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Membrane transporters catalyze the transport of small solute molecules across biological barriers such as lipid bilayer membranes. Experimental identification of the transported substrates is very tedious. Once a particular transport mechanism has been identified in one organism, it is thus highly desirable to transfer this information to related transporter sequences in different organisms based on bioinformatics evidence.
We present a thorough benchmark at which level of sequence identity membrane transporters from Escherichia coli, Saccharomyces cerevisiae, and Arabidopsis thaliana belong to the same families of the Transporter Classification (TC) system, and at what level these membrane transporters mediate the transport of the same substrate. We found that two membrane transporter sequences from different organisms that are aligned with normalized BLAST expectation value better than E-value 1e-8 are highly likely to belong to the same TC family (F-measure around 90%). Enriched sequence motifs identified by MEME at thresholds below 1e-12 support accurate classification into TC families for about two thirds of the sequences (F-measure 80% and higher). For the comparison of transported substrates, we focused on the four largest substrate classes of amino acids, sugars, metal ions, and phosphate. At similar identity thresholds, the nature of the transported substrates was more divergent (F-measure 40 - 75% at the same thresholds) than the TC family membership.
We suggest an acceptable threshold of 1e-8 for BLAST and HMMER where at least three quarters of the sequences are classified according to the TC system with a reasonably high accuracy. Researchers who wish to apply these thresholds in their studies should multiply these thresholds by the size of the database they search against. Our findings should be useful to those who wish to transfer transporter functional annotations across species.
Keywords:Membrane transporter; Functional classification; BLAST; HMMER; MEME; Substrate; TC classification system; Sequence homology
Prokaryotic and eukaryotic genomes each encode for hundreds of membrane transporter proteins that play essential roles for the cellular import and export of ions and small molecules. Furthermore, transporters mediate signal transduction processes catalyzing the export and uptake of signaling molecules. Therefore, the functional classification of membrane transporters is an important task. The available experimental knowledge about transporter function has been compiled in databases such as TCDB [1], TransportDB [2], SGD [3], and Aramemnon [4]. In these databases, the functional classification is normally done according to the hierarchical transporter classification (TC) system [5] adopted by the International Union of Biochemistry and Molecular Biology (IUBMB).
The TC system categorizes transporter sequences according to their class, subclass, (super) family, and subfamily on the basis of functional or phylogenetic information that is based on sequence similarity. An example for this classification would be the PTS Glucose-Glucoside (Glc) super family 4.A.1 that belongs to class '4' group translocators and subclass 'A' phosphate transfer-driven group translocators. Subfamilies might correspond to transported substrates. A particular transporter sequence in such a family is identified by an extra digit to the right as e.g. 4.A.1.1.1.
A very important detail about each membrane transporter is of course the nature of its transported substrate molecule(s). As an alternative to the TC system, one may also classify transporters into different sets according to their substrates. It is presently unclear how such a substrate-based classification compares with the TC classification system. For example, the Aramemnon database lists members of five different TC families as phosphate transporters in Arabidopsis thaliana. In fact, many databases ignore the fourth digit (subfamily) of the TC system that normally refers to the main substrate. Schaadt and Helms have recently reported that membrane transporters from Arabidopsis thaliana that either transport amino acids, oligopeptides, phosphate, or sugar molecules can be distinguished from each other based on their amino acid composition [6,7].
An important research question for membrane biology is whether two membrane transporters in organisms X and Y that show a certain sequence similarity will have the same function or not. Previous computational work in this area classified transporters using sequence homology and motif searches [8,9], amino acid composition [10], and substrate specificity [6]. Interestingly, no study has so far critically analyzed the reliability margins of the individual features. In the general context of protein function, the Pfam repository of protein families has become a quasi-standard. Pfam employs so-called gathering thresholds that are manually curated, family-specific, bit score thresholds that are chosen by Pfam curators at the time a family is built. The threshold used recently corresponds roughly to 'safe' E-value thresholds of ~10-2[11]. In the TC system, the standard used for establishing homology between two proteins is 9 standard deviations (SDs). This corresponds to a probability of 10-19 that the degree of similarity observed arose by chance [12]. Chen and colleagues have recently assessed the performance of different orthology detection strategies for eukaryotic genomes [13].
Here, we have selected the three important model systems Escherichia coli (in the following abbreviated as Ec), Saccharomyces cerevisiae (Sc), and Arabidopsis thaliana (At) that belong arguably to the best characterized species in terms of transport processes. Analyzing homolog databases we found that Sc and At have more homologs compared to pairs (Sc, Ec) and (Ec, At) what reflects the smaller phylogenetic distance between Sc and At. According to the InParanoid database [14], 7173 out of the 26207 At genes (27.4%) have homologs in Sc and 2921 out of the 5884 Sc genes (49.6%) have homologs in At. For comparison, 933 Sc genes (15.8%) have homologs in Ec and 822 out of 4149 Ec genes (19.8%) have homologs in Sc. Finally, only 2778 At genes (10.6%) have homologs in Ec and 1168 Ec genes (28.1%) have homologs in At. Along the same lines, the Arabidopsis sequencing project revealed that a much higher percentage of the proteins in the 12 major functional subsets of the At genome had a BLASTP match with E < 10-30 to a protein from Sc (17–50)% than to a protein from Ec (5–32)% [15].
We used three different approaches to transfer transporter functional annotation between the three organisms by relating the level of sequence identity to the functional similarity between the three studied organisms. In this study, we will term this comparison "functional classification". For this, we used the approaches BLAST that generates alignments that optimize a measure of local similarity [16], HMMER that searches for sequence homologs and performs protein sequence alignment using probabilistic methods [17], and MEME that performs motif discovery in protein sequences on the basis of expectation maximization [18]. So far there seem to be no accepted fixed thresholds for the prediction scores of the three tools. Therefore, different studies tend to use their own suitable set of thresholds [11-13,19-21]. Our study establishes a set of thresholds under which the transporter function can safely be transferred between the three model organisms.
Results and discussion
In this work, we perform functional classification of transporter TC families and of transported substrate molecule using datasets from three model organisms. Our aim is to provide a simple guideline to biologists who wish to get a quick information whether available functional information about a transporter in species X may be transferred to another transporter sequence identified e.g. by BLAST search in species Y. Table 1 provides an overview over the main data sets used in this work. Figure 1 lists common TC families between the three organisms and the distribution of transporters among them. Additional file 1: Tables S1-S3 list all used transporters in this study, their TC families, substrates, and their Pfam description.
Table 1. Datasets
Figure 1. Distribution of transporters among the TC families. Common Ec, At, and Sc TC families with member counts. Most families belong to the Electrochemical Potential Driven Transporters (class 2) and the Primary Active Transporters TC classes (class 3). Shared TC families in the searched organism with more than 2 members were used for MEME motif analysis.
Additional file 1: Tables S1–S3. Considered transporters in organisms EC, AT and SC respectively.
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Beside the TC analysis, we also created substrate families of transporters that are annotated to transport the same substrate. For each organism, we collected four large groups of transporters that have been experimentally shown to catalyze the transport of either metal ions, phosphates, sugars, or amino acids. Metal ion transporters account for about 25% of the complete substrate dataset in each organism. Sc contains twice as many metal ion transporters as Ec and At[22]. This can possibly be related with the existence of metallothionein proteins in yeast that function as a metal storage [23]. At contains three times as many phosphate transporters as Ec and four times as many as in Sc. This is probably due to the essential role of phosphate regulating the At root system [24-26]. Sugar transporters in At even account for 50% of the complete substrate dataset which is twice as many as in Ec and Sc. One possible explanation for this is that plants need sugar to complete photosynthesis [27]. Ec and Sc contain twice as many amino acid transporters as At. Figure 2 provides an overview to which TC families the members of the created substrate families belong. We noticed that the transporters for these four substrates are spread over many different TC families.
Figure 2. Distribution of transporters from the four selected substrate families among the different TC families. Distribution of metal, phosphate, sugar, and amino acid transporters among the different TC families in the three organisms; Ec (squares), At (triangles) and Sc (ovals). The size of the symbols indicates the number of members of this class. See Additional file 1: Tables S1-S3 for more details.
Matching TC families
In this work, we used BLAST for aligning all transporter sequences of one organism against their TC analogues in the two other organisms. Then, we calculated the accuracy measures precision, recall, and F-measure (eq. 1–3) for various E-value thresholds. BLAST multiplies the significance of a hit by the total number of residues in the database. Thus, to make the obtained results independent from the size of the searched database we divided the E-values by the size of the DB that we were BLASTing against. In this way E-values from searches against different TC sets or substrates sets are comparable to each other. In the following, we will term the normalized BLAST results "normalized E-values". As an example, we BLASTed Sc transporter YDR342C either against the At dataset (23,567 residues) or against the non-redundant (nr) database of 2011 with 3,877,139,759 residues. Among the matching sequences, we identified the Arabidopsis transporter At3g19940 in both BLAST runs with an E-value of 1e-58 when searching against the At dataset and 1e-53 when matching against nr. This difference of reported E-values matches the ratio of the database sizes.
On the other hand, when computing the accuracy measures, we multiplied the results by the member count of each family and then averaged over all TC families considered in order to account for the different member count of each family, see Table 2. The last row shows the percentage of transporters that remained unclassified at the given threshold. These are transporters from one organism belonging to the shared TC families that do not share sequence identity better than the given E-value to any transporter in the shared TC family from the other organism.
Table 2. BLAST sequence homology search results within TC families in the three organisms
At the strictest threshold of 1e-20, the assignment of TC family has very high confidence but more than 80% of the sequences cannot be assigned for the Ec-At comparison and about half in the Ec-Sc and Sc-At comparisons. When the threshold is made more permissive, the number of correct predictions increased with few false predictions. We found that the precision and recall increased until 1e-8 but at threshold 1e-4 the number of false predictions increased. As expected, the unclassified percentage decreased as the thresholds were made more permissive. Based on this comparison, a rather permissive normalized BLAST threshold of 1e-8 is very acceptable but 1e-4 can still be considered with caution. When using the absolute identity scores of the alignment instead of the extracted E-values, the results were untrustworthy. The TC family prediction of Ec transporters based on Sc transporters annotated more sequences than the prediction based on At transporters at the strictest thresholds. Additionally, the Sc-At analysis resulted in a higher accuracy compared to the Ec-At analysis.
We then applied HMMER to the same datasets as for BLAST and calculated the accuracy measures and the unclassified percentage in the same way. Table 3 shows the results obtained with HMMER. For the purpose of normalization, the results were divided by the number of found hits in the database that was searched against. Overall, the results are similar to those obtained with BLAST. However, HMMER results are slightly more accurate at loose thresholds and cover a wider annotation fraction at the strictest thresholds with few more false positives. The number of correctly predicted TC family members at the medium-strong thresholds of 1e-16 and 1e-8 is always equal or higher than with BLAST. HMMER also missed fewer points (false negatives) compared to BLAST. This is clearly reflected by the higher recall value calculated most of the times. It should be re-emphasized that the E-values are computed by the three programs used here in different ways and are, thus, not directly comparable. Also, we have applied different normalization procedures - as suggested by the developers - to normalize the results to per-residue or per-sequence levels.
Table 3. HMMER results for homology between TC families from the three organisms
The decisions by HMMER appear similar to BLAST between the three organisms. Apparently, HMMER attained slightly higher precision for almost all thresholds compared to BLAST especially at loose thresholds. Additionally, in the Ec-Sc and the Sc-At analysis, HMMER made predictions for a larger fraction of the test set with a noticeably higher recall for thresholds till 1e-8 compared to BLAST. For threshold 1e-4, HMMER predicted a slightly smaller fraction of the test set compared to BLAST but HMMER reported much higher prediction accuracy. Hence, we suggest an acceptable HMMER threshold of 1e-4.
The enriched sequence motifs identified by MEME in sequences from one organism were subsequently searched in test sets of sequences from the other two organisms using the MAST program [28] from the MEME suite. Table 4 illustrates the results based on using motif searches for family classification of transporters. As can be expected, motif based searches performed better in families with many members such as 2.A.1. For loose thresholds, motif based classification showed lower precision compared to HMMER and BLAST but a comparable precision at the strictest thresholds of 1e-20 and 1e-16 as in Ec-Sc and Sc-At analysis. We suggest that motif based methods may be used beneficially in combination with other methods to support transporter classification. At looser thresholds than 1e-8, motif-based searches seem to lead to unreliable results and should be used with high caution.
Table 4. MAST results for the existence of predicted MEME motifs in TC families
Matching substrates families
In a second step, we used the same three methods to test whether annotations about the transported substrate can be transferred from one organism to the other. For this, we created four subsets of metal ions transporters, phosphate transporters, sugar transporters, and amino acid transporters. These are the four largest known substrate families and comprised 72 Ec transporters, 95 At transporters, and 79 Sc transporters, see Table 1.
As shown in Table 5, the results were markedly different from the TC family results. Despite the fact that BLAST reported acceptable prediction precision in the Ec-At and the Sc-At analysis, the program missed classification of many transporters. We noticed that sequences tend to match sequences from their TC families in other substrate families, rather than their analogues in the same substrate family. Thus, the precision for substrate classification is generally lower than for the TC classification, in particular for the Ec-Sc comparison. For instance, the metal transporter (YMR301C) from Sc was falsely matched to about one third of all Ec transporters in the four substrate families irrespective of their substrates since they belong to the same TC family (3.A.1).
Table 5. Homology search results within the four substrate families based on BLAST
Table 6 presents the HMMER prediction results for substrate families from the three organisms. Compared to BLAST, HMMER reported higher prediction accuracy in the Ec-Sc analysis but slightly lower prediction accuracy in Ec-At analysis at the strict thresholds such as in the TC comparisons. In fact, BLAST classified a slightly larger fraction of the test sets than HMMER in almost all runs. HMMER was also affected by transporters tending to match their TC family members in other substrate families rather than their homologues in the same substrate families.
Table 6. HMMER search results within the four chosen substrate families
Table 7 shows MAST search results for MEME motifs from different substrate families. MEME gave weak predictions in all runs but in the Sc-At analysis. However, recall in the medium strict thresholds 1e-16 and 1e-8 in the Ec-Sc analysis is generally acceptable but accompanied with many misclassifications. In the Ec-At analysis the prediction accuracy was generally low. Here, even the strict threshold of 1e-20 is unreliable because it gave wrong assignments of substrates in two out of three analyses.
Table 7. MAST results for the existence of predicted MEME motifs in substrate families
Surprisingly, 22 Sc sugar transporters were correctly classified from 3 motifs predicted by MEME in the At sugar substrate family. To the best of our knowledge, none of the three motifs have been annotated so far in databases such as [29]. Table 8 lists the regular expressions of these three motifs. The motifs were found around positions 420, 150, and 300 of the protein sequences, respectively.
Table 8. Enriched sequence motifs in At sugar transporters
Application of established thresholds to human datasets
Next, we tested these thresholds on four Hs datasets. In comparison to the three model organisms, these datasets are likely much less complete. We used the three tools to align the Hs transporters using a set of transporters from At and Sc and to align Ec transporters using Hs transporters. The results are in line with the comparisons of the three model organisms. When using BLAST and HMMER, only a small fraction was annotated at strict thresholds but more were classified at more permissive thresholds. Using HMMER, about 50% of the transporters remain not annotated even at the loosest threshold of 1e-4 whereas using BLAST many more were annotated but with a very low prediction accuracy. The reason is that the Hs phosphate and metal transporters were not annotated using the At and Sc sets and even did not help in annotating the Ec transporters. However, sugar and amino acid transporters were mostly correctly annotated. Most annotations of Hs transporters were based on matching (Hs, Sc) pairs. In motif searches, two thirds of the Hs transporters were annotated at the threshold of 1e-16 but none were annotated at the strictest threshold of 1e-20, see Table 9. The complete results of matching (Hs, At) and (Ec, Hs) are listed in Additional file 2: Table S4.
Table 9. Results of the three tools matching Hs and Sc sequences
Additional file 2: Table S4. Complete annotation results of the pairs (Hs, At) and (Ec, Hs).
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Additionally, we studied the pairwise global similarity of all organism pairs using the program ggsearch from the FASTA program suite. The results were generally similar to BLAST and HMMER results with a slightly lower accuracy at the loose thresholds and even lower accuracy at the stricter thresholds. Results are listed in Additional file 3: Tables S5-S6.
Additional file 3: Tables S5–S6. Results of FASTA global searches.
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Prediction of TC families in substrate families
Comparison of the two preceding sections shows that substrate families have less sequence similarity on average compared to TC families. Now, we tested the combination of both properties, see Figure 3. We performed this comparison in a systematic way. For this, we named the extracted families in the form "substrate family_TC family". The four substrate families (amino acids, sugars, phosphates, metals) belong to 19 TC families in Ec, 13 in At and 14 in Sc. 7 families substrate-TC are shared between Ec and At, 7 also are shared between Ec and Sc and 11 are shared between Sc and At. Some TC families belong to many different substrate families like the family 3.A.1 that contains members of 4 Ec substrate families. We used BLAST to analyze the affiliation of test sequences toward their TC or substrate families. Here, only the best match of each substrate_TC family is considered. The heatmap in Figure 3 shows the tendency of Sc sequences to match their analogues from At TC or substrate families. Some Sc transporters matched strongly (black rectangles) their actual substrate_TC families from At like sugar_2.A.1, phosphate_2.A.1 and metal_2.A.55. However, most sequences from shared TC families had weaker matches to their TC families rather than their substrate families. Similar results were obtained in the Ec-At and Ec-Sc comparison, see Additional files 4: Figure S4 and Additional file 5: Figure S5. Thus, we suggest that it is beneficial to apply substrate information as a pre-filter for transporter TC family classification. On the other hand, transporters that transport the same substrate but belong to different TC families generally do not share noticeable sequence similarity. TC information can be the stand alone feature used to classify transporters but a little tuning by substrate information elevates the prediction accuracy. Misclassification will occur in the small substrate_TC families not in the big TC families.
Figure 3. Heatmap of BLASTing Sc substrate_TC families against At families. BLAST homology search of 69 Sc transporters against 84 At transporters from 4 substrate families (amino acids, sugars, phosphates, metals) and 13 TC families (Sc) and 12 TC families (At). The grey scale follows a logarithmic scheme where white means no match better than normalized E < e-04 and black means the best matches better than E < e-20. Families generally match their substrate_TC families. However, they may also match TC families from different substrate_TC families.
Additional file 4. Heatmap of BLASTing Ec substrate-TC families against At families.
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Additional file 5. Heatmap of BLASTing Ec substrate-TC families against Sc families.
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Limitations and implications
In some way, our analysis presented here is a bit "circular" since we employ tools to identify sequence pairs belonging to the same TC categories while the TC classification itself was established in part based on phylogenetic analysis that is again based on sequence similarity. However, in a practical use case it is far simpler to run a BLAST or FASTA analysis than to establish a complicated phylogeny. Hence, our results reflect to what extent simple sequence similarity captures the structure of the more elaborate TC classification.
When comparing the results of the four methods (BLAST, FASTA, HMMER3, MEME), the reader should not forget that different strategies are employed by each of the methods to derive E-values for the reported results. Hence, the results of different methods are not directly comparable.
Note that datasets to be used for motif discovery are typically cleaned up for sequence redundancy e.g. using BLASTCLUST with a 25% sequence identity threshold [30]. Here, we did not do this because this would significantly decrease the number of families in the TC dataset that can be used for analysis. Hence, the MEME analysis partially rediscovered sequence similarities.
This work suggests that the current TC system adopted by IUBMB is a more robust classification feature compared to substrate classification. It is quite likely that phylogenetic inference is a more sensitive indicator of homology than simple sequence similarity or identity. Thus, it appears worthwhile to test the performance of phylogeny-based methods to relate the substrate specificities of membrane transporters.
When trying to completely block the transport of a certain substrate across a particular membrane of an organism it is hard to rely only on the TC information because one substrate can be transported by several transporters from different TC families. One possible explanation in fact is that transporters assigned to different sequence families might actually share a similar 3D structure and the structural similarity might provide an indication about the evolution of the transporter function. Such studies require more sensitive search methods like AlignMe [31].
We observed that classifying membrane transporters according to TC families gives more accurate results than classifying them according to substrate families. At the strictest threshold of 1e-20 for normalized E-values, predictions based on BLAST and HMMER result generally in high precision, but a huge fraction of the data remains unclassified. We suggest an acceptable threshold of 1e-8 for both programs where at least three quarters of the sequences are classified with a reasonably high accuracy. Researchers who wish to apply these thresholds in their studies should multiply these thresholds by the size of the database they search against. On the other hand, MEME showed unsatisfactory behavior for thresholds below 1e-8. Prediction of TC families split from substrate families showed satisfactory results implying that the application of substrate information as a pre-filter would improve the prediction results. The analysis and suggested thresholds in this study should be useful to those who wish to transfer transporter functional annotations across species without having to build a new phylogeny such as for the TC system. With respect to substrate annotation, the findings of this work may be combined with those of Schaadt et al. [6] who established amino acid composition for substrate annotation of transporters, and with the work of Saier MH Jr. [32].
Overview of the data
In the training part of this work, we used three sets of membrane transporter sequences from Ec (155), Sc (177), and At (158). In each case, we require that the transporter has been classified in the TC system and that TC/substrate annotations are based on experimental evidence. The sequences and annotations were retrieved from the databases TransportDB [2], SGD [3] and Aramemnon [4], respectively. From TransportDB we downloaded 354 sequences of Ec transporters. Among them, 157 have experimentally confirmed annotations about substrate and transporter class. Sc transporters were extracted from a list of 6752 ORFs downloaded from SGD. 900 transporters existed in verified ORFs among which 788 had a non-hypothetical function. Only 178 transporters had a clear TC family membership which was obtained by BLASTing SGD extracted transporters against the Sc TransportDB by requiring an E-value of 0.0 and a sequence identity of 100%. In Aramemnon, we used the keywords 'transport' and 'carrier' to download 616 transporter sequences from which 159 non-putatives with clear TC classification were extracted. Thereafter, we constructed subsets according to the TC system and according to substrates for later analysis. Obviously, matching a sequence correctly to a particular TC subfamily based on sequence similarity is only possible if this TC subfamily originally contains at least two members (if we take one out for testing, there is at least still one left). Thus, we considered only TC classes with more than one member. Additionally, we also downloaded functional descriptions from the Pfam database [33] for the transporters in the three organisms to assist the substrate information extracted from the individual databases. If substrate information from Pfam conflicted with the original substrate information, the Pfam information was discarded.
The transporters of the three organisms are annotated to 53 (Ec), 29 (At), and 34 (Sc) different TC families. Subclass 2.A (including uniporters, symporters, andantiporters) and subclass 3.A (P-P-bond-hydrolysis-driven transporters) were the most common TC subclasses. In Sc and At, the Major Facilitator Superfamily 2.A.1 accounts for nearly 40% of all transporters while in Ec it is the second largest family after the ATP-binding Cassette (ABC) Superfamily 3.A.1. Shared TC families belong mostly to TC classes Electrochemical Potential Driven Transporters (class 2) and the Primary Active Transporters (class 3).
For the testing part, we created four datasets of experimentally annotated human transporters (Hs). Sugar, amino acid, and metal transporter sets were extracted from the ChEMBL database [34]. Experimentally validated phosphate transporters were obtained from Uniprot [35]. We note that the set of metal transporters contains several proteins that transport several extra substrates besides the metal ion as well.
Prediction tools
The statistical significance of the sequence similarity between an input sequence and sequences in the input set was determined using the well-known tools BLAST [16] and HMMER [17]. The MEME program suite [18] version 4.6.0 was used to identify enriched sequence motifs in sets of transporter sequences from one organism belonging to the same TC family or that transport the same substrate. Later, the MAST program from the MEME suite provided a score when statistically significant motifs were identified in the sequences from the other organisms. Additionally, we used the tool ggsearch36 from the FASTA suite [36] to test whether sequences transporting the same substrate express not only local but also global sequence similarity.
First, we used NCBI BLAST version 2.2.23 and HMMER version 3.0 for pairwise comparisons of all 90 Ec transporters against the 84 At transporters that belong to 14 shared TC families. In the MEME analysis, we used only common At and Ec TC families with two or more members i.e. 71 Ec transporters and 77 At transporters belonging to 7 TC families. Next, we aligned the 98 Ec transporters belonging to 18 TC families against 131 Sc transporters. Ec and Sc shared 14 TC families that could be searched by MEME involving 87 transporters from Ec and 127 from Sc. Finally, we used BLAST and HMMER to compare 157 Sc transporters from 23 TC families against 141 At transporters. At and Sc shared 12 TC families involving 130 transporters from At and 120 from Sc. Repeatedly, we used sequences from different organisms but belonging to the same TC families as inputs and test sets for the classifiers to test the quality of the prediction. For identifying enriched sequence motifs with MEME, the sequences must be grouped into families that are likely to share motifs. Here, we used MEME to determine up to 3 motifs in each shared TC family between each pair of organisms; 7 such TC families for (At- Ec), 14 for (Sc- Ec), and 12 for (At- Sc). BLAST E-values were normalized by the number of residues in the searched database (see Results section). HMMER E-values were normalized by the number of hits.
In order to identify reliability thresholds at which functional information can be safely transferred between organisms, we tested thresholds (1e-20, 1e-16, 1e-12, 1e-8 and 1e-4) for the E-values and evaluated prediction accordingly. We calculated the accuracy measures precision (positive predictive value), recall (sensitivity) and F-measure (equations 1, 2, 3) at each threshold to evaluate the prediction performance (Tables 2, 3, 4, 5, 6 and 7). Precision emphasizes the role of unexpected results whereas recall emphasizes the role of missing classification points. F-measure is a suitable accuracy measure considering precision and recall as we want precision and recall to be evenly weighted. High precision points at a strong prediction boundary while members of other classes rarely match the current class. High recall points at strong similarity within the class members as they rarely match members of other classes. For an actual TC or substrate class, a false negative is a membrane transporter from the class that is predicted to belong to another class, while a false positive is membrane transporter from another class that is predicted to belong to the current class. An example confusion matrix is illustrated in Table 10.
Table 10. An example confusion matrix predicting TC class 3.A.1 members
Ec: Escherichia coli; Sc: Saccharomyces cerevisiae; At: Arabidopsis thaliana.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AB and VH conceived this study. AB wrote scripts, compiled data-sets, and performed the data analysis. AB and VH analyzed the data and jointly wrote the manuscript. Both authors read and approved the final manuscript.
We thank Nadine Schaadt and Ruzianisra Mohamed for their helpful comments and careful reading of our manuscript. AB was supported by a predoctoral scholarship from the German-Jordanian University.
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Tian W, Skolnick J: How well is enzyme function conserved as a function of pairwise sequence identity?
J Mol Biol 2003, 333:863-882. PubMed Abstract | Publisher Full Text
Eide DJ: The molecular biology of metal ion transport in Saccharomyces Cerevisiae.
Annu Rev Nutr 1998, 18:441-469. PubMed Abstract | Publisher Full Text
Peterson CW, Narula SS, Armitage IM: 3D solution structure of copper and silver-substituted yeast metallothioneins.
FEBS Lett 1996, 379:85-93. PubMed Abstract | Publisher Full Text
Williamson LC, Ribrioux SPCP, Fitter AH, Leyser HMO: Phosphate availability regulates root system architecture in Arabidopsis.
Schachtman DP, Reid RJ, Ayling SM: Phosphorus Uptake by Plants: From Soil to Cell.
Plant Physiol 1998, 116:447-453. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Shin H, Shin H-S, Dewbre GR, Harrison MJ: Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments.
Plant Journal 2004, 39:629-642. PubMed Abstract | Publisher Full Text
Williams LE, Lemoine R, Sauer N: Sugar transporters in higher plants–a diversity of roles and complex regulation.
Trends Plant Sci 2000, 5:283-290. PubMed Abstract | Publisher Full Text
Gribskov M, Bailey TL: Combining evidence using p-values: application to sequence homology searches.
Bioinformatics 1998, 14:48-54. PubMed Abstract | Publisher Full Text
Marsico A, Scheubert K, Tuukkanen A, Henschel A, Winter C, Winnenburg R, Schroeder M: MeMotif: a database of linear motifs in α-helical transmembrane proteins.
Nucleic Acids Res 2009, 38:D181-D189. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Frith MC, Saunders NFW, Kobe B, Bailey TL: Discovering Sequence Motifs with Arbitrary Insertions and Deletions.
PLoS Comput Biol 2008, 4:e1000071. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
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Paulsen IT, Sliwinski MK, Saier MH Jr: Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities.
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Proc Natl Acad Sci 1988, 85:2444-2448. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


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No sense taking it easy after returning from a concussion.
Shelby County's Ben Axline was back in the lineup last week against Seneca after missing the North Hardin game. All he did was lead the Rockets with seven catches for 139 yards, score two TDs and deliver the two biggest hits of the game.
In fact, Axline, a junior, has been involved with the three biggest hits this season.
Board will review Collins items
The Shelby County Public Schools board will meet Thursday at 7 p.m. at Clear Creek Elementary.
The board will consider:
If you've ever wondered why so many people spoil their children, it's simple. They get two big payoffs.
First, the obvious: it's easier just to get through the moment by indulging children's demands, rather than stopping to teach them a lesson. And the more subtle reason is that by living for and through the child, they can avoid all their own feelings, issues and responsibilities. It's like a drug—a pain-killing escape from reality.
Too many cooks sure didn't spoil the broth Monday night, with delicious food, sparkling spirits, a great jazz band, door prizes and even cornhole.
Add to that a thoroughly mellow crowd, and you have the makings a great evening.
I keep telling myself that I do like snakes…and really I do, just not in the kitchen.
Yes, there was a snake in the kitchen one afternoon when I came home from work. My cat, Parc, helped me wrangle it.
The desire to build a conference center in Shelby County is about to take on an entirely new visual concept.
Leon Mooneyhan, former superintendent of Shelby County Public Schools and chief executive officer of the Ohio Valley Educational Cooperative (OVEC), will present plans Monday for a City Center, which offer significant conference space and a larger community theater in downtown Shelbyville.
Well, we can certainly say it was a pleasant summer season, so I am definitely not complaining…but it wasn't the best year for tomatoes. Cool and wet is good for some but not for the prized tomato.
Shelbyville Police said Thursday they were not aware of charges being filed in the investigation of a fight earlier this month at the skate park in Shelbyville.
SPD Chief Robert Schutte had said Wednesday that the altercation involving several juveniles had been referred to Shelby County Attorney Hart Megibben for evaluation.
"I don't know anything more than I did the other day," Schutte said.
The SCHS boys' golf team battled Collegiate and the rain Tuesday in the team's final match of the regular season, and the Rockets got the better of both.
Shelby hammered Collegiate, 150-215, setting the mark for the team's best match score of the year.
Josh Griffin led the Rockets again, this time shooting an even-par 36. Behind him were a tightly grouped set of Tayler Adams 37, Turner Mayton 38 and Jared Redmon 39. It was the first time this year the Rockets' finished a match with all four players in the 30s.
Information was gathered from previous years of The Shelby Sentinel, The Shelby News and The Sentinel-News. You can reach the writer at email@example.com. If anyone has an old photo that they would like to run with this column bring it and the information into The Sentinel-News office or e-mail it to the writer at email@example.com. We are also looking for mystery photos.
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subset: commoncrawl


text:
Page 3 Archives for September 2011
A sperm swimming
A cat chews on a piece of fur
A moustache sits on the head of a vehicle
Mike Tyson plays with two pigeons
A lady with sunglasses covered in mud
A stop sign inspired by Journey
A pregnant woman uses her stomach as a table to eat food
A boy puts on eye curler, while a girl pretends to shave
zach galifianakis plays the piano
A clerk in a convenience store stops a would-be thief by slamming him on the head with a folding chair. That would be a disqualification, but the ref was distracted as usual.
Summit High School finally got around to having a football team, and what do they do on their very first play, ever? Run it all the way back. This is a moment this school never will forget.
It's eco-friendly, and I bet Al Gore loves it. It looks like an inconvenient booth.
This is an annual activity in El Salvador, done in remembrance of a 1922 volcanic eruption. I wonder what they'll be doing this in remembrance of, the next night?
We posted the taco restaurant fight earlier this week. Check out this alternate angle that captures a couple extra parts of the fight. My favorite is 1m25s when the dude misses his punch and runs head first into the counter.
"You're gonna flip over our newest minitrucks!" In all seriousness, this guy was lucky to walk away from this accident. It appears the only thing broken was his scooter.
The cat is indoctrinating the first recruit into its army of evil. He has higher hopes for future recruits.
The evil, chanting cat's motives were not entirely clear before, but thanks to our new translation, you can see that he had some truly evil tricks up his sleeve.
The best part of this crash is the arm drag the woman performs as she's crashing. Really, it's a tale as old as time. A man chases after a woman only to end up having her drag him down with her.
We first saw this clip about 2 years ago and it suddenly started going around again this week. Can anyone think of another news blooper more embarrassing than this one?
This is probably the happiest you will ever see someone get for breaking their hand.
We posted on Facebook a couple days ago a simple yet awesome beach prank and these guys already managed to pull it off on this chick. Adding the drink was a nice touch guys, well done.
Well, I failed this. You get 15 seconds to find which one of these pug dogs is not the fake.
This is a great prank by notorious prank caller Tom Mabe, but I'd be a little worried leaving the store with that dude in the red cap outside. He doesn't seem to be a fan of pranks.
This guy picks probably the least appropriate time to get sucked into his cell phone. Thanks to his phone's autocorrect, his last tweet read "Puck The Police!"
This up and coming gymnast just took out his one fan.
Meet Techno Viking's great uncle, The Jaeger Meister. He would've been a part of the fellowship of the ring, but he heard there was a monster jam going on that day.
Skippy was hired to produce a commercial for Furniture Warehouse and is a perfect example on why you shouldn't always go with the low bidder.
If this dude defines winning as how many punches you can block with your face than I guess he is right, he did win.
The dude in yellow gets out of his car and with three guys lying on the ground the first thing he checks is the scratch on his front bumper.
It's the old good cop/bad cop routine but this time played out by just one guy.


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Glacius - Desktopography 2013
"As winter approaches, the legendary awakes on the mountain top and reveales his pure ice strength to the world"
My piece for this year Desktopography. Unfortunately I was a kinda late with the deadline and due the amount of submissions and control this year It didn't get accepted, but still going to release it though.
Thank you for watching!


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June 6, 2012
Role Models are Important
On Twitter recently I saw @tammybutow talking about a new project she's launched to highlight female role models in I.T. Tammy's project is affiliated with Go Girl, an I.T. career showcase for high school girls. Her idea is to collect photos of women with I.T. careers and compile them into a video to inspire the students. When I was in high school, I thought computer programmers were just guys in neckties who worked at IBM. I don't remember any girls in the Computer Club, and I certainly didn't join myself. I briefly considered a dual CS major in college but chickened out for fear I wasn't smart enough. I had no idea that one day I'd know lots of cool, smart, funky women who work in I.T. with jobs like "Programmer," "Game Designer," "User Experience Director," "Project Manager," and even "Business Systems Analyst." (That last one is actually ME!)
So anyway, this is an issue I feel pretty strongly about. I don't want my nieces growing up to think that computers aren't something girls work with. So I gathered up all the women in the office yesterday morning and corralled them into a photograph. (There are actually more than five, but this is all I could round up on short notice.) I present to you the inspiring women of Sitback!
Full size version here. Thanks to Kunaal for the photograph!
From left to right, that's Lindsey (Project Manager), Alison (Group Account Manager), me (Business Systems Analyst + GEEK!), Leanne (User Experience Director), and Elly (Producer). Woo! Go women of Sitback!
Updated to add: I can't believe I forgot to mention the most inspiring woman in I.T. I know!


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|Search | About | Preferences | Interact | Help
|Find any book at the best price.
› Find signed collectible books: 'Celebrate Through Heartsongs'
Mattie J.T. Stepanek is an award-winning poet whose struggle with a rare form of muscular dystrophy has touched the lives of people nationwide. Celebrate Through Heartsongs, his fourth inspiring collection of poetry, features works written between the ages of three and eleven, and continues to spread Mattie's message of universal hope, peace, courage, and love. Fully illustrated by the poet, the collection will appeal to people of all ages, religions, and beliefs.
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› Find signed collectible books: 'Heartsongs'
Language:Chinese.Paperback. Pub Date: 2002 Pages: 32 in Publisher: Hyperion Mattie JT Stepanek began writing poetry at the age of three In Heartsongs. Mattie explores the uncensored reality of living with a rare form of muscular dystrophy and associated with the grief with the loss of his three siblings to the same life-threatening condition. Heartsongs is a collection of the early poems and artwork of this gifted. courageous. award-winning poet.
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› Find signed collectible books: 'Heartsongs 2/3 Part Treble'
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› Find signed collectible books: 'Heartsongs and Journey Through Heartsongs: & Journey Through Heartsongs'
Title: Journey Through Heartsongs <>Binding: Hardcover <>Author: MattieJ.T.Stepanek <>Publisher: HyperionBooks
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› Find signed collectible books: 'A Heartsongs Collection: Heartsongs and Journey Through Heartsongs'
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› Find signed collectible books: 'Heartsongs Collection (Library Edition): The Poetry of Mattie J. T. Stepanek'
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› Find signed collectible books: 'Heartsongs Collection: The Poetry of Mattie J. T. Stepanek'
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› Find signed collectible books: 'Heartsongs: Inspirational Poems that Inspire Life'
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› Find signed collectible books: 'A Heartsongs Treasury - 3 Copy Slipcase'
3 Volume boxed set. Titles are: Heartsongs, Journey Through Heartsongs, and Hope Through Heartsongs.
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› Find signed collectible books: 'Hope Through Heartsongs'
The celebrated child Mattie J.T. Stepanek continues his internationally inspiring poetry with Hope Through Heartsongs. For those who haven't followed Stepanek's rise to fame, this young phenomenon writes poetry that is wise beyond his years and has appeared on The Oprah Winfrey Show and Larry King Live. Indeed, as Gary Zukav marvels in the book's foreword, Stepanek's wisdom seems to extend beyond what most human beings master in a lifetime. "A heartsong is something deep inside each of us," Mattie Stepanek explains in his introduction. "It's our sense of why we are here and how we can keep going. It's like a purpose. It may be to live as a mommy or a daddy, or a firefighter or a delivery person, or a child with a disability who teaches others about patience and love and acceptance."
As a child living with muscular dystrophy who has lost all three of his siblings to the same disease, Stepanek knows the landscape of loss and grief. How easy it would be to wallow in despair, yet Stepanek has chosen hope--not the wide-eyed hope of the innocent, but the hope consciously chosen in the face of human suffering and troubling global problems. Many believe that global healing has to start with the little people and move its way up to the leaders. What better place to begin than with the words of this amazing child?
You may be tall--Gail Hudson
I may be small,
But inside we are the same
length of strength
--Mattie J.T. Stepanek, June 1998
› Find signed collectible books: 'Journey Through Heartsongs'
Mattie J. T. Stepanek takes us on a Journey Through Heartsongs with more of his moving poems. These poems share the rare wisdom that Mattie has acquired through his struggle with a rare form of muscular dystrophy and the death of his three siblings from the same disease. His life view is one of love and generosity and as a poet and a peacemaker, his desire is to bring his message of peace to as many people as possible.
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› Find signed collectible books: 'Just Peace: A Message of Hope'
"I was touched by the depth of passion and awed by the firm resolve with which Mattie Stepanek pursued a dream that has evaded men and women throughout history. What began as a casual discourse, not too different from others I have had with inquisitive young people who have reached out to me, became a treasured and enlightening friendship that changed my life forever. With the purity of heart that only a child can possess, and the indomitable spirit of one who has survived more physical suffering than most adults will ever know, Mattie convinced me that his quest was not inconceivable. Inspired by his enthusiasm and without reservation, I committed to a partnership with him. . . . These words of wisdom and inspiration came from the most remarkable person I have ever known." --Jimmy Carter
Sometimes the most important messages come from the most unlikely places. Mattie J.T. Stepanek, a 13-year-old boy, made a difference before he died with his Heartsongs poetry. He continues to impact the world through Just Peace. This poet, best-selling author, peace activist, and prominent voice for the Muscular Dystrophy Association fervently believed in and promoted world peace not just as a concept, but as a reality.
Mattie was working on this manuscript with Jimmy Carter when he died in June 2004. His mother, Jeni, who edited the material and wrote a preface for the book has published it at her son's request. Just Peace explores Mattie's concept of the world and all people as a unique mosaic of gifts. War and injustice shatter the mosaic, which can only be made whole again by planning and actively pursuing peace. The young visionary's essays, poetry, and photographs appear throughout the book. Jimmy Carter has written a special foreword for the book.
Just as important to the book and enlightening to the reader are Mattie's many correspondences. Central to these are his personal e-mails to and from former president Jimmy Carter, Mattie's peace "hero" and role model. The Nobel Peace Prize winner met Mattie, considered him an angel, messenger, and hero in his own right, and was genuinely affected by Mattie's passion and drive. Just Peace is an intimate portrait of a president, a young man of hope, and peace itself.
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› Find signed collectible books: 'Loving Through Heartsongs'
Language:Chinese.HardCover. Pub the Date: January. 2003 Pages: 70 in Publisher: Hyperion attie JT Stepanek has won the hearts of millions with his inspiring poems from his first two New York Times bestsellers. Now he's ready to spread a little love with Loving Through Heartsongs. a beautiful collection of poems written about innocence and trusting love from a child's perspective. Mattie's struggle with muscular dystrophy has never kept him from feeling deep love for his family. friends. country. and faith-heartfelt emotions that are reflected throughout these pages.
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› Find signed collectible books: 'Reflections of a Peacemaker: A Portrait in Poetry'
I write about anything that touches the essence of my existence. What I witness, what I feel, what I think, what I fear, what I treasure. I write about life, which is our greatest gift." --Mattie Stepanek, 1990-2004
Mattie J. T. Stepanek lived and died a child, but he had the spirit of a giant. Affected by a rare and fatal neuromuscular disease, Mattie lived almost fourteen years but in that time became a poet, best-selling author, peace activist, and a prominent voice for the Muscular Dystrophy Association. Before his death in June 2004, his five volumes of Heartsongs poetry sold more than a million copies.
Reflections of a Peacemaker: A Portrait Through Heartsongs is the final collection of Heartsongs that Mattie was working on when he died. It includes the last poem Mattie penned along with a special collection of unpublished poetry, photographs, and artwork spanning the decade from when he began writing Heartsongs at age three. Culled from the thousands of poems, essays, and journal entries Mattie left behind, the entries in Reflections of a Peacemaker create a portrait of Mattie in his own words. In these poems he explores disability, despair, and death but also the gifts he finds in nature, prayer, peace, and his belief in something "bigger and better than the here and now." The poems are grouped by theme such as playful, stormy, sacred, and final Heartsongs, with each section introduced by a personal tribute from the likes of Maya Angelou, Oprah Winfrey, Larry King, and former President Jimmy Carter.
In the words of Mattie's mother, Jeni Stepanek, who has published Reflections of a Peacemaker at her son's request, "In reading these poems we enter Mattie's world and gain insight through a child who somehow balanced pain and fear with optimism and faith."
More editions of Reflections of a Peacemaker: A Portrait in Poetry:
› Find signed collectible books: 'Canciones En Mi Corazon: Poemas E Ilustraciones De Matthew Joseph Thaddeus Stepanek, "Mattie (Spanish Edition)'
Poesía que surge del corazón, de los sentimientos, de las intuiciones, del dolor, y de los sueños y que es, al mismo tiempo, un mensaje de paz y esperanza.
Mattie empezó a escribir poesía y cuentos a los tres años. Parte de su obra refleja la dura e ineludible realidad de vivir con una extraña forma de distrofia muscular y el dolor que conlleva la pérdida de sus tres hermanos a causa de tan grave enfermedad. La mayoría de sus poemas, sin embargo, proclaman la esperanza llena de ingenuidad, la profunda sabiduría y el delicioso humor de la infancia.
Que Mattie nos transmita ese alentador mensaje nos enriquece, pero no sólo eso, también nos emociona, pues él es un ejemplo patente de que a pesar de las circunstancias más adversas vale la pena afrontar la vida con optimismo.
En un ser como él, que por las circunstancias mencionadas bien podría estar sumido en el desaliento, causan emoción y admiración su optimismo, su fe en Dios y el valor que concede al don de la vida. Y así nos llega a sus lectores.
Mattie podría quejarse, podría lamentarse pero no lo hace. Por el contrario, convierte su situación en algo que lo impulsa hacia la esperanza y a inspirar en los demás ese mismo don, plantando en sus corazones una semilla que les puede llevar a construir un mundo mejor, un mundo de amor y de paz.
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Maria Montessori Charter Academy
3175 Sunset Blvd Ste 100
Rocklin, CA 95677
Phone: 916-630-1510
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"I grew up mainly with Death, but the base I received from the good old Thrash. The bad and rough has always been one of my favorites, not for nothing that my first record of that kind was "Endless Pain" Kreator. The Black instead I only lived like a vision fleeting, and I have not consciously interested in it right now then. Today I enjoy every group that mixes the two styles in order to let him vent the public and I think that Americans Weaponizer fulfill so egregious arduous task. The quartet is dedicated with great fervor to the metal fast and enthralling, producing scales that basically recall school Lemmy, Venom, Hellhammer and especially Possessed. The rhythmic imperious and sharp breaks the eardrums even more robust, the drum work is pretty darn clean and precise, the voice is a dream for every fanatic of pure hatred violent and petty. I have a vision: I see the mass of flesh composed of a public deprived of his will that breaks the bones to the sound of "Warbastard" broken nose"


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Previous Concerts by The Canterbury Orchestra - 2006
Saturday 17th December 2006 at 3:30pm
St. Mildred's Church, Canterbury
|A Journey to Christmas
|Overture - Lustspiel
|Keler Bela
|Les Patineurs (Skater's Waltz)
|Emil Waldteufel
|A Reading
|In a Persian Market
|Albert Ketelbey
|Oxford Street (from London Again Suite)
|Eric Coates
|Mary Poppins Selection
|Richard Sherman & Robert Sherman
|A Reading
|A Dream of Christmas
|Albert Ketelbey
|We Three Kings Of Orient Are
|A Christmas Festival
|Leroy Anderson
|O, Come All Ye Faithful
|I'm Dreaming of a White Christmas
|Irving Berlin
|We Wish You A Merry Christmas
Soloist: Sue Chivers (Soprano)
Readings: Ken Pickering
Saturday 25th November 2006 at 7:30pm
St. Peter's Methodist Church, Canterbury
The Music of Mozart
Celebrating the 250th anniversary of Mozart's birth.
|Overture to Don Giovanni
|Piano Concerto No. 24 in C minor
|Symphony No. 35 in D (The Haffner)
Soloist: David Rudduck (piano)
Conductor: John Myhill
Leader: Roberta Johnson
Saturday 10th June 2006 at 7:30pm
King's Hall Theatre, Herne Bay
Faces of Mozart
Celebrating the 250th anniversary of Mozart's birth
|Overture II Seraglio
|Mozartiana: from Suite 4
|Variations to Variations and Rondo ala Turca
|Mozart arr Myhill
|Symphony No. 41 in C major (Jupiter)
Soloist: Nick Peck (piano)
Saturday 18th March 2006 at 7:30pm
St Mary the Virgin, Ashford, Kent
Concert in Ashford Parish Church
|Mendelssohn's Violin Concerto
|Schubert's Overture Rosamunde
|Beethoven's Symphony No. 4 in B flat
Soloist: Floriane Peycelon
Next Concert
Into the 19th Century
7:30pm Saturday 27th June, Colyer-Fergusson Hall, University of Kent
Tickets £12 full and £5 students and children available from the Gulbenkian Box Office
This year's summer concert features two works by Beethoven, the first of which is the fine Coriolan Overture written in 1807 for the tragedy of the same name by the German author Heinrich Joseph von Collin about the ancient Roman leader Gaius Marcius Coriolanus. The Overture is programmatic in that the music follows the themes of the play, with Beethoven using a minor theme to reflect warlike intent and a gentler theme in the relative major to represent the pleas by Coriolanus' mother for him to abandon his attack on Rome. The programme continues with Mendelssohn's acclaimed Violin Concerto, one of the most popular and frequently performed works in the violin concert repertoire. Premiered in 1845, the Concerto was written over a period of seven years during which Mendelssohn sought advice from his friend Ferdinand David. Although the Concerto follows a traditional form, it has several innovative elements, such as the early entrance of the solo instrument at the beginning of the work and the close melodic and harmonic connection between the three movements which run together in a style known as through-composed. We are delighted to feature the Orchestra's Leader, Amanda Wyatt, as the soloist. The final work in the concert is Beethoven's Symphony No.1, which served to announce his talents to Vienna in 1800. Like the previous work in the programme it predominantly follows the established structure of the day, but has some innovative features that hint at the magnificence of the symphonies to come, such as the ambiguity of key in the opening bars, the accelerated tempo of the Menuetto, and the unusual use of the traditional sonata form in the first and last movements.
Interested in playing with the Canterbury Orchestra?
Call Nicky Pound, Canterbury Orchestra Manager on 01304 812755 or email us to find out whether we have vacancies.


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Sunday's Hymn
Come, Gracious Spirit, Heav'nly Dove
Come, gracious Spirit, heav'nly Dove,
With light and comfort from above;
Be Thou our Guardian, Thou our Guide,
O'er every thought and step preside,
O'er every thought and step preside.
The light of truth to us display,
And make us know and choose Thy way;
Plant holy fear in every heart,
That we from God may ne'er depart,
That we from God may ne'er depart.
Lead us to holiness, the road
Which we must take to dwell with God;
Lead us to Christ, the living way,
Nor let us from His pastures stray,
Nor let us from His pastures stray.
Lead us to God, our final rest,
To be with Him forever blest;
Lead us to Heav'n, its bliss to share,
Fullness of joy forever there,
Fullness of joy forever there.
Other hymns, worship songs, sermons etc. posted today:
- Update, 8:00pm: Jesus, Thy Blood and Righteousness at Rosemary at Home
- I'd Rather Have Jesus at threegirldad
- It Is Well With My Soul at Hopeful Morning
- Jesus Is a Mighty Saviour at Tried With Fire
- Trusting and Hoping at The Happy Wonderer
- Great God of Wonders at Field Stone Cottage
- I Lift My Heart to Thee at Whatever Things
- Hail the Day That Sees Him Rise at Conjubilant with Song
- His Name Is Jesus at Theology for Girls
- Lord's Day 23, 2011 at The Thirsty Theologian
- Voices From the Past — June 5, 2011 at The Upward Call
Have you posted a hymn (or sermon, sermon notes, prayer, etc.) today and I missed it? Let me know by leaving a link in the comments or by contacting me using the contact form linked above, and I'll add your post to the li


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Jewelry and Metal Arts Subject Guide
Jewelry Design
- 21st century jewelry: the best of the 500 Series (2011)
- Dreaming jewelry (2010)
- Jewelry by artists: in the studio 1940-2000: selections from the Daphne Farago Collection (2010)
- 7000 years of jewelry (2008)
- Mourning art & jewelry (2004)
- The splendor of ethnic jewelry: from the Colette and Jean-Pierre Ghysels Collection (1994)
- Erte: art to wear: the complete jewelry (1991)
Jewelry Making Techniques
- Gemstone settings: the jewelry maker's guide to styles & techniques (2012)
- Color, texture & casting for jewelers: hands-on demonstrations & practical applications (2011)
- The new encyclopedia of jewelry-making techniques (2010)
- The ultimate jeweler's guide: the illustrated reference of techniques, tools & materials (2010)
- Mixed-media jewellery (2009)
- Metals: materials for inspirational design (2004)
- The colouring, bronzing, and patination of metals : a manual for the fine metalworker and sculptor: cast bronze, cast brass, copper and copper-plate, gilding metal, sheet yellow brass, silver and silver-plate (1983)
Jewelry Illustration
Current Subscriptions
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- Current Obsession
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- Gender in jewelry: an exploration of how gender is perceived through jewelry [online thesis] (Tessa Orton, 2013)
- The landscape of man and nature: a series of jewelry (Francesca MacKie, 2012)
- Perceived value : body adornment made out of speaker wire (Shin-Chi Chen, 2010)
- The projection of nature onto city life: a series of metal sculptures of Yu Jean Choi (Yu Jean Choi, 2010)
Articles and Reference Resources
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Surnames Helmandollor to Helmchen Free Family Genealogy
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Note: I've already posted a spoiler-free overall review of The Clone Wars: Season 6, but for these individual episode reviews, full spoilers are included.
Click Here for More Clone Wars: Season 6 Episode Reviews
Even if you initially felt that somehow Fives could make it through this story alive, by the third episode, it became clear this was heading towards a grim outcome - not just the massive one we know from Revenge of the Sith, but in the short-term, it became clear that Fives simply could not be allowed to live. What he was finding out had to be kept a secret until Revenge of the Sith, and Palpatine would not allow someone with that information to keep walking around.
Still, even with the inevitably of it all hanging over it, Fives' death was very sad to behold. This was a character who truly represented the everyman (everyclone?) grunt on the series, going back to the pivotal episode "Rookies" in Season 1, which really elevated The Clone Wars from its initial few offerings and showed what the series was capable of. We'd seen his buddies in Domino squad fall one by one, but Fives was still standing, and watching him finally go out was suitably effective and emotional, backed by a stirring piece of music by Kevin Kiner.
[When I first watched this episode, I was all set to write about Fives' death marking the end for every single one of the "Rookies" - until Dave Filoni offered up this reveal from the episodes that were never completed. Hmm!]
Before Fives met his end, we got another good "man on the run" installment, as Fives, drugged and increasingly out of it, was pursued though the streets and skyways of Coruscant. Now we can certainly connect some dots as to what happened when Palpatine and Fives were left alone in that room, given we saw Fives was injected with something by Nala Se and he later reveals Palpatine simply told him of his involvement in Order 66 - all of which would explain why he attacked the Chancellor. But, given how we were with Fives and his POV for almost the whole time here, I wish we'd actually seen that happen. I know it would cross a line this show never did completely, 100% revealing Palpatine is a villain, but still… We know, right?!
Amongst the sadness, Fives' journey through Coruscant included some amusing touches, such as a look at a clone bar, and the drunken, off duty guys who befriended Fives. I also loved hearing some in the crowd go, "Oooooh!" when Shaak Ti did that huge, Jedi leap forward while chasing Fives - because come on, if you saw that in person, you'd be pretty impressed, right?!
However, the entire time, we also got a great look at Fives' paranoia, fueled by what he'd seen, what he'd learned, the drugs in his system, and his inability to get anyone to just easily believe what he was saying. It added to the tragedy at hand to see this guy going to such a fragile mental place before the end.
Anakin clearly didn't believe Fives' story at all, dismissing him as a rambling mad man in his final moments, but still, it was frustrating seeing the Jedi at large again seem duped, with Mace Windu declaring, "so… I guess it's over then," hearing the "parasite" story Palpatine offered. There are a lot of reasons to understand why the Jedi and Republic simply could not stop using the Clone Troopers, as they are in the middle of a full scale war and this is their army, but that idea could have been articulated a bit more directly, or we could have seen other Jedi outwardly show a bit more concern... though Yoda did seem to have his misgivings.


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That goes without saying, I think. But what is really happening is that, and as great as the education system is America now, there would be far to many teachers, and it would then suck to be a teacher.
Isn't it funny how teachers are so admired, but at the same time, so disrespected? Teaching is not something that you do because you -needs a job man-, it's only successful if it comes from your heart. Maybe that sounds cheesy, but it's true.
I can hear it now, "Don't you talk to me like that you little shit, I use to work at IBM!"


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We created Pandora to put the Music Genome Project directly in your hands
It's a new kind of radio –
stations that play only music you like
Eye Of The Tiger
Features of This Trackelectric rock instrumentation
disco influences
acoustic rhythm piano
extensive vamping
thru composed melodic style
minor key tonality
melodic songwriting
dirty electric guitar riffs
an aggressive male vocalist
These are just a few of the hundreds of attributes cataloged for this track by the Music Genome Project.show more
Similar Tracks
2 go to where u can change ur password
3 say ur crushes' name 10 times
4 close ur eyes
5 now put random password without knowing what it is.
6 close ur phone.
7 now try to remember what password u randomly chose.
8 realize uve been trolled and try to jailbreak ur phone
9 realize u can't, and become depressed and never do these stupid challenges!
say your crushes name
close your hand
pick a school day
say your name
post this on 15 other songs and your crush will ask you out on the day you picked


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Jeff Lytle: Many memories center around news plant on Central Avenue
They said it would be a cold day when the Daily News breaks ground on its new headquarters.
They were right.
It dipped down to 40 degrees Thursday, the day we marked the beginning of construction at Immokalee Road between U.S. 41 and Goodlette-Frank Road.
Another weather marker of the milestone came when Jim Syoen, our naplesnews.com forecaster, told the gathering, "I do the interactive weather.''
Puzzled stares would have been his response to that in 1969, when our current headquarters was built on Central Avenue, in 1980 when it grew horizontally and even 1990 when it was expanded vertically.
The groundbreaking, while focused very much ahead, prompts a look back at a building that served us so well at the former center of the universe, five blocks north of Naples' historic Four Corners.
We stood on its roof to watch the Naples Fire Department torch the tenement shacks that made up the McDonald's Quarters slum next door to the north. We stood up there to watch fireworks and shuttle launches, except one, for Challenger, which we recall only too well watching from the side steps.
Its smells have changed. The odor of rubber cement is gone from a time when "cut and paste" meant cutting stories with scissors and pasting them together with paste.
No more photographic darkroom chemicals — or darkroom. No more smoke.
Sounds are different, inside and out. No more typewriters and wire-service bulletin bells, and no more roosters from the slum crowing at dawn — which we heard while arriving in the early a.m. when we were a p.m.
Our expansions meant enduring the din of jackhammers and managing to work with office equipment half-covered with plastic for protection from falling pieces of ceiling material and summer rain.
We used to drive across railroad tracks on Central Avenue to get to work from the west and ford the Central Avenue "river" after summer downpours.
We launched our first newsroom computer system on primary election day 1982. No pressure.
We marveled when cable TV was installed in order that, among other things, our newsroom could monitor CNN.
We would use it as a hurricane shelter — kids, dogs and all — thinking it was the most formidable bunker in the world. Now we see it differently. We see it as the old Southwest Florida International Airport terminal — swell, until we got a preview of the new one.
We remember when a mailed threat to a former editor led to locks on strategic doors inside and out. Until then, our newsroom and entire plant were wide open for wandering bands of civic groups' publicity chairpersons, fishermen with prize catches, Jungle Larry, the guy with shuffleboard scores and the PR clown from the circus who would come in every spring and whack us with a rubber hammer.
Our building looked inviting to an escaped convict weary of hiding in the mangroves and getting eaten alive by mosquitoes. He came to surrender. He asked first, before we called the cops, to have his picture made — as evidence of the beating he was sure he was about to get at the jail.
A member of the Ku Klux Klan walked into the newsroom about noon one Saturday — just as the former "Sunday" paper was going to press — to pass out business cards and advise of a demonstration, with white hoods and all, on a busy street later that day.
Then there is the huge, gold chandelier in our lobby. It was put there in the last — and I do mean last — expansion, sending a signal of either unusual taste or misplaced grandeur.
Our real estate editor, Jim Lockhart, who has worked here since 1980 and contributed mightily to this story of a building, adds this about what he remembers: "Having former editor Alan Horton show me around the partially completed upstairs newsroom and asking if maybe there should be an elevator."
Thousands of you have been inside. Many, many more have had contact with us here via mail, phone, fax and e-mail — and thus share a connection to this special place.
And so we head north, to today's center of the universe. It will be a cold day when we ever move from there.
- - -
Jeff Lytle is editorial page editor of the Daily News. His e-mail address is firstname.lastname@example.com. Call him at 263-4773.
Subscribe to the Naples Daily News
Need Help? Call us at 1-888-878-6842.
Monday-Friday: 6am-6pm / Saturday: 7am-3pm / Sunday: 7am-2pm


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A clear-cut call for a third runway at Heathrow was made by the Airports Commission today.
Rush-hour commuters complained of "pizza oven" temperatures on Tubes and buses as Londoners braced for the hottest July day in almost a decade.
Claims that controversial adverts which asked commuters "Are you beach body ready?" were offensive and shamed women have been dismissed by the watchdog.


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Aye aye, fit like?
Welcome, one and all, to the Flying Pig Productions website.
Here you will find a cornucopia of information and related thingamybobs and hoojimaflips about the finest comedy sketch team in the sunny and cosmopolitan metropolis of Aberdeen, Scotland.
So join us, and explore the world of Archie & Davie, Mither & Faither, Ruaridh Duguid, Maurice The Liar, Pishy Pishy Watson and others too numerous to mention.
See us live in 2015
We have two very special live dates coming up, at HMT in Aberdeen on July 18th and at the King's Theatre Edinburgh on July 25th click on the posters for more info and to order tickets.


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If the problem persists contact your website administrator. Please check logs for further debugging.
Wegmans Tuscan Garbanzo Beans & Kale
|Olive oil
|1⁄4 Cup (4 tbs) (Wegmans)
|Mire poix
|8 Ounce (Food You Feel Good About Cleaned and Cut)
|4 Clove (20 gm), finely chopped (Food You Feel Good About Peeled)
|Garbanzo beans
|46 Ounce, drained, rinsed (Italian Classics)
|Diced tomatoes
|28 Ounce (Wegmans)
|Crushed red pepper
|1 Teaspoon
|Dried thyme leaves
|1 Teaspoon
|Bay leaves
|Coriandar powder
|1 Teaspoon
|1 Teaspoon
|Cracked black pepper
|1⁄2 Teaspoon
|Tomato sauce
|24 Ounce (Seasoned Tomato Sauce)
|3 1⁄2 Cup (56 tbs)
|Kale greens
|16 Ounce (Food You Feel Good About Cleaned & Cut)
|Garlic flavored oil
|1⁄4 Cup (4 tbs) (Wegmans Basting Oil, Herb and Garlic)
1. Heat olive oil in a large stockpot on medium. Add mirepoix and saute, stirring, about 6 minutes, until veggies begin to soften. Add garlic and cook 1 minute.
2. Add garbanzo beans and stir. Then tip in the can of diced tomatoes with juice and add crushed red pepper, thyme, bay leaves, corriander, salt, and pepper. Saute for 2 minutes to release the flavors.
3. Add seasoned tomato sauce and water. Bring to simmer on medium high heat. Reduce heat to medium low and simmer stirring occasionally, for about 20 minutes.
4. At the end of 20 minutes, add kale. Cook, stirring occasionally, another 10 minutes.
5. Add basting oil (herb and garlic oil); stir. Cook 2 minutes.
6. Remove bay leaves and ladle the stew/soup into bowls and sprinkle each serving with shredded Parmigiano-reggiano cheese if you like. Omit cheese if vegan and top with fresh herbs. Serve with crusty bread.


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I used to bend to the will of my partners. Fold, cave, however you want to say it, I used to put them first and myself always last and it lasted until il I was used up and they either moved on or I did because I was simply too burned out to continue.
I've learned a lot though, in the last few months, first and foremost that I'm stronger than I ever imagined. Second, I'm starting eliminate negative people from my life, those who take and drain my time, energy, and resources and who give nothing back in return. Third, I'm learning to put myself first and take care not to spread myself to thin. It's great that I want to save the world but I can't do much for anyone if I burn out.
Since September, I've begun a relationship with one of my closed and dearest friends. A girl who I've loved for years but could never pursue because life is complicated like that sometimes. She is the sweetest, nicest, most giving person I know. Since being with her, I am once again in touch with my creative side. Something I haven't given much thought to in many years. I've been getting into photography. She edits my photos and does a tremendous job at that. She is also teaching me to play the mandolin which I'm enjoying immensely.


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The perils of being an industry leader means that when it comes to recruitment we are constantly searching for people who have the drive to not just carry on our good work, but will become intrinsic to our future successes.
We believe that the only way to become better and stronger is to secure talent and passion. Whether it's analytics that you love or search, copywriting, design, social or paid media, we want to hear from people who want to grow.
Award winners don't win awards by chance. If you fancy winning our next one with us, take a look at the current vacancies we have on offer, or email us at [email protected]
Meander through London's developing Kings Cross, past the various journalist hangouts and the tasty tapas restaurant next door and you'll arrive at the Greenlight offices. Bare bricked, spacious and stylish, a walk through our doors means you're met by today's digital leaders.
Whilst we all work hard, we like socialising just as much. Choose a song on the juke box, pour yourself a premium blend coffee and get comfortable. Because once people join the Greenlight family, we find they tend to stick around for a while.
First and foremost we value people who want to work as hard as us, who want to achieve and want to develop. You should have a real curiosity and interest in the industry as well as be excited to work for our clients. Our agency ethos relies on the development of good relationships. Relationships that win clients and keep them. Relationships that harvest a productive working environment. We value energy, drive, creativity and high-powered thinking.
Recruitment is a two-way process. At Greenlight we're incredibly focused on making sure that we hire the right people for the right positions. There's nothing worse than leaving an interview without truly understanding the company or the role. This is why we've designed a unique interactive recruitment process which can open up a real dialogue between us and you.Read More
We want to make life easier and better for our employees. When it comes to benefits and perks we're happy to go above and beyond the basics to take care of our Greenlight staff. From child care supplements and gym membership, to a tasty bacon sarnie to get you through a Friday morning, we are on standby to give all our employees that extra bit of TLC where its needed.Full Details
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When Greenlight's HR team invited me to a daylong assessment centre, rather than a standard interview, I have to admit I was a little daunted! However, by the end of the day I was left in no doubt that this was a better way to recruit for both employers and candidates than the usual half hour slot.
The role entails responding to inbound inquiries, following up on marketing campaigns and outbound calling to target prospects across a number of different verticals.View Position
We are looking for a talented and enthusiastic Web Developer to join our team here at Greenlight Digital. As a Web Developer, you'll be responsible for working on a key online platform, working on both backend development as well as developing the highly responsive front end UI. You should be proficient with online technologies and coding best practice, and be able to confidently advise non-technical people (internally and clients) about the best solution/technologies/approach and feasibility of projects.View Position
We are looking for a top-notch digital designer to join our small team of designers in our new offices in Kings Cross. The right candidate will be a creative and enthusiastic individual, with proven excellent design and front-end development skills, who is ready to create great solutions in collaboration with our experienced development, copywriting and social media teams. They will be given the opportunity to work on a wide range of digital projects for our extensive client list, as well as occasionally providing support to our internal marketing team.View Position
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We're looking for a great storyteller who will uncover original data to tell informed stories as well as enhance editorial content. This person will also work closely with the content and engagement strategists to brainstorm ideas and turn them into compelling stories, infographics and interactives. An average day might include: working with a writer to crunch data and discuss a trend or story, writing an original data-driven story and/or working with the design teams to create a clever visual content piece.View Position


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No. 2: Alastair Cook
Jul 7, 2015 Joining Matthew Hayden as opening partner in the former Australian captain's Ashes XI: an unflappable run-getter from England
Jul 7, 2015 Joining Matthew Hayden as opening partner in the former Australian captain's Ashes XI: an unflappable run-getter from England
Jul 5, 2015 Ryan Harris is not the only one to finish his career on 27 Tests. How much do you know about this select club of cricketers?
The Investec Ashes 2015
Jul 4, 2015 He has not played a Test in two years but Steven Finn is back in England's Ashes planning and believes the best is yet to come
Jul 4, 2015 How well do you know the records of players in Australia and England's Ashes squads?
The Test
Jul 4, 2015 Simon Jones' career was blighted by injury but he was a key part of the attack who brought the Ashes home for England a decade ago and his story remains a compelling one
Jul 3, 2015 With the red Dukes ball, against Australia's current batting line-up, Stuart Broad has a much better record than James Anderson
Jul 3, 2015 England allrounder Ravi Bopara on being awarded a benefit year by Essex, his autographing ethics, and putting up with Jonathan Trott's velcro-heavy superstitions
Jul 2, 2015 Lillee and Thommo, Illy, Boycs and Snow: the '70s was a fine time for a youngster captivated by England-Australia Test matches
The Investec Ashes 2015
Jun 30, 2015 Ahead of what is likely to be another heated Ashes series James Anderson says he has cooled his own fiery approach
The Investec Ashes 2015
Jun 29, 2015 At the age of 33, Johnson does not expect to be back for another Ashes campaign and, since being forced to miss last year's World Twenty20 with a toe injury, he has played every game as if it is his last


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In 1986 Kae made her debut as a suiyuu in anime when she landed the part of a nursery school child in the series Maison Ikkoku.
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Up for sale in the Houston showroom is a true survivor 1971 GMC Sierra Grande 1500. The C series pickup was Chevrolet and GMCs... More
1979 GMC 1/2 Ton Step Side Truck. New V8 Crate Engine, Auto Trans, Vehicle shows 51000 miles. This Vehicle just had a high quality... More


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Re: Mohela and Direct Loans
Not a problem - MOHELA will report the information that is in the account as it was sent to us. So, in your case, if the prior servicer sent to us 1996, then MOHELA will report what was sent to us, 1996. We will report what was included in the file to us, from the prior servicer. This is not a new loan, it is a transfer of servicing of a pre-existing account. Does this help?
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