text:
- Research article
- Open Access
The serine/threonine kinase 33 is present and expressed in palaeognath birds but has become a unitary pseudogene in neognaths about 100 million years ago
© Lautwein et al. 2015
- Received: 19 January 2015
- Accepted: 13 July 2015
- Published: 22 July 2015
Serine/threonine kinase 33 (STK33) has been shown to be conserved across all major vertebrate classes including reptiles, mammals, amphibians and fish, suggesting its importance within vertebrates. It has been shown to phosphorylate vimentin and might play a role in spermatogenesis and organ ontogenesis. In this study we analyzed the genomic locus and expression of stk33 in the class Aves, using a combination of large scale next generation sequencing data analysis and traditional PCR.
Within the subclass Palaeognathae we analyzed the white-throated tinamou (Tinamus guttatus), the African ostrich (Struthio camelus) and the emu (Dromaius novaehollandiae). For the African ostrich we were able to generate a 62,778 bp long genomic contig and an mRNA sequence that encodes a protein showing highly significant similarity to STK33 proteins from other vertebrates. The emu has been shown to encode and transcribe a functional STK33 as well. For the white-throated tinamou we were able to identify 13 exons by sequence comparison encoding a protein similar to STK33 as well.
In contrast, in all 28 neognath birds analyzed, we could not find evidence for the existence of a functional copy of stk33 or its expression. In the genomes of these 28 bird species, we found only remnants of the stk33 locus carrying several large genomic deletions, leading to the loss of multiple exons. The remaining exons have acquired various indels and premature stop codons.
We were able to elucidate and describe the genomic structure and the transcription of a functional stk33 gene within the subclass Palaeognathae, but we could only find degenerate remnants of stk33 in all neognath birds analyzed. This led us to the conclusion that stk33 became a unitary pseudogene in the evolutionary history of the class Aves at the paleognath-neognath branch point during the late cretaceous period about 100 million years ago. We hypothesize that the pseudogenization of stk33 might have become fixed in neognaths due to either genetic redundancy or a non-orthologous gene displacement and present potential candidate genes for such an incident.
- Serine/threonine kinase 33
- Aves
- Pseudogene
- Evolution
- Genetic redundancy
- Non-orthologous gene displacement
Serine/threonine kinase 33 (stk33) was first discovered in the course of comparative genome analyses of human chromosome 11p15.3 and its orthologous region in the mouse [1–3]. This region is highly syntenic (see Additional file 1: Figure S1) and known to be associated with several diseases including different types of cancer [4–8]. Stk33 is predominantly expressed in testis, certain brain regions and embryonic organs such as brain, heart and spinal cord in human and mouse, implying that it might be involved in spermatogenesis and organ ontogenesis [9]. Its protein product has been classified as a member of the calcium/calmodulin-dependent kinase group [1]. STK33 has been shown to co-localize with the intermediate filament protein vimentin in tanycytes of mouse, rat and hamster [10] and kinase assays demonstrated that STK33 is able to phosphorylate vimentin in vitro [11]. STK33 and vimentin are associated in vivo and this interaction is not mediated by any additional protein as demonstrated by co-immunoprecipitation and co-sedimentation assays [11]. Thus, STK33 might be involved in the dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin [11]. Furthermore, stk33 has attracted attention since it has been identified as an essential component for the survival of KRAS-dependent cancer cells by high-throughput RNA interference (RNAi) screens [12–14]. It has also been associated with pancreatic cancer [15]. However, the role of stk33 in KRAS-dependent cancer cells remains disputed because other research groups were not able to find a correlation between the knockdown or inhibition of stk33 and KRAS-dependent cancer cell survival, neither using RNAi nor small molecule inhibitors [16–19]. Nevertheless, recent studies found an association of stk33 with colorectal cancer [20], hepatocellular carcinoma [21], hypopharyngeal squamous cell carcinoma [22] and lung cancer [23].
To date, stk33 has been found only in the subphylum Vertebrata. So far it has been found in multiple vertebrate classes including mammals, reptiles, amphibians and fish, but not in birds (A phylogeny of all known STK33 proteins has been built, see Additional file 1: Figure S3). Neither the analyses of the International Chicken Genome Sequencing consortium [24], nor our own screening of the chicken (Gallus gallus) genome for stk33 revealed a functional copy (see below). So far, this was the only vertebrate class with no indication for a functional stk33. Since stk33 genes are present as well in mammalians as in phylogenetically older vertebrate species (see Additional file 1: Figure S3), we wanted to know whether birds do not have stk33 genes in general or if the chicken is an exception. Therefore, we analyzed the genomes and transcriptomes of more than 30 different bird species using deep sequencing and publicly available next generation sequencing data [25].
The class of Aves comprises about 10,500 extant species [26] and the majority of these species arose from a rapid radiation after a mass extinction event about 66 million years ago within neonaves [27, 28]. It is divided into the clade Palaeognathae, including the flightless ratites and the tinamous, and the clade Neognathae, including all other extant bird species [27, 29, 30].
In this article, we report that stk33 is present and expressed in paleognath birds, but has become a unitary pseudogene within the subclass Neognathae during the cretaceous period about 100 million years ago.
First, we analyzed the chicken (Gallus gallus) genome for the presence of a functional copy of stk33. In human (Homo sapiens) and mouse (Mus musculus) stk33 is located in a highly syntenic region on chromosome 11p15.3 and chromosome 7E3, respectively [2]. In the chicken this region could be identified on chromosome 5, whereas in the chicken no stk33 has been annotated in this region (see Additional file 1: Figure S1). Therefore we screened the whole genome and this region in particular for the presence of stk33. These genomic screens revealed the presence of only eight remnant exons containing both frameshifting indels and multiple stop codons (Fig. 3) in this region and multiple large genomic deletions that have led to the deletion of several exons. Extensive BLASTn searches of the chicken genome did not reveal a duplication or a retrotransposition event for stk33.
This led us to the conclusion that stk33 has become a unitary pseudogene in the chicken, raising the question if the chicken is an exception or if stk33 is missing in birds in general.
Stk33 is present and expressed in the subclass Palaeognathae
Encouraged by our findings that stk33 is expressed in the African ostrich, we investigated if the genomes of other paleognath birds contain a functional stk33 gene. For this purpose we chose the closely related emu (Dromaius novaehollandiae) and the white-throated-tinamou (Tinamus guttatus). For the emu we used transcriptomic Illumina data of the embryonic brain with a total of 51.1 Mio reads (Illumina) and 182,922 reads (454) from adult brain (Table 2). Since the publicly available genomic data [SRA: SRP019803] was of insufficient quality for the generation of a suitable reference sequence, we mapped the emu transcriptomic reads to the ostrich stk33 mRNA sequence and extracted the consensus sequence. The transcriptomic reads were mapped back to this consensus sequence for verification. This led to an mRNA sequence of 2,560 bp in length split into 14 exons. The mRNA sequence contains a single open reading frame of 1,635 bp which shows 94 % identity (E = 0.0) with the ostrich mRNA sequence. The emu ORF encodes a 544 aa protein of 60.1 kDa. Amino acid sequence alignments of the ostrich and emu STK33 proteins show 97 % identity (E = 0.0) within the catalytic domain (Fig. 2). Across the whole protein sequence the similarity is 91 %. The emu STK33 also contains a serine/threonine protein kinase catalytic domain, an ATP binding site and an active site.
Stk33 has become a unitary pseudogene in Neognathae
Since we had evidence that a functional copy of stk33 is missing in the genome of the chicken, we wanted to know whether this is a particular case for galliformes or whether this is generally the case for all neognath birds. Therefore, we examined a total of 28 neognaths (Additional file 1: Table S2) on a genomic level and four on a transcriptional level in order to achieve a good sample distribution over the Neognathae pedigree (see Additional file 1: Figure S2) [29, 27, 30]. The genomic studies were carried out as outlined for the white-throated-tinamou.
All of the 28 neognaths analyzed showed only remnants of non-functional stk33 sequences. Frequent large genomic deletions of several kilobases within the stk33 locus were observed, leading to the loss of several exons. BLASTx searches of the remnant exons showed multiple frameshifting indels and frequent SNPs, leading to the generation of multiple premature stop codons within the stk33 ORF (Fig. 3 and Additional file 2: Figure S5). Even the closest relative to the paleognath birds in our list, the mallard (Anas platyrhynchos) shows a severely degenerated stk33, including the deletion of six exons, several indels and multiple premature stop codons (Fig. 3). In the Anna's hummingbird (Calypte anna) an additional genomic inversion of a 3 kb fragment of the stk33 gene is present, leading to the inversion of the 5′ end of exon 2 (Fig. 3). All birds examined belonging to the order Passeriformes (golden-collared manakin, American crow, Tibetan ground tit, collared flycatcher, zebra finch, white-throated sparrow, medium ground finch), which represents about 60 % of all extant avian species, showed deletions of the exons 3, 5, 8, 12 and 13, several indels and premature stop codons (see Fig. 3 and Additional file 1: Figure S1). All other neognaths show similar severe mutations (see Additional file 1: Figure S1). Interestingly, all neognaths examined share the deletion of exon 3.
We have investigated whether stk33 is still transcribed in spite of the many mutations found in the neognath stk33 genes. We also looked for the possibility that in neognaths stk33 might have potentially acquired new exons in the course of evolution and thus, a functional variant of stk33 might have been restored. For this analysis, we downloaded several transcriptomic SRA data sets (Table 2) of tissues in which transcription of stk33 has been described previously [9, 10]. The data included more than 1.5 Billion reads from chicken (Gallus gallus), about 200 million reads from the white-throated sparrow (Zonotrichia albicollis) and 50 million reads from the collared flycatcher (Ficedula albicollis). In addition, we sequenced cDNA generated from zebra finch (Taeniopygia guttata) brain, yielding 9.5 million reads (Table 2). These data sets were then mapped to the appropriate genomic stk33 target region and to the stk33 mRNA sequence from the African ostrich. In all this data we could not detect any stk33-related sequences. Furthermore, RT-PCR from cDNA using pairs of primers designed to the remnant exon sequences did not yield any products.
In order to exclude that a duplication or a retrotransposition of stk33 occurred during the course of evolution, we performed both transcriptional de novo assemblies and extensive BLASTn searches of the WGS assemblies. These analyses did not reveal any duplicated or retrotransposed stk33 copies elsewhere in the genome, so that we can conclude that stk33 turned into a unitary pseudogene in neognaths and is no longer expressed.
In this study we used a combination of large scale next generation sequencing data analysis and PCR to elucidate the genomic structure and the transcription of the stk33 gene in the phylogenetic class of Aves. In both ostrich and emu, belonging to the phylogenetically older Palaeognathae, we were able to prove the presence of a functional stk33 which is also undoubtedly transcribed. In the third paleognath, the white-throated tinamou, genomic analysis showed the presence of 13 exons with no frameshifting indels or premature stop codons within the coding region. We therefore conclude that the white-throated tinamou, as ostrich and emu, carries a functional copy of stk33. In the future this will need to be proven by transcriptomic and/or proteomic analyses. In summary, we can conclude that stk33 is present and expressed in all paleognath birds.
In neognaths we found only non-functional remnants of stk33. There were no transcripts of these stk33 remnants detectable. Genomic deletions, indels and premature stop codons clearly led to non-functionality of the degenerate stk33 remnants (Fig. 3 and Additional file 2: Figure S5). Furthermore, there is neither evidence for a functional stk33 copy elsewhere in the genome nor are new exons acquired to compensate for those that have been lost. This leads us to the conclusion that stk33 has become a pseudogene in neognaths during the evolutionary history of the class Aves. Although there is ample evidence that no gene duplication occurred, a copy of stk33 could have been retrotransposed back into the genome and somehow become a functional substitute for the original stk33. But since these so called processed pseudogenes are generally considered to be "dead on arrival" [32, 33], due to the lack of a promoter, this scenario would be considered highly unlikely. In addition, all attempts to identify any copies of stk33 elsewhere in any neognath genome failed, so that we are rather confident that this subclass of birds is the only one among vertebrates lacking stk33, as can be inferred from those vertebrates with a present STK33 (see Additional file 1: Figure S3). Such gene loss is a common and important evolutionary process and has been described in bacteria [34], eukaryotes [35] and archaea [36]. It is very abundant in some lineages such as bacteria or tunicates, that have undergone considerable genome reduction during their evolution [34, 37–40]. Birds also have undergone gene loss and genome reduction compared to other tetrapod classes, but not to the same extent as in bacteria or tunicates [41–43].
It is interesting that all neognaths lack exon 3, indicating that this chromosomal deletion may have been the initial mutation leading to the degeneration of stk33. This mutation most likely occurred at a very early stage in the diversification of the class of Aves during the late cretaceous period about 100 million years ago, at the branching point of Palaeognathae and Neognathae (see Additional file 1: Figure S2) [27, 29, 30]. Interestingly, a current large scale comparative genomics study in birds could show that the origin of neognath birds was accompanied by an elevated rate of genomic rearrangements [30]. This and the long time span are in accordance with the drastic changes seen in the stk33 locus in neognaths. There are cases where pseudogenes have gained new functions expressing non-coding RNAs that can have a variety of different regulatory functions [44]. However, in spite of intensive searches we could not detect any stk33 related transcripts in any of the neognath bird tissues analyzed. Therefore, a function of the stk33 pseudogene is highly unlikely. This leads to the question, which gene could have taken over the function of stk33 in neognaths or if its function has become dispensable.
In the following we would like to discuss different hypothesis about how the loss of stk33 could have been tolerated and become fixed in neognath birds. One possibility could be, that the function of the stk33 gene could have become dispensable, leading to the fixation of the pseudogene by genetic drift [45]. We believe this to be unlikely since stk33 has been implicated in critical functions such as spermatogenesis and organogenesis [9] and there is no obvious indication to assume that neognath birds can manage without these functions of stk33 whereas paleognath birds and other vertebrates cannot. Following this assumption, stk33 could have undergone subfunctionalization, a process where a pair of genes that resulted from a duplication event retained different subfunctions of the original gene [46, 47]. If one of these subfunctions should have become dispensable, the corresponding paralogous gene would have acquired the observed deleterious mutations. But since we could not find any evidence for a stk33 duplication event in neognath birds, we can preclude this possibility.
A third possibility is given by the "less is more" hypothesis. It states that gene loss can be an advantageous event and thus can serve as an engine of evolutionary change [48, 49]. Even though this may seem contradictory, since it suggests evolutionary innovation by degeneration, there are several examples of such a positive selection for gene loss [50–52]. One could argue that the loss of stk33 may be beneficial, since it has been identified as an essential component in several types of cancer [12–15, 20–23]. However, the role of stk33 in cancer remains controversial [16–19] and we do not have any indication for a beneficial effect due to the lack of stk33, making this a rather unlikely hypothesis. Nevertheless, analyzing if neognaths display any form of immunity to these types of cancer could represent an interesting approach, since they constitute natural knockouts for stk33.
Another possible explanation for the toleration of gene loss is the phenomenon of genetic redundancy, which can be caused by either gene duplication or convergent evolutionary processes, leading to proteins that are performing the same function, but are unrelated in sequence [53, 54]. Due to the lack of selective pressure on these genes, genetic redundancy would be considered to be evolutionary unstable. However, genetic redundancy appears to be quite frequent in genomes of higher organisms, implying that redundant genes can be evolutionary stable under certain conditions [54–58]. One model describing a stable genetic redundancy takes pleiotropy into consideration. If gene A performs function f1 and gene B performs both functions f1 and f2, but f1 with a slightly reduced efficacy, this genetic redundancy would be considered as evolutionary stable [59]. The presence of different splice variants in ostrich, human and mouse [1] strongly indicates that STK33 has more than one function and that vimentin may not be its only substrate [11]. This is supported by the fact that STK33 can also be found in vimentin-negative cells [11]. If we presume stk33 to be gene B and assume that the function f2 has become irrelevant to the neognath lifestyle, stk33 would have inevitably been lost in neognath birds.
A variant of the latter possibility is that stk33 may have become dispensable, because it has been functionally substituted by a non-orthologous, but functionally analogous kinase in neognaths, a process called non-orthologous gene displacement (NOD). Several examples of such a NOD have been reported [60–64].
Known vimentin phosphorylating kinases and their phosphorylation sites
Ser-38; Ser-71
Protein kinase C
Ser-33; Ser-50
Ser-38; Ser-82
Protein kinase A
Ser-38; Ser-72
MAPKAP kinase-2
Ser-38; Ser-50; Ser-55; Ser-82
Aurora kinase B
Ser-457; Ser-458
The phosphorylation sites of two kinases, Plk1 and p37, are located outside the amino-terminal head domain [79, 80] and thus probably are no candidates that could substitute stk33. Three other kinases (Pak1, CDK1, Aurora kinase B) are also not suitable candidates since they only have a single phosphorylation site [73–78]. Even though MAPKAP kinase 2 and CAMKII have at least one phosphorylation site within the same region that is phosphorylated by STK33, they both also phosphorylate Ser-82 [68, 69, 71, 72], making them unlikely substituents for STK33. The three remaining kinases, Rho-kinase, protein kinase C and protein kinase A all have 2 phosphorylation sites within the same region as STK33 (Table 1) [65–67, 70]. However the vimentin alignment clearly shows that Ser-50, which is phosphorylated by protein kinase C [66, 67], is not conserved in birds (Additional file 1: Figure S4), making it a rather unlikely candidate. Thus Rho-kinase and protein kinase A represent the best candidates to compensate for the loss of stk33 in neognath birds, since their phosphorylation sites are within the same region as those for stk33 and are highly conserved in birds (Additional file 1: Figure S4).
Further studies about stk33 on the genomic and protein level, especially about the exact phosphorylation sites of STK33, are necessary to further elucidate its function and to give additional indications towards the actual substituent. Without further knowledge all hypothesis remain speculative.
Here, we elucidate and describe the genomic structure and the transcription of the serine/threonine kinase 33 (stk33) in paleognath birds, such as the African ostrich (Struthio camelus), emu (Dromaius novaehollandiae) and the white-throated tinamou (Tinamus guttatus) using both large scale next generation sequencing data analysis and PCR. We were able to show that all three paleognath birds analyzed carry functional and expressed copies of stk33 and thus, we concluded that stk33 is present and expressed in paleognath birds in general, as it is the case in all other vertebrate classes.
Surprisingly, in neognaths birds we found only degenerate remnants of stk33 that have become non-functional by genomic deletions, indels and premature stop codons (Fig. 3 and Additional file 2: Figure S5). Furthermore, no transcripts of these remnant exons could be detected. Interestingly, all neognath birds share the deletion of exon 3. These results lead us to the conclusion that stk33 became a unitary pseudogene in the evolutionary history of the class Aves during the late cretaceous period about 100 million years ago, at the branching point of Palaeognathae and Neognathae that was accompanied by an elevated rate of genomic rearrangements in neognaths [27, 29, 30]. We hypothesize that this pseudogenization of stk33 might have become fixed in Neognathae due to either genetic redundancy or a non-orthologous gene displacement (NOD). Rho-kinase and protein kinase A have been identified as the most promising candidate genes by comparing their vimentin phosphorylation sites to those of STK33.
Animal material
All procedures concerning animals were performed in accordance with the published Directive 2010/63/EU of the European Parliament under a protocol approved by the local Administration District Official Committee. Moreover, all efforts were made to minimize the number of animals and their suffering.
Tissues expected to have high stk33 expression were obtained from three bird species: zebra finch (Taeniopygia guttata), chicken (Gallus gallus) and African ostrich (Struthio camelus).
Adult male zebra finch (Taeniopygia guttata) were obtained from a local pet store, anaesthetized with carbon dioxide, killed by cervical dislocation and used for the preparation of brain and testis. Fertilized chicken (Gallus gallus) eggs were obtained from a local breeder and incubated for 6–17 days at 38 °C. After incubation eggs were opened and either whole embryos or the embryonic brain were isolated. Testes and complete heads from the African ostrich (Struthio camelus) were obtained from a local breeder. Ostrich heads were cut in half for preparation of the Cerebellum. After preparation all material was stored at −80 °C until further use.
Sample preparation and sequencing
Tissue material was homogenized using a syringe and cannula. RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany), High Pure RNA Tissue Kit (Roche, Penzberg, Germany), PureLink® RNA Mini Kit (Life Technologies, Carlsbad, USA) or the GTC-method [81]. Subsequently, the RNA quality was tested using the 2100 Bioanalyzer (Agilent, Santa Clara, USA). DNA was isolated with the peqGOLD Tissue DNA Mini Kit (PeqLab, Erlangen, Germany).
1.5 μg of total RNA isolated from ostrich cerebellum and testis were each used for the library preparation using the TruSeq RNA Sample PrepV2 Kit (Illumina, San Diego, USA) and a 100 bp paired-end run was performed on an Illumina HiSeq 2000 (IMSB, Mainz, Germany). For zebra finch adult brain RNA sequencing, library preparation was carried out by GENterprise Genomics (Mainz, Germany) using 1.5 μg of total RNA and samples were sequenced in a 150 bp paired-end run on an Illumina HiSeq 2500 (IMSB, Mainz, Germany).
Handling of SRA data
Overview of all next generation sequencing data sets used in this study. SRA studies SRX736627, SRX738984 and SRX738987 were carried out at the IMSB Mainz. All other sequencing data was downloaded from the NCBI Sequence Read Archive
SRA study
Read count
Read layout
African ostrich (Struthio camelus)
Illumina HiSeq 2000
100 bp paired
African ostrich (Struthio camelus)
Illumina HiSeq 2000
100 bp paired
African ostrich (Struthio camelus)
Illumina HiSeq 2000
100 bp paired
Emu (Dromaius novaehollandiae)
Illumina HiSeq 2000
80 bp paired
Emu (Dromaius novaehollandiae)
Illumina HiSeq 2000
80 bp paired
Emu (Dromaius novaehollandiae)
generic sample
274 bp single
White-throated sparrow (Zonotrichia albicollis)
Illumina HiSeq 2000
100 bp single
Chicken (Gallus gallus)
Illumina HiSeq 2000
100 bp single
Chicken (Gallus gallus)
Illumina HiSeq 2000
35 bp paired
80 bp paired
40 bp paired
Chicken (Gallus gallus)
Illumina HiSeq 2000
40 bp paired
36 bp paired
80 bp paired
Chicken (Gallus gallus)
Illumina HiSeq 2000
100 bp paired
Collared flycatcher (Ficedula albicollis)
Illumina Genome Analyzer IIx
100 bp paired
Japanese quail (Coturnix japonica)
Illumina HiSeq 2000
100 bp paired
Zebra finch (Taeniopygia guttata)
Illumina HiSeq 2500
150 bp paired
Trimming of Illumina data
Sequencing reads were trimmed for quality and adapter sequences using CLC Genomics Workbench (CLC bio, Aarhus, Denmark). Quality trimming was done using an error probability limit of 0.01 and a maximum of 1 ambiguous base. All sequences below a read length of 15 bp were discarded.
De novo assemblies and mappings
De novo assemblies of genomic and transcriptomic reads were conducted in CLC Genomics Workbench (CLC bio, Aarhus, Denmark) using standard parameters. Stk33 contigs were identified using local BLASTn/BLASTx searches against a stk33 database containing all available stk33 sequences.
The feature "map reads to reference" of the CLC Genomics Workbench (CLC bio, Aarhus, Denmark) was used to map transcriptomic reads to transcriptomic contigs and "large gap read mapping" was employed to map transcriptomic data to genomic contigs. For intraspecies mappings a similarity of 98 % for at least 95 % of the read length was required, for interspecies mappings this similarity was reduced to 80 % similarity across 80 % of the read length. Mismatches were given a penalty of 2 and insertions or deletions were given a penalty of 3.
Genomic studies
Stk33 is located in a highly syntenic region spanning from st5 to ric3 (Additional file 1: Figure S1). In order to obtain the genomic target regions for stk33 of the different birds we downloaded the annotated genomic contigs containing the region between genes st5 and tub from UniGene. If these were not available we downloaded all assembled genomic contigs of the respective genome assembly project from the NCBI genome database (Additional file 1: Table S1) and identified contigs located in the stk33 target region by local BLASTn searches with the genomic stk33 contig from ostrich as reference. Putative exons of stk33 were identified by comparison with the ostrich exons. Premature stop codons were identified by local BLASTx searches of single exons with the full length STK33 protein sequence from ostrich as reference.
PCR and Sanger sequencing
cDNA was generated employing SuperScript® III reverse transcriptase (Invitrogen, Carlsbad, USA). Traditional PCRs were carried out using GoTaq® Polymerase (Promega, Fitchburg, USA) or Q5® High-Fidelity DNA Polymerase (NEB, Ipswich, USA). Takara LA Taq® DNA Polymerase (Takara, Otsu, Japan) was used for long Range PCRs. Primers were purchased from Invitrogen (Carlsbad, USA) and Sigma Aldrich (St. Louis, USA). DNA was denatured at 98 °C for 30 s, annealing was carried out at 60 °C for 30 s and extension was performed at 72 °C with an extension time of 1 min/kb. RACE PCRs were done using the GeneRacer™ Kit (Invitrogen, Carlsbad, USA). Sanger sequencing was carried out by StarSeq (Mainz, Germany).
Availability of supporting data
All sequencing data generated in this study is available from the SRA-Archive (http://www.ncbi.nlm.nih.gov/sra) under the accessions numbers SRX738987 for the zebra finch, SRX738984 and SRX736627 for the African ostrich. Stk33 sequences generated have been deposited into the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/). Accession numbers are available from Additional file 1: Table S2. Due to GenBank limitations all sequences have also been included in Additional file 3.
We thank Benjamin Rieger and Dr. Steffen Rapp for their bioinformatics support. We are grateful to Sabine Fischer and Dr. Romina Petersen for their helpful comments on the manuscript.
Financial support by the NMFZ (Naturwissenschaftlich-medizinisches Forschungszentrum) from the University Medical Center, Johannes Gutenberg-University Mainz is greatly acknowledged.
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


text:
Movie Description:
The vampire has three days to prolong the life. During this time it has to tempt virgin Charlotte and catch her soul. Unfortunately for the vampire Charlotte has a friend that does a task more difficult.
About movie:
Original Title: Embrace Of The Vampire
Country: USA
Released: 1995
Language: English
Run time: 93 min
Studio: The Ministry of Film, General Media Entertainment, New Line Cinema
Director: Anne Goursaud
Actors: Martin Kemp, Rebecca Ferratti, Glori Gold, Harold Pruett, Charlotte Lewis, Seana Ryan, Sabrina Allen, Alyssa Milano, Rachel True, Jordan Ladd, Robbin Julien, Christopher Utesch, David Portlock, Gregg Vance, John Riedlinger
Genre: Horror, Thriller
File info:
Quality: DVDRip
Format: AVI
Video: XVID 852×454 25.00fps
Audio: MPEG Audio Layer 3 48000Hz stereo 256Kbps
Size: 1217 Mb
Download Embrace Of The Vampire (1995) DVDRip

subset: commoncrawl


text:
Hello every one.
I trying to add NFS Mount on the Teradata Server.
I am logged in as root in the Linux and am trying to mount it with the below command
"mount -t nfs NFSSERVERNAME:/Disk /desitnation_Folder"
when I use this command I get the below error message, what should be the next step I can go with.
"mount server reported tcp not available, falling back to udp"
If your nfs client has this:
# mount -t nfs NFSSERVERNAME:/Disk /desitnation_Folder
mount server reported tcp not available, falling back to udp
mount: RPC: Program not registered
and this:
# rpcinfo -p nfsserver-ip-address
No remote programs registered.
you may have tcpwrappers in the nfs server, so, in the nfs server edit /etc/hosts.allow and add:
portmap : ip : ALLOW
portmap : ALL : DENY
nfs-server : ip : ALLOW
nfs-server: ALL : DENY
do not restart anything, just mount your filesystem in the client


text:
French protesters clash with police over labour reforms
Clashes broke out on the streets of France on Thursday during fresh protests over labour reforms, just a day after beleaguered President Francois Hollande was forced into an embarrassing U-turn over constitutional changes.
A nationwide strike shut the Eiffel Tower, disrupted train services and saw dozens of schools closed or barricaded by students.
Riot police flooded the streets and clashed with protesters in the western cities of Nantes and Rennes, among 200 demonstrations expected across the country.
Police said around 10 people had been arrested in the capital, where demonstrators marched under banners reading "We want better" and "A giant leap forward to the 19th century".
Adding to Hollande's miserable week, a separate strike by air traffic controllers threatened travel chaos for thousands of passengers, while drivers faced more than 400km of tailbacks on motorways around Paris.
The Socialist government is desperate to push through reforms to France's controversial labour laws, billed as a last-gasp attempt to boost the flailing economy before next year's presidential election.
But protests by unions and students turned violent last week and demonstrators vowed an even bigger turnout on Thursday.
They are angry over plans to make it easier for struggling companies to fire workers, even though the reforms have already been diluted once in a bid to placate employers.
Hollande's government was still reeling from his decision Wednesday to abandon constitutional changes that would have allowed dual nationals convicted of terrorism to be stripped of their French citizenship.
The measure had been derided as ineffective and divisive, including by left-wing rebels within the Socialist party — many of whom also oppose the labour reforms.
Least popular president
Already the least popular president in France's modern history, Hollande is seeing his numbers continue to fall, with another poll on Wednesday showing he would not even make it to the second-round run-off in the presidential election.
Hollande, 61, has vowed not to run again if he cannot cut the country's stubbornly high unemployment figures — long stuck at around 10% — and he hoped the labour reforms would encourage firms to hire more staff.
But pressure from the street and parliament's back benches caused the government to water down the proposals so that they apply only to large firms.
Some reform-minded unions have given their support to the changes, but last week's demonstrations saw cars burned in Paris and more than 30 people arrested as protesters clashed with police, who responded with tear gas.
A video of an officer punching a 15-year-old boy on the sidelines of a protest in the capital went viral and fuelled further anger.
The officer was taken into custody and questioned on Thursday, police and judicial sources said.
A recent opinion poll found that 58% of the French public still opposed the measures.
Labour Minister Myriam El Khomri said this week that she understood why "such a profoundly reformist text has raised questions and requires debate," adding: "It is not a blank cheque for companies."
Bosses are also unhappy, particularly over the removal of a cap on compensation paid for unfair dismissal, and the scrapping of plans that would have allowed small- and medium-sized companies to unilaterally introduce flexible working hours.
Parliament is set to vote on the reforms in late April or early May.
Air travel chaos
Aviation authorities told airlines to cancel 20% of their flights from Paris Orly airport on Thursday and a third of flights from the Mediterranean city of Marseille as air traffic controllers went on strike again.
Paris' Charles De Gaulle airport was not expected to be affected by the 36-hour walkout over job cuts and a lack of investment in new technology.
The Airlines for Europe lobby group said it was the 43rd strike by French air traffic controllers since 2009. It was due to end at 5am (11am Malaysia) on Friday.
A strike last week disrupted travel plans for thousands of passengers as airlines were forced to cancel up to a third of flights.


text:
روزی کی نوکریوں کی ایپلیکیشن۔
Ajax Messages
The family of Al Ameen has a history of "Four Generations". Textile production started in 1920's & 1947 saw major diversification into trading. Today, Al Ameen is a multi-dimensional organization specializing in varied services as a distributors, importers, manufacturers & manufacturer's representat ...مزید پڑھیں
Territory Sales Manager
Engro Foods
Motivational Speaker & Blogger
Living Upbeat
Marketing Manager
Digital Online Devices
Digital Marketing Manager
IT Services Group
Head of HR, Training & OD
Senior Team Lead
Konext PVT. LTD
Business Excellence Specialist
Nestle Pakistan
Social Media Marketing Manager
Recruitment & Training Executive
ACS Synergy (Pvt) Ltd.
Insolito Associates
Chief HR Officer
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text:
"Papa, I can always tell we're about to go on a trip because my tummy tells me so."
We'd been on the road for no more than ten minutes. The car swerved and pulled to the right. A semi-truck had passed on the left. The accelerator tried to keep things moving forward.
My tummy had agreed. I could feel every bounce of the road. The spring, shocks, and tires did a great job dampening the breaks in the road. The trio, however, made the ride like a camel in desert.
The car rides high on two axles. As a family we were perched atop swaying in the March wind. Like the truck before, the wind tossed us as we rode down the highway. Swerving, bouncing, and breezing down the road was going to be the course for the day.
We are destined to part far away. It is Spring Break. The road was a ticket to a foreign land. The end of the road promised something new and different.
This is what the tummy was saying. "You are going to be a different person when you come home. Your experiences will teach you new things about who you are and where you come from. Who are you?"
The lengthy conversation is what makes it feel weird. Normally the tummy just says "I'm hungry." Rarely is this part of the body so insightful.
The beginning of a trip is like the first hill of a roller coaster. This car ride is no different than the ride in the amusement park.
There is a bump in the road. "What was that tummy? What did you say?"


text:
Heritage Category: Listed Building
Grade: II
List Entry Number: 1105512
Date first listed: 07-Nov-1985
Statutory Address: CHURCH COTTAGE
The above map is for quick reference purposes only and may not be to scale. For a copy of the full scale map, please see the attached PDF - 1105512 .pdf
The PDF will be generated from our live systems and may take a few minutes to download depending on how busy our servers are. We apologise for this delay.
This copy shows the entry on 19-Jan-2019 at 10:10:01.
Statutory Address: CHURCH COTTAGE
The building or site itself may lie within the boundary of more than one authority.
County: Devon
District: West Devon (District Authority)
Parish: Thrushelton
National Grid Reference: SX 44746 87572
Legacy Record - This information may be included in the List Entry Details.
Reasons for Designation
Legacy Record - This information may be included in the List Entry Details.
Legacy Record - This information may be included in the List Entry Details.
3/341 Church Cottage -
Poorhouse and sexton's cottage now in use as a house. Circa early/mid C17, partly rebuilt in the early C19. Stone rubble and cob with a Delabole slate roof gabled at the left end, hipped at the right end, formerly thatched. Stone stack at left end, large C17 rendered stack with brick shaft on ridge. Single depth plan 3-rooms wide which may originally have had a cross passage. The right-hand end of the house is C17. A 1680 glebe terrier refers to the division of the house into 2, one consisting of "one lower room which is a wood house and one chamber over it which is planked". This probably refers to the extant 2 right-hand rooms, the first floor room being heated from the C17 stack and formerly reached by an external stair at the right end. A circa early C17 ground floor fireplace to the same stack heats the middle room which was presumably the hall of the second 1680 property which may have been 2 rooms and a through passage. In the early C19 the entire left-hand end of the house was rebuilt or remodelled with Tudor style fenestration. C20 renovations and repair. 2 storeys. Asymmetrical 3-window front with off-centre shallow gabled stone porch leading into the stair hall. All the windows on the front elevation are 3-light casements with timber arched lights and diagonal leaded panes, except ground floor window right which is a 2-light casement with 6-panes per light inserted in a blocked doorway. Windows throughout have slate sills and the ground floor windows have flat stone arches. Interior Ground floor timber lintel with straight cut stops and exposed joists of slender scantling. The circa early C17 fireplace to the middle room is massive with chamfered granite jambs and a chamfered timber lintel with runout stops. First floor right has a large fireplace with a timber lintel, axial beam and exposed joists introduced in the late C20. Pegged tie beam trusses have a probably later collar. A circa early C19 architect's plan and elevation of the house exists in the parish records.
Listing NGR: SX4474687571
The contents of this record have been generated from a legacy data system.
Legacy System number: 92584
Legacy System: LBS
This building is listed under the Planning (Listed Buildings and Conservation Areas) Act 1990 as amended for its special architectural or historic interest.
End of official listing


text:
Know Your Prescription Drugs
It is not just illegal drugs that pose potential threats to one's life; prescription drugs can sometimes be just as deadly. Today I had an experience that I will never forget, and I must say that I am lucky to be alive.
Saturday, March 27, 2010 started out as smooth as any other morning. I started my morning with a cup of coffee while I checked my email, a practice that I do practically each and every morning. Everything was normal that is until I had driven about 20 miles from my house. What happened next is a real testament to the angel or angels that watch over me.
It all began at 5:30 that quiet Saturday morning as I was driving to Dallas. To get to Dallas from Fort Worth I have to navigate three serious highways—Interstate 830, Texas State Highway 121 and Interstate 635. On Highway 121 there is road construction for about the last four miles before my exit to merge on to I-635. I was traveling in the left lane when I began to merge over three lanes of traffic to my exit. I had successfully merged two lanes and somehow I failed to continue to continue merging and I missed the exit altogether.
A few minutes later I noted that I was on DFW Airport Property, lost and delirious, I had no idea how I got there. I was in front of a guard shack where two people, one standing and one sitting, were looking at me as though they were mystified at what they saw. Not to mention there were cars behind me, all blowing their horns. It was like a dream and I didn't know what I was doing there. I reached for my wallet as if to pay the person sitting in the guard shack, but he stopped me short of pulling out my wallet and said, "Just take the ticket man."
I don't remember if I took the ticket or not. The next thing I remember was sitting on the shoulder of the Interstate, fumbling through my cell phone looking for my wife's cell phone number to call her. My first thought was I'd had a stroke.
Finally I got through to my wife and I told her that I was lost and that I thought I'd had a stroke. Just then, I looked up and in plain view in front of me was a sign that read, "Fort Worth exit 1 mile".
"I'm not lost any more", I said to my wife, "I'm coming home," and then I hung the phone up.
I do not remember driving home, nor do I remember even turning into my subdivision. I only remember seeing my wife standing on the lawn next to the driveway as I backed my car in. I remembered the worried look on her face as she helped me out of the car; I slowly walked upstairs and got in the bed.
I do not remember anything of what occurred next, I only have my wife's account of the story; this is what she said:
After seeing that there was something else wrong with me besides sleepiness my wife awakened me. I asked, "Where are we going?" "To the hospital," she answered. She put my shoes on, I took them off; she buttoned my shirt, I unbuttoned it and lay back down on the bed and went to sleep.
Her persistence paid off and she rushed me over to the hospital where I was immediately admitted under the pretense that I'd had a stroke. I underwent an EKG, MRI CAT scan and I was given a heart monitor to monitor my heart activity.
After pre-examination, it was first believed that I had a Transient Ischemic Attack (TIA) or a mini stroke. TIA may be a warning signal before a stroke occurs. A stroke is like a TIA but, a stroke may cause permanent damage to the body. TIA happens when blood flow to the brain is decreased. When this happens, oxygen going to the brain is also decreased. TIA may lead to loss of certain body functions controlled by the part of the brain affected by the TIA. The effects of a TIA last for a few minutes to an hour. All effects are gone in less than 24 hours and do not cause permanent damage to the brain or body. TIS is caused by high blood pressure or diabetes. Other causes may be high amounts of cholesterol (fat) in the blood or smoking.
When the results of my MRI CAT scan were studied it was determined that I did not have TIA or a stroke. What I had experienced was one of the side effects of a medicine called Zolpidem which is another form of Ambien. Zolpidem is prescribed for insomnia.
The Medicine Data Sheet states that someone taking Zolpidem can experience sleep walking if a full eight or more hours of sleep are not achieved when taking Zolpidem. The doctors all agreed that I had experienced a serious case of sleep walking, or sleep driving even though I had been awake for more than one and a half hours prior to this episode.
My brain was still performing normal bodily functions but the part of the brain responsible for memory was disabled, therefore I do not remember a thing. For me, it was one of the scariest experiences of my life.
Before taking medication, be sure to thoroughly read the Medicine Data Sheet, and get to know your prescription drugs. It may save your life.


text:
We've had plenty over the past couple of months in particular, but, there's another Kardashian Konspiracy to add to the list.
Khloe Kardashian and Tristan Thompson are at the forefront of this one. Fans think that they've figured out what Khloe and Tristan are calling their daughter, who is due very soon.
Eagle-eyed followers have noticed that Khloe has been posting a lot of floral pictures lately (roses in particular) which leads them all to believe that she is calling her daughter Rose.
This wouldn't be the strangest guess, after all, nothing the Kardashians do or post is by accident.
Last weekend, Khloe celebrated her baby shower which was an all-pink affair and there were many roses at that too. Perhaps, Khloe is just showcasing the incredible floral display at her event. They did probably cost the same as a mortgage so we'd be flaunting them too, Khlo.
Other options that have been doing the rounds besides Rose are Rosie and Grace. Many of her followers have been commenting saying that there's a connection between roses and grace. Others are not so convinced. They think Grace or Rose are too normal for a Kardashian.. Can't argue there.
Khloe tweeted during the week to tell fans that she is due very soon and that she is getting 'more excited' by the day.
Until that big reveal, may the brain-wracking continue..


text:
4 words:
This is a draft of a foreword I am writing with a colleague...
Over the last two decades, governments around the world have acknowledged that changes in the environment affect human activities in ways that are increasingly important. Supplies of essentials--shelter, water and food--can be affected by decadal cycles in climate and disasters that are either more intense, more frequent, affecting more people, or often, all of the above. Governments are acknowledging that the environment is no longer a reliable invariant, resources are not inexhaustible, and we can no longer count on "business-as-usual."
Much financial support has been given to the scientific research community by governments to observe and investigate Earth processes, trends, abrupt events, and disasters. Governments are especially interested in what might be "tipping points," or bifurcations in chaotic systems like the climate. Fundamental scientific inquiry--discovery--will always remain one driver of such studies, however relevancy has become increasingly important. Governments need Science (and here Science is the larger enterprise of the natural and social sciences) to actively contribute towards solutions to the problems caused by the complex, non-linear interactions between the changing environment and our social structures.
Other communities also are becoming focused on this need for integration of Scientific research with the policy process. The international development community is concerned with sustainability of agriculture, water management and desertification issues, as well as urban planning--all topics in which global change is a factor, and for which Science should have significant input. In the non-governmental world, conservation efforts are becoming aware that ignoring climate change may make resource allocation decisions about particular locations and species moot.
National and international donors are also becoming focused on global change. The coming decade of global environmental change research will result in increasing pressure to ensure connections with the decision needs of Governments. Member states of the United Nations are striving to achieve the Millennium Development Goals, which are inextricably linked to closer integration of Scientific information with policy development.
For many scientists and research institutions, this is a new context. It is also a new context for many science funding agencies, which have not previously had to manage the interactions between different areas of science and policy that this approach implies. There are very real needs for scientific input into the policy and research management worlds. There are examples of past successes like the 1987 Montreal Ozone Protocol. However, in retrospect, despite the significant difficulties associated with the science underlying the negotiation of that protocol, the scientific community is well aware that the larger issue of global environmental change is much more complex than the ozone-related subset. Most of the simple Science problems have already been addressed.
The global environmental change science landscape is evolving rapidly. Earth systems science has done well in transitioning to team-based work, within Earth systems science, but it is just starting to tackle team-based work with social scientists, and with non-scientists like policy and media specialists. Other disciplines, like engineering and medicine, have worked in this interactive mode for much longer (centuries in some cases), and will certainly respond to this new challenge effectively. The global environmental change science community and its institutions must adapt to this new mode of collaboration if they are to contribute to a sustainable future.
Future climate-related crises may actually decrease the amount of funding available for global environmental change science. Three factors contribute to this possibility: first, extreme events such as the hurricane-related damages on the Gulf Coast in the United States cause enormous reorganization in government funding structures, and the disciplines able to respond on short (and therefore perceived as relevant) timescales will generally benefit: medical services, social services, reconstruction logistics, et cetera. The ability of Science to provide answers on timescales of hours, days or even weeks is limited--the immediate value of the information currently provided is extremely low. Second, these extreme events are usually geographically restricted compared to many global change issues. Paradoxically, regional and local crises can have greater impact on funding priorities than global ones, even if the local events are exacerbated by global change. A third factor contributing to the possibility that funding will decrease for global environmental change research is that this science is framed within fundamentally different time-scales from those governing political and public interest. Without a concerted effort by the Scientific community at consciousness-raising and education on global environmental change issues, phenomena that occur on decadal timescales (not to mention century, millennial, or beyond) will have little 'traction' in the political and popular world that determines funding.
If Science does not rise to the occasion, it will become irrelevant in the policy decision chain addressing sustainability, and possibly worse, lose ground in funding for basic science.
Ensuring Science's relevance will require increased internal coordination between the natural and social sciences and their governance structures, and ongoing dialogue with important external social actors: the policy sector, business and industry, and labour. These types of stakeholder interactions will require adaptive management, which has fundamental implications for all levels, including the ultimate government funding agencies. Project management (by scientists, by the grantee institutions, as well as by the overseeing granting agencies) will have to allow for flexibility in project objectives, and for changes in the basic metrics used for scientific research and productivity. Budgetary allowances will have to be made to accommodate the increased need for science communications, additional dissemination modes, and interactions specialists.
The historic reluctance of science to engage in this process is, of course, related to the perceived trade-off between Science's long-term credibility and the relatively short half-life of policy interests. A carefully tended dialogue with governments on what is currently "policy relevant," and what the related policy impact indicators are, must be maintained.
This volume explores the interactive frontier between science and policy, and looks at bridges between the two in the context of transformative research carried out in the Americas, primarily under the sponsorship of the Inter-American Institute for Global Change Research, an international treaty organisation. In spite of the difficulties in organizing new research networks, there are several successes in the included Chapters that demonstrate that the interactions described above between Science and policy can indeed change local and national government policies. Interestingly, one of the Institute's greater achievements has been the formation of a cadre of academic administrators in the region who are now capable of administering multi-institutional, multi-currency, and multi-disciplinary research projects, and who are now pursuing funding from regional development agencies.
The processes of science and policy-making almost always differ from our preconceived notion: rather than being linear processes that lay out a question, analyse it, and propose a solution based on that analysis, they are both complex non-linear iterative processes that deal with multiple, interlinked, and changing questions. Closer cooperation between two such processes cannot be expected to be simple. All actors must learn something about the peculiarities of the other's culture.
We look forward to that conversation.


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A first time bride who`s age is 60 years old got everyone chanting after the emergence of her wedding photos.
"60 years old and a first time bride… never been married!…..Dang!!..she looks good at 60!!….#goodgenes..#healthyliving..
I remember the first time we met and she told me her age…I was like#hollymolly!!! #abasi mbok …..#unbelieveable……she went on to tell me how shes been waiting for 'mr right' since when she was in her 30's and he never came. And so two years ago, he finally appeared and proposed in 2016…..#God is good…#delayisnotdenial my dearest aunty sarah!..i celebrate with you and may God bless your union…. from@bridesnmoreikeja….#it'snevertoolate…..more pictures of our youngest and latest bride in town coming"


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Eden Prairie workshop to help build better parents
December 18, 2017 by James
The early years of a child's development can often be some of the most influential in a person's life, and an Eden Prairie event will help parents to make the most of this relatively short period of time.
Parents and caregivers alike who are raising a young child and who come to the 'Parents Are Hypnotists, Too!' workshop will learn about how their words and actions can have an impact on their son or daughter's developing brain. It will be facilitated by Roberta, and the organizers noted that she is both a hypnotist and the founder and former administrator of a Montessori school.
The organizers went on to say that the class is best suited to parents of a child who is an infant, right up to those who are age 10. The people who participate will have an opportunity to learn new ways they can have a positive influence on their son or daughter, and anyone with questions has been asked to forward them to firstname.lastname@example.com. Flyers that promote a function like this to the local community can be crafted by a printing services provider.
It will be held on February 6 at The FARE Hypnosis Center, which is located at 8353 Crystal View Road. The registration fee to take part in the event is $25.


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by Hugh Martin & Judy Garland
Jul 3, 2015
Love Lyrics
Song Meaning
Verse 1
Love can be a moment's madness
Love can be insane
Love can be a life of sadness and pain
Verse 2
Love can be a summer shower
Love can be the sun
Love can be two hearts that flower as one
Verse 3
It can be, fine and free
But that kind
Is not so very easy to find
Verse 4
Love can be a dying ember
Love can be a flame
Love pledged in September
May be dead in December
You may not even remember it came
Verse 5
Love can be a joy forever
Or an empty name
Love is almost never ever the same
Verse 6
Love can be an evil-doer
Love can be a fog
Love can make you feel like you were a dog
Verse 7
Love can be a snow-capped mountain
Love can be the truth
Love can be an endless fountain of youth
Verse 8
It can be ecstasy
But it's true
It doesn't always happen to you
Verse 9
Love can be a four-score failure
Love can bring you fame
Love fresh as the morning
May be wild when it's 'borning
And then without any warning, it's tame
Verse 10
Oh love can be a sweet endeavor
Or a dirty shame
Love is almost never ever, the same!
Lyrics Licensed & Provided by LyricFind
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Cut It Up Challenge #74 - Stuck on You
Friday, June 26, 2015
I'm free! At least for the weekend :) Hope you find you're free to play with us in our new Cut It Up Challenge! June invites us to use a sticker on our project (and remember to use a die!) The challenge runs from today through July 8th.
The winner of this challenge will receive a $20 gift certificate from our fabulous sponsor The Robin's Nest ($30 gift certificate if a product from their on-line store is used!)
For my card, I used a Doodlebug sticker (the multi-colored dots) and added punched butterflies. The stitched cuts in the base were made from Paper Smooches Stitched die and the word die cut is from Papertrey Ink.
Be sure to check out the awesome projects the Design Team has to share:
Kathy - You Are Here
Labels: challenges , Cut It Up Challenges , Summer , Thank You
9 comments :
This card is so bright and cheery! Love the stitched die with the butterflies flying along in a rainbow effect!ReplyDelete
This is such a happy card, The butterflies on their stitched trail just make me smile.ReplyDelete
Oh so Pretty!!!!! This is one of my Fav's! LOVE the colors and the pretty Butterflies! Have a Crafty Day!! :)ReplyDelete
Scrappers Anonymous
What gorgeous use of bflies! AND, perfectly lined up -- fabulous card~ReplyDelete
So happy, colorful and cheery. I love all of the butterflies! CIU DTReplyDelete
I love all of the butterflies. Great card.ReplyDelete
- AnonymousJuly 3, 2015 at 7:32 AM
This is so cool! I love how the butterflies are flying in formation!ReplyDelete
What a bright and beautiful card! Love it!!!ReplyDelete
This card is so fresh and pretty! I love your take on my sticker challenge :)ReplyDelete
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text: Thank you to everyone that made the Fair Housing Symposium 2017 a huge success. Y.I.M.B.Y. (Yes! In My Backyard: Negotiating the Changing Landscape of Fair Housing) covered important topics such as fair housing issues for persons with criminal records, local nuisance ordinances and fair housing issues for domestic violence and sexual assault survivors. The symposium also featured current issues surrounding housing discrimination in the LGBTQ community. The Arizona Fair Housing Partnership is pleased and honored to have our guest speakers and panelists share their expertise on these significant issues.


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- Burnt Mills Sunoco
Barry G Customer since 2013CJ, Brian and the team are superb. They are so helpful and patient. We love to use this station for our services, they also happen to be one of the cheapest gas stations in our area.2 months ago
Joe G Customer since 2014CJ and his staff have been my auto sevice provider for over ten years. Quality work, reasonable prices and honest assessments of what work is needed. I trust them completely.11 months ago


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|Clockwise from upper left: Shepard & Tali, Jean & Scott,
Apollo & the Midnighter, Mulder & Scully.
Whether it's a day you mark or not, have a great weekend!
|Clockwise from upper left: Shepard & Tali, Jean & Scott,
Apollo & the Midnighter, Mulder & Scully.
Thanks! Valentine's Day back to you, too ;-)
I only recognize 2 out of your 4 couples. (Ack! Gotta get a comic fix.) The ones I *knew* are Jean & Scott and Mulder & Scully.
Have a great Valentine's weekend too :-). Night OwlReplyDelete
Haha, that's awesome...happy Valentine's day to you too! :)ReplyDelete
I only knew the same two as D7ana. Do Shepard and Tali come from separate lines, or is their height difference always that discrepant?ReplyDelete
There is supposed to be a bit of a height discrepancy between the two, but you're right - not by that much - they are from different lines. Shepard is from PlayArts and Tali is from DC Direct (the DC Direct Shepard was ugly, so I passed on the matching figure from that line).Delete
*Love, exciting and new... yes we're action figures, but we need love toooooo.*ReplyDelete


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Whether care homes should call residents by affectionate terms of endearment such as 'sweetie' or 'darling' caused quite some debate in the national media this week.
However, the advice from the Care Quality Commission to refrain from using what they call "patronising language" is nothing new and it was about six years ago that we first looked carefully at the regulator's recommendations at The Old Vicarage and came to the conclusion they were right, overly affectionate names could be seen as demeaning by some of our residents.
My view is that in some towns and cities in the UK, people will happily call each other 'love' and 'duck' and that's fine, but it may not be acceptable to some or even a few of our residents and, therefore, we encourage our team not to do so. We ask our team to use a resident's preferred name, which might be completely different from their real name, a nickname, perhaps. We find this an informal and friendly approach, where no-one is going to get upset or embarrassed.
Ultimately, every resident should feel comfortable and as a matter of course, we talk to our managers to ensure everyone on the care team is clear on this policy. We firmly believe it's the proper way to talk to residents and while people should feel as much at home as is possible when they go into care, they also need to feel respected at all times.
Dave Lock
The Old Vicarage Care Home, Clay Cross, Derbyshire


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this is a commissioned painting done for a friend's friends. it is 36" x 24", acrylic & collage on canvas. i used remnants (receipts, maps, tickets, etc.) of their trip to thailand and china as well as photos they took to create a layered and textured collage. there are probably 6 or 7 layers of different elements, combined using acrylic mediums and gels, laser toner transfers, paper and paint. the first pics are detail shots of various stages in the process. the last three pics are the main stages of the process. first there is the collage foundation. second is the layering of printed acetate images on top of the foundation. third is the painting and adding of textures.
this is the finished painting (below).
Monday, April 23, 2007
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Job Description:
Position Overview
We are seeking a strong, full-stack developer with a sharp, creative design eye; has experience creating a seamless bridge between the front-end and the back-end design. This role requires excellent interpersonal skills for working with business sponsors, analysts, project managers, content editors
The developer will bridge the gap between design and technical implementation, taking an active role on both sides and defining how the application looks as well as how it works.
• Develop ( JAVA ) systems including coding, testing, and debugging.
• Develops Javascript, HTML5, and CSS3 of user-facing features as per design components and information architecture
• Has familiarity and extensive experience with popular Content Management Systems (CMS)
• Ensures the technical feasibility of designs
• Optimizes application for maximum speed and scalability
• Utilizes the results from usability testing to help identify and fix defects.
• Collaborates with business and IT decision-makers to discuss system requirements and brainstorm ideas for new
• Creates and maintains a repository of common assets and reusable code
• Wields knowledge of Search Engine Optimization (SEO) to develop search-friendly websites and interfaces
• Has awareness of the "state of the art" and assess new trends and emerging technologies in and related to UI/UX design
Required Knowledge/Skills, Education, and Experience
• Experience in communicating with users, other technical teams, and management to collect requirements, evaluate alternatives and develop processes and tools as needed to support the organization
• Strong analytical skills and technical aptitude
• Excellent communication skills with positive energy
• Experience with Agile development necessary
• Bachelor's degree in Computer Science, Software Engineering or a related field of study
• Minimum five to ten years of experience in software development
• Knowledge of multi-tier architectures
• Experience using web services (REST, APIs and JSON)
• Experience with database technologies
Preferred Knowledge/Skills, Education, and Experience
• Expertise using JAVA,HTML5, CSS3, XML, XSL, jQuery, JavaScript, and
other common JavaScript frameworks
• Good understanding of advanced JavaScript libraries and frameworks,
such as AngularJS, ReactJS, etc.
• Strong focus on user experience and simple design solutions
• Strong ability to learn and apply established style guides to design and development
• Experience with Git or other software version control system
• Knowledge of Java application servers such as Apache, etc.
• Experience with testing methodologies such as TDD and BDD
Requisition Number: 223023


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JM Financial is bullish on Coal India has recommended buy rating on the stock with a target price of Rs 350 in its research report dated May 31, 2018.
Coal India (CIL)'s net profit – adjusted for a one-time gratuity provisioning – stood at INR 62bn (+42% YoY), resulting in FY18 profit growth of 3% YoY (despite a profit decline of 16% in 9MFY18). Notably, the price hikes taken in Dec'17 are reflected in FSA realisations, which grew 19% QoQ to INR 1,403/T, while e-auction realisations improved 31% YoY to INR 2,112/T. FY18 profit growth is led by a) 6.5%/13% YoY growth in FSA /e-auction volumes and b) e-auction realisations growing 20% YoY. The positive impact of coal re-stocking by power plants is clearly evident in higher off-take growth for CIL (6.8% in FY18). We factor offtake growth of c.7% in FY19-20 for CIL vs. management's target of 8.6% in FY19. While FY18 profit growth has been flattish due to wage provisioning, we estimate high growth in FY19 profits (c.20%), despite factoring only 7% offtake growth (vs. management's target of 8.6%) and lower e-auction prices. With all the negatives of wage provisioning and degradation already factored in FY18 earnings, the benefit of growing volumes and price hike would reflect in FY19/20.
We maintain BUY with a DCF-based TP of INR 350, while CIL currently trades at 5x FY20 EV/EBITDA and P/E of 11.8x FY20.
Disclaimer: The views and investment tips expressed by investment experts/broking houses/rating agencies on moneycontrol.com are their own, and not that of the website or its management. Moneycontrol.com advises users to check with certified experts before taking any investment decisions.
Source: moneycontrol.com


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Since Halo: Reach has come out, people have wondered what exactly these rare, golden Ranger Elites actually are meant for, and quite simply, what they do. BOB is the strongest Elite in the campaign, and killing him yields rewards. Killing a BOB gives you a unique medal on Bungie.net called Seek and Destroy. The description of this medal tells you to quite simply, "wipe them out."
These Elites are hidden throughout every single campaign mission. Exodus and New Alexandria have no walking BOBs found yet, but instead they do have many unique locations for Banshee BOBs. Banshee BOBs can be hijacked only on New Alexandria after activating the Pelican and Phantom Easter Egg switch. When you jack a Banshee BOB, or destroy one, no enemy comes out of them. They are registered as Banshee BOBs and do count as a BOB kill but instead you don't get a medal for killing them. This is proved through the weekly challenge on the 19th of January which told us to kill the BOB in a Banshee flying fast and low on New Alexandria. Click here for proof and challenge information
"Walking" BOBs have unique spawns on every campaign mission, and some even spawn behind you for a few seconds as you progress through some of the missions. BOBs don't stay around for long, they disappear in thin air if you don't kill them quick enough or you wait too long to engage them. They are very strong, and on Legendary they can take multiple Laser blasts and get sniped over five times without even dying. They are not to be taken lightly. They are unique in their coloring and they tend to flee from every and all battles.
BOBs have many rumors surrounding them. Some say they are simply a reference Easter Egg to the BOBs from Marathon. Some say that they, when killed in certain order, unlock the infamous "BOB" Helmet. Some say that they do something, we just don't know what yet. Either way, killing BOBs is challenging, and hunting them down, is pretty fun. They appear on all difficulties, though some only appear on Legendary. The BOB helmet origin started when IGN visited 343 and pictures of the helmet below appeared.
The helmet seen above was seen in Bungies armory on July 7th, 2011 while they were livestreaming Bungie vs The World. Any previous theories of it not being on the disc are all false. Bungie wasn't playing on any different Xboxs than anyone else and they had the same Halo: Reach disc we do, except they can unlock any armor as they please do to them being the creators of the game.
For those of you who want to search for BOBs, or just want some extra fun in the campaign, the following videos, which aren't mine, will aid you in doing so. Keep an eye on this page as it will be updated frequently with more BOB information and more BOB locations!


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Tidy up around the workplace is most likely the exact opposite thing you need to consider when you get the chance to work. Or, then again perhaps you consider it, yet have a million different needs on your plan for the day consistently. Actually, numerous entrepreneurs go up against the undertaking of cleaning the workplace themselves. Not exclusively would this be able to be an overwhelming, particularly when you have a huge organization, yet it likewise removes time from the business you'd lean toward the run.
We know you realize that a spotless office is critical. In this way, how about we take a gander at a portion of the reasons why it really matters:
Initial introductions: People base their conclusions of your organization off what they see. An office space that is not as much as satisfactory at first look is one that clients consequently start to scrutinize the nature of items and administrations of. We've every single made judgment in view of our observations. It's human instinct. Not exclusively would you like to make a decent initial introduction, however when individuals leave, you need the notoriety of your organization to be one that they say nothing but good things about. It's intriguing: A perfect space influences individuals to need to be there. It makes an inviting situation for the two representatives and clients. We should consider it for a moment. Who needs to burn through 40 hours every week in a grimy work space?
Our Office Cleanz Specialists are gifted at protecting the nature of your furniture and gear. Think about whether it's justified regardless of the venture? Office Cleanz: office cleaning services Singapore offers the comfort of calm cleaning arrangements and a 100 percent fulfillment ensure. In case you're not upbeat, we'll do it again for nothing. Contact Our Experts for proficient, green-accommodating business cleaning arrangements, today.


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Two of Cranford's food establishments are asking customers for some feedback so they can get a better idea of how to serve their clientele. Both Southside Roast and Dinner in a Pinch have posted brief surveys on their sites for customers to fill out. Southside Roast is giving away a $10 gift card to one lucky respondent, while Dinner in a Pinch is offering a free appetizer to anyone who fills it out. So take five minutes and fill out the surveys. Don't you love it when businesses are constantly looking for ways to improve?
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Hotel Mansion Lions in Eastbourne - 3 HOTEL INFO stars
This is what awaits you:
- 1 bottle of mineral water
- Parking directly by the hotel
Style of hotel and ambience
Guests start their holiday at the family-run hotel Mansion Lions in Eastbourne with a warm welcome and a picturesque view on Eastbourne Pier. Spacious and elegantly furnished rooms guarantee a pleasant stay in a unique ambience. In the restaurant, guests look forward to mouth-watering dishes à la carte. Moreover, a magnificent private garden with picnic area is available.
Special features of the hotel
Relax in the lounge/bar or watch the world go by from the sun room with a seafront view or stroll along the Grand parade and explore the nearby attractions but on your return you can be assured of a comfortable and friendly atmosphere. Begin the day with a hearty cooked breakfast and after a busy day exploring return to a relaxed drink in the lounge followed by dinner in the elegantly furnished restaurant. The restaurant with its executive chef's varied menu offers a relaxed dining experience with high quality service which will bring you back day after day.
Hotel data
Hotel built in 1877, Number of floors 4, Total number of rooms 106, Number of single rooms 23, Number of double rooms 52, of which have 2 separate beds 32, of which have a double bed 30, of which are suitable as three-bed rooms 3, Hotel closed from 2011-01-02, to 2011-02-14, Reception manned 24 hours a day, Earliest check-in 14:00, Latest check-out 10:00, English-speaking staff available during the day, English-speaking staff available at night, Hotel, Holiday hotel, Beach hotel, family-run hotel, No-smoking hotel, No-smoking room 106
AddressShow map
1 Grand Parade, BN21 3YS Eastbourne, United Kingdom
- Station (Eastbourne) 1.2 km
- City centre (Eastbourne) 0.51 km
- Airport (London Gatwick Airport (LGW)) 54.17 km
Service times
Reception open:
12:00 AM to 12:00 AM
Reception manned at weekends:
12:00 AM to 12:00 AM
Earliest check-in:
Latest check-out:
- Visa
- Eurocard/MasterCard
- American Express
Accepted payment methods
Hotel facilities
- Reception (lobby)
- Elevator
- Restaurant(s) 1 open from 07:30.00 until 09:30.00 and from 18:00 until 20:00.00
- Hotel bar
- Garden/park
- Garden terrace
- Fire alarm
- Smoke detectors
- Breakfast from 07:30 o'clock to 09:30 o'clock
- Vegetarian food available
- Wholefood available
- Diabetic food available
- Hallways equipped with fire exstinguishers
- Hotel complies with country/state/local fire laws
- Audible smoke alarm in hallways
- Emergency exits for each floor
- Building meets all current local, state and country building codes
- Hotel staff trained in first aid
Room facilities
- Easy access for wheelchairs
- Satellite TV
- Desk
- Seating area
- Telephone on the desk
- Fridge in 5 rooms
- Windows for opening
- Make-up mirror
- Hairdryer
- Bathroom with window
- Emergency information
- Plan of emergency exits
In-house services
- Safety deposit boxes at reception
- Ice machine
- Express check-out
- 1 bottle of mineral water
- Parking directly by the hotel
- Pets not allowed
Hotel location
Evaluations of Mansion Lions
The staff were very friendly and helpful.
There was nothing to dislike.


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Dear Diary, another practice in the hall today. Nothing new.
Dear Diary, I got pulled away from practice today. My sister was going to the dentist. But why did I need to go? Dad told me it was so he could kill two birds with one stone and take us both. So I believed him.
Well that was a mistake! Turns out it was all a trick. We left us as soon as Bella was finished, without me getting a check up. Turns out we going to get my 11 year old shots! Great just what I needed.
The shot didn't hurt too much, but Dad totally owes me a pony!