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MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by targeting mRNAs for translation repression or mRNA degradation. Many miRNAs are being discovered and studied, but in most cases their origin, evolution and function remain unclear. Here, we characterized miRNAs derived from repetitive elements and miRNA families expanded by segmental duplication events in the human, rhesus and mouse genomes. We applied a comparative genomics approach combined with identifying miRNA paralogs in segmental duplication pair data in a genome-wide study to identify new homologs of human miRNAs in the rhesus and mouse genomes. Interestingly, using segmental duplication pair data, we provided credible computational evidence that two miRNA genes are located in the pseudoautosomal region of the human Y chromosome. We characterized all the miRNAs whether they were derived from repetitive elements or not and identified significant differences between the repeat-related miRNAs (RrmiRs) and non-repeat-derived miRNAs in (1) their location in protein-coding and intergenic regions in genomes, (2) the minimum free energy of their hairpin structures, and (3) their conservation in vertebrate genomes. We found some lineage-specific RrmiR families and three lineage-specific expansion families, and provided evidence indicating that some RrmiR families formed and expanded during evolutionary segmental duplication events. We also provided computational and experimental evidence for the functions of the conservative RrmiR families in the three species. Together, our results indicate that repetitive elements contribute to the origin of miRNAs, and large segmental duplication events could prompt the expansion of some miRNA families, including RrmiR families. Our study is a valuable contribution to the knowledge of evolution and function of non-coding region in genome.
Citation: Yuan Z, Sun X, Liu H, Xie J (2011) MicroRNA Genes Derived from Repetitive Elements and Expanded by Segmental Duplication Events in Mammalian Genomes. PLoS ONE 6(3): e17666. doi:10.1371/journal.pone.0017666
Editor: Marc Tjwa, University Hospital Frankfurt, Germany
Received: September 12, 2010; Accepted: February 10, 2011; Published: March 16, 2011
Copyright: © 2011 Yuan 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 the National Natural Science Foundation of China (Project No. 61073141 and No. 60971099). 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.
Several hypotheses for the origin and formation of new microRNA (miRNA) genes have been proposed [1]–[3]. MiRNA genes may originate from inverted duplication (mainly found in plants) [4]–[7], tandem duplications (also called local duplication) [8]–[10], segmental duplications (SDs) [9],[11], random hairpin structures located in intronic or intergenic regions [12], or from repetitive elements especially transposable elements (TEs) [13]–[20]. Repetitive elements are multiple copies of DNA sequences present in the same genome and also classified as tandem arrays or interspersed repeats [21], [22]. Tandem arrays include satellites, telomeric repeats, subtelomeric repeats, microsatellites and minisatellites. Interspersed repeats encompass TEs (DNA transposon, LTR retrotransposons and non-LTR retrotransposons) and processed pseudogenes [21], [23]. Some SDs (intrachromosomal duplications) are also referred to as low copy repeats [24], [25]. Repeat sequences are prevalent in the genomes of all plants and animals. For example, repeat sequences make up more than 50% of the human genome [24], and TEs account for 45%, 40%, 15–22%, 12%, and 8.6% of the human, mouse, fruit fly, nematode and chicken genomes, respectively [24], [26]–[28]. In some plants, TEs constitute up to 90% of the genome [27].
TEs are attracting more attention than before because more and more evidence shows that TEs play a major role in shaping the structure and function of the genome. TEs, when inserted and integrated upstream of a gene, may change the expression pattern of the gene [29]; in an exon they may produce a new protein domain [29], [30]; and when inserted into an intron TE may to produce a de novo protein [31], [32]. There is also some evidence to suggest that repeats may have contributed to the birth of miRNAs. That some miRNAs are derived from genome repeats in both sense and antisense directions was first documented in Arabidopsis thaliana [33]. In animals, repeat-derived miRNAs (RdmiRs) were first discovered in the human, mouse and rat genomes. Initially, only 7, 9 and 10 miRNA genes containing repeat sequences were found in the human, mouse and rat genomes, respectively [19]. Later, Piriyapongsa et al reported 68 human miRNA genes that share sequences with TEs, and discovered 55 TE-derived miRNAs among 462 human miRNA genes (miRBase 8.2) [18]. Now a lot more evidence is available to support the hypothesis that miRNAs could be derived from TEs in plants and animals [13]–[20].
Copy-number variant (CNV) is defined as a DNA segment at least 1 kb in length, in which copy number differences have been observed by comparison of two or more genomes [34]. Some CNVs, when fixed in a population, give rise to partial SDs [35], [36]. SDs are segments of DNA >1 kb in length that occur in two or more copies per haploid genome, with the different copies sharing >90% sequence identity [37]. Although previous reports have mentioned that novel miRNAs can be produced from miRNA gene duplication [4]–[10], there is little known about the miRNAs that are produced and expanded in SD events. We hypothesized that, like protein-coding genes, miRNAs (including RrmiRs) may also duplicate and expand accompanying large genomic SD events in their evolutionary history. We tested this hypothesis and found that SD pair data are helpful in identifying some miRNA paralogs. We have defined a target SD as the SD that is duplicated from another SD, the source SD. A source and its target are defined as a SD pair.
In this study, we present a systematic study for the miRNAs derived from repetitive elements and expanded in the SD events in human, rhesus and mouse.
Materials and Methods
A genome-scale combinational method for detecting homologous miRNA
The miRNAs used in this study were downloaded from miRBase 16 (Sept 2010) [38]. The current miRBase contains 1,048 miRNA genes and 1,223 mature sequences from human, 672 miRNA genes and 1,055 mature sequences from mouse, and 466 miRNA genes and 488 mature sequences from rhesus. As in miRBase [40] and in previous reports [16]–[18], [39], the hairpin sequences of miRNAs are referred to as miRNA genes [16]–[18], [39] and their loci as microRNA gene loci [40]. SD data are all from the UCSC Genome Browser [41]. To utilize the SD pair data from the mm8 mouse genome assembly, we translated the coordinates of the miRNA genes from the mm9 mouse assembly to the mm8 assembly using the liftOver utility [42] provided by UCSC Genome Browser [41]. Because of a gap in the corresponding genomic region in the mm8 assembly, 7 miRNAs from the mir-290 family could not be mapped. These miRNAs are not included in the following analysis. Some in-house scripts were also used to process data.
We developed a combinatorial method to identify homologous miRNA genes. First, we obtained the whole genome pairwise alignment (human vs rhesus and human vs mouse) data from the UCSC Genome Browser [41]. These alignment data produced by the LASTZ (a replacement for BLASTZ [43]), chain and net program (http://hgdownload.cse.ucsc.edu/admin/jksrc.zip). We then used the alignment data to map the human miRNA genes to the rhesus and mouse genomes with the LiftOver utility [42] to obtain the genome coordinates of potential miRNA genes in the two genomes. Next, we filtered out the potential miRNA genes that were shorter than 39 bps or longer than 215 bps (These two values were determined based on the current miRBase sequence data for animals). The sequences of the preliminary potential miRNA genes were retrieved and classified using the MiPred program [44]. The region where several genome coordinates of miRNA genes overlapped (mapped from the human miRNA coordinates usually from the same miRNA family), was considered to be the only valid miRNA genome coordinate and is the only one used in the present study. Finally, potential miRNA paralogs in SD pairs were identified and classified with MiPred [44]. The newly identified orthologous and paralogous sequences of human miRNA genes were named according to the miRBase naming criteria [38].
Genome-wide analysis of repeat-derived miRNAs
Repeats were annotated with the RepeatMasker program [45] and the genomic positions of repeats were taken from UCSC Genome Browser [41] using the Table Browser [46]. The coordinates of mature miRNAs were calculated according to their host miRNA genes. We used these coordinates to identify all miRNA genes overlapping with repeat sequences with Galaxy [47] and in-house scripts. The data were analyzed using a suite of functions written in R (version 2.9.0) [48]. If the coverage density of repetitive elements was at least 50% in a miRNA gene or 100% in one of the associated mature miRNA sequences, then the miRNA gene was considered to be a RdmiR [18]. To conveniently analyze the data, the miRNAs with a low coverage density of repeats (coverage density of repetitive elements >0% and <50% in the miRNA gene and <100% in its mature miRNA) were called possible repeat-derived miRNAs (PRdmiRs). All RdmiRs and PRdmiRs were determined by this criterion, regardless of whether the miRNA genes were locate on the same strand or on the opposite strand to the overlapping repetitive element. In this study, the RdmiRs and PRdmiRs are both referred to as repeat-related miRNAs (RrmiRs). The other miRNAs with no overlapping repeats are the NRdmiRs.
All the coordinates of the annotated genes of the three organisms were downloaded from the knownGene and refGene tables in the UCSC Genome Browser [41], non-protein coding gene information was removed, and the difference in distribution of RrmiRs and NRdmiRs was determined. As in previous reports [49], [50], all the miRNAs were classified into intragenic and intergenic miRNAs. The minimum free energy (MFE) of the hairpin structures for the three pre-miRNA types (RrmiRs, PRrmiRs and NRdmiRs) from the three species was calculated using the RNAfold program [51]. If the sequences from the three species were identical, only one from each miRNA family was retained.
Calculation of conservation scores and miRNAs analysis in duplication data
Per-site conservation scores between 0 and 1 were calculated by phastCons program [52] based on the 17-species multiz alignment data [53] downloaded from the UCSC Genome Browser [41]. The 17-species include human, chimp, rhesus, mouse, rat, rabbit, dog, cow, armadillo, elephant, tenrec, opossum, chicken, frog, zebrafish, tetraodon and fugu. Because the only available conservation data for human are at Galaxy [47], and because they also include information for rhesus and mouse, we used the human miRNA genes to calculate the conservation scores for the NRdmiRs, PRdmiRs and RdmiRs. The total score and the average score of each pre-miRNA were calculated according to the single-nucleotide conservation score. To determine the lineage-specific miRNA families, the conservation scores of miRNAs, the paralogs and orthologs of miRNAs from miRBase, the new miRNAs discovered in rhesus, mouse and the other species were all taken into consideration.
To investigate if RrmiRs expansion accompanied SD events, we compared the genomic coordinates of the RrmiRs and SDs. CNV data have also been used to analyze the evolution of miRNAs. Human CNV data, which contain 1,445 copy number polymorphisms (CNPs), is from Redon et al. [36]. The UCSC liftOver program was used to convert human hg18 assembly coordinates for the CNV data to the hg19 assembly coordinates. The mouse and rhesus CNV data used in the analysis were obtained from the published data [54], [55].
Target prediction, functional enrichment analysis and functional network construction
The 3′UTR sequences of reference protein-coding genes of human, rhesus and mouse were downloaded from the UCSC Genome Browser. If there were multiple variants of the 3′UTR sequences, then the longest one for each of the protein coding genes was retained. Common targets of the conservative RrmiR families were predicted using the PITA [56] and miRanda programs [57]. Enriched Gene Ontology (GO) terms for the targets were analyzed with the Bioconductor package topGO [58]. To identify which of the functions were validated by biological experiments, we searched the literature in Pubmed (up to 8 December 2010) for the miRNA entities. We integrated this information with the data from miRTarBase release 1.0 that curates experimentally validated microRNA-target interactions [59], and constructed functional interaction networks using Cytoscape v2.7.0 [60].
Novel orthologs and paralogs of human miRNAs identified in the rhesus and mouse genome
The rhesus and human genomes diverged ∼25 million years ago and have 93% identity [61]. The mouse and human genomes diverged ∼75 million years ago and more than 90% of their genomes can be divided into corresponding regions of conserved synteny [62]. Because the divergence rate is low, the orthologous sequences of many functionally important elements (including the miRNA genes) can be aligned and identified and their cross-species conservation can be investigated [62].
We have identified 228 novel miRNA homologs in the rhesus genome (Mmul1.0 assembly) and 22 novel miRNA homologs in the mouse genome (mm9 assembly) (Text S1) using the method described in the Materials and Methods section. Based on the SD pair data, we found 12 novel miRNA paralogs in the human genome and two miRNA paralogs in the mouse genome; however, none were found in the rhesus genome (Text S1). There is an SD pair (chrX: 1314235–2068238 (+) and chrY: 1264235–2018238 (+)) in the corresponding pseudoautosomal region of the two sex chromosomes and the DNA sequences of the two segments are identical. Interestingly, the known miR-3690 gene [63] is located in the SD region on chrX: (1314235–2068238 (+)). No miRNA genes have yet been found on Y chromosome [40], but, using the SD pair data, we identified hsa-mir-3690-1and hsa-mir-3690-2 on both the X and Y chromosomes (Hsa-mir-3690 is documented in miRBase 16 and one new paralog of it has been found. We have renamed them according to the miRNA name criteria (Text S1)). Two duplicate copies of hsa-mir-3690 are located in an intron of the CSF2RA gene in the pseudoautosomal region of the X and Y chromosomes (Figure 1). In the same intron of CSF2RA gene (Figure 1), we found 15 duplicate copies of hsa-mir-3690 which was first documented in miRBase 16; only 2 of the copies were identified by MiPred [44] as potential miRNA genes, 7 were classified as pseudo miRNA genes and others were not miRNA genes (data not show). The composition and order of the genes in the the pseudoautosomal regions that contain the SD pair are shown in Figure 1. From this we can infer that duplicate copies of hsa-mir-3690 arose before crossover (which creates SD pairs) occurred in the pseudoautosomal regions of the X and Y chromosomes.
(A) chrX: 1314235–2068238 (+). (B) chrY: 1264235–2018238 (+).
Repeat related miRNAs
We have found 278 miRNA genes (226 RdmiRs and 52 PRdmiRs) in the human genome that overlap with repeats (Table S1). A recent paper that focused on TE-derived miRNAs, reported 68 human miRNA genes that shared sequences with TEs and 55 miRNA genes that were TE-derived [18]. Most of the TE-derived miRNAs that were reported in the earlier work were also identified as RdmiRs in the present study. Exceptions to this were hsa-mir-130b and hsa-mir-648 that we classified as NRdmiRs, and hsa-mir-659 that was classified as a PRdmiR. In our study, we used the data processed by the updated RepeatMasker program and the data from the updated miRBase. This may explain the discrepancies between our results and the results from the earlier study. In addition to miRNAs that overlap with TEs, we found many miRNA genes that overlap with other types of repetitive elements (Table S1). We also identified 141 RrmiRs (115 RdmiRs and 26 PRdmiRs) in the rhesus genome and 168 RrmiRs (141 RdmiRs and 27 PRdmiRs) in the mouse genome (Table S1).
RrmiRs not only differ in terms of the coverage density of repeat sequences, but also in the number of different repeats from which they are derived (Figure 2 and Table S1). In the two primate species, most of the RrmiRs are derived from TEs that include DNA repeats, LINE, SINE and LTR while in the mouse genome, and most RrmiRs are from simple repeats followed by LTR, SINE, and LINE (Figure 3). The most abundant repetitive element types from which the human and rhesus miRNAs are derived are the DNA transposons (Figure 3), and the most abundant of those are the MADE1 elements belonging to the TcMar-Mariner family (Table S1). It is remarkable that the 42 human miRNA genes and the 25 rhesus miRNA genes that share sequences with MADE1 elements are all members of the miRNA-548 family (Table S1). In mouse, the most abundant repetitive element type from which the miRNAs are derived is the simple repeats ((CA)n and (TG)n). These simple repeats have produced the largest miRNA family (mir-467 family) in mouse during the evolutionary process.
Differences between NRdmiRs, PRdmiRs and RdmiRs
The genomic distribution of NRdmiRs, PRdmiRs and RdmiRs in human, rhesus and mouse is shown in Figure 4. There are no length data available for the Y chromosome of rhesus, making it impossible to draw it and the miRNAs mapping to the genome scaffolds or to the unplaced contigs also have not been plotted. As described earlier [64]–[68], the miRNA genes in animals tend to occur in clusters (Figure 4). We classified miRNAs into intragenic miRNAs and intergenic miRNAs and found that NRdmiRs and RrmiRs show significant different distributions in protein-coding genes compared to intergenic regions (human: chi-square test p-value = 0.004131; rhesus: chi-square test p-value = 0.03475; mouse: chi-square test p-value = 2.1e-07).
To further explore the differences between the RdmiRs, PRdmiRs and NRdmiRs, we calculated their MFE structures using the RNAfold program [51] to determine the MFEs of hairpin structures for the three pre-miRNAs types in the three species. We found that the NRdmiRs, PRdmiRs and RdmiRs have significantly different MFE values (Figure 5, Kruskal-Wallis chi-squared = 13.2434, df = 2, p-value = 0.001331). The MFE values for NRdmiRs are significantly different from those for PRdmiRs (W = 97326.5, p-value = 0.007105, Wilcoxon rank sum test) and RdmiRs (W = 422230.5, p-value = 0.006562, Wilcoxon rank sum test). No significant difference in MFE values was found between PRdmiRs and RdmiRs (W = 21293.5, p-value = 0.1866, Wilcoxon rank sum test).
The open circles indicate outliers.
We evaluated the sequence conservation of the miRNAs based on the whole genome alignments of human, rhesus, mouse and 14 other vertebrate species by calculating the per-site conservation probability of the human pre-miRNAs. However, hsa-mir-1268 (derived from AluJo), hsa-mir-1299 (derived from CER), hsa-mir-3673 (derived from (TA)n), hsa-mir-3669 (derived from (CATATA)n) and hsa-mir-3683 (derived from SATR1) were not included in the calculation, because the orthologs of these miRNAs are not found in any of the other aligned animal genomes. Hsa-mir-1268 and hsa-mir-1299 do have homologs in chimpanzee (ptr-mir-1299) and orangutan (ppy-mir-1268 and ppy-mir-1299) that are documented in miRBase 16. This indicates that these two miRNAs are primate-specific miRNAs, and suggests that hsa-mir-3673, hsa-mir-3669 and hsa-mir-3683 may be human-specific miRNAs. The average conservation scores of NRdmiRs, PRdmiRs and RdmiRs are 0.4320636, 0.3406138 and 0.1585666 respectively, Figure 6 indicating significant differences in their conservation (Kruskal-Wallis chi-squared = 64.5498, df = 2, p-value = 9.62e-15). There is no significant difference between the conservation scores of NRdmiRs and PRdmiRs (W = 22320, p-value = 0.2510, Wilcoxon rank sum test). The significant differences in conservation scores are between PRdmiRs and RdmiRs (W = 7364.5, p-value = 0.001523, Wilcoxon rank sum test) and between NRdmiRs and RdmiRs (W = 117123.5, p-value = 1.110e-15, Wilcoxon rank sum test) (Figure 6).
The open circles indicate outliers.
Lineage-specific characters of the RrmiR families
Recent studies indicate that repetitive elements (especially TEs) have driven genome evolution in diverse ways, some of which tend to be lineage-specific [69]–[72]. For example, the mir-548 family (derived from MADE1 element) is primate-specific [16] and the mir-1302 family (derived from MER53 element) is a placental-specific miRNA family [20]. Similarly, we have found quite a few RrmiR families that are lineage-specific. Some larg families, the mir-506, mir-1972, mir-3118 and mir-3179 families are primate-specific. Although, in the present study, the mir-1285, mir-1289 and mir-3116 families are only appear in human, the mir-1285 and mir-3116 families are actually primate-specific miRNA families, and the mir-1289 family is not limited to primate species, but is also found in horse (eca-mir-1289). We also identified one murine-specific families, mir-1906 family, and some large families, for instance, mir-1195, mir-1937, mir-3470 and mir-3471 families are mouse-specific (Table S2). Three miRNA families that are the results of lineage-specific expansion were found in the mouse genome: the mir-466 and mir-467 families derived from simple repeats and the mir-297 family derived from SINE and LTR repetitive elements (Table S1 and Table S2). Although the mir-1255 family is a large miRNA family that includes members from primate and horse, no homologs have been found in mouse. In addition, there are quite a few small lineage-specific families, such as mir-1268, mir-1299, mir-3673 (human), mir-3669 (human), and mir-3683 (human) that may be expanded as more data become available (Table S2).
MiRNAs in segmental duplication and the expansion of some miRNA families promoted by segmental duplication events
It is well known that gene duplication can involve inverted, local (tandem duplication) or segmental duplication events. That many miRNA genes originated from inverted duplication, and that miRNA families expanded through local duplication (tandem duplication), has been well characterized [4]–[10]. However, little is known about the expansion of miRNA genes and miRNA families through segmental duplications.
SD pairs are found as arrays of local duplications or dispersed between or within chromosomes. In the human genome, 55 NRdmiR genes, 4 PRdmiR genes and 22 RdmiR genes are located completely within SDs (Table S3). These 81 miRNA genes (distributed at 90 loci in the genome) include 69 miRNA genes in 59 SD pairs, and 12 miRNA genes for which the homologs have not been found in the corresponding SD pairs (Table S3 and Table S4). We also found that 20 of the miRNA genes (distributed at 22 loci in the genome) overlap with SDs in the rhesus genome. Except for mml-mir-372, which is not completely located within its corresponding SD, all the other rhesus miRNA genes are located in SDs (Table S3). The 20 rhesus miRNA genes include 16 miRNA genes in SD pairs, while the homologs of the other 4 miRNA genes have not been discovered in the corresponding SD pairs (Table S3 and Table S4). In the mouse genome, we only found 53 miRNA genes completely located in SDs and two (mmu-mir-367, mmu-mir-669g) partially overlap with SDs. Among the 55 mouse miRNA genes (Table S3), there are 46 miRNA genes in SD pairs and 9 miRNA genes that are found in only one SD of the corresponding SD pair and no homolog in the other SD of the pair (Table S3 and Table S4). It is interesting that most mouse miRNA genes in SDs and in SD pairs are derived from simple repeats in chromosome 2 (Table S1, Table S3 and Table S4).
When a miRNA gene is in a SD pair, we have identified three situations that may occur: (1) the miRNA gene has at least one paralog, (2) the miRNA gene has a homolog that is not a miRNA, and (3) the corresponding homolog segment is absent in the other SD of the same SD pair. Based on the SD pair data, we identified 12 potential miRNA genes in human and two potential miRNA genes in mouse (Text S1). The paralogs of only 25 miRNA genes in SD blocks from the three species were not detected in SD pairs. Two possible explanations and evolutionary scenarios might explain these results: (1) these regions originally harbored miRNAs that duplicated from one of the SDs of the SD pair and one of the genes has since degenerated, or (2) the miRNAs in the SD regions were gained after the SD duplication event.
When a miRNA family has only one miRNA gene in an SD pair, this implies that no homologs of the gene have been identified in the other SD of the SD pair. Then the expansion of this miRNA family could not have been promoted by early SD events in the evolutionary process. But if the members from a miRNA family are present in the two SDs of an SD pair, it is likely that the miRNA family was expanded by the early SD events. We found NRdmiR and RrmiR families that may have expanded by SD events. There are 16 known miRNA families (and 17 miRNAs that have not yet been classified to a known miRNA family in miRBase or Rfam) in the human genome, 5 known miRNA families in the mouse genome and 6 known miRNA families (and 4 miRNAs that have not yet been classified to a known miRNA family) in the rhesus genome that expanded by SD events (Table S4, Table S5). Some RrmiRs were also duplicated by SD events in their evolutionary history. In the human genome, RdmiR genes from 4 known families and some PRdmiR genes from the mir-3179 family (hsa-mir-3179-1, hsa-mir-3179-2, hsa-mir-3179-3) expanded by SD events, in addition to the transposition effect in the past evolutionary history (Table S5). In the rhesus genome, some genes (mml-mir-3118-1, mml-mir-3118-4) from only one RdmiR family, the mir-3118 family, expanded by SD events (Table S5) and in the mouse genome, the RdmiR genes from three known miRNA gene families duplicated via SD events. We have listed some RdmiR genes from the mir-467 family and some PRdmiR genes from the mir-467 family that also expanded by SD events (Table S5). These events reflect one of the evolutionary mechanisms responsible for the expansion of miRNA families under the SD model. No common miRNA families that have been expanded by SD events were identified in all three genomes, but we found that the mir-3118 and mir-3156 families in human and rhesus expanded by SD events genome.
CNV data will also help us investigate the evolution of gene families including non-coding gene families. Some CNVs that are fixed in the population and some duplication type CNVs contain SDs [35], [36]. These SDs are often variable in copy number and can be referred to as CNVs [25]. A copy number polymorphism (CNP) refers to a CNV that occurs in more than 1% of the population [34]. The CNV data also show that duplication events have contributed to the expansion of RrmiRs. We found 85 NRdmiRs, 8 PRdmiRs and 28 RdmiRs that map to 79 locations of the human CNP data (Table S6), and 26 miRNAs (17 NRdmiRs, 3 PRdmiRs and 6 RdmiRs) that map to SD pair blocks located in CNP blocks (Table S4 and Table S6). Only one NRdmiR gene in rhesus and one RdmiR gene in mouse overlap with an SD pair and CNV block (Table S4 and Table S6). These results provide further evidence for the hypothesis that miRNA genes expanded by duplication events and indicate that the copy number of some miRNAs varies in different species.
Functions of the common RrmiRs in the human, rhesus and mouse genomes
Among the known miRNA families, we found 19 RrmiR families that are common to the human, rhesus and mouse genomes (Here we provisionally define hsa-mir-3174, mml-mir-3174 and mmu-mir-3174 as belonging to the mir-3174 family, because they have not yet been classified in miRBase or Rfam), 64 RrmiR families common to human and rhesus, 24 RrmiR families common to human and mouse and 19 RrmiR families common to rhesus and mouse (Figure 7, Table S2). These miRNA families are highly conserved across the species, suggesting that selective pressure may have driven the acquisition and retention of special functions.
(A) Venn diagram. (B) An image of the detailed list. The miRNA families are indicated on the left of the heatmap.
To investigate the functions of 19 of the conserved RrmiR families, we used a computational method to classify their target gene according to their function in the cells. We found regulation of transcription, central nervous system development, and negative regulation of biological process to be the most significantly enriched GO terms in the target genes of the miRNAs from the 19 selected RrmiR families from mouse, rhesus and human (Figure S1 and Table S7). Protein complex assembly and nervous system development were the most common biological progress terms for human and rhesus, while negative regulation of cellular process and cell proliferation were the most common biological process terms for human and mouse (Figure S1 and Table S7).
We reconstructed functional networks by literature mining. Although very few functional studies of RrmiRs are available, experimentally validated miRNA-target interactions for quite a few RrmiRs (including miRNAs from the most common RrmiR families, Table S8) have been well documented in the miRTarBase Release 1.0 [59]. In addition, we manually curated from the literature and 17 new miRNA-target pairs for which the functions have been well studied. The functional networks of miR-92b (PRdmiR, mir-25 family, derived from GC rich tandem repeats), miR-28 (RdmiR, mir-28 family, derived from LINE), miR-151 (RdmiR, mir-28 family, derived from LINE), miR-421 (RdmiR, mir-95 family, derived from LINE), miR-1271 (RdmiR, mir-1271 family, derived from LINE), miR-340 (RdmiR, mir-340 family, derived from DNA transportable element) and miR-378 (RdmiR, mir-378 family, derived from SINE) have been reconstructed (Figure 8). In the miR-378 network (Figure 8A), ERBB2 is a transcription factor of the miR-378 gene and it's host gene PPARGC1B which encodes PGC-1β [73] and HNE, which it also appears, could downregulate miR-378 and induce the expression of its target gene, SuFu [74]. The expression of miR-378* increases during breast cancer progression and miR-378* induces the Warburg effect in breast cancer cells by inhibiting the expression of two PGC-1 partners, ERR and GABPA [73]. In the miR-28 network (Figure 8C), ASF/SF2 expression is modulated by miR-28 and miR-505 (not show here) which are negatively controlled by LRF to influence the proliferation and survival of mouse embryonic fibroblasts [75]. In Figure 8E, how miR-151 exerts this function by targeting RhoGDIA to activate Rac1, Cdc42 and Rho GTPases is shown [76]. In addition, miR-151 can function synergistically with its host gene FAK to enhance HCC cell motility and spreading [76]. Although the functions of the 7 RrmiR families displayed in Figure 8 have only been validated in human miRNAs, because they are common miRNA families in human, rhesus and mouse, they are likely to have the same functions in the other mammals. We found many RrmiRs are in cancer cells (in vitro) or in tumor tissues and several studies have shown that some RrmiRs are expressed in the central nervous system [77]–[80]. This experimental evidence roughly validates the functional enrichment results generated computationally.
(A) miR-378 (RdmiR, mir-378 family). (B) miR-340 (RdmiR, mir-340 family). (C) miR-28 (RdmiR, mir-28 family). (D) miR-1271 (RdmiR, mir-1271 family. (E) miR-151 (RdmiR, mir-28 family). (F) miR-421 (RdmiR, mir-95 family). (G) miR-92b (PRdmiR, mir-25 family).
As yet, little is known about the origin of most miRNAs and miRNA families in mammals. Here, we characterized miRNAs derived from repetitive elements and some miRNA families expanded by SD events in several mammalian genomes. We have found 226 RdmiRs and 52 PRdmiRs in the human genome, 115 RdmiRs and 26 PRdmiRs in the rhesus genome and 141 RdmiRs and 27 PRdmiRs in the mouse genome. Although most reports on RrmiRs have mainly concentrated on TEs, there are a few reports on miRNAs derived from tandem array sequences using computational methods and biological experiments [15], [19], [81]. In this study, we identified a number of miRNAs derived from tandem repeats and low-complexity repeats (Table S1). The most striking instance is the large mir-467 family in the mouse genome, which was derived from simple repeats. Many miRNA genes derived from repetitive elements have been identified, but the current methods used to identify miRNAs always discard the segments that map to repetitive elements annotated in the genomes. Although this may sometimes be valid, some recently identified miRNAs, such as the mmu-mir-2134 family, the mmu-mir-2135 family and hsa-mir-3172, were documented in miRBase 14 and/or miRBase 15, but because they are derived from tandem repeats (rRNA repeats, tRNA repeats), they have been removed from the more recent miRBase 16. Strictly speaking, it cannot be proven that all the potential miRNAs derived from repetitive elements are not miRNAs just because their function is, as yet, unknown. Recently, a growing body of literature (Table S8) has made it possible to validate the functions of PRdmiRs and RdmiRs, many of which were found to be expressed in tumors. We strongly suggest that small RNA fragments that map to genome regions annotated as repetitive elements should not be discarded before biological experimental data are available to verify them.
NRdmiRs, PRdmiRs and RdmiRs have some significantly different characteristics: (1) their distribution between protein-coding regions and intergenic regions is biased, (2) there are obvious differences in the MFEs of their secondary structures, and (3) because most RdmiRs are relatively young, they are relatively less conserved than NRdmiRs and PRdmiRs in vertebrates. This result agrees well with a previous report [18]. However, we did find 19 RrmiR families that were conserved in human, rhesus and mouse. We also identified many RrmiR families that are lineage-specific or that undergo is lineage-specific expansion. An example of this is the mir-467 family that is hugely expanded in mouse.
As in our previous study [20], here too we found some miRNA families (including RrmiR families) that may originate from and expand by repetitive elements. In addition, we discovered that miRNA families can also expand by SD events. Examples of this are the mir-297, mir-466, mir-467, mir-548 [16], mir-1302 [20], mir-1972, mir-3118 and mir-3179 families (which are all RrmiR families listed here) (Table S5). Our results show the complex evolutionary dynamics of some miRNAs. CNV regions may contain hundreds of genes, disease loci, functional elements and SDs. The association of CNVs with SDs has been observed in the human genome [35], [36], and Redon and his colleagues have found that nearly a quarter of the CNV regions were associated with SDs in human genome [36], while the SD-mediated non-allelic homologous recombination mechanism accounts for about a quarter (<28%) of CNVs formation [35]. Many miRNA families were produced from duplications as tandem repeats of small fragments or as large fragment segmental duplications (synonymous with copy number variation in some time). We analyzed all human miRNA family members that mapped to CNP regions and found that the distance between the loci on the same chromosome of members of one miRNA family is ∼1000-nt or more. We found 26 human miRNA genes that mapped to both SD pair blocks and CNP blocks (Table S6). In rhesus and mouse, although we found associations of SDs with miRNA genes in their genomes, only one of their miRNA genes is in both an SD pair block and a CNV block respectively (Table S6). These results provide further evidence that duplication events promoted the expansion of miRNA genes, including RrmiR genes, in the human and other primate genomes, and indicate that the copy number of some miRNAs varies in different species. In line with previous reports that most mouse duplications are distributed in discrete clusters of tandem duplications [54], we found that the miRNA genes clustered in SD blocks and were distributed mainly on chromosomes 2, 12 and 13 (Table S3), and that nearly all the miRNA genes in SD pairs tended to cluster and were located on chromosome 2 (Table S4). In mouse, although 70% of the CNV blocks were completely located in SD regions, a recent study reported that most mouse duplications are depleted of genes [54]. This could account for the finding that there are very few mouse miRNA genes associated with CNVs, and indicates a significant difference between human and mouse miRNAs. For rhesus, more evidence is needed to explain some of the reported phenomena.
In conclusion, we have presented evidence for two possible mechanisms for the origin and evolution of miRNA genes in mammals. Our main results suggest that repetitive elements contribute to the de novo origin of miRNAs, and that large SD events may also accelerate the expansion of miRNA families, including RdmiRs. Our work also shows how SD pair data can be used to identify miRNA paralogs. Our results indicate that some RrmiRs undergo species- or lineage-specific expansion and, while some are conserved in mammals, they are less conserved in other vertebrates compared to NRdmiRs. Moreover, we have provided both computational and experimental evidence for the functions of some common RrmiR genes that have become fixed in the three mammals studied.
Supporting Information
Text S1.
Figure S1.
The topological GO graph for the enriched GO biology process terms of the target genes of common RrmiRs in the human, rhesus and mouse genomes. (A) human (B) rhesus (C) mouse.
Table S1.
List of miRNA genes that overlap with repeats.
Table S2.
List of RrmiR families from the human, rhesus and mouse genomes.
Table S3.
List of miRNAs that overlap with segmental duplications.
Table S4.
List of miRNAs located in segmental duplication pairs.
Table S5.
List of miRNA families located in segmental duplications in the human, rhesus and mouse genomes.
Table S6.
List of miRNAs locate in the copy-number polymorphism and copy-number variant blocks.
Table S7.
The enriched GO biology process terms for the target genes of common RrmiRs.
Table S8.
List of RrmiR-target pairs validated by biological experiments mined from various sources.
Author Contributions
Conceived and designed the experiments: ZY XS HL JX. Performed the experiments: ZY. Analyzed the data: ZY. Contributed reagents/materials/analysis tools: ZY. Wrote the paper: ZY XS.
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Pony Island
4th Jan, 2016
Daniel Mullins Games
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Platforms: Mac, SteamOS, PC (Microsoft Windows)
Pony Island is a suspense puzzle game in disguise. You are in limbo, trapped in a malevolent and malfunctioning arcade machine devised by the devil himself. It is not a game about ponies.
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Reviewing this game is a bit tricky biz. Because it is more of a piece of interactive art, than a game. And every spoiled bit of info ruins a byte of experience. So i regret watching the trailer. But on the other hand - it's a real pain in the ears to make someone not so open-minded to play a game called "The Pony Island". I still can't persuade the tow of my video-game-junkie friends to try ti (I've told them not to watch the trailer). T... Read More
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Fuse Creativity System Project Guide: Fun Party Themes, Idea for Handmade Cards, And More Paper Crafts from Fiskars
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"I like this vintage style Santa graphic, but I read that the designer used a cutting machine to cut a red vinyl decal to afix to the cream painted wood board. I don't know what kind of cutting machine she is talking about and I wonder how pricey it is? I'd like an alternative to getting that Santa on there."
"This wall decor is very chic with any message you write on it! And it couldn't be easier. However, another option to using foam core board would be to actually buy an old thrift store bulletin board and spray paint it black. I think it would give the wall art a little bit more of a lived-in rustic look as the cork board adds texture and interest."
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Pool Party Invitations
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Missile Defense: DOD's Report Provides Limited Insight on Improvements to Homeland Missile Defense and Acquisition Plans
GAO-14-626R: Published: Jul 17, 2014. Publicly Released: Jul 17, 2014.
The Department of Defense (DOD) was directed to report to the congressional defense committees on the status of efforts to improve the homeland ballistic missile defense capability of the United States and a description of the results of two planned flight tests. The Missile Defense Agency (MDA) is developing the Ground-based Midcourse Defense (GMD) system to defend the United States against a lim...
Department of Defense's Waiver of Competitive Prototyping Requirement for the Army's Indirect Fire Protection Capability Increment 2, Block 1 Program
GAO-14-643R: Published: Jun 11, 2014. Publicly Released: Jun 11, 2014.
The Department of Defense's (DOD) rationale for waiving the competitive prototyping requirement in the Weapon Systems Acquisition Reform Act (WSARA) of 2009, as amended, for the Army's Indirect Fire Protection Capability (IFPC) – Increment 2, Block 1 program addresses one of the two bases provided in the statute; namely that the cost of producing competitive prototypes exceeds the expected lif...
Department of Defense's Waiver of Competitive Prototyping Requirement for the Army's Armored Multi-Purpose Vehicle Program
GAO-14-521R: Published: Apr 25, 2014. Publicly Released: Apr 25, 2014.
The Department of Defense's (DOD) rationale for waiving the competitive prototyping requirement in the Weapon Systems Acquisition Reform Act of 2009, as amended (WSARA), for the Army's Armored Multi-Purpose Vehicle (AMPV) program addresses one of the two bases provided in the statute; namely that the cost of producing competitive prototypes exceeds the expected life-cycle benefits (in constant dol...


text:
I was legal (don't worry). He was a swim coach from a neighboring team when I was on swim team at school. It was incredible, but we had to share our love in utmost secret, for obvious reasons. Anyone else have an experience like this?
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Wireless Mouse Issue
I have a wireless Logitech mouse that works great except for 1 issue. Whenever I use it the onscreen keyboard will not popup at all. To get the onscreen keyboard to work again I have to turn off bluetooth. As soon as I turn bluetooth off it works great.
Any ideas?
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text:
LibertyEF Fonts
<link href="//db.onlinewebfonts.com/c/4dd30cbd7ed98611d256025040e6ca1a?family=LibertyEF" rel="stylesheet" type="text/css"/>#2 Using @import CSS directive, put the following line in add to your css file.(http | https)
@import url(//db.onlinewebfonts.com/c/4dd30cbd7ed98611d256025040e6ca1a?family=LibertyEF);#3 Use font-face declaration Fonts.(http | https)
@font-face {font-family: "LibertyEF"; src: url("//db.onlinewebfonts.com/t/4dd30cbd7ed98611d256025040e6ca1a.eot"); src: url("//db.onlinewebfonts.com/t/4dd30cbd7ed98611d256025040e6ca1a.eot?#iefix") format("embedded-opentype"), url("//db.onlinewebfonts.com/t/4dd30cbd7ed98611d256025040e6ca1a.woff2") format("woff2"), url("//db.onlinewebfonts.com/t/4dd30cbd7ed98611d256025040e6ca1a.woff") format("woff"), url("//db.onlinewebfonts.com/t/4dd30cbd7ed98611d256025040e6ca1a.ttf") format("truetype"), url("//db.onlinewebfonts.com/t/4dd30cbd7ed98611d256025040e6ca1a.svg#LibertyEF") format("svg"); }
Copyright (c) 1994 Elsner+Flake Designstudios. All Rights Reserved.
Gill Sans Alt One WGL W01 BdIt
Monotype Imaging Inc.Author
Eric GillTrademark
Gill Sans is a registered trademark of The Monotype Corporation in the United States and/or other countries.Copyright
Font software Copyright 1990, 1991, 1998 Adobe Systems Incorporated. Typeface designs Copyright The Monotype Corporation. All rights reserved. Gill Sans is a registered trademark of The Monotype Corporation in the United States and/or other countries.Description
NOTIFICATION OF LICENSE AGREEMENT This typeface is the property of Monotype Imaging Inc. and its use by you is covered under the terms of a license agreement. You have obtained this typeface software either directly from Monotype Imaging or together with software distributed by one of the licencees of Monotype Imaging. This software is a valuable asset of Monotype Imaging. Unless you have entered into a specific license agreement granting you additional rights, your use of this software is limited to use on up to five (5) workstation for your own publishing use. You may not copy or distribute this software. If you have any questions concerning your rights you should review the license agreement you received with the software or contact Monotype Imaging for a copy of the license agreement. General information:www.fonts.comFor license terms and usage rights, please visit our web site at:www.monotypeimaging.com/html/type/license.htmlLizenzbedingungen und Nutzungsrechte finden Sie auf unserer Webseite:www.monotypeimaging.com/html/type/license.htmlPour plus d'informations concernant les droits d'utilisation et les questions contractuelles, veuillez consultez notre site:www.monotypeimaging.com/html/type/license.htmlPara obtener informacin acerca de los trminos de licencia y los derechos de uso, visite nuestro sitio en el Web en:www.monotypeimaging.com/html/type/license.htmlPer le condizioni contrattuali ed i diritti d'uso, visitate il nostro sito web all'indirizzo:www.monotypeimaging.com/html/type/license.html
Flower Sketch W95 Regular
Natsuko HayashidaAuthor
Natsuko HayashidaTrademark
Flower Sketch is a trademark of Natsuko Hayashida.Copyright
Copyright (c) 2010 by Natsuko Hayashida. All rights reserved.Description
Copyright (c) 2010 by Natsuko Hayashida. All rights reserved.
New Aster LT W01 Semibold
Linotype GmbHTrademark
New Aster is a trademark of Simoncini S.A.Copyright
Copyright 1989, 1995, 2002 Adobe Systems Incorporated. All Rights Reserved. 1981, 1995, 2002 Heidelberger Druckmaschinen AG. All rights reserved.License
NOTIFICATION OF LICENSE AGREEMENTYou have obtained this font software either directly from Linotype GmbH or together with software distributed by one of Linotype's licensees.This font software is a valuable asset of Linotype GmbH. Unless you have entered into a specific license agreement granting you additional rights, your use of this font software is limited to your workstation for your own use. You may not copy or distribute this font software. If you have any questions regarding your license terms, please review the license agreement you received with the software.General license terms and usage rights can be viewed at www.linotype.com/license.Generelle Lizenzbedingungen und Nutzungsrechte finden Sie unter www.linotype.com/license.Pour plus d'informations concernant le contrat d'utilisation du logiciel de polices, veuillez consulter notre site web www.linotype.com/license.Linotype GmbH can be contacted at:Tel.: +49(0)6172 484-418
Soleil W01 Light
Wolfgang HomolaAuthor
Wolfgang HomolaTrademark
Soleil is a trademark of Wolfgang Homola.Copyright
Copyright (c) 2011 by Wolfgang Homola, distributed by TypeTogether. All rights reserved.Description
Complete End User License Agreement (EULA) available in this: http://www.type-together.com/resources/eula/TT-EULA.pdf
Dyspepsia V3
2.000 2004Company
Ray LarabieAuthor
Ray LarabieCopyright
1999 Ray Larabie. This font is freeware. Read attached text file for details. Info & updates visit www.larabiefonts.com. Donations gratefully accepted at www.larabiefonts.com/donation.html. Also visit my commercial type foundry at www.typodermic.com. This font was updated in 2004.Description
If you would like to help support Larabie Fonts please consider making a donation. It costs a lot to keep the site running and free from irritating pop-up or banner ads. Any amount is appreciated and goes right back into making maintaining the site and developing more free fonts. Visit www.larabiefonts.com for details.
Kartoon Kutz 4 NF W95 Regular
Nick's FontsAuthor
Nick CurtisCopyright
Copyright (c) 2004-2010, Nick Curtis. All Rights Reserved. Unauthorized duplication or redistribution is expressly prohibited.License
By using or installing this font data, you (or you on behalf of your employer) agree to be bound by the terms of this Agreement. This Agreement constitutes the complete agreement between you and Nick's Fonts.NUMBER OF USERS: In consideration for the license fee paid, Nick's Fonts grants to you only, the Licensee, the non-exclusive, nontransferable right to use and display the font data. If you are using this product for your work, this agreement applies to your employer. Nick's Fonts may be used on up to five (5) CPUs at your site connected to any number of printers or other image-producing devices (regardless of resolutions) at your own site. You may make one (1) copy of the Nick's Fonts font data solely for backup purposes provided the copyright and trademark notices are reproduced in their entirety on the backup copy. YOU MAY:-Send a copy of any Nick's Fonts font data along with your documents to a commercial printer or other service bureau to enable the editing or printing of your document.-Use this font data to embed fonts within PostScript files or PDF files for distribution, viewing, and imaging to third parties.-Use this font for personal projects or for projects for which you are paid including, but not limited to, artwork, logotypes, personalized apparel, word art and web graphics.YOU MAY NOT:-Modify, adapt, translate, reverse engineer, decompile, disassemble or create derivative works based on the Nick's Fonts font data without Nick's Fonts' prior written consent, except for modifications made strictly for your personal use or, if this license was purchased by your company, for use solely within your company.-Sell this font data AS FONT DATA to a third party under any circumstances including, but not limited to, rubber-stamp alphabets, die-cut alphabets, rub-on alphabets, or digitized fonts for embroidery. COPYRIGHT. The Nick's Fonts font data is copyrighted; unauthorized copying of Nick's Fonts is expressly forbidden. You may be held legally responsible for any infringement of Nick's Fonts' intellectual property rights that is caused or encouraged by your failure to abide by the terms of this Agreement. Additionally, Nick's Fonts reserves all rights not specifically granted to Licensee.TERMINATION. This Agreement will immediately and automatically terminate without notice if you fail to comply with any term or condition of this Agreement. If this Agreement is terminated, you agree to destroy all copies of the font data in your possession. DISCLAIMER AND LIMITED WARRANTY. For a period of 90 days after delivery, Nick's Fonts warrants that the font data will perform in accordance with the specifications published by Nick's Fonts. NICK'S FONTS MAKES NO OTHER WARRANTIES, EXPRESS OR IMPLIED. THE WARRANTIES OF FITNESS FOR A PARTICULAR PURPOSE AND MERCHANTABILITY ARE SPECIFICALLY EXCLUDED. NICK'S FONTS DOES NOT WARRANT THAT THE FONT DATA IS FREE FROM ALL ERRORS AND OMISSIONS. This warranty gives you specific legal rights. You may have other rights, which vary from state to state. NICK'S FONTS SHALL NOT BE LIABLE FOR ANY DIRECT, INDIRECT, CONSEQUENTIAL, OR INCIDENTAL DAMAGES (INCLUDING DAMAGES FROM LOSS OF BUSINESS PROFITS, BUSINESS INTERRUPTION, LOSS OF BUSINESS INFORMATION, AND THE LIKE) ARISING OUT OF THE USE OF OR INABILITY TO USE THE NICK'S FONTS FONT DATA. Because some states do not allow the exclusion or limitation of liability for consequential or incidental damages, this limitation may not apply to you. GOVERNING LAW. This agreement is governed by the laws of the United States of America and the State of Virginia.
CCToBeContinued W00 Bold Italic
Active ImagesAuthor
John RoshellTrademark
CCToBeContinued is a trademark of Active Images.Copyright
Copyright (c) 2011 by Active Images. All rights reserved.License
Active Images Font License AgreementPlease read this license agreement carefully before you install your fonts. By installing fonts sent to you by ACTIVE IMAGES, you accept this license agreement. If you do not accept the terms and conditions of this license, do not install the fonts in question.1. LICENSE Active Images is the exclusive owner of the electronically transferred computer lettering fonts. We grant you, and you accept, a non-exclusive license to use the electronically transferred fonts, but you are not the owner of them. Your right to use the fonts under this license agreement is called the license. By fonts, we refer to the electronically transferred fonts and any embodiment or use that you make of the fonts in any form.2. AUTHORIZED USER AND USE You alone, as the purchaser of the fonts, are authorised to use the fonts under the license for personal, business or distributed projects. With few exceptions: fonts are licensed for use on 5 CPUs and/or 1 output device. Additional licenses are available on a ratio of 5 CPUs to 1 output device. Call us if you are not clear on whether you require additional licenses. Any other duplication or use by any other person or entity constitutes software piracy, a criminal offense.3. LIMITED WARRANTY Active Images makes no warranties, express or implied, regarding the electronically transferred computer software, its merchantability or its fitness for any particular purpose.Active Images warrants the magnetic media to be free from physical defects for 90 days, after purchase. Active Images will replace the disk free of charge should it fail within this time. These fonts are sold for use only on the computer platform indicated on the packaging. Use of these fonts on any platform other than that for which it was originally intended is not recommended and invalidates any warranty.Active Images reserves the right to terminate this License at any time and void your license if you fail to comply with the terms and conditions of this License.If you have any questions about the License Agreement, our return policy, security and on-line ordering, or anything else, please feel free to contact us at email@example.com, or at the telephone number below.ACTIVE IMAGES8910 Rayford DriveLos Angeles, CA 90045Tel: 310 650-7904www.activeimages.com
Fighting Spirit TBS Bold V1
Style : BoldPackages
Fighting Spirit TBSStyle
1.00 February 21, 2009, initial releaseCopyright
Typeface (your company). 2009. All Rights ReservedDescription
This font was created using Font Creator 5.0 from High-Logic.com
Mf Queen Leela V1
Mf Queen LeelaStyle
Misti's Fonts (Misti Hammers)Author
Misti's Fonts (Misti Hammers)Trademark
Mf Queen Leela is a trademark of Misti's Fonts (Misti Hammers).Copyright
Copyright (c) 2014 by Misti's Fonts (Misti Hammers). All rights reserved.Description
Copyright (c) 2014 by Misti's Fonts (Misti Hammers). All rights reserved.License
Free for personal use. Commercial use requires donation.


text:
BH-KatiesMum (14-11-2014)
These are mine today, day 4 of the revlon polish
Last edited by Snooze; 14-11-2014 at 09:16.
Yay! I'm so excited about this thread!! These are mine atm.. it's actually a lot pinker on the glitter nail than the photo looks.
"Insert witty signature here"
This was last week.
"Insert witty signature here"
oh I love sparkles. I think I add sparkles on top of every nail polish ... just coz it always looks better
FrothyFrog (14-11-2014)
ImageUploadedByThe Bub Hub1415921632.349221.jpg
Some sparkly purple nails for me...this colour looks amazing in sunlight, it distracts me while I'm driving lol
~Marigold~ (16-11-2014)
Pregnant for the first-time?
Not sure where to start? We can help!
Our Insider Programs for pregnancy first-timers will lead you step-by-step through the 14 Pregnancy Must Dos!


text:
Yahoo is testing a new research aid called SearchPad. It allows users to take and save notes on various areas of research, from trips to product research to pretty much anything you want!
SearchPad can detect when your searches on Yahoo look like research. It will prompt users conduct such behavior to see if they want to take notes.
Here's a video demonstrating SearchPad:
Related Reading:
Yahoo! Adds eBay Application to Home Page Test
Yahoo Tests 'Glue Pages' in India
Related reading
Can Pinterest crack (and monetise) visual search?
Pinterest made a clear declaration of intent last week with the announcement that Randy Keller, Google's former head of image search, has joined the photo sharing site as Head of Search.
What we can learn from the latest trends in voice search
Voice search is already a fast-rising trend in the search industry, and its latest developments indicate that it's starting to win ground in daily use.
How can you successfully set up an efficient business blog on a very tight budget?
Setting up online presence for your business can be overwhelming. So much to plan and so much to invest (in terms of both time and money).
What is influencer marketing, and why should it be part of your digital strategy?
If I tell you I have the secret formula for any entrepreneur to succeed, you may not believe me. But if Neil Patel, New York Times best selling author and a person The Wall Street Journal calls 'the web's top influencer' tells you, "My secret formula is the greatest", you will probably believe him.


text:
METAA quick note about posting (this is a general notice to assist our readers as well as those who contribute content) (self.haiti)
submitted by DarthMedicus[M] - announcement
Haiti's Terra Incognita: Grande Colline (A documentary on some of Haiti's remaining cloud/rainforest) (youtu.be)
submitted by MajorLzrDiaspora
OPINIONHORROR IN HAITI PRISONS (secretsofaidsandebola.blogspot.be)
submitted by joelsavage
MEDIA😍 Garcelle (i.reddituploads.com)
submitted by TortugasVeryOwn
MEDIABeautiful of Cap-Haitien, Haiti (facebook.com)
submitted by Upcultured
QUESTIONFitbit Compatibility in Haiti (self.haiti)
submitted by baldred05
MEDIAMichael Brun X Lakou Mizik - Gaya (ft. J. Perry) (youtu.be)
submitted by MajorLzrDiaspora
HUMAN INTERESTRaising Haiti Executive Director Meets with APF Leadership (raisinghaitiinitiative.tumblr.com)
submitted by APFUSAFoundation
QUESTIONCan someone from here help me with a postcard w/ a little note on it? The note should say " Stanly, Elena's love for you reached (city,country) or anything.This is for a surprise. I will be forever greatful if you could help. Thank you so much. I am just trying to fulfill his dreams of travelling.😑 (i.reddituploads.com)
submitted by elenaobiedo
HUMAN INTERESTRainn Wilson • Three Legs Program (youtube.com)
submitted by APFUSAFoundation
MEDIAPierre Garçon Escorts Miss Haiti On Return From Miss Universe Pageant (redskins.com)
submitted by DarthMedicus
OPINIONCharles Ortel: 'Clinton Robin Hood in Reverse Must Be Punished' (dadychery.org)
submitted by DarthMedicus
NEWSParty of Haiti president-elect wins majority of parliament - News (jamaicaobserver.com)
submitted by DarthMedicus
HUMAN INTERESTRaising Haiti Foundation — Meet Magguie Bellabe (raisinghaitiinitiative.tumblr.com)
submitted by APFUSAFoundation
OPINIONHaiti's exploitation by the Clintons? Who is really pulling the strings? Can Donald Trump help? (youtube.com)
submitted by lotusQ
MEDIAGood job Miss Haiti! You're a winner our eyes! Bon Bagay! 🇭🇹 💙❤🇭🇹 (instagram.com)
submitted by MajorLzrDiaspora
NEWSHaiti president-elect says Trump business background to help ties (cnbc.com)
submitted by MajorLzrDiaspora
HUMAN INTERESTCommunity Leaders Meet to Implement the Three Legs Program in Medor (raisinghaitiinitiative.tumblr.com)
submitted by APFUSAFoundation
HUMAN INTERESTRaising Haiti Foundation • Medor: Pioneering the Three Legs Program (raisinghaitiinitiative.tumblr.com)
submitted by APFUSAFoundation
QUESTIONAgriculture grants for Haiti (self.haiti)
submitted by schmack1001
MEDIALaunching a weekly email news service, Ayiti Weekly (ayitiweekly.com)
submitted by crisco_disco4
QUESTIONTravelling to Haiti (self.haiti)
submitted by shalyar
BUSINESSFirst Bus MADE IN HAITI by Haitian Engineers Unveiled (pressvue.com)
submitted by odingamu
NEWSThe DEA Nabs Haiti's Guy Philippe: Rebel, Drug Runner, and Politician. (thedailybeast.com)
submitted by ZmajLee


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Mayor Jackie Biskupski, Council Member Lisa Adams, and Sugar House Community Council Chair Amy Barry will take part in a ceremonial "demolition" of the abandoned Ute Car Wash structure on 2011 So
City News
Salt Lake City initiates courtesy dismissal program for city parking pay stations - June 30th, 2016
Today, Mayor Jackie Biskupski announced Salt Lake City would begin a one-time courtesy dismissal program aimed at individuals who receive tickets due to errors made at city parking pay stations o
Construction begins on Phase 2 of the 1300 South Reconstruction - June 29th, 2016
On Tuesday, July 5, the 1300 South Phase II Reconstruction Project will begin.
Salt Lake City hosts international rugby competition at newly opened Regional Athletic Complex - June 29th, 2016
The Salt Lake City Regional Athletic Complex (RAC) will host the Women's Rugby Super Series, presented by USA Rugby, Rugby Utah, and the Utah Sports Commission, from July 1 through July 9.
Mayor Jackie Biskupski and Office of Diversity and Human Rights Statement on U.S. Supreme Court's decision on Immigration Executive Actions - June 24th, 2016
Today, Mayor Jackie Biskupski and Yolanda Francisco-Nez, Coordinator of the Office of Diversity and Human Rights for Salt Lake City, released a statement of disappointment on the Supreme Court's
Applications available for Salt Lake City's 2016 Leadership Utah Fellowship - June 24th, 2016
The Salt Lake City Human Rights Commission and the Salt Lake City Mayor's Office of Diversity & Human Rights (ODHR) invite Salt Lake City residents to apply for the 2016 Leadership Utah Fello
Final public engagement workshop, an online survey, and a meeting of the Homeless Services Site Evaluation Commission set for this week - June 20th, 2016
Salt Lake City's ongoing efforts to address homelessness in Salt Lake City with a unique and compassionate model continue this week with a series of events designed to e
Salt Lake City to host final public engagement workshop on homeless resource centers criteria on Monday - June 17th, 2016
Residents are invited to the last of four public workshops designed to help prioritize criteria for two new homeless resource centers to be located in Salt Lake City.
City Budget discussion may wrap up on Tuesday at Council Meeting - June 13th, 2016
Council 2016 priorities helping shape decisions What: City Council Work Session & Formal Meeting When: Tuesday – June 14, 2016
SLC Council statement on weekend mass shooting in Florida - June 12th, 2016
PRESS RELEASE CITY COUNCIL of SALT LAKE CITY FOR IMMEDIATE RELEASE June 12, 2016 Contact: Dan Weist, Director of Communication 937 305-8891


text:
Written on: 11/06/2013
The ordering part was extremely easy, fast and intuitive. It is so easy to select the parts that you require and which you do not - as easy as a mouse click! I have ordered a new computer with parts that I required (minus a graphics card) and it was so painless and even better - the website actually helps you by letting you know whether or not you need a more or less powerful PSU, saving YOU money!
I am glad that PC Specialist actually double check and do thorough testing on the system before dispatching it! I have received my computer in perfect state - there was a special filling preventing all the parts from being damaged in delivery.
I have opened my new computer and was amazed at how everything was packed extremely well, no lose wires, bolts, etc. The build quality is great! I am pretty confident in this service and will surely use it again for my next gaming rig!
Already recommended to my friends!
As rated by our community of reviewers
" From Laptop to PC - UKGC Best Choice Ever! " Read More
" Terrible customer service " Read More
" Palicomp Carbon Intel TFT 22 " Read More
Written by KHLondon
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This re-issue form Lindberg presents a detailed model of a Dodge L700 semi tractor with a Shell Oil tanker trailer. The kit comes injected in red and includes shell decals. Pictorial assembly instructions guide the building process.
Royal mail at £3.50, or Express at £6.95.
Full details at checkout.
Some items cannot be sent via Royal Mail due to their restrictions.
To see shipping costs simply add the item to your basket and choose your country from the drop down list on the view cart page.
Use this form to ask us a quick one sentence question about this specific product. For more in depth queries, please feel free to email or call us.
Our system will drop you an automated e-mail as soon as this product comes back into stock.


text: Videos Suzie Cooney: This Woman Rips! By Standup Journal Posted on Sep 7, 2014 Share Tweet Share Share Email NEW SURF VIDEO Summer Fun on Maui.. Small surf, BIG FUN – Suzie Related Items: Share Tweet Share Share Email Retailer Locations Subscribe Submit a Photo Submit a Video BECOME A BETTER PADDLER Give us your email and get 5 instant actionable tips, plus exclusive discounts, news and stories ! ! Subscribe Something went wrong. Please check your entries and try again.


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SAR-controlled adaptive off-time technique without sensing resistor for achieving high efficiency and accuracy LED lighting system
Liu C.-H.,National Chiao Tung University |
Hsieh C.-Y.,National Chiao Tung University |
Hsieh Y.-C.,National Chiao Tung University |
Tai T.-J.,Richtek Ltd. |
Chen K.-H.,National Chiao Tung University
IEEE Transactions on Circuits and Systems I: Regular Papers | Year: 2010
A successive approximation register (SAR) is utilized to control adaptive off-time order to regulate accurate light-emitting diode (LED) current and improve efficiency of LED driver. The proposed SAR-controlled adaptive off-time technique without the external sensing resistor can sense the current flowing through LEDs during the turning on of the N-type power MOSFET, as well as provide adaptive off-time depending on the input voltage and the numbers of LED, to obtain an accurate LED current. Experimental results show the inductor current ripple is kept within ±15% of the DC current. As a result, line regulation is guaranteed this proposed design. © 2010 IEEE. Source
Lin R.-L.,National Cheng Kung University |
Liu S.-Y.,Leadtrend Inc. |
Lee C.-C.,Richtek Ltd. |
Chang Y.-C.,National Cheng Kung University
IEEE Transactions on Industry Applications | Year: 2013
This paper presents the Taylor series expression based light-emitting-diode (LED) model for the analysis of the LED driver system. In order to precisely describe the nonlinear V-I curve of LEDs, Taylor series can be utilized to express the Shockley equation of LEDs for the derivations of the dc and small-signal equivalent models. Based on the proposed LED models, the LED array with M paralleled strings of N LEDs in series can be constructed. Furthermore, the equivalent resistances of the N × M LED array can be derived. Finally, one LED array with eight paralleled strings of 15 LEDs in series is constructed to verify the equivalent small-signal resistance with the measured results. © 1972-2012 IEEE. Source
Hung C.-Y.,Richtek Ltd. |
Hung C.-Y.,Tunghai University |
Kao T.-C.,Richtek Ltd. |
Kao T.-C.,Tunghai University |
And 7 more authors.
Electronics Letters | Year: 2014
A simple scheme is proposed to increase the hold voltage and not change the original trigger voltage of a silicon-controlled rectifier (SCR) to enhance its latch-up immunity without changing the device dimensions. It is found that using the lightly doped P-diffusion instead of the highly doped P+ diffusion as the P emitter of the anode can increase the hold voltage of an SCR. © 2014 The Institution of Engineering and Technology. Source


text:
Doggy Day Care in Menlow, TX
Drop off your dog at your sitter's home in the morning, and pick them up in the evening.
Very Great with dogs and puppies.
Italy , TX, 76651
Dog Boarding, Drop-In Visits, Doggy Day Care, Dog Walking
About: I grew up with 4 dogs. I cared and loved all of them. I raised 2 of them in their puppy years and would wake up as early as it took to keep them ha... (more)
fun, outgoing,student, playful nice
Waco, TX, 76705
Dog Boarding, House Sitting, Drop-In Visits, Doggy Day Care, Dog Walking
"Faith is extremely attentive with our dog. She followed all our instructions and goes above and beyond in taking care of our sweet pet family member. I HIG... (more)"
Experience exceptional service and a personalized match
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|West, TX
|Wiggins, TX
Don't let your 9-to-5 stop you from being a great pet parent. When you're heading to work, drop off your beloved pet with a loving dog sitter near you. Your dog will get the love and care they're used to at home, and you'll get photo updates and the peace of mind that comes from finding great care for your pet. We're proud to offer a wide range of pet care, including puppy day care and pet day care for all kinds of pets. We know you love your pets, but you can't always be there, which is where our loving sitters come in. Forget kennels, find a loving neighbor who will give your dog the one-on-one attention they deserve. That way, when you head to work, you know your dog's in good hands.


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Brian Medved
Age: 45
Employer/occupation: owner of WI Home Solutions
Education: B.A., University of Wisconsin-Whitewater
Contact: firstname.lastname@example.com; brianmedved.com
Why are you the right person for the job?
I have a background in construction, management, loss prevention, leadership roles in volunteer organizations and fundraising, this experience has allowed me to lead others and we have accomplished amazing results in a short amount of time. I am a critical thinker that comes up with innovative solutions to complex problems. What is the long-term answer for closing the district's budget gaps?
The board has done a good job closing the budget gap by using the tools provided by ACT 10 and paying down debt. There needs to be five-year, 10-year, etc., planning for budget items to avoid deficits. The board needs to focus on competitive bidding for health care insurance as a way to create savings for the district. A focus on health and wellness programs is a great way to keep staff healthy and reduce costs. My experience in construction should be a compliment to the existing board; I have many ideas to reduce maintenance costs and future building needs.
What is a solution for the district's issue of limited building space and growing enrollment?
Enrollment seems to be leveling off and may even go down in the next several years. Having taken a tour of the schools and being able to see the issues of space first hand, I have several ideas to make better use of space and some possible future expansions for the district. I know that this can be accomplished with minimal expense and fit within our current budget constraints. I will focus on saving for the future because I do believe that our enrollment will increase as the district gets recognized as one of the best in the country.
Jill North
Age: 58
Employer/occupation: hand therapist for private practice hand surgeon at Elmbrook Hospital
Education: occupational degree from Mount Mary College
Contact: (262) 628-4491; email@example.com
Why are you the right person for the job?
I am committed to providing excellent public education. I have been involved in the schools and community for the past 17 years. My experience and involvement gives me a perspective on what is happening in our schools. I positively interact with all groups and am a creative problem solver.
What is the long-term answer for closing the district's budget gaps?
Schools are locked into budgets through revenue caps. The per student cost of education was capped in 1993. Twenty years have passed and any allowable increases along with state cutbacks monetarily challenge school districts to deliver current education or develop any new programming. Capital improvements cannot be funded unless other areas are cut or a referendum is passed to increase spending. Explore additional ways of producing revenue outside of the tax base and state aid. Continue to nurture and foster relationships with school and community groups. These groups use the school buildings but also give back monetarily to the district.
What is a solution for the district's issue of limited building space and growing enrollment?
I am a firm believer in small class sizes. The district has a guideline/policy for class size. We are over the guideline in several elementary grade levels.
Specialty classrooms can be used to deliver grade level education however this means art and music travel from room to room. I feel this is temporary solution to space. Ultimately, building an elementary school is the solution to easing the space needs and getting back to the class size policy. The board will need to feel the pulse of the community in judging whether to put the referendum question out there.
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Poppies Samui
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Week 20 - 16 October 2015 to 22 October 2015
... br>
Massaman curry was on the menu. We had most of the ingredients, three were missing, so we decided to go to the local Tesco Lotus Express, across the way from the fresh produce market. Funny how one goes shopping for just three items and you end up with 11 or so in your basket, which is of course exactly what happened. I was alerted to this fact when Adri asked "can I bring you a basket?" On the way back we stopped off at a banana stall and bought a small stalk ...
Week 10 - 7 August 2015 to 13 August 2015
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Koh phangan 23rd may- ?
... cliff edge shack, it feels like you are intruding in somebody's home. The place is decked in reggae colouring including reggae coloured fairy lights. So the three of us order a drink and sit in the most chilled out bar In the world. We are the only people there. The reggae boys demonstrate various tricks including how to breathe fire by holding the lighter in yer gob , swallowing the gas then light it. ( something I never taught at school) ) and we head home. - see ...
Fishing village night market
We spent the morning relaxing at the resort until 1, when we got the shuttle bus into town. After lunch we did some more shopping, of course. We headed to a place called bo phut or fishermans village for a night market. We weren't expecting anything spectacular and were ...
Day 56 - Ko Samui
Scott and I arrived in Ko Samui yesterday morning (31st July). We are staying at Samui beach resort which is lovely. Considering we paid no more than £15 a night for this hotel, it is on the beach front with private sunbeds, a swimming pool and nice clean bungalows with wifi that occasionally works. The sun is beaming here on the east coast and the water is luke warm. As soon as you are waist deep you are surrounded by ...