0.3M NaOH for 20min at 37°C. Then 3.6N sodium bisulfite and 10mM hydroquinone solution were added. Samples were incubated in 1min at 95°C then 12hr at 50°C. Salts were removed using the Wizard DNA Clean-Up System (Promega) and desulfonated in 0.3M NaOH at room temperature for 5 min. Then, the Spp1 enhancer region was amplified by PCR and cloned into pGEM-T Easy Vector System (Promega). The inserts were sequenced to identify the methylated and unmethylated sites. The primer sequences used for amplification are summarized in Additional file 1.
Sequence data of CCl4-treated and control samples from this study has been deposited to the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under accession no. SRA048978 and SRA048984, respectively.
To develop early-stage liver fibrosis, mice were administered 2 ml/kg CCl4 for 2 weeks (see Methods for details). To determine whether this treatment induced liver fibrosis, we used blood samples to investigate two enzymatic activities of well-known fibrosis-indicative serum parameters--Alt and Ast. We found that CCl4 treatment increased enzymatic activities of both Alt and Ast (Figure (Figure1A).1A). Furthermore, we found that well-known fibrotic markers, such as αSma, Col1a2, and Timp1, were up-regulated in the CCl4-treated livers (Figure (Figure1B).1B). To confirm histopathological change in the livers following CCl4 treatment, we used MT and Sirius-red staining, which are commonly used to detect collagen accumulation in liver tissue. Although MT staining did not reveal histological changes, Sirius-red staining revealed an increased distribution of collagen (Figure (Figure1C).1C). Given that MT staining is known to detect continuous ECM whereas Sirius-red staining specifically detects collagen [16], these results suggest that 2 weeks of CCl4 treatment induces fiber production, but not fibroblast growth. This suggests that treatment with CCl4 induced the development of early-stage liver fibrosis.
To investigate the effect of CCl4 treatment on DNA methylation status in early-stage liver fibrosis, we attempted to profile the genome-wide DNA methylation of the CCl4-treated liver tissues. The genomic DNA was fragmented, and the methylated DNA fragment was precipitated by MBP. The methylated DNA fraction was then subjected to high-throughput sequencing using a SOLiD 5500xl. These sequence reads were then mapped onto the mouse genome (NCBI37/mm9) using the MethylMiner methylated DNA enrichment kit and Lifescope. We obtained 7,612,236 reads from the CCl4 sample, and 24,584,122 reads from the control [17]. To find the significant peak, these data were further analyzed using the ChIP-seq tool in Genomics Workbench with the mouse genome annotated using the BioMart website. The peak-finding algorithm included the following four steps: 1) Calculate the null distribution of the background sequencing signal; 2) Scan the mappings to identify candidate peaks with a higher read count than expected from the null distribution; 3) Merge overlapping candidate peaks; 4) Refine the set of candidate peaks based on the count and the spatial distribution of forward and backward reads within the peaks. The estimate for the null distribution of coverage and the calculation of the false discovery rate (FDR) were based on the window size and maximum FDR (%) parameters. The window size specifies the width of the window that is used to count reads during estimation of the null distribution as well as during subsequent scanning for candidate peaks. Maximum FDR indicates the maximum proportion of false positive peaks that are acceptable among the called peaks. In this study, when only MBP samples were used, each negative binomial distribution was fitted to the counts from the low coverage regions. This distribution was used as the null distribution to obtain the number of windows with a particular count of reads expected in the absence of significant binding. By comparing the number of windows with the specific count we expected to observe under the null distribution and the number we actually observed in our data, we can calculate FDR for a given read count and window size as the "fraction of windows with a read count expected under the null distribution/fraction of windows with the observed read count". In this study, we set window size and FDR to 300 bp and 1%, respectively. We identified 125 and 88 peaks in the CCl4-treated and control samples, and summarized all peaks with CpG annotation and several statistic values, including FDR, normalized difference, and Wilcoxon filter p-value, in Additional file 3.
The pie chart in Figure Figure2A2A indicates the methylated sites in each genomic element, such as the transcription regulatory region including the promoter and the 3' flanking region, the gene body including the exon and intron, and the intergenic region (See Figure Figure2A2A legend for detail definition of these terms). Methylated sites in the gene body, the transcription regulatory region, and the intergenic region, were assigned (23.0/12.0%, 10.2/9.0%, and 66.0/79.0% in CCl4-treated/control samples, respectively) (Figure (Figure2A).2A). We found that almost methylated sites are specific to each sample (Additional file 3). Therefore, we considered CCl4-specific and control-specific methylated sites as CCl4-induced "hyper" and "hypo"methylation sites, respectively. The chromosomal distribution of the MBP peaks revealed that the number of control-specific methylation peaks was greater than that of CCl4-specific peaks in most chromosomes (Figure 2B-D). These results indicate that the onset of CCl4-induced early-stage liver fibrosis might be associated with global hypomethylation.
Then, the functions of the genes with methylated sites, which are assigned to the gene body and the transcription regulatory region, are classified using in silico analysis software, IPA (See for details Methods for IPA analysis, and Discussion for the functional aspect of methylated sites within intergenic region). We found that cirrhosis, fibrosis, and HCC were identified only in the control sample (Figure (Figure3A).3A). Previous studies have indicated that the progression of liver fibrosis leads to cirrhosis and eventually to HCC, a primary hepatic cancer [18]. Furthermore, it has been known that aberrant regulation of gene expression is at the basis of many diseases, such as cancer [19]. These results suggest that aberrant DNA methylation causes dysfunction of gene transcription and may affect the onset of fibrosis and its progression to post-fibrotic diseases.
Using IPA, we then re-analyzed the annotated genes in the promoters, which is important for gene transcription. Intriguingly, liver fibrosis was detected as the predominant hepatotoxic function in the control sample (p-value = 1.06 × 10-3-6.37 × 10-3) (Figure (Figure3B).3B). Moreover, none of the liver diseases detected in control samples were detected in the CCl4-treated samples. IPA re-analysis revealed that this fibrotic function in the control sample was associated with one gene--Spp1.
In IPA, the p-value is calculated using a right-tailed Fisher's exact test to assess the probability that the association between the focal molecules in the experiment and a given function is owing to random chance. Smaller the p-value, lesser is the probability of random association. In general, p-values < 0.05 indicate a statistically significant, nonrandom association. Therefore, these results suggest that CCl4-induced hypomethylation of a regulatory region, such as the promoter or enhancer of Spp1, may result in the onset of liver fibrosis and its progression to post-fibrotic diseases such as cirrhosis and HCC through an increase in Spp1 expression.
The hypomethylation peak was located approximately 18 kbp upstream of the transcription start site (TSS) of Spp1 (Figure (Figure4A,4A, red). We placed this MBP-seq peak into the UCSC genome browser and found that the hypomethylation site upstream of Spp1 has several chromatin features identified from studies by other groups, including: 1) mono- and trimethylation of histone 3 lysine 4 in the liver (H3K4 me1 and H3K4 me3) (Figure (Figure4A,4A, orange) [20]; 2) the binding of RNA polymerase II and related acetyltransferace p300 in the liver and in the MEL leukemia cell line (Figure (Figure4A,4A, light blue) [21]; and 3) it is a DNase I hotspot in the liver (Figure (Figure4A,4A, green) [22]. Previous studies have demonstrated that these epigenetic features are observed in the enhancer region [20,23-25], and that hypomethylation of an enhancer predominantly induces mRNA expression [26-28]. Thus, these epigenetic annotations lead to the hypothesis that this hypomethylated site may function as an enhancer that regulates Spp1 expression.
To confirm this hypothesis, we performed biochemical assays. Considering with the technical limitation of MBD-IP assay, a bisulfite modification assay was carried out to verify at a single-locus-based resolution (See Methods for details). Complementary using these assays, we confirmed that the site annotated by the sequence database was actually hypomethylated by CCl4 treatment (Figure (Figure4B4B and and4C).4C). We then examined whether the methylation on the upstream of Spp1 TSS will affect transcriptional activity. Luciferase assay were performed using in vitro methylated upstream of Spp1 TSS. This in-vitro-methylation assay revealed that methylation on upstream of Spp1 TSS down-regulated its mRNA levels (Figure (Figure5A).5A). We then performed a qPCR assay and revealed that Spp1 expression was increased by treatment with CCl4 (Figure (Figure5B)5B) and 5-dAza-C (Figure (Figure5C),5C), both of which induce hypomethylation upstream of Spp1 TSS. These results allow us to conclude that the upstream of Spp1 TSS functions as enhancer of Spp1. These results suggest that CCl4 treatment might up-regulate Spp1 expression in early-stage liver fibrosis through hypomethylation of the Spp1 enhancer.
In silico analysis and functional validation of hypomethylation suggest that Spp1 expression might cause fibrosis-induced liver diseases, such as those shown in Figures Figures3A3A and and3B;3B; this hypothesis is in agreement with previous studies [29]. Thus, to obtain functional insight into Spp1 in the end-stage liver disease HCC, we analyzed the mRNA level using the human cancer microarray database Oncomine. Analysis of studies by Mas et al. [11] , Chen et al. [12] , and Wurmbach et al. [13] revealed that Spp1 expression was higher in HCC than in normal liver tissues (Figure (Figure6),6), suggesting that Spp1 might play a crucial role in HCC development as well as in the onset of fibrosis.
The DNA methylation status of well-known genes associated with fibrosis progression, such as Rasal1, Fli1, and Thy1, has been reported to increase along with fibrosis progression, which induces proliferation of fibroblasts and the production of collagen [9,30,31]. Furthermore, 5'-Aza, an inhibitor of DNA methylation, reportedly attenuates the progression of renal and liver fibrosis in vivo and in vitro [7,8]. These reports indicate the importance of hypermethylation of the genome in the progression of liver fibrosis.
In contrast to these previous reports, our analysis revealed that DNA methylation status was significantly reduced in CCl4-induced early-stage liver fibrosis. Detailed analyses revealed that in early-stage liver fibrosis, the Spp1 enhancer was hypomethylated and Spp1 expression was up-regulated. Previous findings from genetically engineered Spp1-knockout mice have shown that Spp1 plays an important role in both progression and reduction of liver injury and fibrosis [32,33]. Although the precise mechanism underlying the role of Spp1 in fibrosis remains unknown, Spp1 is a pivotal cytokine/chemokine generated by the Kupffer cells in response to liver damage [34] that induces inflammation [35], which is a contributing factor in liver fibrosis. Our results indicate that an epigenetic alteration, DNA hypomethylation of the Spp1 enhancer, may precede the up-regulation of Spp1 expression and induce the onset of CCl4-induced early-stage liver fibrosis. Spp1 is also a known enhancer of tumor development and metastasis [36]. Using the Oncomine microarray database, we demonstrated that Spp1 expression level is increased in HCC. It has been documented in primary gastric cancers, that major phenotypic change in cancer-associated myofibroblasts is a global reduction in DNA methylation [17]. This may also be indicated in liver, the importance of hypomethylation of the Spp1 enhancer in the progression of HCC.
Unlike Spp1, we found that almost DNA hypomethylation induced in early fibrosis was assigned in intergenic regions (Figure (Figure2A).2A). Recently, it has been reported that DNA hypomethylation causes genomic instability and alteration of gene transcription in several human cancers (i.e. colorectal and prostatic adenocarcinoma, breast cancer, intestinal type-gastric carcinoma, and HCC) [37,38]. These results indicate that DNA hypomethylation in intergenic regions may trigger the progression of cancer through genomic instability of cancer-related genes in addition to transcriptional regulation of those genes in the onset of liver fibrosis. On the other hand, 5'-Aza treatment successfully reduces cancer in mammals, involving human, suggesting that carcinogenesis results from DNA hypermethylation as well as DNA hypomethylation [38-40]. Therefore, how these distinct DNA modifications commonly progress cancer is most important study for epigenetic effects on diseases in the future.
Although hypermethylation occurs during the progression of liver fibrosis, our results indicate the importance of hypomethylation in the onset of liver fibrosis. According to our results as well as other reports, it appears that DNA methylation status may change from hypo- to hypermethylation during the progression of liver fibrosis. Thus, hypomethylation in early-stage liver fibrosis may contribute to the onset and/or development of HCC.
α-Sma: α-Smooth muscle actin; Alt: Alanine aminotransferase; Ast: Aspartate aminotransferase; CCl4: Carbon tetrachloride; Col1a2: Collagen, type I, alpha 2; ECM: Extracellular matrix; Fli1: Friend leukemia integration 1; HCC: Hepatocellular carcinoma; Rasal1: RAS protein activator like 1; Spp1: Secreted phosphoprotein 1; Thy1: Thymocyte differentiation antigen 1; Timp1: Tissue inhibitor of metalloproteinase 1
The authors declare that they have no competing interests.
YK and NI performed the animal experiments and biochemical analyses. TW performed the high-throughput sequencing and bioinformatic analysis. WO and CY supported these analyses. All authors wrote, read, and approved the final manuscript.
The pre-publication history for this paper can be accessed here:
Table of primer sets used in this study. These qPCR primer sets were used in Figures 1B, 5B and C.
mRNA levels of reference genes. Cyclophilin were used to normalize genes in Figure 1B and 5B. Gapdh were used to normalize genes in Figure 5C.
Statistics and annotation of MBP-seq peaks. This table was generated by Genomics Workbench, and used in Figures 2, 3, and 4A.
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Put a unique twist on this year's fall fashions. PANDORA's Shimmering Droplets collection can add the perfect finishing touch to any look.
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RT @BridalMusings: Need a little romance in your Tuesday?
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Take $5 off some of our favorite ALEX AND ANI collections. Offer ends tomorrow.
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Every love story is unique – the diamond that represents it should be, too. Discover… https://t.co/by4Z73oXZV/p>
RT @ScottKay: "Coming together is a beginning; keeping together is progress; working together is success" - #HenryFord https://t.co/5mCLXLD…/p>
Are you thinking about popping the question? Follow the link in our bio to see new arrivals!… https://t.co/LXd8S0woZK/p>
Beautiful custom engagement ring shared by our happy customer, @allllysaurus. Like this post to congratulate the #bridetobe!
Design Your Own Diamond Ring: http://bit.ly/2bPGV2Q
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Buy an ESSENCE COLLECTION bangle bracelet and select charm for only $75. Ends August 31st! https://t.co/bQz3Q8eZ81 https://t.co/Q125gcX8TX/p>
On a scale from 1-10 what do you rate this double halo engagement ring? Tell us in the comments… https://t.co/DStmrFKZbO/p>
Refresh your weekday look with Tous Jewelry.
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Are you on Instagram? Follow us @reedsjewelers.
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We are proud to introduce the Liberty Copper Collection from ALEX AND ANI. Each piece of this collection features original copper preserved from the centennial restoration of the Statue of Liberty.
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Congrats to #reedsbride @brookie6353. Her Forevermark engagement ring is stunning. Like this post if you agree!
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RT @Perfect_Wedding: 9 things every woman should do before she gets married >> https://t.co/LTjGBCvUqy https://t.co/xeqZU4aVCr/p>
RT @Perfect_Wedding: 7 wedding dress errors to avoid https://t.co/rHQDdZ2nHN https://t.co/fFGIESe8MQ/p>
A ring as unique as your love! This stunning ring features a 1ct natural yellow diamond accented by a double halo and a crisscross white & yellow gold band.
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#TrendoftheWeek is now live! Will you be the lucky winner of this Infinite Love Necklace?! https://t.co/r3KpeZU4qT https://t.co/m6AYbTxrE7/p>
Tomorrow's Trend of the Week giveaway will feature this romantic Infinite Love Diamond Necklace. Share this post if you would love to add it to your jewelry collection!
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Comedy film 'Oconomowoc' sets local premieres
The comedy "Oconomowoc," the feature-film debut of Lawrence University graduate Andy Gillies, has its first Milwaukee-area showings at four theaters in the next two weeks.
Filmed in the title western Waukesha County community, "Oconomowoc" centers on a twentysomething, played by fellow Lawrence alumnus Brendan Marshall-Rashid, who moves back home with his mother and stepfather and agrees to help a friend rebuild his mishandled T-shirt business. »Read Full Article(3)
AT&T drops Al Jazeera America news channel just before debut
Al Jazeera America, the first all news cable network since Fox News Channel debuted in 1996, begins broadcasting at 2 p.m. Tuesday. But shortly before its debut AT&T U-verse announced it would not carry the channel in a contract dispute, according to The Wrap.
It is also not carried by Time Warner Cable. It will be carried by DirecTV (Channel 358) and DISH (Channel 215). It will be in less half as many TV homes as CNN and Fox. »Read Full Blog Post(104)
New Berlin grad makes Betty White leap for joy on 'Tonight Show' segment
New Berlin — New Berlin West High School graduate Kevin Bania appeared on the Aug. 8 "Tonight Show with Jay Leno" in a segment called "Does This Delight Betty White?" »Read Full Article
Milwaukee Film Festival announces music movie program
Film and music are twin siblings from another mother. Because they are so alike in creative spirit, it's not surprising when filmmakers explore the musical impulse, and vice versa.
This is particularly evident from the 2013 Milwaukee Film Festival's eight-film Sound Vision lineup, announced Tuesday. »Read Full Blog Post(1)
On Tap Today: Wanted - Seeking Milwaukee teen pop fans to 'Call Me Maybe'
Carly Rae Jepsen, pictured here opening for Justin Bieber at the BMO Harris Bradley Center last October, will co-headline the Riverside Theater tonight with boy band the Wanted. Journal Sentinel photo by Mike De Sisti. »Read Full Blog Post


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This is the sixth installment of many of the Skinnee Labs TIM Comparison 2011. For most of the installments, results will be released in two or three TIM sets roughly every two or three weeks and today we have three TIMs to show. Arctic Silver 5 is thermal compound for CPU and GPU. Thermal compound helps in transferring the heat from the CPU cores to the heatsink by filling up the microscopic valleys between processor cores and heatsink. Without the use of thermal paste a processor will always run hot, that will reduce its life. Arctic Silver is the biggest name in thermal paste. For the longest time, Arctic Silver 5 was known as the pinnacle of thermal paste and its sibling, Ceramique, was also highly regarded for being the best performing paste without any conductivity/capacitance qualities. You can think yourself the CPU manufacturer Intel ships its own thermal paste with boxed Intel CPUs, now you can think how important is the thermal paste for the CPU and GPU. Intel's stock Thermal Interface Material is not available on online stores, so we have only one option left is to buy aftermarket thermal compounds. Things changed a few years back, however; Arctic Cooling MX-2 came on the scene and exceeded Arctic Silver 5's performance while also not being capacitive. Even so, a quick glance at the number of user reviews at Newegg shows Arctic Silver makes the most popular pastes on the market with AS5 and Ceramique (although Ceramique has now been EOL'd). Currently Arctic Silver is considered to be the best thermal paste for CPU and GPU chip doesn't matter if it is of desktop's or laptop's. In this tutorial I going to review the Arctic Silver 5 thermal paste and tell you about it pros and you will be able to decide whether to buy it or not.
Continue reading "How To Apply Arctic Silver 5 To Your Processor"


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Regional Advanced Training Course on In Situ Preservation of Underwater Cultural Heritage, 19-26 October 2011
The regional training course, organized by UNESCO Bangkok Office under the Norwegian Government project on "Safeguarding the Underwater Cultural Heritage of Asia and the Pacific", will take place at the Khao Laem Ya-Moo Koh Samet National Park, Rayong Province, Thailand.
The training is being implemented in collaboration with the Underwater Archaeology Division of Thailand, to which the Asia-Pacific Regional Field Training Centre established under the project has been attached.
Fifteen national experts from nine countries (Cambodia, Indonesia, Kenya, Kyrgyztan, Malaysia, Pakistan, Philippines, Sri Lanka and Thailand) who are graduates from earlier Foundation Courses will undergo training in the different methods of in situ preservation through lectures and practical exercises. Experts from ICOMOS-ICUCH and the National Museum of Denmark will share their expertise with the participants.
For more information please contact Mr Ricardo Favis and consult the dedicated webpage of the UNESCO Bangkok office.
<- Back to: Underwater Cultural Heritage


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cool I checked it out. and I haven't heard the Bled. Is silent treatment their best album?
Digging: Taking Back Sunday - Tidal Wave
fuck no
band sucks so idc but yea
The Silent Treatment is their second best, imo. I love their vocalist.
didnt read ur review i just dont like this album
i feel like i've missed ur last 3 reviews being on the front page.
@Calc, just bad luck. I feel like everytime I submit a review half the site does the same so my reviews get bumped off the home page pretty quick lol oh well
Cool I'll check it out now
who negged? I will destroy you!
"i feel like i've missed ur last 3 reviews being on the front page."
Me too. I thought maybe you were taking a break from reviewing.
no i've just slowed down. i'll still be reviewing but more like once a week instead of every day lol
bonus track for this, make it right, is one of their best songs.
haven't heard the bonus tracks so my review was just based on the 13 tracks i've heard :P
shit band
Digging: Bryan MacLean - Ifyoubelievein
^ shit user ;)
hey dont be mean to andcas thats not nice
cool i'll check them out thanks
Least favorite NFG... Though they played "Hold My Hand" on the "Sleep When I Die" tour that I saw recently. Fantastic show!
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


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Teen has close call when smartphone has meltdown
A 13-year-old girl in Texas had a close call after her smartphone had a meltdown.
Ariel Tolfree woke up to the smell of burning plastic and smoke. She found that her Samsung Galaxy S4 slipped under her pillow when she was sleeping and it started melting through the bedding.
"The whole phone melted, the plastic the glass — you can't even tell it was a phone."
Samsung says its phones are safe and points out the battery in the girl's phone was a replacement, not a Samsung original.
The company is investigating what happened and has offered to replace the phone and damaged bedding.


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Cherokee Grand Hotel, Cherokee
- Free WiFi and free parking
Main amenities
- 90 smoke-free guestrooms
- Outdoor pool
- Free casino shuttle
- 24-hour front desk
- Coffee/tea in a common area
- Air conditioning
- Daily housekeeping
- Lobby fireplace
- Front desk safe
For families
- Cribs/infant beds (surcharge)
- Rollaway/extra beds (surcharge)
- Sofa bed
- Microwave
- Refrigerator
- Connecting/adjoining rooms available
What's around
- Oconaluftee Islands Park (1 mi / 1.6 km)
- Eastern Cherokee Agency (1.2 mi / 1.9 km)
- Cherokee Welcome Center (1.4 mi / 2.3 km)
- Museum of the Cherokee Indian (1.5 mi / 2.4 km)
The place was great except the water didn't get hot at all it was like Luke warm ugh.
Clean and quaint hotel within eye shot and easy walking distance of the casino, for a…
- Asheville, NC (AVL-Asheville Regional) 71-min drive; 55.8 miles/89.7 km
- Free self parking
- Free casino shuttle
Cherokee Grand Hotel, Cherokeefrom NZ$111
- Single Room, 2 Double Beds, Non Smoking
- Deluxe Room, 2 Queen Beds
- Standard Room, 1 Queen Bed
- Standard Room, 1 King Bed
- Deluxe, One King Bed, Non-Smoking
- Deluxe Room, 1 King Bed
- Standard Room, 2 Queen Beds
- Deluxe Room, 1 King Bed, Jetted Tub
- Standard Room, 1 King Bed, Smoking, Jetted Tub
- Deluxe Room, 1 Queen Bed, Smoking
- Deluxe Room, 1 Queen Bed
- Deluxe Room, 2 Queen Beds
- Deluxe Room, 1 King Bed, Smoking
Recommended hotels
At a glance
Key facts
Hotel size
- This hotel has 90 rooms
- This hotel is arranged over 4 floors
- Check-in time 4 PM-5:30 AM
- Check-out time is 11 AM
- Express check-out
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
Travelling with others
- One child (16 years old and younger) stays free when occupying the parent or guardian's room, using existing bedding
- Pets not allowed
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
- Free casino shuttle
Other information
- Smoke-free property
- No alcohol served onsite
In the hotel
- Coffee/tea in a common area
- Outdoor seasonal pool
- Outdoor pool
- 24-hour front desk
- Number of buildings/towers - 3
- Elevator/lift
- Safe-deposit box at front desk
- Fireplace in lobby
In the room
- Air conditioning
- Coffee/tea maker
- Iron/ironing board
- Down comforter
- Blackout drapes/curtains
- Sofa bed
- Sofa bed size double
- Premium bedding
- Pillowtop mattress
- Private bathroom
- Shower/tub combination
- Designer toiletries
- Hair dryer
- Cable TV channels
- Desk
- Free WiFi
- Free local calls
- Refrigerator
- Microwave
- Daily housekeeping
- Connecting/adjoining rooms available
Cherokee Grand Hotel, Cherokee's small print
Also known as
- Cherokee Grand
- Cherokee Grand Hotel
- Inn Of The Seven Clans Hotel Cherokee
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Only registered guests are allowed in the guestrooms.
Children under 18 years old are not allowed in the swimming pool without adult supervision.
The name on the credit card used at check-in to pay for incidentals must be the primary name on the guestroom reservation.
Optional extras
Early check-in can be arranged for an extra charge of USD 0.00
Late check-out can be arranged for an extra charge of USD 0.00
Rollaway beds are available for USD 10.00 per stay
Cribs/Infant beds are available for USD 10.00 per stay
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
More great choices
Recent reviews
Good 3.8 /5 from 1116 reviews
No bath/hand soaps in the hotel and no alternative available either. The reply was that hopefully the shipment will be here tomorrow. They didn't think that it was necessary to buy something in the meanwhile from supermarket
Over all great visit. The only thing is the hotel needs to update there website , we booked a deluxe double queen room , which was a great room no problem there, but the website advertised it would have two queen beds plus a sleeper sofa which i understood the reason why it did not. But still false advertisementbecause it did not have sleeper sofa bedbut everything else was wonderful clean rooms clean pool clean hotel. I will stay again
Nice and relax
We paid $175 for this room. The room was ok, but in the morning when we needed to shower there was no soap. I went to front desk and asked girl if I could get some soap she said they were out. Her response to me was " just use the shampoo, it's the same thing." REALLY!! She made no apologies, just excuses that they didn't get their delivery. Great management would have gone to a store and made sure that their guest were able to shower with soap. I requested some type of reduction in my bill due to the fact we were not gonna be able to shower. She said she'd pass it on to management. When my husband paid by credit card there was no adjustment made. Therefore I rate it poorly, they obviously don't care that much about their guest. For the price we paid they could have put in a .50 bar of soap.
We stayed 2 nights and it was perfect. Big, clean rooms, comfy beds, large bathroom and tub. Walking distance to Harrah's Casino. We couldn't have asked for more.
Is the description of this hotel not correct? Tell us


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As a Bumble and Bumble Stylist you will motivate and manage team to build deep relationships with customers, share our brand story with everyone and our salon heritage" dedicated to the craft, culture and commerce of hairdressing" you will have great communication skills and a "flair for hair" demonstrating and selling products that are as important as a scissor and comb, with the ability to work towards and achieve retail sales targets.
You will work as part of a dedicated team, where you will lead sharing ideas for building and sharing our brand story , whilst working together to ensure the smooth and efficient running of this busy counter.
As a Bumble and bumbles tylist you will receive ongoing training and development to polish your hair styling skills, whilst helping you further develop your commercial expertise.
Applicants must have the ability to manage and lead a team, strong communication skills, NVQ in hairdressing would be good but not essential if you have a flair for hair!
applicants must have previous retail experience with a love of all things hair, with a creative flare for hair styling


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carbon strategy Companies serving Marshall Islands
Current filtering criteriaYou can remove a filter by clicking on the x
The CarbonNeutral Company
based in London, UNITED KINGDOM
Over 13 years experience in carbon offset programmes which reduce emissions, increase revenue, manage costs and engage stakeholders. Every organisation – from services though to manufacturing - produces global warming gases as a by-product of doing business. And there's a CO2 'footprint' too, from customers using products and services – taking a flight, driving a car, ...
based in Elsternwick, AUSTRALIA
Our services evolve around life cycle assessments and life cycle thinking. The overarching aim is to help clients understand and quantify environmental issues. A whole-of-life approach is essential if we are to make better decisions and become more sustainable.A detailed understanding of the relevant topics and contributors enables our clients to undertake positive ...
GP Strategies Corporation
based in Columbia, MARYLAND (USA)
GP Strategies Corporation (NYSE:GPX) is a global performance improvement company and a leader in sales and technical training, e-learning solutions, management consulting, and engineering services. With more than 40 years of leadership in training, consulting, and engineering services, GP Strategies has the proven processes, best practices, and lessons learned to bring ...
The Carbon Desk
based in London, UNITED KINGDOM
CarbonDesk is an independent broker specialising in carbon emissions. In addition to trading we offer unbiased advice to industrial and financial traders in the emissions arena. The company is developing option and derivative based strategies for clients on both the buy- and sell-side of the market but mostly focusing on small to medium sized industrial clients. As we ...
Carbon Neutral
based in Perth, AUSTRALIA
Carbon Neutral is a not-for-profit carbon consultancy and offset provider. We assist clients to measure, reduce and offset greenhouse gas emissions. As a registered environmental charity, we have a strong focus on developing biodiverse conservation plantings and carbon sinks which deliver significant environmental co-benefits. Now in its tenth year Carbon Neutral is ...
Asia Carbon Pte Ltd
based in Singapore, SINGAPORE
Asia Carbon Global provides an integrated and seamless approach to the business of energy, environment and sustainable development. We focus on initiatives that include the flexible market mechanisms of the Kyoto Protocol as well as the fast-growing opportunities presented by the voluntary markets. Our global network is coordinated through our headquarters and regional ...
Less Carbon Ltd.
based in Providencia, CHILE
Less Carbon is a new and innovative company dedicated to originating, developing and trading carbon credits from forestry and land use projects in Latin America. Its strategy is to develop a portfolio of high quality projects which yield deliverable carbon credits while clearly demonstrating social and environmental benefits to stakeholders. Carbon credits from these ...
Carbon Disclosure Project
based in London, UNITED KINGDOM
Carbon Disclosure Project is an independent not-for-profit organization holding the largest database of primary corporate climate change information in the world. Thousands of organizations from across the world's major economies measure and disclose their greenhouse gas emissions and climate change strategies through CDP.
Carbon Clear Limited
based in London, UNITED KINGDOM
Carbon Clear Limited is a world leader in carbon management. We help organisations address the challenges posed by climate change. We work with hundreds of organisations to: measure their carbon footprint, reduce their carbon emissions, source high quality carbon offset credits, comply with environmental legislation, communicate their achievements.
Carbon Trade Exchange Ltd
based in Sydney, AUSTRALIA
Carbon Trade Exchange is a global electronic trading platform serving the compliance and voluntary carbon markets.Carbon Trade Exchange (CTX) has been established to aid the growth and transparency of the carbon markets and become the world's most inclusive trading platform. CTX maximises the environmental benefits of carbon trading by bringing unrivalled liquidity and ...
Renewable Carbon Management LLC
based in St. Cloud, MINNESOTA (USA)
Renewable Carbon Management, LLC (RCM) sells and licenses the NaturTech Composting System for converting organic materials into value added commodities. We also provide strategic partnerships and operating expertise for the lowest cost and highest environmental performance available.
Carbon Market Data
based in London, UNITED KINGDOM
Carbon Market Data is a research company and market data vendor offering information, consulting and technology services to a wide range of organisations in the world. Carbon Market Data aims to help the financial community assess the new business risks and opportunities presented by climate change. By designing new web-based carbon information solutions, we help to ...
Carbon Planet Limited
based in Adelaide, AUSTRALIA
Carbon Planet Limited is a global carbon management company whose mission is to enable every individual and business on the planet to manage their contribution to the defining issue of our age, global warming. Carbon Planet has been working with businesses around the world, helping them quantify the risks and explore the opportunities emerging in the carbon constrained ...
CRS Carbon Revenue Services S.A.
based in Rome, ITALY
CRS Carbon Revenue Services started work as a consulting company due to new needs and opportunities which arose from the Kyoto Protocol. Since its establishment, research in the area of Emission Reduction Activities, resulting in implementation of frontier energy efficiency businesses, CDM Projects have been developed for investors, funds, public institutions and private ...
Global Clean Energy Corporation
based in UNITED ARAB EMIRATES
Global Clean Energy Corporation develops energy and environmental projects with a special focus on renewable energies and carbon abatement. Further we develop CSR strategies and advise on low carbon technologies and offsetting opportunities.
Cleaner Climate Ltd.
based in The Rocks, AUSTRALIA
Cleaner Climate is a vertically integrated carbon solutions provider. We provide a full suite of services designed to assist clients in transitioning to the low carbon economy with an emphasis on carbon origination, development and sustainability strategy.Qualifications * United Nations Environment Program Preferred Partner * Provider to the Organisation for ...
Zeroemissions (Abeinsa)
based in Palmas Altas - Sevilla, SPAIN
Zeroemissions is a company integrated into Abeinsa which, in turn, is Abengoa`s industrial engineering and construction business unit. Zeroemissions`s aim is to provide global solutions for climate change through the promotion, development, and trade of carbon credits, corporate carbon strategy, carbon offset services and innovation in technologies for greenhouse gas ...
GHG Accounting Services
based in Vancouver, BRITISH COLUMBIA (CANADA)
GHG Accounting Services Ltd. ("GHG Accounting") provides specialized carbon and sustainability consulting and greenhouse gas accounting services to corporations, local government agencies and not-for-profit organizations. In particular, GHG Accounting offers services in connection with (i) emissions reporting and footprint inventory quantification, (ii) quantification, ...
CK Connect Co2
based in Liverpool, UNITED KINGDOM
CK Connect co2 are trading experts who specialise in the worldwide carbon trading markets. Carbon trading enables businesses to reduce their carbon footprint, those with higher than the recommended emissions can buy permits from businesses with low emissions or alternatively can buy carbon credits issued to emission reducing projects.
Prominent Systems, Inc
based in Covina, CALIFORNIA (USA)
Prominent Systems, Inc. is a corporation that specialized in the complete range of activated carbon. We produce and sell a wide variety of activated carbons for all kinds of applications. Our manufacturing plant are located in Indonesia, Vietnam, and China. Our products lines include coconut shell, bituminous coal base, wood base, and Lignite base Activated Carbon.Coconut ...


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'Candid Camera' aims to make new generation smile
LOS ANGELES (AP) - Smile, "Candid Camera" is making a comeback.
Starting Monday, TV Land will air a new version of the hidden-camera show that first debuted in 1948.
The 10-episode series is produced by Peter Funt, son of the original show's creator and host, Allen Funt.
Peter Funt will co-host with Mayim Bialik of "The Big Bang Theory." Guest stars including Megan Hilty, Reggie Jackson and Carnie Wilson will help pull off the gags.
The original show's catchphrase was "Smile, you're on 'Candid Camera,'" which is how people learned they had been pranked. Versions of the show have returned to TV several times since its initial run.
In its first week, new episodes of "Candid Camera" will air at 8 p.m. EDT Monday through Thursday. The show will then air weekly on Tuesdays, starting Aug. 19.
See more from "The Big Bang Theory" in this AOL.com slideshow:
See Gallery
Also on AOL.com:
As if orangutans snuggling isn't cute enough . . .
Bizarre beach trend hits the shoreline
Big dog saves tiny Chihuahua from jaws of coyote


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Winter must be cold and snowy (said no one, ever). How's about a season in Latin America instead?Read More »
All Posts
Below Zero Teaser
A sneak-peek at some snowy freeriding action up north this winterRead More »
Foilboarding Tips: Water Start to Transition
Episode numero two of the Liquid Force foilboarding tips and tricks video seriesRead More »
A Ways to Kite
Shifty moves and skate-inspired tricks just south of the borderRead More »
High Fives (in Brazil)
It's all about sharing the stoke, both on and off the waterRead More »
The Kiteboarder Magazine for Android Users
Back issues of TKB are now available for free on your favorite deviceRead More »
Teammates Episode 2
Candid recordings and hilarious outtakes amongst teammates at this year's Red Bull King of the AirRead More »
Girl Gone Kiting
When was the last time you went on an overnight snowkiting trip, alone?Read More »
Tkb Light Wind Gear Test Application Form
Tkb will be in San Diego testing all the new 2015 Light Wind Gear from participating brands, March 15-22, possibly longer, depending on weather conditions. If you would like to join us for testing, can commit to meeting us at ...Read More »
Fall Sessions
One chill edit and a bonus chuckle at the very endRead More »
Collegiate Kiteboarding Comps Take Off in Florida
The CKA continues to rock the East Coast with stops in Marathon Key and MiamiRead More »
SnowSports Industries (SIA) Product Highlights 2015/16
Twenty-two new products snowkiters might find useful in the coming yearRead More »
Maxime Nocher Joins the F-One Team
A young, hard-working rider from the South of France with an impressive collection of titles.Read More »
All The Way
Nothing ventured, nothing gained. If you're going to try, go all the wayRead More »
The IKA KiteFoil Gold Cup is Back
One of the world's fastest wind-powered watercrafts finds its place in racing (again)Read More »


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The Orthodox Church of St. Matthew's Multi-Cultural Festival
Welcome to Columbia's biggest and best multi-cultural festival, where we bring the world to you! Please join us the first weekend in October for our 2016 festival.
Oktoberfest Happy Hour
Friday, September 30; 6-9 pm
Multi-Cultural Festival
Saturday, October 1; 11 am – 8 pm
Sunday, October 2; 11:30 am – 6:30 pm
The Orthodox Church of St. Mathew
7271 Eden Brook Drive
Columbia, MD 21046
located in the Kings Contrivance Village Center


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Sean Kingston's Passenger Recalls Minutes Before Crash
The girl who was behind Sean Kingston when the jet ski crashed against a Miami beach bridge on the weekend has recounted minutes into the disaster. Cassandra Sanchez, Sean's longtime friend, told TMZ that the rapper was thinking of turning the jet ski around at last minute but was driving "really fast" that he lost control of the vehicle.
"Both jet skis pulled out of the dock at the same time and we were going so fast we just blasted past them," Cassandra said. "When we turned a corner and crashed ... they were so far behind us they didn't even see us crash." She remembered yelling to Sean before the crash, "We can't fit under there! Are we going to try and go under there? Sean stop!"
Their other friends quickly rushed in to help and signaled an off-duty Coast Guard employee who then rescued them in a boat. They pulled Sean who was "coughing up blood, foam and pink stuff". Cassandra recalled, "...when he finally opened his eyes, he kept repeating the words, 'I'm hurting. I'm hurting'."
Cassandra has been discharged from the hospital although she said that her ribs and jaw are still sore. Meanwhile, Sean is still hooked to a tube and stays in ICU to be stabilized. His rep told MTV, "He is aware of what is going on around him," adding that the singer "is sedated but fully conscious." He would be kept in the hospital for a couple more weeks.
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Anthony Ha
Anthony Ha is a senior writer at TechCrunch, where he covers media and advertising. Previously, he worked as a tech writer at Adweek, a senior editor at the tech blog VentureBeat, and a local government reporter at the Hollister Free Lance, where he won awards from the California Newspaper Publishers Association for breaking news coverage and writing. He attended Stanford University and now lives in Brooklyn.
Disclosure: I own shares in VentureBeat.CrunchBase profile →
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Hotels in Sarnico, Italy
Search & Compare Sarnico Hotels
Trending now in Sarnico
Hotels in Sarnico
- Most bookedCocca Hotel Royal Thai SPA
115 people looked at this hotel in the last hour
Most recent review
"A friendly hotel with great spa facilities. Away from the busier parts of Lake Iseo."
A Traveller, Aug 2016, GB
115 people looked at this hotel in the last hour
Most recent review
"Lovely small hotel with great view of wonderful lake"
A Traveller, Sep 2015, GB
10 people looked at this hotel in the last hour
Most recent review
"Great Staff. Great location. Wonderful few Days. Look forward to doing it all again!"
A Traveller, Oct 2014, GB
Hotels within 5 miles of Sarnico
18 people looked at this hotel in the last hour
Most recent review
"Couple of nights of luxury on our tour of the lakes. You need to have a car to get to this hotel which is high on a hill overlooking the lake and for exploring the area. Weather was perfect so outdoor pool area was superb and outdoor eating area overlooking the lake was fabulous. Food in restaura..."
A Traveller, Sep 2016, GB
18 people looked at this hotel in the last hour
Most recent review
"The hotel is in a good position in Paratico and only a short walk along the promenade and over the bridge to Sarnico where you can get the ferry to other places on the lake.The swimming pool is set in lovely mature gardens and is quiet and peaceful.The hotel staff were superb and went out of thei..."
A Traveller, Jul 2016, GB
- Hotel EurovilNo Guest Reviews yet
17 people looked at this hotel in the last hour
Most recent review
"Hotel was fine, room was large and quiet. Staff was very helpful and spoke English. Breakfast was included and was very good. Pictures on the internet of the hotel were deceiving, showed people on beach by the lake. The hotel is across the street, not on the lake, there is a beach area which w..."
A Traveller, Jul 2016, US
14 people looked at this hotel in the last hour
12 people looked at this hotel in the last hour
Most recent review
"Perfecto! It was the most amazing hotel room and amazing please to stay in."
A Traveller, Sep 2016, US
11 people looked at this hotel in the last hour
Most recent review
"A truly lovely stay in a beautiful family run hotel. Worthwhile asking for a room with a view over the lake - our view was fantastic."
A Traveller, Jul 2014, US
- Hotel StazioneNo Guest Reviews yet
4 people looked at this hotel in the last hour
Best hotels in Sarnico
If you are looking for Sarnico hotels in a particular area, you can use the search feature at the top of the page to search by neighborhood, landmark, address or zip code, or you can search by using the built-in map feature. You can also browse hotels in Sarnico by type, amenities, and star-rating so that you can find the best hotel. Currently, top rated hotels in Sarnico are:
- Bed & Breakfast Centro Storico
- Cocca Hotel Royal Thai SPA
- Hotel Sebino
- Podere Castel Merlo
- Relais Mirabella
The location of your Sarnico hotel is important. If you are traveling to Sarnico, &StateorCountry for business, you may want to consider getting a hotel close to the airport or near your meeting venue. By doing so, you can really cut down on transportation costs and time. You may also want to book a hotel that offers free wifi just in case you need to get some work done.
If you are traveling for vacation, figure out which attractions or landmarks you want to see and book a hotel that is within close proximity. Although hotels near attractions might cost a little more, you may find that spending those extra few dollars is worth the convenience. You can even find hotels that align with your interests. For example, if you are looking for a relaxing vacation, a spa hotel may be the perfect choice for you.
You'll also want to determine which type of accommodations will suit your needs before booking a hotel in Sarnico. If you're traveling with the kiddos, a family friendly hotel will offer larger rooms and more sleeping arrangements. For those traveling for an extended period of time, booking a hotel suite with a kitchenette is a smart move and will feel a little more like home. If you are budget conscience and just need a place to rest your head, a hostel or motel may be all you need.
If you are looking to score the best discounts and deals, use the filter to sort by price from low to high. You can book a hotel in Sarnico for as low as $51 per night. If you are looking to cut down on expenses, look for hotels that include free breakfast and parking. Some hotels even offer free shuttle service to and from nearby places. You can also sort by star rating, proximity, and guest rating.
Before you book, check out user reviews. Hotels.com has 126 reviews in Sarnico. We show reviews from Expedia and TripAdvisor so you know that you are reading genuine, reliable reviews. Be sure to download the Hotels.com app so you can book your reservations on the go, view amenities, and save the hotels you want to view and compare later.


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There isn't much in common between the WWE and the English Premier League.
One involves men in tights throwing each other around a squared ring, whilst the other involves 22 men kicking around a ball of air for 90 minutes.
However, you could compare WWE wrestlers to the Premier League clubs, and this is what I'm going to try to do here.


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- Ensemble:
- Format:
Somebody That I Used to Know: 2nd Percussion
2nd Percussion
By Gotye
Concert Band
2nd Percussion. By Gotye. By Luiz Bonfa (1922-). Arranged by Victor López. Instructional. Part. 1 pages. Published by Alfred Music - Digital Sheet Music (AX.00-PC-0015419_PC2).
Item Number: AX.00-PC-0015419_PC2
Internationally acclaimed, this bouncy Gotye tune will be a high point on your beginners' concert. Reinforcing staccato style with a bit of percussion support, if you are looking for a selection for your band that the whole world knows, look no further, because you just found it! Infectious! (2:00).
About Digital Downloads
Digital Downloads are downloadable sheet music files that can be viewed directly on your computer, tablet or mobile device. Once you download your digital sheet music, you can view and print it at home, school, or anywhere you want to make music, and you don't have to be connected to the internet. Just purchase, download and play!
PLEASE NOTE: Your Digital Download will have a watermark at the bottom of each page that will include your name, purchase date and number of copies purchased. You are only authorized to print the number of copies that you have purchased. You may not digitally distribute or print more copies than purchased for use (i.e., you may not print or digitally distribute individual copies to friends or students).
Customers Who Bought Somebody That I Used to Know: 2nd Percussion Also Bought:
New York Mining Disaster 1941
The A Team


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Hotels near Franz Josefs S-Bahn
Search hotels near Franz Josefs S-Bahn in Vienna
Get Secret Prices on select hotels
These prices aren't available to everyone.
Trending now in Vienna
- Most bookedVienna Grand Apartments CityNo Guest Reviews yet
145 people looked at this hotel in the last hour
Most recent review
"Fabulous top quality spacious apartment with attention to detail. Fully fitted kitchen with stocked fridge and modern coffee maker including a dishwasher. Beautiful bathroom and well stocked with plenty of toiletries. Extremely comfortable bed and luxury bedding. Staff were extremely welcoming..."
B, Sep 2016, GB
43 people looked at this hotel in the last hour
Most recent review
"it was very convinyet to travel were we had to go with the train that is a minite away.brekfest was exelent."
A Traveller, Nov 2015, IE
25 people looked at this hotel in the last hour
Most recent review
"The owner of the hotel was very accomodating and cordial. I would stay here again considering its location and amenities"
A Traveller, Jun 2016, US
18 people looked at this hotel in the last hour
Most recent review
"We were a group of 3 travelers meeting in Vienna: one arriving at 8 am and Roland the host was good enough to let her in early to the apartment. We booked 2 apartments next to each other which was perfect: the apartments were bright and well furnished. Roland also provided tourist information and..."
A Traveller, Aug 2015, AU
- Luxury Design Home StroheckgasseNo Guest Reviews yet
17 people looked at this hotel in the last hour
11 people looked at this hotel in the last hour
Most recent review
"The apartment is a very good choice for staying in Vienna. It is very well situated, just 3 blocks from the metro station in a quiet neighborhood but with all the services you may need (restaurants, supermarkets, etc.). I didn't like the key handover procedure. Nobody was waiting for us and we ha..."
A Traveller, May 2014, US
- CheckVienna - LiechtensteinstrasseNo Guest Reviews yet
7 people looked at this hotel in the last hour
5 people looked at this hotel in the last hour
Most recent review
"At first I booked a single room here because it was rather cheap. It was a great deal, especially with all that surrounding area. Only 30 min on foot to get to the city centre, which I appreciated as well. The hostel itself is an old somehow vintage building with a nice beergarden, where I spent ..."
A Traveller, Sep 2015, IE
5 people looked at this hotel in the last hour
- Vienna apartment one mariannengasseNo Guest Reviews yet
4 people looked at this hotel in the last hour
Map of hotels near Franz Josefs S-Bahn
- St. Stephen's Cathedral
- Vienna State Opera
- Stephansplatz
- Vienna International Centre
- Hofburg Imperial Palace
- University of Vienna
- Messe Wien Exhibition and Congress Center
- Wiener Stadthalle
- Schoenbrunn Palace
- Vienna City Hall
- Wiener Prater
- Karlsplatz
- Spanish Riding School
- Schwedenplatz
- International Atomic Energy Agency
- Belvedere
- Vienna University of Technology
- Viennese City Park
- Gasometer
- Naschmarkt
Hotels in Vienna near Franz Josefs S-Bahn
Are you looking for a cheap Franz Josefs S-Bahn hotel, a 5 star Franz Josefs S-Bahn hotel or a family friendly Franz Josefs S-Bahn hotel? You just landed in the best site to find the best deals and offers on the most amazing accommodations for your stay.
When you search for hotels near Franz Josefs S-Bahn with Hotels.com, you need to first check our online map and see the distance you will be from Franz Josefs S-Bahn, Austria.
Our maps are based on hotel search and display areas and neighborhoods of each hotel so you can see how close you are from Franz Josefs S-Bahn and refine your search within Vienna or Austria based on closest public transportation, restaurants and entertainment so you can easily get around the city. All the hotels details page show an option for free or paid onsite parking.
If you wish to see the hotels with the highest featuring discounts and deals near Franz Josefs S-Bahn, simply filter by price/ average nightly rate. We recommend you filter by star rating and read our genuine guest reviews so you can get the best quality hotel with the best discount.
One of the new features on Hotels.com guest reviews is that also show reviews from Expedia for Franz Josefs S-Bahn hotels and the TripAdvisor Franz Josefs S-Bahn hotels reviews so you can make sure that you checking with a reliable source. See the review scores on our Vienna hotel information pages.
Make the most out of your family vacation when you book your accommodation with Hotels.com – book your hotel near Franz Josefs S-Bahn, Vienna after reviewing the facilities and amenities listed for each hotel.
After booking your hotel near Franz Josefs S-Bahn, expect to receive your reservation confirmation in the mail in less than 10 minutes. The confirmation email contains more information on all nearby attractions, local directions and weather forecast, so you can better plan the days during your trip.
After getting the best hotel rates you can still save more by winning 1 free night! That's right, book 10 nights in any hotel near Franz Josefs S-Bahn, Vienna and after you sign up for the Welcome Rewards program, you are eligible hotel you receive 1 night free*
The best hotel deals are here: We have Franz Josefs S-Bahn hotel deals, Franz Josefs S-Bahn last minute deals and offers to get you the cheapest Franz Josefs S-Bahn hotel with our lowest price guarantee.
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Bill Fox, left, Seven Ag Consulting, Yuma, Ariz., discusses citrus pest and disease issues with Bob Zimmerman, center, B & Z Nursery, Porterville, Calif., and Michael Rogers, University of Florida, Lake Alfred, Fla.
Michael Rogers, University of Florida entomologist.
Californians attending the Bayer meeting include from left: Ted Batkin, Citrus Research Board, Visalia; Marilyn Kinoshita, Tulare County Ag Commissioner, Tulare; and the CRB's Brian Taylor, also of Visalia.
Researchers collect yellow sticky pest traps looking for the Asian citrus psyllid.
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text:
A GROUP of Philadelphia police officers spends its free time investigating claims of disorderly conduct, harassment and criminal mischief, knowing the cases don't have a ghost of a chance of making it to court.
Still, the officers set up video cameras at each scene and spend hours poring over evidence, looking for just one shadow, listening for just one voice.
Once, a voice said "Mommy, I'm here," another time, "F--- you!"
Even disembodied voices have trouble with authority.
By day, the officers protect the mayor, police headquarters and the streets, but by night, they patrol the paranormal as members of the group Olde City Paranormal.


text:
Convert Corel Draw Vector Files to SWF and upload to Magento
This project was awarded to Quicketch for 1000 ZAR.Get free quotes for a project like this
Awarded to:
Skills Required
Project BudgetR210 - R1750 ZAR
Total Bids1
Project Description
This project is small and should be easy. It contains 2 steps. Step 1 export Clipart to swf. Step 2 upload 26 swf sets to Magento.
We have 26 Corel Draw Vector images ready. These images must be exported from either Corel Draw or Adobe Illustrator to a simple swf (flash) file. No animations just the layers of each one of the 26 Vector images. So a black and white clip art image might have 2 layers i.e. Black and White. This one Corel Draw Vector image must be exported twice as layer 1 and layer 2.
Once all 26 vector images are exported please then log into Magento backend and upload via the PD designer. If a vector image that had 2 layers exported to swf then you need to upload 2 layers so that the image can have different colours that you can set through the T shirt design tool here: [url removed, login to view]
To see how to export the Corel or Adobe Illustrator Vector images you can read how to here:
How to create vector (SWF) cliparts:
[url removed, login to view]
How to Add vector (SWF) clipart to Magento:
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