text:
- Research article
- Open Access
Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists
© Burki et al; licensee BioMed Central Ltd. 2010
- Received: 4 March 2010
- Accepted: 2 December 2010
- Published: 2 December 2010
Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).
167 genes were selected for phylogenetic analyses based on the representation of at least one rhizarian species for each gene. Concatenation of these genes produced a supermatrix composed of 36,735 amino acid positions, including 10 rhizarians, 9 stramenopiles, and 9 alveolates. Phylogenomic analyses of this large dataset revealed a strongly supported clade grouping Foraminifera and Acantharea. The position of this clade within Rhizaria was sensitive to the method employed and the taxon sampling: Maximum Likelihood (ML) and Bayesian analyses using empirical model of evolution favoured an early divergence, whereas the CAT model and ML analyses with fast-evolving sites or the foraminiferan species Reticulomyxa filosa removed suggested a derived position, closely related to Gromia and Phytomyxea. In contrast to what has been previously reported, our analyses also uncovered the presence of the rhizarian-specific polyubiquitin insertion in Acantharea. Finally, this work reveals another possible rhizarian signature in the 60S ribosomal protein L10a.
Our study provides new insights into the evolution of Rhizaria based on phylogenomic analyses of ESTs from three groups of previously under-sampled protists. It was enabled through the application of a recently developed method of transcriptome analysis, requiring very small amount of starting material. Our study illustrates the potential of this method to elucidate the early evolution of eukaryotes by providing large amount of data for uncultivable free-living and parasitic protists.
- Phylogenomic Analysis
- Celestite
- Long Branch Attraction
- Approximately Unbiased
- Plasmodiophora Brassicae
Over the last few years, the phylogenomic approach was successful in untangling several aspects of the early evolution of eukaryotes. Most eukaryotic diversity has been assigned to one of few supergroups [1] and new relationships between these large assemblages have emerged. For example, the unexpected close evolutionary affinity of Rhizaria to two of the "chromalveolate" groups, stramenopiles and alveolates (the SAR group, or Harosa in [2]), was recovered in several phylogenomic analyses [3–6]. Even orphan lineages that have been very challenging to place within the eukaryotic tree, such as the telonemids and centrohelids, or the breviate amoebae, have recently been shown to be related to haptophytes and centrohelids or to Amoebozoa, respectively [7–9]. However, several question marks remain, notably concerning the placement of the root [10], the monophyly of some supergroups [11], and the relationships within and between the supergroups, especially where uncultivated protists dominate.
The supergroup Rhizaria, composed of several phyla that are difficult to maintain in laboratory cultures, is a good example of the persisting uncertainties for the relationships between the major members of this assemblage. Although a few rhizarians can be isolated and cultivated [12], the majority is known only from environmental sequences [13] or single-specimens extractions [14, 15]. Consequently, rhizarians are represented in sequence databases almost entirely by ribosomal rDNA [16]. A few protein sequences of actin, α-tubulin, β-tubulin, RNA polymerase II, and polyubiquitin are available for selected taxonomic groups [17–20] but for other lineages, such as radiolarians, only the actin-coding gene has been sequenced, which is in sharp contrast to the great diversity of the group and its ecological importance. Recently, five small rhizarian cDNA libraries have been sequenced (3 Cercozoa and 2 Foraminifera), partially filling the gap in comparison to other supergroups, and one genome project (Bigelowiella natans) is in progress (http://www.jgi.doe.gov/sequencing/why/50026.html).
According to the current consensus, Rhizaria are composed of three highly diverse and possibly monophyletic phyla, Cercozoa, Foraminifera, and Radiolaria (including Acantharea, Polycystinea and Taxopodida, but excluding Phaeodarea that were shown to branch among Cercozoa [21]). The Rhizaria comprise also the parasitic Phytomyxea and Haplosporidia, as well as various marine filose and reticulose protists, including Gromiida and Filoreta, sometimes considered members of Cercozoa [22, 23]. The relationships between these groups are uncertain, due to the lack of resolution observed in the SSU and LSU rDNA as well as the few available protein trees. The most controversial is the position of Foraminifera, whose fast evolving SSU rDNA sequences branch either close to Haplosporidia and Gromiida [19, 24] or as sister group to Radiolaria [13, 25, 26]. The weakly supported grouping of Foraminifera and Radiolaria observed in some SSU and LSU rDNA trees led to the creation of the infrakingdom Retaria [26, 27]. Another source of information came from the insertions of one or two amino acids at the monomer junctions in the highly conserved protein polyubiquitin. These insertions have been found in Cercozoa and Foraminifera but not in all other eukaryotes studied to date, including radiolarians [17, 23]. It has been argued that the ancestor of polycystine and acantharean Radiolaria could have lost the insertion, but the lack of insertion could also be explained by contamination of DNA samples by non-rhizarian protists [23].
To test the Retaria hypothesis and to shed light on the relationships between most of the deeply branching rhizarian groups, a protocol was developed to prepare cDNA libraries suitable for 454 sequencing from a handful of cells collected from environmental samples. We obtained and analyzed more than 670,000 ESTs from 2 marine acantharean Radiolaria (Astrolonche sp. and Phyllostaurus sp.), 2 parasitic Phytomyxea (Plasmodiophora brassicae and Spongospora subterranea) and Gromia sphaerica, a giant marine testate protist that is capable of producing macroscopic bilaterian-like traces [28]. Phylogenetic analyses of 167 genes support the Retaria hypothesis and suggest that this group may be most closely related to Phytomyxea and Gromia. Moreover, our study confirms the presence of polyubiquitin insertion in some Acantharea and reveals another possible rhizarian-specific signature in one of the ribosomal proteins.
Dataset construction
The phytomyxean P. brassicae and S. subterranea are parasites of the plant genera Brassica and Solanum, respectively, and the in vitro callus samples were prepared according to an unpublished protocol (Bulman et al. submitted). Consequently, an unknown amount of plant contamination was expected in the phytomyxean ESTs. An initial blast examination showed that many of the phytomyxid-callus contigs had high similarity to plant sequences and were thus possibly derived from the host cells. We took advantage of the large amount of data available for Brassica and Solanum to filter out these plant sequences and simplify data searching for constructing the single-gene alignments (see Methods).
A total of 167 gene alignments with at least one rhizarian species represented in each were constructed for phylogenetic analyses. Based on recently published results suggesting close evolutionary affinities between Rhizaria, stramenopiles and alveolates, forming the so-called SAR group [3, 5, 6], representatives for these 3 groups were included. The full dataset comprised 10 rhizarians, 9 stramenopiles, and 9 alveolates. In order to reduce the risks of artifacts, 11 green plant taxa were chosen to root our phylogenies because 1) of the availability of complete genomes for many lineages, thus considerably reducing the amount of missing data in the outgroup; 2) they have evolved more slowly comparatively to most of the SAR species; and 3) their relative evolutionary proximity to the SAR group in the tree of eukaryotes [4]. However, an alternative outgroup, haptophytes, was also tested as it was proposed to be more closely related to the SAR group (data not shown) [7]. We did not select it for the final analyses because only medium-sized EST datasets are available for a limited number of species, except for one complete genome (Emiliana huxleyi), and the intra-Rhizaria relationships remained identical to the trees rooted using the green plants (see below). Each single-gene dataset was thoroughly tested by bootstrapped maximum likelihood (ML) analyses for deep paralogy or suspicious relationships possibly indicative of lateral gene transfer (LGT) or contamination. The acanthareans are known to harbor zooxanthellae symbionts and polycystine radiolarians are hosts of prasinophytes, dinoflagellates and other alveolates [29]. Accordingly, non-acantharean sequences were expected to be found. Out of the 167 selected genes, we could identify 1 sequence related to haptophytes in Astrolonche and Phyllostaurus, 2 sequences of dinoflagellate origin in Astrolonche, 5 and 2 sequences of general plant affinity in Astrolonche and Phyllostaurus, respectively, and, surprisingly, 25 sequences in Astrolonche and 21 sequences in Phyllostaurus clearly belonging to streptophytes (angiosperms). It is not clear to us why streptophyte sequences were present in our acantharean dataset, but one possible explanation could be that the samples were contaminated with a small amount of pollen. All these contaminant sequences were removed from our alignments. The curated protein alignments were concatenated into a supermatrix amounting to 36,735 unambiguously aligned amino acid positions (global percentage of missing data: 40%; see Additional file 1 for details) that was subjected to phylogenetic analyses.
Phylogenetic analyses of the supermatrix
To better evaluate these differences, a topology comparison analysis using the approximately unbiased (AU) test was performed [30]. Both trees in figure 1 (P = 0.916) and in figure 2 (P = 0.084) were not rejected at the 5% significance level. This test was based on the comparison of trees obtained with 2 non-nested models, "LG" (Figure 1) and "CAT" (Figure 2), using the "LG" empirical matrix. Hence, if the topology in Figure 2 had been rejected, it would not have been very informative because the "CAT" model could still have inferred the true tree. In the present case, however, the LG-based AU test kept the "CAT" tree among the trees possibly correctly describing the relationships within Rhizaria, thus strengthening the branching pattern showed in Figure 2. In addition, a topology with Acantharea alone in a sister position to the rest of Rhizaria was also tested in order to estimate the likelihood of the basal branching of Radiolaria seen in some SSU trees (see [16] for a discussion). This topology was strongly rejected (P = 7e-09), further supporting the association of Foraminifera and Radiolaria.
Evaluating the branching order within Rhizaria
Actin phylogeny
Rhizarian signatures
Interestingly, we identified a new insertion of 2 and 4 amino acids in the 60S ribosomal protein L10a, a characteristic also apparently unique to Rhizaria. A phenylalanine (F), an asparagine (N), and a serine (S) followed by a lysine (K) were inserted at position 104 in G. sphaerica, R. filosa, B. natans, and Paracercomonas sp., respectively (Figure 8B). In G. sphaerica, the sequence contained 2 additional inserted amino acids, i.e. a valine (V) and a glycine (G). Unfortunately, this gene was not present in the acantharean dataset and several attempts to amplify it by PCR failed. Blast searches against GenBank-nr and dbEST revealed no other known rpl10a gene containing this insertion.
This study provides the first robust evidence for a relationship between Foraminifera and Acantharea, a member of Radiolaria. This result is rather surprising, taking into account the considerable differences in morphology, composition of the skeleton, and lifestyle between these groups. Radiolarians have intracellular celestite (SrSO4) (in Acantharea) or siliceous (in Polycystinea) skeleton consisting of strontium sulphate and are holoplanktonic. In contrast, the foraminiferal skeleton (when present) is extracellular, agglutinated or calcareous, and the majority of foraminiferans are benthic. Pseudopodia morphology is also markedly different: radiolarians possess stiff, ray-like pseudopodia called axopodia, while foraminifers are defined by the presence of fine, anastomosing granuloreticulopodia. Still, there are also common cell characteristics shared between these 2 groups, the importance of which must be re-evaluated in view of our data. For example, the network of fine reticulopodia observed in some radiolarians exhibits bidirectional streaming, and is used for capturing prey and locomotion in a similar manner as the foraminiferal granuloreticulopodia [29]. Further studies of proteins involved in pseudopodial formation in both groups are needed to examine these properties at the molecular level. In that respect, it is interesting to note that our acantharean ESTs contained an unusual beta-tubulin strongly resembling the highly diverging type 2 sequences reported in Foraminifera by [33], as well as a less diverging isoform weakly grouping with a new foraminiferan beta-tubulin type (here named "type 3") found in R. filosa cDNA library (Additional file 2).
The clustering of Foraminifera and Acantharea observed in our analyses partially confirms the Retaria hypothesis [26]. Although multigene data for the 2 other main groups of Radiolaria, Polycystinea and Taxopodida, are still unavailable, we predict that they will also group with Foraminifera. This relationship is suggested by the phylogenetic position of three fast-evolving sequences of polycystinean actin as sister to foraminiferan actin paralogue 2 [19] as well as by the grouping of Foraminifera with environmental clones assigned to Polycystinea and Sticholonche in an analysis of combined SSU and LSU rDNA [25, 34]. However, the branching order of these groups was uncertain and Foraminifera may in fact branch within the radiolarian clade, suggesting that Radiolaria (Radiozoa) could be paraphyletic [25, 34, 35]. The next challenge will be testing whether this surprising pattern arises due to an artifact of LBA, and testing the monophyly of radiolarians.
Further effort is also required to resolve the relationships among the rhizarian groups. Thus far, all phylogenetic studies of this supergroup have recovered Radiolaria alone, or together with Foraminifera as the most basal clade [16]. The latter topology was supported by our LG and WAG-based tree reconstructions (Figure 1), but not by the Bayesian inference with the CAT model (Figure 2). Instead, this method suggested that Retaria are closely related to Gromia and Phytomyxea, to the exclusion of Cercozoa. Although this association received only low support with the full dataset, it was strengthen by the experiments with the foraminiferans or the fast-evolving sites removed, as well as by the AU and cross-validation tests. The removal of R. filosa was particularly informative in indicating that this species alone could have attracted Retaria at the base of Rhizaria in the "LG" tree, due to its high rate of evolution. Indeed, when it was not included, the topology suggested by the "CAT" model was robustly recovered. Acanthareans, on the other hand, seemed to be less prone to LBA as they consistently branched in a derived position when both foraminiferans were removed. The fast-evolving sites removal analysis also convincingly supported the grouping of Retaria, Gromia, and Phytomyxea, and was in agreement with the properties of the CAT model; it has been shown that this model infers homoplasies better than empirical models (such as the LG model used in the RAxML analyses) [36]. Therefore, it might be interpreted that for our complete dataset, the CAT model detected and correctly interpreted more of the saturated positions that were misleading in the RAxML analysis. Within this group, the position of Gromia could not be inferred with precision as it branched either as sister to Retaria or Phytomyxea. However, the grouping of Gromia with Phytomyxea was recovered only when Acantharea were absent or R. filosa was included in the analyses with the "LG" model. Moreover, the better fitted CAT model robustly placed Gromia in a sister position to Retaria. Interestingly, the association of Foraminifera, Acantharea, Gromia and Phytomyxea has never been described, although SSU and actin trees showed generally unsupported relationships between some but not all lineages [37, 38]. In addition, other lineages, such as Haplosporidia or Filoreta also belong to this group and will likely be crucial for resolving the internal branching order.
Finally, our study clarifies the question whether acanthareans and polycystines truly lack the rhizarian-specific polyubiquitin insertion, as previously reported [22]. To explain the apparent absence of the insertion in these two groups, it has been proposed that it was lost in radiolarians, or was acquired after their divergence [22]. In our EST data, both acantharean species feature polyubiquitin sequences with the insertion, suggesting that the sequences presented in [22] were not of acantharean origin, but perhaps originated from unidentified symbionts.
Our multigene analysis elucidates the relationship between two important rhizarian phyla, Foraminifera and Radiolaria (Acantharea), which has been a matter of recent controversy. Because Acantharea do not fully represent the radiolarian diversity and genomic data for other important groups (Polycystinea and Taxopodida) is still missing, we cannot rule out the possibility that Radiolaria are paraphyletic. Nevertheless, our study strongly indicates that a basal position of Radiolaria with respect to the rest of Rhizaria is highly unlikely. Instead, our analysis suggests a novel grouping including Foraminifera, Radiolaria, Gromia and Phytomyxea. Within this group, Gromia might be most closely related to Foraminifera and Radiolaria, but its specific phylogenetic position will depend on other important lineages such as Haplosporidia or Filoreta.
Collecting and isolation of specimens
G. sphaerica was collected near Little San Salvador Island in the Bahamas at about 720 m depth (24°34.5'N; 076°00.1'W) and total RNA prepared as described in [28].
Acanthareans were collected during May-June 2008 at the outlet of the Villefranche Bay, Mediterranean sea (43°41'N; 7°18'48E). Plankton samples were taken using a plankton net (mesh diameter 20 μm) drawn vertically from the depth of 200 to 0 m. Concentrated samples were immediately brought to the lab and processed. Living acanthareans were picked from the plankton with needles, washed with filtered seawater and placed in RNAlater solution (Ambion). The solution was allowed to penetrate into the cells for 24 hours at 4°C, after which the samples were kept frozen until further processing. In total about 300 cells of Phyllostaurus and 50 cells of Astrolonche were collected and used for library preparation.
The 2 phytomyxean samples were prepared from in vitro grown callus consisting of S. subterranea infected Solanum tuberosum cells and P. brassicae infected Brassica rapa cells, respectively. Full details of the generation, growth and characterization of these callus lines will be detailed elsewhere (Bulman et al. submitted). Briefly, sections of S. subterranea or P. brassicae root galls were surface sterilized and placed on MS media. Segments of white/green multiplying cells were transferred to new media as they proliferated. Fresh green callus cells were harvested and transferred into RNAlater (Qiagen).
Preparation of cDNA libraries for 454 sequencing
The G. sphaerica cDNA library was prepared from approximately half of an individual, as described in [39], with minor modifications (described below) that ensured enrichment of the data with protein-coding sequences. Briefly, the methodology involves cDNA synthesis and amplification using SMARTer cDNA synthesis kit (TaKaRa BIO/Clontech, Mountain View, CA), normalization using Trimmer kit (Evrogen, Moscow, Russian Federation), fragmentation by sonication, end-polishing, ligation of adaptors, and amplification of the 454-ready sample. The design of the adaptors ensures that the sequencing proceeds only from the cDNA breaks introduced by sonication, rather than from original termini, which helps to reduce the amount of adaptor-derived sequences in the resulting data.
For acanthareans, we originally prepared the libraries from 100 Phyllostaurus and 20 Astrolonche cells using the same protocol without normalization, but after Sanger-sequencing 24 randomly picked clones per species we found that the libraries consisted predominantly of non-coding sequences, most likely representing 3'-UTRs of the original transcripts. To enrich our libraries with the coding regions, we amplified the SMARTer kit-synthesied cDNA with a long primer (5'-AGTGGACTATCCATGAACGCAAAGCAGTGGTATCAACGCAGAGT-3') at a concentration of 0.1 μM, instead of using the one included in the kit, which resulted in preferential amplification of the longest cDNA fragments due to mild PCR-suppression effect [40]. Moreover, after fragmentation and adaptor ligation, only the 5'-ends of the original cDNAs were amplified and subjected to sequencing, by using the primer annealing to the ligated sequencing adaptor [39] and the primer matching to the unique sequence of the template-switch oligonucleotide used during the cDNA synthesis (5'-GCCTCCCTCGCGCCATCAGCCGCGCAGGTACGTATCAACGCAGAGTACGCGG-3'). The libraries were sequenced using 454 GS-FLX. The latest version of the cDNA preparation protocol, adapted for the latest 454 version (Titanium), is available on Matzlab website [41].
For the 2 phytomyxean species, cDNA libraries were constructed by Vertis Biotechnology AG (Germany) according to their Random-Primed (RPD) cDNA protocol. Frozen cells were ground under liquid nitrogen and total RNA isolated from the cell powder using the mirVana RNA isolation kit (Ambion). Poly(A)+ RNA was prepared from total RNA. First-strand cDNA synthesis was primed with an N6 randomized primer and second-strand cDNA was synthesized according to the classical Gubler-Hoffman protocol [42]. Double stranded DNA (dsDNA) was blunted and 454 adapters A and B ligated at the 5' and 3' ends. dsDNA carrying both adapter A and adapter B attached to its ends was selected and amplified with PCR using a proof reading enzyme (24 cycles). For 454 sequencing the cDNA in the size range of 250 - 600 bp was eluted from a preparative agarose gel.
Contig assembly and sequence alignment
All newly generated reads were assembled into contigs using the Newbler assembler with default parameters, generating the following number of contigs larger than 100 bp: G. sphaerica, 24,433; Astrolonche sp., 6426; Phyllostaurus sp., 5056; P. brassicae, 27,333; S. subterranea, 14,531. To filter plant sequences out of the phytomyxean datasets, the phytomyxid-callus contigs were compared to plant cDNA sequences using the BLAT tool [43] and those with very high similarity were discarded (e-value threshold < 1e-50). The 27,333 P. brassicae contigs (containing B. rapa) were compared against 2,529,141 Brassicaceae ESTs (NCBI as of 25 March 2009, http://www.ncbi.nlm.nih.gov/) and resulted in 24,166 contigs showing nearly identical hits. The 14,531 S. subterranea contigs (containing S. tuberosum) were compared to 1,000,784 Solanaceae ESTs (NCBI as of 25 March 2009, http://www.ncbi.nlm.nih.gov/), giving 8,282 contigs with a nearly identical hit. Manual inspection (using blastn) of a subset of the filtered sequences confirmed that these were indeed likely to originate from the plant host cells. The remaining 3,167 P. brassicae contigs with low or no hit against the Brassicaceae ESTs and the 6,249 S. subterranea contigs with low or no hit against the Solanaceae ESTs were used in subsequent screenings.
Blast searches against databases containing the 5 species above and taxa downloaded from GenBank (http://www.ncbi.nlm.nih.gov/) and JGI (http://genome.jgi-psf.org/) were performed to retrieve the genes of interest (see Table S1 for the list of species). These genes corresponded in part to genes that we used in previous phylogenomic studies [3, 4, 7], but also to 55 genes representing additional members of large protein families that were not previously included in our alignment, such as more minichromosome maintenance proteins (MCM) or more proteasome subunits, but also several new ribosomal proteins and proteins for which a broad sampling was available (see Additional file 3 for a list of the newly added genes). In total, 202 single-gene alignments were constructed, automatically aligned with Mafft [44], using Gblocks [45] to remove ambiguously aligned positions (with half of the gapped positions allowed, the minimum number of sequences for a conserved and a flank position set to 50% of the number of taxa plus one, the maximum of contiguous non-conserved positions set to 12, and the minimum length of a block set to 5) and followed by manual adjustment when needed with BioEdit [46]. The orthology and possible contamination in each gene was tested by ML reconstructions with 100 bootstrap replicates using RAxML 7.2.2 (LG substitution matrix) [47], and visual check of the resulting individual trees. Out of the complete set of 202 genes, 23 showed evidence for deep paralogy and were therefore discarded. In addition, we also excluded 12 extra genes as they did not contain any species of Rhizaria. Our final dataset contained 167 genes, which represented 36,735 amino acid positions after concatenation, and 39 species belonging to Rhizaria, stramenopiles, alveolates and green plants (outgroup). The single-gene and concatenated alignments are available at http://www.fabienburki.com. A table listing all genes and a detailed view of the missing data repartition for each taxa can be found in Additional file 1. The 167 trees constructed from the final selection of genes are available in Additional file 4. Since many of the Phytomyxea contigs were short due to the relatively small number of contigs that were obtained from the phytomyxid parasites, 35 genes were chosen as targets for further sequence acquisition. PCR primers were designed based on the short contigs and longer DNA sequences were obtained by RT-PCR or 3'RACE using the callus RNA as template (as in [48]). Scafos 1.2.5 [49] was used for performing the concatenation process of the single-genes.
The actin and beta-tubulin alignments were built by retrieving from GenBank sequences belonging to all rhizarian (actin) or eukaryotic (beta-tubulin) groups, and adding to them the sequences identified in the datasets generated in this study. 72 rhizarian and 6 stramenopiles (outgroup) sequences were included in the actin tree; 119 eukaryotes were analyzed for the beta-tubulin tree. We also searched our datasets for the 1 or 2 amino acids insertion at the monomer-monomer junctions carried in most rhizarian species. Finally, the construction of the single-gene alignments revealed a rhizarian specific insertion of 2 amino acids in the 60S ribosomal protain L10a.
Phylogenetic analyses
ML analyses were performed using RAxML 7.2.2 [47] in combination with the LG amino acid replacement matrix [50]. The best ML tree was determined with the PROTGAMMA + F implementation in multiple inferences using 10 randomized parsimony starting trees. Statistical support was evaluated with 100 bootstrap replicates. Bayesian analysis using the WAG + G + F model (4 gamma categories) was done with the parallel version of MrBayes 3.1.2 [51]. The inference consisted of 1,000,000 generations with sampling every 100 generations, starting from a random starting tree and using 4 Metropolis-coupled Markov Chain Monte Carlo (MCMCMC). 2 separate runs were performed to confirm the convergence of the chains. The average standard deviation of split frequencies was used to assess the convergence of the 2 runs. Bayesian posterior probabilities were calculated from the majority rule consensus of the tree sampled after the initial burnin period as determined by checking the convergence of likelihood values across MCMCMC generations (corresponding to 50'000 generations). PhyloBayes 3.1 [52] was run using the site-heterogeneous mixture CAT model with the rates-across-sites heterogeneity handled by a Dirichlet process (ratecat). 2 independent Markov chains with a total length of 19'000 cycles were performed, discarding the first 2'000 points as burnin, and calculating the posterior consensus on the remaining trees. Convergence between the 2 chains was ascertained by examining the difference in frequency for all their bipartitions (< 0.1 in all analyses). Bootstrap CAT proportions were obtained after 5000 cycles with a conservative burnin of 1000 on 100 pseudo-replicates generated with Seqboot (Phylip package [53]). Manual verification of 10 replicates showed that the burnin is generally between 500-700 cycles. For each replicate, trees were collected after the initial burnin period and a consensus tree was computed by readpb (PhyloBayes package). Consense (Phylip package [53]) was then used to calculate the bootstrap support based on these 100 consensus trees. Due to limited size of single-genes for parameter estimation under non-parametric models such as CAT, the PhyloBayes-based actin and beta-tubulin phylogenies were ran under the LG model.
The site-removal analysis was performed using PAML [54] to identify the fast-evolving sites, as implemented in the AIR package [55]. Because the topology chosen to estimate the site-wise rates strongly influences the results [32], rates were calculated for the 105 possible different topologies describing the evolutionary relationships of the 5 rhizarian lineages, and sorted according to the mean of the rates estimated on all topologies. 5% to 90% (10% intervals between 10% and 50%, 5% intervals between 55% and 90%) of the fastest evolving sites were then removed (percentage of the total rate distribution), and bootstrapped ML analyses were run with each of these 14 shorter alignments. PhyloSort [56] was used to search the pseudo-replicate trees for the relationships of interest. Phylobayes analyses with the CAT model were also done on each reduced alignment.
The statistical model comparison was done using the cross-validation (CV) method available in PhyloBayes 3.1 [52]. A learning and a test sets were generated by randomly splitting (no replacement) the original alignment into 10 replicates made of 90% and 10% of the original sites, respectively. Each of the learning and test alignments amounted to 33,062 and 3673 positions, respectively. A MCMC run was performed for each replicate under a fixed topology (either the "CAT" or "LG" tree) for a total of 5000 cycles ("CAT") or 1500 cycles ("LG"). Due to technical reasons associated with this test in PhyloBayes, a discrete gamma distribution with 4 categories was used for modelling the rate heterogeneity across site (i.e. dgam instead of ratecat). This slightly different model should not affect the conclusions given the big difference between CAT and LG. The lower number of cycles under "LG" was due to a much greater computational time per cycle as compared to when the "CAT" model was used. The first 500 and 150 points were discarded as burnin for the "CAT" and "LG" runs, respectively, and the remaining points used to compute the cross-validation log-likelihood.
Topology comparisons were conducted using the approximately unbiased (AU) test [30]. For each tested tree, site likelihoods were calculated using RAxML 7.2.2 with the LG model and the AU test was performed using CONSEL [57].
Accession numbers
454 reads generated in this study for G. sphaerica, Astrolonche sp. and Phylostaurus sp. were deposited in GenBank under the study accession SRP004044.1, and for P. brassicae and S. subterranea under the study accession SRP003604.2. The RACE products for P. brassicae and S. subterranea were deposited in GenBank under the accession HO772678-HO772709. The new Maullinia ectocarpi polyubiquitin sequences were deposited in Genbank under the accession HQ366774-HQ366778.
We thank John Dolan and Collette Febvre from Biological Station in Villefranche Sur Mer for help in collecting and identifying acanthareans, as well as 2 anonymous reviewers for valuable comments and suggestions The project was supported by the Swiss NSF grant 31003A-125372, National Oceanic and Atmospheric Administration Office of Ocean Exploration Grant # NA07OAR46000289 (''Operation Deep Scope 2007'') to MVM, and a G. & A. Claraz Donation. FB is currently supported by a prospective researcher postdoctoral fellowship from the Swiss National Science Foundation and by a grant to the Centre for Microbial Diversity and Evolution from the Tula Foundation. The phylogenetic analyses were performed on the freely available Bioportal at the University of Oslo (http://www.bioportal.uio.no) and the Vital-IT (http://www.vital-it.ch) Center for high-performance computing of the Swiss Institute of Bioinformatics.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
New York poised to require bachelor's degrees for RNsA century ago, nursing students learned their trade by trial and error while working in hospitals. Today, 45 percent of registered nurses have bachelor's degrees, and most have at least an associate degree.
Nationally, there is a growing movement to require all RNs to earn a bachelor's degree, and the profession is looking to New York to be the first state to mandate it.
"New York is really a linchpin here in terms of what will happen in the rest of the country, and I believe if we can pass it there will be a cascading effect of other states following," said Barbara Zittel, the former executive secretary to the state Board for Nursing. Zittel retired so she could lobby for the mandate.
Last year, the Institute for Medicine published a report on the future of nursing that set a goal of increasing the number of baccalaureate nurses to 80 percent by 2020.
The push is on.
One New York hospital, North Shore LIJ, requires newly hired nurses to earn a bachelor's within five years.
Locally, hospitals havve almost completely stopped hiring licensed practical nurses, opting for registered nurses who have at least an associate's degree. Then hospitals urge associate-level RNs to go back to school for a bachelor's.
On Friday, Russell Sage College will announce it is slashing tuition for RNs who want to earn a bachelor's degree. "We asked what can we do as an institution to support the advancement of registered nurses," said Glenda Kelman, chair of nursing at Sage.
This is the school's answer: Instead of $40,000, the program will now cost $24,000.
Sage will also offer on-site courses at St. Peter's Hospital and Glens Falls Hospital.
Glens Falls Hospital and Albany Medical Center have also partnered with Excelsior College in Albany, an online school, to provide courses at a reduced rate -- between $9,000 and $24,000 depending on the student's experience level.
Research has shown that patients fare better when cared for by nurses with higher education levels. According to a 2003 study, for each 10 percent increase in the number of hospital nurses with bachelor's degrees, death rates decrease 5 percent.
In New York, more than half of RNs have bachelor's degrees, slightly higher than the national average.
New York licenses about 8,000 new RNs each year, and 5,000 of them are prepared at the associate-degree level. On average, about 20 percent of associate-degree nurses go on to earn a bachelor's, Zittel said.
Legislation was introduced in the state in 2005 that would have required new nurses to earn a bachelor's degree within 10 years of receiving their nursing license. It would take that long to earn a bachelor's degree if a student took one course a semester. During those 10 years, nurses can practice as RNs.
Currently licensed nurses would not be subject to the law, and even students on the waiting list of nursing schools would be grandfathered.
The Legislature has never voted on the bill, but nursing leaders plan to renew their lobbying efforts. The bill now has the backing of community college leaders, who initially resisted it, Zittel said. Unions still oppose it because they are concerned grandfathered nurses will demand financial assistance for a bachelor's education even though they are allowed to practice without one.
Zittel believes that won't happen based on the experience in pharmacy and physical therapy, which now require doctorates to practice. Most grandfathered practitioners continued to practice at their original education level, she said.
New York has been chosen as a pilot site to implement the recommendations in the IOM report. On Thursday, nursing leaders will gather at the Foundation of New York State Nurses in Guilderland at a conference convened by the NYS Action Coalition. The coalition is funded by a grant from the Robert Wood Johnson Foundation and AARP.
Susan Reinhard, a nurse and senior vice president of policy at AARP, will be a keynote speaker. Reinhard said nurses are taking more responsibility as health care is focusing on preventing illness and reducing hospitalizations.
"Nurses need to be better prepared to do the teaching and the care coordination that is required to help people manage their chronic conditions, rather than getting into a situation where they need to go back into the hospital," Reinhard said. "It's a different skill set that many nurses have been taught or been practicing."
Reach Cathleen F. Crowley at 454-5348 or email@example.com.
Improving nursing
The Institute of Medicine's "Future of Nursing" had eight recommendations:
1. Allow nurses to practice to the full extent of their training and remove restrictions like collaborative agreements between nurse practitioners and physicians
2. Expand opportunities for nurses to lead quality improvement efforts
3. Implement nurse residency programs
4. Increase the proportion of nurses with a baccalaureate degree to 80 percent by 2020
5. Double the number of nurses with a doctorate by 2020
6. Ensure that nurses engage in lifelong learning
7. Prepare and enable nurses to be leaders
8. Build an infrastructure for the collection and analysis of inter-professional health care workforce data
Education levels of New York registered nurses:
52 percent have bachelor's degree or higher
44 percent have an associate's degree
4 percent have a diploma from a hospital-based program
Source: NYS Board for Nursing

subset: commoncrawl


text:
November 18, 2008
Suntop Homes, Built 1939
The stitching itself wasn't complicated. It's almost all backstitch in blue floss. (My husband picked the floss color for me in an attempt to get as close as possible to true blueprint ink.) How much easier can it be? Famous last words.
I cut up an old bedsheet for the background fabric, which worked out nicely. I reversed the image on my computer and printed it on photo paper, then transferred it onto damp fabric. This was a new technique for me and worked pretty well, although I had to do some tweaking and definitely learned some things for the future.
My biggest headache was struggling with fabric tension, as I was using a larger Q-Snap style frame and having some difficulty with it, and the weave of the fabric was causing my lines to look not so straight despite my following the printed lines exactly. The obsessive-compulsive side of me took over and turned this into a monster of a project, but I finally stepped back, looked at it from a distance, and pronounced it acceptable. I'm not happy with the puckering where I did the long and short stitch on the thicker lines--another tension issue--but I am certainly not redoing it. Ha!
This was definitely a nice change of pace from what I've been working on, and even though my critical eye can get caught up looking for all the flaws, when I can step back and be objective I'm really quite pleased with myself. My husband framed it in a nice black frame for me, and I printed out a Wikipedia entry that gives background info on the building, which I attached to the back of the frame. I'm hoping it will be a hit when Christmas rolls around.
11 comments:
- Tweed Delights said...
Truly amazing! :-)
- November 19, 2008 at 4:59 AM
- Heather - CROQZine.com said...
Saw this via a twitter link to the craftster post. AMAZING!!! Awesome!
- November 19, 2008 at 2:02 PM
- Redhoon said...
I saw this through a link sent by a friend, and it is truly amazing! I had to post a link to it on my blog. Very well done!
- November 20, 2008 at 12:29 PM
- Rachelle said...
- This comment has been removed by the author.
- November 21, 2008 at 12:41 AM
- OnePrettyThing said...
Oh how AMAZING! I love it so, SO much! I found a link to this in my email tonight and wow! So creative! I'll be linking to this if you don't mind!
- November 21, 2008 at 12:44 AM
- Muddlepud said...
Thank you so much, everyone. I am truly flattered by the attention this has gotten here and at Craftster. :)
- November 21, 2008 at 3:22 PM
- goody-goody said...
That is amazing--truly an original handmade gift!
- November 21, 2008 at 8:12 PM
- Maitreya said...
What a fantastic idea! It turned out great.
- December 2, 2008 at 12:11 AM
- Junie Moon said...
I saw this featured on One Pretty Thing and had to pop over to say what a brilliant idea this is. I love your project.
I'm thinking I will borrow your idea and get an architectural rendering of my husband's college (The Citadel) and do one for him for Christmas. Thank you for the inspiration and sharing your project with us.
- June 5, 2009 at 3:03 PM
- Anne said...
That is so fabulous looking. I may have to try that for my husband.
- June 6, 2009 at 7:35 AM
- fabrik-BILDschön said...
this is awesome!
greetings from germany
- August 18, 2010 at 2:48 AM


text:
In the Detail
- Lightweight frame for easy carrying Corner protectors and kick plate to protect against wear and tear
- Strong, polyester fabric for durability Water-resistant Printed with a stylish, hexagonal, geometric design
- Expandable main compartment Top, side and grab handles for easy handling One large, soft front pocket and one rear pocket Lightweight, locking trolley handle to suit any height
- Elastic packing straps and hanger loop to keep clothes crease-free Mesh pocket ID tag so your bag will always find its way home
- Four wheels, with 360 degree rotation for easy mobility Lightweight wheels
- TSA combination lock, built into the suitcase
- 10-year international warranty
- Colour Oxygen Black
- Features Lightweight, Expandable, Pockets, Grip Handles
- Dimensions 81 x 48 x 30 (cm)
Packing Capacity ? The internal space available, taking into account the trolley system, wheels, etc101 (L)
- Expanded Capacity 110 (L)
Maximum Volume ? The external space the case will take up, including the wheels, handles, etc116/128 (L)
- Weight 2.5 (kg)
- Material Polyester
- Wheels 4 Wheels, 360 Degree Rotating Wheels
- Locks TSA Fixed Combination
- Product Code 4081124015
Oxygen is rated out of 5 by 12.
Rated 5 out of 5 by harry from beautifully made like all y antler cases will outlast me....
Date published: 2019-04-07
Rated 5 out of 5 by Vesi from Just what I needed - ready to explore the world! I already had the smaller cabin size case and was very pleased with it so when I needed a larger one knew I would like the same and was most relieved to discover I could purchase it online as I was not able to find it in any stores ( I had purchased my smaller one in a store ). Extremely lightweight and easy to manoeuvre, spacious and expandable in size and lovely blue colour makes it easier to spot when collecting after a flight - please do not stop producing these cases!
Date published: 2019-01-25
Rated 5 out of 5 by Disneyhloiday from Great Suitcase Very light could not find one lighter of the same size. Bought two to go to Florida with the family and was so impressed bought one more came off the plane after the round trip and still looked like new.
Date published: 2018-12-07
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SKU: 4081124015Oxygen


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text:
Trip report E.W.Dijkstra, Nuenen & London, 10 Dec.'84 - 10 Jan.'85
(Sunday 23 Dec. 1984) We left the USA on Monday 10 Dec. 1984, but had left Austin the day before --courtesy Jayadev Misra, who saw us to the Airport at noon-- for Atlanta, where we stayed for 23 hours with a nephew and his family. (A very nice alternative to an awkward connection!) With our hosts we climbed the Stone Mountain on Monday morning; my sandals were inadequate and in Nuenen I have bought in the mean time a pair of flexible hiking boots. (also in Texas, sandals were not always the most ideal footwear.) During the KLM flight from Atlanta to Amsterdam, a high-pitched noise from the projector prevented us from sleeping during the movie; at Schiphol we almost missed part of our luggage, two suitcases being delivered on the wrong belt. Tuesday morning, 09:30, Marcus --our eldest son-- collected us at Eindhoven Airport.
During the days to come "And did you like your stay in Austin?" would be the most frequent question we had to answer. This was an opportunity to make up the balance sheet. Certainly most of it: most of UT, most of the countryside, most of the food, most of our house, most of our pattern of life (that is, to the extent it has been established), and most of the KMFA. At the same time, we had to admit the initial problems had sometimes been overwhelming, e.g. to register my new car it had to be inspected, to get it inspected I had to drive it through the town, to be allowed to drive it I had to be insured, and in order to get the insurance it had to be registered! (This is now a nice example of a deadlock situation, but as long as it lasted it was hardly funny.) and the problems with getting our house (more or less) finished and the airconditioning (more or less) wired according to specification had, of course, been out of proportion.
Our major adjustment problems so far seem to have been three.
(i) This one is trivial in nature, but was nevertheless tiring, time-consuming, and sometimes frustrating: the way in which goods are divided over different shops differs from what we were used to. And if you start thinking about the amount of information involved, it is suddenly not surprising at all that it takes a lot of time, effort, and experience to learn the new partitioning.
(ii) We were faced with a different interface between the industry and its customers: the last two or three phases of quality control are left to the customer. Well, that takes some getting used to! One side of the coin is that if you return a vacuum cleaner or handmixer, or point out that your airconditioning does not work, you get a new vacuum cleaner or handmixer and the control of your airconditioning is rewired; the other side of the coin is that your handmixer collapses an hour before the dinner guests arrive, you have to go to the shop at least twice instead of once, and that you have to leave the country before you have had the opportunity to verify that this time the airconditioning really works. (The last rewiring was done three days before our departure: I could only pray that the Bösendorfer won't dry out during our absence.)
Aside I now know the origin of "user satisfaction" as a quality criterion for software, and am grateful for that insight. I always found it a ridiculous criterion because it is technically useless: in 1960, Jaap Zonneveld and I implemented ALGOL 60 under the --evidently most inspiring-- slogan "The user be damned." It worked beautifully. (End of aside.)
(iii) In our innocence we thought that 22 years in the Dark South of the Netherlands --worse: only 15 minutes from the Belgian border-- had been a lesson that would last for life. But we were definitely unprepared for the high level of ubiquitous incompetence we would face in Austin. (When I try to tell with my most stoic face that on my car insurance papers, only 4 of the 17 characters of the chassis "number" are wrong, people here think I am kidding and don't believe me. When, early December, after we had ordered an ironing board, Sears delivered a hair dryer, we were no longer amazed.) A quarter of a century ago I learned that "The Backward Country" is a reference to the USA. I was surprised to learn that even in clerical and technological areas the nickname is still appropriate.
(Wednesday 26 Dec. 1984) Back in the Netherlands, life was as busy as expected. Ria went to her mother in Amsterdam, together we went twice to my mother (who had her 84th birthday on the 20th). We paid two visits to our dentist, I a visit to my physician for my medicine and to have the arteries in my neck checked --they were okay--. Ria did a lot of shopping, I some. We went to the bank in person, but confusion over the IRS I could clear up over the telephone. The first Thursday morning I helped my son Marcus move. Etc.
So far, I worked twice with Carel Scholten and twice with Netty van Gastern. With Netty I screened what she had written since her visit to Austin. The first day with Carel was devoted to what we had done during the preceding months; the second day was devoted to our book, on the manuscript of which I had started in the mean time. I wrote a long letter to my nephew Sybrand in Kortrijk, who had sent me a beautiful proof (which I recorded one evening in EWD904). It is so short and so beautiful that I may use it next month as an example of what can be achieved by formal means when I have to give a talk at the Symposium that will be staged on the occasion of Carel Scholten's retirement at Philips.
I attended last week's session of the Tuesday Afternoon Club --this week the THE is closed--. I showed them some nice proofs and drew their attention to the problem of establishing whether one given expression is a syntactic descendant (or: substitution instance) of another given expression. In the presence of symmetric, associative operators, the problem seems in general difficult, and I could convince them why it would be nice to know how difficult the problem really is. I had a long discussion with Martin Rem --more about science politics than about science itself--, and I paid three other visits to the THE.
In the Department of Mathematics and Informatics I found all the familiar tensions still very much alive. The mathematicians are still the champions of mediocrity and sabotage the informatics curriculum by giving the informatics students a fake course instead of teaching them what a proof is. (For one of their courses the examination was such a caricature that 170 out of 175 students passed the test!) The problem is that in the joint department the mathematics branch is almost as overstaffed as the informatics branch is understaffed. Were this not the case, the computing scientists would have taught their students mathematics themselves. Nothing shows that my departure has reduced the tensions; the problem is that these mathematicians are afraid. But by behaving in the way they do they disqualify themselves so blatantly that they can only hasten their own dismissal. (I see no future for socialist mathematics.)
(Sunday 6 Jan. 1985) In the mean time I worked another two Mondays with Netty and another two Fridays with Carel. (In between I had worked on the manuscript of our book.) I wrote a number of letters and spent a day on the completion of a refereeing job on which I had started shortly before I left Austin. (The paper was much too fat.) I had every reason to be grateful to Burroughs for having left me the copying machine in Nuenen: without attending to my mailing list I made several hundred copies this month.
And the social activities continued. On 1 Jan. 1985 Femke joined me on a trip to my mother (to wish her a Happy New Year and to say goodbye). On 3 Jan. 1985 Ria and I went to Holland. While she visited her mother in Amsterdam I visited friends in Aerdehout. After having collected Ria at her mother's retirement home, we went for dinner to friends in Voorburg.
We left them at 22:20. That night the winter started: we were caught by a snow storm and it took us almost three hours to reach Nuenen. Today, around noon, Femke called from Groningen asking us what to do about frozen water pipes. (Our house in Nuenen being better designed, it was a new experience for her.) Tomorrow I have to fly from Amsterdam to London, and I hope the best! (What else can one do?)
(Saturday 12 Jan. 1985) On Monday morning, Ineke van der Meiden took me to the Eindhoven railway station; the car ride took almost 40 minutes instead of the usual 12. Trains were still moving, but the trip to Amsterdam took an extra hour. Our planning had been sufficiently pessimistic: we caught our plane which flew on time. In London, Netty van Gasteren and I were collected by a BP driver, who took us to the BP Training Centre (very nice, but for the absence of decent blackboards).
The next two days, Netty and I addressed in turn an audience of about 20 people about the streamlining of the mathematical argument; it was a new audience, we used some old examples and some new ones. It was a nice audience --five of the people never opened their mouths-- but rather mixed, and that is a problem nowadays. Decades ago, when needing an example familiar to all, one could always fall back on Euclidean geometry, but that is no longer a common background today. (With for some triangle A meaning "it is isosceles" and B meaning "it has two equal angle bisectors" the proof of A⇒B used to be trivial, the proof of B⇒A used to be a notorious teaser, but only two people remembered that! Needless to say, we proved A≡B.) The audience seemed very pleased with our performance (more pleased, in fact, than we were ourselves).
Next morning, Netty gave me a wake-up call at 06:30. At 07:00 the taxi came to take us to Heathrow where our ways parted: I flew first to Schiphol, were Netty arrived shortly before I boarded the flight to New York. The PanAm flight from there to Austin was delayed by 1½ hour. When I arrived --past midnight, after about 24 hours of travel-- I was releaved to see Ham Richards waiting for me and annoyed to learn that my suitcase (which I had seen through customs in New York) had not made it. When I woke up this morning, it had started snowing. I phoned PanAm about my suitcase; it had arrived in Austin but I did not get the impression that PanAm was very eager to deliver it at Robbie Creek Cove.
While we were in Nuenen, Ria's bicycle was stolen when Rutger had used it to go to Eindhoven. (He had to walk back in the middle of the night.) I read a four-volume history of The Low Countries (with many illustrations): a long history of hunger, misery, violence and dishonesty. I could not escape the impression that my country's history as I learned it in my youth had been somewhat rose-coloured.
During our absence, the FM radio programs had changed: the German music program included more modern composers in the evening and the Flemish had become even more nationalistic; the Dutch radio had a new announcer with a very vulgar diction. We heard Moussorgsky's "Pictures at an Exhibition" thrice, and so with Handel's "Messiah" --once sung in German!--.
Austin, 12 Jan. 1985
prof.dr. Edsger W.Dijkstra
transcribed by Tristram Brelstaff


text:
Alien By Thierry Mugler Mini Edt .2 Oz
This Perfume was created by Thierry Mugler in 2005 and makes an amazing gift for any occasion. This spectacular feminine scent includes a blend of Amber Woods Orange Blossom Green Notes and Vanilla.
Designed For Women
This product is the original authentic name brand and NOT a knockoff or imitation.


text:
For each issue, Surgical Products
relies on its readers to share what
innovations and technology they are
interested in, and this month they
had questions about Image Stream
Medical's EasySuite 8.0.
To learn more, Surgical Products
connected with Laura Holly, Image
Stream Medical's director of marketing.
Holly: Today, the trend is toward moreminimally invasive, image-guided pro-cedures. We see these kinds of proce-dures particularly in the cardiac, vascular,neurology, and spine areas – aortic valvereplacements (TAVR), endovascularaneurysm procedures (EVAR), and treat-ments of vertebral compression frac-tures, for example. Similar image-guidedtherapies are becoming more commonin thoracic, trauma, and oncologicalapplications as well. Successful out-comes for these highly complex proce-dures rely on the precise coordination ofmulti-disciplinary. It's vital that each teammember, whether in the room or else-where in the hospital, has access to thehigh-quality visual information they needand can quickly and easily communicatewith the rest of their colleagues in real-time during the procedure.
Holly: Surgical teams will immediately notice that EasySuite's real-time
collaboration features are completely
integrated into their control system.
Establishing a two-way video conference with colleagues with exceptionally
high quality audio and video is now
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the right room displays, dimming the
room lights, or adjusting their music.
Making it this easy to use ensures that
clinicians can take advantage of these
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flexibility to do ad-hoc conferences and
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clinicians is a big win.
Holly: Creating an environmentwhere surgical teams, comprisedof in-room and remote clinicians,have access to the informationthey need when they need it –without breaking scrub or withoutwaiting – leads to more timelyclinical decisions and ultimatelymore efficient and better care. Inenvironments where every momentis important, this kind of efficiencyis key.
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Click to subscribe to this magazine


text:
1. Three 'Algerians' arrested after naked teenager found bound, gagged and gang-raped underneath the Eiffel Tower
Three illegal immigrants were in custody in Paris today after a teenager was found tied up and gagged after allegedly being gang raped underneath the Eiffel Tower.
The horrifying attack took place after the 19-year-old was lured on a Facebook 'date' in the French capital. [...]
The woman was dragged underneath a bush, where a blanket had been laid out. She was assaulted by the men, and then gagged, beaten and tied up. [...] (A jogging couple) found the woman in deep shock, naked and crying.
2. Global News: The United States appears to have led a coalition of bombers over Syria, which paved the road to allow Islamic State fighters to take an entire area of Syria.
BEIRUT – U.S.-led coalition jets bombed a Syrian army position at Jebel Tharda near Deir al-Zor airport on Saturday, killing dozens of Syrian soldiers, Russia and a war monitoring group said, paving the way for Islamic State fighters to overun it.
Syria's army general command said in a statement that the air strike was "conclusive evidence" of U.S. support for Islamic State, noting that the strike was "dangerous and blatant aggression".
Some other sources on this action include RT News and Almasdarnews.
3. Unprovoked brutal beating by eight "southern looking" people on a German train.
The original German story is here, and a full English translation can be found at this site.
A short excerpt:
They hadn't done anything except that they accidentally walked into a photo in progress. This provoked eight young men so much that they jumped the two 20 year olds and beat them senselessly. The victims managed to get out of the train at the next stop at Bahnhof Rathaus Steglitz. The attackers fled on the train unrecognized towards Oranienburg. [...]
When the victims tried to defend themselves, an incredibly violent scene unfolded: One man from the group took one of the 20 year olds into a choke hold. Several of the others then hit and battered him. An uneven fight. The other victim was kicked in the face several times. At Bahnhof Steglitz the two 20 year old men could escape the train - with visible injuries. No trace of their attackers.
According to statements of the victims and their female friend the unknown suspects were between 17 to 18 years old and looked southern.
4. Muslims on the Greek island of Lesbos chant "jihad jihad jihad"
Pamela Geller has the timeline on this event which contains additional exposure of tactics intended to inflame and disrupt the locals of the Greek island.
5. German government fails to recognize fake passports used by "refugees".
A state attorney general has said he wants to seize records from the Federal Migration Office over the possible use of fake passports by refugees entering Germany. The issue has stirred tensions in Merkel's coalition.
Germany's Federal Migration Office (BAMF) has been accused of severe security flaws after it was revealed that officials failed to detect counterfeit passports used by refugees entering the country.


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Email: Clash of the Titans
Dec 19, 2016
By: Todd Jenkins
The IMG Academy Ascenders took on the Norcross GA Blue Devils in the 2nd round of the City of Palms Classic Tournament. IMG was set to defend their USA Today fifth ranking against the 15th ranked Norcross team. Highly ranked guard Trevon Duval scored the first points of the game with a 3 pointer, however the biggest matchup was between IMGs twin towers Emmitt Williams and Silvio De Sousa versus Norcross Rayshaun Hammonds and Lance Thomas.
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Evan Rachel Wood Rips Through 4 Non Blonde's 'What's Up' [VIDEO]
'True Blood' star Evan Rachel Wood is known for being an edgy actress. But did you know she can sing, too?
She has had roles in musical fare like 'Across the Universe,' and once covered Justin Bieber, but Wood is largely celebrated for her acting work. However, the new mom totally blew our minds when she belted out 'What's Up,' the smash, '90s alt rock hit by 4 Non Blondes, at an event in Los Angeles.
In case you need a memory jog, the band was lead by Linda Perry, who reinvented herself as a songwriter for Pink and Christina Aguilera through the years.
ERW, looking cute in menswear chic with black pants and a red blazer, the latter by Moschino, was accompanied by piano playing while she roared through the song.
The song has some big notes and Wood hit 'em all. We're totally feeling it.
Oh '90s nostalgia is back and in a big way. Evan Rachel Wood reminded us of that.


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The film will see the VICKY DONOR actor Ayushmann Khurrana and the DELHI BELLY boy Kunal Roy Kapoor sharing the screen space. Earlier, Rohan had worked with Kunal Roy Kapoor as the producer when Kunal had directed political satire THE PRESIDENT IS COMING some years ago.
Says Rohan, "I realized that Kunal has great potential as an actor and that's why we got him on board as well. Ayushmann and Kunal are new age actors who have a definite connect with today's audience."
CHECK OUT: Abhishek Bachchan - I have never cashed in on my name
Speaking on association with Bhushan Kumar, Rohan says, "We have done many films with Bhushan Kumar and he is an extremely passionate producer. It is amazing to see the kind of clout he enjoys in the industry, but in spite of that, he has the passion to make those kinds of films that he believes in."
Bhushan Kumar too is quite contented to work with Sippy Junior. Says Kumar, "Rohan is one director who has a great sense of music. The experience of working on DUM MAARO DUM with him was great. And even now, he has given me a lot of freedom to get involved in the music of this film."
Well, one is eagerly looking forward to watching the duo - Ayushmann and Kunal - weave their magic onscreen.


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Tipped, a new site launched last week, provides people with the chance to view and share tips on the best places in their local areas, from bars and restaurants to shops and hotels.
Tipped provides listings for more than 50 towns and cities throughout the UK, and its users can provide their own reviews of places they have visited, or search through the directory for reviews of nightspots, hotels etc in their local area.
It is also a social network – users can upload their own pictures, create profiles, as well as connecting with other reviewers and keep up with tips from friends and contacts.
Other users can say whether or not your tips have been useful, and over time this can enhance your reputation as a reviewer on the site.
Having only launched last week, the reviews on the site mainly cover London, but it could build itself into a valuable resource if enough people start to use the site and leave their own reviews.
One unique selling point for this kind of site is that it has been designed with mobile users in mind. It has been optimised for mobile, and reviews can also be sent to your mobile, complete with a Google map of the venue. which could be very useful when on the move and looking for a place to go.
The site looks good, and is nice and easy to use – descriptions of the various bars, restaurants etc are accompanied by contact info, pictures and transport info, and the location is shown via Google Maps.
There is some competition in this area – welovelocal, for instance, which launched earlier this year, provides reviews and listings of local bars/restaurants, as well as having a similar social network element, though it doesn't have the same mobile access.
Tipped, which is based in London, was founded by Joel Brazil, ex-head of mobile for Microsoft UK's online services group, and the site has already attracted around 4,000 unique users through beta testing and word of mouth.


text:
Hi, I am developing an application that uses the SSML specification. In my SSML files, I make a reference to the file synthesis.dtd at http://www.w3.org/TR/speech-synthesis/synthesis.dtd. I did notice that with Microsoft Internet Explorer (v8 and v9, don't know other versions) it is impossible to access, receceiving a nice error '403'. No problems with others browers like Mozilla Firefox or Google Chrome. Any ideas? Thanks.Received on Friday, 5 November 2010 21:34:46 UTC
This archive was generated by hypermail 2.3.1 : Tuesday, 6 January 2015 21:07:42 UTC


text:
I have written on this topic before, but I feel I need to write about it again; whilst the majority of people in the Western world are not particularly wealthy, we find again and again that prices of this, that and the other are going up, whilst wages are being frozen, people are losing their jobs and there is real uncertainty in many people's lives in just how they are going to make ends meet and what plans they are to take in life.
The people who helped cause the financial meltdown and the subsequent economic chaos we find ourselves in are living privileged lives, sheltered from the very mess they helped to create. When the banks messed up, we bailed them out. So, in effect, they failed but didn't fail! Nice for some. Many millions of people in the United Kingdom are feeling the serious consequences of this economic mess, and are having to make all sorts of budget cuts to all sorts of things they were used to, and we are seeing that things like gas and electricity, the price of petrol, the price of food, transport and many other things are going up month after month, whilst wages and benefits remain the same, or in some cases are being cut.
Shouldn't a Christian stay out of politics and economics, you might say? Well, I believe we must speak out where there is injustice, and I believe we have the right to challenge, in a lawful way, the way our country is run. It is not being run well at this time, but no matter how hard the ordinary person is squeezed, the wealthy and powerful and those who rule and make laws seem to live well beyond the means of the ordinary person. And whilst many of us are dreading the next round of price rises, the people at the top seem not to care, primarily because those at the top have wealth and high-paying careers to shelter them from the worst of it.
All over the world people are campaigning against sharp practises by the wealthy, corporations and governments that don't care or who seem impotent to do anything really radical, like tackle the high prices of goods and services, the price of utilities, and the rising tide of unemployment. The ordinary person has no real say in how their countries are run, if they ever did, but at this time the gulf between the wealthy and powerful and the person at or near the bottom is very wide indeed.
There is a moral dimension to all of this, and if we are to be honest we need to look at and explore the moral dimension. From one angle, price rises are wonderful; if you are benefitting from them, that is! From another angle, the price rises are something that many millions of people around the world are dreading. I refer specifically to the UK: it wouldn't be so bad if it was just food and household goods that were increasing, but it is so many other things as well, like the cost of petrol, the cost of public travel especially train travel, the ever-rising cost of utilities like gas and electricity prices; there seems no end in sight, and yet our politicians, of whatever political persuasion, and the people who run and own these companies seem unable to do anything about it, and seem less to care, perhaps because they are all wealthy and well-provided for. It's the same old story, it seems. So let's come back to the moral issue at stake. The moral issue is that these price rises are, when it boils down to it, an issue of greed. And I believe implicitly that God does not countenance injustice and does not turn a blind eye to injustice for ever. Some people may get away with their sin this side of Heaven, but will certainly not on the other. There is immorality, greed and injustice woven into these price rises and they are at worst making some people's lives worse than they really need to be. There will be an answer to this injustice; mark my words.
It seems that, as ever, we in Britain have the highest prices for food, general goods and services, train travel, petrol and gas and electricity prices; why is this? Will we ever get a real answer from any politician about why we are always paying more for almost everything than any other Western European country, without the usual bluster and waffle and patronising tone all around? We can but hope. But deep down, if we are honest, we know that we are being ripped off and taken for a ride, often by the very people who are supposed to be fighting our corner. If we can't get rid of capitalism and I personally don't want to, then it needs changing. Firstly, if you have and make lots of money, you can pay more tax; if you are poor or make little, you pay less tax. It's that simple really. We need to redistribute wealth fairly this way, unless we want anarchy on the streets; not in a 'hippy-dippy' or Communist way or anything that has links to any political ideology, just a fair and simple redistribution that makes sense and is easy to administer. Secondly, we need to truly work together, governments, bosses, corporations, businesses and the regular person on the street in making our finances work, and perhaps we all need to stop living beyond our means and stop living on borrowed credit. Thirdly, we all need to find a purpose in life; I believe that goes beyond normal concerns but is very much the most important consideration in being human.
There is another problem I feel with Britain too; it seems that too much power and wealth and influence is concentrated in the hands of a relatively few people and also especially in the South East of England. This reflects history and tradition, but isn't it time we began to live in a modern nation, one that is based on genuine equality and fairness, as opposed to privileged status and unfairness?
When people give to charity, people from the Western nations to countries like India, Africa, Eastern Europe and Latin America, no one questions the fact that richer people are giving to poorer people; why should they? But why, when people are talking about redistributing wealth from the richest to the poorest within wealthy Western nations, is there a complete reluctance? Why are many wealthy people in the West so greedy and selfish? The only aim of charity, in any circumstance, is to redistribute wealth and resources from those who have to those who don't have.
What is the Christian answer? Well, as I wrote, I believe Christians need a purpose in life, even if at first that is just being obedient to God and walking in His ways. We also need to ask why there needs to be, in some circles, absolute unlimited wealth, and for others near-wretched poverty. Just what is the purpose of such wealth creation if it only benefits a handful of people? If a person is living in poverty, or struggling financially in any way, then maybe, and for the first time, they need to pray and ask God for help; He really can help! I'm not being religious here or anything like that, I'm just telling you the truth; God can, and does, work in the real world. For Christians, we need to lean hard on Jesus and to ask in prayer that we can satisfy our need before our greed and we need to be honest about ambitions and understand quite simply that if we make a lot, we need to put a lot back, and if we don't make a lot or if we are struggling and poor we can as I said ask God to make our lives better without the need necessarily to have millions and millions of pounds, even though I believe there is nothing fundamentally wrong with being wealthy provided you use some of that wealth wisely and to help others and you are prepared to pay a fair rate of tax.
Finally, I don't believe it's necessary to be wealthy to be happy, and it's not necessarily a prime requisite for being a Christian either. True happiness, and contentment, go beyond wealth creation and is a spiritual kind of wealth, one which lasts and no one can steal from you. I'm not suggesting either that being poor or wretchedly poor makes you super-spiritual in some way either; who wants to live in poverty after all? It's beyond that too. God will provide, and is able to provide all your needs, regardless of whether you are rich or poor. It's having faith in God that enables us to see the bigger picture, and will teach us that money is not the most important thing in the world.


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Rhododendrons have nearly finished by now but there is one real glory in R. Polar Bear with its pure white, very scented, big trusses of lily-like flowers. It is a strong grower, eventually forming a small, umbrella-shaped tree with a good trunk. It does need moisture and shade to prevent it cooking at this time of year, but if you can afford the space it is a real treat.
Another big evergreen with a superb scent is Magnolia grandiflora. Starting now, it will produce enormous individual white flowers until the frost. Often grown against walls, the flowers are lovely from upstairs windows. However, it is hardy in SE England and can be grown effectively as a free-standing tree. Goliath has rounded leaves, Exmouth�s are longer and Edith Brogue has a reputation as the hardiest.
Our hydrangeas start to flower now. Hydrangea Haven has been established as the obvious way of bringing summer colour into rhododendron beds and woodland gardens but there is a hydrangea for any garden, The H. serrata varieties are the most compact and ideal for small gardens and tubs. Examples are Preziosa, which is pink, aging to purple-red and Tiara, pale blue and early-flowering. More about Hydrangeas in theAugust section.
Few plants flower all summer. Fuchsias are an exception and F. magellanica Tricolor is quite outstanding as it is hardy and a very effective foliage plant, with silvery green leaves with cream and pink tinges, as well as long, thin flowers of red and purple. Ideal planted with Lacecap hydrangeas, silver-leaved plants like artemesias and also Verbena bonariense.


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The Impact of the Banking Act 2004 on the Performance of the Banking Industry in Ghana
The world of work and organization has become increasingly demanding and turbulent. There are numerous challenges currently facing by the banking sector in Ghana (Laulajainen, 2003). These are globalization, responsiveness to customers, increasing revenue and decreasing costs, building organizational capability, change and transformation, implementing technology, attracting and developing human capital, and ensuring fundamental and long-lasting change. Thus, levels of competition among organizations have increased. As in the banking industry of Ghana, the banking sector are offering different add on services to enhance their competitive advantage despite of the promulgation of Banking Act 2004. Some banks in Ghana offer internet banking, some also offers high interest earnings, some also offer low cash loan, and etc. With this head-to-head battle among banks in Ghana, banks are creating ways to become efficient despite of the promulgation of Banking Act 2004. Basically the Banking Act 2004 is an act that amends and consolidates the law relating to banking, to regulate institutions which carry on banking business and to provide for other related matters. This Act cover the issues about Ghana banking regulatory systems, licensing of banks, capital and reserves, liquidity, ownership and control, restrictions on lending investment, power of supervision and control, accounts and audit, and other issues related in banking business of Ghana.
Aims and objectives of the study
While the research questions only refer to the information that the researcher intended to question. The objectives, however, will focus on the necessary problems and objectives that should be clarified in order to gather the intended information and also be able to derive specific information that are not limited by the previous questions. With these objectives, the study will be able to attain the necessary information that can help derive further conclusions and proper recommendations. The study intends to determine the impact of the Banking Act 2004 to the banking industry of Ghana. The study intends to get the appropriate data to help in making the proper assessment. There are other aims and objectives of the study. This includes
- To determine what Ghana's banking sector do to meet the needs of their clients.
- To determine the things done by Ghana's banking sector to give satisfaction to their clients in accordance to the promulgation of Banking Act 2004.
- To determine and assess the perception of banking sectors pertaining to Ghana Banking Act.
- To identify how successful the banking sector of Ghana is and how the Banking Act 2004 contribute to the success of the bank.
The study shall use survey using questionnaires to gather pertinent data (Creswell, 1994). The questionnaire will be made in such a way that the Ghana banking sectors will spend less time in answering it. Moreover, the study shall also use previous studies and compare it to its existing data in order to provide conclusions and competent recommendations. Survey can be done either by personal survey, telephone survey, self administered questionnaire, mailed questionnaire, and email questionnaire.
The study will initially gather information that will serve as introductory part of the study. These kind of information helps the reader what the study is about, what it intends to do and what will be its result. The study will then gather related literature to prove the need for conducting the study. The literature review can help in determining what are the studies already done, what study needs to be corrected. The study will then determine the methods and means for data to be gathered and analyzed. In this part the data is being readied to be gathered and analyzed but the method to gather it will first be determined. The next part of the study is gathering, presenting and interpreting the data. In this part the validity of the hypothesis and ideas about the study will be proven. The last part of the study will be the part where conclusions and recommendations will be stated. In this part final statement about the study will be done.
Creswell, JW 1994, Research design: qualitative and quantitative approaches, Sage, Thousand Oaks, California.
Laulajainen, R 2003, Financial geography: a banker's view, Routledge, New York


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Ghost Team One (2013) - Synopsis
When roommates Brad and Sergio accidentally arouse the dead, they team up with sexy amateur ghost hunter, Fernanda, to expose the evil inside their home. But just as the guys start to get close to their hot new partner, they discover that a horny demon wants in on the action. Now, cameras are up, and more than fear is rising in this outrageous romp where only one thing is certain: someone is gonna get screwed.


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(Published in City Express, The New Indian Express, dated 10 March 2012, retrieved from http://expressbuzz.com/entertainment/reviews/paan-singh-tomar/371311.html)
Cast: Irrfan Khan, Brijendra Kala, Mahie Gill, Vipin Sharma
Director: Tigmanshu Dhulia
Rating: 3.5 stars
Only Irrfan Khan could have pulled off a role that requires him to be petulant, commanding, beseeching and awe-inspiring at once. He's proved what a fine actor he is with films as varied as The Warrior, where he plays a very different wronged hero from Paan Singh Tomar,The Namesake, where he plays a gentle father and husband, and movies like Life in a Metro, where he plays a fast-speaking, almost comic character. As an army man turned athlete turned rebel – he insists on being called baaghi, not dacait – he carries Paan Singh Tomar, leaving the viewer troubled as s/he walks out of the cinema.
It takes a performance like this to move an urban audience from indifference to the violence of the ravines, to anger at the systemic failures that force promising young men into crime. At the beginning of the film, we relate most to an official who brusquely says no family dispute in the Chambal Valley gets sorted out without a couple of killings. Towards the end, our empathy is entirely with 'Paan Singh Tomar gang', headed by a man who says he won't surrender, on the basis of a sporting principle – when you start a race, win or lose, you must finish it.
However, the film flounders for a focal point. Paan Singh's constant refrain that no one gave a toss when he was a star athlete, but the entire nation knows his name now that he's a baaghi, and a dedication at the end to "our unsung sports heroes" seems to indicate the movie is about athletes who didn't get their due. But the corrupt cops, and the intricate portrayal of the change in Paan Singh's attitude towards the system, suggests this is about the rise of dacoits.
Tigmanshu Dhulia's painstaking efforts are obvious – the research is thorough, the film was mostly shot on location, and the recreation of the athletic meets is remarkably realistic. A serious story is punctuated by moments of hilarity – the errands Paan Singh's wife (Mahie Gill) assigns their children to get them out of the house when she wants to make love, Paan Singh's jibes at an overweight journalist (brilliantly played by Brijendra Kala), the manner in which the gang evades border checks, and idiosyncratic scenes where a policeman is made to apologise to his uniform, a kidnapped son yells at his father for trying to bargain over the ransom, and a prospective bride's family agrees to an alliance because the groom's father has had his picture in the papers. From the jokes by the campfire, to the hot-headed young men, every aspect of the film rings with veracity.
There are poignant moments too – where Paan Singh is surprised softy ice creams are available in India, and wants to know their price, where he can't resist an interview because he's finally getting attention, and where he refuses to believe the army can be corrupted, even while buying army-issue guns. There are moments of cinematic excellence, but the profusion of parallels between his steeplechase days and his running over hurdles to escape the police begins to grate. Ok, dude, we get it.
I wish they'd shown us why Paan Singh became so notorious. The canvas appears rather too small – there's been no real crime against important people, the gang colludes with the police, Paan Singh's army connections take him a long way; so why does a family dispute merit an attempted encounter?