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Traditionally, researchers match a microRNA guide strand to mRNA sequences using sequence comparisons to predict its potential target genes. However, many of the predictions can be false positives due to limitations in sequence comparison alone. In this work, we consider the association of two related RNA structures that share a common guide strand: the microRNA duplex and the microRNA-target binding structure. We have analyzed thousands of such structure pairs and found many of them share high structural similarity. Therefore, we conclude that when predicting microRNA target genes, considering just the microRNA guide strand matches to gene sequences may not be sufficient — The microRNA duplex structure formed by the guide strand and its companion passenger strand must also be considered. We have developed software to translate RNA binding structure into encoded representations, and we have also created novel automatic comparison methods utilizing such encoded representations to determine RNA structure similarity. Our software and methods can be utilized in the other RNA secondary structure comparisons as well.
Citation: Chen X, Shen L, Chou H-H (2014) MicroRNA-Target Binding Structures Mimic MicroRNA Duplex Structures in Humans. PLoS ONE 9(2): e88806. https://doi.org/10.1371/journal.pone.0088806
Editor: Junming Yue, The University of Tennessee Health Science Center, United States of America
Received: November 12, 2013; Accepted: January 15, 2014; Published: February 13, 2014
Copyright: © 2014 Chen 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 is supported by National Science Foundataion Grant DBI-0850195, (http://www.nsf.gov/awardsearch/showAward?AWD_ID=0850195). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
MicroRNAs (miRNAs) are small noncoding RNAs about 21 nucleotides (nt) in length that regulate gene expressions. miRNAs target messenger RNAs (mRNAs) and trigger either their translational repression or degradation [1]–[4]. After pre-miRNA processing, mature miRNA duplexes are loaded into the Argonaute (Ago) proteins within the RNA-Induced Silencing Complex (RISC) [5]–[7]. The assembly activates RISC, which is then directed to target mRNAs [8]–[10]. Before RISC assembly, small RNA duplexes, which include both miRNAs and small interfering RNAs (siRNAs) in some species, can be sorted according to their structure features [11]–[14]. This sorting process is species-specific and determines into which Ago protein will each duplex be loaded. In Drosophila, if mismatches happen on the central 9–10th locations of a guide strand counted from the 5′ end, the small RNA duplex will be loaded into its Ago1 protein [11]. Otherwise, it will be loaded into Drosophila Ago2 protein [11]. However, it has not been observed that human has a strict small RNA sorting system. Loading of miRNAs into human Ago proteins seems random but predictable — among the 4 human Argo proteins, Ago2 is the most abundant and it interacts with the majority of miRNAs (60%) [15], [16]. A recent study showed that the human miRNA hsa-miR-451 only associates with Ago2 rather than the other Ago proteins [17], which suggested that there may be some human miRNA sorting rules yet to be discovered.
After loading, the unwinding of small RNA duplexes in RISC goes through two different pathways that are either slicer-dependent or slicer-independent [18]–[20]. Slicer-dependent unwinding, which cleaves the passenger strand, requires extensive base-pairing. Human Ago2, the only human Ago protein known to have slicer activity, can cut a perfectly base-paired passenger strand between the 10–11th locations [15]. The nicked passenger strand is then degraded. Most of the human miRNA duplexes have mismatches in this location, i.e., their guide and passenger strands are not perfectly complementary [1], [21]. Therefore, most human miRNAs will instead go through the slicer-independent unwinding [22]. Comparing to the slicer-dependent unwinding, the slicer-independent unwinding is much slower at producing activated RISCs [18].
In the past few years, there were extensive studies on the human miRNA target recognition mechanism. Most of these studies were based on sequence comparisons [23]–[25]. However, considering just miRNA matches to mRNA sequences is not sufficient to correctly predict miRNA targeting sites, resulting in many false-positive candidates [26]–[28]. The miRNA-RISC assembly process has been shown to determine the inhibition efficiency that a miRNA can exert on its target genes [18]. It is not clear how the assembly process affects the inhibition efficiency. It could be that the assembly efficiency determined the amount of activated RISC complexes that can inhibit the target genes, or it could be that the assembly process determined the guide strand structure whose efficiency in binding to target genes is governed by thermodynamics. We hypothesize that the miRNA-RISC assembly process affects the miRNA-target recognition efficiency at the guide strand structural level, i.e., miRNA target prediction precision may be improved by considering sequence matches as well as RNA structure comparisons.
In this work we have tested our hypothesis by studying the correlation of known miRNA duplex structures with known miRNA-target binding structures. All selected miRNA duplexes have experimentally validated guide strand and passenger strand sequences, allowing their duplex structures to be inferred. All selected miRNA-target binding structures have also been experimentally validated. The selected miRNA duplexes were then precisely paired with the validated miRNA-target binding structures — the same miRNA guide strand must be shared between the two structures in any pair. The similarity between the paired structures was then measured by a common RNA structure comparison method, and we have found that 69% of the pairs exhibit high similarity. An alternative statistical measurement was also applied and it revealed that most paired structures share common structural patterns that could not have been generated randomly. Further analysis indicated that this close resemblance of miRNA-target binding and miRNA duplex structures is unrelated to the evolutionary age of the miRNAs. Therefore, our study concludes that the miRNA-target binding structure closely mimics the miRNA duplex structure, and this relationship may need to be considered to improve miRNA target prediction.
Selection of paired miRNA duplexes and miRNA-target binding structures
Since our purpose is to compare the structure similarity between miRNA duplexes and miRNA-target binding structures, it is required to collect these structures and pair them together for this study. Human miRNA duplex sequences were selected from release version 18 of the public database miRBase, which is the most comprehensive miRNA database and includes 1527 published human miRNA duplexes (http://www.mirbase.org) [29]. Some mature miRNA duplexes in this database have experimentally verified sequences of both strands but others have only a single verified strand (Figure 1A). To infer the duplex structure of a mature miRNA, we need both strand sequences. As a result, 547 mature miRNA duplexes having both strand sequences were selected from miRBase (Table S1). Human miRNA-target binding structures were selected from the experimental results of Helwak et al. [30]. They provided a high confident data set of 18502 miRNA-target binding structures from 413 miRNAs (Figure 1B and Table S2). Because each miRNA normally have multiple target genes, the set of miRNA-target binding structures is significantly larger than the number of participating miRNAs.
(A) miRBase includes mature miRNAs with either two known strands like hsa-let-7a-1 (2 red sequences) or only one known strand like hsa-miR-1-1 (1 red sequence). To infer the miRNA duplex structure, we have selected only mature miRNAs with both strand sequences. (B) miRNA-target interaction data were obtained from the paper of Helwak et al. In this example, hsa-let-7a-5p is the guide strand of hsa-let-7a-1 (colored blue), and its validated binding with one of its targets, KIAA0284, has the structure as shown.
Intersecting the set of 547 miRNA duplexes having both strand sequences and the set of 413 miRNAs forming the 18502 validated miRNA-target binding structures produced 321 unique miRNA duplexes (Figure 2 and Table S3). The uniqueness here refers to the duplex structure, not the name of a duplex. For example, the longer pre-miRNA sequences of hsa-let-7a-1 and hsa-let-7a-3 are different, thus their different names, but their shorter mature miRNA duplexes are the same; both have the same guide strand hsa-let-7a-5p and the same passenger strand hsa-let-7a-3p. To avoid redundancy, only one of the duplicated mature miRNA duplexes was retained in the 321 unique miRNA duplexes. The guide strand within a miRNA duplex was determined according to the miRNA-target binding structure data — the strand that binds to target mRNAs was identified as the guide strand.
(A) The data selection workflow of miRNA duplexes and miRNA-target binding structures from miRBase and the paper of Helwak et al. (B) The selection criteria and the resulted numbers of miRNA duplexes and miRNA-target binding structures. At the end, 321 miRNAs with both strand sequences and validated miRNA-target binding structures were selected and 17919 miRNA duplex and miRNA-target binding pairs were selected for pairwise comparisons.
After the 321 unique miRNA duplexes were identified, the miRNA-target binding structures associated with them were also selected. Several miRNA duplexes have the same guide strand but different passenger strands (they are considered different among the 321 duplexes). The miRNA-target binding structures associated with the same guide strand were thus paired to all these miRNA duplexes. For example, the guide strand hsa-let-7a-5p has 310 different target bindings (Table S2). hsa-let-7a-5p is shared by two different miRNA duplexes hsa-let-7a-1 and hsa-let-7a-2. Therefore, 620 structure pairs, the first 310 associated with hsa-let-7a-1 and the other 310 associated with hsa-let-7a-2, were produced. In total, 17919 miRNA duplex and miRNA-target binding structure pairs were produced (Figure 2 and Table S4).
Global features of miRNA duplexes and miRNA-target binding structures
As mentioned in the Introduction, the way a small RNA duplex will unwind within RISC depends on its mismatch locations. If mismatches happen on the central 8–12th locations of its guide strand, the small RNA duplex will follow the slicer-independent unwinding pathway. Alternatively, if its passenger strand is perfectly complementary to its guide strand, the small RNA duplex will take the slicer-dependent unwinding pathway [18]–[20]. Therefore, mismatch patterns on the guide strand generally have been used in RNA duplex studies [18], [31]–[33]. However, considering only mismatches on the guide strand ignores subtle signals on the passenger strand that may also be important. For example, the pre-miRNA hsa-miR-1-1 on Figure 1A has 2 mismatched adenines on its guide strand shown in red color. When counting only mismatches on its guide strand, both adenines will be counted as 1 mismatch despite their different mismatch patterns on the passenger strand. Therefore, to better compare the global features of miRNA duplexes and miRNA-target binding structures, we have decided to consider the base-paired locations on both strands.
Figure 3A illustrates the base-pairing distribution of the 321 miRNA duplexes we have selected. The distribution can be visualized in 3 different ways: a 3-D surface graph, a 2-D contour map and a 1-D projection of the base-pairing counts onto the guide strand axis. Note that the 1-D projection adds up all base-pairing counts along the passenger strand axis that are projected onto the same guide strand location, thus the height of the 1-D projection is higher than the 3-D graph. The similar set of graphs obtained from the 18502 miRNA-target binding structures is provided in Figure 3B. We can see that on miRNA duplexes there is a noticeable dip of base-pairing counts roughly in the central 10–13th locations of the guide strand. The guide strand seed region (the 2–7th nucleotides) has lower base-pairing counts among miRNA-target binding structures than among miRNA duplexes; most base-pairing counts in this region is below 80% for miRNA-target binding structures. This supports the notion that seed region interactions with targets are not canonical for many miRNAs [30]. The other locations on the miRNA-target binding structures also have lower base-pairing counts than the duplexes, but their counts remain high at over 60% throughout most of the structures.
(A) The distribution corresponding to the 321 miRNA duplexes that is drawn in a 3-D surface graph, a 2-D contour map and a 1-D projection onto the guide strand locations. (B) The distribution corresponding to the 18502 miRNA-target binding structures obtained from the Helwak et al. paper that are drawn in similar graphs.
The analysis above supports the validity of our method to count the base-paired locations on both strands instead of the mismatched locations only on the guide strand, because it reveals many important miRNA features as follows. Among miRNA-target binding structures, the seed region of the guide strand seems to have mixed canonical and non-canonical match patterns, which is consistent with existing knowledge [34], [35]. In addition, miRNA duplexes tend to have lower base-paring counts in its central region, which is also consistent with previous studies [36], [37]. Noticeably, non-seed region base-pairing counts are significantly lower on miRNA-target binding structures than on miRNA duplexes. A possible explanation is that a miRNA must be packaged well in double-stranded format within pre-miRNA before it can be successfully processed and incorporated into RISC. Significant mismatches anywhere along the duplex can disturb the stability of its structure and ruin its function. However, once incorporated into RISC, the miRNA guide strand does not require perfect base-pairing beyond the seed region to target genes. The global features of miRNA duplexes and miRNA-target binding structures seem to suggest that they are not very different in the seed region but are noticeably different in the non-seed region. What we need to find out next is if such distinction holds at the individual structure pair level.
Pairwise structural comparisons between miRNA duplexes and miRNA-target bindings
To study if the distinctive base-pairing distributions of miRNA duplexes and miRNA-target binding structures hold at individual miRNA level, we systematically compared the 17919 pairs of miRNA duplex and miRNA-target binding structure that share a common guide strand. The RNAforester software was applied on the 17919 pairs to obtain the relative similarity score between the two members in each pair. Remarkably, nearly 69% of the pairs (12006/17919) have high similarity scores above 0.7 in a range of 0–1 (Figure 4A). The score cutoff value 0.7 was noted by RNAforester authors to indicate significant structure similarity [38]. In each of the 12006 pairs with high scores, the miRNA duplex structure is similar to its miRNA-target binding structure (Figure 4D, left). A closer way to look at this phenomenon is to individually consider the top 200 guide strands from the selected 321 miRNA duplexes that have the most number of targets, thus they have received the most number of structure similarity scores. Figure 4C reveals that most of their similarity scores are above 0.7 (green color). Among all 249 guide strands of the selected 321 miRNA duplexes that have received more than one similarity scores, 78% have received more scores above 0.7 than below, and only 14% have received more scores below 0.7 than above (Figure 4D, right). This indicates that miRNA-target binding structures generally mimic the associated miRNA duplex structure sharing the same guide strand.
(A) The similarity scores of the 17919 structure pairs were calculated by RNAforester and ordered from high to low; scores above 0.7 are considered similar. (B) The histogram of the length-scaled Hamming distance values of the 17919 structure pairs divided into 100 bins (bar chart) with the mean value at 0.354 (red line); the expected mean value is at 0.50 for random structure pairs (magenta line). (C) The similarity score ratio for each miRNA duplex. Many miRNA duplexes were paired with multiple target bindings and thus received multiple similarity scores; the top 200 miRNA duplexes are shown here and are ordered by the count of scores received. Green: similarity score ≥0.7 and considered similar; Purple: similarity score <0.7 and considered different. Note the Y-axes on the four subgraphs were not drawn to the same scale. (D) Overall similarity ratios. Left: among the 17919 structure pairs considered, 69% are similar with RNAforester scores ≥0.7; Right: among the 249 miRNA duplexes that received more than 1 similarity scores, 78% have more scores ≥0.7, 14% has more scores <0.7 and 8% have equal counts.
We have developed an alternative method to compare the structure similarity within a pair of miRNA duplex and miRNA target binding structure that share the same guide strand. In this novel method, the guide strand match pattern in each structure was converted into a series of numbers: matched bases were set to 1 and unmatched bases were set to 0 (Figure 5). As a result, two series of binary numbers were generated from each pair of structures. Subsequently, we calculated the length-scaled Hamming distance between these two series of numbers: distance values closer to 0 indicate the two series are very similar, distance values closer to 1 indicate the two series are almost exactly opposite, and distance values near 0.5 indicate the two series are likely unrelated. The mean of the 17919 distance values is 0.354 (Figure 4B, red line), and the 1st quartile and 3rd quartile are at 0.272 and 0.435 respectively. Z-test was applied to test whether 0.354 is significantly different from the expected mean value of 0.50 if the two number series in all 17919 pairs were completely random and unrelated to each other (Figure 4B, magenta line). The Z-test revealed a p-value less than 0.0001, thus we can conclude that most paired structures share a similar pattern and this similarity is not random. Taken together, our data suggest that the two types of structures, a miRNA duplex and its associated target binding structures, tend to be very similar when compared individually.
(A) The miRNA duplex hsa-miR-16-1 and its corresponding dot-bracket and number notations. (B-D) The miRNA-target binding structures of hsa-miR-16-1 with 3 different target genes and their corresponding dot-bracket and number notations. (E) The similarity score and distance value between hsa-miR-16-1 duplex structure and each of its 3 miRNA-target binding structures that are calculated separately using the dot-bracket and number notations. The similarity scores and distance values visually correspond well to the perceived structure similarities in this example.
The high pairwise structural similarity is unrelated to miRNA evolution
miRNAs are ancient. In the animal kingdom, miRNAs were present at the dawn of metazoan [39]–[42]. Two miRNA expansions were observed at the basin of bilaterian lineage and vertebrate lineage [41]. With the advent of next-generation sequencing, more miRNAs were discovered from various species. These studies suggested that the number of miRNAs in each species is closely correlated with its morphological complexity [36], [37]. So far, more than one thousand miRNAs have been found in humans according to the data from miRBase [43]. Some miRNAs are highly conserved whereas others are younger — they emerged just recently [44]. A total of 304 unique guide strands can be identified from the 321 miRNAs involved in the 17919 pairs selected for structure comparisons (see Figure 2 and Materials and Methods). We have selected for analysis 15 representative metazoan species that are from shorter to longer evolutionary distances from the humans. The number of known homologs among the 304 human miRNA guide stands in each of the 15 species is summarized in Figure 6A. None of the 304 guide strands has homologs in Amphimedon queenslandica, and only one, hsa-miR-100, has homologs in both bilaterians species and Nematostella vectensis — this is consistent with previous studies [45]. Most human miRNA guide strands have homologs in the other four vertebrates: Mus musculus, Gallus gallus, Xenopus tropicalis and Daniorerio. The number of miRNA guide strands that have homologs in protostomia decreases, which is also consistent with the notation that when some miRNAs became important in a particular lineage, they are rarely lost in the descendant lineages [39], [46].
(A) The 321 selected miRNA duplexes contain 304 distinct guide strands; their homolog counts in each of the other 15 species is listed. (B) The similarity scores received by each miRNA guide strand are drawn in a boxplot with the median value identified by a blue line; the guide strands are then ordered according to their medians. The number of the other species that contain homologs to each human guide strand is also identified by a red dot. There is no discernable correspondence between the similarity score distribution in blue lines and the evolutional ages of miRNAs in red dots.
When the 304 human miRNA guide strands are individually considered, about 85% of them have homologs in less than 6 other species (Table S5). The logical question to ask is whether the conclusion we have drawn in the previous section, that a miRNA duplex and its associated target binding structures tend to be similar, is only applicable to younger miRNAs? To answer this question, the similarity scores of the 17919 pairs of miRNA duplex and miRNA-target binding structures were mapped onto the 304 unique guide strands, which are then ordered and summarized in Figure 6B. For guide stands that received multiple mapped similarity scores, their score distributions are represented by boxplots and their median values are used in the ordering which goes from the least similar to the most similar (blue lines). Superimposed on Figure 6B is the count of the other species that have homologs to each of the ordered 304 human miRNA guide strands (red dots). From this figure it can be concluded that the similarity between miRNA duplex and miRNA-target binding structure is not correlated to miRNA evolution age.
The traditional understanding about miRNA is that the passenger strand is excluded during duplex unwinding and only the guide strand is needed to guide the activated RISC to target mRNAs; the passenger strand does not seem to play any role in the targeting process. However, recent studies in plants showed that the passenger strand may affect miRNA-triggered transitivity, a production of secondary siRNAs through antisense transcription [47],[48]. Further study revealed that the asymmetry in miRNA duplex structure is actually triggering the transitivity and the asymmetry is determined by both the passenger and guide strands [49]. These novel observations contradict the traditional guide-strand-centric understanding and suggest that passenger strands can still be functionally important before being excluded from RISC. Our data reveal that the miRNA-target binding structure mimics the miRNA duplex structure when they share a common guide strand. Therefore, we conclude that the passenger strand sequence, which helps determine the miRNA duplex structure, must also be considered in order to more precisely predict the targets of miRNAs at the structural level. Our conclusion coincides with the notation that the passenger strand may also be functionally important.
Target recognition is a binding process between the miRNA guide strand and targets. It is initialized by base-pairings in the seed region of the guide strand. Subsequently, compensatory base-pairings in the 3′ region of the guide strand, especially in the 12–17th locations, may enhance target binding [1], [50], [51]. In Figure 3B, the base-pairing distribution reveals that the seed region has both canonical and non-canonical matches. The high frequency of base-pairings in the non-seed region suggests that matches in this area may still be critical in target binding. On the other hand, the base-pairing distribution of miRNA duplexes (Figures 3A) reveals that they have a higher base-pairing frequency both in the seed region and the non-seed region. We can conclude from the global feature analysis that the base-pairing distributions of miRNA duplexes and miRNA-target binding structures are somewhat similar but they are not a simple mirror image as suggested in the literature [18].
Furthermore, tens of thousands of pairwise comparisons between miRNA duplexes and miRNA-target binding structures that share a common guide strand lead to a conclusion — the secondary structures of miRNA duplex and miRNA-target binding closely resemble each other (Figure 4). We can hypothesize that there are certain structural preferences of each miRNA guide strand which are carried forward from its binding with the passenger strand to its binding with the targets. The structural preferences are likely due to the physiochemical properties of the guide strand rather than evolutionary conservation because similar preferences can be observed from miRNAs of vastly different ages (Figure 6).
Our rationale is that miRNAs must form stable duplexes for Dicer cutting and RISC incorporation. Therefore, they require more stable overall base-pairing distribution as exhibited in Figures 3A. If too many mismatches happened anywhere on a duplex, the pre-miRNAs would not form a stable double-strand and that might have prevented mature miRNA production. When the guide strand binds to mRNA targets, it may encounter two possibilities: it may either bind more specifically to its targets with enhanced base-pairing in its 3′ non-seed region, or it may bind to more targets by tolerating more mismatches in this region. From our data, it seems that both possibilities are evident: the base-pairing distribution in the 3′ non-seed region is more dispersed as seen in Figure 3B, but the individual target binding structures are also specific, i.e., they mimic the miRNA duplex structures as seen in Figure 4. The guide strand may have certain physiochemical preferences to binding targets in its non-seed region that may enhance its potency. Also, non-specific guide strands might have interrupted too many genes and were quickly eliminated from the population during evolution.
We can draw the following conclusions from our study. First, it is not sufficient to consider just the guide strand sequence when predicting novel miRNA targets. The passenger strand, though previously considered irrelevant in miRNA target prediction, must also be considered to facilitate more precise miRNA target predictions at the structural level. Second, when binding to targets there are certain physiochemical preferences beyond the seed region of miRNA guide strands, which must also be considered to enhance miRNA target prediction precision. Taken together, our analysis on the pairwise structural comparison between miRNA duplexes and miRNA-target binding structures provides some new insights that may improve the precision of miRNA target predictions.
Materials and Methods
Structure encoding of miRNA duplex and miRNA-target binding
The secondary structures of miRNA duplexes were inferred using the hybrid-min software [52]. The miRNA-target binding data identified the guide strand of each miRNA. Oriented by the identified guide strand, the secondary structure of each miRNA duplexes was encoded into the dot-bracket and number notations (Figure 5A). The dot-bracket notation uses parentheses and dots to represent base-paired and mismatched bases; this notation was used by the RNAForester software to compare RNA structure similarities [53]. The number notation uses 1 and 0 to represent base-paired and mismatched bases; this notation was used in the Hamming distance calculation followed by the Z-test to also compare RNA structure similarities. There is a difference between the two notations: the dot-bracket notation encodes both strands of a miRNA duplex but the number notation encodes only the guide stand. The miRNA-target binding structures from the paper of Helwak et al. [30] were also determined and encoded into the dot-bracket and number notations in the same way (Figures 5B-D).
The Perl program mirna_structure_notations_v2.pl was created to automatically encode the structures of miRNA duplexes and miRNA-target bindings into the dot-bracket and number notations. Usage instructions can be obtained by running this program without any command-line argument. This program can also produce a global summary matrix counting the base-paired locations on the guide stand and passenger strand from all input structures. For example, if the 3rd base on the guide strand is base-paired with the 4th base on the passenger strand in a particular input structure, then the count at matrix location (3,4) will be increased by 1. Such matrices were used to create Figure 3.
Pairwise comparison of miRNA duplex and miRNA-target binding structures
RNAforester is one of the structure comparison methods we have used [53]. This software compares two RNA secondary structures in the dot-bracket notation and determines their similarity score [53]. The Perl program mentioned above can convert the structures of miRNA duplexes and miRNA-target bindings to this notation. However, RNAforester only compares single-strand RNA foldings, not RNA duplex structures. Therefore, structures such as miRNA duplexes or miRNA-target bindings need to be converted into single-strand RNA folding representations. This is customarily accomplished by connecting together the dot-bracket notations obtained from the two strands of a duplex structure with a minimum of 3 dots to mimic a loop between them (see Figure 5) [54]. In our tests, we have found that the RNAforester similarity score is not sensitivity to the length of the loop (data not shown). To execute RNAforester solely for RNA secondary structure comparisons without also considering the primary sequence similarities, its parameter bm (base match score), br (base mismatch score) and bd (base indel score) were all set to 0, whereas its parameters pm (pairing match score) and pd (pairing indel score) were both set at the default values 10 and -5, respectively.
The miRNA duplex and miRNA-target binding structure in each selected pair share a common guide strand. The shared guide strand can serve as an anchor to determine the similarity between the two RNA secondary structures. We have created a novel method that can also determine RNA structure similarity. The Perl program mentioned above can convert the secondary structures of miRNA duplex and miRNA-target binding into a series of binary numbers. On the guide strand, a base-paired location is converted to 1 and an unmatched location is converted to 0. Subsequently, we calculated the length-scaled Hamming distance between the two series of numbers. If the two numbers on the same location is the same, 0 is added to the sum; otherwise 1/n is added to the sum, where n is the length of the guide strand. The sum of all values represents the distance between the two series of binary numbers. If the two series of numbers are the same, their distance should be 0. However, if they are complete reversals of each other, their distance should be 1. Any two random series should have an expected distance close to 0.50 because at each location there is equal probability to add or not to add 1/n to the sum, so the sum should approach 1/n×n/2 = ½. We have obtained 17919 distance values from the 17919 structure pairs. The mean of our distance values is 0.354, and the 1st quartile and 3rd quartile are at 0.272 and 0.435 respectively. This distribution is determined to be significantly different from the expected mean value of 0.50 for completely unrelated structure series after performing a Z-test (p-value<0.0001).
The relationship between structure similarity and miRNA evolution
To determine if the similarities of miRNA duplexes and miRNA-target binding structures are related to miRNA evolutionary age, homologs to human miRNAs from the other 15 species were also selected from miRBase [55]. The miRNA names reflect their association to specific miRNA families. For example, the four miRNAs dre-miR-21 (Daniorerio), gga-miR-21 (Gallus gallus), mmu-miR-21 (Mus musculus) and hsa-miR-21 (Homo sapiens) all belong to the miR-21 family. We took a human miRNA-centric approach for the evolution study and considered only miRNAs from the other species that can be associated to the 321 human miRNAs selected for our study. Because only the guide strand is shared between a miRNA duplex and its miRNA-target bindings, we considered only guide stands in the evolution study. Some consolidation of guide strand data was performed. If two human miRNAs share the same guide strand, only one guide strand was retained for the study. Conversely, if both strands of a miRNA duplex are functional guide strands, both were retained for the study. For example, the two strands hsa-let-7a-5p and hsa-let-7a-3p of the human miRNA hsa-let-7a-1 are both guide strands, but they have different homologs in the other 15 species — 13 other species contain hsa-let-7a-5p homologs but only 5 other species contain hsa-let-7a-5p homologs.
After the consolidation, 304 distinct human miRNA guide strands were retained for the evolution analysis. The non-human miRNAs were initially assigned to the 304 human guide strands based on their name. If a non-human miRNA can be assigned to different human guide strands, they will be assigned in duplicate to simplify the analysis. Because some miRNA strand names are incomplete or incorrect, all non-human miRNAs assigned to a human guide strand were subsequently sequence-aligned to the guide strand to validate the assignment. If a non-human miRNA aligned poorly to the assigned guide strand, it was manually inspected and its assignment was removed when necessary. The counts of human miRNA homologs that can be found in the other species are listed in Table S5.
Supporting Information
Table S1. 547 mature miRNA duplexes selected from miRBase that have both strand sequences.
Table S2. 18502 validated miRNA-target binding structures that were collected from the paper of Helwak et al.
Table S3. 321 unique miRNA duplexes from the intersection of the set of 547 miRNA duplexes having both strand sequences and the miRNAs involved in the set of 18502 validated miRNA-target binding structures.
Table S4. Dot-bracket and number notations for 17919 pairs of miRNA duplex and associated miRNA-target binding structure. Note that the two structures in each pair share a common guide strand. Also listed in the last two columns of the table are the RNAforester similarity score and the Hamming distance value for each pair of structures.
Table S5. The main part of the matrix listed the number of homologs of the 304 distinct human miRNA guide strands that can be found in each of the other 15 species; the 15 species were ordered according to their evolutionary distance from humans. The total number of human miRNA guide stands that have homologs in each species is summarized on the right side of the table under the column "Count of human siRNAs having homologs in each species". The number of species covered by each of the 304 distinct human miRNA guide strand is also summarized at the bottom row of the table labeled "The number of species covered".
Program S1. Perl Program (downloadable Perl code). The Perl program mirna_structure_notations_v2.pl used to generate the dot-bracket and number notations on Table S4.
We thank Dr. Xun Gu for the discussion about miRNA evolution, and our colleagues Hyejin Cho and Jennifer Chang for their assistance. We also thank a prior anonymous reviewer for the suggestion to use the Helwak et al. data set, which dramatically improves both the quality and quantity of the miRNA-target binding data that are analyzed in our work.
Author Contributions
Conceived and designed the experiments: XC HC. Performed the experiments: XC HC. Analyzed the data: XC LS HC. Contributed reagents/materials/analysis tools: HC. Wrote the paper: XC HC.
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As part of this project, the company is also strengthening its partnership with Sacmi Heavy Clay, which has, right from the start, played a key role in the ambitious plans to technologically revamp the long-standing brickworks acquired by Cerro Negro in 2015.
These latest orders placed with Sacmi Heavy Clay complete the ambitious "Planta Dual" project. The latter will allow the Argentinian firm to perform a real-time switch from brick production to roofing tile production (and vice versa), providing an effective response to market dynamics.
Sacmi Heavy Clay has been a pivotal partner in the turnkey revamp of the existing tunnel kiln (begun in 2016, now being installed), and has now completely redesigned the handling system for the roofing tile drying racks, including the below-press conveyor (RAT), up to the point where the dried tiles are transferred to the kiln car loading system.
While the automatic kiln car handling system allows rapid switching from one type of production to another, the fully renewed control logic optimizes the working of the entire machine set. This is another example of Sacmi excellence when it comes to designing and supplying latest-generation control systems.
Another part of the order regards modernization of the loading and unloading of the cars for the chamber dryer. These were automated and equipped with cutting-edge detection systems (laser reader) to ensure maximum flexibility and precision of operation. Completing the order are two drying chambers for masonry bricks. These have been fully modernized to combine increased productivity and quality with minimal energy consumption.
At the root of the customer's investment decision lies not just the technological excellence of individual Sacmi HC solutions, but also Sacmi's ability to work in close contact with the local team. This has ensured a fast, customized response that meets the specific needs of an existing production plant, making it a pivotal local player in terms of efficiency, flexibility and output quality. Currently being built at Sacmi's manufacturing facilities, this new plant will be delivered and started up by the end of autumn.
Sacmi Imola S.c.r.l.

subset: commoncrawl


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One of my favorite reference sources is a book of WWII uniforms I got a few months back. It's especially good because it's full of (neutral) gray mannequins posed with various soldier uniforms on. Here I've taken the uniform of a Nepalese Gurkha, replaced the mannequin with a robot, and set him in the sketchy beginnings of some kind of cockpit. You know, I was once on a flight when they let the autopilot land the plane. It was a horribly rough landing, and they only told us about putting our lives in the hand of a robot after it was over. Traitors…
Autopilot engaged
Jan 20
Posted on January 20, 2012, in Robots For All. Bookmark the permalink. Leave a comment.


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So turns out it's the end of another year already. Huh. This keeps happening. I guess it's a good thing; better than the alternatives?
At the beginning of this year, I wrote a blog post about 2012 and what it might be like and what I hoped to do in it. Let's see how that went …
- From my list of things to do in 2012 (in blog post):
- make pizza from scratch (we did this a lot; this was a good idea 🙂
- watch Inception – finally watched this a few months ago, with C. I found it less twisty and confusing than I had been led to believe it was. However, I have also watched Primer so my standard for 'twisty and confusing' is pretty high.
- Clear 'My Life Would Suck Without You' on Expert level (DDR). I haven't got good scores on Expert level … but I have successfully cleared the level.
- Start a joint savings account with C. Hurrah for having well-organised household finances!
Partial successes:
- read Paul through Mediterranean Eyes – it's brilliant and I've read a lot of it, but I don't think I've actually finished it. Oops. I would very, very highly recommend it though – fantastic commentary on 1 Corinthians (which as I understand it is one of the 'Pauline' letters that is actually thought to have been written by Paul …) with loads of cultural and literary insights.
- write a program in Python – I did start re-doing some stuff in Python. But this was really about getting back into programming – and in my current job I have been writing programs in various languages (mostly C#, also bits of SQL, ASP, javascript, etc) so I was happy to let this one slide.
- make chocolate cheesecake brownies – I honestly can't remember if I've done that this year or not. However, I have made lots of delicious baked goods – including homemade pain au chocolat for Christmas – so I'm counting that as a partial success.
- get good photos of C and me. Arg. If you know me in real life and we're both somewhere, please take photos! We have good digital cameras and have friends with good digital cameras, we should be able to do this … it's just so easy to put off.
- write an essay about something and put it on the blog. Hm. Thesis-writing took over, I think …
- go to a 'High' Church service (defined as one with incense and a language I don't speak, like Latin or Russian). I found that moving house and therefore having to find a new Church anyway took up all of my going-new-religious-places energy – I find it hard to go to new/different religious venues unless I am going with someone who can let me know how things work. I'd still really like to go to a really formal/liturgical/'bells and smells' service, but being realistic I will find it hard to go on my own so I don't know if this will happen.
- memorise 1 Corinthians 13. Frankly, I totally forgot. But I have started a project to read through the whole Bible in the space of a few months (keeping it vague …), which kind of ticks some of the same mental boxes for me.
- learn a piano piece. Kept getting put off, and then we moved and the piano didn't.
Hmmm. Overall, I'm pretty happy with how that went :).
Other thoughts about 2012:
- I commented at the beginning of the year that 2011 had been odd in that there hadn't been a big change in my life around September (whereas previously the academic year had dictated the rhythm of my years). In September 2012, I started a new job – so in a way it feels back to life as normal there. I do programming-y stuff, and my coworkers are nice.
- I successfully completed the graduate course I was studying for! 🙂 handed in in August but didn't have confirmation until the end of November. That was really bloody hard work (mostly because it is a BIG jump going from doing A level essays to doing graduate-level essays/thesis, and this year I had to actually write and complete the thesis) but I am pleased with how it turned out. I'm also pleased that by doing it I've worked on a serious project involving writing – I like writing, but tend to only write recreationally, as it were.
- C and I moved out and moved quite far away from where we had been living, for my new job. It's really nice to be renting a place of our own 🙂 although living with family is fantastic (and I do mean that honestly) it's really neat to have our own little place, and be able to host get-togethers and stuff. We've also become a bit more foodie-ish – I think having his own kitchen has been a big thing for C, we have some great kitchen things (mostly received as wedding presents) so we have good equipment, and it's a way to get interesting food on our (pretty basic) budget. Come visit, there might be homemade foccacia!
- We have had friends and family to stay. I am loving this. It is SO COOL to be able to say "yes, come, please, we have floorspace and sleeping bags and board games and we will feed you delicious things". I am so lucky to have super friends to invite, and ones who are willing to travel here!
- We bought a car! This is exciting. Although C and I still walk nearly everywhere (travel to work, food shopping, library visits, most other shopping is all done on foot), there are some places you can't walk, like when you want to drive to visit people who live hundreds of miles away.
- We joined a new Church – kind of a necessity when you move hours away from the old one. The people seem nice. I went to a study group in the Autumn and learnt virtually nothing about Philippians but met people, which was the idea, and we went to Church today to help with packing up Christmas decorations (lots of beautiful candles = lots of candle wax to clean up …) and we're gradually starting to get to know people.
- I joined a new dance group which is cool. It's good exercise, and nice to meet people outside of work.
2012 has been a really good year for me, and for us. I am very thankful that so very much has gone right for us this year. There were some tricky bits*, but overall this year we have had lots of smooth bits and lots of good bits and not many tricky bits.
How was your 2012?
(*Top tip: if you've had tonsillitis for a few days and it hasn't got any better and the walk-in-clinic person says they were going to give you antibiotics but now they're not going to, DON'T ACCEPT IT. GET THE ANTIBIOTICS. It may save you becoming Really Extremely Ill Indeed because you got quinsy which affects your breathing and leads to having to stay in hospital where lots of exciting things happen like oxygen masks and intraveneous antibiotics. Also, if symptom checkers say "can you do this" and you technically can but it causes agonising pain, count that as "I can't do this" I am very grateful for the NHS, but I wish the non-A&E bits of the NHS we visited were a bit more competent, like the A&E bit we visited was.)


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Late February is always a busy time around here. It's still winter in southern Oregon. That means cool temperatures, especially in the mornings. And before Spring arrives, this is the time to welcome back our farm crew and ready our Certified Organic farm for the long growing season ahead.
Here's a short list of what we do during our farm's opening week. Continue reading "9 ways we get the farm ready"


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"i'm a fool when you smile at me, i'm convinced-i get caught up in you and i can't resist/i know you lied to me, it just drives me nuts, that one kiss could convince me to believe your bluff"
i don't think it's a coincidence that the "marriage" is between brat and snoop-as people have been saying for years that she jacked his flow, but it was also obviously an homage to "ain't no fun", my favourite misogynistic youth anthem. i'm sure it was missy's genius ass that put all these pieces together.
let's take all of our love muffins rollerskating, already.


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You're all invited to Soho House for a fun-filled day celebrating all things Bollywood! Activities will include Henna, Indian food stalls, Music, Bouncy Castle, Crafts, sport activities, face painting and much more!
Access to the gardens, visitor centre and exhibition is free.
Tours half price. Some charges apply for activities.
Tour times: 11.30, 1pm & 2.30pm
Charges may apply for some activities.


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QB Mason Rudolph of Northwestern has a top two and is rapidly closing in on decision time. LSU and Oklahoma State are the favorites. "I'll probably make my decision in the next two or three weeks and then I'll camp at the school I plan on going to to get more familiar with their coaching staff," he said. Rudolph did not name a leader. He picked up an offer from Virginia last Wednesday, his sixth. The Cavaliers join LSU, Virginia Tech, Oklahoma State, Ole Miss and Louisville. Of those that offered, Louisville is the only school that has not visited Rudolph this spring. Other schools to come by were Alabama, Tennessee, Vanderbilt, Notre Dame, Appalachian State, Arkansas and North Carolina. "South Carolina has not been by," said Rudolph. "I am pretty sure Georgia is planning to come by later this week. I've talked to pretty much every school that has been by to see me."
LB Christian Miller of Spring Valley Friday announced a decommitment from the Florida Gators. Miller had committed to the Gators in mid February. He is still considering the Gators along with Alabama and Tennessee.
OL David Sharpe (6-8 290) of Jacksonville holds 14 offers including Clemson and USC. His others are Florida State, Florida, Alabama, Georgia, North Carolina, Duke, Miami, Purdue, Wisconsin, Michigan State, Ohio State and Georgia Tech. He has visited Florida and Florida State and will get to Clemson and USC this summer. Sharpe does not have favorites but does have interest in USC and Clemson. "I like South Carolina, I like coach (Steve) Spurrier Jr.," he said. "He wants to get me up there and see the campus. He said I'll fit in well with the program. I've been talking to coach (Jeff) Scott and he wants to get me up there also." Sharpe has never been to USC or Clemson. He said all the schools are recruiting him about the same. He'll make his decision after the season. "I'm looking for some place where I'm comfortable," he said. Sharpe grew up a Florida fan but said that won't factor into his decision.
DL Zeke Rodney of South Pointe picked up an offer from Toledo after the Rockets paid him a visit during spring practice. His other offers are SC State, Boston College, Louisiana-Lafayette, Marshall, Charlotte and Appalachian State. Clemson, Kansas State, Tennessee, Boston College and Indiana have also visited Rodney recently. He was happy with the offer. "We didn't talk much but they want me to come up for a visit," he said. Clemson assistant Jeff Scott visited Rodney at practice and invited him to a camp over the summer. "I plan on going," said Rodney. Florida State and Indiana are camps he plans to attend. "I camped at Carolina last summer so I probably won't this year," said Rodney. "I will go there for a visit though." Rodney does not have any favorites.
WR Terry Googer of Atlanta will not visit USC this Saturday. At one time Googer was thought to be visiting with a group of prospects from Georgia. Googer will try to visit this summer.
Northwestern DB Corey Seargent was offered Thursday by Charleston Southern.
WR Quadarius Fair of Andrew Jackson is moving away from the area and won't be playing football there this season according to coach David Moore. Fair was not out for spring practice due to the pending move. Fair is also a basketball prospect and may be pursuing that.


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The three day weekend made it possible to get away! Luke and I left for Spokane on Saturday morning after getting breakfast bagels at Moscow Bagel. The bagels were really yummy and gave us energy to drive behind all the super super slow drivers on the two lane road to Spokane. We met up with my Aunt Grace and cousin Katie at the Riverfront Park Ice Skating Rink.
Go Luke go!
Katie takes to ice like a fish takes to water!
Cousins! The rink was having a Beach Party, which explains the Leis.
After we were done Ice Skating we all went back to Aunt Grace's house where we met up with Jenny, and Allie and played with all of Katie's toys.
Katie riding Butterschotch the mechanical horse, who scared the tiny pink mechanical poodle that Jenny was holding.
Luke,sitting in the tiny kids chair. What a picture.
Katie prefers to ride Butterscotch sidesaddle while wearing a cape and holding her sword.
Who knew Jenny was so good at performing the Super-woman stunt?
We (Luke) also helped break in Katie's Wii this weekend. We learned two new games: Dance, Dance, Revolution and High School Musical Sing-a-long. So much fun!


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Atrial Flutter and Radiofrequency catheter ablation questions...
I'm a student, and we're suppose to interpret a rhythm strip. I have a rhythm strip for a patient who has atrial flutter. And the pt. had radiofrequency catheter ablation to correct the atrial flutter. I was able to observe the procedure in the cath lab at a local hospital.
My questions are:
Does the patient receive any cardiac medications to correct the flutter before catheter ablation is attempted to permanently cure the atrial fluter? If so, what meds are used?
Lastly, I understand the P waves in atrial flutter are not measurable, that's what I read in the book. The ORS complex is measurable and it is sually less than 0.10 sec. Is it possible to measure the QT interval in atrial flutter? I am trying to find the end of the T wave in my rhythm strip, but I can't find it. The example in the book said that QT intervals can't be measured in atrial flutters.
I was merely observing that day during the clinical, so I didn't get to see his medication lists before the procedure to see if he had any cardiac medications.
About Animaniacs, BSN
Joined: Feb '04; Posts: 108; Likes: 4
Specialty: 6 year(s) of experience in Med-Surg/TelemetryOct 31, '05Pts in A.Flutter are treated with meds before ablation is attempted. Just think..meds before anything invasive. Sounds like this pt was unable to convert with the use of medications. Depending on pts hx and dx the meds could be betablockers, calcium channel blockers, digoxin, and/or amiodarone. This pt should also be on a anticoagulant, such as coumadin or lovenox. This is held for the procedure, of course.
You are correct about the inability to measure a T wave or QT in A.Flutter. The 12-lead is able to come up with those measurements. Just because you can't measure it on the 5-lead, doesn't mean there isn't one. Remember..the Qwave is the beginning of the QRS (ventricular contraction) and the T wave represents the ventricles repolarizing-getting ready for another contraction. Atrial Flutter is a dysrhymthia of the atrium, not the ventricles.
Hope this helps!!:wink2:


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COLOR: Black
Midi dress with straps and button fastening in the front. Features a tie belt around the waist and side pockets.JOIN LIFECare for fiber: TENCEL®Lyocell. TENCEL® is derived from wood grown in sustainable forests that guarantee reforestation.HEIGHT OF MODEL: 178 cm.


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Two Texans killed in I-55 accident
SPRINGFIELD. Ill. (WRSP) —
Authorities in Montgomery County are investigating a crash that killed two people from Texas.
It happened on Interstate 55 at mile post 52.
One car sideswiped the other causing it to lose control and overturn.
Both the driver and the passenger were ejected from the vehicle.


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No matter where you live, or what kind of a home you have, there are a number of common disasters that can always strike in your home. Often it is a matter of doing whatever you can to try and keep these disasters at bay, and that can be easier said than done. However, as … Continue reading 3 Home Disasters That Can Be Averted
Keeping those memories alive…
Life is all about making memories and memories can be made in many different ways. It's not just about those big milestone events such as birthdays, graduations or weddings, it's also about those times where you burst into fits of laughter with your best friend, when someone who made a big impact on your life … Continue reading Keeping those memories alive…
Feeling Uninspired? Here are Some Places to Look for a Fresh Perspective
Sometimes when we are halfway through a creative project, we can run out of inspiration and what once was a joy can slowly become a grind to get finished. There are some typical places you might head for inspiration, but there are some more unusual ideas that you should consider next time you find yourself … Continue reading Feeling Uninspired? Here are Some Places to Look for a Fresh Perspective
A Healthy Space Equals A Healthy Outlook!
There's something about rearranging your living space that does a lot for your state of mind. Once you've got your own personal style sorted out you can really reflect yourself in your surroundings, or you can create a haven of an interior based on a single theme you've always loved. After all, being able to … Continue reading A Healthy Space Equals A Healthy Outlook!
6 Things That Confirm You're An Adult
Nothing is more terrifying than the thought of growing up and becoming an adult. That's something we all learned from watching Peter Pan too many times, our fingers crossed as we silently wish Neverland is a metaphor for real life. Of course, deep down, we know this isn't true. It's why you've got a "Don't … Continue reading 6 Things That Confirm You're An Adult
Five Things to Check when Spring Cleaning Around Your Home
When the weather turns warm and Bank Holidays arrive, you might want to make the most out of the time you have at home and clean out your conservatory, give your house a spring clean, or even deal with things that need to be fixed. Once you move the furniture, you might come across some … Continue reading Five Things to Check when Spring Cleaning Around Your Home
Being Stylish Whilst Being Yourself
When it comes to decorating the interior of your house, there's so many themes, colours, and designs to choose from. You can easily go minimalist if you're on a budget, with white washed walls and one painting in your living room. Or you can go cluttered but homely, with pencil pots, stacks of books, and … Continue reading Being Stylish Whilst Being Yourself
Building A Fantasy Land In Your Back Garden
Your garden. The place where weeds are likely to overgrow, the fence is dark and leaning, and the shed hasn't seen the light of day in at least a year. And all this adds up to a sense of mystery and adventure, just like walking through the woods and discovering hidden sounds, sights, smells, and … Continue reading Building A Fantasy Land In Your Back Garden
More Truths About Cohabiting You Never Thought About – Part 2
Moving in with the person you love might sound like a dream, but the harsh reality tells a different story. In fact, a lot of couples are very clear about the difficulties they've experienced – and continue to experience – when they decided to share a home. There's no point beating around the bush. When … Continue reading More Truths About Cohabiting You Never Thought About – Part 2
Renovating our conservatory…
It has been agesssss since I have done a renovation post, in fact the last one I did was in June 2016 when we renovated our bedroom (which has a lovely new king size bed now 😬), so I've decided to share our conservatory makeover with you, which to be fair we have only just … Continue reading Renovating our conservatory…


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Free Court Index Search - Private Investigator Blog Free Court Index Search A free search of a court index that's more flexible than the one at the county court site is a desirabl...
Sunday, September 04, 2005
~ cell phone number directory anyone? ~
The inclusion of cell phone numbers in published directories is increasing in Europe and a majority of American wireless companies endorse the development of U.S. directory. Read the article


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Are Video Games Dangerous?
This is the million dollar question constantly being asked by all concerned individuals especially parents with kids. Are video games dangerous? Is it possible to get addicted to playing video games? If yes, what are some of the signs that indicate that one is addicted?
Let's start off by saying that video games are designed to be addictive in the first place. By this I mean video game designers are constantly coming up with new innovations including storylines to make their video games stand out. After all if they are not interesting in the first place who will buy them talk less of play them. There are various strategies video game designers use to keep us engrossed with playing. Some of the clever features include breaking a game into levels of difficulties (starting off with the very simple to the complex), trying to beat the highest score or even beat the computer game. All these features can leave you playing for hours.
While video game addiction is not widely accepted as a problem in many circles (due to a lack of sufficient medical research), evidences yet abound to proof that it is a problem. In 2005 a Korean man dropped dead after playing an online computer game for 50 hours. A circuit Judge in 2009 vacated his seat after it was discovered that he crashed his car because of a night of video games with a former defendant. A youth was involved in murder after hours of playing the video game "Grand Theft Auto.
The most common sign of addiction is a strong compulsion to play for long hours while isolating family members and friends in the process. This can lead to things like not having enough sleep, missing or not eating healthy meals, feeling agitated when unable to play and a break-down in relationships. With kids it can be playing games rather than doing their homework.
But videogames can help kids improve hands-eyes coordination, learn problem solving and analytical skills. Playing video games like Dance Central, Wii Fit can keep us healthy. Video games can also be a means of relaxation encouraging and building interaction between family members
So where does this leave us? We strongly feel that video games are not dangerous but the overuse of it is what leads to addiction. Just like alcohol or any other thing in life there is a need to exercise self control. Parents need to keep an eye on their kids especially the type of video games they play. Gamers need to understand that they need to take regular breaks and not stay glued to their gameplay. Also remembering to take regular break at least 5mins for every 45 – 60 mins of gameplay can help reduce impact of game addiction.
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[Tru Blu Tuesday] What Dzokhar Tsarnaev Deserves
William Roper: So, now you give the Devil the benefit of law!
Sir Thomas More: Yes! What would you do? Cut a great road through the law to get after the Devil?
William Roper: Yes, I'd cut down every law in England to do that!
Sir Thomas More: Oh? And when the last law was down, and the Devil turned 'round on you, where would you hide, Roper, the laws all being flat? This country is planted thick with laws, from coast to coast, Man's laws, not God's! And if you cut them down, and you're just the man to do it, do you really think you could stand upright in the winds that would blow then? Yes, I'd give the Devil benefit of law, for my own safety's sake!
– Robert Bolt, A Man for All Seasons
When two explosions rocked the country last week on April 15th in Boston, we were sent spiraling into sadness and anger. It is to be expected that a nation so heartbroken would respond with passion, and so it should. However, in our passion we must not forget the qualities which made us great. One of those qualities, as etched in the Constitution over and over again, is the defense of the minority – the smallest minority of all being the individual. This concept oozes from such clauses as the right to free speech, the right to bear arms, the protection from unlawful search and seizure, and the right to due process of law. These rights are not limited to the pure among us. They are not set aside for only the good citizens. They are for everyone.
Yes, that includes the devil himself, should he ever show his unsightly face in this country. Today nineteen year old Dzokhar Tsarnaev wears the devil's mask. In this past week he has become the face that so many now instantly recognize and despise. Yet despite the awful crime with which he has been held suspect, this young man is still under the protection of the supreme law of the land.
What protections are those? The Fifth Amendment for your review (emphasis added):
No person shall be held to answer for a capital, or otherwise infamous crime, unless on a presentment or indictment of a Grand Jury, except in cases arising in the land or naval forces, or in the Militia, when in actual service in time of War or public danger; nor shall any person be subject for the same offense to be twice put in jeopardy of life or limb; nor shall be compelled in any criminal case to be a witness against himself, nor be deprived of life, liberty, or property, without due process of law; nor shall private property be taken for public use, without just compensation
And the first section of the Fourteenth Amendment:
Section 1. All persons born or naturalized in the United States, and subject to the jurisdiction thereof, are citizens of the United States and of the State wherein they reside. No State shall make or enforce any law which shall abridge the privileges or immunities of citizens of the United States; nor shall any State deprive any person of life, liberty, or property, without due process of law; nor deny to any person within its jurisdiction the equal protection of the laws.
Because Tsarnaev is, in fact, a citizen of these united States, he has every claim to these clauses. Furthermore, until he is proven guilty through due process, he is to be presumed innocent. As difficult as that may be, considering the aforementioned passion behind this case, he is still entitled to them. This entails that he be read his Miranda rights, be provided legal defense, tried before a jury of his peers and provided every other accommodation required by the Constitution.
Why should we do this? Why give a person, someone suspected of the most atrocious of crimes, the protection of law? Because in protecting him, we protect ourselves. If we cut down the laws, as William Roper would have done, in order to get after the devil, we will have nothing with which to protect ourselves when he turns back towards us. "But I haven't done anything wrong" you might say. Oh? But how would we know that without the law to determine justice? When there is no respect for law and order, the line between right and wrong is virtually erased and the good among us are no more innocent than the evil. Under such a horrid circumstance, we are all Dzokhar Tsarnaev.
Dzokhar Tsarnaev derserves what we all deserve. The right to due process of law.


text:
If you're a Bay Area resident who's too terrified to watch the utter destruction of San Francisco via earthquake and an epic tsunami in the new movie "San Andreas," well, you may be in the minority.
"San Andreas" is the No. 1 film in the country, a statistic buoyed by high ticket sales in San Francisco theaters.
San Francisco's AMC Metreon Theater was the No. 4 grossing theater in the country for "San Andreas" and the three SF theaters showing the film led to a 71 percent increase in moviegoer traffic in the city, according to the Hollywood Reporter.
"Some of the naysayers said people living in these places would be too scared to see 'San Andreas,'" Dan Fellman, president of domestic distribution at Warner Brothers, said in the publication. "Obviously, they were wrong."
If a rising panic sets in while checking out your backyard being destroyed on the big screen, keep in mind that seismology experts have charged the film's quakes, which measure over the size of 9 on the Richter scale, with faulty science.
"Even if the entire San Andreas were to break all at once from north to south you wouldn't get anything larger than an 8-ish earthquake," Jean Paul Ampuero, an assistant professor at Caltech, told NBC News.


text:
The object of the picture tag is to:
1) Choose the 4th folder where you store your pictures on your computer
2) Select the 4th picture in the folder
3) Explain the picture
4) Tag 4 people to do the same
I tested on my computer what the 4, 4 photo would be. Then when I went to upload, I got my folders in a different order and a different photo. Here are the gentlemen snuggling with Thomas.


text:
guy lipmore
'Only A Game'. - Poem by guy lipmore
Just a simple game of Monopoly,
Over seven hours of 'fun' for the family.
A bloody trouncing they got on the night,
Blew them out of sight.
An emphatic win, just a fact,
So just to recap.
Twelve houses, thirteen hotels, plus over seven grand,
As predicted and planned!
A master class of wheeling and dealing,
Astute purchasers and canny selling.
Like five grand for Old Kent Road,
They mocked, but it was clever and bold.
Lip-More was the king of finance,
The others did not stand a chance.
Yes, business is a ruthless game,
However, he felt no shame.
There was no guilt on his conscience,
Regards to battering his opponents.
Their wounds they are licking,
After their absolute kicking.
For them it was bit galling,
They were feeling raw after their mauling!
They still won't admit,
And just get over it.
Topic(s) of this poem: family
Poet's Notes about The Poem
Comments about 'Only A Game'. by guy lipmore
Ratnakar Mandlik (11/13/2015 11:39:00 AM)
Business is a ruthless game
However, he felt no shame
There was no guilt on his conscience
Most realistic observations penned in the form of this poem. Thanks for sharing.10 points. (Report) Reply
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text: This video by buzzfeed has been a hit on social media. It shows make up artists applying make up to men for the first time. The mens reactions are priceless. I think they really liked the way they looked with make up and the hair extensions. This video shows me that man or woman, make up really help make people look better. Even if it is just a bit of foundation.


text:
Learning lab: What bacteria can teach us about communication and teamwork
Regular readers may remember "What's bugging us: Cockroaches rival humans in ability to make decisions as groups", a post that signaled my return to blogging following a brief break.
I now return to blogging at the end of a similar break. Therefore, it seemed somehow fitting to do so with a post that explores a related theme. This one concerns not cockroaches, but an even lowlier lifeform: germs.
Not only do cockroaches work well in groups, but so, too, do bacteria, according to "A Biologist's Listening Guide to Bacteria", a recent story on National Public Radio, which featured an interview with Bonnie Bassler, a professor of molecular biology at Princeton University, who has made some intriguing discoveries regarding marine bacteria.
Bassler's work demonstrated the capacity of these tiny organisms to actually communicate with each other. In studies done on glow-in-the-dark bacteria, Bassler and her assistants learned that
It turns out that when one of these bacteria is all alone, it doesn't glow. After all, that would be a waste of effort because nothing could ever see such a tiny amount of light. But it does send out chemical signals that say, hey I'm here ... and it listens back for other bacteria sending the same signal.Okay, folks, if cockroaches and bacteria can communicate and work together, what's up with us humans? Hmmm?
When enough bacteria are doing this, they know they have a quorum. All of a sudden, they light up and do all sorts of other things to act in concert, like a super-organism.
"So they turn on and off 100 different genes, to let them turn off behaviors that are good when you're alone and turn on genes that are good when you are a community. And for reasons we don't understand, the gene that lets them make this beautiful blue light is one of the genes they turn on," Bassler says.


text:
HOUSTON—City workers took to the steps of City Hall Monday to address the City of Houston's wage theft ordinance and other agreements with the city.
City employees including security officers and janitors have filed multiple complaints with the Inspector of General Security officers. The workers claim that the city has been underpaying them for years and is in violation of multiple city wage ordinances.


text:
April 22,2018
Walking in my P U R P O S E all 2O18. 🦋
As I follow purpose, blessings are following me!! It's the chain of command. When you do what you're CALLED to, favor has NO choice but to follow in pursuit of you! -Jess 💙
This true blue outfit 💙 was a fun color blocking adventure lol. The off white peplum top is from Forever 21…super comfortable and cute. The royal blue pencil skirt is from Dillard's as well as my Jessica Simpson pumps. Catch this! These accessories (earrings & necklace) are from Walmart! 🙌🏽 Let's not sleep on them amen? Lol Lastly, my Michael Kors watch and then a pair of gold bangles with a teal ring both from Forever 21. Email firstname.lastname@example.com for all styling inquiries!


text:
Goblin Valley, Utah, USA
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Keywords: 1997, colorful, differential erosion, earth science, entrada sandstone, eroded, eroding, erosional feature, geography, geology, goblin valley, goblin valley state park, hoodoo, jurassic deposit, landform, landscape, mushroom-shaped pinnacle, north american, red siltstone, rock formation, rock pedestal, rounded, sandstone, scenic, sedimentary rock, spheroidal weathering, toadstool rock, united states, utah, valley of the goblins, weathered
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text:
Special sex is an amazing experience which everybody takes pleasure in. Females like sex equally as much, otherwise even more, compared to individuals! This may break just what you believed you comprehended, yet it's exact. However have you uncovered of current that your lovemaking has lost a little of it's dazzle? Never stress or give up on it! There are a variety of points which you can perhaps carry out in order to recover it to its former splendor and also you can perhaps be giving her leg rattling climaxes in no time!
Probably the least fascinating location that you can make love is your bed room. If you want to enliven the whole sexual experience, sex furniture is a fantastic method to go. Sex furniture is generally really discreet and also blends in well with the other points in your house. Unlike flat surface areas like a bed or a mattress, this kind of furniture is most likely to allow you explore a broad range of settings that you would certainly have previously not assumed achievable. You are a whole lot more likely to discover fun sex settings which are most likely to stimulate her clitoris and also g-spot in something like a sex swing instead of on a bed.
You might have heard this before, yet I merely can not emphasize sufficient just how much ladies like being kissed by their enthusiasts. It is something so important and also fundamental but yet it appears to be overlooked by a great deal of men. Attempt a few of these attractive moves on your lady if you intend to crackle up your sex life. Suck on an ice cube or sip on a chilly beverage prior to kissing her. This will get her hot in no time flat! Or maybe attempt introducing some actual sweetness to a kiss by consuming strawberries or putting a small amount of honey in your mouth right prior to securing lips. It's likewise crucial not to neglect to kiss her while in the act of having sex.
One certain cornerstone of extraordinary sex is establishing the scene and also creating an atmosphere around you. Pay really very close attention to the sights and also appears regarding you. Maybe attempt installation a couple of tinted light bulbs to provide the location a sexy background. It has been located in studies that the shade purple can emphasize a lady's orgasm. This is a truly fast method to rev up her sensual energy. Whilst you are at it, why not play some songs to offer a sound track to your session? Prior to beginning sex, ladies appreciate a moment of affection and also affection. Attempt sipping on a few glasses of wine and also hearing something hot and also steamy on the CD player. Explore her body and also locate her pleasure areas before a fire location if your have one.
Take your time and also do not rush right into sexual activity. This could be among the greatest mistakes you can make if you intend to get your woman DEFINITELY switched on. The majority of the time, a lady will take a bit to find about to the idea of sex and also end up being aroused, where as a male can do the precise very same point in a heart beat. For several ladies, a sensuous massage therapy will get her electric motor running hot. This certain type of massage therapy, in contrast to a normal one, is one where you will focus more on her sensual areas. Take your time to discover where these are. They can differ from woman to woman. Enjoyable sex settings that have worked for a loved one previously might not help your existing woman. Be actually sexy and also mindful toward her and also she will soon allow you understand when it's time to take it to the following degree. All ladies actually enjoy it when their clitoris is stimulated. This can be with a hand or with foreplay yet there is likewise a brand-new method to do it. The Womanizer is a brand-new and also amazing clitoral stimulator that makes use of light air bursts to stimulate the clitoris. The majority of ladies climax after just a few mins. That is amazing. This short article regarding womanizer has all the information you need. Go there, check out the wonderful short article and then look for the very best offers on this exciting new product.
You can actually provide her something to review with her close friends if you attempt a few of these suggestions out. Women speak about sex with their partners far more compared to men do, think me. If you want to actually inject some seasoning right into you enjoy life, try out a few of these methods when points following fume and also heavy with your fan.
Make it so that your actions show up to have an all-natural circulation and also rhythm. A great method to figure out just what turns her on is to introduce a couple of sex playthings right into your sexual activity. It will be a truly excellent method for you to uncover just what turns her on and also enter sync with her sexuality. If she possesses a vibrator, you can learn a whole lot from her if you get her to reveal you just how she uses it. Take notice of the speed, angle and also the type of stress that she makes use of to pleasure herself and then mimic these techniques on her using your hands, tongue or your johnson!
Interior prostate massage therapy is likewise a really effective device for any kind of woman to have in her bag of sexual tricks. Its purpose is to lengthen climaxing, due to its extraordinary feeling for men, several a savvy woman use this method to really speed climaxing up when essentially, they are ready for sex to end.
She is most likely to discover sex a boring or monotonous affair if you are short of a bit of creative thinking in the bed room. Utilizing your imagination, I bet you can consider some fun sex settings to attempt. While her body is not something you should look over, her mind is one of the most crucial sex organ and also the one that you must seek to stimulate one of the most. Look directly right into her eyes and also love her with your words and also touch. She will be a whole lot more available to sex if your tickle her fancy a little. Something that can get her actually hot is a little bit of attractive sensual literary works. If you want, you might like to attempt reviewing her a couple of web pages from this grown-up material prior to going off to bed. When you arrive, she could have a little something more than rest on her mind.


text:
We stopped for lunch on a saturday and although we were seated right away, our waiter was a scattered mess and from the looks of it our table was not the only one feeling the pain. It took him a long period of time to...More
1 - 10 of 358 reviews
We stopped for lunch on a saturday and although we were seated right away, our waiter was a scattered mess and from the looks of it our table was not the only one feeling the pain. It took him a long period of time to...More
This restaurant had impeccable service. A great adult environment that was also family friendly. The raspberry sauce is amazing! I wouldn't skip the cotton candy spun on site as a great treat for the whole family!
Doesn't look like much from the street, but was surprisingly good. Salads were fresh and and my turkey sandwich was good. Popcorn is served to each table to munch on. Service was good and efficient. Inside or outside dining available.
This is a good options to go with kids right in the middle of the main street. They give out popcorn at the table when you arrive. There is a fairly varied menu and goods kids menu. The server was fantastic. Very sweet and nice....More
We stopped in around 11:30 am on a Sunday. The inside, where we were seated, was empty. Our waitress was friendly to begin with and we got drinks and placed our order quickly. Then it went downhill. Two more tables were seated and it took...More
Circus Cafe has always been sort of an awkward focal point in my opinion. I lived in Saratoga for awhile and I never visited. On my most recent trip we decided to pop in for a drink mid-afternoon. I was surprised by their huge drink...More
Good food, good atmosphere, great location and the kids love it. They also have outdoor seating which is nice and the popcorn is good
A fun place to be! The food was outstanding. I order the spicy shrimp Veracruz, the best Ive ever had. Our server was fantastic. We will definitely go back when we are in NY again. I highly recommend circus cafe for all ages
It was very busy and hot but we sat outside at a table in the shade. Food was okay, nothing special.
We just needed something to hold us over until dinner so we stopped at the Circus Cafe. We had a Mediterranean Antipasto plate and quesadilla. The Mediterranean plate was pita bread, homemade hummus (YUM!), kalamata olives, roasted red peppers and cucumbers. There was a good...More


text:
Thewindows 10 logonui.exe application erroris a kind of structure which is used by House windows and other Microsoft appropriate applicationand suppliers.Companies use codes to distinguish what brought on the trouble. This can be by means of a technological description and/ora numeric mistake amount. In certain circumstances the problem might have much more factors in the format. Any additional hexadecimal computer code contains the deal with of the memory places where instruction(s) have been filled throughout the duration of the problem.
Plus, This is usually the consequence of poor gateway. You may have to restart your router and your personal computer to be able to continue with the up-date.Generally, this really is the result of a malware that may be blocking Windows Update or WSUS from obtaining Microsoft Hosts.Residuals of your infection strike may have also messed together with your House windows Upgrade Program registry. Which means you will not have consent gain access to Microsoft servers.


text:
According to CTD, police were deployed at all entry and exit points of Punjab's capital to curb the threats of more attacks looming after the Lahore blast.
Right as police approached a vehicle to search, armed men inside the vehicle opened fire. Police effectively responded, and in retaliatory fire, four terrorists were killed, while three others managed to flee the scene in the dark of night, spokesperson of CTD added.
CTD claimed to have recovered a huge quantity of arms and ammunition from the custody of the terrorists who are said to be affiliated with the banned outfit of Tehreek-e-Taliban.
Meanwhile, a heavy contingent of police has been called in the area to launch search operations for apprehending the absconded accomplices of the killed terrorists.


text:
|The stupid question is the question not asked
Re^5: A nice little question for the insaneby crenz (Priest)
|on Apr 06, 2005 at 11:37 UTC ( #445262=note: print w/replies, xml )
|Need Help??
Yes, you're right. However, I don't know the scope of the original poster's problem, so I thought I'll just supply the link so that the OP can think about whether the modules might already do what (s)he needs to do -- or not.
In Section Seekers of Perl Wisdom


text:
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Punchbowl Falls is a photograph by Inge Johnsson which was uploaded on December 10th, 2011.
Punchbowl Falls on Eagle Creek, Oregon... more
Wall Art
Home Decor
Punchbowl Falls
Inge Johnsson
Photograph - Digital Capture Watermark Not On Actual Print
Punchbowl Falls on Eagle Creek, Oregon
Eagle Creek is a creek in Hood River County, Oregon, United States. The creek drains into the Columbia River in Multnomah County, with its outlet on the Columbia River Gorge. It is in the Mount Hood National Forest. The Eagle Creek Trail, the most popular trail in the Gorge, follows the creek. Tunnel Falls and Punch Bowl Falls, located on Eagle Creek, are remote and scenic waterfalls along the creek. The Historic Columbia River Highway crosses the creek's outlet to the Columbia River. A substantial hike through some occasionally rough terrain is required to get to the creek's namesake waterfall. Punch Bowl Falls has been featured in inspirational posters and other media. Eagle Creek Trail was built by the CCC and WPA in the 1930s for the Pacific Crest Trail, probably to cross the Columbia River at Bonneville Dam. The PCT was rerouted to its current route some 1.5 miles (2.4 km) further east sometime by the early 1970s to connect with the Bridge of the Gods across the Columbia River.
December 10th, 2011
Viewed 7,423 Times - Last Visitor from Beverly Hills, CA on 06/22/2018 at 3:16 AM
Sales Sheet
More from Inge Johnsson
Comments (68)
Beautiful image. Congratulations.
EyeCatcher Image!.......Congrats, Inge!......L/F....Carry On!
Splendid beauty.
You've done it again, Inge. This is just superb. L/F
Such a beautiful setting and capture!
Wow! Beautiful capture f/v L
So Lush and beautiful! v
Love the colors....L
A true paradise on this beauty, Inge! Love the lush greenery, the rocks, moss, and the beauty of the flowing water. Superlative presentation. It is a pleasure to see so much variety and beauty in the works that are submitted for FEATURING on our WFS site. It is an honor to FEATURE this piece of work on our Homepage of our site. Thanks for sharing and being active in our group membership. Liked Forever, Elvis
So lush and beautiful!!!! :)))fv
Nature at its finest! Breathtaking! v
Beautiful shot Inge! f/v
Tough shot to do with the sun hitting the trees, but you certainly pulled it off! Nice shot. Someday I will hike into here and give it my best shot.
Started to day Inge ..
This is beautiful! I hope to see and experience this waterfall someday.
A perfect image! V/f.
Again...?? Well deserved Inge!! I grew up about 45 minutes from here - lots of beauty - you've captured it! Congrats...
Yes, again :-D Thanks. Fantastic place. And even better now that log is gone.
I can see why this sold! Congratulations! It's a gorgeous shot! :) V/F
Thanks Patricia.
Inge, Stunning! Congrats on your sale!
Thank you!
simply wonderful
Many thanks. Wonderful place.
Yes, perfect!
Thanks so much, Skip!
Gorgeous landscape, beautiful light and color, congratulations on your sale!!
Thank you, Christina!
Fantastic image, Inge!! v
Lynn, thank you :)
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