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Global target mRNA specification and regulation by the RNA-binding protein ZFP36
- Neelanjan Mukherjee1Email author,
- Nicholas C Jacobs1,
- Markus Hafner3,
- Elizabeth A Kennington2,
- Jeffrey D Nusbaum3,
- Thomas Tuschl3,
- Perry J Blackshear2 and
- Uwe Ohler1Email author
DOI: 10.1186/gb-2014-15-1-r12
© Mukherjee et al.; licensee BioMed Central Ltd. 2014
Received: 8 January 2013
Accepted: 6 August 2013
Published: 8 January 2014
ZFP36, also known as tristetraprolin or TTP, and ELAVL1, also known as HuR, are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embryonic kidney cells, and examined the combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1.
Targets bound and negatively regulated by ZFP36 include transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance upon ZFP36 overexpression independent of effects from confounding features. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences ELAVL1 interacts with to stabilize transcripts.
ZFP36-RNA target specificities in vivo are quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to unravel in vivo combinatorial regulation by RNA-binding proteins.
Regulation of gene expression is a complex process coordinated at many steps. Post-transcriptional regulation is controlled through RNA-binding proteins (RBPs) and non-coding RNAs (ncRNAs) interacting with RNA regulatory elements (RREs). Dynamic and combinatorial interactions of RBPs and ncRNAs with these RREs determine the functional outcome of specific steps of RNA processing, such as splicing, polyadenylation, export, stability and translation [1]. Additionally, interactions between RBPs and RREs govern messenger RNA (mRNA) stability and translational efficiency.
AU-rich elements (AREs) are conserved cis-regulatory elements, originally discovered in the 3′ untranslated region (UTR) of labile mRNAs, encoding cytokines and early expressed immune response genes [2, 3]. The most commonly defined AREs are the pentamer AUUUA, which is often found tandemly arranged, and the nonamer UUAUUUAUU, which is a pentamer flanked by uridylates [4–6]. AREs are generally considered instability elements, since they typically interact with RBPs that subsequently recruit mRNA degradation machinery [7, 8]. Examples of ARE-binding proteins include the ELAV family, ZPF36 family, HNRNPD/AUF family, TIA1, TIAR and KSRP. Mice lacking the AU-rich region containing multiple AREs residing in the 3′ UTR of the tumor necrosis factor α (TNFα) transcript develop inflammatory disease, demonstrating their primacy in gene regulatory mechanisms [9].
Tristetraprolin (ZFP36) is the founding member of the TIS11 family of RBPs. It interacts with AREs through tandem CCCH zinc fingers (TZFs) [10]. Mice deficient in ZFP36 also exhibit inflammatory disease phenotypes, mostly explained by deregulation of TNFα mRNA stability and biosynthesis [11]. The interaction of ZFP36 with AREs in the 3′ UTR of targeted mRNAs, such as TNFα, promotes mRNA degradation [12]. ZFP36 is a nucleo-cytoplasmic shuttling protein that is predominantly cytoplasmic at steady state [13]. The RNA-binding specificity of ZFP36 resides in the TZF domain, which interact with low nanomolar affinity with synthetic RNA substrates containing the nonamer UUAUUUAUU [14]. The NMR structure of the nonamer sequence bound to the TZF domain from human ZFP36L2 revealed that the zinc fingers symmetrically bind to UAUU half-sites, with the 5′ uridylate unbound [15]. There have been many detailed in vitro investigations of ZFP36–mRNA interaction (reviewed in [16]); however, in vivo studies have only determined the mRNA pools associated with ZFP36 and have not defined individual binding sites at high resolution [17, 18].
Although many ARE-binding RBPs recruit mRNA degradation complexes, the ELAV/Hu family of proteins binds to AREs and promotes mRNA stability and translation [2, 3, 19–21]. Both in vitro and in vivo studies have determined that Hu proteins interact with AREs, as well as U and CU-rich sequences [4–6, 22, 23]. These sequence preferences were substantiated in vivo using photoactivatable ribonucleoside cross-linking and immunoprecipitation (PAR-CLIP) to define high-resolution interaction sites for the ubiquitously expressed family member ELAVL1 [7, 8, 24–26]. Due to T-to-C transitions indicative of RBP–RNA interaction sites [10, 27], PAR-CLIP gives a higher resolution of binding sites than earlier methods [11, 28, 29]. A commonly postulated mechanism through which ELAVL1 promotes mRNA stability is competition with ARE-binding RBPs that promote degradation. Like ZFP36, ELAVL1 is a nucleo-cytoplasmic shuttling protein, but under normal conditions is predominantly nuclear localized [12, 30], consistent with numerous intronic binding sites found in PAR-CLIP experiments [13, 24–26]. Simultaneous temporal and spatial competition for binding sites by ELAVL1 and ZFP36 is important when responding to stimuli, particularly immune activation, upon which ELAVL1 redistributes from the nucleus to the cytoplasm [14, 31] and ZFP36 is induced and cytoplasmically localized. There is evidence for competitive binding and displacement of ZFP36 with increasing amounts of ELAVL1 and/or MAPK signaling influencing the combinatorial regulation of the TNFα mRNA by ELAVL1 and ZFP36 [15, 32]. However, the extent to which similar competitive mechanisms exist transcriptome-wide is unknown.
Conflated direct and indirect relations are a concern, particularly when integrating multiple genomic measurements to understand complex gene regulatory mechanisms. An example of a relation relevant for post-transcriptional regulation is that longer transcripts contain more binding sites for trans-acting factors. Consequently, significant correlations between 3′ UTR length and miRNA-dependent downregulation have been found in numerous datasets [16, 33]. Overexpression of ZFP36 in the absence of stress has been demonstrated to promote stress granule assembly [18], potentially causing non-ZFP36 specific effects in transcript abundance. Partial correlation analysis quantifies the direct dependence between two variables (for example, number of binding sites or expression changes) when accounting for other variables (for example, UTR length and mRNA abundance). It has recently been applied to investigate genome evolution [34, 35], as well as chromatin state and transcriptional regulation [36]. Here we apply it to account for generic transcript features when inferring functional relations between the number of binding sites and gene expression changes due to perturbing the expression of the RBP.
There are numerous examples of combinatorial binding of RBPs resulting in specific splicing decisions [37–40], but this has not been as extensively studied for mRNA stability. Given the numerous ARE-binding RBPs, the principles underlying the integration of RBP binding and concomitant regulatory outcomes have remained elusive. We therefore determined the in vivo mRNA-binding sites of ZFP36. The interaction sites and sequence preferences were compared to similarly acquired in vivo HuR interaction data, specifically contrasting AU-rich sequences with predominantly U- and CU-rich sequences. Our results indicate active competition between ZFP36 and ELAVL1 for commonly used AU-rich sequences and define the characteristics of these combinatorial regulatory events.
Results and discussion
ZFP36 preferentially binds to 3′ UTRs of mRNAs encoding regulators of gene expression
The majority of ZFP36 mRNA binding sites (70%, 2,314) mapped to the 3′ UTR, and very few binding sites were located at the 5′ UTR or the protein coding region (Figure 1C). Approximately one-quarter of the mRNA binding sites were intronic (871), which is conceivable since ZFP36 is a known nucleo-cytoplasmic shuttling RBP [13]. However, the 3′ UTR sites had a substantially higher T-to-C conversion fraction and specificity relative to ZFP36 sites from other annotation categories including introns, 5′ UTRs and protein coding regions (Additional file 3: Figure S1B). We observed closely spaced ZFP36 sites within 3′ UTRs (Figure 1D), indicative of multimerization of ZFP36 and supported by results from 2-hybrid analysis and immunoreactive bands migrating at twice the expected number of kilodaltons of ZFP36 in cell extracts (PJB, unpublished data). Of the 1,148 transcripts with more than one ZFP36 site in the 3′ UTR, 228 had two sites within 70 nucleotides, the range for which proximity of ZFP36 sites is above background. The statistically significant enrichment for ZFP36 sites in the last 100 nucleotides of the 3′ UTR (Figure 1E) was intriguing given its ability to promote deadenylation [43]. This spatial bias was not observed for the sites of all other investigated 3′ UTR binding RBPs (Additional file 3: Figure S1D) and thus not due to biases in library generation, though it may be related to AU-richness proximal to polyadenylation sites [44]. Of the 2,143 genes with ZFP36 sites, 64% exclusively had 3′ UTR sites, 23% exclusively had intronic sites and only 8% contained both 3′ UTR and intronic sites (Figure 1F). Genes with 3′ UTR sites were less likely to also have intronic sites in contrast to ELAVL1 [25]. The strong preference for 3′ UTR binding was anticipated for an RBP predominantly involved in cytoplasmic regulation of mRNA stability and translation.
In vivoZFP36 binding specificity
Previously reported in vitro ZFP36 binding studies quantified the binding of numerous RNA probes to the 73 amino acid portion of ZFP36 containing the TZF using fluorescence anisotropy measurements [48]. For each probe, we compared the experimentally determined association, K, and disassociation constant, K d , with the SNR values calculated from our ZFP36 PAR-CLIP data matching experimental RNA probe and in vivo binding site sequences. We observed a very strong correlation with K d values (Figure 3C). This strong concordance allowed us to calculate predicted K d values from the PAR-CLIP derived SNR ratios and thus perform in silico experiments with specific sequences. The relation between SNR and K d was non-linear. For example, larger differences between the SNR values for the nonamer (UUAUUUAUU) and the octamer (UUAUUUAU) translated to smaller differences in the predicted K d values for high affinity targets (Figure 3D). When inserting a spacer in the octamer between the UUAU half-sites bound by the TZF domain, a single nucleotide disruption by any nucleotide resulted in similar increases in predicted K d values. However, multiple continuous disruptions with non-uridylates exhibit quite large increases in predicted K d values, whereas disruptions with uridylates had little to no change (Figure 3D). These results highlight the quality and complementary value of the PAR-CLIP method, as well as the detailed in vitro biochemical analysis of ZFP36 substrate specificity.
Specificity of ZFP36 function
To evaluate the regulatory function of ZFP36, we used microarray analysis to compare transcript abundance before and after doxycycline-induction of EGFP-ZFP36 fusion protein expression in HEK293 cells otherwise expressing little endogenous ZFP36 (Additional file 4: Figure S2A,B and Additional file 5: Table S2). We detected 2,784 genes with statistically significant expression differences by comparing overnight doxycycline-induced cells to mock-induced cells. Doxycycline treatment of the parental cell line did not yield any statistically significant differences defined by Bonferroni corrected P < 0.01 (Additional file 4: Figure S2C). Only 45% of the differentially expressed genes decreased as a result of ZFP36 overexpression (downregulated genes). The large number of upregulated genes was likely due to secondary effects from prolonged ZFP36 overexpression (Additional file 4: Figure S2D). (Detailed characterization of genes with ZFP36 and ELAVL1 sites with numerous other motif contributions for significantly upregulated and downregulated genes are shown in Additional file 4: Figure S2H.) The 1,254 significantly downregulated genes decreased by approximately 3.5 fold on average. The breadth and degree of expression differences resulting from EGFP-ZFP36 overexpression were distinctly greater than the typical fine-tuning associated with most RBPs and miRNAs. This is particularly striking in comparison to ELAVL1 where only 15 genes exhibited greater than a twofold change due to ELAVL1 knockdown (Additional file 4: Figure S2G from [25]).
Downregulated genes were significantly enriched for AU-rich sequences in an unbiased analysis of the contribution of 3′ UTR motif elements to expression changes (Additional file 4: Figure S2E) [50]. Thus, we examined the association between the number of counts per 3′ UTR of specific AREs and EGFP-ZFP36-induced changes in mRNA abundance. The RREs described by the ZFP36L2:ARE NMR structure, the UAUUUAUU octamer and the UAUU half-site, had the strongest association with ZFP36-induced changes in mRNA abundance. The UAUUUAUU octamer had a stronger association with ZFP36-induced changes in mRNA abundance than the UUAUUUAUU nonamer (Additional file 4: Figure S2F). The UAUU half-site had a stronger association with ZFP36-induced changes in mRNA abundance than the AUUUA pentamer (Additional file 4: Figure S2F).
To illustrate functional effects, genes are frequently stratified by a variable of interest (such as the presence or absence or number of binding sites), followed by a statistical test for significant phenotypic differences (such as expression changes) between the subsets. For instance, we observed strong associations between the number of ZFP36 sites in the 3′ UTR of a gene and its downregulation due to EGFP-ZFP36 overexpression (Figure 4B), between 3′ UTR length and EGFP-ZFP36-induced changes in mRNA abundance (Figure 4C), and between ZFP36 sites and 3′ UTR length. These effects were corroborated by the strength of Spearman correlations between all three variables (Figure 4D, left). However, pairwise correlations are not able to quantify direct and indirect relations for more than two variables and thus we employed partial correlation analysis. Comparing the correlation and partial correlation of ZFP36 sites and 3′ UTR length with EGFP-ZFP36-induced changes in mRNA abundance, most of the relation between ZFP36 sites and EGFP-ZFP36-induced changes in mRNA abundance was independent of 3′ UTR length and vice versa (Figure 4D). Secondary effects from prolonged ZFP36 overexpression, such as spontaneous stress granule assembly [52] and changes in the expression levels of other RBPs, were potential explanations for the independent contribution of 3′ UTR length to EGFP-ZFP36-induced changes in mRNA abundance.
We determined which transcript regions contained the most independent information for explaining EGFP-ZFP36-induced changes in mRNA abundance, without having to lose statistical power by separating transcripts into mutually exclusive categories, such as binding within introns only or 3′ UTRs only. EGFP-ZFP36-induced changes in mRNA abundance correlated negatively with the number of ZFP36 3′ UTR sites and positively with the number of intronic ZFP36 sites (Figure 4E, left). Partial correlation analysis revealed that ZFP36 sites outside of 3′UTRs, including introns, did not contribute any additional independent explanation of EGFP-ZFP36-induced changes in mRNA abundance (Figure 4E, right). Here, the strong negative association or avoidance between the number of intronic and 3′ UTR sites per gene (like the results in Figure 1F) acts as a mediator in explaining the spurious effects between intronic ZFP36 sites and EGFP-ZFP36-induced changes in mRNA abundance. This result does not rule out that ZFP36 sites from other transcript regions have other functional effects, but we only utilized 3′ UTR binding sites for further analysis.
We compared our ZFP36 PAR-CLIP and overexpression data from human HEK293 cells with global mRNA stability changes after serum stimulation for ZFP36 wild-type (WT) or knockout (KO) mouse fibroblast cell lines [53]. Genes with increased mRNA half-lives in the ZFP36 KO vs WT mouse cells were significantly enriched for genes with ZFP36 PAR-CLIP sites in 3′ UTR (P = 0.003) and genes significantly downregulated upon EGFP-ZFP36 overexpression (P = 0.001), but were not significantly enriched for genes significantly upregulated upon EGFP-ZFP36 overexpression (P = 0.1) (Additional file 6: Figure S3A). Interestingly, ELAVL1 RIP-chip targets from human Jurkat T-cell activation were significantly enriched in genes with increased mRNA half-lives in the ZFP36 KO vs WT mouse cells, even though ZFP36 RIP-chip targets from activated mouse macrophages were slightly but not significantly enriched [18]. Genes with ELAVL1 sites in the 3′ UTRs were similarly enriched, but genes without ELAVL1 or ZFP36 sites in their 3′ UTR did not have any enrichment. These results provide independent validation of the ZFP36 bound and regulated genes in our study using a system not reliant on ectopic overexpression of ZFP36. The enrichment of ELAVL1 gene is further support for the potential of ELAVL1 to effect expression of ZFP36 targets.
Combinatorial regulation of mRNAs with AREs by ZFP36 and ELAVL1
To assess specific sequences within ELAVL1-bound sites and the strength of their contribution to the EGFP-ZFP36-induced changes in mRNA abundance, we performed the same analysis using ELAVL1 sites containing CUUUC, UUUUU or AUUUA. The AUUUA ELAVL1 sites (−0.21) accounted for more EGFP-ZFP36-induced changes in mRNA abundance than the CUUUC ELAVL1 sites (−0.07) (Figure 5C,D). This result was consistent with the observation that ELAVL1 specific sites were characterized by predominantly U- and CU-rich sequences, while overlapping ELAVL1 and ZFP36 sites were characterized more by AU-rich sequences (Figure 5E). The exact same analysis using an independently derived ELAVL1 HEK293 PAR-CLIP library yielded similar results (Additional file 7: Figure S4D-F), supporting the robustness of our findings. EGFP-ZFP36-induced changes in mRNA abundance were better explained by the ARE nonamer and octamer compared to U8 or U9. ELAVL1-knockdown-responsive mRNAs had the reciprocal pattern (Additional file 6: Figure S3B). Finally, transcripts containing 3′ UTR sites for both ELAVL1 and ZFP36 had a greater concomitant decrease upon ELAVL1 knockdown and decrease upon ZFP36 overexpression, relative to transcripts containing 3′ UTR sites for only ELAVL1 or ZFP36 or no sites (Figure 5F, shifted down and to the right). Taken together, there is substantial potential for ZFP36 and ELAVL1 to compete for the same pool of binding sites, while exerting a more targeted influence on a subset of sites with factor-specific sequence preferences.
Our global in vivo ZFP36–mRNA interaction experiments support previously reported biochemical in vitro ZFP36 affinity measurements, demonstrating the remarkable precision and quantitative characteristics of ZFP36 PAR-CLIP results. ZFP36 targets identified in this study were enriched for ZFP36-dependent changes in mRNA stability in mouse fibroblast cells. ZFP36 interacted with mRNAs encoding proteins important for the immune response and cancer. Many of these are other RBPs central to these two biological processes, specifically other ARE-binding RBPs such as the positive-stability regulator ELAVL1. There was extensive overlap between the interaction sites of ZFP36 and ELAVL1. Over 80% of ZFP36 sites in 3′ UTRs overlapped with ELAVL1 target sites. The preference for ZFP36 and ELAVL1 to bind and regulate mRNAs encoding RBPs suggests a complex interconnected network of post-transcriptional regulation that needs to be reconciled with respect to known ZFP36 and ELAVL1 protein expression, localization and modification dynamics, particularly in response to immune activation [54].
Partial correlation analysis accounting for confounding features demonstrated that a higher number of ZFP36 sites corresponded to stronger downregulation upon EGFP-ZFP36-induced changes in mRNA abundance. While the two proteins bind AREs, ZFP36 clearly shows a preferences towards AU- vs simply U-rich or CU-rich sequences relative to ELAVL1. This was corroborated at the level of mRNA stability, since ELAVL1 sites containing AUUUA were best able to explain EGFP-ZFP36-induced changes in mRNA abundance.
In summary, this study provides a resource for the precise transcriptome-wide RNA interactions of two ARE-binding RBPs with antagonistic functions. The precise sequence definition and function of AREs are an important question that will greatly benefit from global interaction data for these and other ARE-binding RBPs. Furthermore, we have integrated multiple genome-wide assays with computational analyses to advance our understanding of the sequence basis and extent of combinatorial gene regulation at the RNA level, in particular the ARE-mediated regulation by two of the most well-characterized RBPs.
Materials and methods
Cell lines
Doxycycline-inducible FLAG/HA-tagged ZFP36 HEK293 cell lines were produced utilizing the Flp-In™ T-REx™ 293 Cell Line system (Life Technologies, USA).
Flp-In™ T-REx™ HEK293 cells were generated with a FLAG/HA-tagged human ZFP36 or an EGFP-tagged human ZFP36. Both cell lines were cultured in DMEM supplemented with 10% tetracycline-reduced fetal bovine serum and appropriate selection antibiotics. For induction of FLAG/HA-tagged or EGFP-tagged ZFP36 protein, cells were treated for 16 hr with 1 μg/ml doxycycline (Sigma, USA).
The protocol was performed as detailed in [41] for a FLAG-HA-tagged ZFP36 and sequenced using an Illumina platform. The primary dataset comprised previously derived ELAVL1 PAR-CLIP and ELAVL1 knockdown data [25]. This was integrated with ZFP36 PAR-CLIP data. An independent ELAVL1 PAR-CLIP dataset for HEK293 cells was also used to substantiate our results. Specifically, we used the union of all ELAVL1 T1 PAR-CLIP libraries. PUM2, QKI and FMR1 HEK293 PAR-CLIP libraries were investigated to compare spatial binding biases in 3′ UTRs [55, 56]. Precise analysis parameters for the PAR-CLIP libraries above are provided in Additional file 1. Binding site coordinates for ELAVL1 HEK293 CLIP and HeLa PAR-CLIP were downloaded from doRiNA [57].
Briefly, reads from the ZFP36 and ELAVL1 deep-sequencing library were stripped of the adaptor sequence using Cutadapt [58]. Reads that were less than 20 nucleotides in length or contained an ambiguous nucleotide were discarded. The remaining reads were aligned to the human genome (hg19), with up to one mismatch allowed and ten alignment locations, with Bowtie version 0.12.7 [59]. Reads with T-to-C mismatches were reconciled [42] to retain only those that mapped to a single genomic location for the minimum number of mismatches. Annotation was performed as described in [56]. Full site- and gene-level data for the primary data compared are in Additional file 2: Table S1 and Additional file 5: Table S2, respectively.
Motif affinity
The SNR values were calculated for each probe sequence in Table one in [48] appearing at least once in 3′ UTR ZFP36 binding sites. The SNR was calculated as in [42], and is defined as the number of instances per nucleotide in a given set of PARalyzer interaction sites, divided by the number of instances per nucleotide in the background set. In this case the background was the sequence of the longest annotated 3′ UTR for a gene. The SNR values were plotted against the K and K d values from [48]. The formula for the power curve fit of the SNR to K d was used to predict K d values for other sequences.
Microarray samples and analysis
EGFP-ZFP36 cells were passaged at 1:10 dilution in 15 ml of media in ten T75 flasks. They were allowed to grow for three days to 60% confluence. They were then treated with doxycycline (Sigma D989) to a final concentration of 1 μg/ml. The same volume of sterile water was used as a control. The cells were incubated for 16 hr at 37°C, in 5% CO2. The flasks without doxycycline were about 80% confluent. Cells with doxycycline were about 40% confluent and there were more floating cells and rounded cells. The cells were harvested by trypsinizing. Cells from five flasks per treatment were pooled. They were centrifuged at 500 g for 5 min, and resuspended in growth medium to final concentrations of 1 × 107 cells/ml. Fluorescence-activated cell sorting (FACS) was done using a FACSAria II from Becton Dickinson (San Jose, CA). Cells were excited at 488 nm and sorted based on their GFP fluorescence at 525/550 nm.
The FacsDiva software version 6.1.3 was used for analysis. Doublet discrimination gates were set on forward scatter and side scatter to ensure the sorting of single cells. The sort gate was set on a histogram of GFP + cells for a fluorescence signal of greater than 103 log units. Control cells were sorted in the same way, except that fluorescent cells were excluded (Additional file 3: Figure S1B). The sorted cells were collected into DMEM with 10% FBS in 6 ml polypropylene tubes at approximately 2 × 106 cells per tube. They were pelleted at 500 g for 20 min at 4°C and washed in PBS, then transferred into 1.5 microfuge tubes at approximately 4 × 106 cells per tube. These were centrifuged at 500g for 20 to 30 min at 4°C. The supernatant was removed, and the pellets were snap frozen in liquid nitrogen and stored at −80°C. This experiment was repeated on five independent days, using cells from passages 17, 20, 23, 26 and 27. RNA was isolated from approximately 3 × 106 to 6 × 106 cells using the GE Illustra RNAspin mini kit. To control for doxycycline effects, parental HEK293 cells were passaged at 1:4 or 1:5 dilution in 15 ml medium in four T75 flasks. They grew for 2 days to approximately 60% confluence. They were then treated with doxycycline in the same way, that is, 1 μg/ml for 16 hr at 37°C in 5% CO2. Control cells were treated with the same volume of sterile water. The cells were harvested by trypsinizing, and processed and used for the microarray in the same way as for the GFP + cells. The same experiment was performed on five different days, and comparisons were made using five doxycycline treated vs five vehicle (water) treated samples, with each experiment done as pairs of treated and untreated cells. The samples were stored at −80°C. The integrity of the RNA was confirmed by running 3 μg of each RNA sample on 1.2% agarose/formaldehyde gels.
Then 1.5 μg of RNA from each sample was provided to the NIEHS Microarray Core Facility for analysis, using an Affymetrix Human Genome U133 Plus 2.0 gene chip (Affymetrix, Santa Clara, CA). As directed in the Affymetrix 3′ IVT Express kit protocol, 100 ng of total RNA was amplified. Then 12.5 μg of amplified biotin-RNA was fragmented and 10 μg were hybridized to each array for 16 hr at 45°C in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip Hybridization, Wash and Stain Kit and user manual. Arrays were scanned in an Affymetrix Scanner 3000 and data were obtained using the GeneChip® Command Console Software (AGCC; Version 1.1). The data were normalized using gcrma [60] and then filtered to retain only unambiguously mapping probes and minimal expression level (mean log hybridization value for replicates within a group > 4.5). Bonferroni corrected P values for fold changes were calculated using Gene Pattern [61]. Gene set enrichment analysis [62] was used to compare HEK293 data to ZFP36 mouse fibroblast mRNA stability data. Human–mouse mappings were done using an appropriate microarray chip. The probes were ranked by differential mRNA half-life score, with higher scores indicating a larger slope in ZFP36 KO than ZFP36 WT cells (for details refer to [53]).
Partial correlation
Analysis of spatial patterns
To visualize binding along transcripts, cluster data was taken from PARalyzer output and annotated. Only 3′ UTR, 5′ UTR, coding and intron annotation categories were utilized. The transcript segment associated with the annotation category of each cluster was selected using a GENCODE version 18 gene transfer format file [63]. For each gene, we used the transcript that had the longest segment matching the binding site of the cluster. The location and relevant distance along the transcript segment were then calculated. For each of the relevant annotation categories, a density plot was created utilizing each of these distances. Permutations of the observed data were used to create the background data distributions. For each cluster we created a random site along the length of its associated transcript segment repeated 100 times. The cluster plot function in the R LSD package [64], was used to plot the observed and permuted data. The LSD cluster plot function creates a graph in which the black center line is the median, and the thick gray lines separating the color gradients around the black line represent the upper and lower quartiles. The color gradient continues until the 10th and 90th percentiles.
Data availability
Both ZFP36 PAR-CLIP sequencing and overexpression microarray datasets have been deposited into GEO [GEO:GSE53185].
AU-rich element
Dulbecco's modified Eagle's medium
green fluorescent protein
- miRNA:
- ncRNA:
non-coding RNA
nuclear magnetic resonance
photoactivatable ribonucleoside cross-linking and immunoprecipitation
phosphate-buffered saline
RNA-binding protein
RNA regulatory element
- siRNA:
small interfering RNA
signal-to-noise ratio
tumor necrosis factor
tandem CCCH zinc finger
untranslated region
wild type.
TT and UO acknowledge support from an award by the US National Institutes of Health (R01-GM104962). NM is supported by EU Marie Curie IIF (EU). PJB acknowledges support from the Intramural Research Program of the NIH, NIEHS. We would like to acknowledge Jack Keene for very insightful early discussions and suggestions.
Authors' Affiliations
- Keene JD: RNA regulons: coordination of post-transcriptional events. Nat Rev Genet. 2007, 8: 533-543. 10.1038/nrg2111.View ArticleGoogle Scholar
- Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A: Identification of a common nucleotide sequence in the 3′-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci USA. 1986, 83: 1670-1674. 10.1073/pnas.83.6.1670.View ArticleGoogle Scholar
- Shaw G, Kamen R: A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986, 46: 659-667. 10.1016/0092-8674(86)90341-7.View ArticleGoogle Scholar
- Xu N, Chen CY, Shyu AB: Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay. Mol Cell Biol. 1997, 17: 4611-4621.View ArticleGoogle Scholar
- Zubiaga AM, Belasco JG, Greenberg ME: The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Biol. 1995, 15: 2219-2230.View ArticleGoogle Scholar
- Bakheet T, Williams BRG, Khabar KSA: ARED 3.0: the large and diverse AU-rich transcriptome. Nucleic Acids Res. 2006, 34: D111-D114. 10.1093/nar/gkj052.View ArticleGoogle Scholar
- Shyu AB, Belasco JG, Greenberg ME: Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 1991, 5: 221-231. 10.1101/gad.5.2.221.View ArticleGoogle Scholar
- Chen CY, Gherzi R, Ong SE, Chan EL, Raijmakers R, Pruijn GJ, Stoecklin G, Moroni C, Mann M, Karin M: AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell. 2001, 107: 451-464. 10.1016/S0092-8674(01)00578-5.View ArticleGoogle Scholar
- Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G: Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements. Cell. 1999, 10: 387-398.Google Scholar
- Lai WS, Stumpo DJ, Blackshear PJ: Rapid insulin-stimulated accumulation of an mRNA encoding a proline-rich protein. J Biol Chem. 1990, 265: 16556-16563.Google Scholar
- Taylor GA, Carballo E, Lee DM, Lai WS, Thompson MJ, Patel DD, Schenkman DI, Gilkeson GS, Broxmeyer HE, Haynes BF, Blackshear PJ: A pathogenetic role for TNFα in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Cell. 1996, 4: 445-454.Google Scholar
- Carballo E: Feedback inhibition of macrophage tumor necrosis factor-α production by tristetraprolin. Science. 1998, 281: 1001-1005.View ArticleGoogle Scholar
- Phillips RS: Members of the tristetraprolin family of tandem CCCH zinc finger proteins exhibit CRM1-dependent nucleocytoplasmic shuttling. J Biol Chem. 2002, 277: 11606-11613. 10.1074/jbc.M111457200.View ArticleGoogle Scholar
- Blackshear PJ, Lai WS, Kennington EA, Brewer G, Wilson GM, Guan X, Zhou P: Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates. J Biol Chem. 2003, 278: 19947-19955. 10.1074/jbc.M301290200.View ArticleGoogle Scholar
- Hudson BP, Martinez-Yamout MA, Dyson HJ, Wright PE: Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d. Nat Struct Mol Biol. 2004, 11: 257-264. 10.1038/nsmb738.View ArticleGoogle Scholar
- Brooks SA, Blackshear PJ: Tristetraprolin (TTP): interactions with mRNA and proteins, and current thoughts on mechanisms of action. Biochim Biophys Acta (BBA) – Gene Regul Mech. 2013, 1829: 666-679. 10.1016/j.bbagrm.2013.02.003.View ArticleGoogle Scholar
- Emmons J, Townley-Tilson WHD, Deleault KM, Skinner SJ, Gross RH, Whitfield ML, Brooks SA: Identification of TTP mRNA targets in human dendritic cells reveals TTP as a critical regulator of dendritic cell maturation. RNA. 2008, 14: 888-902. 10.1261/rna.748408.View ArticleGoogle Scholar
- Stoecklin G, Tenenbaum SA, Mayo T, Chittur SV, George AD, Baroni TE, Blackshear PJ, Anderson P: Genome-wide analysis identifies interleukin-10 mRNA as target of tristetraprolin. J Biol Chem. 2008, 283: 11689-11699. 10.1074/jbc.M709657200.View ArticleGoogle Scholar
- Jain RG, Andrews LG, McGowan KM, Pekala PH, Keene JD: Ectopic expression of Hel-N1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3 T3-L1 adipocytes. Mol Cell Biol. 1997, 17: 954-962.View ArticleGoogle Scholar
- Fan XC, Myer VE, Steitz JA: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation. Genes Dev. 1997, 11: 2557-2568. 10.1101/gad.11.19.2557.View ArticleGoogle Scholar
- Peng SS, Chen CY, Xu N, Shyu AB: RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 1998, 17: 3461-3470. 10.1093/emboj/17.12.3461.View ArticleGoogle Scholar
- Levine TD, Gao F, King PH, Andrews LG, Keene JD: Hel-N1: an autoimmune RNA-binding protein with specificity for 3′ uridylate-rich untranslated regions of growth factor mRNAs. Mol Cell Biol. 1993, 13: 3494-3504.View ArticleGoogle Scholar
- Tenenbaum SA, Carson CC, Lager PJ, Keene JD: Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci USA. 2000, 97: 14085-14090. 10.1073/pnas.97.26.14085.View ArticleGoogle Scholar
- Lebedeva S, Jens M, Theil K, Schwanhäusser B, Selbach M, Landthaler M, Rajewsky N: Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR. Mol Cell. 2011, 43: 340-352. 10.1016/j.molcel.2011.06.008.View ArticleGoogle Scholar
- Mukherjee N, Corcoran DL, Nusbaum JD, Reid DW, Georgiev S, Hafner M, Ascano M, Tuschl T, Ohler U, Keene JD: Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability. Mol Cell. 2011, 43: 327-329. 10.1016/j.molcel.2011.06.007.View ArticleGoogle Scholar
- Kishore S, Jaskiewicz L, Burger L, Hausser J, Khorshid M, Zavolan M: A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nat Meth. 2011, 8: 559-564. 10.1038/nmeth.1608.View ArticleGoogle Scholar
- Hafner M, Max KEA, Bandaru P, Morozov P, Gerstberger S, Brown M, Molina H, Tuschl T: Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition. RNA. 2013, 19: 613-626. 10.1261/rna.036491.112.View ArticleGoogle Scholar
- Natarajan A, Yardımcı GGR, Sheffield NC, Crawford GE, Ohler U: Predicting cell-type–specific gene expression from regions of open chromatin. Genome Res. 2012, 22: 1711-1722. 10.1101/gr.135129.111.View ArticleGoogle Scholar
- Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB: CLIP identifies Nova-regulated RNA networks in the brain. Science. 2003, 302: 1212-1215. 10.1126/science.1090095.View ArticleGoogle Scholar
- Fan XC, Steitz JA: HNS, a nuclear-cytoplasmic shuttling sequence in HuR. Proc Natl Acad Sci USA. 1998, 95: 15293-15298. 10.1073/pnas.95.26.15293.View ArticleGoogle Scholar
- Atasoy U, Watson J, Patel D, Keene JD: ELAV protein HuA (HuR) can redistribute between nucleus and cytoplasm and is upregulated during serum stimulation and T cell activation. J Cell Sci. 1998, 111: 3145-3156.Google Scholar
- Tiedje C, Ronkina N, Tehrani M, Dhamija S, Laass K, Holtmann H, Kotlyarov A, Gaestel M: The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU–rich element–dependent translation. PLoS Genet. 2012, 8: e1002977-10.1371/journal.pgen.1002977.View ArticleGoogle Scholar
- Arvey A, Larsson E, Sander C, Leslie CS, Marks DS: Target mRNA abundance dilutes microRNA and siRNA activity. Mol Syst Biol. 2010, 6: [http://www.nature.com/msb/journal/v6/n1/full/msb201024.html]Google Scholar
- Freudenberg J, Wang M, Yang Y, Li W: Partial correlation analysis indicates causal relationships between GC-content, exon density and recombination rate in the human genome. BMC Bioinformatics. 2009, 10: S66-10.1186/1471-2105-10-S1-S66.View ArticleGoogle Scholar
- Kim SH: Correlated asymmetry of sequence and functional divergence between duplicate proteins of saccharomyces cerevisiae. Mol Biol Evol. 2006, 23: 1068-1075. 10.1093/molbev/msj115.View ArticleGoogle Scholar
- Gat-Viks I, Vingron M: Evidence for gene-specific rather than transcription rate–dependent histone H3 exchange in yeast coding regions. PLoS Comp Biol. 2009, 5: e1000282-10.1371/journal.pcbi.1000282.View ArticleGoogle Scholar
- Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, Blencowe BJ, Frey BJ: Deciphering the splicing code. Nature. 2010, 465: 53-59. 10.1038/nature09000.View ArticleGoogle Scholar
- Pandit S, Zhou Y, Shiue L, Coutinho-Mansfield G, Li H, Qiu J, Huang J, Yeo GW, Ares M, Fu X-D: Genome-wide analysis reveals SR protein cooperation and competition in regulated splicing. Mol Cell. 2013, 50: 223-235. 10.1016/j.molcel.2013.03.001.View ArticleGoogle Scholar
- Zarnack K, König J, Tajnik M, Martincorena I, Eustermann S, Stévant I, Reyes A, Anders S, Luscombe NM, Ule J: Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell. 2013, 152: 453-466. 10.1016/j.cell.2012.12.023.View ArticleGoogle Scholar
- Zhang C, Frias MA, Mele A, Ruggiu M, Eom T, Marney CB, Wang H, Licatalosi DD, Fak JJ, Darnell RB: Integrative modeling defines the Nova splicing-regulatory network and its combinatorial controls. Science. 2010, 329: 439-443. 10.1126/science.1191150.View ArticleGoogle Scholar
- Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M, Jungkamp A-C, Munschauer M, Ulrich A, Wardle GS, Dewell S, Zavolan M, Tuschl T: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell. 2010, 141: 129-141. 10.1016/j.cell.2010.03.009.View ArticleGoogle Scholar
- Corcoran DL, Georgiev S, Mukherjee N, Gottwein E, Skalsky RL, Keene JD, Ohler U: PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol. 2011, 12: R79-10.1186/gb-2011-12-8-r79.View ArticleGoogle Scholar
- Fabian MR, Frank F, Rouya C, Siddiqui N, Lai WS, Karetnikov A, Blackshear PJ, Nagar B, Sonenberg N: Structural basis for the recruitment of the human CCR4–NOT deadenylase complex by tristetraprolin. Nat Struct Mol Biol. 2013, 20: 735-739. 10.1038/nsmb.2572.View ArticleGoogle Scholar
- Pesole G, Liuni S, Grillo G, Saccone C: Structural and compositional features of untranslated regions of eukaryotic mRNAs. Gene. 1997, 205: 95-102. 10.1016/S0378-1119(97)00407-1.View ArticleGoogle Scholar
- Leppek K, Schott J, Reitter S, Poetz F, Hammond MC, Stoecklin G: Roquin promotes constitutive mRNA decay via a conserved class of stem-loop recognition motifs. Cell. 2013, 153: 869-881. 10.1016/j.cell.2013.04.016.View ArticleGoogle Scholar
- Hodson DJ, Janas ML, Galloway A, Bell SE, Andrews S, Li CM, Pannell R, Siebel CW, MacDonald HR, De Keersmaecker K, Ferrando AA, Grutz G, Turner M: Deletion of the RNA-binding proteins ZFP36L1 and ZFP36L2 leads to perturbed thymic development and T lymphoblastic leukemia. Nat Immunol. 2010, 11: 717-724. 10.1038/ni.1901.View ArticleGoogle Scholar
- Papadaki O, Milatos S, Grammenoudi S, Mukherjee N, Keene JD, Kontoyiannis DL: Control of thymic T cell maturation, deletion and egress by the RNA-binding protein HuR. J Immunol. 2009, 182: 6779-6788. 10.4049/jimmunol.0900377.View ArticleGoogle Scholar
- Brewer BY: RNA sequence elements required for high affinity binding by the zinc finger domain of tristetraprolin: conformational changes coupled to the bipartite nature of AU-rich mRNA-destabilizing motifs. J Biol Chem. 2004, 279: 27870-27877. 10.1074/jbc.M402551200.View ArticleGoogle Scholar
- Meisner N-C, Hackermüller J, Uhl V, Aszódi A, Jaritz M, Auer M: mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure. ChemBioChem. 2004, 5: 1432-1447. 10.1002/cbic.200400219.View ArticleGoogle Scholar
- Sood P: Cell-type-specific signatures of microRNAs on target mRNA expression. Proc Natl Acad Sci USA. 2006, 103: 2746-2751. 10.1073/pnas.0511045103.View ArticleGoogle Scholar
- Rounbehler RJ, Fallahi M, Yang C, Steeves MA, Li W, Doherty JR, Schaub FX, Sanduja S, Dixon DA, Blackshear PJ, Cleveland JL: Tristetraprolin impairs Myc-induced lymphoma and abolishes the malignant state. Cell. 2012, 150: 563-574. 10.1016/j.cell.2012.06.033.View ArticleGoogle Scholar
- Stoecklin G, Stubbs T, Kedersha N, Wax S, Rigby WF, Blackwell TK, Anderson P: MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay. Nat Struct Mol Biol. 2004, 23: 1313-1324.Google Scholar
- Lai WS, Parker JS, Grissom SF, Stumpo DJ, Blackshear PJ: Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts. Mol Cell Biol. 2006, 26: 9196-9208. 10.1128/MCB.00945-06.View ArticleGoogle Scholar
- Kratochvill F, Machacek C, Vogl C, Ebner F, Sedlyarov V, Gruber AR, Hartweger H, Vielnascher R, Karaghiosoff M, Rülicke T, Müller M, Hofacker I, Lang R, Kovarik P: Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation. Mol Syst Biol. 2011, 7: [http://www.nature.com/msb/journal/v7/n1/synopsis/msb201193.html]Google Scholar
- Hafner M, Lianoglou S, Tuschl T, Betel D: Genome-wide identification of miRNA targets by PAR-CLIP. Methods. 2012, 58: 94-105. 10.1016/j.ymeth.2012.08.006.View ArticleGoogle Scholar
- Ascano M, Mukherjee N, Bandaru P, Miller JB, Nusbaum JD, Corcoran DL, Langlois C, Munschauer M, Dewell S, Hafner M, Williams Z, Ohler U, Tuschl T: FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature. 2012, 492: 382-386. 10.1038/nature11737.View ArticleGoogle Scholar
- Anders G, Mackowiak SD, Jens M, Maaskola J, Kuntzagk A, Rajewsky N, Landthaler M, Dieterich C: doRiNA: a database of RNA interactions in post-transcriptional regulation. Nucleic Acids Res. 2012, 40: D180-D186. 10.1093/nar/gkr1007.View ArticleGoogle Scholar
- Martin M: Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011, 17: 10-12.View ArticleGoogle Scholar
- Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10: R25-10.1186/gb-2009-10-3-r25.View ArticleGoogle Scholar
- Wu Z, Irizarry RA, Gentleman R, Murillo FM, Spencer F: A model based background adjustment for oligonucleotide expression arrays. J Am Stat Assoc. 2004, 99: 909-10.1198/016214504000000683.View ArticleGoogle Scholar
- Gould J, Getz G, Monti S, Reich M, Mesirov JP: Comparative gene marker selection suite. Bioinformatics. 2006, 22: 1924-1925. 10.1093/bioinformatics/btl196.View ArticleGoogle Scholar
- Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005, 102: 15545-15550. 10.1073/pnas.0506580102.View ArticleGoogle Scholar
- GENCODE project. [http://www.gencodegenes.org/releases/18.html]
- LSD: Lots of Superior Depictions. [http://CRAN.R-project.org/package=LSD]
- Mukherjee N, Lager PJ, Friedersdorf MB, Thompson MA, Keene JD: Coordinated posttranscriptional mRNA population dynamics during T-cell activation. Mol Syst Biol. 2009, 5: 288-View ArticleGoogle Scholar
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Ottilio Valentino Grassi had been born on 10 May 1723 in Udine, son of Valentino Grassi (1680-1762) and his wife Livia. In 1742 Ottilio Grassi began his six-year apprenticeship in Vienna. In 1760 he was appointed master goldsmith and in 1761 he took the oath as "Wiener Bürger" (Viennese Citizen). His first son Anton Mathias was born on 26 June 1755 in the "Blumenthalisches Haus" am Hof (Stadt No. 310, today Am Hof 6a) and baptized in the Schottenkirche with his maternal grandparents officiating as godparents:
(A-Ws, Tom. 34, fol. 247v)
Ottilio Grassi's next child, Joseph Mathias, was born on 22 April 1757 and like his older brother he got his middle name from his grandfather (and godfather), the goldsmith Mathias Winterhalter (1696-1763):
Consequently Ottilio Grassi's two daughters got their middle name from their maternal grandmother Margaretha Winterhalter, née Poser (1715-1775): Elisabetha Margaretha Grassi was born on 29 October 1759 and Eleonora Margaretha on 24 September 1761. The next child of the Grassi family was Johann Gottfried Grassi, born on 12 January 1766, who was named after his godfather, the K.K. Hof Cammer Registraturs Adjunct Johann Gottfried Anhalt (who in 1754 had been Ottilio Grassi's best man). The last child of the Grassi family was born on 7 April 1769 and christened Johann Christoph after his godfather, the K.K. Hofkammer Concipist Christoph von Keßler. Keßler was a legendary freemason who in 1782 seems to have been responsible for Joseph and Anton Grassi joining the Viennese Masonic lodge "Zur Beständigkeit".


text: My 14yo begs to eat at Jersey Mike's, so when we saw this one, we had to stop. Their subs are very fresh and oh so good. We had mini subs which are 5-1/2". I had the chicken salad which was very good. It is a little pricey. My only real complaint is that they serve Pepsi products, rather than... More


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This is the November 16, 2012 news archive. View today's Packers rumors and news.
Green Bay Packers
Green Bay Packers News and Rumors
James Jones' past drops wind up working out for Packers
profootballtalk.com Updated Nov 16, 2012
Packers wide receiver James Jones hasn't dropped a ball yet this season. That's worthy of noting for a player who led the NFL in drop percentage over the last three seasons. Plenty of those drops caused frustration in Green Bay, but there's not muchShare Tweet
Final Word: NFC NorthESPN.com Nov 16, 2012Five nuggets of knowledge about Week 11: History rules: The Green Bay Packers have won 12 of their past 13 games against the Detroit Lions and 14 of their past 16, dating to the start of the 2004 season. Their most recent loss came in 2010, a game quart…Share Tweet
60 Minutes says Rodgers' reaction proves that he's sensitiveprofootballtalk.com Nov 16, 2012The ticking stopwatch finally struck "go" time. Last week, Packers quarterback Aaron Rodgers expressed disappointment regarding the manner in which he was portrayed in a 60 Minutes profile. On Thursday, Rodgers reiterated his concerns. In responseShare Tweet
'Playbook': Packers vs. LionsNov 16Will the Detroit Lions defense be able to slow down Aaron Rodgers and the Green Bay Packers? "Playbook" explains why the Lions will face a tough challenge in Week 11.


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Sol House Mallorca mixed by Ibiza Rocks
- Sun terrace
- Outdoor Pool
- Free WiFi
- Low Deposit Options Available
- Interest FREE Payment Plans
The recently renovated Sol House Mallorca mixed by Ibiza Rocks boasts an ideal location only 50m from the magnificent Calvia Beach and in the heart of the lively tourist and ... Continue reading
The recently renovated Sol House Mallorca mixed by Ibiza Rocks boasts an ideal location only 50m from the magnificent Calvia Beach and in the heart of the lively tourist and shopping area of the famous Magaluf. Close to the hotel you will also find sports facilities, the nautical sports area, five golf courses and numerous trendy restaurants and bars to make your stay unforgettable.
The bus to the city centre departs only 50m from the hotel, and if you'd like to explore the city of Palma it is a short drive of around 16km away. The Palma de Mallorca international airport is 26km from the hotel.
Sustainable Tourism Tax is payable in resort from 1st July 2016 up to a maximum of 2 Euros plus VAT per adult per night, depending on the hotel rating.
- Entertainment staff
- Evening entertainment
- Golf course (within 3 km)
Other facilities
- Sun terrace
- Terrace
- Air conditioning
- Car hire
- Facilities for disabled guests
- Family rooms
- Free WiFi
- Heating
- Lift
- Newspapers
- Non smoking rooms
- Non smoking throughout
- Safety deposit box
- Vending machine (drinks)
- Vending machine (snacks)
Room facilities
All rooms have been fully renovated and sport a fresh, modern design and amenities to ensure you have a comfortable and enjoyable stay.
They feature either two single or a double bed, private furnished balcony, free Wifi, 42" flat screen TV (Spanish and international channels), telephone, air conditioning, safe, mini fridge and a fully equipped private bathroom with shower and hairdryer.
Other Information
24-hour-reception, 24 hour wake-up call, currency exchange, laundry and ironing service (extra charge), private parking (extra charge), car hire (extra charge), free Wifi throughout the hotel (from Summer 2016)
+ 25% off any attraction option at Katmandu Park or Golf Fantasia with much fun for all ages!
+ Free bus transfer to Katmandu Park or Golf Fantasia from your hotel on select days or a special taxi fare of just 1€ per person per way to either Katmandu Park or Golf Fantasia!
+ Height, age and weather restrictions may apply on some attractions.
+ Please ask at your hotel´s front desk to get full details!
Valid for 2017 for the hotels Sol Wave House, Sol House Mallorca Mixed by Ibiza Rocks, Sol Guadalupe, Sol Barbados, Meliá Calviá Beach, Me Mallorca, Sol Beach House Mallorca, Sol Palmanova, Sol Lunamar
Price Overview
The prices shown below show the cheapest holiday offers for the Sol House Mallorca mixed by Ibiza Rocks in Magaluf. Please use the price links to see all holiday deals available for that month.
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To make booking a holiday at Sol House Mallorca mixed by Ibiza Rocks in Magaluf easier for you, we provide a range of different payment options. To find out if you can book Sol House Mallorca mixed by Ibiza Rocks with a low deposit holiday option, please click the "Price and Availability" button above.
Anyone in the 18-30 age bracket whose holiday mission is fun, sun and soaking up sangria (or all manner of alcoholic beverages) will have the time of their lives in Mallorca's raucous premier party resort of Magaluf. The resort is situated about 10 miles (16km) west of Palma, the island's capital, has become one of Europe's most popular destinations for young British travelers eager to experience the famous nightlife in Magaluf. It sports hundreds of bars, discos and clubs, and has a wide choice of budget accommodation and restaurants. Magaluf and its wide sandy beaches such as Mallorca Beach, Magaluf Beach and Palma Nova Beach, is particularly packed during June, July and August with young holidaymakers who are known locally as 'gambas' (red prawns), especially if they stint on the sun cream. During the off-season it is much quieter with many of the wilder entertainment venues closed, and in recent years has been drawing older clientele and families during this period.
Resort Rating
Daytime activities in Magaluf are centred on the long sandy beach, where most of the party-goers sleep off their holiday nightlife escapades in the sunshine. The more energetic can take advantage of various watersports, like jet skiing, donutting, kite surfing or even scuba diving. The waterpark and a wild west theme park are also popular options, offering thrills and spills. Excursions to other resorts on Mallorca, and into the capital, Palma, are also available. Many visitors prefer to hire a car or moped and explore the island on their own steam.
Magaluf is a resort favoured by young budget holidaymakers, and it therefore has an abundance of fast-food outlets. Those in self-catering accommodation find they are never very far from a familiar name like McDonalds, Burger King or Pizza Hut. The resort also has a wide selection of restaurants, the majority catering to British tastes. There are several restaurants that offer Sunday roasts and other favourites like bangers and mash, fish and chips and shepherd's pie. For variety there are Indian, Chinese, Mexican, Italian and even some Spanish restaurants too.
It is the pulsating nightlife that brings holidaymakers to Magaluf, and therefore there is no lack of bright lights after dark, the party swinging into, and beyond, dawn the next morning. Most start the evening in one of the plethora of bars and move on to dance or theme parties hosted by top DJs in the numerous clubs along the famed Punta Ballena strip. Touts line the strip seducing customers into their establishments with special offers on drinks and cocktails. The most renowned of Magaluf's clubs is the massive BCM, which uses around three million gallons of bubbles to cover its floor on its famous foam party nights. Other well-known names are Carwash, Bananas and Boomerangs. A popular alternative to drinking and clubbing is an evening at the Pirates Adventure theme dinner and show.
The promenade and streets in the centre of Magaluf are lined with dozens of shops selling beachwear, souvenirs and other Spanish vacation souvenirs geared for holidaymakers. Better shopping can be found in Palma, an easy taxi or bus ride away. Every Monday there is a market in Calvia, six miles (10km) inland; good buys here include porcelain, jewelery and leather goods. There is also a popular market in Inca each Thursday for those who want to go farther afield. There are good supermarkets for shopping in Palma Nova and Magaluf, they stock all the well-known international brands, as well as local produce. Most things are good value, particularly alcohol and cigarettes.
Magaluf is geared mainly for young Brits looking for a loud and lively holiday; it's not a great destination for those after peace and tranquillity. Although it has improved in recent years, the skyline of Magaluf remains characterised by 1960s and 70s apartment blocks and the resort is packed with salesmen; the street vendors are best avoided but the 'PRs' outside the bars and restaurants can be worth chatting up as they offer free drinks. Visitors should be aware of the pickpockets on the beach at night.
Essential Information
Time Zone
Plug TypeVoltage 220V 50Hz
€1 = £0.85
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Photos of the Magaluf area
Panoramio is a photo-sharing network designed for users to upload their own travel photos and place them on the map, giving you a better idea of what to expect from a location before you visit. We decided to use Panoramio because we want you to see images that reflect real life, not just the glossy photos that the brochures provide.
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Hotel address : Cala Blanca 2, Magaluf, Majorca, Balearic Islands, 07181
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Questions and Answers about Sol House Mallorca mixed by Ibiza Rocks
If you can't find what you're looking for or if you have a question about this specific hotel, please get in touch via the Add a Question button below. Our knowledgeable team will aim to respond to your question within 72 hours. In the meantime, previously asked questions can be found below and answers to some of the most commonly asked questions can be found on our FAQs page.
is this hotel a family and couples or is it stags hens and group hotel 1 Answer
As with most hotels in Magaluf, this is geared towards the younger market, especially in high season, June July and August
Added 1 year, 8 months ago.
By Mandy, loveholidays.com team member


text: The Brooklyn Nets Are Cleaning House Patrick Redford1/10/16 12:02pm253EditPromoteGo to permalinkThe Brooklyn Nets are the most aimless team in the NBA. They're old, bad, and they don't have any of their draft picks, on account of trading too much for Kevin Garnett and Paul Pierce, then watching them tailspin into irrelevancy. This morning, they fired coach Lionel Hollins and "reassigned" GM Billy King.


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Revolving door to 14-2
The Patriots' devastating loss to the Ravens in last year's playoffs signaled change was necessary. So coach Bill Belichick began to alter the team's landscape. When they take the field next Sunday for this year's playoffs, the Pats will feature 25 players who weren't on last year's roster. Five starters on each side of the ball are new. Here is a breakdown of some key positions:
Defensive line
Key departures: Jarvis Green (signed elsewhere), Ty Warren (IR), Ron Brace (IR)
Key replacements: Myron Pryor, Gerard Warren, Kyle Love
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The upsurge in high quality cameras in smartphones has resulted in a decline in sales of compact cameras. However, the camera industry has responded by making newer camera models that are more advanced and generally provide more options than a smartphone camera could. There isn't really a lack of choice when it comes to camera market today, so finding the …Read More »
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The MSCS program is now open for all F1 students!
Apply now for Winter 2017!
News about the WASC Accreditation.
Learn technological skills with humanistic values. Classes available onsite and online. The Master of Science in Computer Science at Sofia University provides students with a broad background in software development and other core disciplines of computer science. It also gives them the opportunity to further their knowledge in foundational and applied topics. Located in Palo Alto, the heart of the Silicon Valley just minutes away from top companies such as Google, Apple, HP and Facebook provides the students with an environment rich in opportunities.
The program features:
- Onsite and hybrid/online formats
- Combine technological development with transpersonal values and skills for the practicing professional
- Promote creativity, cultural sensitivity and mindfulness
- Bring essential skills and values to the next generation of well-balanced leaders in the workplace
- Small classes and close interaction with faculty
- Faculty that are experts in their field, and bring the cutting edge technology and real-world computing experience to the classroom.
Concentrations Offered:
- Artificial Intelligence & Machine Learning
- Human Computer Interaction
- Holistic Leadership & Management
- Transformative Computing
- Transformative Unmanned Aircraft Systems Computing
- Big Data and Data Mining
- Software Design and Process Excellence
- Wearable/Mobile Application Development
- Data Science
Required Course Work: 48 quarter units = 36 trimester units (1 – 2 year program)
- 5 Core (4 CS courses and 1 Transpersonal Studies course)
- 13 Electives (11 CS courses and 2 Transpersonal Studies course).
In addition to contemporary coursework in their disciplines, students can expect to take courses in transpersonal psychology, a full-spectrum psychology that encompasses a continuum of human experience and behavior. Some examples of courses are "Theories of Personality and Personal Growth", "Positive Psychology", and "Mind and Body Interaction in High Stress Work Environments".
Tuition: Estimated total cost of tuition for the degree: $36,000.00
Contact: firstname.lastname@example.com or 650-493-4430


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"Hoss Man (JIRA)" <j...@apache.org>
|[jira] [Commented] (SOLR-2366) Facet Range Gaps
|Mon, 27 Jun 2011 22:00:49 GMT
[ https://issues.apache.org/jira/browse/SOLR-2366?page=com.atlassian.jira.plugin.system.issuetabpanels:comment-tabpanel&focusedCommentId=13055793#comment-13055793 ] Hoss Man commented on SOLR-2366: -------------------------------- bq. Guess my main point with the examples was to suggest that a facet.range.spec should not require facet.range.start and facet.range.end, but that the first and last values in the spec list should be taken as start and end, instead of requiring start and end in addition. ... bq. Simply document that facet.range.spec is mutually exclusive to the parameters gap,start,end and other. I respect your argument, but i think if this new "spec" param is going to be mutually exclusive of facet.range.other as well as all of the existing mandatory facet.range params (facet.range.gap, facet.range.start, and facet.range.end) then it seems like what you're describing really shouldn't be an extension of "facet.range" at all ... it sounds should be some completley distinct type of faceting ("sequence faceting" ?) with it's own params and section in the response. ie... {noformat} facet.seq=fieldName f.fieldName.facet.seq.spec=0,5,25,50,100,200,400,* f.fieldName.facet.seq.include=edge {noformat} (where facet.seq.include has same semantics as facet.range.include ... except i don't think "edge" makes sense at all w/o the "other" param concept ... need to think it through more) Otherwise it could get really confusing for users trying to udnerstandwhat "facet.range.*" params do/don't make sense if they start using facet.range.gap and then switch to facet.range.spec (or vice-versa) ... ie: "how come i'm not getting the before/after ranges when i use 'facet.range.spec=0,5,25,50&facet.range.other=after' ?") > Facet Range Gaps > ---------------- > > Key: SOLR-2366 > URL: https://issues.apache.org/jira/browse/SOLR-2366 > Project: Solr > Issue Type: Improvement > Reporter: Grant Ingersoll > Priority: Minor > Fix For: 3.3 > > Attachments: SOLR-2366.patch, SOLR-2366.patch > > > There really is no reason why the range gap for date and numeric faceting needs to be evenly spaced. For instance, if and when SOLR-1581 is completed and one were doing spatial distance calculations, one could facet by function into 3 different sized buckets: walking distance (0-5KM), driving distance (5KM-150KM) and everything else (150KM+), for instance. We should be able to quantize the results into arbitrarily sized buckets. I'd propose the syntax to be a comma separated list of sizes for each bucket. If only one value is specified, then it behaves as it currently does. Otherwise, it creates the different size buckets. If the number of buckets doesn't evenly divide up the space, then the size of the last bucket specified is used to fill out the remaining space (not sure on this) > For instance, > facet.range.start=0 > facet.range.end=400 > facet.range.gap=5,25,50,100 > would yield buckets of: > 0-5,5-30,30-80,80-180,180-280,280-380,380-400 -- This message is automatically generated by JIRA. For more information on JIRA, see: http://www.atlassian.com/software/jira --------------------------------------------------------------------- To unsubscribe, e-mail: firstname.lastname@example.com For additional commands, e-mail: firstname.lastname@example.com
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There are lots of things for seniors to do at the DeCorso Community Center!
Various exercise classes, games, and local trips are held on an ongoing basis. View the to see a full list.
The monthly newsletter on page 2 lists upcoming activities such as trips, dinners, luncheons and other events for the current and future months. Some require advanced registration. If you have any questions or want to confirm that a program is running, please contact the center.
Please be familiar with the Trips, Dinners and Activities Policies before purchasing a ticket!
The center wishes to offer new and different opportunities to engage seniors in the community, and is always looking for volunteers to help run its great events and activities. Those with ideas for programs and wishing to help can call 908-665-0046 or CLICK HERE to provide your input online.


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October 10, 2007 — Press Item
October 16, 2007 — Press Item
On Tuesday Democrats introduced the RESTORE Act, which will revise FISA legislation passed in August.
October 12, 2007 — Press Item
October 8, 2007 — The Daily Whip
Mortgage reform; MEJA
The report released today by the House Budget Committee and Chairman John Spratt provides concrete evidence that Democrats, in fact, are the party of fiscal discipline in Washington.
This important legislation – the Mortgage Forgiveness Debt Relief Act – is yet another concrete example of how this Democratic House Majority is focused on common-sense, bipartisan solutions and working to address the priorities of the American people.
Suspensions; railroad safety; taxes
September 28, 2007 — The Weekly Whip
Defense, Government Accountability, and Infrastructure
September 30, 2007 — The Daily Whip
Majority Leader Hoyer delivers a speech on fiscal responsibility and the upcoming appropriations fight at the National Press Club on Friday, September 28.
Iraq, Burma and Other Suspensions
October 2, 2007 — The Daily Whip
Improving Gov Accountability, MEJA
The President's incomprehensible veto of this bipartisan, fiscally responsible legislation – which is supported by strong majorities in the House and Senate, 43 governors, industry groups and the American people – not only violates his own campaign promise in 2004, but also demonstrates a stunning lack of compassion for some of the most vulnerable members of our society.
House Majority Leader Steny H. Hoyer (MD) will address the National Press Club today to discuss the upcoming appropriations battle and larger fiscal issues.
September 26, 2007 — The Daily Whip
Flood Insurance
During Hispanic Heritage Month, we celebrate the numerous contributions Hispanic-Americans have made to our nation.
Congressional Democrats hold the political high ground in their battles with President Bush over the budget and children's health — and plan to press their advantage to the hilt.
Hoyer will address the National Press Club today on appropriations and fiscal responsibility.
September 25, 2007 — The Daily Whip
Continuing Approps, Popcorn Lungs
The House passed the CHIP reauthorization on Tuesday night.
Fiscal Responsibility
In This Section:
Email Updates
Latest From the House Floor
On Thursday, the House will meet at 10:00 a.m. for Morning Hour debate and 12:00 p.m. for legislative business. First/Last votes expected: 2:30 – 3:30 p.m. "One Minutes" (15 per side)


text:
Farming Is a Way of Life for One Tyler County Family
Wheeling Park Speech Team Talks About Going the Distance
WHEELING — Bill Cornforth stood recently on the J.B. Chambers Performing Arts Center stage speaking with two of his team's most important ...
Four Locals Claim Wrestling Titles
Jesuit Stuns West Liberty In Three Overtimes
WHEELING — Wheeling Jesuit coach Danny Sancomb could do little but immediately offer a sheepish grin, only partly because his team came out on ...
Pianist To Perform For Music Club
The Thursday Music Club will meet at St. James Evangelical Lutheran Church, 1409 Chapline St., Wheeling, at 11 a.m. Thursday. President Lynn Welsh will conduct the ...
Folmar Marks Centennial
Clara Pearl Wilson Folmar will celebrate her 100th birthday on March 6. She was born the third of seven children on March 6, 1917, to Earl and ...
Giving Post 89 Second Chance
Let us not forget that the private club Wheeling officials have agreed to consider declaring a public nuisance is not just any organization. It is owned by American ...
Be Cautious With New Test Program
Battling Drug Abuse By the Grim Numbers
Diversity Not Always Respected
One puzzling aspect of America's emphasis on diversity is that we still allow class action lawsuits. They assume we're all exactly the same as each ...
Finding the Money to Extend I-68 to Marshall County
Annual Home & Business Expo Scheduled at Penitentiary
Madolin V. Rush, 93, of Hundred, died Saturday, February 25, 2017 at her residence. She was born April 9, 1923 at Hundred, a daughter of the late Arley C. Sole and ...
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Critic's Choice
Britt Daniel, the frontman for Spoon, scheduled to arrive at the 15th Street Tavern on Friday, March 9, has watched the indie-rock growth cycle from point-blank range. His Texas-based group emerged during the mid-'90s, when the genre was really buzzing. Spoon put out one fine disc after another on a variety of labels -- tiny (Saddle Creek), mid-sized (Matador) and jumbo (Elektra) -- whose dimensions mirrored the style's growth curve. But as the popularity of the sound ebbed, so did the band's fortunes, and its recent signing with Merge, a tastemaking imprint that generally flies beneath the industry radar, is unlikely to change this equation. But such concerns show no sign of bothering Daniel, whose work on the just-released CD Girls Can Tell represents his best to date. New songs like "Believing Is Art," "The Fitted Shirt" and "Chicago at Night" are angular and idiosyncratic yet wonderfully melodic, and the lyrics are clever and evocative: e.g., "Don't say a word/The last one's still stinging" from "Everything Hits at Once." Such quality means that fans in the know will be able to experience the thrill of loving something their less-cool cohorts don't know exists -- one of the most appealing qualities about indie rock since its birth. That, my friends, is called the circle of life.
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Born: October 30,1963
Spouse: --01-21
Zodiac: Scorpio
Biography: Sanjeev bhaskar, obe (born 31 october 1963) is a british indian comedian, actor and broadcaster, best known for his work in the bbc two comedy series goodness gracious me and as host of the kumars at no. 42. he also presented and starred in a documentary series called india with sanjeev bhaskar in which he travelled india and visited his ancestral home in today's pakistan. in 2003, he was listed in the observer as one of the 50 funniest acts in british comedy.Read on Wikipedia


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Tempting Dr. Alexander
Lexi Arabella
Smashwords Edition
Copyright 2013 by Lexi Arabella
This work of fiction is intended for mature audiences only. All characters portrayed in this ebook are eighteen years of age or older.
Smashwords Edition, License Notes
This ebook is licensed for your personal enjoyment only. This ebook may not be re-sold or given away to other people. If you would like to share this book with another person, please purchase an additional copy for each person. If you're reading this book and did not purchase it, or it was not purchased for your use only, then please return to Smashwords.com and purchase your own copy. Thank you for respecting the hard work of this author.
The bad news: my parents were forcing me to see a psychologist after they came home early one day and found me in bed, my tits spilling out of my bra and my ass up in the air, with Danny Wilson completely naked behind me.


text:
If you're taking photographs or videos on rough ground, or in limited space the Hama monopod Star 78 can go where tripods cannot. The single leg monopod can be wedged between rocks, pushed into small gaps or stuck into sand to allow you to get the perfect photo in difficult circumstances. The monopod's 3-dimensional pan head and spirit level add extra professionalism to your photography, giving you more angles, perfect levels and shake-free results. The Hama Star 78 monopod has a quick release coupling allowing a speedy change from camera to camcorder, so you don't miss any magic moments. The monopod has its own storage and transportation bag. ?
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text:
Submit a Critic ScoreClose
|Uncorked Wine & Spirits
|USD 60.29 $60.29 750ml
|Belladonna LebensArt
€62.50 750ml
Orin Swift the Prisoner Napa Valley 2010
Winemaker's Notes:
Dark red, almost garnet hue with bright red on the rim. Ripe cherry and cedar on the nose with hints of dried rose petal and sweet oak. Lovely entry that leads into a fat and savory mid palate. Ripe fig and black currant dominate the finish with a breadth of soft, well integrated tannins. 44% Zinfandel, 26% Cabernet Sauvignon, 18% Syrah, 9% Petite Sirah, 2% Charbono, 1% Grenache
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+ Vintage
Editorial Reviews for Orin Swift the Prisoner Napa Valley
Sweet and dusty on the nose, this shows aromas of tight red fruits flecked with mineral and allspice notes. On entry the texture is quite silky with black cherry fruit that is threaded with a contrasting medicinal edge. There is lovely purity and aromatic fruit, violet and sweet spice notes that are wrapped up in a restrained, slightly chewy package. The finish is long and pure, exhibiting excellent tension and focus in the mouth and right through the end. A bit reticent today but really very fine. 94pts
External Reviews for Orin Swift the Prisoner Napa Valley
Grandiose Nase – betörend schwarzbeerig, kräuterwürzig und mineralischfrisch, Veilchen, Lakritze, Brombeere, auch schwarze Kirschen, Pfeffer, schwarze Oliven und dunkle Pflaumen entströmen dem Glas. Im Mund dann von unerhörter Rasse, Eleganz und geschliffener Frucht. Spannende, salzig-mineralische Aromatik mit Noten von geräuchertem Fleisch, Lakritze, einem würzigen Zimttouch und Nelken. Typisch im Finale der wilde Unterholzton und die superfeinen geschliffenen Tannine.
Interestingly enough, even though many of the 2010 Perrin et Fils selections from the southern Rhone were scheduled to be bottled right after my visit, the 2010 Beaucastel had already been put in bottle. This is a gorgeous wine, a classic blend of 30% Grenache, 30% Mourvedre, 10% Syrah, 10% Counoise and the balance the other permitted varietals in the appellation. Deep purple, with loads of bouquet garni, beef blood, blackberry, kirsch, smoke and truffle, this wine is full-bodied, rich and showing even better than it did last year. I still think it needs 3ndash;5 years of cellaring, and it should last for 25ndash;30 years, as most of the top vintages of Beaucastel do.
Interestingly enough, even though many of the 2010 Perrin et Fils selections from the southern Rhone were scheduled to be bottled right after my visit, the 2010 Beaucastel had already been put in bottle. This is a gorgeous wine, a classic blend of 30% Grenache, 30% Mourvedre, 10% Syrah, 10% Counoise and the balance the other permitted varietals in the appellation. Deep purple, with loads of bouquet garni, beef blood, blackberry, kirsch, smoke and truffle, this wine is full-bodied, rich and showing even better than it did last year. I still think it needs 3andndash;5 years of cellaring, and it should last for 25andndash;30 years, as most of the top vintages of Beaucastel do.
Vivid ruby. Fresh raspberry and cherry on the intensely perfumed nose. Sappy and sweet, with deep red fruit preserve flavors and hints of succulent herbs and rose pastille. Packs a punch but also shows impressive energy and balance. The raspberry note carries through on the finish, which also features a suave, slow-building note of star anise.
Dark, dense and very closed now, this has a tremendous core of crushed plum, linzer torte and blackberry confiture waiting in reserve. Ample singed cedar and mesquite, warm paving stone and black tea notes lurk in the background and glide through the finish. Features serious grip, but wonderful integration. Should cruise in the cellar. Best from 2016 through 2035.
Marc Perrin, of the family that has owned Beaucastel since 1909, says cool nights in August were key to quality in 2010, helping to preserve acidity in the grapes. The blend is a mix of approximately 30 percent Grenache, 30 percent Mourvèdre, 10 percent Syrah and 10 percent Counoise with small amounts of the remaining nine permitted varieties. Grapes are destemmed, flash-heated, then macerated in vats for two weeks. Fermentation takes place in a mix of cement and steel tanks. 7,500 cases made.
Bright ruby. Sexy, spice- and mineral-accented aromas of red and dark fruit preserves and garrigue. Juicy and expansive on the palate, offering vibrant black raspberry and bitter cherry flavors, a hint of smokiness and intense minerality. Tannins come on late and are quickly sucked up by this wine's intense fruit. Rich and lively, with excellent finishing clarity and length.
All Prices
Uncorked Wine & Spirits
West Milford, NJ, NJ (220 mi)
Chateau De Beaucastel Cotes Du Rhone Coudoulet De Beaucastel Blanc 2010 »
$60.29 750ml USD 60.29
Belladonna LebensArt
Hamburg, HH (4,500 mi)
€62.50 USD $ 750ml EUR 62.50
Kölner Weinkeller
Köln, DE (4,400 mi)
2010er - Château de Beaucastel Chateau de Beaucastel rouge Magnum -Subskr.. »
€130.00 USD $ 1500ml EUR 130.00
Flickinger Wines
Chicago, IL (570 mi)
2010 Ch. de Beaucastel Chateauneuf du Pape »
$159.99 1500ml USD 159.99
Kindred Spirits & Wine
Chateau De Beaucastel Chateauneuf-du-pape 2010 »
$1379.88 750ml USD 1379.88
Food Pairings for Orin Swift the Prisoner Napa Valley
Barley Risotto with Mushrooms & Gremolata Recipe
Quick Gorgonzola Pasta Sauce Recipe
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Pork Chops Marinated with Bay Leaves & Lemon Recipe
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Ham, Gruyère & Honey-Mustard Palmiers Recipe
Basic Buttermilk Pancakes Recipe
Dietary Information: Organic
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text:
This is the logo for "La casa dei Freaks":
an unconventional art gallery located in Milan.
The main inspiration came from the big mustache
of the owner of the gallery.
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Comments about Peacock Poster Board, 6 Ply :
I use this board in the classroom for displays and teaching aids. I like it because it's heavier than regular poster board and the colors are bright!
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text:
10 Things:
10 things I enjoy watching
1. CSI (on tv)
2. News! News! (on tv)
3. kids playing
4. Sunsets
5. People
6. Just shoot me (on tv)
7. Will and Grace (on tv)
8. Futbol (Viva Real)
9. Baseball
10. people I love *heart*
10 favorite places
1. Libraries
2. Breton in GB3
4. Velasquez Park
5. Greenhills when its not crowded
6. Old Powerbooks in Arnaiz
7. AA Retreat House
8. my old bed in my mom's bedroom
9. my room
10. in the arms of the man i love (wishing out loud )
All curses must end…even if it's Boston…The Red Sox won the World Series after 86 years defeating the Cardinals in a sweep 11-9 , 6-2 , 4-1, 3-0.
I couldn't sleep last night. I got home at 2am from my usual wednesday night out and was not able to sleep till 5am and woke up at 10am…sarap…think i need my sleeping pills again…
took a bunch of quizzes while I was waiting for my mind to sleep and here's one funny test that I took
The Zodiac Match
(TestDate The Right Sign For Your Personality) <—-is there even such a thing????? Belinda, the best Zodiac Match for your personality is Cancer
Cancer, the Crab (June 22 to July 22):
This emotional and imaginative partner is just your type. Initially, a Cancerian may catch your
eye because of their romantic ways and dedication to you. But as you get to know them, you're even more likely to be drawn to your Cancer's practicality and ability to nurture you. People born under this sign tend to be true-blue, loyal partners who are willing to devote their hearts and souls to their close relationships. Because of the deep investment they'll put into others, Cancerians can be easily hurt and may seem overly sensitive at times. However, more often, Cancers are simply warm and compassionate mates. In the bedroom, you may find the Crab a bit hard to coax out of its shell — at least at first. Cancers can be somewhat timid lovers in comparison to other signs. Typically, a Cancer's nature is to value family above all else. As a result, if you can make a Cancer happy and earn that familial title, you can be sure that this partner will always be there for you.
I don't think I know a lot of people who were born under this sign…checking my calendar…there's no one that I know who's birthday fall between June 22-July 22 not even any female friends ***scratches head*** ..let's try checking any cute celebrities who's birthday falls under that sign….as if….hehehehehe….brad pitt john travolta? ben affleck…kevin spacey? gabriel bryne? christian bautista…


text:
The Real Reason People Moved Last Year
The Real Reason People Moved Last Year
According to the U.S. Census Bureau, the No. 1 reason 36 million people (who were one year and older) moved from 2012 to 2013 was because of the desire for a new home.
Forty-eight percent of those who moved last year cited housing as the main reason that contributed to their decision to move, followed by 30.3 percent who cited family; 19.4 percent who said employment; and 2.3 percent who said "other."
"Picking one reason can be difficult as moves are often motivated by many different, and oftentimes competing, factors," says David Ihrke, a demographer for the Census Bureau and the report's author. "For instance, if one's primary reason for moving is to be closer to work or having an easier commute, they may have to sacrifice other preferences. This could include forgoing cheaper housing options or settling for a different neighborhood. If they mainly want cheaper housing, they may have to deal with a longer commute."
The survey also found gender differences among moving motivations. For example, males tended to cite job-related reasons more so than females as a reason for moving.
Source: "No. 1 reason people move," HousingWire (June 5, 2014)
Read More


text:
Sklz Glide Pad Natural Turf Movement Mat
The Glide Pad uses a proprietary sliding mechanism to simulate the feel of making real divots in real grass. The movement of the pad reduces shock and injury as you swing through and gives authentic feedback regarding your shots to provide a truer ball flight. It can be used at home or the driving range for indoor and outdoor use.
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text:
- Tutorial Films -
There are a huge number of tutorial films to guide, instruct and inspire your crafting…
Ribbon Work Tutorials Goldwork Tutorials Embroidery Tutorials
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"Jersey Shore" is ending after Season 6, which the gang filmed over the summer of 2012, so the trailer for the new season feels a little more poignant than usual. Obviously much of the season will focus on Snooki's pregnancy, but the new footage shows Mike returning from rehab and the whole group really savoring their last summer together.
Before the new trailer, though, the Macaroni Rascals took a walk down memory lane with a recap/clip show called "Jersey Shore: Gym, Tan, Look Back." If you watch the show, you've already seen everything the gang reminisced about: the bromance between Pauly and Vinny, Snooki meeting Jionni, the coining of the phrase "GTL."
But there were also a few things we were reminded of, including Vinny's awesome purple pants, and the fact that they went to Miami (be honest: that season was so boring you forgot about it too).
The final season of "Jersey Shore" premieres Thursday, Oct. 4 at 10 p.m. on MTV.
What did you think of the clip show? What do you think of the new trailer?


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USM Vision Weld Inspection System
USM Vision offers a complete digital weld inspection workflow, from the inspection plan creation to the data sharing that can be done wirelessly. USM Vision has been developed to meet the market need to transition from radiographic to ultrasonic weld inspection according to the international codes and standards.
USM Vision Asset Owner Brochure
Pricing available upon request
Features and Benefits
- Optimizing the use of specialized inspection personnel
- Increasing productivity i.e. lean manufacturing/construction
- Reducing the current weld inspection radiographic constraints: radiation, consumables and chemical waste management
- Simplifying the ultrasonic weld inspection and testing process
- Complete digital inspection workflow
- Offline Inspection plan creation
- Data sharing that can be done wirelessly
Product Downloads
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Graham Cracker-Currant Cake
|1 1⁄2 Cup (24 tbs)
|Graham cracker crumbs
|2 Cup (32 tbs)
|Baking powder
|1 1⁄2 Teaspoon
|Chopped walnuts
|1 Cup (16 tbs)
|Red currant jelly
|1 Cup (16 tbs)
|Sweetened whipped cream
|1 Cup (16 tbs)
Mix sugar with egg yolks.
Add crumbs and baking powder; mix well.
Add 1 teaspoon water and nuts.
Beat egg whites until stiff peaks form.
Fold into crumb mixture.
Pour into two 8-inch foil-lined cake pans.
Bake at 350 degrees for 25 minutes.
Turn cake out of pan; remove foil.
Cool to lukewarm.
Fill layers with red currant jelly; frost top and side of cake with sweetened whipped cream.
Refrigerate for at least 24 hours.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size
Calories 460 Calories from Fat 195
% Daily Value*
Total Fat 22 g33.4%
Saturated Fat 3.9 g19.7%
Trans Fat 0 g
Cholesterol 97.4 mg32.5%
Sodium 139 mg5.8%
Total Carbohydrates 61 g20.3%
Dietary Fiber 2.5 g10.2%
Sugars 49.2 g
Protein 8 g15.6%
Vitamin A 4.3% Vitamin C 0.35%
Calcium 11.2% Iron 7.2%
*Based on a 2000 Calorie diet