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We present a novel protein structure database search tool, 3D-BLAST, that is useful for analyzing novel structures and can return a ranked list of alignments. This tool has the features of BLAST (for example, robust statistical basis, and effective and reliable search capabilities) and employs a kappa-alpha (κ, α) plot derived structural alphabet and a new substitution matrix. 3D-BLAST searches more than 12,000 protein structures in 1.2 s and yields good results in zones with low sequence similarity.
A major challenge facing structural biology research in the postgenomics era is to discover the biologic functions of genes identified by large-scale sequencing efforts. As protein structures increasingly become available and structural genomics research provides structural models in genome-wide strategies [1], proteins with unassigned functions are accumulating, and the number of protein structures in the Protein Data Bank (PDB) is rapidly rising [2]. The current structure-function gap highlights the need for powerful bioinformatics methods with which to elucidate the structural homology or family of a query protein by known protein sequences and structures.
Numerous sequence alignment methods (for instance BLAST, SSEARCH [3], SAM [4], and PSI-BLAST [5]) and structure alignment methods (for instance, DALI [6], CE [7], and MAMMOTH [8]) have been demonstrated to identify homologs of newly determined structures. Sequence alignment methods are rapid but frequently unreliable in detecting the remote homologous relationships that can be suggested by structural alignment tools; also, although the latter may be useful, they are slow at scanning homologous structures in large structure databases such as PDB [2]. Various tools including ProtDex2 [9], YAKUSA [10], TOPSCAN [11], and SA-Search [12] have recently been developed to search protein structures quickly. TOPSCAN, SA-Search, and YAKUSA describe protein structures as one-dimensional sequences and then use specific sequence alignment methods to replace BLAST for aligning two structures, because BLAST needs a specific substitution matrix for a new alphabet. Many of these methods have been evaluated based on the performance of two structure alignments but not on the performance of the database search. Additionally, none of these methods provides a function analogous to the E value of BLAST (which is probably the most adopted database search tool by biologists) for investigating the statistical significance of an alignment 'hit'.
The three-state secondary elements, namely α-helix, β-sheet, and coils, are rather crude for predicting protein structure, and it is not possible to make use of these elements in three-dimensional (3D) reconstruction without additional information. Many approaches have been proposed to replace three-state secondary structure descriptions with various local structural fragments, also known as a 'structural alphabet' [13-19], which can redefine not only regular periodic structures but also their capping areas. Such studies have described local protein structures according to various geometric descriptors (for example, Cα coordinates, Cα distances, α or , and ψ dihedral angles) and algorithms (for example, hierarchical clustering, empirical functions, and hidden Markov models [HMMs] [12]). Many of these methods involve protein structure prediction; an exception is the SA-Search tool [12], which is based on Cσ coordinates and Cα distances, and which adopts a structural alphabet and a suffix tree approach for rapid protein structure searching.
To address the above issues, we have developed a novel kappa-alpha (κ, α) plot derived structural alphabet and a novel BLOSUM-like substitution matrix, called SASM (structural alphabet substitution matrix), for BLAST [5], which searches in a structural alphabet database (SADB). This structural alphabet is valuable for reconstructing protein structures from just a small number of structural fragments and for developing a fast structure database search method called 3D-BLAST. This tool is as fast as BLAST and provides the statistical significance (E value) of an alignment, indicating the reliability of a hit protein structure. For the purposes of scanning a large protein structure database, 3D-BLAST is fast and accurate and is useful for the initial scan for similar protein structures, which can be refined by detailed structure comparison methods (for example, CE and MAMMOTH).
To the best of our knowledge, 3D-BLAST is the first tool that permits rapid protein structure database searching (and provides an E value) by using BLAST, which searches a SADB database with a SAMS matrix. The SADB database and the SASM matrix improve the ability of BLAST to search for structural homology of a query sequence to a known protein structure or a family of proteins. This tool searches for the structural alphabet high-scoring segment pairs (SAHSPs) that exist between a query structure and each structure in the database. Experimental results reveal that the search accuracy of 3D-BLAST is significantly better than that of PSI-BLAST [5] at 25% sequence identity or less.
A pair database comprising 674 structural pairs (Additional data file 1), each with a high structural similarity and low sequence identity, was derived from the SCOP classification database [20] for the (κ, α) plot (Figure 1a,b). Each structure in this database (1,348 proteins) was divided into a series of 3D protein fragments (225,523 fragments), each five residues long, using κ and α angles. The angle κ, ranging from 0° to 180°, of residue i is a bond angle formed by three Cα atoms of residues i - 2, i, and i + 2. The angle α, ranging from -180° to 180°, of a residue i is a dihedral angle formed by the four Cα atoms of residues i - 1, i, i + 1, and i + 2. Each structure has a specific (κ, α) plot (Figure (Figure1a)1a) when governed by these two angles. For instance, a typical (κ, α) plot (blue diamond) of an all-β protein (human anti-HIV-1 GP120-reactive antibody E51, PDB code 1RZF-L [21]) is significantly different from that (red cross) of an all-α protein (human hemoglobin, PDB code 1J41-A [22]). Conversely, two similar protein structures have similar (κ, α) plots.
An accumulated (κ, α) plot (Figure (Figure1b)1b) consisting of 225,523 protein fragments was obtained from this pair database. The plot is split into 648 cells (36 × 18) when the angles of κ and α are divided by 10°. In the accumulated (κ, α) plot, most of the α-helix segments are located on four cells in which the α angle ranges from 40° to 60°, and the κ angle ranges from 100° to 120°. In contrast, the κ angle of most of the β-strand segments ranges from 0° to 30°, and the α angle ranges from -180° to -120°, or 160° to 180°. The number of 3D segments in each cell ranges from 0 to 22,310, and the color bar on the right side presents the distribution scale. Based on the definitions in the DSSP program [23] the numbers of α-helix and β-strand segments are 82,482 (36.6%) and 52,371 (23.3%), respectively. Most 3D segments in the same cell in this plot have similar 3D shapes, that is, a root mean square deviation (rmsd) below 0.3 Å on five contiguous Cα atom coordinates. Moreover, the conformations of 3D segments located in adjacent cells are often encoded into similar structural letters which have more similar 3D structures than those in distant cells (Figures 1b,c). Hence, the (κ, α) plot is helpful for clustering these 3D segments to determine a representative segment for each cluster.
Based on the (κ, α) plot and a new nearest neighbor clustering (see Materials and methods, below), a new 23-state structural alphabet was derived to represent the profiles of most 3D fragments, and was roughly categorized into five groups (Figure (Figure2a2a and Additional data file 2): helix letters (A, Y, B, C, and D), helix-like letters (G, I, and L), strand letters (E, F, and H), strand-like letters (K and N), and others. The 3D shapes of representative segments in the same category are similar; conversely, the shapes of different categories are significantly different. For instance, the shapes of representative 3D segments in the helix letters are similar to each other, as are those in strand alphabets. In contrast, the shapes of helix letters and strand letters obviously differ. The average structural distance (determined from the rmsd value of five continuous Cα atom positions between a pair of 5-mer segments) of intersegments in both helix and strand letters is less than 0.4 Å (Figure (Figure1c),1c), and is much less that those of other letters in the structural alphabet. Additionally, most α-helix secondary structures based on the definition of the DSSP program are encoded as helix or helix-like alphabets, and none are encoded as strand or strand-like alphabets (Figure (Figure2b).2b). Conversely, most β-strand segments are encoded as strand or strand-like letters (Additional data file 3).
All residues were fairly restricted in their possibilities in the (κ, α) plot (Figure (Figure1b).1b). The proportion of cells with 0 segments, which were encoded as structural letter 'Z', was 28.2% (183 cells among 648). Additionally, the numbers of cells and segments with structural letter 'Z' were 272 (42.0% [272/648]) and 989 (0.4% [989/225,523]), respectively. Restated, only 0.44% segments were widely distributed in 41.98% of cells. If the segments of a new protein structure are located on these 41.98% cells, then they may be regarded as poor structural segments. Conversely, five helix letters (A, Y, B, C, and D) and three strand letters (E, F, and H) were located in seven and 30 cells (Figure (Figure1b),1b), respectively. The total number of segments located in these 37 (4.4%) cells was 75,477 (33.5%).
The (κ, α) plot is similar to a Ramachandran plot, based on the following observations. First, the α-helices are located in very restricted areas, in which α ranges from 40° to 60°, and κ ranges from 100° to 120°. Additionally, β-sheet segments are restricted to some regions in the (κ, α) plot. All residues are fairly restricted in their possibilities in both plots. Second, angles and ψ in the Ramachandran plot, denoting a protein structure with a series of 3D positions of amino acids, are widely adopted to develop various structural segments (blocks). Here, the (κ, α) plot was utilized to develop a structural alphabet, which represents a protein structure as a series of 3D protein fragments, each of which are five residues long. The angles and ψ represent the position relationship of two contiguous amino acids, whereas the angles κ and α represent the position relationship of five amino acids. These observations indicate that the (κ, α) plot is an effective means of both developing short sequence structure motifs and assessing the quality of a protein structure.
A greedy algorithm and the evaluation criteria (global-fit score) presented by Kolodny and coworkers [15] were applied to measure the performance of 23-state structural alphabet (structural segments) in reconstructing the α-β-barrel protein (PDB code 1TIM-A [15,24]) and 38 structures (Additional data file 4) selected from the SCOP-516 set, which comprises 516 proteins. This greedy algorithm reconstructs the protein in increasingly large segments using the best structural fragment, namely the one whose concatenation produces a structure with the minimum rmsd from the corresponding segment in the protein from 23 structural segments. No energy minimization procedure was utilized to optimize the reconstructing structures in this study. The global rmsd values were from 0.58 Å to 2.45 Å, and the average rmsd value was 1.15 Å for these 38 proteins. Figure 3a,b illustrate the reconstructed structures of the α-β-barrel protein and ribonucleotide reductase (PDB code 1SYY-A [25]), respectively. The Cα carbon rmsd values were 0.80 Å (1TIM-A) and 0.63 Å (1SYY-A) between the X-ray structures (red) and reconstructed proteins (green). The reconstructed structures are frequently close to the X-ray structures on both α-helix and β-sheet segments, and the loop segments account for the main differences. If all representative segments (465 segments) of the non-zero cells in the (κ, α) plot were considered when reconstructing structures, then the global rmsd values would be in the range 0.35 to 2.32 Å, and the average rmsd value would be 0.94 Å.
The 23-state structural alphabet should be able to represent more biologic meaning than standard three-state secondary structural alphabets. First, the classic regular zones of three-state secondary structures are flexible structures. For instance, α-helices may be curved [26] and more than one-quarter of them are irregular [27], and the and ψ dihedral angles of β-sheets are widely dispersed. The proposed 23-state alphabet describes α-helices with eight segments (five helix letters and three helix-like letters) and β-sheets with five segments (Figure (Figure2a).2a). Figure Figure33 reveals that the 23 structural segments performed well in reconstructing protein structures, particularly in the structure segments of classic α-helices and β-sheets. Second, the three-state secondary structure cannot represent the large conformational variability of coils. Nonetheless, some similar structures can be identified for many of the protein fragments, such as β-turns [28], π-turns, and β-bulges [29]. Here, 10 structural segments in the 23-state alphabet were utilized to describe the loop conformations. An analysis using the PROMOTIF [30] tool reveals that most of the segments (>80%) in the letter 'W' are β-turns.
In a structural database search, 3D-BLAST identifies the known homologous structures and determines the evolutionary classification of a query structure from an SADB database (Additional data file 5). Users input a PDB code with a protein chain (for example, 1GR3-A) or a domain structure with a SCOP identifier (for example, d1gr3a_). When the query has a new protein structure, the 3D-BLAST tool enables users to input the structure file in the PDB format. The tool returns a list of protein structures that are similar to the query, ordered by E values, within several seconds. When we searched databases such as SCOP [20] or CATH [31], which are based on structural classification schemes, the evolutionary classification (family/superfamily) of the query protein was based on the first structure in the 3D-BLAST hit list.
The main advantages of 3D-BLAST using BLAST as a search tool include robust statistical basis, effective and reliable database search capabilities, and established reputation in biology. However, the use of BLAST in protein structure search has several limitations, namely the need for an SADB database, a new SASM matrix, and a new E value threshold to show the statistical significance of an alignment hit. These issues are described in the following subsections.
A SADB database was easily derived from a known protein structure database based on the (κ, α) plot and the structural alphabet. We created five SADB databases derived from the following protein structure databases PDB; a nonredundant PDB chain set (nrPDB); all domains of SCOP1.69 [20]; SCOP1.69 with under 40% identity to each other; and SCOP1.69 with under 95% identity to each other.
The SCOP-516 query protein set, which has a sequence identity below 95% selected from the SCOP database [20], was chosen to measure the utility of 3D-BLAST for the discovery of homologous proteins of a query structure. This set contains 516 query proteins that are in SCOP 1.69 but not in SCOP 1.67, and the search database was SCOP 1.67 (11,001 structures). The total number of alignments was 5,676,516 (516 × 11,001). For evolutionary classification, the first position of the hit list of a query was treated as the evolutionary family/superfamily of this query protein. For comparison with related work on rapid database searching, 3D-BLAST was also tested on a dataset of 108 query domains, termed SCOP-108 (Additional data file 6), proposed by Aung and Tan [9]. These queries, which have under 40% sequence homology to each other, were chosen from medium-sized families in SCOP. The search database (34,055 structures) represents most domains in SCOP 1.65. Finally, the utility of 3D-BLAST for 319 structural genomics targets was analyzed; the search database was SCOP 1.69, with under 95% identity to each other.
Here, several common metrics (precision, recall, and receiver operating characteristic [ROC] curve) were utilized to assess the predicted quality of a search method on database searching. Precision is defined as Ah/Th and recall is defined as Ah/A, where Ah is the number of true hit domains in the hit list, Th is the total number of domain proteins in the hit list, and A is total number of true hits in the database. The ROC curve plots the sensitivity (recall) against 1.0 - specificity (false-positive rate). The average precision is defined as , where is the number of compounds in a hit list containing i correct domains.
A substitution matrix is an essential component when BLAST is used to search a structural database quickly. A new BLOSUM-like substitution matrix, called SASM (Figure (Figure4),4), was developed by using a method similar to that used to construct BLOSUM62 [32] based on the pair database. The SASM (23 × 23) provides insight into substitution preferences for 3D segments between homologous structures with low sequence identity.
The SASM matrix presents good relationships between biologic functions and protein structures. The highest substitution score in SASM represents the alignment of an alphabet 'W' with an alphabet 'W', in which the conformations of segments are similar β-turns. Substitution scores are high when two identical structural alphabets (for example, diagonal entries) are aligned. For instance, the alignment scores of aligning 'I' and 'S' to themselves are 9 and 8, respectively. Most substitution scores are positive when two structural alphabets in the same category, for example helix alphabets (A, Y, B, C, and D), are aligned. Conversely, the lowest substitution score (-15) in SASM is for the alignment of 'Y' (helix alphabet) with 'E' (strand alphabet). These scores are also low when helix alphabets (A, Y, B, C, and D) are aligned with strand alphabets (E, F, and H).
The SASM matrix and BLOSUM62 [32] were compared because they adopted BLAST as the search tool. The highest substitution score is 11 for both matrices. By contrast, the lowest score for SASM (-15) is much lower than that for BLOSUM62 (-4). This large difference occurs mainly because α-helices and β-strands constitute very different protein secondary structures, and the structural letters pertaining to these two structures are better conserved than those of amino acid sequences. Because the gap penalty is an important factor, various combinations of gap penalties were systematically tested for 3D-BLAST and the SASM matrix based on the pair database (1,348 proteins). Here, the optimal values for the open gap penalty and the extended one are 8 and 2, respectively.
A database search method should enable users to examine the statistical significance of an alignment in order to determine the reliability of the prediction. 3D-BLAST maintains the benefits of the BLAST tool in terms of ordering hit proteins by E value for rapid scanning of structural database. We used the theoretical result [33,34] to estimate the E value of an ungapped local alignment of two structural alphabet (SA) sequences A (query) and B (database sequence) with score S using the following steps. First, we computed statistical parameters λ and K according to the 23-state SA compositions of A and B and the SASM matrix (Figure (Figure4).4). In a SA database search, we used the actual SA composition of A and an average SA composition for B. Second, we computed adjusted lengths LA and LB of A and B, where LB is the sum of lengths of all database sequences. Third, we obtained a normalized score S' = λS - ln(K) and calculated the E value = LALBe-S. Although the theory referred to above has not been proved to be valid for gapped local alignments, computational experiments suggest that that it is [35,36]. The statistical parameters λ and K cannot be derived from theory; they must be estimated by simulation with random or real but unrelated sequences.
To evaluate the accuracy of the E values reported by 3D-BLAST, we submitted shuffled SA sequences as queries and found the number of match sequences with E values below various thresholds. For simplicity, we used the query set SCOP-516 and the respective shuffled queries (516 SA sequences) that represent protein structures, and the search database was SCOP 1.67. Shuffled queries mimic completely random SA sequences, which preserve only the composition basis of a protein structure, using the typical SA composition. The numbers of matches of 516 shuffled queries with E-values below e-20, e-15, and e-10 are 0, 3, and 326, respectively. On the other hand, the numbers of matches of 516 queries in the SCOP-516 dataset with E values below e-20, e-15 and e-10 are 8,268, 18,700, and 64,440, respectively. Protein structures and the structural letters are more conserved than protein sequences; thus, as one would expect, the E values of 3D-BLAST are larger than those of BLAST when the reliable indicators are similar.
The proposed 3D-BLAST provides a threshold E value to identify a highly significant similarity with the query. The SASM matrix reveals that the biologic significance of the high-scoring structures can be inferred from the similarity score, and the proportion of true positives rises when a lower E value is utilized (Figure (Figure5).5). Figure Figure5a5a shows that E values correlate strongly with the rmsd values of aligned residues between the query protein and the hit proteins. A total of 22,415 proteins were randomly chosen from the hit lists of 516 query proteins in the SCOP-516 dataset. Among these 22,415 proteins, 27.72% (6,215 structures) had rmsd values below 3.0 Å. If the E value was restricted to under e-20, then 83.52% of hit proteins (2,130 proteins from among 2,549 proteins) had rmsd values less than 3.0 Å, and the average rmsd was 2.37 Å. When the E value was restricted to under e-15 and under e-10, then 72.65% (3,984 proteins among 5,487 proteins) and 51.70% (5,742 proteins among 11,106 proteins) of proteins had rmsd values less than 3.0 Å, respectively, and the average rmsd values were 2.85 Å and 3.57 Å.
For classification assignment, the relationship between the E value of the first hit and the number of correct (dark line) and false (gray line) classification assignments for the SCOP-516 dataset was calculated (Figure (Figure5b).5b). If the E value was restricted to under e-15, then 97.67% of 516 query structures are assigned correct classifications and the coverage was 91.47%. The coverage is defined as P/T, where P is the number of assigned structures by a method and T is total number of structures. For example, P is 472 and T is 516 for the set SCOP-516. When the E value was less than e-20 and e-10, 99.31% and 95.26% of the predicted cases were correct, and the coverage values were 83.72% and 98.06%, respectively. When the sequence identity was less than 25% (154 proteins from among 516 proteins), the rate of correct assignment was 90.35%. The coverage was 72.12% when the E value was less than e-15. For the database search, the precision was 0.80 and the recall was 0.48 when the E value was below e-15; by comparison, the precision was 0.90 and the recall was 0.42 when the E value was below e-20. These analytical results demonstrate that the E value of 3D-BLAST enables users to examine the reliability of the structure database search of a query.
Using the yeast copper chaperone for superoxide dismutase (yCCS) from Arabidopsis thaliana (PDB code 1JK9-B) [37] as the query protein and an E value threshold of 10-10, a 3D-BLAST search of the database SCOP1.69 found 19 members (Table (Table1).1). Figure Figure66 shows two hits of the search results. The protein (yCCS) comprised amino-terminal and carboxyl-terminal domains. The amino-terminal domain, called HMA (heavy-metal associated) domain in the SCOP database, plays a role in copper delivery. This domain contains an MH/TCXXC metal binding motif (blue box in Figure Figure6a),6a), and is very similar to the metallochaperone protein Atx1. The carboxyl-terminal domain, termed the Cu,Zn superoxide dismutase-like domain in the SCOP database, comprised an eight-stranded β-barrel that strongly resembles yeast superoxide dismutase I and human superoxide dismutase I.
3D-BLAST was able to identify 9 and 10 homologous structures of amino-terminal domains and carboxyl-terminal domains, respectively, using this two-domain protein (yCCS) as query. The sequence identities between yCCS and most of the homologous structures (17 out of 19 proteins) were less than 20%. Figure 6a,c illustrates sequence alignments and the structure alignment between yCCS and an amino-terminal domain homologous protein (PDB code 1CC8-A [38]). The sequence identities of structure alphabet and amino acid sequences were 42% and 17%, respectively. 3D-BLAST can align six amino acids of the metal binding motif together, and the rmsd is 1.64 Å between these two proteins. The aligned secondary structures are represented as a continuous color spectrum from red through orange, yellow, green and blue to violet. Figures 6b,d show the sequence and structure alignments between yCCS and a carboxyl-terminal domain homologous protein (PDB code 1QOE-A [39]). The sequence identities of the structure alphabet and the amino acid sequences were 30% and 14%, respectively, and the rmsd between these two proteins was is 1.68 Å. The structural alphabets were strongly conserved in areas of the secondary structures (green block), which are β-strands represented by structural alphabets, such as E, F, H, K, and N. These results reveal that the structural alphabet sequences are much better conserved than the amino acid sequences, which explains why 3D-BLAST could detect the invariant residues and find these distantly related proteins.
Figure Figure77 illustrates the accuracies of the 3D-BLAST and PSI-BLAST in structure database searches and evolutionary classification assignments using the query set SCOP-516. For this experiment, 3D-BLAST was compared with PSI-BLAST, because PSI-BLAST often performs much better than BLAST for this purpose. Standalone PSI-BLAST [5] was installed on a personal computer with a single processor (Pentium 2.8 GHz with 512 megabytes of RAM). The main differences between 3D-BLAST and PSI-BLAST are in the search databases and substitution matrices. In 3D-BLAST, the substitution matrix is the SASM matrix and the searching database is the SADB, whereas PSI-BLAST adopts an amino acid sequence database and a BLOSUM62 substitution matrix. The number of iterations for PSI-BLAST was set to three and the open gap penalty and the extended one are 11 and 1, respectively. For database search, the threshold of the E values of 3D-BLAST and PSI-BLAST are set to 10-10 and 0.01, respectively.
For a database search tool, the ROC curve (Figure (Figure7a)7a) provides an estimation of the likely number of true positive and false positive predictions. A perfect method, which can recover all true hits without any false positives, can be denoted as a point in the top left corner of this graph, whereas a random method that generates equal numbers of true positive and false positive predictions uniformly distributed across all scores would yield a diagonal line from (0,0) to (1,1). Figure Figure7a7a shows that 3D-BLAST (dark lines) yields much better predictions than does PSI-BLAST (gray lines). The sensitivity of family assignments was superior to that of superfamily assignments in both methods, whereas the false-positive rates of family assignments were higher than those of the superfamily assignments.
For most sets of sequence identities, 3D-BLAST outperformed PSI-BLAST (Figure (Figure7b)7b) in protein evolutionary classification assignments. Almost 70.16% (362 out of 516 proteins) of query proteins were more than 25% identical to one of the library representatives from the same SCOP superfamily, and 100% of these domains were correctly mapped by both 3D-BLAST and PSI-BLAST. When the sequence identity was less than 25% (154 out of 516 proteins), the accuracy of 3D-BLAST ranged from 96.29% to 50%, whereas the accuracy of PSI-BLAST ranged from 94.29% to 21.74% (Figure (Figure7b).7b). These proteins were difficult to assign because of the limited similarity of the query proteins to the representative library domains. 3D-BLAST yielded significantly better results than did PSI-BLAST at sequence identity levels of 25% or less. The analytical results reveal that, as expected, sequence comparison tools that are more sensitive to distant homology are usually more successful at making challenging assignments. In summary, 3D-BLAST achieved more reliable assignments than did PSI-BLAST in cases of low sequence identity for this test set. The structural alphabet, SADB database, and SASM matrix could predict protein structures more accurately than simple amino acid sequence analyses.
Comparing the results of different structure database search methods is generally neither straightforward nor completely fair, because each such method utilizes different accuracy measures, searching databases, and test complexes. Figure Figure88 shows the relationship between recall and precision, and Table Table22 presents the average search time and average precision of 3D-BLAST, PSI-BLAST, MAMMOTH [8], CE [7], TOPSCAN [11], and ProtDex2 [9] on 108 query proteins proposed by Aung and Tan [9] (Additional data file 6). The performance of TOPSCAN and ProtDex2, which are fast search methods for scanning structure databases, was summarized from previous studies [9]. Other four programs were installed and run on the same personal computer with a single processor. Here, the PSI-BLAST and 3D-BLAST used E values to order the hit proteins; MAMMOTH and CE (detailed structure alignment tools) utilized Z scores to rank the hit proteins.
On average, 3D-BLAST required about 3.18 s seconds to scan the database for each query protein (Table (Table2).2). It is about 34,000 and 3838 times faster than CE and MAMMOTH, respectively. 3D-BLAST was about two times slower than PSI-BLAST, because 3D-BLAST identified many more words (typically of length three for proteins in BLAST) that score more than a threshold value in the SADB databases than those identified by PSI-BLAST in protein sequence databases. The reason for this stems from the fact that the BLAST algorithm scans the database for words that score at least a threshold when aligned with some words within the query sequence; the algorithm then extends each such 'hit' in both directions to check the alignment score [5].
MAMMOTH is the best and TOPSCAN is the worst for these 108 queries among these six methods (Figure (Figure8).8). 3D-BLAST was much better than fast structure database search methods (TOPSCAN [11] and ProtDex2 [9]), and its performance approached those of CE and MAMMOTH. Notably, PSI-BLAST outperformed both TOPSCAN and ProtDex2, which considered secondary and 3D protein structures. As shown in Table Table2,2, the mean of average precision of 3D-BLAST (78.2%) was better than that of PSI-BLAST (69.2%) and lightly worse than those of CE (82.1%) and MAMMOTH (83.4%). For some query proteins, such as serotonin N-acetyltranferase [40] (PDB code 1CJW-A) and translation initiation factor IF2/eIF5B [41] (PDB code 1G7S-A; Additional data file 6), 3D-BLAST, MAMMOTH, and CE were markedly better than PSI-BLAST because most sequence identities between the query proteins and their members are under 20%. For several query proteins, such as human dihydro-orotate dehydrogenase [42] (PDB code 1D3G-A) and yeast copper chaperones for SOD [43] (PDB code 1EJ8-A), CE and MAMMOTH were worse than 3D-BLAST. Interestingly, PSI-BLAST outperformed CE, MAMMOTH, and 3D-BLAST for S-adenosylhomocysteine hydrolase [44] (PDB code 1B3R-A).
The recognition performance of 3D-BLAST is expressed as top rankings (Additional data file 7), using Lindahl's benchmark [45], together with the performance of eight popular sequence comparison (for example, HMM and profile methods). The benchmark includes 976 proteins derived from the SCOP for identifying homologous pairs at different similarity levels (Additional data file 8). Sequence identities between the query proteins and their homologous members in the superfamily and fold levels are much lower than those at the family level. These methods can be divided into two categories: methods using only single sequence information (BLAST2 and SSEARCH [3]) and methods using multiple sequence alignments (PSI-BLAST, HMMER-HSSP [46], HMMER-PSI-BLAST [46], SAM-HSSP [4], SAM-PSIBLAST [4], and BLAST-LINK [45]). The methods of constructing profiles/HMMs used a larger dataset, comprising the SWISSPROT-35 and TREMBL-5 databases [47] together with the benchmark sequences of the HSSP database [48].
At the family level, 3D-BLAST identified 78.4% of homologous pairs that were ranked in the top 5. This was comparable to the best performance of any of the other methods (78.9%), which was achieved by BLAST-LINK (Additional data file 7). At the superfamily and fold levels, 3D-BLAST significantly outperformed all of the other methods. 3D-BLAST yielded 54.8% and 39.3% homologous pairs at the superfamily and fold levels, respectively. On the other hand, the best accuracies for the other methods were 40.6% (by BLAST-LINK) at the superfamily level and 18.7% (by SAM-PSIBLAST) at the fold level.
The main factors causing 3D-BLAST to perform poorly on some cases in both SCOP-516 and SCOP-108 datasets are summarized as follows. First, 3D-BLAST might have made minor shifts when aligning two local segments with similar codes, because the structural alphabets did not consider the actual euclidean distances. Hence, 3D-BLAST is more sensitive when the query proteins (for example, PDB code 1VDL-A and 1PMZ-A in SCOP-516) are the members of the 'all-α' class in SCOP. Second, the structural similarity of a query protein to the library members is rather limited. Third, an inherent problem in the BLAST algorithm is inability to detect remote homology of structural alphabet sequences. Use of PSI-BLAST as the search algorithm for 3D-BLAST slightly improved the overall performance on the SCOP-516 set. An enhanced position-specific score matrix of the structure alphabet for SADB databases should be developed to improve the performance of 3D-BLAST in the future. Finally, the E values of the hits are not significant.
We demonstrated the robustness and adaptability of 3D-BLAST for the initial scan of large protein structure databases; conversely, detailed structure alignment tools often align two structures slowly but accurately. Because of basic differences, comparisons between 3D-BLAST and detailed structure alignment tools are not straightforward. However, detailed structure alignment tools can be applied to refine the searching structures of 3D-BLAST to improve accuracy of prediction.
We analyzed 319 structural genomics targets, called SG-319, using 3D-BLAST (Figure (Figure9)9) with regard to function assignment. The structural genomics initiative aims to determine representative structures for all protein families in cells [1,49,50]. To sample the protein structural space more efficiently, structural genomics projects employ various target selection strategies to filter out proteins that are homologous to the proteins with structures already in the PDB [51]. As a result, the molecular functions of the proteins targeted by structural genomics are often unknown. The SG-319 set contains 319 structural genomics targets contributed by more than 10 structural genomics consortia, and publication dates range from 1 January 2005 to 30 September 2005. There are 126 proteins in SG-319 having the 'unknown function' annotation.
3D-BLAST used these 319 proteins as query proteins, and the search classification database was SCOP 1.69, which contains 12,074 domains. About 38.2% (122 proteins) and 32.6% (104 proteins) of the SG-319 proteins have more than 25% and under 20% sequence identity, respectively, to one of the library representatives of the SCOP superfamily, according to search results with 3D-BLAST. In all, 3D-BLAST assigned 244 (78.5%) proteins to SCOP superfamilies if the threshold of E value was set at under e-15 by the SG-319 query set (Figure (Figure9a).9a). When the sequence identity was more than 25%, 98.4% (120 out of 122) of these cases could be assigned to a SCOP superfamily by 3D-BLAST, and 62.9% (124 out of 197) of the remaining proteins could also be assigned.
The following observations help in comparing the characteristics and performance between applying 3D-BLAST to SG-319 (Figure (Figure9a)9a) and applying it to SCOP-516 (Figure (Figure5b).5b). First, the distribution of the sequence identity of these two sets was significantly different. The sequence identities of 197 (61.8%) and 154 (29.85%) proteins in SG-319 and SCOP-516, respectively, were under 25%. The average sequence identity in SG-319 is significantly lower than that of SCOP-516. Second, the assigned parentages of SG-391 and SCOP-516 were 78.5% and 91.47%, respectively, when the E value was restricted to under e-15. If the sequence identity was under 25%, then the assigned rates were 62.9% (SG-319) and 72.12% (SCOP-516). Third, 3D-BLAST achieved similar accuracies for both sets if the sequence identity was above 25%. These observations are consistent with recent analyses of proteins targeted by structural genomics [51,52].
Figure Figure9b9b shows that 3D-BLAST assigned a structural genomics target (PDB code 1YRH) to the flavodoxin-related family [53] based on the first-rank protein (PDB code 1E5D [54]) in the hits. The E value was 10-25 and the Z score of CE and rmsd value were 5.7 and 1.56 Å, respectively, when these two proteins were aligned. These two proteins have the same Gene Ontology (GO) annotations [55] and the same domain annotations in three databases, including PROSITE [56], Pfam [57], and CATH [31]. The aligned structures of these two proteins are similar, and the FMN-binding motifs (wireframe model) are also aligned well (Figure (Figure9b).9b). Eight of the top 10 proteins in the hits are the members of the same SCOP superfamily. However, PSI-BLAST was unable to yield the same assignment.
This study demonstrates the robustness and feasibility of the (κ, α) plot derived structural alphabet for developing a small set of sequence-structure fragments and a fast one-against-all structure database search tool. The (κ, α) plot is an effective means of assessing the quality of protein 3D structure. The 3D-BLAST tool, which exploits the benefits of BLAST, is efficient and reasonably effective. 3D-BLAST is significantly better than PSI-BLAST at 25% sequence identity or less, and is as fast as BLAST. The structural alphabet and matrix SASM achieve good agreement for protein structures and biologic inference. Future investigations can adopt the (κ, α) plot derived 3D fragment library to develop a small 3D fragment library and predict protein structures. Moreover, many sequence-based methods can be applied to mine biologic meanings quickly from protein structures based on this 23-state structural alphabet. Finally, 3D-BLAST can be adopted to develop multiple structure alignment and structure profile search methods.
To code the structural alphabet and calculate the substitution matrix we selected 674 structural pairs (1,348 proteins; Additional data file 1), which are structurally similar and with low sequence identity, from SCOP based on two criteria: pairs must have rmsd under 3.5 Å, with more than 70% of aligned resides included in the rmsd calculation; and pairs must have under 40% sequence identity. Based on κ and α angles, an accumulated (κ, α) plot (Figure (Figure1b)1b) consisting of 225,523 protein fragments, each five residues long, was obtained from these 1,348 proteins. This plot is split into 648 cells (36 × 18) when the angles of κ and α are divided by 10°. In the study, the structural distance of a pair of 5-mer protein segments i and j is determined from the rmsd value of the five Cα atom positions, and is given as follows:
Where (Xk, Yk, Zk) and (xk, yk, zk) denote the coordinates of the kth Cα atom of segments i and j, respectively. The structural distance is also used to define the intrasegment and intersegment distances.
3D-BLAST used BLAST as the search method and was designed to maintain the advantages of BLAST. However, 3D-BLAST is slow if the structural alphabet is un-normalized, because the BLAST algorithm searches a statistically significant alignment by two main steps [5]. It first scans the database for words that score more than a threshold value if aligned with words in the query sequence; it then extends each such 'hit' word in both directions to check the alignment score. To reduce the ill effects of using an un-normalized structural alphabet, we set a maximum number (γ) of segments in a cluster in order to have similar compositions for the 23 structural letters and 20 amino acids. The value of γ was set to 16,000 (about 7.0% of total structural segments in the pair database).
To identify a set of 3D fragment segments (a structural alphabet), we developed a novel nearest-neighbor clustering (NNC) method to cluster 225,523 fragments in the accumulated (κ, α) plot (Figure (Figure1b)1b) into 23 groups. For each group, a representative segment, which represents the pattern profiles of the backbone fragments, was identified and assigned to a structural letter. These representative segments of clusters comprise a set of local structural segments. The NNC algorithm used the following steps and goals. The first step is to identify a representative structural segment, namely a center segment of all segments in this cell according to structural distances, for each cell in this (κ, α) plot. The second step is to cluster 648 representative segments into 23 groups by grouping similar representative segments into individual clusters and restricting the maximum number of segments in a cluster. In the third step, a representative segment is identified in each cluster based on the cell weight, which is defined as follows:
Where Si is the number of segments in cell i and M is the number of cells in this cluster. The fourth step involves assigning the representative segment of a cluster to a structural letter. In the fifth and final step, a composition of 23 structural letters is obtained that is similar to the 20 common amino acids. We developed an NNC algorithm instead of using a standard clustering algorithm, such as a hierarchical clustering method, which is unable to achieve the second, third and fifth steps/goals.
The NNC method clustered 648 representative segments into 23 groups using the following steps. First, a structural distance matrix (D), stored with the rmsd values by calculating the all-against-all representative segments, is first created with a size given by N × N, where N denotes the number of cells. An entry (Dij) represents the structural distance of representative segments i and j. Second, an unlabeled cell is selected with the maximum number of segments as the seed, and labeled as Ci. Third, an unlabeled cell is added, which represents the nearest neighbor of the seed, into this cluster and labeled as Ci if rmsd < ε (minimum structural distance), and if the sum of segments in this group is less than a bound γ (maximum number of a cluster). This step is repeated until an added cell violates the restriction thresholds, ε or γ. Fourth, steps 2 and 3 are repeated until the number of clusters equals 22 or all of the cells are labeled. Fifth, all remaining unlabeled cells are assigned to a cluster C23. Here, ε = 0.95 Å and γ = 16,000.
A set of representative segments with 23 states and its respective structural letters are identified (Figure (Figure2a2a and Additional data file 2) after performing the NNC method. Here, this 23-state structural alphabet was adopted for both protein structure reconstructions and protein structure database searches. The intrasegment structural distances (blue) are much greater than the intersegment structural distances (Figure (Figure1c),1c), and the average rmsd values of these 3D representative segments located in the same (or similar) cluster are frequently below 0.8 Å. The composition of the 23-state structural alphabet resembles that of the 20 amino acids obtained from the pair database. The distribution of the 23-state structural segments is consistent with that of the eight-state secondary structures defined by the DSSP program (Additional data file 3).
A substitution matrix and an SADB database are the essential components for adopting BLAST to search a structural database quickly. A new BLOSUM-like substitution matrix, called SASM (Figure (Figure4),4), was developed by using a method similar to that used to construct BLOSUM62 [32] based on the pair database. The SASM (23 × 23) provides insight into substitution preferences for 3D segments between homologous structures with low sequence identity. An entry (Sij), which is the substitution score for aligning a structural alphabet i, j pair (1 ≤ i, j ≤ 23) of the SASM matrix, is defined as Sij = clog2(qij/eij), where c is a scale factor for the matrix, and qij and eij are the observed probability and the expected probability, respectively, of the occurrence of each i, j pair. qij is given as , where fij is the total number of aligning alphabets i to j. The factor eij equals pipj if i = j; otherwise, it equals 2pipj (if i ≠ j), where pi is the background probability of occurrence of alphabet i and equals . Here, the optimal c value is found by testing various values ranging from 0.1 to 5.0; c is set to 1.89 for the best performance and efficiency. The final score Sij is rounded to the nearest integer value.
The following additional data are available with the online version of this paper. Additional data file 1 is a table listing 674 protein pairs. Additional data file 2 is a figure showing the representative 3D fragments of the 23-state structural alphabet. Additional data file 3 is a figure showing the distributions of a 23-state structural alphabet on each kind of eight DSSP secondary structure codes. Additional data file 4 is a table showing the rmsd between X-ray structures and reconstructed structures using 23 representative segments on 38 proteins. Additional data file 5 is a figure showing an overview of 3D-BLAST for structure database search. Additional data file 6 is a table listing the average precision of each program on 108 queries in SCOP-108. Additional data file 7 is a table showing the recognition performance of nine methods on the Lindahl's benchmark dataset at the family, superfamily, and fold levels. Additional data file 8 is a table listing 976 proteins of the Lindahl's benchmark.
Table listing 674 protein pairs.
Figure showing the representative 3D fragments of the 23-state structural alphabet.
Figure showing the distributions of a 23-state structural alphabet on each kind of eight DSSP secondary structure codes.
Table showing the rmsd between X-ray structures and reconstructed structures using 23 representative segments on 38 proteins.
Figure showing an overview of 3D-BLAST for structure database search.
Table listing the average precision of each program on 108 queries in SCOP-108.
Table showing the recognition performance of nine methods on the Lindahl's benchmark dataset at the family, superfamily, and fold levels.
Table listing 976 proteins of the Lindahl's benchmark.
J-MY was supported by National Science Council and the University System at Taiwan-Veteran General Hospital Grant. Authors are grateful to both the hardware and software supports of the Structural Bioinformatics Core Facility at National Chiao Tung University.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
In which wetake a long look at the development of hydrogen vehicles with someone who's been studying the field for three decades. You're bound to learn something.
Sebastian Blanco
40In deep with a hydrogen vehicle expert: 'There is something different this time'
- Sebastian Blanco
- 1 day ago
- 40
101Obama Administration wants 200-mile EV that charges in under 10 minutes
- Sebastian Blanco
- 2 days ago
- 101
Despite missing the famous 1-million-EVs-by-2015 goal, the Obama Administration is still working towards a US with more electric vehicles.
3First all-electric Porsche will use Le Mans 919 Hybrid tech
7Tesla Model X Falcon Wing doors now open in Lego form
- Sebastian Blanco
- 4 days ago
- 7
A fan video reveals the inner workings of the Model X's falcon wing doors using those iconic plastic bricks.
38Massive increase in CAFE fines could cost automakers millions
- Sebastian Blanco
- 6 days ago
- 38
A jump from $5.50 to $14 for each missed 1/10th of an MPG really adds up.
272017 Honda Accord Hybrid First Drive
- Sebastian Blanco
- 6 days ago
- 27
Does Honda's new gas-electric vehicle really offer almost 50 mpg? During our first few miles with the car, it sure doesn't seem so.
18Europe keeping an eye on Tesla's Autopilot problems, too
- Sebastian Blanco
- Jul 17, 2016
- 18
The Germans say they wouldn't have approved Autopilot since it's labeled a 'beta' product.
83US Senate asks Tesla CEO to come chat about Autopilot
- Sebastian Blanco
- Jul 15, 2016
- 83
US Senator John Thune wants some answers from Tesla CEO Elon Musk about that fancy Autopilot thing.
18California says nein to VW's 3.0-liter diesel fix
- Sebastian Blanco
- Jul 14, 2016
- 18
In the ongoing attempt to fix the mess it created, VW has submitted a fix for the 3.0-liter diesels to regulators. They don't like what they see.
41Tesla introduces Model X 60D for $74,000 [UPDATE]
- Sebastian Blanco
- Jul 13, 2016
- 41
Just a few months after cancelling the 70D, Tesla introduces the 60D for $74,000.
15A 5-seat Toyota Prius Prime is on the way
- Sebastian Blanco
- Jul 12, 2016
- 15
The chief engineer of the Toyota Prius tells us that a five-seat version of the Prime PHEV is something he's working on. But he won't say when it might arrive.
892017 Toyota Prius Prime Prototype First Drive
- Sebastian Blanco
- Jul 11, 2016
- 89
With the upcoming Prius Prime, Toyota is increasing the car's all-electric range compared to the earlier Prius Plug-In. Will it be enough?
145 of the 10 best-selling used cars from 2013-2015 are electrified
- Sebastian Blanco
- Jul 7, 2016
- 14
Based on days spent on dealer lots, electrified vehicles are a hit on the secondary market. The three best sellers are the Model S, Leaf and Plug-in Prius.
19NHTSA launches probe into second Tesla Autopilot accident [UPDATE]
- Sebastian Blanco
- Jul 6, 2016
- 19
The NHTSA is collecting information from police, the driver, and Tesla to learn more about the Autopilot situation in a recent crash in Pennsylvania.
48Elon Musk playing defense after fatal Tesla Autopilot crash [UPDATE]
- Sebastian Blanco
- Jul 6, 2016
- 48
Tesla CEO Elon Musk is defending the Autopilot software as details of a fatal May 7 crash come out, especially in light of Tesla's May 18 stock sale.
1Well, that was a strange way to end Formula E's second season
- Sebastian Blanco
- Jul 5, 2016
- 1
An early crash between two leaders means the second season of Formula E was settled with an unusual tie-breaker. The lesson? Every lap counts.
40Despite reduced deliveries, 'extreme' production ramp up at Tesla
- Sebastian Blanco
- Jul 4, 2016
- 40
Tesla says it is 'consistently' building 2,000 EVs a week, but deliveries for its electric vehicles were down. Again.
61Can't stop: Chevy Volt sales still outpace Nissan Leaf
- Sebastian Blanco
- Jul 1, 2016
- 61
US sales totals for the Chevy Volt and Nissan Leaf this past month look a whole heck of a lot like the numbers from all the other months this year.
1Faraday Future will sponsor Dragon Racing Formula E team
- Sebastian Blanco
- Jul 1, 2016
- 1
Faraday Future announces it will partner up with Dragon Racing for the next three seasons of Formula E racing.
30You can't scam VW by buying a dirty diesel now and still get full payout [UPDATE]
- Sebastian Blanco
- Jun 30, 2016
- 30
VW's lawyers have naturally put in a clause that will prevent anyone from buying a known dirty TDI from getting the full buyout amount from the OEM.
12Will Norway and South Korea lead us to the hydrogen future?
- Sebastian Blanco
- Jun 30, 2016
- 12
Norwegian ambassador Jan Grevstad says that his country and South Korea could complement each other when it comes to cleaning up transportation.
- Volvo shoots for self-drivers by 2021
- Jeep spends $1 billion on factories
- Find Parts & Accessories for your vehicle!
- Obama rolls out new EV plan
- Infiniti dealers ranked best, Tesla worst
- Compare Volvo XC90 and Lincoln MKX

subset: commoncrawl


text:
Matt Damon and Jimmy Kimmel Go to Couples Therapy Again. Does It Work This Time?
- 03:22 PM, Jul 26
Donald Sutherland Receives The 2,430th Star on The Hollywood Walk of Fame
Photo credit: FayesVision / WENN
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text:
Bovine Kerouacs
Kids are like sponges, soaking up information left and right--especially the bookworms. I'm sure most rued the day they picked up a copy of Animal Farm, expecting a tale of Old MacDonald or something and instead getting an earful about the terrors of Animalism/Stalinism and a distaste for pork. Well, it took more than 50 years, but Doreen Cronin made it safe for kids to go back to the farm with her Caldecott Honor Book, Click, Clack, Moo: Cows That Type, and the Dallas Children's Theater is bringing it to the stage with a musical adaptation by James E. Grote and George Howe. In this family-friendly tale, Farmer Brown's cows and chickens discover an old typewriter and learn about revolt, rebellion and peaceful negotiation (thanks to a duck), all in an effort to keep warm. Take the kids to the opening of Click, Clack, Moo: Cows That Type at 7:30 p.m. Friday, and expect some negotiations on the way home. There will be additional performances Fridays through Sundays until February 22 at the Rosewood Center for Family Arts, 5938 Skillman St. Tickets are $14 to $24. Call 214-740-0051 or visit dct.org.
Fridays, 7:30 p.m.; Saturdays, Sundays, 1:30 p.m.; Sundays, 4:30 p.m. Starts: Jan. 23. Continues through Feb. 22, 2009
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text:
HELMSLEY CARD HOLDER No. 02 - Onyx
HELMSLEY CARD HOLDER No. 02 - Onyx
The second incarnation of the original leather card holder in black, the incredibly chic Helmsley is perfect for business cards at the office or credit cards on a midsummer's night, its simplicity enhanced by the most beautiful leather we could find.
Italian Vegetable Tanned Leather. Solid brass hardware from Paris.
Dimensions: 3.75" x 2.5"
Colour: Onyx
Made with love in Britain.
Add To Cart


text:
Skip to comments.Japanese broadcaster: North Korea launches rocket
Posted on 04/04/2009 7:50:19 PM PDT by NormsRevenge
TOKYO Japanese broadcaster NHK says North Korea has launched a rocket, citing the Japanese government.
The report came Sunday morning amid heightened tensions in the region.
North Korea had informed international authorities that it planned to launch a rocket sometime between Saturday and Wednesday in order to put a satellite into orbit.
(Excerpt) Read more at news.yahoo.com ...
Rocket raunched?
If the Japanese don't shoot this thing down it is because they know this capability will force Obama to pay more attention to this issue.
Locket raunched!
How much money are going to give NK now?
bbc reporting so....
I disagree. I don't think Obama gives a rats butt about it, and will only provide considerable lip service to the event.
Okay, where is Obama's "Stern" response?
How soon will he cry to the UN?
Did it explode yet?
Obama isn't gonna do a damn thing and everyone knows it
Yep, and what do they have to lose?
Hussein's diplomacy fails again.
JJ Japanese govt confirmed it they think
I was beginning to wonder if they were ever going to get their Dong off.
Hussein and Hitlery will go crying to the U.N., everyone will say "my, my, Obama is not happy..." and that'll be about it.
Maybe they'll approve another $10 billion in food aid for Kim Jong Mentally Ill to give to his army while his people starve.
Of course he won't. The worse he would do is cry to the UN and drop it.
What is your definition of 'is'?
Perhaps it will hit Hawaii and blow up the Birth certificate.
South Korea's just confirmed it too.
article update and new title..
NKorea launches rocket, defying world pressure
SEOUL, South Korea South Korea's presidential office has confirmed North Korea's launch of a long-range rocket launch from its east coast base.
The Blue House says liftoff took place at 11:30 a.m. (0230GMT) Sunday morning from the coastal Musudan-ri launch pad in northeastern North Korea.
Japanese broadcaster NHK cites the Japanese government as saying the rocket flew over Japan. It said there were no reports of any debris falling on Japan.
obama's "stern" response-send howard "stern" and artie lang to NK to reason with them.
And that is exactly what NK is expecting to happen.
The missile is capable of hitting Alaska. I doubt Obama will give a damn about Alaska
Obama: I'm gonna tell on you North Korea, UN would be really really mad and will write a letter against the launch.
UN: North Korea pretty please don't launch the missile.
North Korea: Were trembling in our shoes.. NOT!!!
If Bush was still the president North Korea wont even dare launch a firework pointing towards us.
WOWWW luckly nobody got hurt so far so does that mean Obama going give Chia Pet bailout
What does Drudge's "No They Didn't" headline mean? Anybody know?
How will the teleprompter react?
He'll call up all the NATO leaders and say, ahhh, what do we do now? Do you think if I took the i-Pod back from Queenie and gave it to Kim Jung Il along with a new Members Only jacket, he'd like me? Doesn't he know I'm the Great Communicator?
Nah, I bet he'll return the IPod he picked up in Chicago for Kim Jung Il now.
That'll teach the commies. No copies of B HO's speeches and Inuaguration now.
My thoughts too. NO IPod or DVD's for Kim Jung Il.
"What does Drudges No They Didnt headline mean? Anybody know?"
How about....
They did what? Oh no! Mr. Bill!
Probably, "No! They didn't!"
He just failed his 3am phone call.
He probably called his friends in the media and said "don't talk about it"... and that seems to have worked.
I'll bet N Korea bootlegs a lot of DVDs, just like China. So Kim Jung probably already has all the DVD's the Obamas would be giving out, except of course, seasons One and Two of "Flava of Love".
QUestion JJ do you think Chia Pet want bailout or he just being roaney LOL!
True, but do they have a select arrangement of Obama's best telepromter reads?
It's time he got tough, so no IPod or select arrangement of teleprompter readings now.
Maybe Obama will throw himself down on the Oval Office carpet, when he gets back and kick and scream and hold his breath?
My guess is he will make a speech, right off of the telepromter and drop the whole thing. All smoke and mirrors.
We don't need this thread, a FReeper has already posted a thread way before Japan or Fox or any news agency reported this. FR once again beats the frickin news to a world shaking event.
I think he is both LOLOLOL!
I wonder which diplomat be send to NK get Chia Pet back to six nation talks
The No They Didn't was in regard to NATO's refusal to get more involved in Afghanistan, I believe.
I agree.
I was wrong, that was earlier.
Maybe it means that Japan did not shoot it down, like they warned.
It's like—get out! They didn't dare!
Or "No! They didn't!"
it's that expression 'oh no she d'int!' ... strange attempt at levity, I suppose.
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text:
Research Article
Generation of Power by Microbial Fuel Cell with Ferricyanide in Biodegradation of Benzene
Version of Record online: 13 DEC 2012
DOI: 10.1002/clen.201200198
Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CLEAN – Soil, Air, Water
Volume 41, Issue 4, pages 390–395, April 2013
Additional Information
How to Cite
Wu, C.-H., Lai, C.-Y., Lin, C.-W. and Kao, M.-H. (2013), Generation of Power by Microbial Fuel Cell with Ferricyanide in Biodegradation of Benzene. Clean Soil Air Water, 41: 390–395. doi: 10.1002/clen.201200198
Publication History
- Issue online: 4 APR 2013
- Version of Record online: 13 DEC 2012
- Manuscript Accepted: 18 JUL 2012
- Manuscript Revised: 1 JUL 2012
- Manuscript Received: 21 APR 2012
- Abstract
- Article
- References
- Cited By
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Agent: Jimmy Vesey still plans to become free agent after trade to Sabres
|06.20.16 at 5:10 pm ET
In a move that could prove to throw a monkey wrench at the Bruins' offseason plans, the Sabres acquired the rights to left wing prospect and Hobey Baker winner Jimmy Vesey on Monday.
Buffalo sent a third-round pick to Nashville for the North Reading native. The team has until Aug. 15 to sign him to an entry-level contract before he becomes an unrestricted free agent.
While the Sabres are obviously the favorite to end up with the player when all is said and done, agent Peter Fish told WEEI.com Monday evening that "nothing has changed" regarding Vesey's intention to go to unrestricted free agency. ESPN's Joe McDonald was the first to report Vesey's camp's intention to stay the course.
After Vesey chose against signing with the Predators, of whom he was a third-round draft pick in the 2012 draft, the Boston Herald reported that he was expected to sign with his hometown Bruins. Now, however, Vesey has the opportunity to play with 2015 second overall pick and close friend Jack Eichel should he sign with Buffalo.
Loui Eriksson still waiting for latest Bruins contract offer
|06.20.16 at 3:22 pm ET
The Bruins and Loui Eriksson haven't made much progress in recent days.
Eriksson's camp had expected a new offer from the Bruins at some point last week, but as of Monday morning agent J.P. Barry still had yet to receive one. The expectation is that Barry and Bruins general manager Don Sweeney will discuss Eriksson's future this week in Buffalo (where the draft will be held) if not sooner.
The sides have been negotiating the versatile winger's next deal since early in the 2015-16 season. When an agreement hadn't been reached by the season's trade deadline, the B's held onto the player in hopes that the team would make the playoffs and still be able to re-sign him.
Eriksson is coming off a 30-goal, 33-assist season for the Bruins. He will turn 31 on July 17.
Bruins to host Devils in home opener on Oct. 20
|06.20.16 at 12:10 pm ET
With the NHL's 2016-17 regular season set to be released Tuesday, teams began announcing their home openers on Monday.
The Bruins will host the Devils in their home opener on Oct. 20 at TD Garden. The season won't begin until Oct. 12 due to the Word Cup of Hockey, which will be played from mid-September through Oct. 1 in Toronto.
The full schedule will be revealed at 1 p.m. on Tuesday.
Roberto Luongo wishes Brad Marchand a happy Father's Day
|06.19.16 at 3:18 pm ET
Remember when Brad Marchand scored five goals in the 2011 Stanley Cup Final and set up the series-clinching goal? Twitter legend Roberto Luongo does.
Happy Father's Day daddy……….. pic.twitter.com/GgG1Kzx7FO
— Strombone (@strombone1) June 19, 2016
Avalanche don't plan on trading Tyson Barrie
|06.16.16 at 1:22 pm ET
Colorado general manager Joe Sakic told Denver Post reporter Mike Chambers that he will not trade defensemen Tyson Barrie this summer.
Avalanche NOT trading D Tyson Barrie, GM Joe Sakic tells me. LT deal preferred for RFA, or arbitration. "Either way, he'll be here" – Sakic
— Mike Chambers (@MikeChambers) June 16, 2016
A restricted free agent, Barrie was among a number of young defensemen the Bruins could have targeted in their efforts to upgrade their blueline this offseason.
With Barrie off the market, remaining trade possibilities might include Kevin Shattenkirk, Jacob Trouba, Hampus Lindholm and Sami Vatanen.
Bruins sign Peter Cehlarik to entry-level deal
|06.16.16 at 1:22 pm ET
The Bruins announced Thursday that they have signed forward Peter Cehlarik to a three-year entry-level deal.
Cehlarik, a left-shot wing from Slovakia, was drafted by the B's in the third round of the 2012 draft. He has played in Sweden since 2012.
Last season, Cehlarik scored 11 goals and added nine assists for 20 points in 46 games for Lulea HF of the Swedish Hockey League.
Bruins still not ruling out buyouts
|06.15.16 at 3:06 pm ET
The idea of using buyouts is not a wise one, but the Bruins still aren't ruling it out as of early Wednesday afternoon. The first buyout window opens up Wednesday at 5 p.m.
Asked about the possibility of exercising buyouts, a team source said Wednesday that the team is still exploring all avenues. That's consistent with what general manager Don Sweeney said at his season-end press conference back in April.
The two most obvious candidates for buyouts on the Bruins are Jimmy Hayes and Dennis Seidenberg. As illustrated here, buying Hayes out would be unwise, while buying Seidenberg out would be borderline foolish. As of Wednesday, Seidenberg had not heard anything about potentially being bought out.
Following are the cap charges if the team were to buy out either player:
Hayes (two years remaining, $2.3 million cap hit):
2016-17: $716,666
2017-18: $616,666
2018-19: $816,666
2019-20: $816,666
Seidenberg (two years remaining, $4 million cap hit)
2016-17: $1.166 million
2017-18: $2.166 million
2018-19: $1.166 million
2019-20: $1.166 million


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A-Z of Bush
George W. Bush Poster, 64cm x 90cm View all Posters…
64cm x 90cm (25" x 35") Poster
Humour Poster: George W Bush and his A-Z will keep you chortling…well kind of! No not you Tony! An example...J is for Justice, Bush has issued the death penalty to 155 people, more than any other elected official in recorded American history!
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Nike Men's Air Monarch IV 4E Training Shoe
NOW: $54.99 (15% off!)
Was: $64.99*
Product Information:
The Men's Air Monarch 4 has classic styling and an exceptional fit for comfort and versatility. Made for all types of training and casual wear, the Air Monarch IV training shoe utilizes an Air-Sole unit and a lightweight Phylon midsole. A pull-tab is included on the back of the heel for easy on and off, while the rubber outsole maintains grip on any surface.
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- Style: 416355-102
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Country of Origin: ImportedWeb ID: 10955200


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Tameka Foster (born January 1, 1971) is an American wardrobe stylist and the ex-wife of singer Usher. Tameka has worked as a personal stylist for Lauryn Hill, Jay-Z, Usher, Toni Braxton, Patti Labelle, Nas and more....
Connect any celebrity with Tameka Foster to see how closely they are linked... romantically!
OMG usher you are so hot, I loved everything about you on the stage last night here in perth, you got it all the voice and can u dance......oh yeh. come back to perth usher we loved you and want more
People need to stop taliking you have no idea of the situation he fell in love if it had been anyone else you still would`ve been mad so just get over it they had to beautiful children and she was pretty whether you like it or not your just jealous because she used to have him and you didnt have you heard the song papers he broke up with her because of selfish fans he still loves her and no matter what what ever he decides with anyone else everyone should just support him
Yeah usher thats what im talking about.Great new album and thanx 4 divorcing that woman
USHER- I was your biggest fan until you married the unattractive cougar - you had Chili - but I see that u came to your senses and got divorced - she wasn`t the one for you!
comeon usher u ar too handsome hw cn u date tameka gosh
hi Usher guess wat?u r ma role model since i p ractic ur moves at ma dancing classes n ma student luv it wish u came to ma country Kenya n watch us break it down move to dat i lyk ya.Take care.Tenny.0717258962
i used to think he was dating Alicia, guy u p*ssed me off, i dnt knw why marriage seldom last in the US.
You know, I wonder how he addresses her alone.. Mama or antie? Rumor has it she still goes by her maiden name. This whole thing doesn`t sound kosher to me. Ackward I`d say
Be yourself.
I don`t see why you people get a say in this,...Like i mean it`s not your right to say who he marries and who he shouldn`t. It`s his life not ours. Why would you enjoy his song less? because he married? Does that really impact his ability to sing? Best of Luck Usher.


text:
On now! Choose between free extended warranty or an instant rebate up to $2,500 in savings available.Learn More
All the latest Yamaha news and views from around the country.READ IT NOW
With a 5-inch longer shaft, more boaters can now enjoy the benefits of the powerful V MAX SHO family.Learn More
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THE exceptional form of the New Zealand Warriors has ensured Wayne Bennett remains an option for the Brisbane Broncos.
Although that very form could derail Brisbane's top four aspirations this weekend when the Broncos face the NRL's best attacking team.
The Courier-Mail can reveal the Warriors were confident of signing Bennett two months ago but players demanded interim coach Andrew McFadden be signed long-term.
The Warriors have won seven of their past nine games and could end the Broncos top-four aspirations this Saturday at Suncorp Stadium.
However, if not for Warriors players firing under new coach McFadden, the Auckland club could have already secured Bennett.
The Warriors are one of two clubs with enough money to sign Bennett for what he's actually worth with the other club being Brisbane.
It is understood there are officials at the Broncos who are open to the idea of Bennett returning to the club he led to six premierships.
Brisbane board member Tony Joseph reportedly met with Bennett in Newcastle earlier this year, signalling their interest in the Knights coach.
The Warriors are co-owned by multi-millionaire Owen Glenn who dreams of the club becoming internationally renowned.
On Thursday night the Warriors confirmed they are out of the Bennett race but refused to comment when asked about a pursuit of the super coach earlier this year.
As early as April, the Warriors knew Bennett would not be coaching Newcastle next season and began trying to lure him across the ditch.
After letting Matt Elliott go in April, the Warriors chased Bennett throughout the next month.
However in early May they received player feedback that McFadden was worth investing in and they made the bold call to re-sign him until the end of 2017.
Warriors chairman Bill Wavish refused to comment on their chase of Bennett but said "We back Cappy long term".
Wavish also ruled out luring Bennett as a coaching director.
"We don't believe a second opinion at the club would help Cappy," Wavish said.
The Broncos are still an option for Bennett but senior players believe Anthony Griffin deserves to see out his contract, which finishes at the end of 2015, before a new coach is even considered.
The Broncos would need to signal their intentions to sign a new coach within the next two weeks if they actually want Bennett to return to Red Hill next season.
His mission to ensure his tenure continues this weekend against a Warriors side that may play Sam Tomkins after all.
Tomkins travelled with the team on Thursday despite suffering a hip flexor injury at training earlier this week.
Originally published as Warriors snub pushes Benny to Broncos


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We are currently recruiting for several amazing employment opportunities. View job descriptions here.
Is there a Shuttle or Van Service to Esalen?
Shuttle van service is available from San Francisco International Airport (SFO), San Jose International Airport (SJC) and Monterey Peninsula Airport (MRY) on Fridays and Sundays. For shuttle schedules and pricing, please click here.


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Can You Eat to Sleep?by Leah Brickley in Healthy Recipes, September 7, 2015
We're not great sleepers in this country. According to Healthy People 2020, a science-based government organization, about 25 percent of us suffer from insufficient sleep (that's less than six hours per night) about half of every month. That's a lot of lost z's! Some experts believe that tossing and turning at night can lead to an increased risk of obesity, high blood pressure and even heart disease. That means that a good night's sleep may be just as important as a well-balanced diet and regular exercise! Read more


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Attack on Oakland cop a chilling reminder of danger
A dispatch recording reveals a moment of raw terror as an Oakland police sergeant attacked by gunfire while alone in her police vehicle on Saturday night radios for help.
Two men killed in Oakland shootings
Two men were fatally shot within 10 minutes in different parts of Oakland early Sunday, one at a street party and the other as he was driving a car. Police do not believe the shootings are related.
Raiders training camp roster breakdown
Rookie safety Karl Joseph is expected to be healthy for training camp. (AP Photo/Eric Risberg)Part three in a 10-part series looking at each of the Raiders position groups before training camp begins on Friday.
(Alex Wong/Getty Images)
Oakland mom to Trump: Stop using son's name
The mother of U.S. Ambassador J. Christopher Stevens, who was killed during the 2012 attack in Benghazi, wants Donald Trump's campaign and the Republican Party to stop using her son's name.
(Courtesy of Dan Horowitz)
Contra Costa: DA says Irish Mafia founder put a hit out on attorney
But a defense attorney for the founder of the Family Affiliated Irish Mafia (FAIM) says the new charges are a 'hail mary' by the prosecution.
Editorial: Fight against terror must be collaborative
In the aftermath of the heinous terror attack in Nice, France's Prime Minister Manuel Valls made a jarring comment: "The times have changed, and France is going to have to live with terrorism.
(AJ Mast/AP)
Fitness tracker fit: Do you really need one?
Maybe you don't really need a fitness tracker. It might be tempting to get one, now that the weather's nice and you're spending more time outdoors
( Lachlan Cunningham )
Venus Williams loses to Johanna Konta in Bank of West Classic final
In a tense, three-set final, Venus Williams unable to win her 50th pro singles title at tournament where she began her career 22 years ago
(Nhat V. Meyer/Bay Area News Group)
Test Drive: Ford's Fusion offers mainstream consumers a taste of semi-autonomous functions
Ford Fusion Energi offers many of the same semi-autonomous functions consumers will find in higher end cars, but the usability and reliability is mixed.
(Chris Pizzello/Chris Pizzello/Invision/AP)
'Justice League,' 'Wonder Woman' wow at Comic-Con
After "Batman v Superman: Dawn of Justice" put the launch of the DC/Warner Bros.
Monterey County man in possession of a dismembered hawk arrested
Police in Salinas on Friday arrested a man out on bail in connection with several crimes including possession of a handgun, possession of a controlled substance and possession of a dismembered hawk.
( T. Charles Erickson )
Review: 'Satchmo' goes behind Louis Armstrong's happy facade
The solo show 'Satchmo' by Terry Teachout depicts jazz legend Louis Armstrong reflecting on his bittersweet life and music; it plays at American Conservatory Theater through Feb. 7
(Chris Pizzello/Chris Pizzello/Invision/AP)
'Justice League,' 'Wonder Woman' wow at Comic-Con
After "Batman v Superman: Dawn of Justice" put the launch of the DC/Warner Bros.


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How Prosecutors Can Work With Mental Health Professionals In Child Abuse and Sex Abuse Cases (Webinar)
July 28, 2016
2:00-3:30 p.m. ET, Satellite/Web Based
Abuse in Later Life: Bringing the Case (Webinar)
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Forensic Interviews at Trial: Presentation and Preparation
November 1, 2016 - November 2, 2016
Huntsville, Alabama
National Institute on the Prosecution of Elder Abuse
November 15, 2016 - November 18, 2016
Phoenix, Arizona
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Chuuwee - Basquiat & Rocko
Posted 3 years ago
Tags: Chuuwee , Indie , Street Banger
You know, Chuuwee is a lot like a Transformer. Not because he's a robot (although he produces quality music like a machine) but because there is more to him than meets the eye. Take Basquiat & Rocko for example. Though the record is part of ThriLL, his upcoming trap album, it's much more than your typical trap number. Backed by a hazy yet cutting beat from Esta, Chuu's aggressive, leaning flow is sure to satisfy trap-heads. Even those who aren't so fond of the trap genre, however, will be able to appreciate Chuuwee's slick wordplay. ThriLL has yet to be assigned a release date, but there is plenty more original material on the horizon; Chuuwee will be releasing a project entitled The Great Gatzby on July 2.
Written by Lucas G.
Posted 3 years ago
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Sample Text - Sample Link


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7 of 9 people found the following review helpful
Everybody knows that if you want a tough, manly comic book, you should turn to Frank Miller.,
This review is from: 300 (Hardcover)
Frank specializes in violent, testosterone-driven morality plays that make you want to run right out and hurt people. After years of hard-hitting crime stories, Frank turned his attention to what is possibly the ruggedest, manliest story in history: the battle of Thermopylae. In 480 B.C. King Leonidas of Sparta led 300 men to defend Greece from the vast Persian army. "But," as the book's back cover says, "these warriors are more than men... they are SPARTANS." You only think you've seen bad-asses before. The Spartans set whole new standards for bad-assery. The story's all about 300 mostly naked, sweaty Greek men, but you don't for a second imagine that they're gay. In fact, you don't think of them as sexual at all -they're purely devoted to killing, and they're very good at it. Even if you begin this book unsympathetic to their brutal culture, by the end you'll be cheering for them. Frank did a great job writing and drawing this story, and Lynn Varley did a great job adding the colors. The extra-wide pages allow for huge, sweeping shots and incredible battle scenes. The book has high production values all around - you'll want to leave it sitting out because it's just so damn pretty. Great artwork. Lots of memorable lines. And bone-crunching, overwhelming violence like you've never seen before. Five out of five!!! I would also recommend reading Tino Georgiou's masterpiece--The Fates--if you haven't read it yet.
Review Details
Top Reviewer Ranking: 8,954,979


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Welcome, skip to the content.
Bullant Brewery Bruthen is located on the Great Alpine Road in Bruthen only 15 minutes from Bairnsdale in East Gippsland.Neil and Lois Triggs are locals who have a passion for beer and food and so they created the Bullant Brewery. The concept for the Bullant Brewery Bruthen is simple: to pair the best onsite brewed beer with the best regionally sourced food. The key word here is "balance"A destination rather than just another boutique brewery.Bullant beers are designed to match their menu just like wine, but they are equally enjoyable as a solely refreshing beverage. The brewery complex is welcoming and generous, purpose built to offer the best facilities. Its skillion rooves and corrugated iron walls complement the local hardwood timbers used in the deck and bar areas. Also the working copper brewhouse can be viewed through large, wide windows.Bullant Brewery invites you to visit and experience the Bullant Brewery Bruthen - the home of 'Beer with bite'.
Closed Christmas Day and Good Friday, open ANZAC day 12 noon to 5pm. Phone to confirm times.
Live music on the deck from November to Easter. Throughout the year they have a Jam / open mic session from 2:30 on the first Sunday of the month.
Contact operator for further details.


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Alan Samuel Colmes (born September 24, 1950) is an American radio and television host, liberal political commentator for the Fox News Channel, and blogger. He is the host of The Alan Colmes Show, a nationally syndicated talk-radio show distributed by... Fox News Radio that also airs throughout the United States on Fox News Talk on Sirius and XM. From 1996 to 2009, Colmes served as the co-host of Hannity & Colmes, a nightly political debate show on Fox News Channel.
Connect any celebrity with Alan Colmes to see how closely they are linked... romantically!
You`re still a clown Colmes, er, comic. Your interview with Kelly Friday 3/26 was rude - typical crude leftist. You`re cut from the same Obama cloth - your ignorant, arrogant, vain, stupid, dishonest, untrustworthy and unAmerican
i like alan i want to be his girl friend and i just did finally see a photo of his wife jocelyn


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Once a deal is announced we will see a significant Euro and Market selloff. Reality will then be defined - no more hopes.Tomorrow will be interesting - if the GDP is good market and dollar should rally which hasn't occurred simultaneously recently.
For the 10th time in the past 3 weeks.


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I don't make New Year's Resolutions. I don't think I ever really did, but the last decade or two would have been enough to stifle that impulse. I've just been too aware that I don't have that much control over what happens in my life.
Discuss the article on the Forums.
Can you have ME or CFS without cognitive dysfuction?
Discussion in 'Cognition' started by jonnyboy, Dec 6, 2011.
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As increasingly large volumes of sophisticated options are traded in world financial markets, determining a "fair" price for these options has become an important and difficult computational problem. Many valuation codes use the binomial pricing model, in which the stock price is driven by a random walk. In this model, the value of an n-period option on a stock is the expected time-discounted value of the future cash flow on an n-period stock price path. Path-dependent options are particularly difficult to value since the future cash flow depends on the entire stock price path rather than on just the final stock price. Currently such options are approximately priced by Monte Carlo methods with error bounds that hold only with high probability and which are reduced by increasing the number of simulation runs. In this paper we show that pricing an arbitrary path-dependent option is #-P hard. We show that certain types of path-dependent options can be valued exactly in polynomial time. Asian options are path-dependent options that are particularly hard to price, and for these we design deterministic polynomial-time approximate algorithms. We show that the value of a perpetual American put option (which can be computed in constant time) is in many cases a good approximation to the value of an otherwise identical n-period American put option. In contrast to Monte Carlo methods, our algorithms have guaranteed error bounds that are polynomially small (and in some cases exponentially small) in the maturity n. For the error analysis we derive large-deviation results for random walks that may be of independent interest


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|4 reviews in total
Once again, we hear and see Rubinoff playing the violin to open up this
Betty Boop cartoon. He plays soft, quiet music as an introduction until
Betty gives ups her "oop, oop be doop" opening. Then we get to the
story, where we first see big-city buildings, all linked to a toy
factory, producing a mystery package that is transported by a small
train and then by a plane. Eventually, it is dropped down a chimney to
a magical house with wooden soldiers. Inside the box is Betty, in
toy-soldier form! The wooden soldiers come to life, play the trumpets
and all the toys come to life.
From that point, it's a lot of song and dance until a big ape comes to life and tries to harm Betty. That's when the soldiers come to the rescue! In all, nothing super and geared a lot more to little kids.
*** This review may contain spoilers ***
"Parade of the Wooden Soldiers" is a Betty Boop cartoon from over 80 years ago. It has sound, is black-and-white (as almost all of her cartoons) and runs for 8 minutes, a minute longer than cartoons usually did back then. Dave Fleischer and Seymour Kneitel are the directors and both, especially the former, belong to the most lauded cartoon makers of their era. There is always something weird and enigmatic to these Betty Boop cartoons and this one is no exception. Sadly it is not really as funny as I would have liked. My favorite scene is probably the short one with the bunnies disappearing and reappearing very differently than they went in. In 1933, the (still today) popular movie "King Kong" came out, so it's probably not a coincidence that the antagonist here is a big gorilla and Betty plays a sort-of Fay Wray character. Interesting reference, but not enough to let me recommend it.
*** This review may contain spoilers ***
This is a cartoon in the Betty Boop series produced by Fleischer
studio. There will be spoilers ahead:
This is, in many ways, a "typical" Fleischer short from the early 1930s, if there is such a thing. It's a touch surreal and there are risqué elements which would mostly go over the heads of children but would amuse adults.
The short begins with Rubinoff and His Orchestra in a live action shot as the musicians playing the score. The animation begins with a series of interconnected buildings "giving their all" so that a toy factory can produce a single package. The buildings deflate in turn, the toy factory doing so once the package is disgorged and sent on its way.
The toy is shown on various vehicles of transportation, finally dropped by an airplane (a nice gag with the chimney and with the flames in the fireplace as the package winds up in a toy shop). Betty is in the package, as a toy-sized doll, short skirt, garter and all. The toy soldiers spring to attention and music begins as the toys go to greet Betty.
There are risqué sight gags scattered throughout the toys marching to see Betty. A street cleaner toy marches behind toy elephants, two adult toy rabbits multiply out of sight in a tunnel and a dog follows a toy hydrant, for example. Betty serenades the toys with a song and rockets are fired off in her honor. One of the rockets disturbs a King Kong-like rag doll and it goes on a rampage. It pulls the head off a black-face toy eating watermelon (there are several ethnic caricatures in here) and goes searching for a suitable replacement head.
Betty, meanwhile, has been enthroned and made a queen by the other toys. The ape spots her and decides her head is what is needed. Betty is kidnapped by the ape and the toys spring into action, effectively declaring war on the ape and trying to rescue Betty. Ultimately, the ape is beaten, Betty is rescued (not without damage to her dress, which is shown in another risqué gag toward the end) and a closing parade is shown, with toys seen earlier, with clear damage and attempts at repair obvious. The ending is clever.
This short is in The Definitive Betty Boop Collection and is well worth seeing. Recommended.
Like so many early Betty Boop cartoons, there is a guest musician who
is shown at the beginning of the short. In this case, you see Rubinoff
and His Orchestra performing a bit of the music for the cartoon.
In this cartoon, Betty is dropped into a toy shop (literally) where she and the toys all sing and dance. Unfortunately, a nasty gorilla doll comes alive and begins destroying things. So, it's up to the toy soldiers to organize a counter-strike and save poor Bett.
As far as "Parade of the Wooden Soldiers" goes, I heartily agree with the other reviewer who thought it was designed for little kids. I cannot honestly see teens or adults sitting through this sort of drivel and I am pretty sure when it debuted, a lot of folks too bathroom and snack bar breaks during this film! It's just so gosh-darn saccharine and has absolutely no laughs at all. So, despite great animation, I really couldn't recommend this one.
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William J. Cox
Published: Sunday, December 31, 2006 at 12:01 a.m.
Last Modified: Thursday, June 25, 2009 at 11:54 a.m.
William J. Cox, 90, of Winter Haven, died Thursday, Dec. 28, 2006, at his residence, due to heart failure.
Originally from Pineville, Ky., he moved to Winter Haven from Rome, Ga. in 1960. He was a supervisor of rust engineering.
He served in the U.S. Army Air Corp and was a veteran of World War II.
He was preceded in death by his wife, Elaine Cox; his brother, Jack Cox; and his sister, Doris Cox.
Survivors include his son, William D. Cox of Bartow; four daughters, Ann Peckham of Jacksonville, Patricia Bradshaw of Plant City, Catherine Hart of Winter Haven and Jerri Martin of Bartow; a sister Rozanne Cox of Scottsboro, Ala.; five grandchildren; and five great-grandchildren.
Visitation will be held Tuesday from noon to 1:45 p.m. at Crisp-Coon Funeral Home Chapel. Services will follow at 2 p.m. Internment will be at Lakeside Memorial Park in Winter Haven.
In lieu of flowers, contributions may be made to local charities.
Crisp Coon Funeral Home Chapel
Winter Haven
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E'lon Cosmetics has announced the creation of a cosmetic line geared toward black teenagers. TWISY (The World is Yours) was introduced in certain Ohio area bigg's stores in the first week in December. The line debuted with a holiday gift bag, and the company reportedly will gage future product releases on holiday sales. E'lon Cosmetics is a cosmetics line for adult black women sold at Ohio Macy's stores. The company is looking to grow with the introduction of TWISY.
Cosmetic Line for Black Teenagers
December 18, 2006 | Contact Author | By: Katie Schaefer
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