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Genomewide comparative analysis of alternative splicing in plants
Communicated by Vicki L. Chandler, University of Arizona, Tucson, AZ, March 15, 2006 (received for review September 6, 2005)
Alternative splicing (AS) has been extensively studied in mammalian systems but much less in plants. Here we report AS events deduced from EST/cDNA analysis in two model plants: Arabidopsis and rice. In Arabidopsis, 4,707 (21.8%) of the genes with EST/cDNA evidence show 8,264 AS events. Approximately 56% of these events are intron retention (IntronR), and only 8% are exon skipping. In rice, 6,568 (21.2%) of the expressed genes display 14,542 AS events, of which 53.5% are IntronR and 13.8% are exon skipping. The consistent high frequency of IntronR suggests prevalence of splice site recognition by intron definition in plants. Different AS events within a given gene occur, for the most part, independently. In total, 36–43% of the AS events produce transcripts that would be targets of the non-sense-mediated decay pathway, if that pathway were to operate in plants as in humans. Forty percent of Arabidopsis AS genes are alternatively spliced also in rice, with some examples strongly suggesting a role of the AS event as an evolutionary conserved mechanism of posttranscriptional regulation. We created a comprehensive web-interfaced database to compile and visualize the evidence for alternative splicing in plants (Alternative Splicing in Plants, available at www.plantgdb.org/ASIP).
Alternative splicing (AS) is an important posttranscriptional regulatory mechanism that can increase protein diversity and affect mRNA stability (1, 2). Relative to the predominant transcript isoform, different types of AS have been observed, including exon skipping (ExonS), alternative donor (AltD) or acceptor (AltA) site, and intron retention (IntronR) (Fig. 1; reviewed in ref. 3). AS has been extensively studied by EST/cDNA-based analysis in mammalian systems, and 35–60% human genes were suggested to be alternatively spliced (4–9). In humans, ExonS is the most common type (58% of the total number of AS events are ExonS events involving a single exon, and 11% are ExonS events in which multiple exons are skipped in tandem) (4). IntronR is the least common type of AS in humans (5%) (4); ≈70–88% of the AS events occur in protein coding regions (reviewed in ref. 10), and approximately one-third produce premature termination codons (PTCs) (11). These PTC-containing transcripts are apparent targets for non sense-mediated mRNA decay (NMD) (11). Not all of the predicted AS are real and functional, because many possible sources of false positives exist (10). An AS event has been defined as functional "if it is required during the life cycle of the organism and activated in a regulated manner" (12). To identify functional AS events, conserved AS events between human and mouse were studied (12–17), with the assumption that conservation indicates function. Twenty-five percent of a set of 980 ExonS events in human were found to be conserved in mouse (12). Another study reported that ≈10% of human gene loci with mouse orthologs show conserved AS events (15).
The splicing mechanism in plants is generally conserved compared with mammals (18, 19). However, introns in plant are usually short in length and U-rich (18–20), with a much less apparent polypyrimidine tract near the 3′ splice site than in mammals (21). Our recent genomewide survey on Arabidopsis splicing-related genes revealed variations in SR proteins and hnRNP proteins between plants and mammals, suggesting plant-specific differences in splicing-regulation mechanisms (22). A few AS events were identified experimentally in plants, including genes involved in splicing (23, 24), transcription (25), flowering regulation (26), disease resistance (27), enzyme activities (28, 29), and many other physiological processes and functions (19). A database (PASDB) collecting known alternatively spliced genes in plants is available at http://pasdb.genomics.org.cn (30). Although the prevalence of AS is not yet clear in plants, it is now recognized as playing an important role in the generation of plant proteome diversity (31).
Computational studies of AS in plants have recently been published. Ner-Gaon et al. (32) identified 436 alternatively spliced genes in Arabidopsis by EST-pair alignment. The fraction of IntronR observed in their study was as high as 64%. A sampling of the IntronR events were confirmed by RT-PCR with polyribosome RNA, demonstrating that these IntronR events are not the byproduct of incomplete splicing (32). Iida et al. (33) aligned 248,514 RIKEN Arabidopsis full-length cDNA/EST sequences to the Arabidopsis genome by using a blast-based method. They identified 15,214 transcription units (TUs) containing at least two sequences each and observed alternative splicing for 11.6% of these TUs (33). Three other studies with a smaller collection of EST/cDNA data briefly reported fewer AS events in Arabidopsis (9, 34, 35). All these pioneering studies revealed that a low fraction of genes (5–10%) are alternatively spliced, with IntronR the most prevalent AS type in Arabidopsis. However, none of the above studies included detailed analyses on the position and outcome of AS. Two recent studies with full-length cDNAs reported similar fractions of alternatively spliced genes but conflicting predominant AS types and positions of AS events relative to the coding regions (36, 37). Systematic analysis of AS in other plants has not been reported so far.
Compared with human, the lower fraction of alternatively spliced genes detected to date in plants is possibly due, in part, to lower cDNA/EST coverage. Millions of ESTs were used in human AS analyses (9), whereas less than one-10th of that number were available for Arabidopsis (9, 32–35). The number of publicly available plant cDNA/EST sequences has increased dramatically since the original studies, and, thus, it seemed likely that more AS events would be identified by using current data. Because the rice genome sequence has recently become available (38, 39) and differences are known to exist in the splicing mechanisms of monocot and dicot plants (19), it is of great interest to explore the AS events in rice and compare them with Arabidopsis. In this study, we applied the geneseqer spliced-alignment program (40, 41) to map currently available Arabidopsis and rice full-length cDNAs and ESTs to their respective genome sequences and identified thousands of AS events by exhaustive comparison of the deduced transcription units. The alternatively spliced genes were comparatively analyzed, and a small portion of the AS events were found to be conserved in the two plants. These data strongly suggest that, similar to mammals, AS occurs in plants on a large scale as a mechanism of regulation of gene expression. A user-friendly database has been constructed to store and visualize these AS events, which is frequently updated to reflect increases in cDNA/EST collections in both plants.
Results and Discussion
Genomewide EST/cDNA Alignments in Arabidopsis and Rice.
A total of 95.8% and 85.7% of the current Arabidopsis and rice EST/cDNA collections could be unambiguously aligned to their respective genomes by using the geneseqer spliced alignment program (40). The unaligned ESTs/cDNAs are either from the organelle (chloroplast and mitochondrion) genomes or different subspecies or are short and low-quality sequences. In total, 369,218 Arabidopsis ESTs/cDNAs were matched to the genome and producing 372,772 cognate alignments (see Materials and Methods). As shown in Table 3, which is published as supporting information on the PNAS web site, <1% of the EST/cDNAs have multiple-cognate alignments. A total of 25,231 TUs were identified, 23,856 (94.6%) of which correspond to annotated gene regions and 1,375 (5.4%) to previously uncharacterized gene regions. The average number of ESTs/cDNAs per TU is 14.8. In rice, 283,816 ESTs/cDNAs produced 319,391 cognate alignments, with ≈3% of the aligned EST/cDNAs showing multiple cognate alignments. The high proportion of multiple cognate alignments relative to Arabidopsis presumably reflects recent gene duplications in rice (42). We defined a total of 36,270 rice TUs, 87.7% of which overlap with annotated genes and 12.3% are in previously uncharacterized regions. The average number of ESTs/cDNAs per rice TU is ≈8.8. Rice introns are generally longer and have higher GC-content compared with Arabidopsis introns (see Supporting Text, Table 4, and Fig. 3, which are published as supporting information on the PNAS web site).
Approximately One-Fifth of Expressed Genes Are Alternatively Spliced in both Plants.
As described in Table 3, a total of 21,641 Arabidopsis genes (including 1,375 previously uncharacterized genes that did not overlap any annotated gene) and 30,917 of rice genes (4,466 previously uncharacterized genes) were defined as "expressed genes" by comparing the GenBank and The Institute for Genomic Research (TIGR)-annotated genes with our TUs. In Arabidopsis, 4,707 (21.8%) of the expressed genes display a total of 8,264 AS events (Table 1). Compared with recent estimates of 11.6% based on RIKEN Arabidopsis full-length cDNA sequences (33) and <5% indicated in a TIGR study (35) and other previous estimates (9, 34), our AS ratio is much higher. This increase may be because of the use of (i) more recent, larger EST/cDNA collections and/or (ii) a more sensitive AS detection method. Our Arabidopsis EST/cDNA collection includes with few exceptions all of TIGR's collection and ≈176,000 new sequences not included in the TIGR analysis. As shown in Fig. 2, our list includes 844 of the 909 (92.8%) TIGR-annotated alternatively spliced genes (excluding 279 genes with AS types involving terminal exons not discussed here). Sixty-five genes from the TIGR list are absent in our collection. Among these genes, three AS events actually are presented in our study under different gene names (because of annotation changes), 21 genes are really missed by our method, and the remaining 41 genes are likely false annotations given the lack of cognate EST/cDNA evidence (see Table 5, which is published as supporting information on the PNAS web site). In addition, 10 ExonS cases identified by TIGR lack EST/cDNA evidence, although other types of AS events were identified in Alternative Splicing in Plants (ASIP) for these genes (Table 5). Our study identified 3,863 alternatively spliced genes not identified previously in the TIGR analysis, 2,712 (70%) of which can be attributed to the previously uncharacterized transcript sequences that have become available since TIGR's study, whereas the remaining additional cases are method-specific (detailed data is available upon request).
As for the 1,431 RIKEN-annotated alternatively spliced genes showing AltD/AltA, ExonS, or IntronR, 798 of them (55.8%) were included in our list. We did not perform a detailed comparison between RIKEN′s and our AS list, because only approximately two-thirds of RIKEN′s data set (as available at National Center for Biotechnology Information at the time of this study) were included in our EST/cDNA collection, and ≈200,000 EST/cDNA sequences now available were not included in the RIKEN study (further details available upon request). Another study by using pairwise EST comparisons detected 436 alternatively spliced Arabidopsis genes (32), 418 of which (95.9%) are included in our database.
In rice, the overall AS ratio is very similar to that of Arabidopsis. Approximately 21.2% (6,568 of 30,917) of the expressed genes showed a total of 14,542 AS events (Table 1). The TIGR rice genome annotation project identified 2,538 alternatively spliced genes (43) (http://rice.tigr.org/tdb/e2k1/osa1/expression/alt_spliced.info.shtml) showing all five of the AS types discussed here or alternative initiation or termination sites, which were not considered here (see Materials and Methods). The TIGR gene list is not broken up by AS type, and, therefore, we can only estimate overlap with our determination. Because ≈2,014 (80%) of the TIGR-annotated alternatively spliced genes (including other splicing variation types, roughly 23% as estimated from the TIGR Arabidopsis study by using the same program) are included in our database, we are confident that the vast majority of the TIGR-annotated five AS types considered here also were detected by our method. Thus, in summary, we estimate the occurrence of AS in both Arabidopsis and rice at ≈20% of all expressed genes.
Intron Retention Is the Most Prevalent AS Type in both Arabidopsis and Rice.
Among the five AS types in Arabidopsis and rice, Table 1 indicates IntronR as the most prevalent type (>50% of AS events), followed by AltA, AltD, and ExonS. Alternative position (AltP) is the least prevalent type. IntronR in Arabidopsis recently was demonstrated to be a bona fide AS event instead of merely the product of incomplete splicing in a study by using ribosome-associated mRNAs as template for RT-PCR (32). Although it is possible that some partially processed pre-mRNA are exported to the cytoplasm and associate with ribosomes, it is difficult to distinguish these events from regulated AS events. For both Arabidopsis and rice, ≈10% of IntronR cases in our database derive from transcripts with multiple introns that are all retained. These transcripts may result from sampling unprocessed pre-mRNAs or genomic contaminations in the cDNA library construction. We did not remove these events because they also may be real AS events, but in any case, the general conclusions regarding frequencies of AS types remain the same. IntronR is prevalent in introns of short size and high GC content (see Supporting Text; see also Fig. 4, which is published as supporting information on the PNAS web site).
Compared with AS events in human, where ExonS is the most abundant (4), the high frequency of IntronR in plants may reflect distinct features of plant pre-mRNA splicing. Mammalian genes with long introns and small exons are likely spliced by using the exon definition mechanism (44). This hypothesis is supported by the observation that mutations of mammalian splice sites most frequently lead to ExonS (45). In contrast, the intron definition mechanism may be prevalent in species in which genes typically have small introns (44). As plant introns are generally short and likely recognized by intron definition, failure of splice-site recognition would mostly lead to IntronR. The observed AS frequency differences between plants and human are consistent with this model. As observed AS events are likely a mixture of regulated events and unregulated events (splicing errors), it is not surprising to see a high frequency of ExonS in species in which the exon definition mechanism may be prevalent (human) and a high frequency of IntronR in species in which the intron definition mechanism may be prevalent (Arabidopsis and rice). Our result that short introns and introns with high GC content are more likely to be retained further supports the intron definition mechanism in plants (see Supporting Text). If an intron is too small or has a high GC content, steric hindrance or lack of typical intron-recognition signals may lead to inefficient splice-site recognition and, concomitantly, to a high frequency of IntronR. On the other hand, the exon definition mechanism appears to apply to some plant introns, because the mutation of certain plant splice sites lead to ExonS (46, 47). Comparing Arabidopsis and rice, we note a prevalence of relatively long introns in rice and an ≈5% higher incidence of ExonS in rice. We propose that in both plants, the intron definition mechanism is the predominant mode of intron recognition and exon definition is a more prominent mechanism of intron recognition in rice than it is in Arabidopsis.
Most AS Events Are Mutually Independent.
For most of the identified alternatively spliced genes (3,140 or 67% in Arabidopsis and 3,968 or 60% in rice), only a single AS event has been observed, and for the genes with multiple AS events, most transcripts contain only a single AS event. Because our analysis is based mostly on partial cDNA sequences, the latter observation includes transcripts not long enough to cover multiple AS events in a gene. However, detailed analysis showed that ≈56% of alternatively spliced transcripts covering the locations of multiple alternatively spliced introns in a gene actually only showed a single AS event relative to the constitutive transcript. Of 1,088 Arabidopsis genes and 1,813 rice genes with multiple AS events not limited to IntronR, a small fraction, 115 (10.6%) and 134 (7.4%), respectively, have multiple events within the same transcripts and no single AS event transcripts, suggesting that in these genes, multiple AS events are coordinated (see www.plantgdb.org/ASIP/EnterDB.php for a listing of these genes in ASIP). However, most AS events appear to be mutually independent.
More than One-Third of AS Events May Be Coupled with NMD.
More than half of the observed AS events alter the reading frame and possibly generate an early stop codon (Supporting Text; see also Table 6, which is published as supporting information on the PNAS web site), which renders the alternative mRNA isoforms possible candidates for NMD. PTCs in human were defined as in-frame stop codons residing >50 bp upstream of the 3′ most exon–exon junction (48). Arabidopsis homologs of human proteins involved in NMD and the exon junction complex have been identified (22). It seems that plants also have an NMD-like surveillance system, as suggested by the observation of increased PTC+/PTC− mRNA isoform ratios in a mutant of Arabidopsis UPF3, a homolog of a factor required for NMD in mammals and yeast (49). To investigate whether the 50-bp PTC rule applies in Arabidopsis, we screened a set of 4,868 Arabidopsis genes, for which the annotated longest ORF is fully supported by cDNA evidence and, therefore, is presumably within the constitutively spliced isoform. Fig. 5, which is published as supporting information on the PNAS web site, shows a histogram of the distances between the stop codon and last exon–exon junction in these genes. In 4,675 of the transcripts (96%), the stop codon occurs within the last exon. For 117 of the remaining 193 genes, the distance between the stop codon and the last exon–exon junction is <50 bp. Thus, overall >98% of Arabidopsis genes fit the human pattern of stop codon positioning close to or downstream of the last exon–exon junction. Therefore, we treat as potential substrates for NMD all AS isoforms producing PTCs defined as in-frame stop codons >50 bp upstream of the 3′ most exon–exon junction.
As summarized in Table 6, ≈43% Arabidopsis AS events and 36% rice events produce NMD candidates. These frequencies are similar to the frequency observed in human (35%) (11). Among the five types of AS events, AltP has the lowest incidence of NMD (18–22% of all AltP events), IntronR has the highest (40–48%), and AltA, AltD, and ExonS have frequencies at ≈34–43%. Because NMD is a surveillance mechanism that removes erroneous mRNAs containing PTCs (50), the one-third of AS events that could generate NMD candidates will produce much less protein diversity than might be expected. The NMD mechanism also can be routinely used by the cell to regulate gene expression (51), therefore coupling of AS and NMD could be an important posttranscriptional regulation to adjust the level of transcript isoforms (2).
Conserved Alternatively Spliced Genes.
Gene ontology annotation analysis on the alternatively spliced genes showed consistency between Arabidopsis and rice but no strong biases with respect to occurrence of AS events (see Supporting Text). By further searching Arabidopsis and rice close homolog pairs generated from reciprocal blast best hits (see Materials and Methods), 1,988 (41.7%) of the 4,707 alternatively spliced genes in Arabidopsis were found to have close homologs in rice that are also alternatively spliced and 1,154 of these gene pairs conserved the same AS type (Table 2). Among the five AS types, IntronR is most likely to be conserved, with ≈30% of the intron-retaining genes in Arabidopsis also showing IntronR in rice (Table 2). It should be noted, however, that the conserved alternatively spliced genes do not necessarily conserve the same AS event. For instance, Arabidopsis gene At1g28570 and rice gene LOC_Os01g46120 (GDSL-motif lipases) have similar gene structure and both have ExonS in the ORFs. In the Arabidopsis gene, the second exon is skipped, whereas in the rice gene, the fourth exon is skipped. Details regarding these conserved genes are available from the ASIP database (www.plantgdb.org/ASIP/EnterDB.php).
To identify conserved AS events, we first searched for conserved intron pairs as described in Materials and Methods. A total of 4,142 genes containing 7,640 conserved introns were identified in Arabidopsis, including 352 genes (445 introns) that are alternatively spliced. The frequency of genes containing conserved introns being alternatively spliced is ≈8.5%, much lower than the overall AS frequency (≈22%). As shown in Table 2 (and Table 7, which is published as supporting information on the PNAS web site), within this set of genes we could identify 41 Arabidopsis genes with AS events that are conserved in rice (same type of AS event for the conserved intron pair). Eleven of these events (26.8%) maintained the constitutive reading frame, and 22 (53.6%) produce NMD candidates.
The intron-pair method can identify only conserved introns in coding regions where the flanking exons are well conserved between Arabidopsis and rice. Therefore, we also visually compared a large number of homologous gene structures in Arabidopsis and rice and selected those with the same intron alternatively spliced. A small portion of additional conserved AS events were identified in this way, including U1-70K (U1 snRNP-70K, At3g50670, and LOC_Os10g02630), Tra2/SFRS10 (Transformer-2-β, At1g07350, and LOC_Os03g15890), and polypyrimidine tract binding protein (PTB; At3g01150, At5g53180, and LOC_Os01g43170). For most of the homologous gene pairs with conserved AS events, the AS event has similar effects on translation. For example, both Arabidopsis and rice IDH2 genes have three introns and can use an alternative acceptor site 12 bases downstream for splicing the first intron. A protein with four amino acid residues (VITK) removed will be generated from the alternative mRNA isoform in both Arabidopsis and rice. The PTB genes in both rice and Arabidopsis show transcripts, which include an additional exon in their third introns and produce PTC-containing mRNA isoforms. The mammalian PTB gene can autoregulate its splicing by promoting the skipping of exon 11, which introduces a PTC and leads to NMD (52). This regulation pathway may be conserved in plants. As shown in Supporting Text (and Fig. 6, which is published as supporting information on the PNAS web site), RT-PCR by using mixed Arabidopsis tissues validated the AS events in the atIDH2 and atPTB2a genes.
Interestingly, 30 conserved intron pairs and many more conserved gene pairs have different AS types in Arabidopsis and rice. For instance, At3g60370 and LOC_Os07g30800 (FKBP-type peptidyl-prolyl cis-trans isomerase) are orthologs with similar gene structure. Splicing of the third intron in At3g60370 can use an alternative acceptor site that generates a PTC-containing isoform. Splicing of the third intron in LOC_Os07g30800 alternatively includes an additional exon, which also produces a NMD candidate. Although the two genes have different AS types, it may be that the function of their AS events is conserved.
Based on our large-scale EST/cDNA alignments to the genomes of Arabidopsis and rice, we estimate that more than one-fifth of plant genes are alternatively spliced. Clearly, not all of the thousands of AS events will be biologically functional. Aberrant splicing will inevitably occur during the complicated dynamic splicing process that is happening continuously and under varied physiological conditions. Most aberrant splicing (splicing errors) will be removed by mRNA surveillance mechanisms such as NMD and, thus, are neutral to the organism. In the course of evolution, some splicing variants may be selectively beneficial and, thus, can be fixed as functional AS events.
How to distinguish splicing errors from biologically functional AS events remains an unresolved question. Interruption of protein coding sequences and/or production of PTC are not necessarily good landmarks for splicing errors, because coupling of AS and NMD may be regulated and add another level of regulation to gene expression. Conservation of AS events seems to be a good indication of functional AS events. Several papers addressed conserved cassette exons between human and mouse (12, 15, 17). We have shown that approximately one-quarter of the alternatively spliced Arabidopsis genes undergo the same type of AS in rice, and we identified a small number of conserved events among those. We also experimentally validated a few AS events in Arabidopsis by RT-PCR (see Supporting Text and Fig. 6). The web-interfaced ASIP database can serve as a starting point for the community to perform additional experiments, including high-throughput methods such as oligonucleotide microarrays studies, to identify functional AS events in plants.
Materials and Methods
Data Sources.
Our initial analysis was based on a total of 323,340 Arabidopsis ESTs that were downloaded from GenBank by using the ENTREZ query "Arabidopsis[ORGN] AND gbdiv_est[PROP]." Arabidopsis full-length cDNAs were retrieved from GenBank (query "Arabidopsis[ORGN] AND (FLI_CDNA[KYWD] OR GSLT_cDNA [KYWD])," with some additional sequences obtained from AtGDB (www.plantgdb.org/AtGDB/resource.php) for a total of 62,009 sequences. For rice (Oryza sativa), sets of 298,857 ESTs and 32,136 cDNAs were obtained from GenBank by using similar queries. Updates to our database of plant AS events include more recent sequence depositions, but the analysis results reported here reflect only the sequences available at the time. The five Arabidopsis chromosome sequences were obtained from GenBank (accession nos. NC_003070, NC_003071, NC_003074, NC_003075, and NC_003076). For rice, our analysis was based on TIGR 3.0 of the 12 pseudochromosome sequences (downloaded from http://rice.tigr.org/tdb/e2k1/osa1/pseudomolecules/info.shtml).
Identification of AS in Arabidopsis and Rice.
Arabidopsis and rice full-length cDNAs and ESTs were aligned against their corresponding genomic origins by using the geneseqer (40) with species-specific splice site models (41) and the minimum allowed exon and intron lengths set conservatively to 2 (53) and 20 (54, 55), respectively. More information regarding the minimum intron size is available upon request. unix shell scripts and perl scripts were written to automate the following steps: (i) Loading of geneseqer alignment data was performed, including the predicted exon and intron coordinates and similarity and splice site scores, into a mysql database. (ii) Filtering of the alignment data was performed by using only high-quality alignments (overall similarity and coverage scores at least 0.8). For ESTs/cDNAs matching multiple genomic loci, only the best match (presumed cognate) was used, as described in ref. 34. For highly conserved duplicated genes, there may be multiple locations with equal score. In such cases, all these alignments were used. Exons with individual similarity scores <0.8 and their flanking introns were removed from the remaining alignments. Introns with a local similarity score <0.8 for either the 50 bp upstream or downstream flanking exon sequences also were removed. Redundancy in the sets of qualified exons and introns was eliminated. For any pair of terminal exons sharing the same exon/intron border only the longer exon was retained. (iii) For each intron, its coordinates were compared with each overlapping exon and intron to identify AS events. Two overlapping introns with different 5′ and/or 3′ ends, and all overlapping intron/exon pairs were considered as candidate AS events. For overlapping introns, the intron with the most cDNA/EST evidence was taken to be the constitutive intron. Several cases of AS were distinguished as follows (cf. Fig. 1). Use of an AltD/AltA is defined as an intron differing from the constitutive intron only in the donor or acceptor site. AltP refers to an intron overlapping the constitutive intron but differing in both donor and acceptor site position. ExonS occurs when an exon in one transcript isoform is completely contained in an intron of another isoform. IntronR occurs in a transcript isoform that contains a sequence segment that is precisely spliced out in another transcript isoform (i.e., an intron is fully contained in an exon in our database). (iv) Other transcript variants, including alternative terminal exons and alternative initial exons, were not considered. These events may involve the coupling of other mechanisms such as alternative transcription initiation sites with splicing (56). Candidate exon skipping events involving first or last exons also were removed, because these events also constitute examples of alternative initial or terminal exons, respectively.
Web Interface and Visualization of AS.
Diagrams of different AS types at ASIP are illustrated in Fig. 1. For AltD, AltA, and AltP events, a vertical color bar at the exon/intron border denotes the alternative sites. Multiple alternative sites are distinguished by different colors. For ExonS events, a green box within an intron indicates the position of the skipped exon. To visualize IntronR events, the intron is drawn in a light green color in transcripts from which it is spliced.
Derivation of Putative Transcript Isoforms (PTIs).
To classify AS events with respect to their impact on the translation of the alternatively spliced transcript, it is necessary to deduce the likely full-length alternative transcripts from overlapping ESTs (in addition to experimentally obtained full-length cDNA isoforms). For the purpose of this study, we make the assumption that all AS events within a TU (as defined in ref. 58, with the caveat that because of incomplete coverage, there may be multiple TUs per annotated gene; see Table 3) are mutually independent, and, thus, we derived all possible combinations of alternative exons to generate the set of PTIs for each transcription unit. To do so, we first clustered all exons and introns from our database into sets corresponding to distinct transcription units and then assembled complete gene structures by concatenating compatible exons and introns in the 5′ to 3′direction. This approach is different from TAP (8), which relies on a reference sequence structure, and also from PASA (35), which assembles only observed combinations of exons. With tightly spaced genes, assignment of some ESTs to a particular transcription unit may be ambiguous. However, this ambiguity, did not affect our evaluation of AS, because events involving terminal exons were excluded. The positions of the start and stop codons for each PTI were determined by searching for the longest ORF in the forward direction for PTIs with introns and in both directions for PTIs without introns for which the direction of transcription is ambiguous.
Position and Outcome of AS Events.
To classify AS events relative to their predicted effect on mRNA translation and stability, the constitutive ORF was defined as the longest ORF with the most abundant EST/cDNA evidence in all of the PTIs from the same gene. Any two PTIs differing in only one AS event were then compared. If multiple PTI pairs contain the same AS event, only the pair with the longest ORF was considered for classification of that AS event. If both ORFs from the PTI pair differ from the defined constitutive ORF, then the longer ORF from the PTI pair was redefined as the constitutive ORF for this comparison. To check whether an AS event will generate a transcript that contains premature stop codons and could be degraded by NMD, the distance between the stop codon and the last exon junction was calculated for both PTIs. If one isoform had a distance >50 nt, whereas the other isoform had a distance <50, then the AS event was regarded to produce a NMD candidate. If the AS event produced a PTC in one PTI relative to the other, this stop codon was used to calculate the NMD distance.
Conserved Alternatively Spliced Genes in Arabidopsis and Rice.
To identify conserved AS events between Arabidopsis and rice, all annotated Arabidopsis proteins were matched against all annotated rice proteins, and vice versa, by using blast (59). We used a 1 × 10−20 as the cutoff for blast and labeled the highest scoring hit as "uniquely best" if its value was at least 1 × 10−20 times lower than that of the next highest scoring hit. Reciprocal uniquely best hits were selected as close homolog pairs (potential orthologs). If there was evidence in both genes for the same type of AS event, this gene pair was characterized as conserving AS, although not necessarily the same position and outcome of AS. To further identify conserved AS position, we first derived the set of conserved introns between Arabidopsis and rice by matching all Arabidopsis introns plus 30-bp flanking sequences against rice intron and flanking sequences by using tblastx (value <1 × 10−4 and requiring both flanking exons to have at least 10 bases as part of the blast hits). The subset of these introns that occur in close homolog pairs were defined as conserved introns between Arabidopsis and rice. Conserved AS events were defined as AS events of the same type occurring in conserved introns.
Supporting Information.
For more information, see Figs. 7–9 and Table 8, which are published as supporting information on the PNAS web site.
We thank Wei Huang for help with statistical analysis and Robert Fluhr, Qunfeng Dong, Yuanbin Ru, and Michael Sparks for critical reading of the manuscript. This work was supported in part by National Science Foundation Grants DBI-0110189 and DBI-0321600 and Research Grant IS-3454-03 from the United States–Israel Binational Agricultural Research and Development Fund.
- ‡To whom correspondence should be addressed. E-mail: vbrendel{at}iastate.edu
Author contributions: B.-B.W. and V.B. designed research; B.-B.W. performed research; B.-B.W. contributed new reagents/analytic tools; B.-B.W. and V.B. analyzed data; and B.-B.W. and V.B. wrote the paper.
Conflict of interest statement: No conflicts declared.
- Abbreviations:
- AltA,
- alternative acceptor site;
- AltD,
- alternative donor site;
- AltP,
- alternative position;
- alternative splicing;
- alternative splicing in plants;
- ExonS,
- exon skipping;
- IntronR,
- intron retention;
- non-sense-mediated decay;
- premature termination codon;
- putative transcript isoform;
- The Institute for Genomic Research;
- transcription unit.
- © 2006 by The National Academy of Sciences of the USA
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text:
Learn To Read Variety Pack 9: Guided Reading Level E-F
Support Essential Early Learning Concepts And Skills While Building Reading Confidence
Tied to content areas, these leveled readers make the perfect complement to any beginning reading program. Stories include a boy talking with his hands, a girl demonstrating proper hygiene, and counting to 10 with dancing dinosaurs. The books feature skills such as compound words, state names, and rhyming words. Each book includes either related activities or listings of content-area concepts, sight words, phonics, vocabulary words, and related learning skills.
Pay in 3 easy payments.
Your satisfaction is our priority.
Good Choices for Cat and Dog
Pilgrim Children Had Many Chores
I Can Talk with My Hands
The Little Green Man Visits a Farm
I Need to Clean My Room
Apron Annie in the Garden
Matthew the Magician
This Is the Way
Dinosaurs Dancing
Five Little Monsters
A-Counting We Will Go
- 12 softcover books
- Size: 9" by 6¼"
- 16 pages each
Grades: K-1

subset: commoncrawl


text:
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text:
Maglia, a passion for umbrellas since 1854
Despite his 6.5 feet in height and white hair, Mr. Maglia has got the face of a witty child. He is the last umbrella craftsman in Italy, the fifth "Francesco" in the family, and after a hundred and sixty years he is still manufacturing umbrellas together with his brother Giorgio, in the Milan family business.
The secret of this tradition is in the materials. it takes 70 steps to make an umbrella entirely by hand: they start from the walking stick, then choosing the right wood among rattan, ash, olive, chestnut with and without bark, knotty bamboo and hazel. But also maple, cherry and broom. Then two springs, a double nut and one canola are attached. These pieces are joined with haywire to 8 to 10 steel slats, then they proceed on to the fabric, which is no longer silk, but polyester and cotton. After cutting to size, the fabric is sewn by hand.
The cover can be polka-dotted, Regimental, plain, stripes, Prince of Wales or jacquard, but never printed tissues. Finally the choice of the stick-handle: high-quality hand-stitched leather in different colors. In the "hunting umbrella" niche, they use horns, antlers of the deer and sheep, roots twisted to art by nature and even exotic warthog teeth. The process ends with the steam ironing, open umbrella.
Maglia follows the philosophy of a job well done and perpetuates craftsmanship. Each stage of production is carried out at the Maglia headquarters "old Milan" in Ripamonti Street, where, through a refined execution, the bespoke umbrella is created.
Each year Maglia produces 15 thousand long umbrellas and five thousand telescopic ones, dressed in waterproof fabrics that were made on ties frames.
The company's style is a classic one, following the dictates of the English line: 90 percent of the production is destined abroad, following orders coming from legendary shops such as James Smith & Sons umbrellas in London, well-known department stores in America and Japan, and great fashion designers. In Italy only a few famous but discreet tailors and boutiques carry this product, such as the historical Guenzati e Sacarazzini in Milan, F.lli Cilento in Naples, Flanella Grigia and Sagripanti in Rome and Bocache & Salvucci, world-famous craftsmen shoemakers.
The company is constantly looking for new fabrics. In the last few years, at the urgent request of the Japanese and American market, the company has resumed the manufacture of fine lace and natural fibers parasols with refined handles such as bamboo, horn and silver, adorned with frills, chinesino or trimming.
Maglia's passion has no equal in Italy, like the umbrella museum in Gignese, unique in the world.
|That of Maglia is a passion that has no equal in Italy, like the umbrella museum in Gignese, unique in the world.
Maglia Umbrellas:
- Via Ripamonti 194, Milano (Mi)
- +39 02 55219333
- +39 02 5696977
- [email protected]
- www.ombrellimaglia.it


text:
Jeffrey Reviewed on Jan 29, 2019, 12:54 PM
Got to office a bit early but was attended to immediately with a smile
81-83 flinders st east
Average Pick-Up time: 21-30 minutes
Jeffrey Reviewed on Jan 29, 2019, 12:54 PM
Got to office a bit early but was attended to immediately with a smile
townsville townsville airport
Average Pick-Up time: 11-20 minutes
Alison Reviewed on Jan 14, 2020, 3:20 AM
Great service, have used Budget for our last two car rental
Ron45 Reviewed on Aug 6, 2018, 9:39 PM
All went well and smnoothly. The car was very clean. Staff most helpful.
townsville airport, qld, aust in terminal
Average Pick-Up time: 0-10 minutes
Ron45 Reviewed on Aug 15, 2018, 11:12 PM
Vehicle requested was a small Hiundi and was most suitable for our purpose. Car was clean and interior spotless. Not happy that it cost an extra $ (approx.) $60 to have my wife put onto the hire as an extra driver. It is for this reason I have given the overall value of the rental as average below.apart from that the experience was fine. I have mostly used Thrifty when we travel to Townsville.
terminal building
Average Pick-Up time: 11-20 minutes
Shaun Reviewed on Jan 6, 2020, 1:37 AM
the conditions of the hire were explained to me well. Was not happy that our excess was 4.$k and that we needed to spend another $55 per day to reduce to $600...
Gene Reviewed on Nov 4, 2018, 8:06 PM
I asked my PA to book me a car for a business trip in Townsville and she told me that this price was all inclusive. Checking in they took an extra $400 off my card as it was a debit and not a Mastercard then they wanted an extra $50 to reduce my Premium from $4,000 down to the normal amount There is nothing worse than being taken advantage of and ripped off so I walked up to the other car rental company called national and their price included all insurance and all other costs so I cancelled my Avis booking and what with national instead
We collected 25 reviews in total for all stations in Townsville, Queensland, Australia with an average score of 8.6/10.
Please be aware that some of these reviews may have been translated automatically into English.
You need a great car to take you around Townsville whether you're going for a business trip or a vacation. See the famous sites around the area and park in front of the best restaurants in Townsville. We've collected your most popular questions about car rentals in Townsville. Keep reading to see the dos and don'ts of booking a rental car.
CarRentals.com makes it a cinch to grab the best rates. Just enter your travel dates and go down the variety of rental car suppliers, pick the top rental locations, and find the most wallet-friendly offers in Townsville. You can also:
These tips can help you save even more on your next car rental in Townsville.
From global names like Advantage and Hertz to budget-friendly brands like Economy and Budget, CarRentals.com has some of the best brands in the business. Read through the reviews to get an idea of which one is best for your next trip.
That all depends on your needs and what activities you want to do in Townsville. If you're having a bit of trouble picking the right car, make sure to look over the chart below. We've compared the most popular categories for you:
Typically, the age limit for car rentals in Townsville is 21. However, there might be an additional charge if you are under the age of 25 or for larger or higher category vehicles in particular. Go through the supplier's terms to find what the exact rate will be before you rent your vehicle.
When cruising through Townsville or the rest of Australia, you will need insurance. Almost every supplier needs proof of insurance before you pick up your car. Many countries require driver's insurance. You may be able to buy temporary insurance with the rental agency.
Many major card issuers, like Visa or American Express, give some insurance on your rental, however, there are usually restrictions. For example, you may not be covered if you book a larger or luxury vehicle. Be sure to check your rental car company's policy before you decide.
Most suppliers allow you to choose which policy you want. Here is a list of the typical policies.
Many rental car suppliers offer a range of fuel policies. However, full-to-full is our stand-out choice. It simply means returning the car with the same level of gas as you got it with, so you'll never pay for anyone else's road trips through the Townsville.
If you plan to explore all that Townsville offers, you're going to need unlimited miles. You won't have to worry about extra charges if you decide to drive all over Townsville. Read the fine print to make sure they are actually "unlimited."
Be sure you check out a few things prior to leaving the car rental lot on your Townsville adventure. You should take time to:
Use CarRentals.com to get the best wheels Townsville has to offer. Before you know it, you'll be cruising between the well-known restaurants, renowned landmarks, and all the amazing sites in Townsville.


text:
AnswerConnect is a telecommunications and phone answering company based in Portland, US. They provide virtual receptionist services, so you can have someone available 24/7 to answer your calls, schedule appointments, and do all the other things you'd expect from a receptionist.
The company started operations in 2002, and currently serves businesses of all sizes all over the world.
About AnswerConnect
AnswerConnect provides a wide range of virtual telecommunication services, such as call handling, web chat, call forwarding and more. They work with companies of any size, but realistically virtual receptionist services are best utilized by small to medium-sized businesses that can't justify making a full-time hire in-house yet.
Something that separates AnswerConnect from some of the other virtual receptionist companies is that they assign dedicated agents to your account. They say they also train them in your industry, culture, and business, and the longer you're working with them the better they'll know you and your business.
This means you can have an agent on the end of the line making sure you never miss a sale or inquiry. While your customers are none the wiser that they are talking to a third-party company.
How AnswerConnect Works
To get started with AnswerConnect all you have to do is choose which of their services you're interested in. They will prompt you for your business name and contact number, then you choose one of their plans.
You can either request a toll-free number to use as your dedicated line to their agents or forward your existing number. You'll have access to their online web portal where you set your instructions, keep track of your minutes used, and contact their team if you have any questions.
AnswerConnect offers a range of virtual receptionist services which includes the following core services:
- Inbound call answering – You can have their agents answer your business calls using your scripts and giving your customers the illusion they're talking to your own in-house receptionist.
- Out of hours call answering – You can set specific instructions for out of hours calls.
- Call forwarding – You can set your calls to forward to their agents at specific times or when you're busy.
- Calendar management – Their agents can set appointments and manage your calendar.
- Webchat services – Their agents will set up and manage a webchat box so you never miss an opportunity for a lead on your site.
Plans and Pricing
AnswerConnect currently has six different pricing plans. All of their plans come with a one-off set up fee of $49.99, and their rate per minute ranges from $1.49 per minute for their smallest plan to a very competitive $0.87 per minute for their largest plan.
A complete list of their plans at the time of publishing is:
- Standard – $149 for 100 minutes per month (overage rate of $1.49 per minute)
- Business – $269 for 225 minutes per month (overage rate of $.99 per minute)
- Best Value – $299 for 299 minutes per month (overage rate of $.99 per minute)
- Premium – $549 for 600 minutes per month (overage rate of $.99 per minute)
- Premium (I) – $949 for 1,050 minutes per month (overage rate of $.99 per minute)
- Premium (II) – $1,359 for 1,550 minutes per month (overage rate of $.99 per minute)
All of their plans run month to month and there are no long-term contracts, so you can cancel at any time. A nice feature is that any calls their agents are able to complete in under 30 seconds are free. This means you will not be charged for any misdials or robocalls.
AnswerConnect Alternatives
If you like to try services before you commit to a plan, Gabbyville and Ruby Receptionists are two highly-rated virtual receptionist companies that both offer free trials.
|Ruby Receptionists
|Customer Rating
|not rated
|not rated
|Minimum Monthly Cost
|$299 for 100 minutes
|$229 for 150 minutes
|$99 for 50 minutes
|$110 for 100 minutes
|Price per Minute
|Set-Up Fee
|14-Day Free Trial
|14-Day Free Trial
|30-Day Risk-Free
|Learn more
|Learn More
|Learn More
|Learn More
|Learn More
I asked AnswerConnect if they offer a free trial and they said sometimes they do under "certain circumstances." So, it's worth contacting them and asking for a free trial if you like what you see, the worst is they can say is no!
Your Turn
Have you worked with AnswerConnect? If so, please leave a brief review below to help others with their decision.


text:
I recently came across a striking depiction of the dominant Western worldview, which underlies our conventional way of thinking about violence, especially our assumption that war is natural and inevitable. It is our present-day mythos—our deeply embedded conception of humanity—rendered so starkly that it startles one momentarily into a state of recognition.
I found this remarkable depiction in a book about the history and vitality of peace movements, specifically a chapter on psychology and peace. The authors, Marc Pilisuk and Mitch Hall, observe that we live in a world we have created, both a physical world we have significantly changed and "a symbolic world of mental images that define what we assume to be true." Our prevailing myths are our most comprehensive symbols for identifying our place and purpose in life. They constitute social and political entities, such as nation-states, that "exist only because we believe they are real," that is, "because we invest them with sovereign powers and sacred attachments" and "willingly kill or die for them."[i]
The Western worldview is one of the dominant, although psychologically latent, symbolic constructions of our developed world. Its constellation of beliefs and values articulates inequality, amorality, and force into a mantra of progress that warrants aggression and war. It represents reality as inevitable and renders choice ephemeral.
What are the constituents of this worldview? Here is the list provided by our authors:
- All people are free to compete for success, typically defined as expanded wealth.
- The world's resources exist for exploitation by those best able to take advantage of its gifts.
- Private property is favored by law over either unowned nature or public property.
- Freedom to speak includes the unlimited right to use wealth to influence opinion and public policy.
- Corporations shall have the protection by law afforded to citizens.
- Efficacy is more important than ethics in the attainment and protection of wealth.
- Disparities in wealth of any magnitude are natural and acceptable.
- Poverty is due to deficiencies in the poor.
- Military force is justified to protect corporate interests (often defined as national interests).
- Limited parliamentary democracy (mandating elections while allowing wealth to be used for persuasion) is the much-preferred form of government.
- Psycho-cultural values of power, masculine domination, acquisition, and development are aspects of the natural world order.
- Those not accepting these views or the policies that flow from them pose a danger and must be either trivialized or eliminated.[ii]
The myth blinds us, at the level of the war state, to our everyday, usually nonviolent ways of managing conflict. Unlike the myth of violence, we typically engage our differences with talk of one kind or another, including "ridiculing, persuading, coaxing, arguing, shouting, grumbling," or just walking away. We see people "agreeing to compensate for damages, compromising, reconciling differences, and negotiating settlements . . . . Most individuals cope with bullying, insults, competitive conflicts, and disappointments without resorting to violence or inflicting serious harm on adversaries."[iii]
Once revealed, the Western mythos of war is worth some critical reflection. Perhaps there is a better story to be told? "Fortunately," as our authors insist, "we humans have the capacity to deal with long-term issues with creative dedication and with opportunities to engage with others in building solutions" that advance the cause of peace and justice by peaceful means.[iv]
[i] Marc Pilisuk and Mitch Hall, "Psychology and Peace," in Peace Movements Worldwide, vol. 1, ed. Mark Pilisuk and Michael N. Nagler (Santa Barbara, CA: Praeger, 2011), 53.
[ii] Pilisuk and Hall, 54. See also Marc Pilisuk and Joanne Zazzi, "Toward a Psychosocial Theory of Military and Economic Violence in the Era of Globalization," Journal of Social Issues 62.1 (2006): 41-62.
[iii] Pilisuk and Hall, 55.
[iv] Pilisuk and Hall, 56.


text:
It looks set to be an awards season like no other, with prestige studio titles up against arthouse curios — and the Oscars' baffling new popular film category.
By the time the curtain goes up at the opening ceremony of this year's Venice Film Festival, distributors and awards strategists will have just about picked their jaws off the floor after the Academy of Motion Picture Arts and Sciences unveiled what must rank as one of its most radical rule changes in recent memory. The announcement in early August that the 91st Oscars telecast on February 24, 2019 will include an award for outstanding achievement in popular film came as a bolt out of the blue, taking even seasoned campaigners and studio executives by surprise.
Ostensibly a move by the Academy to make the ailing Oscars telecast seem more relevant to younger viewers, observers say the new category could damage the show's reputation for championing smaller, independent films (which is not always the case — studio films get recognition too). Of greater concern is the possibility that whenever a studio tentpole transcends its genre roots and appears worthy of a shot at the senior Oscar categories, it will be exiled to a populist ghetto.
Eligibility requirements and further details on who will vote for the new Academy Award were not forthcoming at time of writing, but clearly Black Panther is the film that looks likely to be most affected. Marvel Studios/Disney executives are proud of their $1.3bn global smash, and it is understood they have been planning an awards push that encompasses not just crafts and major categories, but best picture and director for Ryan Coogler.
The industry consensus so far is that the Academy is being fuzzy-headed in its good intentions. It took notice when the #Oscarsowhite furore blew up a couple of years ago, and in the past 18 months has made overtures to the world outside, inviting a new intake of younger, more diverse, international entertainment professionals that would account for roughly one-fifth of global membership.
"It effects Black Panther the most in my opinion," says one veteran awards strategist who has worked with the studios for years. Like every expert canvassed by Screen International, they asked to remain anonymous. "If I were Disney, I would be concerned voters pop that film into that new category and leave it off the best picture category. If Paramount gets serious about Mission: Impossible — Fallout, then that gets into the mix there too."
Whatever the case, Coogler's film is regarded by awards observers as one to watch this season. Of the others regarded as potential heavyweights in this early stage populated by known unknowns, there are several studio contenders, the now inevitable presence of the streaming giants, and a serious roster from Megan Ellison's ambitious Annapurna Pictures.
Heavyweight contenders
Among the anticipated early runners are Universal's Venice opener First Man from Damien Chazelle, which sees the youngest-ever winner of the best directing Oscar for La La Land return to the fray. The studio also has US rights on Participant Media's Toronto premiere Green Book from Peter Farrelly, a race drama starring Viggo Mortensen and Mahershala Ali.
Annapurna has If Beale Street Could Talk, another Toronto world premiere that marks Barry Jenkins' follow-up to 2017 best picture Oscar winner Moonlight. Yorgos Lanthimos's Venice world premiere and New York Film Festival opener The Favourite, starring Olivia Colman, Rachel Weisz and Emma Stone, is with Fox Searchlight.
Searchlight is the reigning awards season champions, whose best picture and directing Oscars for Guillermo del Toro's The Shape Of Water could not have come at a better time. If the title of Lanthimos's reportedly Kubrickian period drama proves prescient, Disney chairman and CEO Bob Iger could be purring contentedly about the acquisition of the 21st Century Fox entertainment assets for another year of earnings calls, even if an R rating might preclude The Favourite from playing on Disney's upcoming streaming service.
Early — unreliable — word from those working on Warner Bros' A Star Is Born is positive about Bradley Cooper's directorial debut and Venice world premiere. Cooper and Lady Gaga (in her first major film role) sing live, and the film could make a lot of noise. Friends of Sony are similarly talking up Jason Reitman's The Front Runner starring Hugh Jackman as flawed 1988 presidential candidate Gary Hart. CBS Films has Willem Dafoe as Vincent van Gogh in Julian Schnabel's At Eternity's Gate, which premieres in Venice.
Several strategists agree the influx of new Academy members could influence voting patterns. "This is the year for the focus to be back on diversity with the new Academy members," says one strategist, who notes Black Panther "has something to say and there's a rooting factor for it".
Further films that could benefit from an enhanced awareness of inclusion are: Jenkins' If Beale Street Could Talk, based on James Baldwin's wrenching novel about a pregnant Harlem woman's odyssey to exonerate her incarcerated fiancé; Focus Features' BlacKkKlansman from Spike Lee, whose critically admired Cannes title recounts how an African-American cop infiltrated the Ku Klux Klan in the late 1970s; and Steve McQueen's Toronto-bound heist thriller Widows from See-Saw Films, Film 4 and New Regency, which will open the BFI London Film Festival and features a strong female cast led by Viola Davis. 20th Century Fox distributes.
"I'm hopeful the new younger and more diverse voters will allow for more populist movies and varied movies to be considered," says one top studio executive before the Academy announced its 'popular film' award. In contrast, another campaigner posits the Academy invitations are "cosmetic" and that not everyone will accept membership. "You've got good optics but are these people actually participating — watching movies and voting? It takes a while for all this stuff to percolate."
Open to fresh voices
The same person points out recent Academy screenings of Boots Riley's satire Sorry To Bother You starring Lakeith Stanfield and Tessa Thompson, and Bo Burnham's rites-of-passage drama Eighth Grade show how existing Academy members are open to fresh voices. Recent best picture winners Moonlight and 12 Years A Slave are cases in point. "People respond to storytelling no matter their age or ethnic demographic," they say.
As The Shape Of Water and Birdman have shown in recent years, Venice can kick-start a successful best picture Oscar campaign, while best director winners whose films launched on the Lido include Chazelle (La La Land) and Alfonso Cuaron (Gravity). The latter returns to Italy for the world premiere of ROMA, his first Mexico-set film since his 2001 breakout Y Tu Mama Tambien.
Netflix is handling the black-and-white family saga from Participant Media and held back its films from Cannes after it refused to bow to French distribution window rules. Netflix covets a best picture win and recently signalled its intent by bringing awards guru Lisa Taback in-house. Taback and her team will need to pick a horse and the early indications are Cuaron's film will be the one, although the roster includes the Coen brothers' western anthology The Ballad Of Buster Scruggs, Paul Greengrass's 22 July, which charts the aftermath of the 2011 Norway terrorist attack, and possibly Noah Baumbach's untitled divorce comedy with Scarlett Johansson and Adam Driver and Susanne Bier's sci-fi Bird Box with Sandra Bullock. It is understood Netflix had not finalised theatrical plans for its contenders as of mid-August.
While the company has prospered in the documentary category, Amazon Studios has had the better of it in the other senior sections, earning best actor and best original screenplay awards in 2017 with Manchester By The Sea, and an original screenplay nod for The Big Sick earlier this year. Amazon Studios' present crop includes Steve Carell and Timothée Chalamet in father-son drug drama Beautiful Boy, which could bring Belgian director Felix van Groeningen to the attention of a much wider audience. Mike Leigh's Venice-bound Peterloo and Dan Fogelman's Life Itself, which premieres in Toronto, are also eagerly anticipated.
Annapurna Pictures could pack a punch this season. Its roster includes Nicole Kidman in Karyn Kusama's crime thriller Destroyer, Jacques Audiard's The Sisters Brothers, and the tentatively titled Dick Cheney drama Backseat starring Christian Bale, which Adam McKay is still working on. Sony also has Matthew McConaughey in Yann Demange's '71 follow-up White Boy Rick, while Fox Searchlight could make some noise with Melissa McCarthy in a more versatile role as celebrity biographer, forger and thief Lee Israel in Can You Ever Forgive Me?. The studio might also fancy its chances with Venice premiere The Old Man & The Gun, a curio now that Robert Redford has declared it will be his final acting role.
Focus Features also has the gay conversion therapy drama Boy Erased with Lucas Hedges, Nicole Kidman and Russell Crowe, as well as Saoirse Ronana and Margot Robbie in Mary Queen Of Scots, which had not landed a festival berth at time of writing. A feisty Bleecker Street slate includes Sundance pick-up Colette starring Keira Knightley, Debra Granik's Leave No Trace with Ben Foster and newcomer Thomasin McKenzie, Rachel Weisz in Disobedience, and ensemble drama What They Had starring Blythe Danner, Hilary Swank and Michael Shannon.
Disney's Mary Poppins Returns could catch a wave and stars Emily Blunt in the iconic role. The UK actress also impressed in Paramount'sA Quiet Place, one of several horror films causing a stir, alongside A24's Sundance sensation Hereditary with Toni Collette and Luca Guadagnino's Venice world premiere Suspiria for Amazon Studios, inspired by Dario Argento's 1977 giallo classic.
Finally, Stan & Ollie will close the BFI London Film Festival and stars Steve Coogan and John C Reilly as the famous comedians Stan Laurel and Oliver Hardy. One source said Coogan is outstanding and could go all the way.


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Hold Up, Stephen Bear Confirms That He's Split From Charlotte Crosby After All
Just hours after Charlotte Crosby confirmed her relationship with Stephen Bear was back on, he's now confirmed they've split after all.
"We are better now, we went through a bit of a tough week last week, but we are so much better, thank god," she told Ireland AM. "He's going to see the fight in Las Vegas so I'm going to really miss him."
Well, it looks like the Just Tattoo of Us co-hosts really have called it quits, and it might be for good this time.
"Sadly me and Charlotte have split up," Bear wrote on Twitter. "We have had a good run but we wasn't right for each other."
Sadly me and Charlotte have split up . We have had a good run but we wasn't right for eachother 😢 x
— Bear (@stephen_bear) August 24, 2017
See How Much Charlotte Crosby Has Changed Over 12 Seasons Of Geordie Shore!
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Bear also announced the news to his Snapchat followers.
"I don't normally go on social media to do all this but we've broken up," he revealed. "There's a lot of stuff you don't know, a lot but whatever."
"Before you hear a load of bad stuff about me, I'm use to it anyway," he continued. "I've been a good boyfriend, but now officially, I am single."
Charlotte previousy tweeted that she'd split from Bear, but then backtracked the next day saying she'd just been a "drunk dick".
See How Much Charlotte Crosby Has Changed Over 12 Seasons Of Geordie Shore!
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"The top and bottom of it is I was a massive dick, a massive drunk dick, who decided to go on Snapchat and every other social media, to tell nearly six million people a load of shit," Charlotte insisted at the time.
"Obviously no relationship is perfect, you always have your ups and downs, but I feel like I just did what any other girl does when they see red and I am sorry for that. Me and Stephen aren't actually separated or finished, we just had a bit of a row."
Charlotte is yet to comment on Bear's announcement.
A Complete List Of All Of Charlotte Crosby's Famous Boyfriends...
A Complete List Of All Of Charlotte Crosby's Famous Boyfriends


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No turtle is more critically endangered than the Yangtze giant softshell turtle – with sad news that the last known female of the species died on April 13, 2019, only three individuals are known to exist; two in Vietnam and one at the Suzhou Zoo in China, believed to be more than 100 years old.
Wildlife Conservationists Remain Steadfast In Efforts To Prevent Extinction of the Giant Yangtze Soft Shell Turtle
Whilst field surveys are ongoing of the Yangtze and other river systems for any last remaining wild turtles, no additional wild turtles have been confirmed. Over the past year, WCS has been developing first-of-a-kind hand-held and field-friendly molecular test kit to detect environmental DNA (eDNA) from the Yangtze giant softshell turtle to search for potentially undiscovered animals that may still exist and have evaded detection by traditional methods. Our Yangtze giant softshell turtle eDNA test and methodologies have now been developed and we have shown our methodology can detect the Yangtze giant softshell turtle, as well as a variety of other turtle species we have targeted, and field-validation is now in progress.
Thumbnail softshell turtle image credit: PCalle, WCS
Banner softshell turtle image credit:


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The most common way that students prepare for General Paper is to try to i) read more and ii) memorise content. However, from my teaching experience, I found that this is not the best way to improve in the subject.
i) Read more
Many students have been told to read more, and so, they try to read the news as often as they can, and occasionally squeeze in that extra bit of time reading articles/commentaries they come across on Facebook. But knowing what's happening in the news and knowing how to use them effectively in making arguments are two different things.
ii) Memorise content
Many students also try to memorise case studies and examples as they think including them in their essays will help pull up their grades. However, the merit of an argument is always assessed holistically; merely citing case studies and examples without sufficiently developing a cogent line of reasoning will not impress the examiners.
Without the right strategy, it may be more difficult to see efforts yield results.
What is the best strategy?
I noticed that many students struggle with GP, including those who used to easily score As for English in their secondary schools. This is because GP is not primarily a test of one's language proficiency; it is an assessment of one's ability to i) analyse and synthesize information and ii) effectively articulate his/her positions on different local and global issues with cogent and well-balanced arguments. I call these GP process skills.
The best strategy is to first, focus on developing these process skills, and then work on building up content knowledge and improving the language. Many students do it the other way round and place the most emphasis on developing content knowledge and building up vocabulary.
Unlike content knowledge, it is usually difficult for a student to improve on his/her process skills without the help of a teacher or tutor. I believe that developing GP process skills is a worthwhile investment. These skills will not only enable students to better enjoy and do well in the subject, but also double up as important life skills that can guide them in making sense of themselves and the world beyond the classroom.
Do you need help to build up your GP process skills?
If you have been trying hard to improve your GP but have yet to see results, perhaps it is time to reevaluate your strategy. Feel free to arrange for a free consultation with us and we can work out an action plan which you can take active steps to target different areas of weaknesses.
Click here for more information on our H1 General Paper tuition:


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Best Spas near Rockwall, TX
It is always important to treat yourself to some rest and relaxation! Next time you are looking to reduce stress and rejuvenate your mind and body, one of these spas near Rockwall, TX, will do the trick!
Belfiore Salon & Spa
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When you walk into Belfiore, you are greeted with a warm and calming atmosphere while also being provided with a luxurious experience at their full-service salon and spa.
They can color, cut, shampoo, and style your hair to your personal liking. You can also get a manicure and pedicure while you are there.
For all your spa needs, Belifore offers a variety of body therapies, facial therapies, and massages for a soothing experience. Even have your significant other join you for their Rendezvous for Two spa package, or give the gift of their Girl's Retreat or Gentlemen's Escape package.
After a day at Belifore, you will look and feel amazing!
Every Body Massage
Whether you are looking to get a massage for the ultimate relaxation or as a way of rehabilitation, Every Body Massage has a style of massage for whatever your individual preferences are.
Their Signature Massage is a great way for you to customize your massage just how you want it. Their therapist will focus on what you want to gain out of your session so you get the most out of your massage.
They also have massages ranging from their stress-reducing Aromatherapy Massage to their highly-effective Deep Tissue Massage.
You can also have your friends join with the group packages they offer.
Jour Salon & Spa
A one-stop shop, Jour Salon & Spa provides you with services for all your beauty and spa needs. From waxing to lashes to haircuts and treatments, they can help you with any salon needs you have.
Even book a massage for a comforting and tranquil time. Their Hot Stone Massage helps create positive energy flow and their Hydrating Body Polish uses essential oils during the massage to help exfoliate and refresh the skin.
They also have various spa packages from their Spa for Men to their Full Day of Beauty, which includes a day at Jour spent getting a manicure, pedicure, European Facial, Full Session Massage, and more!
Take a Personal Day!
Book an appointment at one of these spas near Rockwall, TX, and take some much-needed time to yourself with a massage!


text: Since most music lovers enjoy expanding their playlists, I thought a music recommendation thread was in order. If the members of ND dont want this thread here just delete it. ------------------------------------------------------------------------ So I guess I'll start off. A band that I found quite recently is Ikuinen Kaamos, maby you have heard of them. They are a progressive death/black metal band from Finland and their debut album "The Forlorn" is already one of my favorite metal albums. Their second album "Epilogue" is delayed! But I cant fucking wait for it! To me it sounds like early Opeth meets early Ulver. http://www.myspace.com/ikuinenkaamos [ame]http://www.youtube.com/watch?v=zyGTaJBaOZ8&feature=related[/ame] [ame]http://www.youtube.com/watch?v=SaV_3Dq4-b8&feature=related[/ame]


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Description: Selro copper cobra link bracelet; the size chunky copper links are etched as cobras and the links are alternately set with either deep green oval lucite cabochons or fancy copper elements set with a light green rhinestone flanked by pair of faux pearls. Wide fold-over clasp. Attributed Selro.
Size: 7 3/4" x 1 1/4"
Condition: Excellent
Price: $159.00


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We want our students to fully understand the world of possibilities out there and make the most of their talents. We work hard to ensure that all our students have the opportunity to make informed decisions about their future. We aim to help develop students' knowledge, understanding, confidence, and skills so that they make well-informed choices and plans.
We are committed to providing a well-structured Careers and Education programme for all students in Year 7 to 13. We strive to ensure students experience a full, varied and interesting careers journey, having the opportunity to explore their own interests, skills, and passions; understanding the wealth of jobs and education available to them once they leave school. It is integrated into our students' experience across the whole curriculum and is based on a partnership with, students and their parents or carers. We have a rich partnership with local businesses, colleges and universities along with a strong alumni and wider school community to support our Careers Education.
We also work in partnership with Innervate Career Services, an outside agency. They provide one-to-one impartial Career Guidance interviews to Year 11, 12 and 13 students, Group Career Guidance sessions to Year 9 and Destination programmes.
Our programme is a valued area of the curriculum and is guided by the Careers Guidance and access for education and training providers and the eight Gatsby benchmarks identifying the elements of good practice in career guidance.
Key Stage 3
- As early as Year 7, we introduce students to career resources, as they start to identify the types of jobs they are interested in, and the personal qualities and skills required to do each job. Students learn about personal development, planning, and decision making in their PSHE, English and RE lessons.
- During Years 7, 8 and 9 students investigate various aspects of careers education through their PSHE lessons (although there is a crossover into all subjects) and by accessing dedicated careers websites.
- As our students progress through the school, opportunities are developed and include Feature Lectures, webinars, and events dedicated to next steps.
- In Year 9, students make their GCSE choices, and explore how to keep their options open; this is supported by a comprehensive programme delivered through assembly, tutor lessons, PSHE, and parent information events.
- Key stage 3 students have access to educational workshops with STEM companies
Key stage 4
- We encourage all students to consider the career possibilities throughout Year 10 and 11 and provide a wealth of impartial guidance and information on post 16 options.
- All students in key stage 4 have access to feature Lectures talks and webinars, from motivational speakers and industry professionals.
- We encourage key stage 4 students to volunteer within the local community as part of the Duke of Edinburgh Award Scheme
- Our Year 10 Careers Day enables students to investigate a large number of different professions. Engaging with industry professionals based on their career interests through talks, sharing work skills for particular employment sectors. Also exploring all routes e.g. university, college, apprenticeships, Oxbridge, Year in Industry, and less familiar routes such as art, performing art and music.
- Year 10 students have access to educational workshops with STEM companies throughout the year.
- Year 10 students have the opportunity to attend HEP experience days and apprenticeship fairs.
- In Year 11 students focus on making decisions and informed choices for their options after their GCSEs. Well-planned information is provided about our own post 16 provision and external institutions.
- All Year 11 students receive 1:1 meetings with their tutors to discuss their next steps and career aspirations. Alongside, receiving an individual consultation with a member of the Leadership Team and parents to discuss their post 16 next steps and support required.
- Students attend our Year 11 into 6th Form day where post 18 options are discussed and guidance is given on subject choices to support career aspirations. Followed by an open evening for students and parents to explore options available together.
Key Stage 5
- Throughout the year students, receive a wealth of information on post 18 options including higher education and careers opportunities including apprenticeships, internships and lectures/events that will support their aspirations.
- In the summer term, our Year 12 students visit a university open day and attend university fairs (when available). Students are also encouraged to attend further university open days independently.
- In Year 12 students complete a week of work experience and are encouraged to find their own placement in a career sector that interests them. Across the college, students are given the flexibility to partake in additional work experience or placement opportunities.
- We provide workshops, employer encounters, and support, which includes, writing personal statements, interview preparation, UCAS application completion, CV and cover letter preparation, support for competitive courses such as medicine and Oxbridge.
- 1:1 portfolio and interview preparation sessions for art foundation, architecture, design, and creative courses.
- Feature Lectures and talks, which can include studying and working abroad, alternatives to higher education, gap years, CV writing, UCAS and work experience, motivational speakers and industry professionals.
- All students in 6th form are offered 1:1 career interviews with an external consultant.
- In Year 13 students attend a careers evening in January where they engage with industry professionals for 1:1 mock interviews receiving feedback on these and their CVs.
- A stable careers programme – Every school and college should have an embedded programme of career education and guidance that is known and understood by students, parents, teachers, and employers.
- Learning from career and labour market information – Every student and their parents should have access to good-quality information about future study options and labour market opportunities. They will need the support of an informed adviser to make best use of available information.
- Addressing the needs of each student – Students have different career guidance needs at different stages. Opportunities for advice and support need to be tailored to the needs of each student. A school's careers programme should embed equality and diversity considerations throughout.
- Linking curriculum learning to careers – All teachers should link curriculum learning with careers. For example, STEM subject teachers should highlight the relevance of STEM subjects for a wide range of future career paths.
- Encounters with employers and employees – Every student should have multiple opportunities to learn from employers about work, employment, and the skills that are valued in the workplace. This can be through a range of enrichment activities including visiting speakers, mentoring and enterprise schemes.
- Experiences of workplaces – Every student should have first-hand experiences of the workplace through work visits, work shadowing and/or work experience to help their exploration of career opportunities, and expand their networks.
- Encounters with further and higher education – All students should understand the full range of learning opportunities that are available to them. This includes both academic and vocational routes and learning in schools, colleges, universities and in the workplace
- Personal guidance – Every student should have opportunities for guidance interviews with a careers adviser, who could be internal (a member of school staff) or external, provided they are trained to an appropriate level. These should be available whenever significant study or career choices are being made. They should be expected for all students but should be timed to meet their individual needs.
Apprenticeship Guide offering a guide to apprenticeships in England.
All About Careers offers free advice and guidance to young people interested in a range of careers. Also suitable for students, graduates and school leavers seeking internships, placements and graduate jobs. They also have websites for law careers, finance careers, apprenticeships and school leaver programmes.
Cambridge Subject Masterclasses are subject-specific events that offer academically able students in Year 12 (or equivalent) the chance to experience typical undergraduate teaching at the University. Useful whether or not you plan to apply to Cambridge.
Careers Advice in Surrey offers comprehensive information on apprenticeships.
Career Boss offers guides on specific career roles outlining skills and attributes.
Careersbox is a free online library of careers related films, news, and information.
CareerMap is the leading website for 15-25 year olds looking for a pathway into Apprenticeships and early career opportunities.
Careermag offers a complete guide to apprenticeships, courses and university opportunities along with useful articles offering education and career advice.
icould is a small, dynamic charity, providing career inspiration and information for young people.
What you study at sixth form or college can affect your options at university and your future career. Use Informed Choices to help you understand which subjects open up different degrees, particularly at Russell Group universities.
Career-experience programmes for students who aspire to work in investment banking, law, medicine, engineering, politics, and journalism.
Headstart has been established for more than 23 years as a charitable trust providing hands-on Science, Technology, Engineering and Maths (STEM) activities, and engineering taster courses to encourage young people into technology-based careers.
JobCentreGuide an independent guide to JobCentre Plus services, online job searching, CV preparation, and interview technique. They aim to provide you with all the information you'll need to help you find a rewarding job.
LMI for All (Labour Market Information), here you can find out where to get more information and intelligence of the UK labour market.
The Medic Portal in partnership with The Royal Society of Medicine for aspiring medics.
MyBigCareer aim is to provide free one-to-one career guidance to young people from disadvantaged backgrounds and to break down barriers to social mobility. Advice is delivered by professionally qualified advisers volunteering from within the independent sector.
MyKindaFuture is an award winning Social Enterprise that exists to give every young person equal power and opportunity to kickstart their career and shape their future. They work with a large network of schools, colleges and universities to connect students to employers who are looking for their next generation of talent.
The National Careers Service is the publicly funded careers service for adults and young people in England. Launched in April 2012, it brings together elements of previous publicly funded careers services for adults and young people. Its explore careers section is particularly useful to gain an understanding of what a job involves and whether its right for you.
Parental Guidance will give you some of the advice and information you need to support young people during their transition to adult working life. You will find up-to-the-minute and well-researched articles by the country's leading careers writer.
Prospects are owned by the registered charity Higher Education Careers Services Unit (HECSU), providing information to guide and inspire career choices throughout the student journey. Particularly useful for showing what careers degrees can lead to.
The Russell Group represents 24 leading UK universities, which are committed to maintaining the very best research, an outstanding teaching and learning experience and unrivalled links with business and the public sector.
Student Ladder provides information on work placements, graduate schemes and internship opportunities for 16-24 year olds. Application advice on CVs and interviews alongside information, guidance, and advice from GCSE through to graduation.
Success at School is the place for young people to explore careers, get the lowdown on top employers, and search for the latest jobs, courses and advice.
UCAS is an independent charity providing information, advice, and admissions services to inspire and facilitate educational progression.
For long term Gap ideas there are several websites to explore.
Participation in 'out of school' volunteering, clubs and societies are always good. In addition, demonstrating involvement with diverse groups of people and what you have learned from the experiences is impressive in personal statements and future job interviews.
For further information, or if you are business that would like to engage with us in delivering our Careers, Education and Guidance programme, please contact Mrs Joanne Hawkins by email email@example.com or by phone 01932 582577


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IGSO (International Geodetic Student Organization) has its aim in uniting geodesy students from all over the world. At present, 98
universities and schools from 34 countries belong to IGSO.
Every year IGSO organizes the International Geodetic Student Meetings – IGSM. These meetings are not only a wonderful chance to exchange experiences within field of geodesy but also a unique opportunity to get to know other countries' customs and culture, because every year these meetings take place in a different country. Students are given huge possibilities and thanks to them, future advantages.
Official website: https://igso.info


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The Galway Film Fleadh in association with 101 Films International (one of the U.K.'s leading entertainment labels) has announced this year's finalists for The Pitching Competition which takes place on Sunday 14th July at 12 noon in the Galmont Hotel.
Since its inception, the Galway Film Fleadh Pitching Competition has emphasised the fact that good writers with an opportunity to get their first start in the industry. The 10 finalists will pitch their ideas (90-second elevator pitch) to a panel of industry leaders and decision makers.
The winner will receive €3,000 cash award. This is a free event open to the public.
Here are the 10 finalists and their projects.
Mark Dollard – Hellfire
Johnny Schumann – Lorcan
Paul Webster – The Dip
Mark Hennigan – The Future Perfect
Gary White – The Niemba Ambush
Barry Ryan – The Silver Monkey
Mark Kelly – X
Susannah Hunt – Beyond The Pale
Samuel Steele – Groenland
Roisin James – What Would Rocky Do?


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4 Simple Mobile Email Tips
Marketers are spending $51 billion on mobile advertising this year and will up that to $105 billion by 2019, according to Juniper Research. Given those figures, it would only make sense for marketers to do it right.
Viewability should be the top priority, according to the research announced via newsletter on Monday. ShipStation's blog post from Friday expands upon that concept, specifically discussing mobile email.
ShipStation provides the following advice to marketers who want their ads seen:
- White Space Is a Marketer's Friend. Bullet points are easier on the eyes than dense text. Images are friendly, too, but use Alt text in case devices don't render the images.
- Compress Images. They'll load faster.
- Use Emojis Responsibly. Make sure sentences make sense without the emojis, as they may not always render. They can make brands more relatable to certain demographics, but done wrong marketers can seem as though they're trying too hard.
- Employ Responsive Design. Gmail will make the email message fit the screen, but that's not true of all email service providers. [Editor's note: While some Web designers say responsive may be the way to go to gain favor with Google, others are saying keep an eye out for the rise of adaptive design.]
Bonus: Jupiter says mobile marketing provides high visibility and response rates on ads recipients have opted in to see, but marketing that mobile users believe violates their privacy will be treated harshly. Gain opt-in. Also, programmatic and video ads are on the rise on these screens.
What other advice should marketers follow?
Please respond in the comments section below.


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The Eau Claire Chamber of Commerce offers its members and the community numerous programs and services that not only strengthen its members' businesses but also make Eau Claire a better place to live.
Education and Business DevelopmentBusiness and workforce development continues to be an area of focus for the Eau Claire Chamber. Seminars, held throughout the year, help the local workforce obtain the skills they need. The Young Professionals of the Chippewa Valley was created by the Eau Claire Chamber to encourage individuals, ages 21 to 39 years old, to remain in the area's workforce. Area business leaders develop leadership skills and acquire community education through Leadership Eau Claire™.
Governmental AffairsThe Eau Claire Area Chamber of Commerce through its Governmental Affairs Committee is committed to being a proactive voice for the business community. It is the Chamber's goal to keep its members informed on issues that affect them and to facilitate communication between the business community and all levels of government.
Our Future WorkforceThe Eau Claire Chamber has several programs that focus on the future workforce. Partnerships with area school districts enable the Chamber to have programs in place such as Youth Leadership Eau Claire, The Real Life Academy and Excellence in Education.
Public EventsTwo of the Chamber's largest events, Breakfast in the Valley and THE Business Expo, are open to the public. Breakfast in the Valley, which is held annually in June, celebrates the importance of agriculture in the area. THE Business Expo, the area's largest event of its kind, is held annually in October with more than 140 exhibitors.
Chamber Buy Local Bucks The Chamber is proud to have a gift certificate program that helps to keep retail dollars in the local economy. With over 400 participating members, the Chamber Buy Local Bucks program is very successful.
Chamber Hosted TripsThe Eau Claire Chamber of Commerce is pleased to offer its members and community members the opportunity to travel to destinations outside the Chippewa Valley.
Partnership with the U.S. Chamber of CommerceWhen businesses become a member of the Eau Claire Chamber of Commerce, they are also joining the world's largest business federation, the United States Chamber of Commerce. Contact the Eau Claire Chamber for more information about your complimentary joint membership.
To take full advantage of all the Chamber's programs and services, businesses are encouraged to become a member.


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My 20 year old son passed away the summer of 2011. I wanted to get a tattoo in his memory but waited until I found just the right thing. I came across an infinity symbol and loved the design. It's like a figure "8" and considering I have 8 kids, it just felt like the right thing. It symbolizes my eternal love for my son Nate, our love is infinity and not even death can take that away from us. The color blue is a sign of peace, and all I pray is that he is indeed at peace. Blue is also my favorite color.
I have never had a tattoo before so I really didn't know what to expect. Honestly no amount of pain would matter to me because this is something I felt I needed to do. After all, I have delivered several large babies (11 and 12 pound babies) with no pain meds, so I knew I could handle this.
My oldest son (almost 24) and my "baby" (3 1/2) went with me and watched me get the tattoo. The only tears shed were when the tattoo artist drew out the design exactly how I had described and what I wanted. I was overwhelmed with emotions to see it.
The pain wasn't too bad, it was a little cutting and burning sensation at times, especially when he went over the bone I could feel it more. Reminded me of trying to cut into a piece of steak and the fatty part you have to cut a little harder...is how I imagined he was doing on my leg. (I didn't look because for some reason I thought it might make it worse to see when he was going to do it.) I was laughing and telling jokes the whole time. Honestly, halfway through I was thinking about a design if I did get a second tattoo someday.
I survived, it wasn't that bad and maybe someday I might get another one. I really love it and the colors. It's on my lower right ankle: