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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The spatiotemporal regulation of gene expression largely depends on the presence and absence of cis-regulatory sites in the promoter. In the economically highly important grass family, our knowledge of transcription factor binding sites and transcriptional networks is still very limited. With the completion of the sorghum genome and the available rice genome sequence, comparative promoter analyses now allow genome-scale detection of conserved cis-elements.
In this study, we identified thousands of phylogenetic footprints conserved between orthologous rice and sorghum upstream regions that are supported by co-expression information derived from three different rice expression data sets. In a complementary approach, cis-motifs were discovered by their highly conserved co-occurrence in syntenic promoter pairs. Sequence conservation and matches to known plant motifs support our findings. Expression similarities of gene pairs positively correlate with the number of motifs that are shared by gene pairs and corroborate the importance of similar promoter architectures for concerted regulation. This strongly suggests that these motifs function in the regulation of transcript levels in rice and, presumably also in sorghum.
Our work provides the first large-scale collection of cis-elements for rice and sorghum and can serve as a paradigm for cis-element analysis through comparative genomics in grasses in general.
In higher eucaryotes, gene transcription is controlled by a variety of mechanisms such as chromatin modifications or degradation via complementary miRNAs. Gene promoters and their cis-regulatory element composition, however, are the initial checkpoints for transcriptional gene activities and define the potential spatiotemporal expression of a gene. Among other aspects, knowledge of the elementary functional units – transcription factor binding sites – is a prerequisite to understanding regulation of individual genes and their embedding into regulatory networks.
Numerous approaches, both experimental and in silico, have been developed to uncover cis-regulatory elements [1,2]. Chromatin immuno-precipitation combined with microarrays/ChIP-on-chip provides direct experimental evidence for Protein-DNA interactions on genome-scale and is a powerful approach [3]. Yet, ChIP-on-chip is currently not easily applicable in many higher eucaryotes [4]. Other established experimental methods such as staggered promoter deletions or DNAseI footprints provide high-resolution views of single promoters but are infeasible for large-scale analysis. To overcome experimental limitations, computational methods have been developed as time- and cost-effective complements for large-scale motif discovery. These include mapping of known as well as detection of de novo motifs, e.g. [5-8]. In general, two types of data set are used for motif searches as sources of information: a group of functionally related, e.g. co-expressed, genes and orthologous promoter sequences. In the first case, candidate motifs are expected to be enriched compared to a statistical background model. Hence, they can be detected by their over-representation in the respective gene group. In the latter it is expected that non-functional regions will be considerably more diversified compared to functional cis-elements. In a widely applied approach, candidate sites emerge as conserved patterns or phylogenetic footprints from (local) alignments between evolutionary related sequences. Besides the use of single informative sources, several tools have been developed that combine conservation and co-expression information [9-11]. We applied PhyloCon in an earlier analysis [9]. PhyloCon detects motifs in data sets for which promoter sequences of genes co-expressed in one species are complemented with orthologous promoter sequences of one or more related species. In the first step, motif discovery is undertaken between orthologous sequences and initial motifs are generated from local alignments. In the second step, expression data are used to define groups of genes co-expressed in one of the species. Subsequently comparing and merging initial profiles between co-expression groups iteratively refines motifs. The combined application of two sources of information has been demonstrated to provide increased predictive power compared to approaches using only one source, e.g. overrepresentation [9,12].
In contrast to motif discovery from a confined or user-selected set of genes, network-level conservation detects globally conserved motifs from comparison between two genomes. Functional motifs are identified by their unusually high retention in orthologous promoter pairs in comparison to those anticipated from single genome frequencies. An alignment-free implementation of the network-level conservation principle, FASTCOMPARE, has been successfully employed to motif discovery in yeast, nematodes, fruit flies and humans [13]. In our study we adopted FASTCOMPARE to study network-level conservation in sorghum and rice.
A large number of in silico studies to detect de novo cis-regulatory elements have been reported for the bakers yeast Saccharomyces cerevisiae and some of its relatives [14,15]. In yeast, evaluation of biologically meaningful motifs is supported by a plethora of experimentally verified motifs as well as genome-wide ChIP-on-chip studies for transcriptional binding sites. Recently, progress in genome projects of higher eucaryotes, e.g. vertebrates and the genus Drosophila, has boosted motif discovery and our understanding of regulatory networks in these organisms [16]. In higher plants, however, thus far the lack of sequences of evolutionarily closely related plant genomes has restricted large-scale analysis mainly to dicotyledonous plants like the model system Arabidopsis thaliana [17,18]. For the economically and agriculturally highly important monocotyledonous plant genomes, however, until recently only the rice genome sequence was available. This limited comparative genomics approaches to a few hundred gene promoters for which orthologs in monocots have been described and analyzed [19]. With the completion and availability of the sorghum genome this limitation has now been overcome and we are now in a position to undertake genome-scale comparative studies between evolutionarily related monocotyledonous genomes.
Sorghum and rice belong to two different grass subfamilies, the Panicoideae and Bambusoideae, respectively that diverged approximately 60 million years ago [20]. Though genome sizes differ twofold, gene number and order are similar: about 60% of sorghum genes are located in syntenic regions to rice and orthologous relationships are well established by genetic markers as well as whole genome comparisons [20,21]. In addition, transcriptome data for rice that monitor genome-wide expression levels of many thousands rice genes have become available in recent years [22-24]. This now, for the first time, allows us to analyze conserved sequence elements on a genome scale and to detect candidates for transcription factor binding sites between monocotyledonous species.
In this study, candidate cis-regulatory elements in rice and sorghum have been elucidated by two complementary approaches. Firstly, we derived transcriptional networks in rice from correlation matrices of three independent rice expression data sets. Groups of co-expressed rice genes are obtained as maximal cliques of these networks and each gene of a clique is complemented by its sorghum ortholog. PhyloCon was applied to this data set to detect motifs in upstream sequences that are both overrepresented in co-expressed genes and conserved between orthologs. In a complementary approach, candidate motifs were identified by their preferred genome-wide conservation between syntenic promoters following the "network-level conservation" approach of Elemento and Tavazoie [13]. The numbers of motifs two genes have in common correlate with their degree of co-expression. Both methods provide the first large-scale collection of cis-elements for rice and sorghum and indicate promising approaches for cis-element discovery in grasses in general.
PhyloCon as well as network-level conservation use evolutionary conserved footprints for motif discovery. We identified orthologs between Sorghum bicolor and the genome of Oryza sativa ssp. japonica from syntenic regions of the two species [20,25]. Tandem duplications of genes frequently occur in plant genomes and typically comprise approximately one fifth of all genes [25,26]. To avoid complications by tandem duplications, we selected only gene pairs from syntenic regions that were detected as bidirectional best Blastp hits. In total, 15,773 orthologous gene pairs were identified. Comparison of upstream regions that have been deduced from incorrect gene starts can strongly impair motif discovery between orthologs. Thus, we restricted our analysis to those 12,129 gene pairs that fulfilled our stringent criteria for aligned orthologous N-termini (see Methods).
PhyloCon analysis is based on the comparison of conserved sequence profiles from orthologous pairs that are joined via co-expression of genes from a reference genome. In this study co-expressed genes in rice were deduced from three different whole-genome expression data sets: MPSS data for several tissues and abiotic stresses [23], and two oligonucleotide array experiments covering developmental stages, various tissues [22] as well as drought and salt stress conditions [24]. In the following, we refer to the array experiments measuring developmental stages and tissues as YALE-1, while the array describing expression data for stress conditions is termed YALE-2. In total, the experiments comprise 213 individual measurements or arrays, respectively. Figure Figure11 shows an overview of the motif discovery scheme applied in this study.
As it is highly problematic to combine expression data derived from different platforms, functional groups were identified separately for each of the three expression sets. Firstly, we assigned MPSS tags as well as 70 mers of the oligonucleotide arrays to the current rice RAP2 gene predictions and selected genes that show significant expression levels (see Methods). Of the RAP2 gene models, 19,396 had an MPSS tag that unambiguously mapped to one gene model. The oligonucleotide arrays contain a total of 58,404 70 mers of which 27,887 had a unique match to the RAP2 gene annotation. For the YALE-1 experiment, we obtained significant measurements for 13,904 genes while 20,633 genes showed reliable expressions in the YALE-2 arrays (see Methods).
To analyze for co-expressed genes we calculated an all against all Pearson correlation matrix. For each expression set, co-expressed genes were defined as pairs whose Pearson correlation exceeded the 99%-quantile of the background distribution of all correlation coefficients. Background and quantiles were estimated from the all-against all Pearson correlation matrix. Next, an undirected graph with nodes representing genes and edges between them if genes were co-expressed, was constructed. From this graph, co-expressed gene groups were extracted as maximal cliques for each node. To avoid clusters with broad or unspecific expression patterns, we restricted our analysis to nodes with ≤ 100 edges. After filtering for orthologs with congruent N-terminal alignments we determined 4,683 cliques and 6,667 gene pairs for the MPSS data, and for YALE-1 and YALE-2 4263 cliques with 4,395 pairs, and 2,185 cliques with 2,379 pairs. The resulting co-expression groups were subjected to a PhyloCon analysis. Initially, we detected 5,337, 14,754 and 17,068 position specific scoring matrices (PSSM) from the YALE-2, YALE-1 and MPSS derived cliques, respectively. After filtering for simple repeats, the expected frequency in rice upstream regions was determined for the detected PSSM's. For each co-expression group we tested whether the respective PSSMs were statistically overrepresented (see Methods). To obtain a set of non-redundant motifs for each data set, similar PSSMs were subsequently merged to one motif by hierarchical clustering (see Methods). As regrouping of PSSMs to one motif can potentially alter its significance we retested statistical over-representation of the newly formed motifs as described above. This procedure resulted in 1,622 MPSS motifs, 1,500 YALE-1 motifs and 866 motifs from the YALE-2 data set. A list of sites supported both by sequence conservation as well as co-expression is provided (see additional files 1, 2, 3, 4, 5 &6).
PhyloCon deduces motifs from a well-defined subset of co-expressed genes and their orthologs. In contrast, network-level conservation finds motifs by their global, i.e. genome-wide high conservation rate within syntenic promoters. Our approach followed the FASTCOMPARE method as described in Elemento and Tavazoie with some modifications [13,27]. Briefly, we investigated a k-mer search space of 6 mers up to 9 mers. As many transcription factor binding sites tolerate a certain degree of sequence degeneracy, we allowed for up to three variable positions within the k-mers. For exact words, a full enumeration of all possible k-mers was analyzed. For searches allowing variable positions we applied time-effective heuristics. In addition, we included symmetric dyad motifs of the general format X{2,4}-N{2-16}-X{2,4} to cover these binding site patterns. We also tested by a heuristic technique whether specific nucleotides at initially unspecified positions within the spacer of a dyad could result in higher scoring motifs.
The number of occurrences of k-mers per gene has been determined for 2 kb upstream regions of the 12,129 syntenic gene pairs separately for both sorghum and rice genes. To score k-mers, the expected overlap for co-occurrence in syntenic pairs were calculated from single genome frequencies. Ratios of the observed and expected number of co-occurrences were normalized and transformed to z-scores. Motifs with an over-representation of two standard deviations above the mean (z-score ≥ 2) were selected as candidate motifs. This role was also supported by their unusually high degree of global conservation. Of these candidate motifs, many motifs are size variants of one core motif. For example, one of our motifs, AACCCTA, resembles some previously described plant telomere repeats (AAACCCT)n and is highly similar to the telo-box that has been identified in Arabidopsis thaliana as a cis-regulatory element active in meristematic cells [28,29]. Besides the sequence of this telo-box like motif, several size and degeneracy variants (e.g. AAACCC, AACCCTAG) were also highly overrepresented in our dataset. In order to deduce a non-redundant list of significant motifs, only the highest scoring k-mers were retained from size variants. Applying this motif clustering schema, 3,806 k-mers (including 457 dyad motifs) were recovered for further analysis (Table (Table1,1, Additional files 7, 8). As described previously, initial dyad motifs were allowed to converge to higher scoring, more specific motifs. We detected many cases for which a large fraction of spacer positions converted to specific letters (Table (Table2).2). Several dozens of these motifs show a very rare occurrence (3 ≤ n < 10) in the 12,129 syntenic upstream sequences of rice and sorghum but a very large fraction of occurrences in single species are conserved between syntenic pairs (Table (Table2).2). Long and unusually highly conserved motifs may provide a highly specific site or several binding sites in close proximity to ensure a coherent regulation of the respective genes, at least in one biological process or response. Co-functionality of genes sharing such sites is supported by several of our gene groups. The motif CACGNGNTTTGAC is conserved in two WRKY transcription factors and a seven-helix transmembrane protein homolog to the Mlo1 gene from barley. WRKY transcription factors as well as the Mlo1 gene have been experimentally linked to primary pathogen responses [30,31]. In another group, histones H2A and H2B as well as a high mobility group I/Y-2 are present. All these proteins are known to build or dynamically interact with chromatin structures. For the motif GCTCTNCNCNAAGA, conserved occurrences are found for enzymes of the phenylpropanoid- and lignin metabolism, two hydroxyanthranilate-hydroxycinnamoyltransferases and a ferulate-5-hydroxylase.
To validate our motifs derived from network-level conservation and PhyloCon analysis, we compared our findings to previous reports on rice. The PLACE and TRANSFAC databases were searched for matches between our motifs and known sites. PLACE and TRANSFAC currently contain 74 and 55 sequences of rice transcription factor binding sites, respectively [32,6]. However, many sites are redundant between the databases (or even within one database) or represent binding site variations. Hence, the exact number of different motifs is difficult to assess. In our search we also included motifs originally described in other plant species as cross-conservation has been reported for functional cis-elements [33]. Using literature searches as well as public databases containing known rice binding sites, we found 559 of our motifs matching 43 known regulatory sites out of 96 distinct sequences in both databases (see Table Table1,1, ,22 and Additional files 7, 8). In addition, motifs extracted from literature searches but not present in databases have been detected, for instance a perfect match to the ethylene response element GGGCCC and motifs highly similar to the telo-box AAACCCTA reported in Arabidopsis thaliana (see above).
Figure Figure22 depicts two known cis-elements conferring transcriptional ABA responses, the G-box related ABA response element (ABRE) and the coupling element 3 (CE3) as an example of co-conservation [34]. The full list of network-level conserved motifs is provided as additional files (see additional files 7, 8).
Responses to environmental changes and expression patterns in higher eucaryotes frequently result from the combinatorial actions of two or more transcription factors that bind to several distinct cis-elements within a promoter [35]. For functional elements that control transcriptional activities, it is therefore expected that the number of shared elements of a gene pair will correlate with its expression similarity. For rice promoter pairs we analyzed the relation of co-occurrence of motifs detected by network-level conservation and their expression congruency. Expression similarity between a rice gene pair was measured by the Pearson correlation coefficient. To determine particular candidate motifs for a rice gene, all significant motifs were selected that were present in both upstream sequences of a rice gene and its respective syntenic sorghum partner. Pairs were binned according to the number of motifs they have in common, and for each bin we determined the mean Pearson correlation from its members. As shown in Figure Figure3,3, a positive association between the number of shared motifs and the Pearson correlation coefficient for MPSS and YALE2 but not for YALE1 data was detected. Chi-square tests show significant deviations from independency (df = 72; chi-square sums 3128 and 6046 for MPSS and YALE-2, respectively, p-value < 2 × 10–16). Positive correlation was confirmed by a non-parametric, one-sided Wilcoxon rank test (p-value pMPSS < 10–16, pYALE-2 < 10–16). YALE-1 was not significant (p-value pYALE-1~1.0).
Rice genome projects have generated in depth genomic datasets and a comprehensive set of genomic upstream sequences. However, promoter sequences from other grass genomes have become available only sporadically. Comparative or computational biology approaches were therefore restricted to studies of individual pairs of interest and limited by the availability of only a few hundreds of grass promoter sequences. Our knowledge of cis-regulatory elements in monocotyledonous plants by the low number of known transcription factor binding sites is limited to those that have been reported and deposited in plant motif databases. The few dozens of known motifs are in sharp contrast to findings that higher plant genomes typically encode on average more than 1,500 transcription factors [36,37].
With the completion of the sorghum genome, a genome-wide assessment of regulatory sites in rice and sorghum upstream sequences has now become feasible. In this survey, we employed approaches based on two different tools, PhyloCon and FASTCOMPARE. Both tools and approaches have been successfully applied to motif discovery in many non-plant organisms including yeast and mammals. In addition, PhyloCon has previously been applied with success to cis-element analysis in genome survey sequences of Brassica oleraceae vs. Arabidopsis thaliana [13,18].
FASTCOMPARE is based on the 'network-level conservation' principle. This presupposes that regulatory circuitries will be largely conserved between two evolutionary related species and functional network motifs can be detected by their higher global or genome-wide conservation rate compared to non-functional sequences. Evolutionary conservation of functional elements is also assumed for phylogenetic footprinting that discovers motifs from a group of orthologous gene pairs. For the analysis based on PhyloCon, the orthologous groups that are compared and combined result from a prior selection of orthologous mate-pairs by co-expression analysis.
Ab initio analysis of cis-regulatory elements is notoriously error-prone due to small motif sizes and motif degeneracy. Our study was designed to select functional candidate sites and motifs that are associated with transcriptional activity. Co-expression was derived from correlations exceeding the top 1% of background similarities. Additionally, our clique approach required all group members to have a significant expression correlation with all other group members. In our survey, we restricted motif searches to 5'-upstream sequences of size 2 kb (for k-mer searches) or 3 kb (for PhyloCon) (i) to model current knowledge of plant promoter sizes and, (ii) to focus on plant core promoters that presumably contain most functional elements. Though functional enhancers and cis-elements in e.g. mammalian promoters, have been reported up to several tens of thousands bases distant to transcription initiation sites (TIS), plant promoters seem to be more compact [38]. In addition, chance co-occurrences will strongly increase, in particular, for smaller k-mers and degenerated motifs. Upstream sequences of larger size would thus have adverse effects by accumulating false positives or losing statistical power.
Results reported in this study can be divided into two categories: conserved sites and motifs. PhyloCon position specific scoring matrices (PSSMs) are supported by their conservation between orthologous promoters and their simultaneous co-occurrence in genes with expression similarities. Sizes of cis-elements in plants are comparable to non-plant species and typically range between 6 and 12 base pairs [5,6]. The mean size of PhyloCon PSSMs detected in this study was considerably longer (37 bp). Hence PSSMs likely represent concrete conserved sites rather than generalized statistical models for transcription factors. Large sizes for phylogenetic footprints in grasses are consistent with a previous study of 288 maize and rice pairwise and 56 rice, maize and sorghum three-way comparisons, in which a minimum motif size ≥ 20 bp was found to be significant [19]. Such long sites for PhyloCon PSSMs can be composed of two or more motifs and close proximity of these sites is required for functionality in the respective co-expressed group. Alternatively, some of the detected sites could represent signals associated with transcriptional gene activity such as mRNA stability signals or miRNA target sites, for which longer sizes have been reported [39]. Complementary to these long conserved regions, many of the detected network-level conserved motifs represent candidates for transcription factor binding sites. After subjecting the individual detected sites to clustering, in total 3,809 non-redundant motifs were found. The rice genome contains more than 1,600 genes encoding transcription factors and a similar number of cis-regulatory motifs could be expected [37]. However, some of our motifs may still be too specific and one transcription factor may bind to several related motifs. Consistent with this assumption, only for few k-mer positions did we observe sequence variability indicating that scoring functions favor specific k-mers or overrepresented k-mers with an overall low occurrence rate in a genome. Furthermore, many of these motifs were obtained from dyadic motif searches that converged to motifs with highly specified spacer sequences. For these long motifs, similar considerations may apply as for PhyloCon sites discussed above. Taking this into account, the number of motifs reported in this study is close to the number of transcription factors present in rice. On the other hand, our method may have missed transcription factor binding sites that tolerate high degeneracy. Similar findings for highly degenerated motifs have been reported for a FASTCOMPARE analysis in yeast [13]. Nevertheless, our list of motifs up to now provides the most comprehensive analysis of cis-elements in a grass genome.
In previous studies, the functionality of motifs has been confirmed by a variety of approaches. Many surveys have reported an association of motifs with particular biological processes. For large-scale analysis, gene ontologies or metabolic pathways were correlated with particular motifs. In this study, however, we were only able to detect a few such associations, and all enrichments were in very broad biological categories, e.g. 'transcription' (see Methods, results not shown). Missing associations likely result from limitations of the current rice GO annotation. In our search, we found for only 755 RAP2 rice genes (2.7%) at least one GO term belonging to the category 'biological process'. Similarly, only 1,376 rice genes (4.9%) could be mapped on KEGG pathways. In total, a functional annotation has been found for less than 5% of all rice genes. The sparse data basis and low resolution of the current rice GO annotation that mostly assigns top level terms, are the most probable causes for the limited success in detecting significant enrichments.
Several findings support the functionality of our motifs. PhyloCon sites are associated with conservation and co-expression. Despite the limited availability of experimentally verified cis-regulatory elements in grasses, we find numerous matches to known plant motifs or sites in public databases and literature reports. This includes many variants of the ACGT motif, like the G-box or the ABA response element as well as ethylene response elements among others. Interestingly, some top-scoring motifs do not match previously published elements and indicate novel cis-regulatory motifs. The number of motifs two rice genes has in common positively correlates with their expression similarity. This is consistent with the combinatorial nature of transcription regulation [40,35] and strongly indicates that a large fraction of detected motifs are associated with control of transcription. Control may be exerted as transcription factor binding sites or, as discussed previously, as miRNA target sites or signals for mRNA stability.
In summary, motifs reported in this study will provide researchers with a prioritized list of candidates for the gene of interest and can guide experimental designs for numerous sorghum and rice genes. Additional grass genome projects, for instance Brachypodium distachyon, a wheat relative, and maize are well advanced and can be expected to deliver important and information-rich comparative genome templates in the future [41]. This will enable and stimulate whole-genome comparative studies between three and more grass genome sequences. In particular, comparisons between two closely related grasses, maize and sorghum, will allow (i) branch-specific motifs to be accessed and, at the same time, (ii) the identification of motifs common to the monocot clade.
Syntenic blocks between rice and sorghum have been described in [20]. To address possible complications caused by paralogs and tandem duplications, orthologs were selected by bidirectional best BlastP hits located in corresponding syntenic regions. To avoid misassignments caused by erroneous gene models, we restricted our analysis to gene pairs for which pairwise alignments included regions before the 15th amino acid of either protein sequence. In total, 12,192 orthologous gene pairs were selected. Upstream sequences were defined as genomic sequences from the start codon to the start of the upstream preceding gene, with a maximal distance of 3 kb (PhyloCon analysis) and 2 kb (network-level conservation analysis). For sorghum we used gene models version 1.4 of [20], for rice the RAP2 gene annotation was used [25].
We used three different large-scale rice expression data sets, denoted as MPSS [23], YALE-1 [22] and YALE-2 [24].
The 70 mer oligonucleotides used on the YALE-1 and 2 arrays as well as the MPSS tag sequences were remapped to rice gene models of the RAP2 annotation. For both probe sets, only probes that unambiguously identify exactly one gene were used for the analysis. In cases in which no UTR information was available, for the respective transcript 100 bp of the respective 3'-downstream and 5'-upstream genomic sequence were added. For MPSS tags, we analyzed only the probe located nearest to the 3'-end of the transcript as this is expected to be the most informative [42,43]. In total, unambiguous mappings for 22,271 MPSS signatures and for 27,887 oligonucleotide probes were found and used for the subsequent analysis.
Filters were employed to identify signatures or oligonucleotide probes that were expressed abnormally rarely or could be generated by systematic errors. Starting from 22,271 mapped MPSS signatures, a total of 19,396 reliable and significant signatures were selected as described in [42,43]. YALE-1 data was normalized [22] and the strategy described in [44] was employed to derive a threshold for expressed probes. We determined an intensity cut-off of 410 with a 5% false positive rate (see additional file 9). In total, 13,904 rice genes/probes showed significant expression levels. For YALE-2, we adopted filter results from the original analysis resulting in 20,633 reliably expressed probes [23].
For all expression datasets we determined background distributions of pairwise expression similarities from an all-against all Pearson correlation matrix. The 99%-quantile of these distributions was considered as significant for a pair to be defined as co-expressed. Thresholds rmin for Pearson correlation coefficients were r = 0.79, r = 0.88, r = 0.93 for MPSS, YALE-1 and YALE-2, respectively (see additional file 10). In total, for 16,426, 13,223 and 18,820 distinct genes at least one co-expressed rice gene could be identified from the MPSS, YALE-1 and YALE-2 distributions respectively.
Networkx was used https://networkx.lanl.gov/wiki to detect co-expressed gene groups in expression graphs. For an anchor gene its maximal clique was determined. We restricted our search to anchor genes with less than 100 edges. Identical cliques derived from different anchor genes were removed. Finally, 6,677 cliques covering 15,146 genes from MPSS data, 7,456 cliques including 11,412 genes from YALE-1 and 6,681 cliques comprising 8,793 genes for YALE-2 were retrieved.
To generate PhyloCon input data sets, only rice genes having an assigned ortholog in the 12,129 syntenic pairs rice-sorghum pairs were used. Cliques containing less than three pairs were discarded. The final MPSS, YALE-1 and YALE-2 datasets circumvented 4683, 4263 and 2185 cliques respectively, and included 6667, 4395 and 2379 rice genes, respectively.
PhyloCon was downloaded from http://ural.wustl.edu/~twang/PhyloCon/[9]. Parameters for PhyloCon were selected as described previously [18]. Motif profiles reported by PhyloCon were transformed into PSSMs from alignments of all sorghum and rice instances. Transformation as well as statistical tests for motif overrepresentation under binomial distribution was performed as previously described [18]. P-values were adjusted for multiple hypotheses testing applying the Benjamini-Hochberg method [45] to correct for a false discovery rate of 5%. Overall 17068, 14754 and 5337 PSSMs were obtained from the MPSS, YALE-1 and -2 datasets, respectively. Simple repeats like motifs (GC)n were discarded.
Based on sequence similarities motif profiles were clustered. To estimate the similarity of two profiles, multiple sequence alignments of all instances were generated using ClustalW 1.74 (gap opening penalty 1000 and gap extending penalty 0.001). The alignment score was the sum of column scores (match, mismatch and gap scoring 1,-1,-2, respectively) and normalized by size.
A similarity matrix based on all-against all pairwise PSSM alignments were constructed and hierarchical 'bottom up'-clustering was performed using the R package hclust. Clusters were determined by the cut-off corresponding to one-sided 5% significance deduced from the entire similarity matrix.
We followed the protocol of Elemento and Tavazoie [13]. We analyzed motif sizes ranging from 6 to 9-mers. Genome-wide conservation scores were calculated as ratios between observed numbers of co-occurrences in syntenic pairs versus the expected numbers. Transformation to z-scores allows the comparison of scores for motifs of different sizes.
Many transcription factors tolerate some degree of variability for particular site positions. Motifs searched for in this study were therefore represented as regular expressions and the (full) search space is represented by all possible subsets of the sequence space. We applied a heuristic approach to explore the search space. For each exact word, we randomly selected one position to which we added a randomly selected nucleotide. For each round of degeneration, we repeated this procedure four times. For each exact word, we analyzed 100 independent iterations.
Many significant motifs represent size (or regular expression) variations of one common motif theme or may be reported as significant because a subword/-string is significant. We defined a motif to be derived from another motif if it constitutes a (sub-)word of this motif. For variable positions, both motifs had to have overlapping specifities. To reduce redundancy, we ordered motifs according to their score, and removed all motifs from this list that were derived from a higher scoring motif.
We investigated dyad motifs with patterns of type {X}a{N}b{X}a, where X represents a specific letter, N represents any letter from the nucleotide alphabet, and a and b range from 2 to 4 and 4 to 12, respectively. A greedy scheme was applied to test whether more specified versions of the initially unspecific spacer sequence results in a higher scoring motif. For each position in the spacer, we determined the highest scoring representation of all 15 subset variations of the nucleotide alphabet, for instance {A},{AC},{AG} and so on. Next, we replaced each spacer position by its locally highest scoring letter representation starting from the position with the highest score improvement to the second, third and so on improvements. Motifs were re-scored after each replacement. Iterations were repeated as long as the total score of the motif increased.
All statistical tests as well as multiple hypothesis testing corrections were carried out using R routines implemented in the R package stats.
The rice gene ontology (GO) categories were downloaded from the GO database (http://www.geneontology.org/GO.current.annotations.shtml, 2008) and metabolic and molecular interaction pathways were identified in the KEGG database http://www.genome.ad.jp/kegg/pathway.html. Due to different rice annotation versions between these databases and the gene set used in this study, protein sequences corresponding to GO and/or KEGG pathways were remapped to our RAP2 cDNAs by either TBLASTN or BLASTN.
Comparisons against experimentally reported TFBSs were undertaken against all sites from the TRANSFAC (TRANSFAC 7.0, 2005, http://www.biobase.de/index.php?id=transfac) and PLACE databases (PLACE 30.0, 2007, http://www.dna.affrc.go.jp/PLACE/). In total, 96 non-redundant known rice sites were extracted from both databases. A multiple alignment between a known site and all sites included in the corresponding profile was generated and the alignment score was estimated as described above. For network-level conserved k-mers, string pattern matching was employed.
XW and GH carried out the bioinformatic analysis and drafted the manuscript. GH and KFXM conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Motif sites detected by PhyloCon in rice derived from MPSS. Table showes motif sites detected by PhyloCon in rice genes derived from MPSS. The position of sites shown in the third column indicates the distance to the start codon.
Motif sites detected by PhyloCon in rice derived from YALE-1. Table showes motif sites detected by PhyloCon in rice genes derived from YALE-1. The position of sites shown in the third column indicates the distance to the start codon.
Motif sites detected by PhyloCon in rice derived from YALE-2. Table showes motif sites detected by PhyloCon in rice genes derived from YALE-2. The position of sites shown in the third column indicates the distance to the start codon.
Motif sites detected by PhyloCon in sorghum derived from MPSS. Table showes motif sites detected by PhyloCon in sorghum genes derived from MPSS. The position of sites shown in the third column indicates the distance to the start codon.
Motif sites detected by PhyloCon in sorghum derived from YALE-1. Table showes motif sites detected by PhyloCon in sorghum genes derived from YALE-1. The position of sites shown in the third column indicates the distance to the start codon.
Motif sites detected by PhyloCon in sorghum derived from YALE-2. Table showes motif sites detected by PhyloCon in sorghum genes derived from YALE-2. The position of sites shown in the third column indicates the distance to the start codon.
Long motifs detected by network-level conservation analysis. Table gives long motifs detected by network-level conservation analysis. The number of genes in rice and sorghum containing the respective motif in their upstream sequence are listed in the columns '#rice' and '#sorghum', respectively. Observed co-occurrences in syntenic pairs as well as the z-score are given.
k-mer motifs detected by network-level conservation analysis. Table gives k-mer motifs detected by network-level conservation analysis. The number of genes in rice and sorghum containing the respective motif in their upstream sequence are listed in the columns '#rice' and '#sorghum', respectively. Observed co-occurrences in syntenic pairs as well as the z-score are given.
Determination of significant and reliable expression levels in YALE-1. Significantly expressed probes have been determined according to [44]. Background expression is derived from all measurements of 58,404 oligonucleotide probes in 42 experiments. For each expression intensity, the percentage of measurements exceeding the respective expression level has been determined for two classes: (i) probes for which two or more replicates fell below the respective threshold and (ii) probes having higher intensities for 3 or more replicates. The x-axis depicts expression levels measured as the intensity of Cy5 dye, the y-axis the percentage of total measurements. For YALE-1, we found an expression intensity of 410 corresponding to the top 5% of all measurements.
Background distribution for MPSS expression data. Pearson correlations have been calculated for each gene versus all other genes. The correlation matrix has been used as background distribution for genome-wide expression similarities. The 99%-quantile has been numerically determined as significance level for co-expression of a gene pair. As an example, additional file 2 shows the background distribution for the MPSS expression data. X-axis depicts Pearson Correlation Coefficients, y-axis the number of gene pairs. The line marks the obtained 99%-quantile for MPSS at r = 0.79.
We are grateful to Blake C. Meyers for making expression data available prior to publication and to Xing Wang Deng, Xiang Feng Wang and Ligeng Ma for their support and discussions on the expression datasets generated at Yale University. Research in the laboratory of KFXM is supported by grants from the German Federal Ministry of Education and Research within the GABI Future programme (0315055C) and a grant from the Pact for Research and Innovation programme of Saxony-Anhalt.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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- Tap your teeth together. You should hear the same pitch every time you tap your teeth together.
- Go to a piano and find what note corresponds to the pitch coming from your teeth. For example, my teeth give me a Db.
- Hum your way up (or down) from that note to C.
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Contact Info / Websites
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Indian Coal Industry is the world's third largest in terms of production and fourth largest in terms of reserves. Around 70% of the total production is used for electricity generation and the remaining by the steel, cement and other heavy industries. Coal is also used as fuel for domestic purposes.
Despite having one of the largest reserves, the Indian coal industry does not hold a position in the league of global energy suppliers. This can be attributed to the soaring domestic demand. A study conducted by the Indian Planning Commission and the Coal Ministry revealed that India's total coal consumption was expected to increase from 510 mts in 2007-08 to 550 mts by 2008-09.
To meet this requirement, substantial public investment is needed. Even the private players would need to deploy advanced mechanisms to increase production levels.
Recent reports say coal industry in India is facing problems because the coal reserve is on decrease and India might have to import coal from outside.


text:
Oliver were standing on the cursing stone...
The only place where cursing is allowed.
And wow.. I did not realize that he knew that many words...
At least in Osted... haha
Processed today :-D
This photograph is found in the Oliver and the One Photo a Day galleries.
All images are copyright by Jĝrgen Erik Christensen


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Which is the best grip size and weight (the G and the U)for the following racquets...
Discussion in 'Grip' started by DareToSmash, Oct 25, 2008.


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Traveling Luck for Holmeknappen, Hordaland, Norway
Where is Holmeknappen?What's around Holmeknappen?
Wikipedia near Holmeknappen
Where to stay near Holmeknappen
The timezone in Holmeknappen is Europe/Oslo
Sunrise at 08:59 and Sunset at 15:50. It's light
Latitude. 60.5250°, Longitude. 5.1550°
Weather near Holmeknappen; Report from Bergen / Flesland, 27.7km away
Weather : rain mist
Temperature: 4°C / 39°F
Wind: 16.1km/h Northwest
Cloud: Scattered at 600ft Broken at 1200ft
Weather : rain mist
Temperature: 4°C / 39°F
Wind: 16.1km/h Northwest
Cloud: Scattered at 600ft Broken at 1200ft
Satellite map around Holmeknappen
Loading map of Holmeknappen and it's surroudings
Geographic features & Photographs around Holmeknappen, in Hordaland, Norway
- populated place;
- a city, town, village, or other agglomeration of buildings where people live and work.
- farm;
- a tract of land with associated buildings devoted to agriculture.
- Kjedalen (0.5km)
- Porshaugen (3.1km)
- Bjørnestad (3.2km)
- Dalsstøi (3.3km)
- Hestdal (3.6km)
- Mjåtveit (3.8km)
- Myrtveit (3.8km)
- Sjurdal (3.9km)
- Mjåtveitstøi (4km)
- Hestnes (4.1km)
- Leirvik (4.4km)
- populated locality;
- an area similar to a locality but with a small group of dwellings or other buildings.
- Holme (0.4km)
- Hjartåsstøi (0.5km)
- Vollo (2.3km)
- Erstad (2.4km)
- Burkevellene (2.6km)
- Dale (2.7km)
- Sakstad (2.9km)
- lake;
- a large inland body of standing water.
- Dalevatnet (2.5km)
- Siglingavatnet (3.3km)
- Bosvatnet (3.3km)
- Grasdalsvatnet (3.8km)
- Salbuvatnet (4km)
- Asbøvatnet (4.1km)
- cove(s);
- a small coastal indentation, smaller than a bay.
- Skjelviki (2.2km)
- Sletteviki (2.2km)
- Merkjingsviki (2.6km)
- Sørstøi (3.2km)
- Gauviki (3.7km)
Accommodation around Holmeknappen
- point;
- a tapering piece of land projecting into a body of water, less prominent than a cape.
- Laksaneset (1.7km)
- Stigsneset (1.9km)
- Revaneset (2.4km)
- Signalneset (2.8km)
- Olderneset (3.7km)
- hill;
- a rounded elevation of limited extent rising above the surrounding land with local relief of less than 300m.
- Brakstadfjellet (3.2km)
- Krossfjellet (3.8km)
- Kjerrgardsåsen (4.1km)
- cape;
- a land area, more prominent than a point, projecting into the sea and marking a notable change in coastal direction.
- Holmeknappen (0km)
- Ospeneset (4.4km)
- bight(s);
- an open body of water forming a slight recession in a coastline.
- Gardsendviki (0.9km)
- Kjerringbukta (2.5km)
- bank(s);
- an elevation, typically located on a shelf, over which the depth of water is relatively shallow but sufficient for most surface navigation.
- Nordre Hanevikskallen (1.8km)
- Søre Hanevikskallen (2.3km)
- rocks;
- conspicuous, isolated rocky masses.
- Skarveskjeri (4.4km)
- reef(s);
- a surface-navigation hazard composed of consolidated material.
- Niso (2.8km)
- administrative division;
- an administrative division of a country, undifferentiated as to administrative level.
- Meland Herad (1.2km)
- bay;
- a coastal indentation between two capes or headlands, larger than a cove but smaller than a gulf.
- Hauglandsosen (4.3km)
- pond;
- a small standing waterbody.
- Grasdalstjørnet (4.4km)
Wikipedia entries close to
- Hjartås city city o
- Hanevik city city o
- Skjelviki city city o
- Askevatnet lake
- Meland city city k
- Frekhaug city city o
- Nordhordland Bridge landmark bridge
- Askøy city city i
- Alversund Bridge landmark bridge
- Rossland, Norway city city o
- Herdla isle isle
- Kleppestø city city o
- Vikavågen city city o
- Breistein city city o
- Skålevik city city o
- Lindås city city i
Airports close to Holmeknappen
Bergen flesland(BGO), Bergen, Norway (27.7km)
Soerstokken(SRP), Stord, Norway (87.7km)
Floro(FRO), Floro, Norway (125.3km)
Sogndal haukasen(SOG), Sogndal, Norway (136.5km)
Haugesund karmoy(HAU), Haugesund, Norway (140.1km)
Airfields or small airports close to Holmeknappen
Boemoen, Bomoen, Norway (79.6km)
Bringeland, Forde, Norway (108.3km)
Dagali, Dagli, Norway (196.8km)
Photos provided by Panoramio are under the copyright of their owners.


text: My father is my hero, my inspiration and my unconditional love. i will fall short of my vocab if i have to express my feelings for him but more over i want to keep on feeling the love for him rather than express them in words. years ago i came across a poem in Chicken Soup for the Parent's Soul, and, Stories for a Teen's Heart 2. which literary made me cry. have a look.p.s. read it with love :) DADDY`S DAY.Her hair up in a ponytail, her favorite dress tied with a bow Today was Daddy's Day at school, and she couldn't wait to goBut her mommy tried to tell her, that she probably should stay homeWhy the kids might not understand, if she went to school aloneBut she was not afraid; she knew just what to sayWhat to tell her classmates, on this Daddy's DayBut still her mother worried, for her to face this day aloneAnd that was why once again, she tried to keep her daughter homeBut the little girl went to school, eager to tell them all About a dad she never sees, a dad who never calls There were daddies along the wall in back, for everyone to meetChildren squirming impatiently, anxious in their seatsOne by one the teacher called, a student from the classTo introduce their daddy, as seconds slowly passedAt last the teacher called her name, every child turned to stareEach of them were searching, for a man that wasn't there"Where's her daddy at?" She heard a boy call out"She probably doesn't have one." Another student dared to shoutAnd from somewhere near the back, she heard a daddy say"Looks like another deadbeat dad, too busy to waste his day."The words did not offend her, as she smiled at her friendsAnd looked back at her teacher, who told her to beginAnd with hands behind her back, slowly she began to speakAnd out from the mouth of a child, came words incredibly unique"My Daddy couldn't be here, because he lives so far awayBut I know he wishes he could be, with me on this dayAnd though you cannot meet him, I wanted you to knowAll about my daddy, and how much he loves me soHe loved to tell me stories, he taught me to ride my bikeHe surprised me with pink roses, and he taught me to fly a kiteWe used to share fudge sundaes, and ice cream in a coneAnd though you cannot see him, I'm not standing all alone'Cause my daddy's always with me, even though we are apartI know because he told me, he'll forever be here in my heart"With that her little hand reached up, and lay across her chestFeeling her own heartbeat, beneath her favorite dressAnd from somewhere in the crowd of dads, her mother stood in tearsProudly watching her daughter, who was wise beyond her yearsFor she stood up for the love, of a man not in her lifeDoing what was best for her, doing what was rightAnd when she dropped her hand back down, staring straight into the crowdShe finished with a voice so soft, but its message clear and loud"I love my daddy very much, he's my shining starAnd if he could he'd be here, but heavens just too farBut sometimes when I close my eyes, it's like he never went away"And then she closed her eyes, and saw him there that dayAnd to her mother's amazement, she witnessed with surpriseA room full of daddies and children, all starting to close their eyesWho knows what they saw before them, who knows what they felt insidePerhaps for merely a second, they saw him at her side"I know you're with me daddy." To the silence she called outAnd what happened next made believers, of those once filled with doubtNot one in that room could explain it, for each of their eyes had been closedBut there placed on her desktop, was a beautiful fragrant pink roseAnd a child was blessed, if only a moment, by the love of her shining bright starAnd given the gift of believing, that heaven is never too far


text:
Hi! Welcome to my personal finance blog. If you're here to learn about stocks, the stock market, and everything about stock investing, then you've come to the right place. Let me guide you through your stock investment journey from being a learner to being a pro.
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Revoltionizing Real Estate Work Flow
When copier/printer dealers think real estate, color applications have typcially come to mind. After all real estate companies were early adopters of color. But good opportunities now exist for such business to create value with document management.
As one of central Florida's largest real estate companies, ERA Showcase faced two major challenges common to all real estate offices: it needed to manage a mountain of paperwok and it needed to improve its marketing procedures.
Download the full article here.


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Guinea pigs(7 Posts)
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In Welsh writer-director Christopher Monger's 1995's romantic comedy, Hugh Grant has an awfully small adventure in a Welsh small town in 1917. He and Ian McNeice play English cartographers who arrive to measure the height of the local beauty spot Ffynnon Garw, and find that since a mountain must be at least 1,000 feet, this one is 16ft short of a mountain.
The villagers decide to keep the duo in distracted in town, staying on to measure it again, while they drag the extra earth up the hill, er mountain, to make it the required height. Symbolically they even dig up their nationally sacred rugby pitch for earth.
The film's characters are largely caricatures, though they're played with much gusto by a talented ensemble – scene-stealing Kenneth Griffith as the Reverend Jones, Colm Meaney as the feuding publican Morgan the Goat, Tara Fitzgerald as the alluring Betty of Cardiff imported to romance Grant, and Ian Hart as the speechless war victim, Johnny Shellshocked.
It would be nice to report after all his troubles that Grant reaches a new peak with this film, but in fact he gives his usual turn of charming, doddering Englishman once again, and though very skilled it's beginning to wear perilously thin. Though mostly entertaining and amusing, this likeable Ealing studios-style comedy is 16ft short of being a classic, needing to push harder: more laughs, more romance, and more social comment about the first World War and the society of the day would make it a much more compelling experience.
As a movie it's a well-appointed molehill rather than an awesome mountain. But, nevertheless, it remains modestly engaging, pleasant and quietly charming, with appealing actors giving likeable turns. Siskel and Ebert gave it two thumbs up.
The original story is by Ifor David Monger.
Christopher Monger based his script on a family legend told to him by his father and grandfather. In creating his larger-than-life characters, he relied on memories of people he has known in his home village of Taff's Well, near Cardiff. 'Some are composite characters,' he says. 'but I think certain people will recognise themselves.'
Monger wrote the part of the English mapmaker come to measure a Welsh hill for Hugh Grant, who loved it and signed on at once. Expect gentle, old-style humour. Monger says: 'Welsh has no crude swear words, unlike the inferior Anglo-Saxon tongue. The worst insult in Welsh is to call someone Sais. It means Englishman!'
Hugh Grant and Tara Fitzgerald also star together in Sirens (1993).
© Derek Winnert 2014 Classic Film Review 734 derekwinnert.com


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1.2 Weight Loss Drugs Segment by Type (Product Category)
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2 Global Weight Loss Drugs Market Competition by Manufacturers
2.1 Global Weight Loss Drugs Capacity, Production and Share by Manufacturers (2012-2017)
3 Global Weight Loss Drugs Capacity, Production, Revenue (Value) by Region (2012-2017)
3.1 Global Weight Loss Drugs Capacity and Market Share by Region (2012-2017)
3.2 Global Weight Loss Drugs Production and Market Share by Region (2012-2017)
4 Global Weight Loss Drugs Supply (Production), Consumption, Export, Import by Region (2012-2017)
4.1 Global Weight Loss Drugs Consumption by Region (2012-2017)
4.2 North America Weight Loss Drugs Production, Consumption, Export, Import (2012-2017)
4.3 Europe Weight Loss Drugs Production, Consumption, Export, Import (2012-2017)
5 Global Weight Loss Drugs Production, Revenue (Value), Price Trend by Type
5.1 Global Weight Loss Drugs Production and Market Share by Type (2012-2017)
5.2 Global Weight Loss Drugs Revenue and Market Share by Type (2012-2017)
5.3 Global Weight Loss Drugs Price by Type (2012-2017)
6 Global Weight Loss Drugs Market Analysis by Application
6.1 Global Weight Loss Drugs Consumption and Market Share by Application (2012-2017)
6.2 Global Weight Loss Drugs Consumption Growth Rate by Application (2012-2017)
6.3 Market Drivers and Opportunities
7 Global Weight Loss Drugs Manufacturers Profiles/Analysis
7.1 Roche
7.2 GSK group
8 Weight Loss Drugs Manufacturing Cost Analysis
8.1 Weight Loss Drugs Key Raw Materials Analysis
8.2 Proportion of Manufacturing Cost Structure
8.3 Manufacturing Process Analysis of Weight Loss Drugs
9 Industrial Chain, Sourcing Strategy and Downstream Buyers
9.1 Weight Loss Drugs Industrial Chain Analysis
9.2 Upstream Raw Materials Sourcing
9.3 Raw Materials Sources of Weight Loss Drugs Major Manufacturers in 2015
10 Marketing Strategy Analysis, Distributors/Traders
10.1 Marketing Channel
10.2 Market Positioning
10.3 Distributors/Traders List
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text:
Museum of Science and Industry in Tampa
Known as MOSI, it's only an hour from Orlando. Housed in two buildings, the MOSI has standing exhibits, traveling exhibits and some outdoor activities like a zipline and ropes course. Kids will love the Disasterville exhibit with interactive exhibits for hurricanes, fire, tornadoes, earthquakes and more. The Kids in Charge! building has tons of exhibits to keep the younger set happy including a mini Publix, but also some cool things for adults like the bed of nails. There's also an IMAX theater on site. General admission cost is $26.95 for adults and $20.95 for kids, although if you're an Orlando Science Center annual pass holder, you get free admission. IMAX, special exhibits and the zipline course (48 inches and 60 pounds to ride) are extra. Parking is $5. mosi.org


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March/April ivf/fet
View Single Post
March 4th, 2013, 10:02 AM
Join Date: Mar 2013
Posts: 6
J and K - Thanks for your input... and I guess I was pretty naive to think that I would be one of the few NOT to tell our families. I'm thinking once we (hopefully) get that BFP we will just share with our families that we are expecting... there isn't a need to tell them how because if this works for us I'd like to do it again in the future without the questions! I hope that you are feeling better today than yesterday!
Onlybygrace - I agree, it's no one else's business!
kayakr - I hope that your IVF treatment isn't delayed... wishing you and your DH the best!
So, I consider myself a pretty laid back and passive individual, however, this birth control is making me feel pretty moody (mostly just in the mornings). I've actually never been on birth control pills before (I did use the patch when I was much younger) but luckily the BCPs aren't for too long. I'm just thinking... if BCPs are making me feel moody I can't imagine what the other medications will do! I think it's time for me to suck it up and dive into the packet of information my doctor gave me on each medication. Have any of you gotten away with little to no side effects from the other medications?
BTW - I'll be happy to deal with side effects if it gets us that BFP!!! I just like to be informed and prepared.
Have a great day everyone!
View Public Profile
Find all posts by Clover19


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Cute right? This adorable dress is so simple to make, and great for using up fabric scraps! The possibilities with this dress are endless! Imagine doing a black tank top, solid red, red with white polka dots and then another solid red. A Minnie Mouse dress!
You are here: Home / Holidays / 4th of July / Easy Sew Girls Dress {FREE pattern}
Easy Sew Girls Dress {FREE pattern}
February 27, 2013 By Aunesty Janssen Leave a Comment
Filed Under: 4th of July, Sewing, Sewing
About Aunesty Janssen
I am Aunesty Janssen, a mom who is a huge Disney Addict. I have 6 amazing kids, 21 down to 2. With my oldest daughter in CO working for Wells Fargo, my eldest son in the Army, my middle daughter attending college {& a WDW CM}, my middle son a Junior in high school and my two toddlers, the three year old boy, Carson, who is on the Autism Spectrum and Courtney, my large personality two year old.
I love crafts, blogging, baking and spending time with my family.
When I am online, I am usually writing a blog post, or talking on Facebook. You can find me on Aunesty.com, AunestyJanssen.com or Temporary Tourist and few more...
"Even miracles take a little time." - The Fairy Godmother, Cinderella


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Angry Twink Hatred Becomes Hot Twink Passion
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Stannum Boutique Hotel & Spa offers you so much more than just an elegant accommodation; it is really an escape to a modern world with a touch of luxury and technology with the best location in La Paz without the hassle of the inner city. Enjoy the stunning views of the Snow-capped Mountain "Illimani" and the city.
The peculiar location in a modern mall , different views of the mountains and city and personalized service are the perfect combination for maximum comfort , making of each stay a pleasant and memorable experience.
We are located in the best entertainment center of La Paz with easy access to cinema, gym, restaurants, shops, bank , game rooms, ATMs, etc. A few steps from the historic and businesscentre in the heart of the city and relatively close to many touristic attractions. This whole area is the most accessible and safe area of the city.
Exclusive design, elegant and cozy
Visit us!
Stannum Boutique Hotel & Spa has 40 tastefully furnished and equipped rooms to provide maximum comfort to our guests. This great work was developed by Jorge Avila of A2 Architects and Nhora C. Quintero from NCQ Design , well recognized for their expertise in projects in Chile, the Caribbean and the US.
Our luxuriously decorated rooms offer the utmost comfort set in a modern building surrounded by spectacular views of the mountains and the city.
The best entertainment center of the city is located on the ground floor of the tower where the Hotel is located . It has 11 cinemas, food court , restaurants , cafes , games, fitness center, shops, bank, ATM's , parking and muchmore
Meeting Rooms
Spacious and modern spaces for different kind of events where we fully dedicate to the organization and attention that may need. We have several types of layouts like classroom, banquet, auditorium, cocktail, etc, ideal for any event.
Business Center
Open 24 hrs a day, 7 days of the week. Equipped with 4 modern computers. Moreover, we also have printer, ipad, photocopy machine, facsmile, and cellphones (upon request and availability).


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This post is part of Baby Hero's series of profiles for International Women's Month, celebrating women who #MakeItHappen. Su-Mei Thompson is CEO of The Women's Foundation, an non-profit dedicated to improving the lives of women and girls in Hong Kong. Su-Mei is a mother to two girls and an inspiring leader who has become the city's most influential voice on equal opportunities for women.
I was born and brought up in Malaysia. My parents were pretty humble and they had always drilled into me that as an Asian, and as a girl, I was at a disadvantage, so I would need to work harder just to keep up. I am enormously grateful to my mother for how she shaped who I am. I had a strict upbringing with very little molly coddling or extravagance. No money was wasted on toys or Christmas presents. My mother worked really hard and spent her savings on giving me the best education she possibly afford. She instilled in me the importance of hard work and having the biggest boldest vision of what I could be.
I went to boarding school in the UK (Cheltenham Ladies College), earned an Undergraduate degree at Cambridge and a Masters at Oxford. I loved my university days – Cambridge was great fun after being cloistered in all girls boarding school for five years! At Oxford I knuckled down to earn my First class degree, something I am still incredibly proud of.
It might sound feeble but I chose law because my parents wanted me to and in those days, children listened to their parents! (Try telling that to my daughters now, Ha!). I was a classical pianist and accepted to the Performers' Course at Dartington College of Music (UK). They wanted to shelter me from the pressure and competition of life as a career musician and in the end I bent to my parents' will and applied to read law at Cambridge. We also had a tradition of lawyers in the family; both my paternal grandfather and both my parents were UK qualified barristers.
One particular experience in my early career cast a very long arc over the rest of my life. At Linklaters – as a first year associate, I was part of a very small team working on a massive transaction. Our client was acquiring assets from a big mining company, these assets being mines across the whole of South America. It was a male dominated, chauvinistic environment and there I was, this young 24 Asian female traveling the globe and doing extensive due diligence. When I arrived back in London, I went straight from the airport to a meeting with our clients and advisors from all parties - all who were older experienced men. I delivered my findings but also raised some red flags in the deal that made everyone sit up and listen. It was a baptism by fire. That experience has served as a constant reminder to stay fearless even if I am in the minority.
"If working and being a new mom is making you feel miserable and conflicted, then know it IS possible to take a career break and to get back into the game."
From Linklaters to Walt Disney, The Financial Times and then to Christie's, there are takeaways that apply to whatever sector one's in. First of all, don't just run after successful people. I've always gone out of my way to reach out to someone who has experienced a setback. People who are down and out have a way of boomeranging back and they remember who stuck with them through the bad times. Secondly, build a deep connection by meeting people in person, particularly over a meal. I am naturally shy and would rather have lunch at my desk but instead I have breakfast and lunch meetings 4-5 days a week. Thirdly, keep in touch with people particularly when you don't need anything from them. If you maintain contact when you don't have an ulterior motive, it makes it so much easier when you actually need a favor.
At The Women's Foundation, I am responsible for raising awareness and mobilizing action for the challenges women and young girls continue to face. This involves speaking at local and international conferences, researching and publishing opinion pieces, and joining the dots with the work of other organizations in a way that is transformational so that the sum is greater than the parts. The speaking engagements can be nerve-wracking but I've been fortunate to meet some truly inspirational women from Au Sung Suu Kyi to Christine Lagarde to President Ellen Sirleaf Johnson.
TWF is also making a documentary called 'She Objects', which focuses on gender stereotyping by the media. We know from our work that media has the power to create or exacerbate gender stereotypes which are the root cause of so many challenges which women face in the workplace and in the broader community.
"Don't build a life in the context of a career but rather, build a career in the context of a life."
If working and being a new mom is making you feel miserable and conflicted, then know it IS possible to take a career break and to get back into the game. I see so many women hesitate to take a break, and those who take a break lose their self-confidence and rule out getting back into the job market in a significant way. It needn't be that way.
Having my two daughters Tallulah (9 Years) and Allegra (7 Years) made me stop to reflect on what was really important, how was I going to measure my success, and how was I going to balance competing priorities in future. While they were little, I decided to step off the corporate ladder. When I was ready to jump back into the workforce, the right opportunity came along at The Women's Foundation.
When they get a bit older, the two things I'm going to be telling my daughters are – firstly, it is important to do something you enjoy with people you like. If you add it all up, we spend a significant part of our lives at work and with our colleagues, so ask yourself, "Is this the right career path for me? Is this the company where I want to build my career?"
Secondly, don't build a life in the context of a career but rather, to build a career in the context of a life. It is vitally important to have outside interests, a sport or a hobby and equally important to have people in your life outside the office - they will help you maintain perspective in times when things aren't going well at work.
Women tend to be less explicit about their ambitions and face the challenges of balancing family life with a demanding career, all these factors along with discriminatory practices and entrenched old boy's networks continue to impede the progress of women into the C-suite and boardrooms. Sheryl Sandberg articulates the barriers very well in 'Lean In' – she talks about the Leadership Ambition Gap whereby women expect less of their careers than men and how women start to lean back in their careers in anticipation that they are going to be looking after their husband or a family much earlier than they really need to. I completely agree with Sheryl that the single most important career choice a woman makes (if she marries) is her choice of a spouse. In this, she is saying something we've said all along at TWF, that we won't have equality in the workplace until we have greater equality at home.
It's hard to believe that in this shiny metropolis that is Hong Kong, one in five people is living at the poverty line and among them, women are particularly vulnerable.
I really admire Alicia Wieser and Samar Shahreyar, the founders of Baby Hero, for getting Baby Hero off the ground and being so clear and principled about their values and objectives for the business. Yes it's a business but they are really concerned about social impact. In this, they are incredible role models for other entrepreneurs. At TWF, we want more women to found and grow companies. Entrepreneurship is vital to allowing women to become more economically self-reliant and the great thing is that it's possible to be an entrepreneur regardless of age and social background. Extensive studies show that when women are given the right resources in developing countries, their families and communities also benefit.
TWF's emotional heartland is in its grassroots community and leadership programmes -
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"If God had wanted a better you, He would have created a better you, and that's the challenge -- He did."
"Sometimes you wish you were someone else entirely, although not usually anyone you've met personally."
"Only a man is allowed to give up. There is always someone for whom a woman must carry on."
"You can see it as risking betrayal, disappointment and ingratitude, or you can see it as faith, hope and charity."
On the Seventh Day, the Creator sat back, took off his gloves and said,"No fingerprints. They'll just have to find me in the sunsets."
~~ Robert Brault


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This website is for consumers who don't have a local insurance professional to answer their questions about long-term care insurance.
The Talk
Hear Rob Lowe, Maria Shriver & others have "The Talk" about long term care planning.View full post
Barbara's Decision
After buying a long term care policy 10 years ago, Barbara doesn't have to worry about burdening her children with the cost of her care.View full post
Assisted Living
Long term care insurance has allowed her 84-year-old mother to have peace-of-mind after moving into an assisted living facility.View full post
Aug 27
Outside-of-the-Box Thinking About Long-Term Care Planning–One Size Does Not Fit All
As a shoe aficionado, I have observed that long-term care planning is quickly starting to resemble my closet of 100 pairs of shoes.
Jun 20
Phyllis Shelton LTCi Update
with Takacs-McGinnis Elder Care Law 06-20-17
Mar 11
Denial is Not a River in Egypt
People have a 50 percent chance of needing LTC at some point in life, and about 30 percent of people who do may need care for five years or more, according to a new study by LifePlans, Inc. Being in denial about this could be the worst thing you ever do to the people who love you.
Jan 01
What your state lets you keep, effective 7/1/2017
This is a table that shows the minimum assets and income each state allows nursing-home residents and their spouses to keep.
Oct 24
What Does Long-Term Care Cost?
Planning for long-term care is best done with information gathering to make an informed decision. The "deer in headlights" approach doesn't work.
Sep 07
Federal LTC Insurance Program Rate Increase (updated)
The employee education program for the Federal government's group long-term care insurance program in 2002 beat Donald Trump to The Apprentice.
Jun 26
Operation TRIPLE Starfish – LTC, FIAs and GOLD?
Today's turbulent economy is forcing Americans who value their independence over anything else to find new ways to remain financially secure into old age. Long-term care insurance can play a huge role in that picture, but there are two other things you can do of which you may not be aware.
Jun 22
Operation Starfish – Are You That One? Women, Please Act NOW!
Are you that one? Do you have children or other loved ones who will suffer if you don't plan for long-term care? Book a time TODAY as time is almost out to get a policy that doesn't charge women more.
Jan 10
Calling All Single Women Without Long-Term Care Insurance (and their single friends)
If you are a woman who wants to keep your independence as you age, this article is for you.


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- COLD-CURE MOLDED FOAM. This spaceage material encases the interior steel frame and sinuous springs, allowing for body-friendly curvatures to be covered with glove-fitted upholstery. This guarantees absolute comfort and durability.
- LUXURIOUS FEEL AND TOUCH. A layer of soft high resilient polyurethane foam with fibers ensures a luxurious feel and extra softness.
- 3 DISTINCT SIZES AVAILABLE – WITH THE SAME DESIGN. All IMG relaxers are available in different sizes to assure a perfect fit for your comfort requirements.
- ADJUSTABLE HEAD AND NECK SUPPORT Exclusive adjustment system that allows your head and neck to rest in an infinite number of comfort positions. It can easily be adjusted by a handgrip
Recliner Dimensions: 32x40-45x32 (WxHxD)
Around 2 weeks. HQ for IMG is in Corona, Ca very close to our store. Most orders should ship to us in about 48 hours and once we receive it, it takes about 5-7 business days leaving your wait time around 2 weeks
Custom Order:
10-12 Weeks.
What is the weight limit for this chair?.
All IMG chairs have a 300lbs weight capacity, however IMG tells its customers if you can fit in it, you can sit in it.
Does the stem height change/Is it adjustable?
This chair does have three height options. The standard height comes on the chair but can be replaced for a high or low at no additional cost. This change makes the height 1.1 inches taller or shorter from standard. PLEASE NOTE** Changing the height will make any order a Custom Order regardless of color or size. .
The following outlines the extended Warranty Policy for your new IMG product(s).
Please take the time to thoroughly familiarize yourself with its contents. Please also note that all warranty periods stated (in year) below begin from the original date of purchase.
10-years warranty frames
Steel structural frame elements are warranted against breakage for a period of ten (10) years. Wood sofa frames and steel insert frames are also warranted against breakage for a period of ten (10) years.
10-years warranty – molded foam
Molded cushions are warranted against collapsing for a period of ten (10) years. Loss of foam resiliency should not be confused with softening of foam or stretched cover, which are results from normal use. Owners of upholstered furniture should also expect foam and fiber fillings to compact over time.
5-years warranty mechanism
Recliner mechanisms including steel springs are warranted against breakage for a period of five (5) years.
Please note that this does not cover any type of minor sound or noise in the mechanism or unevenness in swivel mechanisms. This is normal and the result of general use.
5-years warranty laminated wood
Laminated Scandinavian recliner parts (for Modern Comfort, Nordic and senior chairs) are warranted against breakage for a period of five (5) years.
Please note that this does not cover any form for minor sound or noise in the laminated wood or unevenness caused by temperature or moisture. This is normal for all living materials.
2-years warranty power
Warranty period for defects of new motors and related parts are two (2) years.
Excluded from the warranty are non-material defects or deviations in the properties and condition of the product, which are negligible for the value and intended use of the equipment. Replacement of spent batteries / rechargeable batteries is also not covered by this warranty.
2-years warranty leather and fabric
IMG leather products are warranted against tearing and cracking for a period of two (2) years. IMG fabric products are warranted for a period of two (2) years against seam slippage, fraying, and dye transfers.
This fabric warranty does not cover tears, flattening of nap, pilling, fading, or shrinking and is not valid when heavy soiling or abuse is evident.
The warranty does not cover normal fading, damage due to improper use of chemical treatments that are not endorsed by IMG, damage from lack of required caution or regular care and maintenance.
The warranty does not include claims for veins, wrinkling, natural markings from healed scars or variations in grain or color. These natural features are proof that only genuine leather has been used. The warranty does not include stretching of leather or fabric. This is normal Warranty Policy and results from normal use.
Exposure of leather and fabric to sunlight or extreme light sources will cause fading and damage and is not covered by the warranty.
2 years warranty Workmanship & other materials and components
All other materials, parts, components, and other furniture products not specifically included in the above warranties and workmanship are warranted against defects for two (2) years.


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Release Date: 2001 |
Sadly I don't agree with you ,everything that Sepuluta has made sepultura after roots sucks
after arise*
Album Rating: 1.5Thought this would have had a couple of reviews. Looks pretty good for a first.
everything that Sepuluta has made sepultura after roots sucks
This years release isn't so bad.
its a point of view. me i love the Load album of Metallica while many others think it sucks. so i appreciate how the reviewer loves the record. not a bad review either.
what in the name of all that is good
Album Rating: 1.0this is unbelievably bad
I really liked the review, however I don't really agree with the mark, I think Nation drags a little towards the end. I believe Dante is their best post Max album
Shocked to see someone who actually enjoyed this release. I personally thought it was horrible and way to bland. That and Against were my two worst
The first Sepultura album i purchased and still is one of my fave of theirs... holds good memories.
Good review man, Pos'd
This is a really bad album
Album Rating: 4.5What hell are u taking about this is one of there best albums, yeah maybe its not there ussual thrash but like its said eariler at least u can understand them..... They had to find a new found sickness to ajust....the leads voice is perfect contrast to the music they bond almost like the band sevendust. They were just playing the rite sound in the wrong year anywayz most new metal artist look upto sepultra as greats with out the bs of beening main stream... X LaTeRz X
Album Rating: 2.0Border Wars is an amazing song. But album is not.
Album Rating: 2.0i'm usually a defendant of the Green era but this is pretty poor
Album Rating: 1.5terrible
Album Rating: 2.0Yeah it sucks ass besides "Border Wars"
Album Rating: 1.5cant remember it lol
Album Rating: 1.5i think ur forgetting deliverance came out the year after
Album Rating: 2.0sylosis...ugh
Album Rating: 3.5I agree with the description. An "underrated album". Good songs, great stuff and versatile!
you have it inside, Max! you should never have quit the band!
Album Rating: 1.5lol
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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Dream Lounge
- (Digipack Packaging)
- Artist: Janina Parvati
- Format: CD
- Release Date: 5/19/2015
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Beautiful melodies, gentle rhythms and ambient soundscapes invite you to calm down leaving all everyday sorrows behind. Imagine yourself lying on a remote beach, watching the endless ocean and fell the peacefulness of your innermost being coming closer.
To play the media you will need to either update your browser to a recent version or update your version of Flash Player.


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... capsules?
I am a senior and part D Medicare does not include benzonatate for coughs. My cough is from scar tissue in the lungs, not a cold.
I'm looking for a substitute on my health ins. formulary. Medicare denied me after I sent an appeal. The Rx is $135. Per month! I am on a SS fixed income.
How can they deny me when my cough is not due to a cold? Any suggestions? I'm desperate as the benzonatate is the ONLY medication that actually works for me. The other narcotic and non-narcotic pill and elixir do nothing.
Thank you!❤
Tessalon - Is there another Rx tablet which can be used as a substitute to benzonatate 200 mg.?
- Asked
- 7 Aug 2016 by Croniccough
- Active
- 23 Jan 2017
- Topics
- tessalon, cough, benzonatate, medicare, formula, scar tissue, scar, tablet, health, lungs, lung, capsule, tissue
... capsules?
Responses (2)
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Benzonatate - I had a bad bronchial cough. I found some 200mg tessalon perles which expired in 2007?
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Companies are looking for ways to get more customers and leads. Why not try social media digital marketing? Learning and trying something new is just trying to keep up. Things change fast in the world of business. Making sure to explore new topics and strategies is just the name of the game.
People have smartphones and tablets and they make it easy to get started with social media applications. Learning how to use digital marketing social media means investing time or finding other options.
Getting information fast means plugging into the social media twitter and other sites. The information flows fast online and coming up with a plan for using the platforms might be outside of the thinking of some business owners. The large numbers of social media users means that it is a factor.
The twitter social media might be different from others. Each social network will have its norms. Getting up to date with the platforms and finding out how they work is essential. Software applications move things forward. Just think about how much software has changed things over the years.
Some businesses might ignore social media as a passing fad. Others might take the wheel and decide to learn how it works and check for the potential upside. Lots of people are using social media accounts and it is an opportunity to connect.


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watch chronograph man Fossil Other
Code: CH2869
€ 117.30 € 159.00 You save € 41.70
This article is no longer produced by parent company and so the purchase does not guarantee the necessary spare parts and technical assistance.
Product Info
Product: watch chronograph man Fossil Other CH2869
Fossil Watch Men Of The Other Collection - Mens. With Dater Chronograph Fossil Watches Collection Of Holiday 2015. The Clock Is Quartz Movement, Of 45 Cm Case Diameter, Cash Material Plated Steel Grey Ip With Steel Strap Plated Grey Ip, water resistance to 10 ATM. New Arrivals in January 2015, Ref: Ch2869
this chronograph will be shipped with the original box Fossil
30 days to return the product
this chronograph is shipped worldwide with UPS
Features chronograph CH2869 Fossil
brand: Fossil
package: original box Fossil
Product Guarantee: International Warranty Fossil
kind: man
category: watches
Subcategory: chronographs
style: trendy
producer code: CH2869
collection: Other
Bracelet Colour: Steel
Case Material: Steel
Case size: 45mm
Watch Bracelet material: Steel
Watch Digits: Hour Markers with stick shape
Watch Shape: Circular
Water Resistance: 100m - 10ATM - 10bar
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New Delhi, Sept. 3: Delhi High Court has asked health minister Anbumani Ramadoss to sign AIIMS degree certificates within 24 hours.
Ramadoss has been refusing to sign the certificates of AIIMS pass-outs over his long-running feud with the institutes director, P. Venugopal.
The minister had blamed Venugopal for the delay in issuing the certificates, following which the Resident Doctors Association had gone on a two-day strike. The protest had thrown patient services in the countrys premier medical institute into disarray and prompted the high court to intervene.
Over 700 such certificates are pending with the institute for the last two years.
Justice S. Ravinder Bhatt said: The Union health Minister shall affix his signature on 49 certificates/degrees as well as such other certificates of students who need these to take admission for higher studies. The minister shall sign the certificates within 24 hours.
The court directed AIIMS officials to immediately forward these certificates to the ministers office so that they can be flown to Chennai for his signature.
The judge also made a reference to the feud between the minister and the director, saying: You are using the court as a tool which I wont allow. You are resorting to shadow boxing.
AIIMS Resident Doctors Association secretary Krishna Kant Mishra hailed the court decision. The high court has solved a big problem.
The BJP was quick to demand the health ministers resignation. It is a major embarrassment for the UPA government, and the minister should resign on moral grounds, it said.
The certificates bear four signatures, that of the AIIMS director (Venugopal), the registrar, the dean and the AIIMS president (now Ramadoss).
But with Venugopal refusing to accept Ramadoss nominee V.P. Gupta as the registrar over his own, Sandeep Agrawal, and the matter pending in the high court, Ramadoss had refused to sign any certificate.


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In this episode, we'll discuss some basic tips on starting your own garden, no matter WHERE you live, as a hedge against inflation, food storage or for a healthier lifestyle! (And anyone who's ever eaten tomatoes grown in THEIR garden can attest to that!). Tips on difficulty, a little history & some basic plants to get you started.
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