text:
- Research article
- Open Access
The vertebrate makorin ubiquitin ligase gene family has been shaped by large-scale duplication and retroposition from an ancestral gonad-specific, maternal-effect gene
- Astrid Böhne1,
- Amandine Darras†1,
- Helena D'Cotta†2,
- Jean-Francois Baroiller2,
- Delphine Galiana-Arnoux1 and
- Jean-Nicolas Volff1Email author
© Böhne et al; licensee BioMed Central Ltd. 2010
- Received: 29 July 2010
- Accepted: 20 December 2010
- Published: 20 December 2010
Members of the makorin (mkrn) gene family encode RING/C3H zinc finger proteins with U3 ubiquitin ligase activity. Although these proteins have been described in a variety of eukaryotes such as plants, fungi, invertebrates and vertebrates including human, almost nothing is known about their structural and functional evolution.
Via partial sequencing of a testis cDNA library from the poeciliid fish Xiphophorus maculatus, we have identified a new member of the makorin gene family, that we called mkrn4. In addition to the already described mkrn1 and mkrn2, mkrn4 is the third example of a makorin gene present in both tetrapods and ray-finned fish. However, this gene was not detected in mouse and rat, suggesting its loss in the lineage leading to rodent murids. Mkrn2 and mkrn4 are located in large ancient duplicated regions in tetrapod and fish genomes, suggesting the possible involvement of ancestral vertebrate-specific genome duplication in the formation of these genes. Intriguingly, many mkrn1 and mkrn2 intronless retrocopies have been detected in mammals but not in other vertebrates, most of them corresponding to pseudogenes. The nature and number of zinc fingers were found to be conserved in Mkrn1 and Mkrn2 but much more variable in Mkrn4, with lineage-specific differences. RT-qPCR analysis demonstrated a highly gonad-biased expression pattern for makorin genes in medaka and zebrafish (ray-finned fishes) and amphibians, but a strong relaxation of this specificity in birds and mammals. All three mkrn genes were maternally expressed before zygotic genome activation in both medaka and zebrafish early embryos.
Our analysis demonstrates that the makorin gene family has evolved through large-scale duplication and subsequent lineage-specific retroposition-mediated duplications in vertebrates. From the three major vertebrate mkrn genes, mkrn4 shows the highest evolutionary dynamics, with lineage-specific loss of zinc fingers and even complete gene elimination from certain groups of vertebrates. Comparative expression analysis strongly suggests that the ancestral E3 ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos.
- Zinc Finger
- Ring Domain
- Zygotic Genome Activation
- Elephant Shark
- Therian Mammal
Three functional mkrn genes, mkrn1, 2 and 3, have been described so far in vertebrates, with mkrn3 being specific to therian mammals. Makorin1 has been well studied in mammals. Its described functions are linked to E3 ubiquitin ligase activity, for example ubiquitination-mediated degradation of capsid proteins as a defence mechanism against virus infection [5]. Mkrn1 is also involved in the control of cell cycle arrest and apoptosis through ubiquitination and proteasome-dependent degradation of proteins p53 and p21 [6]. Mkrn1 targets human telomerase catalytic subunit (hTERT) for proteasome processing during differentiation or cell cycle arrest [7]. Makorin1 might also have transcriptional activity and regulates RNA polymerase II-dependent transcription independently from E3 ligase activity, with either negative or positive effects on gene expression [8]. Changes in mkrn1 expression have been associated with different types of cancer and other diseases, as observed for other RING proteins with E3 ligase activity [9–12].
A close relative to mkrn1 is mkrn3 (aka Znf127 in human and mouse), which has been detected only in therian mammals. Mkrn3 has been first identified in the Prader-Willi syndrome critical region on 15q11.2 in the human genome [13, 14], but with no obvious role in the disease [15]. No function has been assigned so far to Mkrn3. Mkrn3 in fact corresponds to an intronless retrocopy of mkrn1 generated through reverse transcription of an mkrn1 mRNA molecule. The formation of such retrogenes is catalyzed by the reverse transcriptase encoded by autonomous retrotransposable elements. Several other retrocopies of mkrn1 have been identified in mammalian genomes, most of them probably corresponding to pseudogenes. The possible involvement of such pseudogenes in the regulation of the founding source gene mkrn1 has been proposed and debated [8, 13, 14, 16].
The third functional member of the makorin family described in vertebrates is mkrn2. This gene partially overlaps with the raf1 proto-oncogene in antisense transcriptional orientation [17]. Functional data have been only published for the clawed frog Xenopus laevis, where Mkrn2 is a neurogenesis inhibitor acting upstream of glycogen synthase kinase-3beta (GSK-3beta) in the phosphatidylinositol 3-kinase/Akt pathway. The third C3H zinc finger, the Cys-His motif as well as the RING zinc finger are necessary for this anti-neurogenic activity [18, 19].
During the screening of a testis cDNA library from the platyfish Xiphophorus maculatus (poeciliid), we have identified a gene with gonad-specific expression representing a new divergent member of the makorin gene family in vertebrates, which was named mkrn4. Comparative analyses in different species revealed the evolutionary dynamics of this family of RING finger proteins, suggesting an ancestral gonad-specific function and maternal embryonic expression before duplication in vertebrates.
Results and discussion
Mkrn4 is a new member of the vertebrate makorin gene family
In order to identify new potential gonad-specific transcription factors or regulatory proteins expressed in fish gonads, we randomly sequenced clones from a testis cDNA library of the platyfish Xiphophorus maculatus. In silico screening for sequences containing zinc finger domains revealed a clone with similarity to uncharacterized members of the makorin gene family [20] (Genbank accession number HQ377193). Since three functional mkrn genes have been described so far in vertebrates, the newly identified gene was called makorin4 (mkrn4).
Surprisingly, analysis of genome sequence drafts and EST data failed to detect mkrn4 in two murid rodents, the mouse Mus musculus and rat Rattus norvegicus, even after targeted scrutiny of chromosomal segments syntenic to mkrn4-containing loci from other species (on Mmu17 in mouse and Rno20 in rat). Partial mkrn4 sequences were found in other rodents such as Ord's kangaroo rat (Dipodomys ordii), thirteen-lined ground squirrel (Spermophilus tridecemlineatus) and Guinea pig (Cavia porcellus; for this species a deduced sequence could be included into the Mkrn phylogeny), as well as in the American pika Ochotona princeps (lagomorph). No mkrn4-related sequences were identified in the genome draft of another lagomorph, the rabbit Oryctolagus cuniculus, and in the northern tree shrew Tupaia belangeri (order Scandentia). For all these species, no mkrn4 EST sequence was present in databases. Hence, these results suggest that the mkrn4 gene has been lost in murid rodents (mouse and rat). In some other rodent or lagomorph species, mkrn4 may be also on the way of degradation (as suggested by long branch for Guinea pig sequence; Figure 2) or even might have been already eliminated, but genomic sequences of better quality will be required to confirm this hypothesis.
We noticed that mkrn4 was annotated as a pseudogene in the current Ensembl human genome release, under the name of Mkrnp2[24]. Very similar orthologous sequences were identified in syntenic regions of the genome of chimpanzee (chr. 6, 99% nucleotide identity with the human sequence in the coding region), orangutan (chr. 6, 99% nucleotide identity) and macaque (chr. 4, 96% nucleotide identity), a degree of conservation surprising for a pseudogene evolving under neutral selection. Only gorilla did not show any mkrn4 sequence, despite the presence of a large syntenic region on chromosome 6 including several genes surrounding mkrn4 in other primates. However, the draft sequence of this region contains many gaps, especially at the position where mkrn4/mkrnp2 would be expected. Hence, mkrn4 might be absent from draft but present in the genome of the gorilla.
In human, mkrnp2 was annotated as pseudogene due to a premature stop codon at position 53 in the Ensembl transcript. We suggest that the translation start codon was wrongly annotated in the Ensembl prediction. A start codon in frame +3, using the ATG at position 54 of the predicted transcript, leads to a full length protein without any further interruption of the open reading frame. This ATG is conserved between primate sequences and annotated as the bona fide start codon in GenBank entry EAX03829.1 used in sequence alignments and molecular phylogenies (Figures 1 and 2). Primate sequences were reannotated accordingly (Figure 1).
Using primate sequences as query against EST databases in BLAST analysis, no evidence of transcription could be found. We then compared the rate of non-synonymous (Ka) to the rate of synonymous substitutions (Ks) between mkrn sequences in primates (human, chimpanzee, orangutan and macaque) and other vertebrates. The average Ka/Ks value obtained between primate mkrn4 sequences was 0.12, indicating an excess of synonymous vs. non-synonymous substitutions. This value was similar to those obtained between primate mkrn1 (0.16) and mkrn2 (0.11). The average Ka/Ks ratio for mkrn4 between fish sequences (medaka, platyfish and guppy) was 0.27. Taken together, these observations suggest that mkrn4 evolved under purifying selection in primates and fish, as observed for other makorin genes. Mkrn4 might be a functional protein-coding gene in primates but expressed at a very low level, in a particular type of cells or under very specific conditions. Alternatively, mkrn4 might have lost its expression potential after initial functionality in different primate species.
No mkrn4 sequence was found in the genome of more divergent species, including the elephant shark Callorhinchus milii (Chondrichthyes aka cartilaginous fish, a divergent jawed vertebrate) [25] and the invertebrate chordates Branchiostoma floridae (amphioxus) and Ciona intestinalis/savignyi (sea squirts).
Large-scale duplication and the evolution of the makorin gene family
Mkrn1, mkrn2 and mkrn4 are all present in both ray-finned fish and tetrapod lineages, indicating that these genes have been formed through gene duplications at least 450 million years ago early in the vertebrate lineage. Mkrn1 and mkrn2 but not mkrn4 were detected in the genome of elephant shark Callorhinchus milii, a cartilaginous fish. In contrast, we could identify only one makorin gene in amphioxus and sea squirts (non-vertebrate chordates), as well as in other more divergent non-vertebrate species including yeast and rice. In the fly Drosophila melanogaster, four makorin genes were identified, but three of them clearly corresponded to retrocopies of the only bona fide intron-containing mkrn gene. Hence, the mkrn gene family observed in vertebrates is probably the result of two events of gene duplication having taken place between the emergence of vertebrates and the divergence between tetrapods and ray-finned fish. At least the duplication having led to mkrn1 and mkrn2 occurred before the split between cartilaginous and bony fish.
In order to know if gene duplications at the origin of the mkrn family might be linked to rounds of whole genome duplications having occurred at the basis of the vertebrate lineage [26–29], we tested if mkrn genes might be included in large ancient duplicated regions called paralogons having resulted from such events.
These results indicate that mkrn2 and mkrn4 are parts of ancient large duplicated regions present in distant vertebrate species. Hence, these paralogons might have been generated by one of the whole genome duplications having occurred in the ancestral vertebrate lineage, the so called 1R and 2R duplications [26–29]. Both mkrn2 and mkrn4 are called ohnologs (paralogous sequences resulting from whole genome duplications). On the other hand, we can not exclude the occurrence of a very large segmental gene duplication having generated these duplicated blocks.
The region containing mkrn1 shows little conservation between mammals and fish (data not shown). We could not find any evidence for synteny between the mkrn1-containing region and other mkrn regions. It has been previously proposed that mkrn1 and mkrn2 are indeed the result of a single gene duplication event [17].
Retroposition and the evolution of the makorin gene family
In addition to mkrn1, mkrn2 and mkrn4, multiple other mkrn-like sequences are present in mammalian genomes. Some of these sequences correspond to intronless retrocopies of mkrn1. Their functional status concerning expression, regulation and mutual interference has been the source of controversial debate [16, 30].
Mkrn1- and mkrn2-related retro(pseudo)genes in human and mouse genomes.
Gene names
EST support
Human mkrn1
Hsa7 140.152.840-140.179.369
source protein-coding gene with introns
expressed in many adult tissues
Hsa15 23.810.454-23.813.164
protein-coding retrogene
EST data for blood, brain, esophagus, liver, placenta, prostate and skin
Hsa1 43.356.765-43.358.101
no EST support
Hsa2 153.707.170-153.707.338
no EST support
Hsa3 130.647.120-130.646.607
located in intron of ATP2C1 (ENSG00000017260), MKRNP5*
no EST support
Hsa9 99.488.172-99.489.449
RP11-535M15.2, ENSG00000233820, makorin ring finger protein 1 pseudogene
no EST support
Hsa12 88.177.249-88.178.345
no EST support
Hsa12 88.176.895-88.176.996
Hypothalamus BI457939.1
Hsa20 45.092.541-45.093.763
no EST support
HsaX 40.693.508-40.697.060
MKRNP5, MKRN4, ZNF127-Xp, ZNF127L1, ZNF-Xp, ENSG00000238222, OTTHUMG00000024109,
partially processed retropseudogene
no EST support
HsaX 73.380.895-73.382.363
MKRNP1, ZNF127-Xq, RP13-204A15.3, ENSG00000224430
no EST support
HsaX 91.627.539-91.627.653
inside intron of PCDH11X
retro(pseudo?)gene, very short but intact ORF
no EST support
HsaY 5.352.120-5.352.234
inside intron of PCDH11Y
retro(pseudo?)gene, very short but intact ORF
no EST support
Mouse mkrn1
Mmu6 39.347.803-39.370.437
Mkrn1, ENSMUSG00000029922, NM_018810
source protein-coding gene with introns
widely expressed in adult tissue with strong expression in adult gonad and embryonic nervous system [ref. [30] and EST data]
Mmu7 69.563.293-69.564.927
Mkrn3, ENSMUSG00000070527
protein-coding retrogene
EST data for brain, inner ear, pancreas, spinal cord and thymus
Mmu2 27.338.749-27.339.289
no annotation
newly annotated retropseudogene
no EST support
Mmu3 129.843.302-129.845.025
no annotation
no EST support
Mmu5 89.267.034-89.268.427
Mkrn1-ps1, AC160533.6, ENSMUSG00000082389, AF494488
no EST support
Mmu5 111.334.263-111.334.760
no EST support
Mmu13 77.911.041-77.911.846
in Intron of 1110033M05Rik (ENSMUSG00000064138)
no EST support
MmuX 54.923.521-54.923.664
no EST support
Mouse mkrn2
Mmu6 115.551.979-115.568.688
Mkrn2, ENSMUSG00000000439, C81377; 2610002L04Rik
source protein-coding gene with introns
EST data for several tissue
Mmu10 30.093.050-30.095.334
no annotation
no EST support
MmuX 13.504.413-13.506.637
Partially processed retropseudogene (contains only 2 introns)
no EST support
Mmu2 106.753.075-106.754.515
no annotation
no EST support
We detected makorin3 and other retrocopies of makorin1 only in mammals and not in chicken, frog and all five fish genomes analyzed. An mkrn3 sequence was detected in opossum, suggesting an event of retroposition that took place before the split of marsupials from therian mammals. No mkrn3 was found in the current genome draft of the platypus.
Retrocopies of mkrn2 were found in the mouse but neither in the human nor in non-mammalian vertebrate genomes (Table 1). These three retropseudogenic copies of mkrn2 are located on three different chromosomes in M. musculus. No retrocopy of mkrn4 was found in all vertebrate genomes analysed, but three potentially functional retrocopies of the single intron-containing mkrn gene were identified in the fruit fly Drosophila melanogaster. Taken together, these observations demonstrate an intriguing tendency of the mkrn gene family to generate duplicated copies by retroposition.
Structural evolutionary dynamics in the Makorin family
In contrast, this otherwise well conserved structure was not found in Mkrn4 proteins (Figures 1 and 4). While the RING domain and the C-terminal C3H zinc finger are found in all Mkrn4 sequences, the third C3H zinc finger and the CysHis motif are missing and have been probably lost in the ancestral mkrn4 gene early after duplication.
Only fish and Xenopus have kept the first two C3H complete domains, which are both absent from mammalian sequences. In birds, a remnant of one of these domains has been retained in chicken and zebra finch under the form of a C2H domain, the second C3H domain having been maintained only in zebra finch but not in chicken.
These results indicate that the evolution of the Mkrn4 protein is associated with the dynamic loss of zinc finger domains otherwise conserved in Mkrn proteins. This is consistent with a to some extent faster evolution also reflected by the longer branches in the Makorin-like group in the phylogenetic tree (Figure 2). Another example of zinc finger domain loss is visible in mkrn3: this mkrn1-derived retrogene encodes a protein having lost the second C3H zinc finger (Figures 1 and 3) in human and mouse.
In the much conserved Mkrn1 and Mkrn2 groups, the platypus sequences present a reduced number of zinc fingers, like the opossum Mkrn2 sequence. This might reflect a biological reality, but might also be an artifact of local poor sequence assembly quality of the genome drafts for these species.
Evolution of makorin expression in vertebrates
In adult tissue, the whole mkrn gene family showed a clear gonad-biased expression in fish and frog, with particular high expression in ovary (Figure 5). Accordingly, mkrn1 is upregulated during ovarian maturation in flatfish, with strong expression in previtellogenic ovarian follicles [32]. Such a specificity of expression was not observed in birds and mammals, even if mkrn1 has been reported to be strongly expressed in mouse adult testis [4] and mkrn4 showed a slightly testis-biased expression in chicken.
Taken together, these results suggested that the ancestral mkrn gene was preferentially expressed in the gonads in a primitive vertebrate, with conservation of expression pattern after duplication in fish and amphibians but partial relaxation of this specificity and possible extension of functions in birds and mammals.
Mkrn expression during early embryonic stages in fish largely reflected maternally stored mRNA expressed during oogenesis [33, 34], since mRNA levels were very high at very early stages (Figure 6). RNA levels then decreased throughout development, probably due to dilution effect through continuous cell division, and reached a very low level shortly after midblastula transition (MBT), the state of zygotic transcription activation. MBT takes place around stage 11 in medaka [35] and starts at cycle 10 corresponding to 512 cell stage in zebrafish [36] (between 128 cell stage and dome in our experiments). Maternal expression was observed for all three mkrn genes in both medaka and zebrafish, suggesting here again an ancestral expression pattern. As observed in adults, no tissue-specific expression was observed in chicken embryos one day before hatching (Figure 6).
Through the identification of mkrn4, a new member of the makorin gene family present in tetrapods and ray-finned fishes, we were able to gain some insights into the structural and functional evolution of this E3 ubiquitin ligase gene family. We could show that different evolutionary dynamics affect different mkrn genes in vertebrates. While mkrn1 and mkrn2 were detected in all bony vertebrates studied, mkrn4 was found to be absent from rat and mouse, two rodent murids, as well as from rabbit, a lagomorph. In addition, both Mkrn1 and Mkrn2 proteins have retained the ancestral arrangement of zinc fingers, with four C3H motifs surrounding the RING domain responsible for the ubiquitin ligase activity. In contrast, Mkrn4 has lost several motifs early after its formation through duplication, and subsequent lineage-specific zinc finger loss was observed. Taken together, these observations suggest that Mkrn4 is subject to more relaxed evolutionary constraints than Mkrn1 and Mkrn2, with possible development of new general and lineage-specific functions. However, all Mkrn proteins contain the RING domain with adjacent C-terminal C3H zinc finger, indicating that E3 ubiquitin ligase activity and interactions with nucleic acids might be properties common to all Makorin proteins. It appears therefore possible that Mkrn4 function, if still required in rat and mouse, might be performed by Mkrn1 or Mkrn2.
Through comparative genomics, we could find evidence that mkrn2 and mkrn4 have been duplicated together with other genes through a large-scale duplication. These genes are embedded in paralogons, i.e. in large syntenic chromosomal blocks of duplicated genes within a same genome. These paralogons were found in both tetrapods and ray-finned fish, tracing back their origin to at least 450 million years ago. We propose that mkrn2 and mkrn4 duplication was the result of either 1R or 2R, the two events of genome duplication having taken place at the basis of the vertebrate lineage [12, 26–28]. Both 1R and 2R arose before the split between cartilaginous and bony fish; one might therefore expect the presence of both mkrn2 and mkrn4 in the genome of elephant shark. However, only mkrn2 was detected. This might be explained by secondary loss of mkrn4 or incomplete coverage of the elephant shark genome. The molecular mechanism of formation of mkrn1 remains elusive. Involvement of 1R or 2R appears possible (but we could not find any evidence for that) or of a more local event of segmental duplication. Conservation of intron position between mkrn1, mkrn2 and mkrn4 (data not shown) suggests that retroposition was not involved.
After formation of mkrn1, mkrn2 and mkrn4, the makorin gene family was shaped by lineage-specific retroposition-mediated duplications in vertebrates. Such retrogenes are formed by reverse transcription of generally mature mRNA molecules followed by integration of the formed cDNA into a new location of the genome. In mammals, retrogene formation is catalyzed by reverse transcriptase encoded by LINE-1 retrotransposons [37]. Retrogenes were detected for mkrn1 and mkrn2 in mammals but not in other vertebrates, and not for mkrn4. Most of the retrogenes are pseudogenes, but at least one of them, mkrn3, is functional and conserved in therian mammals. Hence, retroposition is an evolutionary mechanism of duplication and functional diversification in the mkrn family. There is at the moment no satisfying explanation for the strong tendency of the mkrn family to generate retrogenes. Germ cell expression, necessary for retrogene formation and transmission to the next generation, might predispose mkrn genes to duplication through retroposition. Goncalves et al.[38] have proposed that the gene expression level in the germ line is correlated with the number of retropseudogenes, since abundant mRNAs are more likely to be taken as a substrate by reverse-transcriptases. However, high mkrn expression is not the only explanation, since no retrocopy was detected for mkrn genes in amphibians and fish, in which they show a strong gonadal expression. Other factors might be involved, for example the affinity of expressed reverse transcriptases for mRNA molecules.
Finally, comparative expression analysis showed a strongly gonad-biased expression pattern for mkrn1, mkrn2 and mkrn4 in medaka and zebrafish (ray-finned fishes) and amphibians, with particularly strong expression in ovary. Even if some mkrn genes showed also preferential gonadal expression in chicken and mouse, expression was much less specific in birds and mammals. Maternal RNA was detected for all three genes in early medaka and zebrafish embryos. Taken together, these results suggest that the ancestral single-copy mkrn gene was gonad-specific in primitive vertebrates, with a possible role in sexual development or gonad functions. This ancestral gene was also possibly maternally expressed and therefore potentially involved in fundamental processes such as fertilization, cell division or germ cell development [34].
Nothing is known concerning the functions of Mkrn proteins in fish and amphibian gonads and embryos, with the exception of a described role for Mkrn2 as a negative regulator of neurogenesis in Xenopus[18]. At the moment it is therefore difficult to propose a function for the ancestral vertebrate Mkrn protein, but results obtained for Mkrn1 suggest a link with the regulation of cell proliferation and apoptosis [4, 6]. An involvement of the ubiquitin pathway in testis and spermatogenesis has been proposed for different organisms and testis-specific transcripts of different ubiquitin-interacting factors have been reported (for example [39–43]). Some data indicated an involvement of the ubiquitin pathway in the maintenance and development of ovarian germ cells in the fly [44] and the zebrafish [45] and during maturation of the ovary in mouse [46]. Several examples of maternally-expressed RING finger proteins with essential functions during development have been reported [47–51]. Multispecies comparative functional analysis of mkrn genes is now required to better understand the evolution of this gene family in vertebrates.
Platyfish (Xiphophorus maculatus, population Rio Jamapa WLC1274, closed colony stock derived from Jp163A), zebrafish (Danio rerio, strain AB/TU) and medaka (Oryzias latipes, strain Hd-rR) were kept under standard conditions at the PRECI aquarium facility of the IFR128 Biosciences Gerland-Lyon Sud (Lyon, France). Fish embryos were raised to the required stages of development in E3 embryo media [52] at 26°C for medaka and 28.5°C for zebrafish. Medaka and zebrafish embryos were staged according to Iwamatsu [53] and Kimmel et al.[54], respectively. Chicken samples (embryonic chicken, one day before hatching and male adult tissue) were provided by B. Pain (Institut de Génomique Fonctionnelle de Lyon, ENS de Lyon, France). Adult Xenopus laevis samples were gifts from F. Vollmar (Department of Cell and Developmental Biology, University of Würzburg, Germany) and C. Schultheis (Physiological Chemistry I, University of Würzburg, Germany). Experimental research reported here has been performed following internationally recognized guidelines (DDSV, Direction Départementale des Services Vétérinaires de Lyon, agreement number A 69 387 0602).
In silico analyses
Protein and nucleotide sequences of mkrn genes were identified using BLAST/BLAT searches against GenBank [55], the current Ensembl genome assemblies ([24] version 57, March 2010) of human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), macaque (Macaca mulatta), mouse lemur (Microcebus murinus), mouse (Mus musculus), guinea pig (Cavia porcellus), horse (Equus caballus), rabbit (Oryctolagus cuniculus), cow (Bos taurus), opossum (Monodelphis domestica), wallaby (Macropus eugenii), platypus (Ornithorhynchus anatinus), chicken (Gallus gallus), clawed frog (Xenopus tropicalis), zebrafish (Danio rerio), medaka (Oryzias latipes), green spotted pufferfish (Tetraodon nigroviridis), fugu (Takifugu rubripes), three-spined stickleback (Gasterosteus aculeatus), sea squirts (Ciona intestinalis and Ciona savignyi), the amphioxus genome (Branchiostoma floridae) [56] and the elephant shark genome (Callorhinchus milii) [57]. Automatic genome annotation was refined manually using EST data and related protein sequences with the help of FGENESH [58].
Nucleotide and protein sequences were loaded into BioEdit [59] and aligned using MUSCLE [60, 61] and CLUSTALW2 [62]. Alignments were checked manually and ambiguously aligned regions were removed before phylogenetic analysis. Phylogenetic trees were calculated based on a 131 amino acid alignment including the RING domain and the downstream adjacent C3H zinc finger. Maximum likelihood reconstruction was done using PhyML 3.0 [63, 64], with 1000 bootstrap replicates under the LG model. Similar results were obtained using the neighbor-joining method as implemented in MEGA4 [65] with bootstrap values of 10.000 replicates. MEGA4 was also used to visualize phylogenetic trees. To establish syntenic relationships between genomes we used Genomicus [66], Synteny Database [67] and information provided by Ensembl Gene Trees [24]. Annotation of conserved protein motifs was performed with Motif Scan [68] and NCBI conserved domains [12].
Ka/Ks calculation was peformed using KaKsCalculator under the MYN model [69] on pairwise nucleotide alignments done with LASTZ [70] and MUSCLE [60, 61]. Indicated Ka/Ks values are averages of values obtained for all possible pairwise comparisons.
RT-qPCR expression analysis
Total RNA was extracted from separated male and female tissues (brain, eyes, gills, heart, intestine, kidneys, lung, liver, muscle, ovary, skin, spleen and testis) and from whole embryos using the TRIReagent (Molecular Research Center, Inc.). After DNase treatment, reverse transcription was performed starting from 1 μg of total RNA using the RevertAid First Strand Synthesis kit (Fermentas) and random hexamer primers. Real-time PCR was done on 2 μl of dilutional series of cDNA starting from a dilution of factor 20 using IQTM Custom SYBR® Green Supermix (Bio-Rad). PCR amplification was monitored with a CFX96 RealtimeSystem (Bio-Rad). After one incubation step at 95°C for 3 min, the thermal cycling protocol was as follows: 95°C for 10 seconds, 55-62°C (primer-dependent annealing temperature) for 20 seconds and 72°C for 15 seconds during 40 cycles. Primers were designed manually and ordered from Invitrogen (Additional file 3). For each primer set, the efficiency of the PCR reaction was measured in duplicate on serial dilutions (factor 10) of the cDNA sample. Real-time PCR efficiencies (E) for each reaction were calculated from the slope of the standard curve using the equation E = 10(-1/slope)-1 as implemented in the BioRad CFX manager V1.5 software, with 100% efficiency as an indicator of a robust assay. All results reported here are averages of two independent reverse transcription reactions, which have been each tested with two PCR reactions. For quantification, data were analyzed by the 2-DDCT method [71], by normalizing to the housekeeping gene rpl7 (ribosomal protein l7). Expression was normalized to eye tissue (fish) or heart (due to lack of eye samples for female frog and chicken).
This work was supported by a Ph.D. fellowship from the French Ministère de l'enseignement supérieur et de la recherche to AB and by grants from the Agence Nationale de la Recherche (ANR), the Institut National de la Recherche Agronomique (INRA, département PHASE), the Centre National de la Recherche Scientifique (CNRS) and the Fondation pour la Recherche Médicale (FRM) (to JNV). We thank B. Pain for providing chicken samples, F. Vollmar and C. Schultheis for Xenopus samples and M. Sémon for helpful discussion on phylogenetic reconstruction and synteny analysis.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<url>http://creativecommons.org/licenses/by/2.0</url>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Mollie Makes issue 32
Sew a teacup garden, hand-print neon napkins, and stitch a Scandi-style horse, in issue 32 of Mollie Makes. Out today!
This issue we're celebrating those special touches that take makes to the next level. We're magicking napkins into conversation pieces, stitching up a storm in leftover fabric and, dare we say it, starting to think about Christmas gifts. We're also having spooky thoughts in prep for Halloween.
Bring the outside in with Liberty-print clouds – the perfect rainy day make – and make light work of Jane Foster's retro lampshade. Read all about a collector of Swedish Dala horses, then try sewing your own. Plus, head to Utrecht for an apartment tour with illustrator Annemoon van Steen.
Available from all good newsagents, supermarkets and our official online store. Sold out? Download it from Zinio, Google Play or Apple Newsstand.
Where to find Mollie Makes overseas
What's on your craft table? Show us on Instagram, Twitter, Pinterest and Facebook.

subset: commoncrawl


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Coming Soon: "When Your Eyes Close" – release date: 7th September 2018….
When Black Dogs Sing
Tanya Farrelly's debut short fiction collection, When Black Dogs Sing,
introduces us to an intriguing range of twenty-first century Irish
characters. A young woman rents a room, to her boyfriend's horror, in the
home of an eccentric, bi-curious photographer; a poet attends a party at
his girlfriend's family home to discover that the invite was made with
nefarious intention; a husband is outraged to discover that his wife is
posing at a life drawing class; two girls break into the flat of an internet
date and stumble on a dangerous secret; a stakeout ends in dire
consequence when a friendship comes undone; a woman leaves home on
a freezing night in the hope of gaining her partner's attention; and in the
title story a mother is haunted by her son's disappearance.
Cover Art by David Sweet: http://davidsweet.co.uk/
The Girl Behind the Lens
A dark psychological thriller about the secrets that destroy us, perfect for fans of THE GIRL ON THE TRAIN and BEHIND CLOSED DOORS.
Photography student Joanna Lacey has always been close to her mother. But when Rachel Arnold turns up on their doorstep telling them that a man's body has been found in the canal, Joanna's world falls apart. The father she never knew was a married man – and now he's dead.
At the funeral, Joanna meets Oliver Molloy, the man who found the body. She's convinced her mother is still keeping things from her and turns to him for help. Never one to say no to a pretty face, Oliver is happy to oblige.
But Oliver is concealing a dark secret of his own, and Joanna soon starts to suspect that something isn't right. Can she uncover the truth before the past destroys them both?
Buy books at: https://www.amazon.co.uk/Tanya-Farrelly/e/B01MS2JHTV/ref=sr_tc_2_0?qid=1488922762&sr=8-2-ent


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A TEENAGER faces jail after committing one of the most horrific animal cruelty offences in city history.
The 17-year-old, who cannot be named for legal reasons, picked up a dog while it was being walked by its owner and threw it from a bridge on to a busy road.
Harry the Jack Russell suffered such serious injuries that he had to be put to sleep by a vet.
This week Milton Keynes magistrates heard how the teenager admitted his crime – which he blames on drink and cannabis – and they adjourned the case until next month for sentencing.
The bench has the power to grant a maximum of six months' imprisonment for a single offence and/or a fine of up to £5,000.
At an earlier hearing they heard how the bare-chested teenager and two friends demanded money from Harry's owner Kevin Wilding in July last year.
When Mr Wilding refused, the teenager grabbed seven-year-old Harry, lifted him above his head and threw him over the side of the bridge above the busy V8 Marlborough Street.
A motorist managed to swerve but Harry suffered head injuries causing brain damage and blindness.


text: http://bugs.winehq.org/show_bug.cgi?id=21603 --- Comment #6 from Robert Riches <rm.riches at verizon.net> 2010-02-06 13:00:19 --- I'll second that the patch resolves the crash. -- Configure bugmail: http://bugs.winehq.org/userprefs.cgi?tab=email Do not reply to this email, post in Bugzilla using the above URL to reply. ------- You are receiving this mail because: ------- You are watching all bug changes.


text: Located in Manitoba, the cross country skiing in Riding Mountain Park is known for two nice XC ski trails. The best Riding Mountain Park cross country skiing based on popularity are considered to be Focus Andy Lake and North Escarpment Trail System. For a detailed Nordic ski trail description and printable trailhead map, just select a Riding Mountain Park cross country skiing trail below.


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- Any of Enju's Precocious Crush fantasies involving Rentaro will fall into this.
- In episode 1, when Rentaro heard about a bean sprout sale (note, the Tendo Civil Security Company is always at Perpetual Poverty), he rushes off to buy them and gave them to Kisara. Kisara's reaction is priceless.
- A sick Miori collapses in Rentaro's apartment in episode 5. Kisara then enters, and collapses from hunger right on top of her. He tells Enju having those two girls in the same vicinity is a bad thing, as evidenced by the Cat Fight moments later when Miori teases Rentaro too much in front of Kisara.
- As the Cat Fight starts, Rentaro and Enju nonchalantly move the dinner table to the next room.
- Kisara's offer to hold hands with Rentaro in episode 6 is foiled when a tiny pair of hands begins to grope her breasts instead. Said hands belong to Enju, who's not happy about how big Kisara's breasts are.
- From the same episode, Kisara's response to Rentaro having a meeting with Miori alone is to intercept Rentaro right when he arrives at the office and instruct him to fill her with bullets after she tear gasses the room. Rentaro, with a look and voice that pretty much says he expected this to happen and is tired of it all, just tells her to wait outside and deal with it.
- This exchange from Episode 8Kisara: Good morning, Satomi-kun, I'm coming in.(Rentaro and Enju wake up)Rentaro: Could you say that before you get in?Kisara: Shut up, I've come to take back Tina today, Huh? Where is she anyway?(Tina crawls out from under Rentaro's blanket)Tina: Good morning. Wasn't last night fun, onii-san? Though it was scary at first, I'm happy that you taught me a lot of things.(Cue shocked looks from Rentaro, Enju, and Kisara)Kisara: I-I'm calling the police...Enju: Tina beat me to it!
- A meta example; the author notes in the light novel have numerous Self-Deprecation, but this one happens to be noteworthy in volume 5's author notes:Shiden Kanzaki: Finally, there are the readers. Using the sweet bait of "lolis, lolis here" and "boobs, boobs here" like a peepshow to entice the readers, then turning the page to show disgusting scenes like heads falling suddenly to the ground or disemboweled guts, known for underhanded marketing methods rare in recent times, the worst writer in all of historyme, Shiden Kanzakistill energetically aiming for renewing the record for being the worst.
Funny / Black Bullet
Go To


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Maxitec has made two significant 11th hour inclusions to its exhibition inventory for the Austech section of Reed Exhibitions' NMW 2008.
The sheet metal machine and software specialist will provide access to the Tensol Origami long-length folding machine from OEMB, which is designed for long length work and the Finn-Power servo electric turret punch press E5 Compact Express for bending, forming, tapping and marking.
Tensol Origami:
The Origami has machines with folding lengths of four metres, six metres, eight metres, 10 meters, 12 metres and 14 metres, as well as customised lengths for the occasional specific need.
This wear-proof, long life folding machine has a hardened reinforcement bar suitable for folding materials, which exceed the hardness of standard steels.
Work depths of these machines vary from (Type H) 1,100mm depth, 1,250mm depth (Type I), 1,500mm depth (Type U).
Much of the performance of these folding machines is based on sophisticated control units. These simple, reliable, intuitive and precise control systems have features including one program allowing possibility of more than 100 lines, touch screen operation, program incremental entries, program fold repetitions, hard disk minimum of 40GB, numeric program names, USB stick for backup and programs restore and ergonomic designed control consol.
In addition, there is the possibility to execute radius with the help of a specific programming macro, possibility to manage the optional tapered back gauge and the programming of the optional pneumatic disappearing back-stop fingers on the BC-04 and BV-99.
Also, high speed pumps can be managed; managing the signalling lights up to 4 positions, laser safety system management, hydraulic shear management, segment management and graphic part representation and all of this can be managed offline.
Finn-Power E5 Compact Express
E5 Compact Express is a compact, high-performance servo electric turret punch press with fully automated material handling capabilities. It is a multi-purpose machine for bending, forming, tapping and marking.
With fully programmable punching and upforming stroke (NC-axis), the E5 Compact Express from Finn-Power is available with 20 ton and 30 ton punching force.
For economic operation, the E5 has low electric connection, consumption and maintenance costs. The compact size and small foot print make it easy to place in every facility.
The machine is well suited to small fabrication shops wanting to introduce some level of automation. It can be easily worked in manual mode just like any conventional turret press.
Ease of manual loading is greatly enhanced by the availability of 4 positioning pins ensuring optimal operator ergonomics. Unloading of processed components is semi-automatic: the machine places the components in a freely programmable position on the table thus making the operator's work much easier.
During automatic operation, sheets are loaded from a loading table to the clamps of the turret punch press. Double sheet detection and centring are performed for every sheet.
Ready sheets are unloaded using a moving table and is carried out simultaneously while the loader is loading the next sheet. Processed sheets can be stacked on an unloading table, which is located below the moving table and is adjusted automatically as the sheet stack grows.
Raw material can be added and unloaded sheets removed while the machine continues to operate. Material flow to and from the machine is from one direction, its loading table works at optimal operating height, it has large loading sheet stack capacity of 350mm stack height and 3,000kg stack weight.


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This part is meant as a help for those people that really want to respect the Chinese etiquette and thus earn a lot of respect from the Chinese.
China is known as a state of etiquette and ceremonies. Many proverbs have been passed down from generation to generation such as 'civility costs nothing' or 'courtesy demands reciprocity' and so on. For instance, there is an interesting short story. Once upon a time, a man went on a long tour to visit his friend with a swan as a gift. But it escaped from the cage on the way and in his effort to catch it, he got hold of nothing but a feather. Instead of returning home, he continued his journey with the swan feather. When his friend received this unexpected gift, he was deeply moved by the story as well as the sincerity. And the saying 'the gift is nothing much, but it's the thought that counts.' was spread far and wide.
Chinese used to cup one hand in the other before the chest as a salute. This tradition has a history of more than 2000 years and nowadays it is seldom used except in the Spring Festival. And shaking hands is more popular and appropriate on some formal occasions. Bowing, as to convey respect to the higher level, is often used by the lower like subordinates, students, and attendants. But at present Chinese youngsters tend to simply nod as a greeting. To some extent this evolution reflects the ever-increasing paces of modern life.
It is common social practice to introduce the junior to the senior, or the familiar to the unfamiliar. When you start a talk with a stranger, the topics such as weather, food, or hobbies may be good choices to break the ice. To a man, a chat about current affairs, sports, stock market or his job can usually go on smoothly. Similar to Western customs, you should be cautious to ask a woman private questions. However, relaxing talks about her job or family life will never put you into danger. She is usually glad to offer you some advice on how to cook Chinese food or get accustomed to local life. Things will be quite different when you've made acquaintance with them. Implicit as Chinese are said to be, they are actually humorous enough to appreciate the exaggerated jokes of Americans.
As is said above, Chinese consider gifts as an important part to show courtesy. It is appropriate to give gifts on occasions such as festival, birthday, wedding, or visiting a patient. If you are invited to a family party, small gifts like wine, tea, cigarettes, or candies are welcomed. Also fruit, pastries, and flowers are a safe choice. As to other things, you should pay a little attention to the cultural differences. Contrary to Westerners, odd numbers are thought to be unfortunate. So wedding gifts and birthday gifts for the aged are always sent in pairs for the old saying goes that blessings come in pairs. Though four is an even number, it reads like death in Chinese thus is avoided. So is pear for being a homophone of separation. And a gift of clock sounds like attending other's funeral so it is a taboo, too. As connected with death and sorrow, black and white are also the last in the choice. Gift giving is unsuitable in public except for some souvenirs. Your good intentions or gratitude should be given priority to but not the value of the gifts. Otherwise the receiver may mistake it for a bribe.
Conservative suits for men with subtle colors are the norm.
Women should avoid high heels and short sleeved blouses. The Chinese frown on women who display too much.
Subtle, neutral colors should be worn by both men and women.
Casual dress should be conservative as well.
Men and women can wear jeans. However, jeans are not acceptable for business meetings.
Revealing clothing for women is considered offensive to Chinese businessmen.
Do not use large hand movements. The Chinese do not speak with their hands. Your movements may be distracting to your host.
Personal contact must be avoided at all cost. It is highly inappropriate for a man to touch a woman in public.
Do not point when speaking.
To point do not use your index finger, use an open palm.
It is considered improper to put your hand in your mouth.
Avoid acts that involve the mouth.
Gift giving is a very delicate issue in China. It is illegal to give gifts to government official however; it has become more commonplace in the business world (more information about gift giving at the end!).
It is more acceptable to give gifts either in private or to a group as a whole to avoid embarrassment.
The most acceptable gift is a banquet.
Quality writing pens as considered favored gifts.
The following gifts and/or colors are associated with death and should not be given:
* Clocks
* Straw sandals
* A stork or crane
* Handkerchiefs
* Anything white, blue or black
Always arrive on time or early if you are the guest.
Do not discuss business at meals.
Do not start to eat or drink prior to the host.
As a cultural courtesy, you should taste all the dishes you are offered.
Sample meals only, there may be several courses.
Never place your chopsticks straight up in your bowl. By placing your sticks upright in your bowl your will remind your host of joss sticks which connotes death.
Do not drop the chopsticks it is considered bad luck.
Do not eat all of your meal. If you eat all of your meal, the Chinese will assume you did not receive enough food and are still hungry.
Women do not usually drink at meals.
Tipping is considered insulting, however the practice is becoming more common.
Bowing or nodding is the common greeting; however, you may be offered a handshake. Wait for the Chinese to offer their hand first.
Applause is common when greeting a crowd; the same is expected in return.
Introductions are formal. Use formal titles.
Often times Chinese will use a nickname to assist Westerners.
Being on time is vital in China.
Appointments are a must for business.
Contacts should be made prior to your trip.
Bring several copies of all written documents for your meetings.
The decision making process is slow. You should not expect to conclude your business swiftly.
Many Chinese will want to consult with the stars or wait for a lucky day before they make a decision.
Present and receive cards with both hands.
Never write on a business card or put it in your wallet or pocket. Carry a small card case.
The most important member of your company or group should lead important meetings. Chinese value rank and status.
Develop a working knowledge of Chinese culture.
Allow the Chinese to leave a meeting first.
Gift giving!
Countries in the world with a Chinese cultural influence, a collectivist attitude, accept gifts with a reserved demeanor. In order not to appear greedy, a gift will not be immediately taken, but refused three times before finally being accepted. Each time it's refused, you as the giver must graciously continue to offer the gift. And once it's taken, tell the person you're happy it's been accepted.
The gift is offered using both hands and must be gift-wrapped; though it won't be opened it front of you. It will be set aside and opened later. This tradition eliminates any concern that the recipient's face might show any disappointment with the gift.
If you're presented a gift, follow the same process of refusing it three times then accept it with both hands. You'll also not open it, but wait until later.
In China, official business policy considers gifts as bribes, which are illegal. Though the policy is softening, there may be times when a gift you offer will absolutely not be accepted. Should you find yourself in this situation, graciously say you understand and withdraw it. Waiting until negotiations have concluded will eliminate the appearance of bribery when a gift is presented.
A good guideline if there's a concern is to offer a gift, saying you're giving it on behalf of your company. It's important to always honor the most senior person, so he will be the individual you actually present with the gift, stating you want him to accept it on behalf of his company. This gesture, company to company, will usually circumvent any problem regarding undue influence. If you have several gifts to present, never give the same item to people of different rank or stature. The more senior the person, the more expensive the gift.
Typically one person is not singled out to receive a special gift, especially in front of a group. If you've established a good working relationship with someone and want to give a gift, arrange a time when the two of you are alone to present it. Then when you do give it, be sure to say it's being offered as a gesture of your friendship, not business.
A gift's value should be commensurate with the level of the business dealings. This applies both to an individual's gift and a corporate gift. There are times when an expensive gift fits the occasion and circumstance, but an overly extravagant one could create complications or embarrassment, as the recipient may not be able to reciprocate.
In Chinese culture symbolism is important, with colors and numbers having special meaning. For instance, at Chinese New Year, Money may be given in a red envelope; it must be even amount, using an even number of new bills.
Red is a lucky color; pink and yellow represent happiness; and the number 8 is the luckiest number. The colors black, white and blue and the #4, or four of anything, are negatively associated with death or funerals. Also included in this category are clocks, handkerchiefs, and straw sandals.
Sharp objects like knives or scissors represent a 'severing of a friendship or relationship'- including a business relationship.
You don't want to inadvertently select a gift that has a negative or unlucky association. And because of the symbolism, it can happen. For instance, a fine writing pen would be a good gift, unless it has red ink.
Early in your business relationships, you may want to make your gift selections from a local store where you'll be given the proper information and direction. At least it's wise to have items gift wrapped once you've arrived in the country, to eliminate incorrect choices for colors and types of paper.
In China, plan a banquet, especially if you are being honored with one.
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- Poster presentation
- Open Access
Growing and equipping the infection control community in Singapore
© Ling et al; licensee BioMed Central Ltd. 2011
- Published: 29 June 2011
- Public Health
- Clinical Outcome
- Lower Cost
- Control Program
- Care Centre
Introduction / objectives
Infection Control Association (Singapore), ICAS, founded in 1999, has 153 members of whom 25 are from countries outside Singapore. To facilitate exchange of information and data on infection control principles and practices, ad-hoc seminars and workshops are held to address topics of interest.
It conducted its 1st basic course in Infection Control in 2002, in collaboration with the Asia Pacific Society of Infection Control (APSIC). This comprehensive program attracted large number of ICPs from the region and is offered once in 2 years. An Advance Course is held on alternate years to meet needs of senior ICPs. ICAS developed 3 national guidelines or standards through consensus working groups from its members and the Ministry of Health – Schools and Day Care Centres; Intermediate and Long Term Care Centres and the Management of MRSA Patients.
In 2009, it celebrated its 10th Anniversary with the launch of its 1st International Congress of ICAS, which helped to foster research in the Infection Control Community. Its recently held 2nd congress was a success with close to 400 registrants for its congress and post-congress workshops.
The effectiveness of infection control programs now demand that our ICPs turn to creative strategies to achieve improved clinical outcomes with conserved resources and lower cost. The challenge for ICAS in the next decade will be to equip its members to be effective agents of change.
Disclosure of interest
None declared.
Authors' Affiliations
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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set database: LEGO 610 super wheel toy Canada versionset number: 610
image courtesy of bricklink
image courtesy of lugnet
image courtesy of LEGO Historian
image courtesy of friedi1959
set name: super wheel toy Canada version
theme: Samsonite
year: 1965
pieces: 304
price: us$10
minifig: none
nice Samsonite set.
Canada version with long box.
come with basic bricks, wheels and specialty bricks.
can be built into vehicles.
released similar as 610 super wheel toy US version and 1610 super wheel toy Sears exclusive by LEGO in 1965.
great playability.
overall design is great.


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Despite the rough patch that started of Beyoncé's new album, she has really pulled it off and showed that she isn't going down slope! With "Run the World (Girls)" not getting the attention that we all would have expected there's plenty of other tracks on Beyoncé's forth album "4" that covers up for Run the World! Check out what Beyoncé had to say about the direction of "4" in an interview with Dazed & Confused Magazine:
"I don't want to hear about 'Single Ladies' or 'Crazy In Love'. I don't want to hear it. I believe that there are certain things that happen and they happen naturally…Honestly, I'm much more interested in showing people the sensitive, the passionate, and the compassionate person that I am. More so than Sasha Fierce."
"But right now, this part of my life is all about embracing change and going to the next level, taking risks and showing my bravery. Not being safe. Not doing the song that everyone else on pop radio sounds like. Basically, what everyone else is doing, I want to do something completely different. I feel like I've earned that right. Risks excite me."
Beyoncé starts of the album with the track "1+1" which impressed everyone when she performed a flawless performance of the track on the American Idol. This track shows off Beyoncé's vocal ability! The song is defiantly a nationwide favourite of the album! It's my personal favourite! Beyoncé then runs into the album with the song "I Care" which has to be another favourite of mine! This song is a Mid-tempo/Ballad with the lyrics such as
"I CARE! I know you don't care too much but I CARE!"
You got Beyoncé singing this lyric with a thumping beat and simple backing vocals saying "Lalalalala, Lalalalala" I'm telling you that this song is a Winner!
The next song that is Just a CLASSIC! It is another Irreplaceable! Do I need to say any more? Do you know what song I'm talking about? I'm talking about "BEST THING I NEVER HAD". The song title within its self says it all! If you haven't heard this song yet then you need to YouTube it Right about………… NOW!
Beyoncé really gives me that old school vibe as well on this album! Tracks like "Party" and "Love on Top" are so refreshing. "Love on Top" vocal's are EXCELLENT! I haven't heard Beyoncé sing like this on an album before! "Love on Top" is defiantly another Level from Ms. Knowles!
The album isn't just about Ballad's though! It wouldn't be a Beyoncé album if she didn't do some up-tempo tracks for everybody to shake their booty too! These tracks are "Countdown" and "End of Time". Man how I wish "Countdown" was the first single! These tracks would have really set this album off! But every artist has made wrong decisions with the first single choices. But Countdown and End of Time are Club Dance floor fillers! Mark my words!
The 3 tracks that she has added to the Deluxe Edition of the album are Flawless. Theirs only two that really stand out for me though. That's "Dance For You" and "Schoolin' for Life". "Dance For you" really gives me that Dangerously In Love vibe, defiantly a Slow Jam Dance floor filler. Defiantly a song you would dance to if you're a newlywed! The song left me speechless when I first heard it! "Schoolin' For Life" really is different for Beyoncé and I think I like it because it's so different! With the lyrics that stands out like
"Who needs a degree when your schoolin' for life!?"
Schoolin' for life will defiantly be one of those songs that you'll see in everybody's Facebook Status!
Wrapping up this album review I want to say that this album has reminded me of her first album, second and third! The Old school vibes and the Ballad's such as "I Was Here" remind me of I Am… Sasha Fierce, "Dance For You" reminds me Of Dangerously In Love and "Best Thing I Never had" reminds me of B'Day. If you're really not sure to buy the album because of "Run the World" I'm here to tell you that the album is REALLY good. It's really a different sound from Beyoncé, it's still R&B but it is very different. It must be a part of your Beyoncé collection!Follow @iMallyUK


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Car Games
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Indestruc2 Tank
Escape Inc
Chronicle Bulletin After Story
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Awesome Planes
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Building Rush Unblocked New towns are under construction. Build production centers and deliver goods to construction sites to meet the revenue target in each level.
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Doris Graf-Smith, Interim Priest
My parents met at St. Luke's Episcopal Church in Miss John Bouye's Bible Class. Mom wore a big hat so Daddy could chase her out to the sidewalk after church! I met Archer at UGA, and he wasn't going to go to church if it weren't the Episcopal Church. Done. Fourteen years later, I got a really really strong nudge to consider Holy Orders. Seminary at Emory's Candler School of Theology was followed by parish pastoring in a flock of churches in the Diocese of Atlanta. The moral of this story is: Go to church; wear a bit hat; and listen to God's call to each of us.
Gwin Hanahan, Associate Priest
Gwin was ordained an Episcopal priest at All Saints Episcopal Church, Atlanta in April 2013, five days before her 65th birthday. Following her ordination, she celebrated her first communion with the Church of the Common Ground, a church community on the streets of downtown Atlanta. Gwin came to St. Matthias', Toccoa after she and her husband Jim came home from Tanzania where they had been invited by Bishop Mdimi Mhogolo to return, Gwin as a teacher-priest and Jim as a physician. Mission work is in their future! Growing up in the then-village of Clemson, SC, as the daughter of a country doctor, Gwin accompanied her dad as he attended patients across three rural counties. Gwin and Jim raised four children in the Episcopal church while Jim built a large family practice in his clinic in Seneca, SC, and Gwin kept the home fires burning, sometimes teaching at the Clemson Montessori School. Their five grandchildren are lights in their lives.


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The Dinosaur Museum
Icen Way
Dorset DT1 1EW
This award-winning museum is very popular with adults and children alike. Facts, fossils, skeletons and reconstructions are presented in an interesting and fun way encouraging visitors to touch and feel the exhibits.
Simple information is displayed at child eye-level, more detailed information higher up. You can put your hand in the dinosaur's mouth and feel its teeth, touch the skin and feathers, and listen to the noises they might have made.
There are life-sized models of all the familiar dinosaurs – Tyrannosaurus Rex, Megalosaurus, Protoceratops and Ornithomimus. Also on display are dinosaur bones, skeletal remains of dinosaurs and other fossils from all parts of the world.
The Dinosaur Museum is close to the Dorset Teddy Bear Museum.
Plan Your Visit
Accommodation - Search & Book through Expedia here:
Opening Dates & Times
All year except 24, 25 & 26 December
7 days a week
April to Oct 09:30 – 17:30 hours
Nov to Mar 10:00 – 16:30 hours
Admission Costs
Go to the official museum website (below) for up to date information.
Contact & Further Information
+44 (0)1305 269 880
Getting There
The museum is in the centre of Dorchester, in Icen Way, just off High East Street, and near the main car-parks. There is pedestrian sign-posting from these car parks. The closest car park is about 44 yards (40 metres) away. The main car parks are about 109 yards (100 metres).
- By Car
Dorchester is centrally located on the intersection of the A35 and the A37 and is within comfortable reach of the M27, M3 and M5 motorways.
- By Rail
There are excellent rail links by South West TrainServices on the Waterloo to Weymouth line stopping at Dorchester South. The Sprinter links Bristol to Dorchester West Station. The Museum is about 10-15 minute walk from either station. For timetables, fares & ticketing go to Web: National Rail Enquiries.
Google Map - Dinosaur Museum


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Transition Eye Shadows For Dark Skin
When you think of makeup, it is your eye makeup in which you play with so many colors. So little practice and good experience is required to get it right. You play with many shades of eye shadows, eye liners, mascara and transition colors. Transition colors? What are they? Have you heard of them before? If no then get to know what it is exactly.
It is a matte color used in between the lid eye shadow and the brow highlight. It is worth spending on it because blending it is easier than any other type of eye shadow. So, how do they look like and what shades will suit my dark skin tone?
Here we found few transition eye shadows that will suit your dark skin tone.
MAC Matte Brown Eye Shadows
There are so many brown shades available from MAC. Few to list are wedge, cork, texture, saddle, soft brown, era, kid, malt, espresso, brown down, hand written, swiss chocolate, folie etc. Among these swiss chocolate, texture, soft brown, espresso, twink, antiqued and folie may suit most of the Indian dark skin tones. These eye shadows have buttery texture, good pigmentation and stay over 5 hours. Check few of these shades which you may like to try.
1. Swiss Chocolate: This has a muted reddish brown shade which will suit medium-dark skin tones or you can say wheatish skin tones well.
2. Texture: It's a warm medium brown or burnt caramel shade that goes with many dark skinned women perfectly.
3. Soft Brown: It's a neutral brown shadow that doesn't show up much. Many women like it but if you have dark skin tone then you must check if it matches your skin or not.
4. Espresso: It is a dark brown color yet a not muddy.
5. Twink: This is a gorgeous rich reddish brown shade with shimmers in it. This will suit almost all skin tones.
6. Antiqued: It is looks like ash brown with bronze as described but it seems like brown with strong red undertones. This one as well goes perfectly with dark skin tones or almost all skin tones.
7. Folie: It is yet another brown shade with red undertones that looks amazing on many skin tones.
Coastal Scents Hot Pot:
A reason to get Coastal Scents eye shadows is that many of their eye shadows are dupes of MAC eye shadows. You can save some bucks in your pocket.
1. Cherry Chocolate: Its shade is same as the MAC twink eye shadow; you can call it as a dupe of MAC twink. So it is also a reddish brown shade with shimmers in it.
2. Amber Bronze: It is a warm, rustic bronze with shimmers in it. It is a dupe of MAC bronze eye shadow.
3. Light Taupe: It is a dupe of MAC charcoal brown and has deep, exotic taupe hue shade but it looks like a dark brown shade that goes with all skin tones.
4. Burnt Umber: It is a dupe of MAC saddle eye shadow and has rustic deep berry hue which looks like a dark brown shade.
Nyx Adorable Palette TAS01:
This has 6 eye shadows that include 3 brown shades which are named as matte dark brown, matte brown and shimmery brown. Matte dark brown is a deeper hue with dark chocolate shade; matte brown is a matte lighter brown shade that looks like a chocolate shade while shimmery brown has shimmers in it and is chalky. These three browns can be well used by dark skin toned ladies.
Inglot Freedom System Eye Shadow D.S. 608:
This is warm brown shade which is neither light nor dark brown. This will suit medium to dusky skin tones perfectly.
L'Oreal Infallible Eye Shadow In Endless Chocolate:
This is a beautiful shimmery chocolate brown shade best suitable for very dark skin tones with 7-8 hour staying power and awesome pigmentation.
Other than these there are yet many brown eye shadows available in the market to choose from. While purchasing choose the right shade that is neither too light nor too dark on you.
Have you tried any Transition Eye shadows Before?
You may also enjoy reading:
- MAC Pro Lip Palette 6 Editorial Oranges On Olive Brown Indian Skin Tone
- How To Get Perfect Indian Brows With A Gel Eyeliner (NYX Dark Brown Gel Eye Liner)
- Chambor Dazzle Eyeliner Pencil in Grey Dazzle Review, Swatches & FOTD
- 10 Colored Eyeliners To Brighten The Eyes
- Rimmel London Scandal Eyes Waterproof Kohl Eyeliner 002 Sparkling Black Review & EOTD
- 10 Must-Have Products From NYX


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- 免費無線網絡
- 免費泊車
- 瀏覽酒店網站
Danke, Yusef A -- and ahlan wa sahlan! We look forward to welcoming you back. Thanks for the kind words: so pleased you felt comfortable in our vibrant African colours and were well-fed . . . we're waiting for your return visit.
On behalf of the whole team,
Anahid & Armin
Well, we hope you'll be back for a second time, Vidale_Rodi . . . Very glad that we were able to assist with your pre-arrival arrangements: we believe that personalised service, tips and advice are the best reasons to choose a boutique hotel like ours', so take this very seriously.
I'm so pleased that you felt comfortable and happy with our location and value. Thanks for your great review and we hope to welcome you back again soon!
All the best from the entire team -- and our very best wishes for the holidays,
我們到非洲南部之後就來這個酒店住了四個晚上。這個酒店很好,很安靜,但我們累的時候,就去附近街區的高級餐廳吃飯。老闆和員工都很好!很熱情、親切。早餐菜單非常豐盛,有很多選擇。在到達之前,我們希望酒店能夠安排人去機場接我們,然後我們就有了一個出色的司機和導遊:Roger Cresswell,他帶我們去Cape Peak和Simonstown進行一天的旅行。我喜歡充滿藝術感的窗戶和傢具。我們一定會再來這裏!服務、工作人員、房間都很棒。
Warm, welcoming and hospitable -- if we got these essentials right for you folks, we're proud and happy, ArtTrekker! Thanks so much for the recognition of our great team. We love hosting friends exploring the world together, all the more so when we're able to offer advice, tips and enablers (like the fabulous Roger Cresswell!) to help you love our gorgeous patch of the world as much as we do.
I'm personally delighted at your description of our "happy artwork and furnishings"--that's just how it's supposed to feel (although missed you telling Armin that we need more art !).
We're over the moon that you'll be coming back -- I promise you lots of new things to look at then . . .
Thanks so much for sharing your experience here; we really do appreciate it.
On behalf of the whole team,
Anahid & Luca
Thanks so much, tomthetinker -- "the best place to stay in Cape Town" is what we're always striving to be, so your endorsement is incredibly gratifying. We really enjoyed hosting you (Florence especially loved your appreciation of her breakfasts!) and hope that we'll see you back again on a future visit. You've given us plenty to live up to, so we'd love you to come see what we're working on for the future!
Many thanks again,
& the Derwent team
Thanks so much for your great endorsement and recommendation, Paul W -- we'd be delighted to have you back to stay again, any time: it was a real pleasure hosting you both. I'm so pleased that you were comfortable and enjoyed our beautiful mountain views and the proximity to Kloof Street -- a real gourmet's playground indeed.
We really appreciate your taking the time to share your experience with other travellers -- it really means a lot.
On behalf of the entire team, our very best regards from Cape Town,
Many thanks, SaGr_10, for sharing your experience at Derwent House with other travellers -- we really do appreciate it, and it is such a help to others.
We love it when our [typically-independent] apartment guests make themselves at home at Derwent House, making the most of the pool, garden and other facilities. It was wonderful to see so much of you, especially at tea-time !
So sorry to learn that you were disturbed by other residents: I am afraid that this is urban living today, and the joy people derive from making noise over new year's eve. Apologies for that.
We look forward to seeing you back as promised!
On behalf of the whole team,


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In this second and last post of the series, I will explain another way that we can use the conscious mind to retrain the other-than-conscious mind; again something I use myself and introduce to my clients. As with the REACT framework™ it is surprisingly simple; I have known clients who 'got it' very quickly, applied it diligently and achieved powerful results quickly. On the other hand, I know some clients have struggled, taking weeks and even months to adopt the practice assiduously in order to get the same potent outcomes.
I once worked with a client who I can best describe as a 'force of nature'; intense, determined and forceful. Along with these came what I experienced during our first few sessions as an almost constant torrent of negativity, pessimism and, at times, despair. I will not breach client confidentially about the source and nature of what had driven some deeply held beliefs into the client's other-than-conscious mind, but suffice to say the timing was in childhood. The client seemed to me to naturally experience any situation in a somewhat negative way, identifying problems and obstacles over possibilities and benefits, relating more to pain and worry over joy and harmony.
After considerable persistence on my part (and resistance on hers), I finally managed to persuade her to close her every day by writing down in a journal three positive things from the day. A very simple concept that took her much effort to implement as it seemed so strange and unnatural to her to recognise positives over negatives. In the end, I think I wore her down and she completed daily night-time homework diligently each evening as she went to bed. Once a week, she identified the three best things in her journal. At the end of the month she identified the three most outstanding entries from that month. I detected changes in her behaviour with the negativity gradually decreasing and then a gradual move to equanimity in her demeanour. Towards the end of the second month, she commented spontaneously that she felt she was a lot happier in herself, explaining that a problem at work the day before, which would previously have thrown her into a tail-spin, had seemed to her to be actually rather funny.
I urge you to try out this second method of training the other-than-conscious mind; I know from my clients you have little to lose and much to gain. Find whatever way, mechanism or device works for you and makes it easy for you to do this work every day. Routine is important. Doing this at the same time each day emphasises the routine nature and gets you into the discipline of the daily recording of three positives.
As you do whatever works well for you, ensure the positives you identify are also expressed in positive terms: 'I completed the new page for my website' or 'I got to speak to the prospect I have been trying to reach for days' or 'My new clients just settled my first invoice'. They don't need to be momentous, ground-breaking or particularly significant. They could include a particularly nice meal, a chat with a friend, or a beautiful sunset. The positives don't need to be things you have achieved they should be what you have experienced. They just need to be positives that are positively expressed. They should not be expressed as the avoidance of a negative: 'That meeting could have gone a lot worse' or 'We haven't heard about our bid, so at least we haven't been thrown out yet' or 'We haven't lost any more customers today'. Remember, this is about training the other-than-conscious mind to recognise positives.
As with the REACT framework™, this is a very simple technique that produces outstanding behavioural changes if you adopt it as a daily habit. I wish you every success; if you want more information or help applying either technique contact me at firstname.lastname@example.com or call me on 07764 658071 or register for the REACT framework™ workshop here.
WTT Results designs and delivers transformational change, enabling businesses and individuals to be the best that they want to be. To discuss how we can help your business, please get in touch as above. www.wttresults.co.uk