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The gap between the number of known protein sequences and structures continues to widen, particularly as a result of sequencing projects for entire genomes. Recently there have been many attempts to generate structural assignments to all genes on sets of completed genomes using fold-recognition methods. We developed a method that detects false positives made by these genome-wide structural assignment experiments by identifying isolated occurrences. The method was tested using two sets of assignments, generated by SUPERFAMILY and PSI-BLAST, on 150 completed genomes. A phylogeny of these genomes was built and a parsimony algorithm was used to identify isolated occurrences by detecting occurrences that cause a gain at leaf level. Isolated occurrences tend to have high e-values, and in both sets of assignments, a sudden increase in isolated occurrences is observed for e-values >10−8 for SUPERFAMILY and >10−4 for PSI-BLAST. Conditions to predict false positives are based on these results. Independent tests confirm that the predicted false positives are indeed more likely to be incorrectly assigned. Evaluation of the predicted false positives also showed that the accuracy of profile-based fold-recognition methods might depend on secondary structure content and sequence length. We show that false positives generated by fold-recognition methods can be identified by considering structural occurrence patterns on completed genomes; occurrences that are isolated within the phylogeny tend to be less reliable. The method provides a new independent way to examine the quality of fold assignments and may be used to improve the output of any genome-wide fold assignment method.
Author Summary
When predicting the structure for a protein sequence, a major step is achieved when an evolutionarily related protein with a known structure is identified. This process is called fold recognition, and has been a major force behind improvement in structure prediction. Moreover, fold-recognition techniques have become increasingly important in recent years because of the huge numbers of protein sequences with unknown structures available through sequencing projects on completed genomes. However, all fold-recognition methods tend to produce either a large number of false negatives (at high confidence scores) or a large number of false positives (at low confidence scores). Here we show that the reliability of a fold-recognition technique can be explored by analysing its predictions across a set of completed genomes. We have developed a method that can indicate false positives in these genome-wide assignment sets. The basic idea behind the method is that a fold assignment on a genome is less reliable if the prediction is not observed on evolutionary related genomes. The ability of the method to discriminate false positives is confirmed by independent tests. The method can be used on the output of any genome-wide fold assignment method.
Citation: Abeln S, Teubner C, Deane CM (2007) Using Phylogeny to Improve Genome-Wide Distant Homology Recognition. PLoS Comput Biol 3(1): e3. https://doi.org/10.1371/journal.pcbi.0030003
Editor: Philip E. Bourne, University of California San Diego, United States of America
Received: July 5, 2006; Accepted: November 20, 2006; Published: January 19, 2007
Copyright: © 2007 Abeln et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding from the Engineering and Physical Sciences Research Council and the Wellcome Trust.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: HGT, horizontal gene transfer; HMM, hidden Markov model; PSSM, position-specific scoring matrix
The prediction of protein structures from sequences is becoming increasingly important, particularly as the gap between the number of experimentally determined sequences (>6,000,000) and structures (<35,000) widens. Knowledge of protein structure is essential to the understanding of biochemical processes. In practical terms, knowledge and prediction of protein structures can aid the discovery of new drugs [1].
When predicting the structure for a target sequence, a major step is achieved when an evolutionarily related protein with a known structure is identified. Since structure is more conserved than sequence, it is presumed that the target sequence has a similar fold to the related protein. This process is called fold recognition and has been a major force behind improvement in structure prediction in recent years [2].
At present there are several ways to recognise a fold for a given sequence. If there is close homology between the target sequence and a known structure in the Protein Data Bank (PDB) [3], a simple sequence search, such as BLAST [4], will be sufficient to identify the fold. To detect more distant homologies we can use position-specific scoring methods such as PSI-BLAST [5] and hidden Markov model (HMM)–based methods such as SAM-T98 [6]. These methods are the least expensive forms of fold recognition and are sequence based. There are more computationally expensive methods that take structural information into account; an example of such a technique is THREADER [7].
Recently, many studies have used fold recognition to look at the structural content of entire genomes with aid of PSI-BLAST [8], HMMs [9,10], or threading procedures [11]. These fold-recognition assignments can produce an occurrence pattern on a set of species for a given family, superfamily, or fold as defined by structural classifications such as SCOP [12] or CATH [13]. Sets of such occurrence patterns have proved to be useful for building a phylogeny of species [14,15], for grouping proteins within a similar pathway [16], and for estimating the ages of folds [17].
A major challenge for all fold-recognition techniques is to discriminate a true homologue from a false positive (specificity) using confidence scores such as e-values. e-Values (expectation values) indicate how likely it is that an alignment with the search sequence would occur by chance in a given database (i.e., they should reflect the chance of a false positive assignment). Previous studies have suggested that analysis of structural assignments on completed genomes may indicate false positives of fold-recognition techniques. Yang and coworkers [15] showed that the number of hits on completed genomes drastically increased above a certain e-value cutoff, which could be explained by a sudden influx of false positives. Furthermore, Winstanley and coworkers [17] showed that occurrence patterns of most superfamilies can explain the phylogeny of the genomes reasonably well, but observed a difference in fitness between patterns from two different fold-recognition techniques. In particular it appeared that, in one set, more assignments were made that occurred on isolated leaves of the species tree.
We propose that false positives in fold-recognition assignments might be identified by considering a phylogeny of species. False assignments in such a set might be expected to occur randomly across the genome tree, whereas true positive assignments to a superfamily should be evolutionary related. Hence, we expect that false occurrences have a stronger tendency to be scattered across the tree than true assignments. This study investigates whether isolated occurrences within a phylogenetic occurrence pattern are indeed more likely to be false positives.
Using phylogeny to improve homology searches is not a new idea. It has been known for a long time that by considering the phylogeny of related proteins one can improve sequence alignments. An example is progressive multiple sequence alignment, where an approximate phylogeny of the sequences is used to aid the alignment of multiple sequences [18].
Assignment of function can be facilitated by phylogenomics: a set of known homologs is used to create a phylogeny of proteins in which speciation and duplication events are marked. These can be used to subclassify the proteins in the phylogeny into specific functions. Several protocols as well as automated procedures based on phylogenomics have been able to improve functional annotation [19–21]. Recently it has also become clear that confidence in a modelled structure increases when homologues of the target sequence give a similar structure prediction [22,23]. Here, precalculated phylogenies of entire genomes are used rather than phylogenies of individual proteins.
In this study we will assess genome-wide assignments obtained by PSI-BLAST searches as well as assignments from the SUPERFAMILY database. PSI-BLAST is an iterative version of BLAST, which uses a position-specific scoring matrix (PSSM) to include information about homologous sequences. The PSSM is used to identify the amino acids that are most likely to occur at a given position in the sequence. During each run, sequence information from all hits, with an e-value below a threshold, is added to the PSSM. Final e-values between the target and each database sequence are based on sequence similarity to the PSSM, which is subsequently normalised for amino acid composition with respect to the entire database.
The SUPERFAMILY database [9] is built with SAM-T99 [6], a procedure to find distant homologues using HMMs. The SAM-T99 program was finetuned with expert knowledge to recognise superfamilies as defined by SCOP. For each search sequence a profile HMM is created from homologues, which are found by a simpler sequence similarity search. The HMM is a statistical model that describes the evolutionary behaviour for a set of homologous sequences. These models are then used to calculate a score for each sequence in the database. The e-values are normalised by the reversed score of the searching sequence.
To assess the assignments, occurrence patterns were obtained for every superfamily from our two fold-recognition sets (PSI-BLAST and SUPERFAMILY). We developed a method that identifies isolated occurrences within such occurrence patterns by considering if an occurrence causes a gain at leaf level in the phylogeny.
This study demonstrates that false positives in fold-recognition assignments can indeed be identified by considering a phylogeny of species: isolated occurrences are shown to have higher e-values (are less reliable) than other occurrences. We formulated criteria to predict false positives based upon these results. The set of predicted false positives were validated by comparisons to overlapping PSI-BLAST assignments and to assignments that changed between different versions of the SUPERFAMILY database. Both tests confirmed that the predicted false positives are far more likely to be falsely assigned than other occurrences.
Analysis of occurrence patterns from genome-wide fold recognition also provides a new way to examine the quality of fold assignments. We show that the frequency of occurrences drastically increases for high e-values (>10−8 for SUPERFAMILY and >10−4 for PSI-BLAST), and that this influx is likely to be caused by false positive assignments. In addition, the accuracy of the fold recognition is demonstrated to differ significantly for the different structural classes as defined by SCOP. In principle, this technique can screen assignments of any existing fold-recognition method for false positives. An extended version of the method is given, which can be applied to assignment sets with a high proportion of false positives. This version might be able to improve the search capacity of any genome-wide fold-recognition technique.
Phylogeny and Accuracy of Assignments
All assignments were generated by searching for SCOP domains on the genes of 150 completed genomes (18 archaea, 97 bacteria, and 35 eukaryotes; see Table S1).
The first set of assignments was taken from the SUPERFAMILY database [24] and contained 1,269 different superfamilies. The database provided about 750,000 structural assignments for our set of genomes (Table 1). One or more structural assignments was made to 48% of the gene sequences in this dataset, with a false positive rate estimated to be lower than 1% [24].
Fraction of Assignments That Can Be Analysed by the Method
The second set consisted of assignments obtained using PSI-BLAST [5]. It included all assignments with an e-value lower than 1.0 for testing purposes. The inclusion threshold to build the PSSM was kept low, at 10−5, to diminish the risk of including false positives within the search profile. About 450,000 structural assignments were made with PSI-BLAST (Table 1). As expected, the coverage of genes was a little lower, with one or more structural assignments made to about 36% of the genes. For more details on both sets of assignments, see Materials and Methods.
Parsimony and gains at leaf level.
We first calculated a phylogeny for the genomes. Occurrence patterns were used to build the tree; this has previously been shown to be more robust than tree-building using the number of assignments or copies [14,15,17]. Figure 1 shows the phylogeny used (see Materials and Methods for more details on the tree-building process).
Losses (left-pointing triangles) and gains (right-pointing triangles) are shown as calculated by the parsimony algorithm. The assignments with a gain at leaf level are isolated within the tree. For each occurrence the log10 e-value (log(ev)), the number of copies (copy), consensus with PSI-BLAST occurrence (cons), and cluster distance (clust) are shown. The src column shows which genomes contain a source sequence for the superfamily by indicating the sequence identity to a SCOP domain.
A parsimony algorithm was used to predict an evolutionary scenario of loss and gain events for a superfamily given this phylogeny. The algorithm minimises the number of loss and gain events in the tree for the occurrence pattern of the superfamily.
Our measure for isolatedness indicates whether an occurrence causes a gain at leaf level in the set of minimal gains and losses. The parsimony algorithm predicts a gain at leaf level (gain at the lowest level of the tree), when it would be more expensive to cluster the occurrence with other occurrences. Hence, an assignment with a gain at leaf level is an isolated occurrence within the pattern. Note that the parsimony algorithm makes the sets of loss and gain events quite robust against small changes in the occurrence pattern. A few deletions within an occurrence cluster will usually not cause a gain at leaf level; this diminishes the effect of deletions and false negatives.
Figure 1 shows the full assignment of gains and losses by our parsimony algorithm for superfamily a.126.1 (serum albumin-like).
Detectable false positives.
The technique developed here can only observe false positive occurrences. However, the same superfamily can be assigned to many different genes on a genome and so one occurrence might cover many assignments. As the number of assignments (or copies) for a superfamily on a genome is known to behave like a power law [8,14], the number of genomic occurrences is much smaller than the number of assignments. In this technique, the e-value for an occurrence is determined by the lowest e-value in the set of copies on the genome covering the occurrence. In effect, if a superfamily has many copies on a genome, one of the assignments usually has a very low e-value, which will overshadow those with higher e-values. This effect obviously reduces the number of assignments that can be screened by our technique (Table 1).
In practise we will restrict the set of predicted false positives to isolated occurrences caused by one copy with a high e-value (see below).
E-value distributions.
To understand the e-value distribution of isolated occurrences, we first consider the e-value distribution of all assignments and the e-value distribution of all occurrences.
Figure 2A shows the distribution of e-values for all fold assignments. A local minimum is seen at the higher e-value end of the distribution for both the SUPERFAMILY and PSI-BLAST distributions. This may mark the point where false positives assignments begin to play an important role. Figure 2B shows the e-value distributions for all occurrences. This distribution is shifted to the left, with respect to the assignment distribution.
(A) e-Value distribution of all assignments.
(B) e-Value distribution of occurrences. The e-value of an occurrence is defined as the lowest e-value of all assignments to a superfamily on a genome.
(C) e-Value distribution of occurrences that cause a gain at leaf level (i.e., predicted false positives). Arrows indicate local minima in the distribution, which might indicate the point where false positive assignments become more dominant.
The e-value for an occurrence is taken from the assignment on a genome with the lowest e-value; hence, assignments with higher e-values from the same genome are not represented in this set. The left shift is, as expected, not seen for occurrences with a single assignment on a genome (dotted lines in Figure 2B), since the effect described above cannot occur for occurrences caused by only one assignment. In fact, the one-copy distribution appears to be very similar to the overall assignment distribution. The fraction of false positive assignments should be very small; false positive occurrences would therefore generally be caused by a single false assignment on a genome. Hence, the fraction of false positives in the one-copy distributions is expected to be higher than the fraction of false positives for all assignments. Indeed, it is observed that the local minima have become more prominent in these distributions.
False positives.
We now compare two independent sets of observations: (1) a set of binary values indicating if an occurrence causes a gain at leaf level; and (2) a set of continuous e-values for every occurrence (for discussion on independence see Evolutionary Caveats). e-Values should indicate how likely it is that an occurrence is a false positive. We can therefore use the e-values to investigate our expectation that isolated occurrences are more likely to be generated by a false assignment.
Figure 2C shows the e-value distribution for occurrences with a gain at leaf level. Comparing it with the other distributions, it is clear that this distribution is shifted to the right. This shift appears to confirm the supposition that occurrences with a gain at leaf level are less reliable. The steep increase in the distribution for the SUPERFAMILY data starts at a significantly lower e-value than for PSI-BLAST, possibly indicating that false positives in the SUPERFAMILY set start occurring at lower e-values. However, the e-values for the two sets of assignments are not necessarily comparable, since, as we described earlier, they are calculated using different estimation techniques.
Ranked distributions.
The e-value distributions for the assignments on the genomes are clearly not normally distributed. To make a comparison between different e-value distributions simpler, we have given a rank to the e-value of each assignment. This ranked e-value distribution of all assignments gives a uniform distribution, which would display as a flat horizontal line (not shown). Similarly, any random subset of this reference distribution is expected to be uniform.
Figure 3A and 3B show that the left-hand side of the distribution of occurrences with a gain at leaf level is approximately uniform and hence can be interpreted as a random subset of the assignment distribution. However, for higher e-values, a sudden increase is observed; this indicates that relatively more assignments with a high e-value are found in occurrences with a gain at leaf level than in the reference distribution. We believe that this set of additional isolated occurrences at high e-values is caused by false positive assignments.
The ranked e-value is obtained by ranking the e-values in the set of all assignments (including multiple copies) and is normalised between 0 and 1.
(A,B) Ranked e-value distributions for occurrences with a gain at leaf level. The peak at the righthand side might indicate a sudden increase in false positives.
(C,D) Ranked e-value distribution for all occurrences. The dotted line indicates that occurrences with a gain at leaf level have been removed, and the striped line shows only consensus occurrences, which have been assigned by both SUPERFAMILY and PSI-BLAST. Occurrences caused by a single assignment (one copy) are shown in colour. Both the distributions for all occurrences and for occurrences caused by a single assignment show an increase at the righthand side, probably caused by false positives. This increase is not seen in distributions either where either predicted false positives are taken away or for consensus occurrences.
The ranked e-value distribution for the set of all occurrences (black line) in Figure 3C and 3D is, as expected, shifted to the left with respect to the reference distribution; the e-value of an occurrence is defined as the best e-value on the genome.
However, at higher e-values, the distribution stops decreasing, and even shows an increase for PSI-BLAST. Moreover, the distribution of consensus occurrences, which are predicted by both SUPERFAMILY and PSI-BLAST assignments, does not show a sudden increase at high e-values (striped line). As it has been reported that the consensus of two or more fold-recognition methods can significantly improve the specificity of individual methods [25], the observed increase is likely to be an artefact because of a sudden appearance of false positives for high e-values.
If the set of "gains at leaf level" is taken away from this ranked occurrence distribution (dotted line in Figure 3), the distribution continues to decrease as expected. This shows that the set of isolated occurrences corresponds in size and e-value distribution to a set of likely false positive occurrences.
As described above, occurrences caused by only one assignment should have a similar distribution to that of all assignments, and, hence, the ranked distribution should appear uniform. The coloured line in Figure 3C shows that this is roughly the case for both the SUPERFAMILY and PSI-BLAST occurrences. However, both distributions show a sudden increase at high e-values. Once again, the distribution of consensus occurrences does not show such an increase (coloured striped line). The set of single occurrences from which isolated occurrences have been removed (coloured dotted line) shows less of an increase than the occurrence distribution.
These results all support that the observed change in distribution for high e-values is created by a sudden increase in false positives, and that our set of isolated occurrences corresponds to these false positives. The results also demonstrate that occurrences with a gain at leaf level have in general worse e-values than other assignments.
Evolutionary Caveats
We used fold-recognition methods to find members of a superfamily. Hence, all assignments and occurrences within a pattern should be evolutionarily related. We propose that an isolated occurrence on a species tree might indicate that that occurrence is a false positive. Below we consider what other mechanisms might cause a bad fit within an occurrence pattern for a superfamily.
Horizontal gene transfer.
An isolated occurrence within the tree might be due to horizontal gene transfer (HGT) between species. This is known to be an important process for the enrichment of diversity on genomes [26]. However, we currently have no reason to believe that instances of HGT would give higher e-values for true superfamily assignments than their non-HGT counterparts. We will in practise only consider isolated occurrences as a false positive when the e-value of the occurrence lies above a certain threshold. These thresholds should match values at which we observe a sudden increase in frequency for the ranked e-values (10−10 for SUPERFAMILY and 10−4 for PSI-BLAST). Vice versa, the extent of HGT might be estimated by the part of the distribution for occurrences with a gain at leaf level, which is independent of e-values. In the ranked e-value distributions, this is the uniform part (Figure 3A and 3B).
Distance to source sequence.
Another possible source of error in the technique is a dependency of e-values on the evolutionary distance between the search sequence and an assignment. The search sequence is the initial sequence from which a search profile (HMM/PSSM) is created. This dependence is likely to be smaller for profile-based methods than for simple sequence search tools such as BLAST. In this study, the search sequences are the "source" domains taken from SCOP.
We have so far assumed independence of e-values and phylogenetic fitness of an occurrence. The above effect would not cause a direct dependence, but together with deletion of domains, false negatives, and random noise in e-values, it could result in higher e-values close to the edges of an occurrence cluster. Two different tests were performed to see if our independence assumption would hold.
First, we examined the relationship between e-values and the distance from an occurrence to its source sequence. A genome is said to contain a source sequence for a superfamily when a very close match to a sequence of a known 3-D structure is found in its genes (see Materials and Methods). The distance to a source was calculated as the height of the lowest common ancestor in the tree between the genome of the occurrence and any of the genomes containing a source sequence for the superfamily.
There exists a very weak correlation between the distance to a source sequence and the e-value of an occurrence, with r2 values of 0.05 for SUPERFAMILY and 0.14 for PSI-BLAST (see Figure S1). The correlation becomes even weaker (r2 = 0.04 and 0.10) when the set of source occurrences is removed. Despite the correlations being very weak, they are significant (p < 2e−16) for both PSI-BLAST and SUPERFAMILY.
To assess the influence of this very weak correlation on our method, we examined whether e-values deteriorated towards the edges of a cluster. The cluster distance of an occurrence is calculated as the distance to every leaf in the occurrence pattern divided by the distance to every leaf in the tree. It is then mediated so that the cluster distance of an occurrence is relative to its pattern. Scores significantly higher than 1.0 reflect bad clustering of an occurrence in the tree.
Figure 4A and 4B show that there is a negligible correlation between the cluster distance and the e-value of an occurrence, and in general we can assume independence of these entities (see Figure S2). This allows us to rule out the possibility that a general correlation between cluster distance and e-values causes the previously observed increase in isolated occurrence at very high e-values.
(A,B) Show there is no correlation between the e-value and cluster distance. The cluster distance indicates whether an occurrence lies within a cluster of occurrences (see Materials and Methods for more details).
Saturation Effects on Patterns
The more occurrences there are in a pattern, the lower the chance that an isolated occurrence can appear. Therefore, if a pattern saturates with occurrences, fewer false positives can be predicted through our gain at leaf level technique. There is a strong anticorrelation (r2 = 0.95) between the number of occurrences in a pattern and potential gains at leaf level. A potential gain at leaf level is a leaf without an occurrence, which would cause a gain at leaf level when added to the pattern.
Occurrence patterns can become saturated through an increase in either true or false positive assignments (see Discussion for consequences of additional true assignments). An increase of false positive occurrences could be created by the inclusion of very high e-values into the set of assignments. This might be desirable to enlarge the capacity of existing fold-recognition methods. However, if false occurrences start to dominate the pattern, our technique would begin to fail.
The problem can be overcome using a slight modification to the technique. The parsimony algorithm is first run on a base pattern that is created from assignments below a strict e-value threshold. Genomes without an occurrence in this base pattern are checked for potential gains at leaf level. Then the set of isolated occurrences becomes the union of all gains at leaf level in the base pattern and all potential gains at leaf level, which have an occurrence with an e-value above the threshold. This modification could cause the algorithm to overpredict the number of false positives if the e-value cutoff is set too low.
Figure 5 shows the results of running this procedure with different e-value cutoffs for the base pattern. Only when using extremely low e-value cutoffs (e.g., 10−20) are a considerable number of isolated occurrences detected for middle-range e-values. Hence, the above method can potentially be used without a huge overprediction of false positives. In fact, comparing the distributions (coloured lines) to the consensus data (striped line), a sensible threshold can be determined with an e-value cutoff of 10−8 for SUPERFAMILY and 10−2 for PSI-BLAST. This version of our method can be used on occurrence pattern sets with a high proportion of false positives.
The predicted false positives are calculated through a base pattern created from assignments with an e-value below the threshold. If an occurrence lies on a genome with either a gain at leaf level in the base pattern or a potential gain at leaf level, it is here defined as an isolated occurrence. A clear overprediction of isolated occurrences is only seen in the set with a base pattern cutoff of 10−20.
Fold Recognition and Structural Classes
At the highest level of the SCOP hierarchy, domains are grouped into seven different classes, predominantly based on secondary structure content. In our analysis, predicted false positives rates for these structural classes differ significantly from one another. For example, the first column in Table 2 shows that the proportion of occurrences with a gain at leaf level is lower for the alpha/beta and multidomain classes than for the all-alpha, all-beta, transmembrane, and small protein classes (SUPERFAMILY).
Proportion of Predicted False Positive Assignments for Structural Classes
Some of these differences, although not all, might be explained by the average chain length of the proteins. Domains from the alpha/beta and multidomain classes have on average longer chain lengths than domains from the all-alpha and small-protein classes. Karplus and coworkers [6] previously observed that HMM-based methods might be less accurate at estimating correct e-values for small sequences; this could result in a higher false positive rate. Note that the multidomain class is different from the other six, as it contains proteins that cannot yet be split up into separate domains based on SCOP classification rules. This class is included here because it shows some evidence for sequence-length dependence.
To investigate in more detail why certain structural classes have higher rates of predicted false positives, we submitted a small number of predicted false-positive gene regions to the meta servers (http://bioinfo.pl/meta and http://genesilico.pl/meta). The servers predicted quite a few of the predicted alpha-class regions to be in coil-like structures. On inspection of the source domain, we observed that the region which generated the assignment often contained a very long alpha helix. This strong helical–helical scoring may explain the slightly higher false positive rate for the all-alpha class.
These results must be interpreted with a little care, as the proportions of isolated occurrences might correlate with saturation of the occurrence patterns. As described above, more saturated occurrence patterns result in fewer potential gains at leaf level. Domains from the class of small proteins have on average a lower age than domains from the alpha/beta class [8,17], and will therefore have on average emptier occurrence patterns. To correct for this, the second column of Table 2 shows the proportion of occurrences with a gain at leaf level that have a high e-value (>10−10 for SUPERFAMILY and >10−4 for PSI-BLAST). These proportions indicate that the all-alpha and small-protein classes still display a higher proportion of false positives than the alpha/beta class for both the SUPERFAMILY and PSI-BLAST assignments.
The second score seems to deviate more per class for SUPERFAMILY than for PSI-BLAST, while the standard deviations for the sample distributions are very similar. This may indicate that there is a higher e-value dependence on secondary structure content or domain length for the former method.
Conventional benchmarking of fold-recognition methods involves blind tests on subsets of protein domains with a known structure [27–29] or on sets of sequences annotated with expert knowledge [30]. Using such a procedure, the ratio of false predictions can be estimated as well as the ratio of false negatives. A similar fashion of benchmarking would be difficult for our method, since the majority of genes have unknown structures, and using all genes on the genomes is essential to the method. We have already shown through e-value distributions that isolated occurrences are less reliable. Below we describe two other independent tests to verify our set of predicted false positives. Predicted false positives are defined as an occurrence with a gain at leaf level, a single copy, and a high e-value (>10−10 for SUPERFAMILY and >10−4 for PSI-BLAST).
Although no actual structures are available for the majority of the genes, a set of likely false positive assignments can be obtained by checking for overlap. For a given gene region, more than one assignment may be obtained. If the assignments are to two different superfamilies, the assignment with the worse e-value is likely to be false.
Translating this to occurrence patterns, we can say that occurrences, which are solely generated by assignments with a stronger assignment in the same region, are likely to be false. We selected these occurrences and compared them with our sets of predicted false positives. Table 3 shows that the overlapping occurrences are found nine times more often in our set of predicted false positives than in general occurrences. Fisher's exact test confirms that this difference in numbers is highly significant (p < 2.2 × 10−16). Moreover, if the ratio of overlapping regions is measured within a set of occurrences above the same e-value threshold as the set of predicted false positives and is compared with this ratio of the set of predicted false positives, the fraction of overlapping regions remains significantly higher for the predicted false positives (p = 2.7 × 10−5).
Overlapping Regions in PSI-BLAST
Unfortunately, this test can only be carried out on PSI-BLAST assignments, because in the SUPERFAMILY database, overlapping regions have already been removed. Instead we can compare two different versions of the database. Our study has been carried out on SUPERFAMILY version 1.65. The more recent version (1.69) is deemed to be more reliable.
We assessed assignments that were present in SUPERFAMILY version 1.65, but which had been removed in version 1.69. Again, we selected occurrences for which all assignments on the genome were removed (Table 4). More than 90% of our predicted false positives in SUPERFAMILY 1.65 had been removed in version 1.69. The fraction of removed occurrences is significantly higher in the set of false positives than in general occurrence (p < 2.2 × 10−16) and significantly higher than in the set of occurrences with a similar e-value (p < 2.2 × 10−16).
Updates in the Superfamily Database
These results both confirm that our set of predicted false positives are significantly more likely to be false than general occurrences.
An Increase in True Positives
As described above, fewer isolated occurrences can be detected as occurrence patterns become more saturated. Such saturation could be caused by an increase in true positive assignments, when fold-recognition techniques become far more sensitive, or when many more protein structures become available. Previous work [8] has shown that the number of occurrences is not uniformly distributed. The distribution peaks at a low and a high number of occurrences, with a minimum in the middle. In addition, recent work by Yan and Moult [31] shows that the number of known (super)families, which occur on very few genomes, is expected to grow. This implies that although some superfamily patterns might become more saturated, the number of superfamilies with emptier patterns will also grow. Hence, this method will remain applicable to a large number of superfamilies.
Biological Relevance
When interested in the fold assignment of a single protein, it is important to keep in mind that an isolated occurrence may appear due to lateral gene transfer or extensive gene loss. Nevertheless, it can be advantageous to visually inspect predicted occurrence patterns of homologous sequences with a weak hit to the protein of interest. Anomalies in such a pattern can give an idea about the reliability of the assignments, and may also indicate false negatives or deletions in cases of a "loss at leaf level."
When working on a specific protein, it is also important to be aware of the differences in aim and methodology between this method and phylogenomics methods used for functional annotation. The aim of this study is to find true homologues in a set of likely homologues, whereas phylogenomic methods aim to subclassify a set of known homologues. The difference in aim results in a distinctively different methodology. For known (close) homologues, it is feasible to calculate a phylogeny of proteins and subsequently assign duplication and speciation events [21], whereas in this work, the proteins of interest have very distant (if any) evolutionary relationships and a precalculated phylogeny of genomes is used instead. Hence, our method is not a substitute for a phylogenomics method, but could perhaps be used as a prefilter in cases where structural assignments are involved (e.g., see Sjolander [20]).
Consensus Data and Meta Servers
Previously we described how the method could be extended to assess assignment sets with a large proportion of false positives using a base pattern. However, rather than assignments below an e-value threshold, consensus occurrences could be used to create the base pattern. In this study, two profile-based searching methods were analysed. Although a large proportion of occurrences is in agreement (84% for SUPERFAMILY and 95% for PSI-BLAST), only about 50% of these occurrences were caused by the same number of assignments. The two fold-recognition methods might recognise different homologues on the same genome. Consensus occurrences appear to be more reliable on evaluation by our method: the proportion of isolated occurrences was significantly lower than for occurrences obtained by a single method, even though consensus occurrence patterns are naturally sparser. The usage of consensus occurrences rather than consensus assignments could therefore provide additional information about the reliability of the assignments. This technique might be used as an additional quality check for meta servers, using consensus data of several genome-wide fold-recognition methods.
This study shows that false positives assigned by fold-recognition methods on completed genomes can be detected by determining isolated occurrences in a phylogeny. We developed a method to identify these isolated occurrences by applying a parsimony algorithm to a phylogenetic occurrence pattern. An occurrence is said to be isolated if it causes a "gain at leaf level" in the most parsimonious evolutionary scenario.
It is shown that in principle, isolated occurrences are less reliable than other assignments: the majority of isolated occurrences have a high e-value (>10−8 for SUPERFAMILY, >10−4 for PSI-BLAST). e-Values are shown to be almost independent of evolutionary distance between the source sequence and the genome of assignment. Deletions and/or false negatives are therefore unlikely to cause the observed high e-values of isolated occurrences. To predict false positives in practise, additional constraints should be imposed. The e-value of the occurrence should be higher than a given e-value (>10−8 for SUPERFAMILY, >10−4 for PSI-BLAST) to minimise the number of isolated occurrences caused by lateral gene transfer. In addition, the occurrence (of a superfamily) should be caused by a single assignment to the genome. Using this technique, more than 1,000 false positives can be predicted for both SUPERFAMILY and PSI-BLAST. Tests with independent means to indicate false positives were performed to validate our predicted false positives; one test was based on overlap of PSI-BLAST assignments, and the other considered changes between different versions of the SUPERFAMILY database. Both tests confirmed that isolated occurrences are more likely to be falsely assigned. The method can be extended to assess sets of assignments with a large proportion of false positives and could be used to enhance the searching power of existing fold-recognition techniques. This technique could therefore provide a way to fundamentally improve the assignment sets of genome-wide fold recognition.
Considering occurrence patterns from genome-wide fold recognition also gives a new way to examine the quality of fold assignments. It was observed that the number of assignments and occurrences on genomes drastically increases for high e-values. The most likely explanation for this phenomenon is a sudden increase in false positive assignments above certain e-values, since the sudden increase is not observed in consensus data, with occurrences predicted by both SUPERFAMILY and PSI-BLAST assignments, or for data where isolated occurrences were removed.
When examining the rate of predicted false positives for different structural classes, a significant variance was observed. Domain length and secondary structure content might cause this dependency between false positive rate and structural class.
Materials and Methods
SUPERFAMILY assignments.
The first set of assignments was taken from SUPERFAMILY database [24] version 1.65 and covers 1,269 different superfamilies as defined by SCOP [12] on 150 completely sequenced genomes. About 750,000 structural assignments were made, with the following restrictions imposed: (1) all assignments have an e-value lower than 10−4; and (2) no other assignment on the same region of the gene is made with a lower e-value. Table S1 shows the genomes and their coverage by assignments.
PSI-BLAST assignments.
The second set consisted of assignments obtained by PSI-BLAST [5] searches on the same 150 genomes. Sequences with less than 95% sequence identity from the ASTRAL [32] database were used to search for structural domains in a nonredundant database created from all genes in the 150-genome set. PSI-BLAST was used with a SEG filter and an e-value cutoff of 1 × 10−5 for inclusion in the PSSM. Assignments with an e-value smaller than 1.0 were included after the final run. The e-values for the assignments were taken from the PSI-BLAST run in which the assignment first falls below 1 × 10−5 (i.e., when it is not yet included for scoring in the PSSM). Note that there was no check for overlap of assignments within a gene. In contrast with SUPERFAMILY, a repeat of a domain from the same superfamily within a gene was counted as a single copy.
Phylogenies of the genomes were created using the SUPERFAMILY occurrence data (the number of copies were not included). A neighbour-joining algorithm was used to create a tree. The branch lengths were then normalised so that all leaves were at an equal distance from the root (1.0), following the method used by Winstanley and coworkers [17].
An occurrence is in this study defined as the occurrence of a superfamily on a genome. An occurrence can therefore be caused by more than one assignment. The e-value of an occurrence is defined as the lowest e-value in the set of assignments covering the occurrence.
Isolated occurrences—gains at leaf level.
A parsimony algorithm is used to find isolated occurrences in a pattern given the phylogeny. This minimises the number of loss and gain events for a superfamily in the species tree [17,33,34]; a detailed description of the algorithm can be found in [33]. Isolated occurrences are identified as occurrences that create a gain at leaf level. The algorithm used a gain penalty that was twice the size of the loss penalty in order to take a high number of false negatives into account. Experimentation with a lower relative gain penalty showed only a small increase in isolated occurrences. This indicates the technique is relatively robust against false negatives. The parsimony algorithm was implemented in Java (J2SE 5.0; http://java.sun.com).
Potential gains at leaf level.
A potential gain at leaf level is defined as a genome without an occurrence for a given superfamily that would cause a gain at leaf level if an occurrence were added to the existing pattern. To calculate potential gains at leaf level, the parsimony algorithm is run for every genome without an occurrence in the pattern.
Identifying isolated occurrences using a base pattern.
A base pattern is created from assignments with an e-value below a given threshold. Subsequently, the parsimony algorithm is run on the base pattern, and potential gains at leaf level are predicted. A set of isolated occurrences can be found as the union of the set of (1) occurrences within the base pattern which cause a gain at leaf level; and (2) all occurrences caused by assignments above the e-value threshold, with a potential gain at leaf level.
Distance to source.
The distance to source for an occurrence is the age of the youngest common ancestor between the occurrence and any genome containing the source domain. A genome is said to contain a source sequence for a SCOP superfamily if it covers 80% of its length and has at least 95% sequence identity. Simple BLAST searches were used to identify the source sequences. No source sequences could be identified for a few superfamilies on our set of genomes.
Cluster distance.
The cluster distance reflects how "far away" an occurrence is from any other occurrence within a pattern, given the phylogeny. The cluster distance is calculated as the sum of distances to every occurrence in the tree divided by the distance to every leaf in the tree. This score was subsequently divided by the average score for each occurrence in the pattern so that the average cluster distance of a pattern became 1.0. A cluster score of 1.0 indicates average clustering; a score of <1.0 indicates tighter clustering. Mediation by the average distance to each leaf was used, since some leaves in the tree lie in tighter clusters and would generally produce lower scores without mediation.
A consensus occurrence is an occurrence that is identified by both SUPERFAMILY and PSI-BLAST. Note that the occurrence does not necessarily have to be caused by assignments to the same gene.
Significance test for structural classes.
A simple sampling procedure was used to determine if the proportion of false positives for a structural class deviates significantly from the overall false positive rate in Table 2. The ratio of predicted false positives was calculated for 500 random samples of the occurrence patterns. For each sample, the number of random genome entries was chosen to match the class size. The resulting distribution of sampled false positives rates was used to determine if the rate for each structural class was significantly lower, falling within the lowest 1% of the sampled distribution, or significantly higher, falling within highest 1%. The number of genome entries for a class depends on the number of superfamilies and ranges from almost 6,000 entries (multidomain) to just over 50,000 entries (alpha/beta).
PSI-BLAST overlapping regions.
Overlapping regions of assignments were identified as assignments with a stronger assignment to a different superfamily on the same gene region and with at least 50% of the weaker assignment covered by the stronger assignment. Overlapping occurrences are occurrences that would be taken out of the dataset if only the strongest assignment within a region was retained.
All figures are plotted using R [35] except Figure 1, which was created by a modified version of a Java applet (http://www.stats.ox.ac.uk/∼abeln/howold). Linear regression was also performed using R. Figure 2 is plotted using a kernel estimate for the density function; the amplitude is then multiplied by the number of data elements in the set to obtain an approximate frequency.
Supporting Information
Figure S1. Correlation between e-Values and the Distance to a Source Sequence
Correlation between e-values and the distance to a source sequence for both SUPERFAMILY (A) and PSI-BLAST (B). The distance to a source sequence for an occurrence is calculated as the distance to the youngest common ancestor between the occurrence and a genome containing a source sequence. The correlation shown is very weak (r-squared is 0.05 for SUPERFAMILY and 0.15 for PSI-BLAST).
Figure S2. The Frequency of e-Values for Different Cluster Distances
(A,B) Show that occurrences with a relatively high cluster distance (>1.01) have a distribution similar to the assignment distribution for most of the e-value spectrum, since the ranked distribution is near uniform. Only the right-hand side of the distribution shows a drastic increase in frequency compared with the reference distribution. This sudden increase is once again likely to be caused by false positives. Moreover, occurrences with a cluster distance >1.20 give a very similar distribution of e-values to occurrences with a gain at leaf level.
Table S1. Structural Assignments to the 150 Genomes
This table shows all 150 genomes for which structural assignments are made. The keys to the genomes as used in Figure 1 are given. The coverage of structural assignments is shown for each genome by the number of genes for which at least one assignment could be made. The last column shows the number of additional copies in the SUPERFAMILY data with respect to PSI-BLAST. For the PSI-BLAST data, any superfamily domain that is repeated within the same gene is counted as a single copy, for the SUPERFAMILY data each repeated superfamily is counted separately.
Author Contributions
CMD conceived and designed the experiments. SA and CT performed the experiments. SA analyzed the data. SA and CMD wrote the paper.
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text:
This case is part of the Giving Voice to Values (GVV) curriculum. To see other material in the GVV curriculum, please visit http://store.darden.virginia.edu/giving-voice-to-values. Giving Voice to Values (GVV) is an innovative curriculum for values-driven management and leadership, designed to be integrated across the core business curriculum as well as to stand alone. This case concerns a high tech product marketing manager who pressures the research team to change results in order to appease the senior vice president, much to the team's dismay.
Publishing Authority:
Darden Business Publishing – University of Virginia

subset: commoncrawl


text:
The Background for the Drawing By Lee Kessler (Artist)
"With the onslaught of the Russian Army and their advance on Austria and the Danube in late March 1945, the Germans evacuated Stalag XVII-B, marching those who could walk, on the road West. After a couple of weeks on the road, we passed a place called Mauthausen. We later learned it was a Concentration Camp, although at the time we knew little about them. Approaching us from the opposite direction was a group of prisoners from this camp who had been working in a quarry. They were Hungarian Jews and were guarded by the S.S. We were halted at the side of the road as these walking skeletons passed. Occasionally we heard the crack of pistols and knew what they were for. Those who fell and were too weak to get up were shot. The prisoners followed a wagon and loaded the bodies.
"I approached one of the bodies of a man shot in the head lying along the side of the road and noticed a crinkled photograph by his hand. As he lie, his arm stretched out as if to be reaching for the picture. I moved off the road for a better look at the photo and I was just about to pick it up, but a guard shouted for me to get back. The picture was of a women and two small children. As I glanced back, I saw that a butterfly had lit on him.
"I was obsessed with the scene. Here was this man, dead by the side of the road. The last thing he looked at was a picture of his family, probably his only possession, and where were they? Dead, or in some other camp? At that moment I could only think that everyone has the right to die with dignity, and here was a poor soul who died with such obscurity.
"Sometime in the 1950s, I started a sketch of a rough outline but put it away, since I felt no one would understand what I was trying to portray. Twenty years later, as I lie in the hospital, a nurse who knew me and my association with art suggested I do art work for therapy. I had my wife hunt for this sketch, bring me my pen and ink, and with the encouragement of the staff, I finished the picture.
"Like other pictures, I put it away feeling that no one but me could really understand it.
"In 1983, a POW Convention in Cleveland, when another POW was being interviewed, he related the story of how he saw a man fall. "While lying on the ground, he pulled a picture from his pocket, and as he kissed it the S.S. guard shot him."


text:
RT @jamesjawa Hondo is super-excited that @cultofcummings, supporters of Jim Cumming, the voice of Hondo, are aboard #TeamHondo for #ThisIsMadness
The Cult of CummingsSince 1995, we are proud to be the first and largest Jim Cummings fan site online. This site is a labor of love of Hillary DePiano. Do you have a photo of Jim Cummings, an article or interview that we don't have posted, a hot tip or other item that we could use on the site? Please email us at [email protected].
- Jim Cummings on Facebook


text:
Very first EP ever released by Grieves. Regardless of the fact that there's no Budo, this still possesses some intense songs that even hold up to his current stuff. Sorry about the crap quality but I don't think there's a link anywhere on the internet with a higher quality. Still well worth listening to if you enjoy hip hop in the slightest.
Artist: Grieves
Album: Every Hell Has It's Springtime EP
Release Year: 2005
Genre: Hip Hop
1. Intro
2. Garbage
3. Fairytale Bullshit
4. Money Game
5. Breathe
6. Dear Friend
7. A Song for Us
8. Outro


text:
The latest Beatles News Briefs, Beatles rumors and Beatles and Sixties Rock and Roll
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Remember the last time you synced all of your Beatles collection from iTunes to your iPod, or backed it up? Took a while for all 280 or so tracks, didn't it? Well, researchers at Cal Tech and the University of Victoria say they can now download all the tracks of the entire Beatles catalog in one tenth of a second.
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Blog Archive
- ► 2009 (31)
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Khatta Meetha BeetrootPosted by Medhaa on Thursday, July 10, 2008
Love the sweetness of Beetroots and would normally have them boiled in salad. But recently I started making curries out of it and loved it. After some trials and errors we loved this one. Hope you guys enjoy it too
1 cup beetroot, cut into small cubes
1 onion, chopped
1/4 cup pureed tomato
10 curry leaves
1 inch ginger, grated
1/2 cup curds
1 tsp coriander powder
1/2 tsp red chili powder
1/4 tsp turmeric powder
salt to taste
coriander leaves for garnish
- Heat 1 tbsp oil in a pressure cooker. Add the curry leaves. Add the onions and cook till they are translucent.
- Add coriander powder, red chili powder and turmeric powder. Cook for a minute.
- Add ginger and tomatoes and cook till the oil separates.
- Beat the curds well and add it to the pressure cooker.
- Add the beetroot, salt and 1/2 cup water.
- Give 2 whistles. Let the pressure release.
- Garnish with coriander leaves and serve with puris or roti.
10 comments:
- Cham said...
I make beet curry but not with much ingredients, but love this veg for color! Looks good Medhaa
- July 10, 2008 at 11:32 AM
- Uma said...
the curry looks so colorful and delicious Medhaa! you changed your template? Looks beautiful, but I liked your previous one better.
- July 10, 2008 at 2:06 PM
- Meera said...
Hey, I loved your new template. It looks pretty. The beetroot curry looks nice.
- July 10, 2008 at 2:10 PM
- KALVA said...
Hmm tomato and beetroot.. never tried it... Nice one dear
- July 10, 2008 at 4:16 PM
- sowmya said...
nice one..i too make curry with beetroote,almost the same recipe..
- July 10, 2008 at 8:10 PM
- Illatharasi said...
Hi Medhaa, beetroot looks yummy. I make the same way, except curd, ginger. Will try adding these this time:)
- July 10, 2008 at 11:20 PM
- Suma Rajesh said...
luv the colour....its good...new recipe to me....
- July 11, 2008 at 2:31 AM
- Anisheetu said...
This is looking really good.. first time here... very healthy recipe and i liked ur blog... :)
- July 11, 2008 at 3:37 AM
- Sunshinemom said...
This looks good! We make it simple with just a coconut garnish - Liked your idea!
- July 11, 2008 at 11:09 AM
- Alpa said...
I love beets too, they're so healthy and earthy aren't they? I will be trying this out soon :)
- July 11, 2008 at 3:48 PM


text:
papermonkey.org {make time for playtime} tag:typepad.com,2003:weblog-1407589 2009-09-01T11:19:41-02:30 {make time for playtime} Card making, cooking, baking and makeup! TypePad Sweet 'n Sassy New Release tag:typepad.com,2003:post-6a00e00994522d88330120a5930b6d970c 2009-09-01T11:19:41-02:30 2009-09-01T11:19:41-02:30 Suzy Hi & hello! I am so bummed that family circumstances have prevented me from stamping lately. I still wanted to give a shout out to the Sweet 'n Sassy Stamps Penguin & Snowman Blog Hop to celebrate their new mini winter release. Christmas comes fast and SnS has such a cute way to prepare for it :) The ladies have made the sweetest cards, I do hope you will check them out :) Start the hop at Belinda Chang's blog. I *think* I will be back soon. I hate to even say it because then some other catastrophe hits and I do not want to tempt fate; family must come first at times like these. I thank you for all your emails and even phone calls. Right in the middle of hurricane Danny I got a call by none other than Deb Felts. I sure needed her rays of sunshine - thank you Deb! But I have been...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/09/sweet-n-sassy-new-release.html What a Summer! tag:typepad.com,2003:post-6a00e00994522d88330120a5382c00970c 2009-08-10T19:46:53-02:30 2009-08-10T20:02:24-02:30 Suzy Hello friends :) I am back! I had intended to post during my move but you know what they say about the best laid plans... Oh my goodness, where do I begin? This summer has been both difficult and magical. We went to Edmonton in Alberta to see Allan's parents and to meet his good friend's fiance, where I got the worst case of food poisoning after eating Vietnamese food - I was so cold my skin burned and my teeth chattered all night lol. Next was Jasper and the Canadian Rockies where we saw a mama bear and cubs, tons of mountain goats, deer and elk - but it was not the wild life but rather the Versa rental car that attacked Allan and I both - the dang door was sharp and gashed our legs. We hiked the Edith Clavell Mountain to see the most beautiful glacier (the top pic above). We hobbled...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/08/what-a-summer.html Spread Your Wings tag:typepad.com,2003:post-6a00e00994522d8833011571ffb5e4970b 2009-07-17T07:00:00-02:30 2009-07-17T07:00:00-02:30 Suzy Hi & hello! Welcome to the Magnolia-Licious DT Blog Hop! This month the design team members thought it would be fun to each have their own take on Edwin, Magnolia Stamps' sweet little boy character. Too often he is used only for masculine cards which puts many people who HATE making masculine cards (like me) in a bind. I thought it would be fun to just let him (and my creativity) be. I think this card would be fine for a boy OR a girl. I see nothing wrong with giving a girl or a woman a card with a boy on it :) I stamped Chasing Edwin from the new Butterflies collection twice using Memento ink onto Neenah 80 lb cardstock. I cut him out (all except for the net) and put him on top of the base image with foam dimensionals: this way it looks like he is running right out of the card...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/07/spread-your-wings.html Strike Up The Band! tag:typepad.com,2003:post-6a00e00994522d88330115710ade33970c 2009-07-15T10:30:00-02:30 2009-07-15T10:30:00-02:30 Suzy Hi & hello! As you read this I am off with Allan to visit his family out west. We are headed to his hometown of Edmonton for a bit, then off to Jasper to hike some gorgeous mountains, and finally to Rosetown, Saskatchewan to visit the farm of his Aunt and Uncle. It is a bit of a whirlwind trip! I do have a couple of posts scheduled ahead of time like this one for the Angels Landing Blog Hop which has hopefully posted :) Today two new adorable digi-stamp sets are being introduced, Marching Band One and Marching Band Two. They are sure to delight music lovers of any age. What is really cool is that the animals come in different sizes, some with jointed arms and legs to cut out for moving pieces. Too fun! I colored the Cat with Clarinet image from the Marching Band One set with Copic...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/07/strike-up-the-band.html Log Roll tag:typepad.com,2003:post-6a00e00994522d8833011571ec0177970b 2009-07-10T10:34:04-02:30 2009-07-10T10:51:32-02:30 Suzy Hi & hello! Happy Friday! Today's card uses Aussie Animals and Pierced Tags from the Angels Landing collection of Digi-Stamps on the new Stampin' Up! In Color Textured cardstocks Melon Mambo and Bermuda Bay. I must confess I wasn't thrilled with the new colors at first, but they are quickly growing on me. Melon Mambo is a confusing name as the color is not a melon at all but rather a hot pink; Bermuda Bay is this lovely vivid shade of turquoise that I just know will become a favorite. I love this size of card - it is tall and thin, yet smaller than you normally see for this type of card (about 6x3.5). I colored the koala bear with Copic markers then adhered him with foam dots to a base of Sassafras Lass Fawnd of You Too patterned paper from the Life is Beautiful collection. The darker...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/07/log-roll.html The Budget Gourmet: Mac 'n Cheeseburger Casserole tag:typepad.com,2003:post-6a00e00994522d88330115719267c7970b 2009-07-06T08:15:05-02:30 2009-07-06T08:15:05-02:30 Suzy The end of the month always brings us an empty fridge with bits and pieces of leftovers - a half can of diced tomatoes, a few veggies that aren't enough of a serving on their own etc. We budget for the month and try to hold out doing our big shopping until the first when we head to Costo and go crazy. Feast or famine. I love making this casserole at times like this. It is good for so many reasons: you can throw almost anything but the kitchen sink in it and it still tastes great; it takes care of those odd bits of veggies languishing in your fridge; it is cheap to make and satisfying, and it disguises veggies so well even the pickiest kids will eat it up. The recipe is from Campbell's Soup. I found it in a magazine last January, made it and loved it but feared the recipe was lost forever...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/07/the-budget-gourmet-mac-n-cheeseburger-casserole.html July's Magnolia-Licious Sketch tag:typepad.com,2003:post-6a00e00994522d8833011570cd17bd970c 2009-07-05T13:57:33-02:30 2009-07-05T13:57:33-02:30 Suzy Hi & hello! A quickie post as we are sanding, painting and priming rooms today in OUR NEW HOME! That's right we bought our first home - it is so exciting and scary at the same time as there is so much to do. I have more news but I can't reveal it just yet (this is not big nor too exciting for anyone but me but I will share eventually anyway lol) :) So onto the fun stuff -the July Magnolia-Licious sketch is up and running. This month my sweet and most talented DT sister Jacque made a lovely layered sketch full of details. For such a sweet, feminine sketch I decided to use Valentine Tilda sitting on the Swirl Key image, both from the Magnolia-Licious store. The images were colored with my usual Copic markers, then mounted on Basic Grey Porcelain patterned paper background with chipoard...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/07/julys-magnolialicious-sketch-.html Circled Borders tag:typepad.com,2003:post-6a00e00994522d8833011571a02813970b 2009-07-02T08:28:26-02:30 2009-07-02T08:38:12-02:30 Suzy Hi & hello! Welcome to my stop on the Sweet 'n Sassy Blog Hop! My sweet 'n simple card uses the new set Beautiful Borders, which was designed by DT member Tami Mayberry. I simply stamped the circle border in Ancient Page Chocolate ink, then die cut a bunch of circle and scallops with my Nestabilities. A couple of K&Co. Flatback jewels add a bit of sparkle to the circles. The circle cut cardstock in the center is a Bazzill Edger cut to fit, I like how it mimics the stamps. Be sure to check out all five of our new stamp sets here. While you are there also check out the Christmas in July Stamp special. All Christmas sets are 15% off, and you get the fantastic Holiday Greetings sentiment set free when you spend $25 or more. There is so much more to the special, check out the link for...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/07/circled-borders.html Win a Bow-Easy at Craft Critique tag:typepad.com,2003:post-6a00e00994522d88330115709a2e7c970c 2009-06-30T11:18:16-02:30 2009-06-30T11:18:16-02:30 Suzy Hi & hello! Head on over to Craft Critique to read my article on the Posi-Bendr Bow-Easy, an inexpensive and handy little tool for creating the perfect bow. Tell us how YOU make the perfect bow and you may just win one of three being given away! Be sure to post on the article for your chance to win, not here :) Good luck!

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/06/win-a-boweasy-at-craft-critique.html Giftcard Card tag:typepad.com,2003:post-68440873 2009-06-24T10:30:00-02:30 2009-06-24T10:43:51-02:30 Suzy Hi & Hello! Happy Wednesday to you! Today the Angels Landing Design Team has made an array of gift card holders that are easy to make and so fun to give. Mine is a sweet and simple card & gift card holder in one: the little train from the Angels Landing Alphabet Train digi stamp set tows along a gift card to someone special. To make the gift card pocket, I used two rectangle Nestabilities of Stampin' Up! Certainly Celery Textured Cardstock sealed with red line sticky tape; the Cameo Coral "lid" is a Scallop Rectangle Nestabilites that was cut then added with dimensional foam tape. The pocket is snug enough that there is no chance of the card slipping out (it is the second from the largest rectangle in the Large Classic Rectangles set) The images were colored with Copics then put...

 This is a summary only. Please visit my site for all the pictures and details!https://blog.papermonkey.org/index/2009/06/giftcard-card.html


text:
The world is full of secrets and we got a handful of them. Some declassified and long-hidden documents have made many revelations that will shock you. So, get ready to boggle your mind by what you will read now.
1. Hitler's Execution by Churchill
Sir Winston Churchill, Prime Minister of Britain at the time of war, planned to execute German leader Adolf Hitler in an electric chair. He was also not in favor of releasing Indian independence leader Mohandas Gandhi in 1943.
2. Operation Northwoods
To provoke the innocent people to start a war with Cuba, the Joint Chiefs of Staff and top military leaders of America created a fake terrorist attack plan on its citizen. However, the plan was rejected by the Kennedy administration and it was kept hidden out of embarrassment.
3. Operation Gladio
Operation Gladio was set up by the CIA and NATO to instill fear in the public and controlling them with the strategy of creating tension. It involved false-flag terrorism. Italian government confirmed the existence of Gladio in 1990 when a judge discovered the network during the course of his investigation.
4. My Lai Massacre
It is one of the most horrifying incidents of violence and bloodshed against civilians during the Vietnam War. The massacre in My Lai village took place in 1968 by U.S. Army soldiers who killed more than 500 men, women & children, which was uncovered years later.
5. Operation Paperclip
More than 1,600 German scientists were secretly hired by U.S. government in Operation Paperclip during the 'Cold War'. President Harry Truman authorized the operation in 1946 to help America's postwar efforts. Secret US government papers revealed this safe heaven given to Nazis.
6. Dunblane Massacre
In 1996, 16 children and some teachers were killed in Dunblane by Thomas Hamilton. The documents submitted to Lord Cullen's investigation were placed under a Closure Order of 100-year. It's been revealed later that there were links between Hamilton and a number of well-known Scots.
7. U.S.S Liberty
Israel attacked the intelligence collection ship named USS Liberty on 8th June 1967, knowing that it was a U.S. Navy ship. It killed 34 U.S. servicemen and injured around 173. Senior officials have known this for years but they kept it hidden for nearly 40 years to save Israel from embarrassment.
8. Plame Affair
In July 2003, a columnist Robert Novak revealed the name of Valerie Plame as a CIA operative. He said two unidentified administration sources have leaked Plame's name. This sparked 2-year-long investigation to know who disclosed covert agent's identity. Lewis "Scooter" Libby, the then-Chief of Staff for Vice President Dick Cheney was found guilty.
9. The Lost Bomb
A B-52 bomber crashed at Thule, carrying four hydrogen bombs. The crash created tension between the United States and Denmark as nuclear weapons were prohibited by the country on its terrain. Denmark was never informed that out of four, one bomb was lost, which may still lie on the seabed.
10. The Templars and the Vatican
In 1307, France's King Philip IV arrested Templar leaders on charges of immorality and heresy. Pope Clement V planned to change the order & forgive the Templars. However, he failed in his endeavor because of the pressure of Philip. The Vatican published secret documents in October 2007 and brought to light the trial of the Knights Templar.


text:
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text: Novice with baby in case of embarrassment! Dong Li was Arale ruthless Tucao: you make me? Public concern number Sina micro plastic, see more exciting original content! Original title: Dong Li Arale ruthless Tucao: do you make me? Sina entertainment news "Daddy where 4" this week will continue to Xiangxi red stone forest journey, daddy adorable baby who enjoy the fun in the countryside landscape. Practice of the "new force Dong daddy with the baby" has let netizen dumbfounding, have been crazy for "parenting Dafa" this week he actively challenge to daughter Arale shampoo, and the original shape of the baby Arale". Can Arale sweet interaction with dad still does not stop, the oversized coat wearing force "Dad asked Dong you make me?" Sweet again full screen. Arale "beggar" other appealing design for female Dong dad in a period of "father 4" red stone forest tour, Arale to perm hair instant noodles appearance, exquisite face with big fluffy xiaobianer very pleasant, but the kids restless nature, no one to help take care of Arale before how long will become a "beggar" shape, unkempt hair arouses pity. After the completion of the task to go home, Dong dad distressed daughter, take the initiative to give the baby wash hair styling. This force has had some experience in Dong, for the baby to test the water temperature, the towel, make massage done very carefully, but the boy's character he still does not change the "pit baby" color, shampoo foam to make her daughter a "unicorn" style, Arale also smiled amused the protest "you think my hair is the king!". Arale and sugar! Ask Dong Li: "you make me?" Dong Li baby skills to get the recognition of the baby, Arale is still being taken care of when Dong dad said, I think with Dong dad better than his mother." Let Dong was encouraged by the sweet, looked at her daughter's eyes smile into a line. But with the baby is not always easy to work, baby shampoo will be accomplished by force Dong accidentally knocked over the water, splash Arale pants, remorse Dong dad immediately without hesitation baby by his coat wrapped in wet. Dad put on Dong oversized coat Arale very adorable beauty, small shoulders twilight, with her father Dong exclusive design of the "Arale", a small peak value once again return to the goddess yan. In the program of gold sentence frequent Arale, this time with dad sweet again jumped out of the interaction. In the affirmation of the father of the East with the baby level, and reveal a clever little expression asked Dad "can you protect me?" Can you fix me?" Dong Li happy to respond to the baby, daddy will always protect you, Dad, of course, make sure you." Sweet interaction between father and daughter so that the scene is full of love. (commissioning editor: Gong Mo Wu) more exciting content, please pay attention to female @ Sina (micro-blog)相关的主题文章:


text:
him sat his sister
Janet, a little neat old woman, with a Highland curch and tartan plaid,
watching the very looks of her brother, to her the greatest man upon
earth, and actively looking out for him, in his silver-clasped Bible,
the texts which the minister quoted or expounded.
I believe it was the respect that was universally paid to this worthy
veteran by all ranks in Gandercleugh which induced him to choose
our village for his residence, for such was by no means his original
He had risen to the rank of sergeant-major of artillery, by hard service
in various quarters of the world, and was reckoned one of the most tried
and trusty men of the Scotch Train. A ball, which shattered his arm in
a peninsular campaign, at length procured him an honourable discharge.
with an allowance from Chelsea, and a handsome gratuity from the
patriotic fund. Moreover, Sergeant More M'Alpin had been prudent as well
as valiant; and, from prize-money and savings, had become master of a
small sum in the three per cent consols.
He retired with the purpose of enjoying this income in the wild Highland
glen, in which, when a boy, he had herded black cattle and goats, ere
the roll of the drum had made him cock his bonnet an inch higher, and
follow its music for nearly forty years. To his recollection, this
retired spot was unparalleled in beauty by the richest scenes he had
visited in his wanderings. Even the Happy Valley of Rasselas would have
sunk into nothing upon the comparison. He came--he revisited the loved
scene; it was but a sterile glen, surrounded with rude crags, and
traversed by a northern torrent. This was not the worst. The fires had
been quenched upon thirty hearths--of the cottage of his fathers
he could but distinguish a few rude stones--the language was almost
extinguished--the ancient race from which he boasted his descent
had found a refuge beyond the Atlantic. One southland farmer, three
grey-plaided shepherds, and six dogs, now tenanted the whole glen, which
in his youth had maintained, in content, if not in competence, upwards
of two hundred inhabitants.
In the house of the new tenant, Sergeant M'Alpin found, however, an
unexpected source of pleasure, and a means of employing his social
affections. His sister Janet had fortunately entertained so strong a
persuasion that her brother would one day return, that she had refused
to accompany her kinsfolk upon their emigration. Nay, she had consented,
though not without a feeling of degradation, to take service with the
intruding Lowlander, who, though a Saxon, she said, had proved a kind
man to her. This unexpected meeting with his sister seemed a cure
for all the disappointments which it had been Sergeant More's lot to
encounter, although it was not without a reluctant tear that he
heard told, as a Highland woman alone could ten it, the story of the
expatriation of his kinsmen.
She narrated at great length the vain offers they had made of advanced
rent, the payment of which must have reduced them to the extremity of
poverty, which they were yet contented to face, for permission to live
and die on their native soil. Nor did Janet forget the portents which
had announced the departure of the Celtic race, and the arrival of the
strangers. For two years previous to the emigration, when the night wind
howled dawn the pass of Balachra, its notes were distinctly modelled
to the tune of "HA TIL MI TULIDH" (we return no more), with which the
emigrants usually bid farewell to their native shores. The uncouth cries
of the Southland shepherds, and the barking of their dogs, were often
heard in the midst of the hills long before their actual arrival.
A bard, the last of his race, had commemorated the expulsion of the
natives of the glen in a tune, which brought tears into the aged eyes of
the veteran, and of which the first stanza may be thus rendered:--
Woe, woe, son of the Lowlander,
Why wilt thou leave thine own bonny Border?
Why comes thou hither, disturbing the Highlander,
Wasting the glen that was once in fair order?
What added to Sergeant More M'Alpin's distress upon the occasion was,
that the chief by whom this change had been effected, was, by tradition
and common opinion, held to represent the ancient leaders and fathers of
the expelled fugitives; and it had hitherto been one of Sergeant More's
principal subjects of pride to prove, by genealogical deduction, in what
degree of kindred he stood to this personage. A woful change was now
wrought in his sentiments towards him.
"I cannot curse him," he said, as he rose and strode through the room,
when Janet's narrative was finished--"I will not curse him; he is the
descendant and representative of my fathers. But never shall mortal man
hear me name his name again." And he kept his word; for, until his dying
day, no man heard him mention his selfish and hard-hearted chieftain.
After giving a day to sad recollections, the hardy spirit which had
carried him through so many dangers, manned the Sergeant's bosom against
this cruel disappointment. "He would go," he said, "to Canada to his
kinsfolk, where they had named a Transatlantic valley after the glen of
their fathers. Janet," he said, "should kilt her coats like a leaguer
lady; d--n the distance! it was a flea's leap to the voyages and marches
he had made on a slighter occasion."
With this purpose he left the Highlands, and came with his sister as far
as Gandercleugh, on his way to Glasgow, to take a passage to Canada.
But winter was now set in, and as he thought it advisable to wait for a
spring passage, when the St. Lawrence should be open, he settled among
us for the few months of his stay in Britain. As we said before, the
respectable old man met with deference and attention from all ranks
of society; and when spring returned, he was so satisfied with his
quarters, that he did not renew the purpose of his voyage. Janet was
afraid of the sea, and he himself felt the infirmities of age and hard
service more than he had at first expected. And, as he confessed to the
clergyman, and my worthy principal, Mr. Cleishbotham, "it was better
staying with kend friends, than going farther, and faring worse."
He therefore established himself and his domicile at Gandercleugh, to
the great satisfaction, as we have already said, of all its inhabitants,
to whom he became, in respect of military intelligence, and able
commentaries upon the newspapers, gazettes, and bulletins, a very
oracle, explanatory of all martial events, past, present, or to come.
It is true, the Sergeant had his inconsistencies. He was a steady
jacobite, his father and his four uncles having been out in the
forty-five; but he was a no less steady adherent of King George, in
whose service he had made his little fortune, and lost three brothers;
so that you were in equal danger to displease him, in terming Prince
Charles, the Pretender, or by saying anything derogatory to the dignity
of King George. Further, it must not be denied, that when the day of
receiving his dividends came round, the Sergeant was apt to tarry longer
at the Wallace Arms of an evening, than was consistent with strict
temperance, or indeed with his worldly interest; for upon these
occasions, his compotators sometimes contrived to flatter his
partialities by singing jacobite songs, and drinking confusion to
Bonaparte, and the health of the Duke of Wellington, until the Sergeant
was not only flattered into paying the whole reckoning, but occasionally
induced to lend small sums to his interested companions. After such
sprays, as he called them, were over, and his temper once more cool, he
seldom failed to thank God, and the Duke of York, who had made it much
more difficult for an old soldier to ruin himself by his folly, than had
been the case in his younger days.
It was not on such occasions that I made a part of Sergeant More
M'Alpin's society. But often, when my leisure would permit, I used to
seek him, on what he called his morning and evening parade, on which,
when the weather was fair, he appeared as regularly as if summoned by
tuck of drum. His morning walk was beneath the elms in the churchyard;
"for death," he said, "had been his next-door neighbour for so many
years, that he had no apology for dropping the acquaintance." His
evening promenade was on the bleaching-green by the river-side, where
he was sometimes to be seen on an open bench, with spectacles on
nose, conning over the newspapers to a circle of village politicians,
explaining military terms, and aiding the comprehension of his hearers
by lines drawn on the ground with the end of his rattan. On other
occasions, he was surrounded by a bevy of school-boys, whom he sometimes
drilled to the manual, and sometimes, with less approbation on the part
of their parents, instructed in the mystery of artificial fire-works;
for in the case of public rejoicings, the Sergeant was pyrotechnist (as
the Encyclopedia calls it) to the village of Gandercleugh.
It was in his morning walk that I most frequently met with the veteran.
And I can hardly yet look upon the village footpath, overshadowed by
the row of lofty elms, without thinking I see his upright form advancing
towards me with measured step, and his cane advanced, ready to pay me
the military salute--but he is dead, and sleeps with his faithful Janet,
under the third of those very trees, counting from the stile at the west
corner of the churchyard.
The delight which I had in Sergeant M'Alpin's conversation, related
not only to his own adventures, of which he had encountered many in the
course of a wandering life, but also to his recollection of numerous
Highland traditions, in which his youth had been instructed by his
parents, and of which he would in after life have deemed it a kind of
heresy to question the authenticity. Many of these belonged to the wars
of Montrose, in which some of the Sergeant's ancestry had, it seems,
taken a distinguished part. It has happened, that, although these civil
commotions reflect the highest honour upon the Highlanders, being indeed
the first occasion upon which they showed themselves superior, or even
equal to their Low-country neighbours in military encounters, they have
been less commemorated among them than any one would have expected,
judging from the abundance of traditions which they have preserved upon
less interesting subjects. It was, therefore, with great pleasure, that
I extracted from my military friend some curious particulars respecting
that time; they are mixed with that measure of the wild and wonderful
which belongs to the period and the narrator, but which I do not in the
least object to the reader's treating with disbelief, providing he
will be so good as to give implicit credit to the natural events of the
story, which, like all those which I have had the honour to put under
his notice, actually rest upon a basis of truth.
Such as do build their faith upon
The holy text of pike and gun,
Decide all controversies by
Infallible artillery,
And prove their doctrine orthodox,
By apostolic blows and knocks.--BUTLER.
It was during the period of that great and bloody Civil War which
agitated Britain during the seventeenth century, that our tale has its
commencement. Scotland had as yet remained free from the ravages of
intestine war, although its inhabitants were much divided in political
opinions; and many of them, tired of the control of the Estates of
Parliament, and disapproving of the bold measure which they had
adopted, by sending into England a large army to the assistance of
the Parliament, were determined on their part to embrace the earliest
opportunity of declaring for the King, and making such a diversion
as should at least compel the recall of General Leslie's army out of
England, if it did not recover a great part of Scotland to the King's
allegiance. This plan was chiefly adopted by the northern nobility, who
had resisted with great obstinacy the adoption of the Solemn League and
Covenant, and by many of the chiefs of the Highland clans, who conceived
their interest and authority to be connected with royalty, who had,
besides, a decided aversion to the Presbyterian form of religion, and
who, finally, were in that half savage state of society, in which war is
always more welcome than peace.
Great commotions were generally expected to arise from these concurrent
causes; and the trade of incursion and depredation, which the Scotch
Highlanders at all times exercised upon the Lowlands, began to assume a
more steady, avowed, and systematic form, as part of a general military
Those at the head of affairs were not insensible to the peril of the
moment, and anxiously made preparations to meet and to repel it. They
considered, however, with satisfaction, that no leader or name of
consequence had as yet appeared to assemble an army of royalists,
or even to direct the efforts of those desultory bands, whom love of
plunder, perhaps, as much as political principle, had hurried into
measures of hostility. It was generally hoped that the quartering a
sufficient number of troops in the Lowlands adjacent to the Highland
line, would have the effect of restraining the mountain chieftains;
while the power of various barons in the north, who had espoused the
Covenant, as, for example, the Earl Mareschal, the great families of
Forbes, Leslie, and Irvine, the Grants, and other Presbyterian clans,
might counterbalance and bridle, not only the strength of the Ogilvies
and other cavaliers of Angus and Kincardine, but even the potent family
of the Gordons, whose extensive authority was only equalled by their
extreme dislike to the Presbyterian model.
In the West Highlands the ruling party numbered many enemies; but the
power of these disaffected clans was supposed to be broken, and the
spirit of their chieftains intimidated, by the predominating influence
of the Marquis of Argyle, upon whom the confidence of the Convention
of Estates was reposed with the utmost security; and whose power in
the Highlands, already exorbitant, had been still farther increased
by concessions extorted from the King at the last pacification. It was
indeed well known that Argyle was a man rather of political enterprise
than personal courage, and better calculated to manage an intrigue
of state, than to control the tribes of hostile mountaineers; yet the
numbers of his clan, and the spirit of the gallant gentlemen by whom it
was led, might, it was supposed, atone for the personal deficiencies of
their chief; and as the Campbells had already severely humbled several
of the neighbouring tribes, it was supposed these would not readily
again provoke an encounter


text:
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text:
Conditions for Participation
Your acceptance to an Indiana State University study abroad program and your participation in the in-country portion of the program is contingent upon the following conditions:
- You must maintain good academic and disciplinary standing with Indiana State University.
- You must observe the Student Agreement, informing you that the Indiana State University Code of Student Conduct extends to study abroad and you are obliged to participate in the program and designed.
- You must pass all required pre-departure and/or prerequisite courses with the grade stated in your acceptance letter.
- You must make full payment of all tuition and fees by the due date on your University Bill
- You must possess a valid passport by the designated deadline required for your study abroad program. In addition, you must have obtained an entry visa for all applicable study abroad destinations, if required, by the deadline established for your program.
- Any student who is a registered sexual offender, has a felony charge pending against him or her, or is on university disciplinary probation is excluded from participating in study abroad programs managed by Indiana State. More information can be found in the Disciplinary Eligibility link to the right.
If you do not meet these requirements, even after you have been accepted, you will not be permitted to participate in your study abroad program, and will forfeit all non-recoverable costs.


text:
My library and archives card collection. January 2014, added the National Archives, London.
Favorite research resources
My favorite research resources, (in no particular order):
- The Internet
- Public libraries and archives
- OCR and how it allows for searchable databases of digitized old newspapers
- Librarians and archivists
- Microfilm readers which let you scan and save to file
- The flash drive
- Transcontinental jet travel
- Google Translate, Prompt, ABBYY Lingvo
- My library card(s) – can you ever have too many?
- The digital camera – my portable scanner
- The oversized computer monitor
- Progressive lens
- My Kobo e-reader and how it lets me read digitized rare books
- The printing press
- The spiral notebook
- Interlibrary loan
- The laptop
- The temporary limbo of the transit lounge, and the thoughts that come to you there
Kobo version of EH
As of January 2012, Embroidering History is available for the Kobo eREADER. Click here to purchase.