text:
Identification and Initial Functional Characterization of a Human Vascular Cell–Enriched Long Noncoding RNASignificance
Objective—Long noncoding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes.
Approach and Results—RNA sequencing (RNA-seq) of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell–enriched lncRNA (Smooth muscle and Endothelial cell–enriched migration/differentiation-associated long NonCoding RNA [SENCR]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5′ end of the FLI1 gene and exists as 2 splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-seq experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration.
Conclusions—SENCR is a new vascular cell–enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.
Although the human genome is 30× larger than that of Caenorhabditis elegans, each species is endowed with a similar number of protein-coding genes, a fact seemingly in support of an abundance of junk DNA within our genome.1 Two major discoveries during the past 10 years challenge this decades-old concept. First, genome-wide RNA expression studies show widespread transcription across the mouse and human genomes with roughly equal amounts of polyadenylated and nonpolyadenylated RNA.2–7 Second, the combined efforts of the ENCyclopedia Of DNA Elements (ENCODE) Consortium and many other laboratories have revealed the existence of millions of codes that punctuate the human genome, most notably codes for transcription factor binding.8–12 These findings, coupled with the notion that much of the human genome is functional with 50% to 90% comprising transcribed sequences,13,14 debunk the concept of junk DNA and point to a genome replete with information essential for human life.
See accompanying editorial on page 1124
Much of the noncoding RNA (ncRNA) in a cell functions to orchestrate basic translation (transfer and ribosomal RNA); however, 2 broad classes of ncRNA expanded greatly at the turn of the millennium, primarily as a result of large-scale transcriptomics projects.2,3,15 These ncRNAs are classified subjectively as either short (processed transcript length <200 nucleotides) or long (processed transcript length >200 nucleotides). Short ncRNAs include small nucleolar RNA and their derivatives that act as guide RNAs to modify ribosomal and transfer RNAs,16 as well as microRNA, small interfering RNA, and PIWI-interacting RNA that use Argonaute proteins to mediate endonucleolytic cleavage of target RNAs.17
Long ncRNAs (lncRNAs) function in a myriad of biological processes and may be classified loosely based on their physical location in the genome. Long intervening ncRNAs (lincRNAs) are a subclass of lncRNAs found between 2 transcription units, and they exhibit similar active chromatin signatures as those found around protein-coding genes.18–20 LincRNAs may display tissue-specific patterns of expression and function principally as scaffold or guide RNAs that facilitate chromatin remodeling in cis or trans to directly influence gene transcription (nuclear lincRNAs) or effect changes in mRNA stability/protein translation (cytoplasmic lincRNAs).20–22 Examples of lincRNAs include the abundantly expressed MALAT1 that functions in processing of mRNAs23 and the epidermal prodifferentiating TINCR.24 A recent report defined very long intervening ncRNAs whose expression correlates with malignancy; these transcripts may encompass previously annotated lincRNAs.25 LincRNAs may also overlap transcriptional enhancers to effect cis-mediated changes in gene expression.26,27
Intragenic lncRNAs represent another subclass of RNA genes that reside on the sense or antisense strand of an overlapping gene. Sense lncRNAs have been reported only sporadically,28 although a recent report contends there exists a large number of ill-defined sense ncRNAs within introns.29 Antisense lncRNAs occur in a significant number of protein-coding genes and may overlap the 5′ or 3′ end of a gene, occur entirely within an intron, or overlap multiple exons.30–32 Antisense lncRNAs whose exons overlap protein-coding (or ncRNA) exons are known as natural antisense transcripts and these can function in cis or trans to negatively or positively regulate gene expression through RNA interactions with chromatin remodeling factors.33 Examples of natural antisense transcripts include the X chromosome inactivating XIST34 and the cell cycle regulator ANRIL.35 Some processed antisense lncRNAs do not overlap sense exons and thus may have unexpected functions (below). The number of human lncRNAs is soaring with the current catalogue of LNCipedia36 listing >32 000 (http://www.lncipedia.org/), a number that exceeds all protein-coding genes. Thus, lncRNAs embody a rapidly growing class of genes with functions related primarily to the regulation of gene/protein expression.
Cellular differentiation requires the coordinated activation of unique gene sets through transcription factors in association with cofactors over discrete cis elements. For example, vascular smooth muscle cell (SMC) differentiation is chiefly a function of ubiquitously expressed serum response factor37 binding a cardiovascular-restricted cofactor called myocardin (MYOCD)38 over CArG elements located in the proximal promoter region of many SMC-associated genes.39 Similarly, endothelial cell (EC) differentiation proceeds, in part, through the FOXC240 and ETV241 transcription factors binding a composite cis element, the FOX-ETS motif, found in promoter/enhancer sequences of several EC-specific genes.42 Normal differentiated properties of SMC and EC further require fine tuning of gene expression through the action of microRNAs.43 Because lncRNAs are prevalent and play key roles in modulating gene expression,44 they too may have functions linked to vascular cell phenotype. Little is known, however, about the expression or function of lncRNAs in vascular cells,45–49 and there is nothing known about human-specific, vascular cell–selective lncRNAs. Accordingly, we performed RNA-seq in human coronary artery smooth muscle cell (HCASMC) as a first step toward understanding the potential role of lncRNAs in human SMC phenotypic control. Here, we report on the identification of 31 lncRNAs, including 1 named SENCR (for Smooth muscle and Endothelial cell–enriched migration/differentiation-associated long NonCoding RNA). We have characterized the expression, splicing, and localization of SENCR and have identified unique gene signatures on its knockdown in SMC. SENCR seems to play a role in maintaining the normal SMC differentiated state as its attenuated expression leads to reduced MYOCD and contractile gene expression with elevations in migratory genes that foster a hyper-motile state. This report outlines the first foray into lncRNA discovery in human vascular cells and establishes a foundation for further inquiry into SENCR biology, as well as the identification, expression, and function of other human vascular-selective lncRNAs under normal and pathological cell states.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Identification and Validation of lncRNAs in HCASMC
We have developed a rigorous workflow for the identification and study of lncRNAs in primary-derived HCASMC using RNA-seq methodology (Figure I in the online-only Data Supplement). A total of 79.41% of filtered reads could be aligned to the human reference genome. Thirty-one lncRNAs met our strict inclusion criteria (Methods in the online-only Data Supplement) with the majority (22/31) falling into the lincRNA subclass (Table II in the online-only Data supplement). Conventional reverse transcription polymerase chain reaction (RT-PCR) showed detectable expression of 21 of 31 lncRNAs in a panel of human cell types, including HCASMC and human umbilical vein EC (HUVEC; Figure 1A). Sequence analysis of the PCR products confirmed the identity of each lncRNA (not shown). The majority of HCASMC lncRNAs are distributed widely across human tissues with several detected in dated human plasma (Figure 1B and 1C). One of the lncRNAs (lncRNA9) exhibited a selective pattern of expression in cell lines (Figure 1A and 1D) and human tissues (Figure 1A and 1B). We refer to this lncRNA as SENCR because of its enriched expression in both smooth muscle and ECs (Figure 1A and 1D) and its proposed function (below).
SENCR Is a Vascular Cell–Selective Antisense lncRNA
RNA-seq alignment, 5′ RACE, and RT-PCR with oligo-dT and strand-specific primers established that SENCR comprises 3 exons and is transcribed in the antisense orientation from within the first intron of Friend leukemia virus integration 1 (FLI1), an important transcription factor programming EC and blood cell formation50 (Figure 2A). There is no overlap between SENCR and FLI1 exonic sequences, indicating that SENCR is not a natural antisense transcript33 (Figure 2A). The longest open reading frame flanked by start and stop codons is 61 amino acids; however, analysis of this and other predicted open reading frames in SENCR failed to reveal any known protein-coding domains, suggesting that this transcript has no or low protein-coding potential (not shown). Primers to exons 1 and 3 of SENCR showed the presence of 2 distinct PCR products (Figure 2B). Sequence analysis confirmed these products as full length (SENCR_V1) and an alternatively spliced variant (SENCR_V2) of the SENCR gene (Figure 2A and 2B). These sequences have been deposited in GenBank under accession numbers KF806591 and KF806590, respectively. We used specific primer pairs to examine SENCR isoform expression in a panel of human tissues and cell lines. Results showed SENCR_V1 to be more broadly expressed than SENCR_V2 (Figure 2C and 2D). In general, there was coincident expression of SENCR with FLI1, suggesting that these transcripts may be under similar transcriptional control processes (Figure 2E and 2F). Quantitative RT-PCR analysis suggested the FLI1 transcript to have higher expression than SENCR (Figure II in the online-only Data Supplement).
Exon 1 of FLI1 shows high conservation across 46 mammalian species; however, much less conservation exists across the 3 exons of SENCR (Figure 3), consistent with the fact that no orthologous SENCR transcripts have yet been found outside human/chimp lineages. Interestingly, exons 2 and 3 of SENCR harbor single nucleotide polymorphisms, suggesting potential deleterious effects on SENCR function (Figure 3). Analysis of ENCODE data on the UCSC Genome Browser (http://genome.ucsc.edu/) supports the enriched expression of SENCR in HUVEC with lower levels in other cell types. Further, there is a prominent HUVEC-associated H3K4me3 mark near exon 1 of SENCR, suggesting the presence of an active promoter (Figure 3). As a first step toward delineating SENCR transcription, we cloned and tested several luciferase reporter constructs. Luciferase assays showed little to no detectable SENCR promoter activity in HUVEC unless sequences encompassing the 5′ FLI1 promoter region were included, although even these reporters showed much lower activity than a control promoter construct (not shown). Collectively, these results define an alternatively spliced, vascular cell–enriched antisense lncRNA that overlaps the 5′ end of the FLI1 transcription factor yet, in its mature form, does not harbor exonic sequences that could undergo Watson–Crick base-pairing with corresponding exonic sequences in FLI1.
SENCR Is a Cytoplasmic lncRNA
Quantitative RT-PCR showed SENCR RNA to be most abundant in HUVEC with undetectable transcripts in HeLa cells (Figure 1D). We used high-resolution RNA fluorescence in situ hybridization51 in these 2 cells types to unambiguously discern the intracellular compartment where SENCR transcripts reside. Consistent with quantitative RT-PCR, no SENCR transcripts were seen in individual HeLa cells (Figure 4A, bottom). However, we observed variably low numbers of SENCR RNA molecules in the cytoplasm of individual HUVEC (Figure 4A, top and middle). We sometimes observed SENCR RNA in the nucleus although this probably reflects either active transcription or unprocessed RNA. The cytoplasmic, low-level expression of SENCR RNA contrasts with the higher-level nuclear accumulation of NEAT1 lncRNA as well as cytoplasmic PP1B mRNA (Figure 4A). Biochemical fractionation followed by RT-PCR further documented cytoplasmic localization of SENCR in both HUVEC and HCASMC. In contrast, the lncRNAs NEAT1 and XIST show predominantly nuclear accumulation in these cell types (Figure 4B; Figure III in the online-only Data Supplement). We next used 2 distinct probe sets to SENCR in HUVEC treated with a control dicer substrate RNA or 2 dicer substrate RNAs targeting different regions of SENCR to further demonstrate the specificity of the signal (Figure 4C). Quantitative analysis of coincident hybridization of each probe set demonstrated a likely underestimate of ≈0.8 copies of SENCR per cell, a value that was approximately halved on SENCR knockdown (Figure 4D). These results establish the cytoplasmic localization of SENCR and indicate its relatively weaker level of expression as compared with housekeeping mRNA molecules (PP1B) and at least one other lncRNA (NEAT1).
SENCR Knockdown Exerts Little Effect on FLI1 mRNA in Vascular Cells
Many lncRNAs that overlap protein-coding genes in the antisense orientation exert cis or trans effects on gene expression through the recruitment of chromatin remodeling factors.52 However, no uniform cis-acting effect on FLI1 or neighboring gene expression was observed on knocking down SENCR with multiple dicer substrate RNAs in HCASMC (Figure 5A–5C) or HUVEC (Figure 5D and 5E), consistent with its cytoplasmic localization. There was also little effect of SENCR knockdown on the nuclear accumulation of FLI1 protein or steady-state FLI1 protein levels (Figure 6G and 6H). Further, knockdown of FLI1 effected no significant change in levels of SENCR RNA (Figure 5F). We occasionally observed mild variation in FLI1 mRNA expression (either up or down) with some dicer substrate RNAs in certain isolates of vascular cells; however, these changes were sporadic and not reproducible when tested by multiple investigators. We therefore conclude that reducing SENCR RNA has little to no cis-acting effect on local gene expression.
SENCR Knockdown Alters the Normal Contractile Gene Program in HCASMC
Several cytoplasmic lncRNAs effect changes in a cell's transcriptome through post-transcriptional control processes.53 As an initial step toward understanding the function of SENCR, we performed RNA-seq in HCASMC after knockdown of SENCR to assess changes in the transcriptome. Most sequencing reads were aligned to the reference genome and scatterplots of replicates showed similar transcript profiles (not shown). Statistical analysis of each set of replicates revealed hundreds of genes that were significantly induced or repressed on SENCR knockdown (Figure 6A; Table III in the online-only Data supplement). Strikingly, many SMC contractile genes showed significant reduction in mRNA expression with SENCR knockdown (Figure 6B; Table III in the online-only Data supplement). Gene ontology analysis using DAVID revealed biological processes associated with this reduced contractile gene signature (Table IV in the online-only Data supplement). Of note, the key transcriptional switch for SMC contractile gene expression, MYOCD,39 was also reduced with SENCR knockdown (Figure 6B), and several dicer substrate RNAs to SENCR validated such downregulation in HCASMC (Figure 6D). We also confirmed reduced expression of several of the SMC contractile genes at both the mRNA level (Figure 6E) and the protein level (Figure 6G). Although the SMC contractile program was reduced with SENCR knockdown, several genes associated with cell migration were induced (Figure 6C; Table III in the online-only Data Supplement). DAVID analysis supported biological processes linked to cellular locomotion with SENCR knockdown (Table V in the online-only Data Supplement). We validated 2 migratory genes (MDK and PTN) at the mRNA level in HCASMC (Figure 6F) and HUVEC (Figure IV in the online-only Data Supplement). Collectively, these data show that reduced SENCR expression compromised the SMC contractile phenotype and promoted a promigratory gene signature.
Attenuated SENCR Expression Confers a Hyper-Motile Phenotype in HCASMC
To ascertain whether the increase in promigratory gene expression on knockdown of SENCR translates into a functional phenotype, we performed 2 independent measures of cell migration. Using a scratch wound assay, we observed hyper-motile HCASMC with SENCR knockdown (Figure 7A and 7B). Many of these cells exhibited reorganization of the actin cytoskeleton with formation of lamellipodia, consistent with a migratory cell phenotype (Figure 7Af, arrows). Importantly, the increase in HCASMC migration could be completely rescued on simultaneous knockdown of either of 2 promigratory genes shown to be induced on knockdown of SENCR (Figure 7C; Figure V in the online-only Data Supplement). To further confirm this accentuated cell migration phenotype on knockdown of SENCR, we used a modified Boyden chamber assay. Consistent with the scratch wound assay, we noted that HCASMC migration was elevated with SENCR knockdown although not as much as that observed with the potent migratory stimulus, PDGF-BB (Figure 7D and 7E). We also observed augmented PDGF-BB–induced cell migration on concomitant knockdown of SENCR (Figure VI in the online-only Data Supplement). Taken together, these results strongly support a role for SENCR in the regulation of HCASMC differentiation and cellular motility.
Contrary to the historical notion of pervasive junk DNA,1 most of the human genome is transcribed signifying a treasure-trove of previously unrecognized functional DNA sequences. These include tens of millions of regulatory elements as well as the expansive class of lncRNA genes. LncRNA genes already outnumber protein-coding genes and they exhibit diverse functions related to gene expression and splicing; protein translation, activity, and trafficking; as well as the formation of specialized microenvironmental niches.54,55 Here, we present the first RNA-seq study in a human vascular cell type for the specific discovery of lncRNA genes. We used strict criteria and discovered 31 previously unannotated lncRNAs, 21 of which we validated in human cell lines and human tissues. In addition, we detected a few lncRNAs in dated human plasma, suggesting that these may have potential utility as biomarkers of clinical disease.56 One of the lncRNA genes, named here as SENCR, shows a selective pattern of expression in cells and tissues with highest levels in human vascular SMC and ECs. We discovered that SENCR undergoes alternative splicing, consistent with widespread splicing of transcripts across the human genome.57 SENCR overlaps the 5′ end of the FLI1 transcription factor in the antisense orientation, but does not seem to regulate local gene expression in cis. Indeed, our extensive RNA fluorescence in situ hybridization and biochemical fractionation studies clearly indicate SENCR to be a cytoplasmic lncRNA supporting an extranuclear function. Using RNA-seq after knockdown of SENCR, we observed uniform decreases in expression of SMC contractile–associated genes as well as attenuated expression of the major transcriptional switch (Myocardin) for the differentiation of vascular SMC.39 However, knockdown of SENCR augments a promigratory gene signature that facilitates heightened SMC migration. Thus, we have uncovered a new vascular cell–enriched lncRNA that seems to function in the maintenance of a normal, nonmotile SMC phenotype.
An analysis of 707 sense–antisense gene pairs annotated in the UCSC genome browser58 shows diversity in structural orientation, with most lncRNAs representing natural antisense transcripts (47.0%), followed by intronic (18.8%), divergent (16.4%), completely overlapping (7.4%), 5′ overlapping (7.1%), and 3′ overlapping (3.4%) lncRNAs (Table VI in the online-only Data Supplement). Much of what is known about sense–antisense gene pairs relates to natural antisense transcripts and effects on local gene expression through such processes as transcriptional interference, double-stranded RNA-mediated events, or the guidance of chromatin remodeling complexes that repress or enhance protein-coding gene expression in cis or trans.33,53,59 SENCR falls within the subclass of 5′ overlapping lncRNAs whose exons do not overlap with those of the sense protein-coding (or noncoding) gene. The terminal portion of intron 1 of SENCR overlaps a region of high homology, likely representing conserved sequences corresponding to the proximal 5′ promoter of FLI1. There is another island of homology within intron 2 of SENCR, suggesting SENCR could be a precursor for conserved small RNA molecules. Although the second and third exons of SENCR overlap the 5′ promoter region of FLI1, there is comparatively weak sequence conservation suggesting SENCR does not sponge critical DNA-binding transcription factors necessary for FLI1 mRNA expression (Figure 3). In fact, SENCR and FLI1 seem to be coexpressed in several cells and tissues, including vascular SMC. This is entirely congruent with our inability to show a consistent effect of knocking down either SENCR or FLI1 on the other gene's level of expression. It is interesting to note that there are little, if any, data on expression of FLI1 mRNA and protein in vascular SMC. Further, the functionality of FLI1 in vascular SMC has not been assessed although an EC-specific knockout of Fli1 showed reduced pericytes and vascular SMC investing the dermal microvasculature.60 In light of FLI1 expression in vascular SMC as reported here, it will be important to directly assess the role of FLI1 in vascular SMC differentiation and function through conditional gene ablation studies.
We know little as to how sense–antisense gene pairs involving lncRNAs are transcriptionally controlled. Presumably, divergent (head to head) sense–antisense pairs share a common promoter as has been described for many bidirectionally transcribed protein-coding genes.61 However, it is completely unclear how other sense–antisense pairs may be transcribed, particularly a lncRNA that is coexpressed with the sense mRNA as shown in this report. Simultaneous expression of FLI1 and SENCR would seem unlikely because of transcriptional collision.62 How then might SENCR and FLI1 be transcribed? Perhaps there are shared promoter elements that facilitate alternating transcription between SENCR and FLI1. Consistent with this idea, no SENCR promoter activity was detected unless sequences encompassing the FLI1 5′ region were included, although the level of activity remained much lower when compared with an EC-restricted promoter (DLL4; not shown). Interestingly, a previous report showed undetectable activity of the FLI1 promoter in cells expressing high levels of FLI1 mRNA.63 This could imply there exists a remotely acting enhancer element critical for alternating transcription of SENCR and FLI1. Another possibility is that SENCR and FLI1 are monoallelically expressed in a mutually exclusive manner.64 Recently, single-cell RNA-seq analysis demonstrated that as much as 24% of autosomal genes exhibit monoallelic expression thus providing support for this hypothesis.65 Clearly, a major task for future investigative work will be to elucidate the transcriptional control of SENCR and other lncRNAs during vascular cell differentiation or pathological conditions.
Elucidating the function of lncRNAs has been hampered by the absence of any obvious lncRNA sequence code. One approach to begin understanding lncRNA function is to reduce the level of lncRNA expression and then evaluate the transcriptome of a cell type.66 In this study, we knocked down SENCR in HCASMC and found that the contractile phenotype of these cells was attenuated with concomitant increases in several promigratory genes leading to enhanced cell motility. The mechanism for such changes in cell phenotype is unknown at this time; however, because SENCR is localized to the cytoplasm it seems unlikely that it acts through direct interaction with DNA or the recruitment of chromatin-modifying complexes to target genes as shown for many nuclear lncRNAs.53,67 It is more probable that SENCR functions in some post-transcriptional capacity to effect the observed changes in gene expression. Because all SMC contractile genes were attenuated with SENCR knockdown, a post-transcriptional mechanism would likely involve the targeting of a protein or RNA that is antecedent to the SMC contractile gene program. One possibility would be that SENCR sponges a low abundant microRNA that otherwise would function to mute the SMC contractile gene program, similar to what has been shown for linc-MD1 in skeletal muscle.68 Other potential post-transcriptional mechanisms of action for SENCR include stabilization, destabilization, or enhanced ribosomal translation of pivotal RNA transcripts, as proposed for other recently defined lncRNAs.24,69–71 The results of this study provide a foundation for exploration of these and other possible mechanisms of SENCR activity using emerging biochemical tools to analyze lncRNA interactions with other macromolecules in the cytoplasm.72
The explosive rise of lncRNAs in human and mouse genomes has profound implications for future research in vascular biology. First, unlike microRNAs, which number ≈1000 and almost universally function through a predictable and well-defined process, lncRNAs number in the tens of thousands and their functions and mechanisms of action will be, arguably, as diverse as those for protein-coding genes. This will necessitate a global effort to define all lncRNAs in the vasculature (especially nonpolyadenylated) under normal and stress-induced conditions and delineate their mode of regulation and function. Second, lncRNAs such as SENCR are poorly conserved and lack easily defined sequences that would imply a clear function in blood vessels. The apparent lack of orthologous mouse lncRNA genes such as SENCR constrains the extent to which experimental analyses can be done in a rigorous and controlled manner to gain functional insights. However, mouse-specific lncRNAs may have limited translational relevance to the study of human development and disease. Structural similarity between lncRNAs having little sequence homology may, nevertheless, exhibit comparable functions across species.73,74 In this context, there is a pressing need to gain insight into the structure of lncRNAs to develop lncRNA codes that would facilitate functional classification across species. As a first approximation of the structure of SENCR, we used mFold (http://mfold.rna.albany.edu) and RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) and found it to exhibit a stable RNA structure (Figure VII in the online-only Data Supplement) with minimum free energies of −486 and −470 kcal/mol, respectively. Another implication of widespread lncRNA genes will be the need for extreme caution and strategic design in the creation of genetically altered mice, especially when targeting the 5′ end of a gene where inadvertent disruption of other sequences such as lncRNAs is likely to occur. The emergence of precision-guided genome editing (eg, CRISPR/Cas9) will be of great value in this context.75 Finally, most genetic variation occurs in non–protein-coding sequence space,76 which is interposed with transcription factor binding sites such as CArG boxes11 and lncRNAs such as ANRIL.35 Historically, there has been a notable lack of understanding as to how noncoding sequence variations associated with disease perturb function in a cell. Now, with increasing efforts devoted to understanding noncoding sequences, there will be an effort to model human single nucleotide polymorphisms associated with vascular disease through, for example, CRISPR/Cas9-mediated point mutations in the mouse genome. In this context, it will be important to know whether the sequence variants in exons 2 and 3 of SENCR confer differential expression, localization, or function in a disease setting. Altered lncRNA expression of TIE1-AS146 and ANRIL48 has already been noted in human vascular disease.
In summary, we have developed a rigorous experimental pipeline for the discovery and study of lncRNAs in human vascular cells (Figure I in the online-only Data Supplement). This approach uncovered many previously unrecognized lncRNAs, including the human-specific, vascular cell–selective SENCR, which we show is an alternatively spliced and weakly expressed cytoplasmic 5′ overlapping antisense lncRNA. Loss-of-function studies support the concept of SENCR acting as a fine-tuner of the vascular SMC phenotype. Of note, SENCR is one of the first 5′ overlapping antisense lncRNAs (as defined here in Table VI in the online-only Data Supplement) to be studied in detail. Future work should aim to elucidate the regulatory control and function of SENCR in models of human vascular SMC and EC development as well as disease-associated processes.
We gratefully acknowledge the expertise of the University of Rochester Genomics Research Center for performing the RNA-seq experiments and analyzing differences in protein-coding genes as well as generating the volcano plot.
Sources of Funding
This work was supported by grants from the National Institutes of Health (HL62572 and HL091168 to J.M. Miano; MH099452 to D. Zheng and partially by HL111770; and 5P01-CA013106 to D.L. Spector) and the American Heart Association (10SDG3670036 to X. Long and 12POST11950002 to R.D. Bell). J.H. Bergmann was funded by a DAAD postdoctoral fellowship.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.303240/-/DC1.
- Nonstandard Abbreviations and Acronyms
- endothelial cell
- ENCyclopedia Of DNA Elements
- Friend leukemia virus integration 1
- human coronary artery smooth muscle cell(s)
- human umbilical vein endothelial cell(s)
- lincRNA
- long intervening noncoding RNA
- lncRNA
- long noncoding RNA
- myocardin
- reverse transcription polymerase chain reaction
- smooth muscle and endothelial cell enriched migration/differentiation-associated long noncoding RNA
- smooth muscle cell
- Received January 11, 2014.
- Accepted February 12, 2014.
- © 2014 American Heart Association, Inc.
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- et al
For the first time, RNA-seq has been performed in human coronary artery smooth muscle cell for the discovery of long noncoding RNA genes. We report the gene structure, expression, splicing, and spatial localization of a new vascular cell–selective long noncoding RNA we call SENCR. Although SENCR has no apparent cis effect on gene expression, there is a compromise in the smooth muscle cell contractile gene program on its knockdown with elevations in many promigratory genes. Accordingly, these cells exhibit a hyper-motile phenotype, which can be reversed by knocking down 2 promigratory genes that are induced with SENCR knockdown. These results report the first novel long noncoding RNA gene selectively expressed in human vascular cells and provide a framework for further study of long noncoding RNA genes during vascular cell development and in disease processes.
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- Identification and Initial Functional Characterization of a Human Vascular Cell–Enriched Long Noncoding RNASignificanceRobert D. Bell, Xiaochun Long, Mingyan Lin, Jan H. Bergmann, Vivek Nanda, Sarah L. Cowan, Qian Zhou, Yu Han, David L. Spector, Deyou Zheng and Joseph M. MianoArteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:1249-1259, originally published February 27, 2014https://doi.org/10.1161/ATVBAHA.114.303240
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- Identification and Initial Functional Characterization of a Human Vascular Cell–Enriched Long Noncoding RNASignificanceRobert D. Bell, Xiaochun Long, Mingyan Lin, Jan H. Bergmann, Vivek Nanda, Sarah L. Cowan, Qian Zhou, Yu Han, David L. Spector, Deyou Zheng and Joseph M. MianoArteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:1249-1259, originally published February 27, 2014https://doi.org/10.1161/ATVBAHA.114.303240


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Saturn, the planet of discipline, limitation, structure and responsibility, turns retrograde every year for about five months. In 2013, the retrograde period will begin on February 18 and will last until July 8. During this time, everything that supports the foundation of your world will be up for review. Sound ominous? Well, don't panic. Saturn is retrograde for a long time every single year. Unlike the personal planets (Mercury, Venus and Mars) the retrograde of outer planets usually don't have a dramatic effect on our day to day life. Still, the symbolism and significance behind Saturn Retrograde is useful to understand since it will be a time to reevaluate personal boundaries, obligations and goals.
Typically, Saturn Retrograde is not the ideal time to begin new commitments, particularly ones that will affect your status and reputation. It is, however, the perfect time to reassess your existing obligations. Are you rising to the occasion or have you taken on more than you can handle? Since Saturn Retrograde will slow the pace down, you'll begin to notice whether or not you're making the grade on current projects. You'll have an opportunity to improve your strategy and organization on a project that's meaningful to you. Alternatively, you may take stock in a venture you're involved in and realize that it's no longer something you want to remain committed to ... and that's ok! But whatever you're wondering about, a Life Purpose Astrology report that focuses on Saturn's location in your chart may be just the thing to clue you in. As long as you're honest about whether or not this endeavor has become a source of pain or obstruction and then act accordingly, you're using Saturn Retrograde exactly as you should.
Put the business on ice
Since Saturn has a natural correlation to one's professional status, it is imperative that Saturn is strong and direct when starting any new business venture. You will also want a strong Saturn when you begin anything you hope will elevate your reputation and level of authority. If you start a new business, or major enterprise that will affect your status, there's likely to be a series of problems attached to it. Success will not be denied while Saturn is retrograde. However it will be quite frustrating to achieve. Whatever you begin will likely require extensive reorganization or a complete restructuring. The foundation of anything is not as solid when started under Saturn's Retrograde -- especially when it comes to business.
This doesn't mean you can't achieve any measure of success during the five month stretch of Saturn napping. In fact, as long as you stick to working on existing projects, they can become extremely polished and as a result, you'll achieve the recognition you deserve. Still, it'll be a good idea to refrain from launching any brand spanking new business pursuit now.
Calendar dates to circle
During this particular Saturn Retrograde cycle in the sign of Scorpio, there will be an emphasis on revising the structures you have built around your financial and intimate life. Do you have a deep seated fear of success or failure? Now is the time to identify any unhealthy pattern connected to achievement that might sabotage you. There will be a few helpful aspects and some extra challenging ones to cross over the next several months. Here's a rundown on what is most important for you to navigate:
March 6 and March 28 are days when exceptional gains are possible if you review your existing commitments and reorganize accordingly. On these dates, Mercury will connect perfectly to Saturn allowing you to make a decision about a project and stick to it. On March 6 Mercury will be retrograde and it signals a definite pull to a past obligation or project that may now yield fruit. On March 28 you might put the plan from March 6 into motion.
On April 22 Venus in Taurus will oppose Saturn in Scorpio and this will be an opportunity to reconsider financial and romantic commitments. Are you taking more than you give or do you feel you give until it hurts when it comes to a relationship? Now is the time to honestly examine how the answer to this question makes you feel.
On April 28 the Sun in Taurus will oppose Saturn followed on April 30 by Mars opposing Saturn. With these aspects you may need to relocate your inner authority. If you feel as if a higher up is stepping on your toes or squelching your personal development, it's time to work through it. That might mean cutting ties from someone who is only holding you back.
Mercury in Taurus will oppose Saturn on May 5 and it'll be time to defend a decision to an authority figure in your life. Although you may feel insecure, stand your ground and trust that you're fully capable of making the right choice. Make a commitment to only surround yourself with people who value your mind.
On June 7 Venus in Cancer will trine Saturn and what you've decided to see through during this Saturn retrograde phase will now begin to show its rewards. A romantic relationship might become more stable -- especially if you've worked on building a solid future together.
When the Sun in Cancer and Saturn link up in a friendly way on June 26, your inner and outer authority will blend perfectly. What you see is what you get and everyone around you will respect what they see.
Finally, on July 1 Venus in Leo will square Saturn. A commitment or financial decision might reach a breaking point. A relationship you hold on to merely out of obligation might leave such a bad taste in your mouth. If the love and fun are gone, you know it's time to move on.
Curious to see how Saturn will affect your life? Try our Life Purpose Astrology report and find out today »

subset: commoncrawl


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Christmas In July
Shimmering Patriotic Christmas Ornament
This shimmering 3 inch Christmas ornament features a tapestry of red, white, and blue stripes, embellished with silver glitter, and tiny white snowflakes. This stunning ornament will absolutely sparkle on the Christmas tree, and would make a fantastic gift as well!
This ornament will arrive gift boxed, with a dated and signed name card.


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I wrote about Philip Lynott when it was his birthday. Today is the 24th anniversary of his death and every year on this date, an Irish rock concert is put on in his memory called "Vibe For Philo."
Long live Philip Lynott!
PS Someone just posted this video of the song "Honesty Is No Excuse" from Thin Lizzy's first and eponymous album in 1971. It's an amazing song with lyrics that seem far beyond the mind of a young man, but Phil couldn't have been more than 22 years when he wrote it.
This is "Old Time" from one of Phil's solo records. The Corrs remade it a few years ago and had a hit with it.
Phil gets some help from Dire Straits' Mark Knopfler with his tribute to Elvis Presley in "King's Call." Incidentally, Elvis would have been 75 on 8 January 2010.
This video cuts off abruptly but it's special because it's his last interview from a few weeks before his death. He does not look good. (Hindsight is 20/20, I suppose. His beloved mother Philomena did not find out about his years-long addiction to heroin until he was dying in the hospital. I remember from Thin Lizzy's episode of Behind the Music on VH1, Philomena said that he'd hidden his drug use by injecting into his feet.)
This video also cuts off but it's a fun one. It was for an Irish show and Philomena is there. Phil is 30 here (he gives his age in the interview; he died at the age of 36). My favorite part is when his mother confirms that her son bought her a house and Phil tells the interviewer that he was trying to keep that quiet so the "relations" won't be after him.
And check out the songs that Phil wrote about some of his great loves: his hometown Dublin, his mom Philomena, and his daughters Sarah (the fella at the end is Thin Lizzy guitarist Scott Gorham) and Cathleen (you can hear a snippet of the song here if you scroll down a bit and here are the lyrics).
"Merciful Jesus, what have I done to ya?" Philomena recalled her son spoke these words to her as he lay dying in hospital (again, Behind the Music). This Thin Lizzy song from the album Black Rose says it all, I'm afraid.
We're still in love with you, Phil.
04 January 2010
Goodbye Philo
Labels: Philip Lynott, Thin Lizzy
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A group of high-living young criminals bankroll their extravagant lifestyle with a series of painstakingly planned bank robberies while a dedicated police officer makes it his personal mission to stop them in Columbia Pictures' taut, edge-of your seat crime thriller "Takers." After years of meticulously planned heists, the crew is convinced by one of their own to risk it all for one last big score, but pulling off the job of a lifetime with a dogged detective and a vicious rival gang on their tail is a tall order, even for these seasoned pros.
A recent No. 1 box-office hit in the U.S., "Takers" stars Matt Dillon, Paul Walker, Idris Elba, Jay Hernandez, Michael Ealy, Tip "T.I." Harris, with Chris Brown and Hayden Christensen. In the film, longtime friends Gordon Betts (Elba), John Rahway (Walker), A.J. (Christensen), and the Attica brothers, Jake (Ealy) and Jesse (Brown) finance an extravagant lifestyle filled with hot cars, hotter women and unlimited cash by staging high stakes bank robberies. They avoid being caught by planning to the tiniest detail, leaving no clues and pulling off one—and only one—job per year. Their latest successful caper, a $2 million heist, attracts the attention of LAPD Detective Jack Welles (Dillon), an old-school cop who has sacrificed his marriage, his child and any semblance of a personal life for the job. Despite a lack of support from the department, Welles is determined to track down the elusive gang before they strike again. As the crew celebrates their latest daring robbery in a chic downtown cocktail lounge, Ghost (T.I.), a former comrade-in-arms recently released from prison, drops in with an irresistible proposal. The only member of the crew arrested for an earlier job, Ghost says he has a plan that will net each of them enough money to hang up their ski masks forever: the robbery of an armored car carrying over $12 million. But it has to happen in five days or the opportunity will evaporate.
Although doing another job right away goes against the crew's strict rules of conduct, the lure of all that money proves too great and they decide to risk a daring daylight hold-up on a crowded downtown Los Angeles street. With just a few days to prepare, the crew sets in motion an intricate scheme that unknowingly puts them on a collision course with a group of ruthless Russian mobsters. In the meantime, Welles is unraveling a complex web of evidence that takes him from a small time arms dealer to the Russians and finally… crew leader Gordon Betts. With the clock ticking down, ancient rivalries, unexpected double-crosses, unknown enemies and just plain bad luck complicate the plan, resulting in a deadly showdown no one saw coming.
Opening soon across the Philippines, "Takers" is distributed by Columbia Pictures, local office of Sony Pictures Releasing International. Visit http://www.columbiapictures.
com.ph for trailers, exclusive content and free downloads. Like them at www.Facebook.com/ ColumbiaPicturesPH and join their fan contests.


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Archive for the 'Politics' Category
Need Protection?
If you need more protection or a protective tip here it is…..don't ride on roads. When riding a bike you are balancing on two thin wheels. They slip on ice, paint, leaves. The get stuck in cracks, hit potholes. Other than your helmet, you have no protection at all. Why deny it? I've hurt myself walking, running, brushing my teeth, cleaning my ears, shaving, putting on my glasses, tying my shoes and playing basketball. I have yet to be injured in a bike to car accident. You want to know why? I will tell you. I don't put my bikes skinny little tires anywhere near an automobile. That is silly.
Wikileaks Cables
The wikileak cables are not going to be found on this site. In fact, I am sick of hearing about them. Back to biking…..No one rides as much as they drive/are driven. It is essentially impossible to compare two activities that share nothing in common, for example scuba diving and competitive running, without using a comparable metric. The miles driven vs. ridden are similar only in that they are distance miles, but they are not equivalent in terms of the danger level. Time of participation is a much better metric. While someone may dive 'only' 100 feet down, and spend about 45 minutes doing it, a runner can go about 4 miles in the same time (I don't run much, but that seems about right). Yet, comparing distance travelled is like comparing a chainsaw to an air conditioner.
Sum of All Sense
To sum it up, most cyclists go 15-20 miles an hour on average (frequently slower, rarely faster). Cars go 30-70 miles an hour on average, with most of the '100 million' miles driven on highways, where it's around 70 mph. Then the ratio for hours of equivalent participation is about 70/15 (taking the more realistic speeds) = 4.7. So, the more realistic comparison of injury rates per hours of participation is 390/4.7 = 83 injuries per approximately 1,430,000 hours of use. That is much more similar to the rates of injury while driving, and is a more fair metric of comparing activities. Stay off the roads people!
Safely Overtake an Angry Cyclist
If you drive on a road occupied by people on bicycles it is likely you have attacked or wanted to attack the cyclist. Confrontations between drivers and cyclist may start out as relaxing, but can quickly take a turn for the worse. Arm yourself with these tips and you can safely overtake a cyclist when the need arises.
- As the angry cyclist approaches you, be ready for action. You must always assume they are approaching to do you harm. Often the angry cyclist may approach you with the bicycle raised above their heads in a striking position.
- Make sudden, fast, wild swinging movements. Make a lot of noise. If you have a can with rocks in it shake it loudly while shouting nonsense in the face of the cyclist.
- Move swiftly from side to side. Don't stand in one spot or you will get struck.
- Move into the strike and grab the bicycle with your right hand.
- Spin the aggressor around where you are directly behind them.
- Strike them hard in the center of the back with your fist.
- As the cyclist goes down, deliver 3 strong strikes with the bicycle to the knee or back of the legs.
- Throw the bicycle far out of reach of the injured cyclist.
- Get in your car and drive to over the bicycle rendering it useless, and get ready for your next confrontation.
Unicycles on Roads
I do NOT condone unicycling, bicycling or tricycling by adults on the road. Kids either for that matter. Unicycling – even by an expert – on the road is particularly dangerous. What stupidity! If it's dangerous with adults, then it's dangerous with children too. You don't have to look further than the recent lawsuit involving a 25-year old unicyclist who ran into an elderly lady, causing the lady to fall and break her hip. Unicycles move at even slower speeds than bicycles and adult tricycles. Unicycles are not safe for road travel. Yesterday while driving to the University, a man in a suit riding a unicycle pulled up beside me. He was commuting to work on a unicycle. How stupid! Why in the world would he want to ride that thing on the road? Take it to the park and enjoy your ride, but stay off my roads.
I Nearly hit a Bike this Morning
On a busy 8 lane street I had a bicyclist pull up in between my truck and another car at the stoplight at one of the busiest intersections in the city like he was on a motorcycle or something. Ridiculous. Just an accident waiting to happen. He perfectly could've use the available bike lane and cross walk. But no, he uses a major throughfare as his preferred route of transportation. And guess who's fault it is when they get hit? Oh If I would have had the opportunity I might have taken a quick lane change. Get off my roads and stick to the shoulder. Your skinny little wheels belong at the park not on the road.
Why they Ride in Europe
European countries recognize the enormous subisidies societies pay for fuel and infrastructure supporting motorized traffic and levy excise taxes accordingly. The lion's share of the cost of fuel in those countries is tax. The cost of licensing and annual renewals is based on engine displacement and makes it prohibitively expensive for the average driver to operate a large, inefficient vehicle. For example, if a person in Norway, one of Europe's top oil producers, wants to buy a new Camaro with a five liter V-8, by the time the car is registered and insured he will have spent more than $200,000. It is not the same people. They ride because they have to. They can't afford the damn cars. We can. Stick to the shoulder and stay off my roads.


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Coachella festival always raises the bar in festival attire for the rest of the summers festivals. This years no different Vanessa Hudgens, Selena Gomez and Kylie Jenner all looked amazing but my favorite look of the weekend is Kendall Jenner. I know the nose ring is totally OTT and its going to be an either love or hate look, but I'm totally obsessed with it and she definitely pulls it off!
What where your favorite trends from Coachella, and will you be copying any this year?


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Tin House the magazine to be publishing an excerpt from a book that Tin House Books is publishing this month? That is the case with "Bright Before Us" by Katie Arnold-Ratliff. Not her fault, of course. Does Tin House often do this?
In any event, I didn't notice the disclaimer on the first page that this was an excerpt, so I found the piece disappointing as a story. As a taste of a novel, though, it's fine.
The narrator, Mr. Mason, has been on a field trip with his very young students, and apparently the class came across a body on the beach. So now that they're all back in school, the principal and parents are rightly concerned--especially because Mason was seen sobbing. So many of the parents are attending class this first day back. Meanwhile, at home, Mason's wife is getting a sonogram, which is a big deal because she has a history of miscarriages. Back at school, the police call Mason with some questions about the "timeline" of the incident at the beach, and two juicy tidbits are revealed: Mason saw the woman jump off the bridge and the woman was his ex-wife. Needless to say the police have some more questions for Mason . . .
So. Not a short story, in my opinion (and it's not claiming to be one, either, I hasten to add). An intriguing sample of a novel, though.
Like a dog that returns to his vomit is a fool that repeats his folly. Proverbs 26:11
Saturday, May 14, 2011
Short Story Month 2011: Tin House -- Katie Arnold-Ratliff (#ssm2011)
2 comments:
- Short Story Slore said...
I just sent in my subscription for Tin House! I am always a little disappointed when I think I'm reading a short story and it ends up being a novel exerpt. It's a great way to find out if you want to read the novel, but I pick up short story magazines to read a short story. Not a stand-alone chapter of a novel.
- Sunday, May 15, 2011 11:46:00 AM
- Clifford Garstang said...
I hear you. But at least there was a note to that effect at the bottom of the story's first page. Unlink, say, The New Yorker, which hedges their bets by calling everything "fiction" so that they don't have to call it a "story" or an "excerpt" or something else.
- Sunday, May 15, 2011 12:51:00 PM
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Ultimate Gaming Chair
It's called the X-Dream GYROXUS, works with a PS3, and sells for about $637:
However the X-Dream GYROXUS gaming chair lets you move along with the action – turn, dive, lift-off, and climb as you make your way through your favorite game – you will really feel it all. Talk about being thrown from your seat! The X-Dream GYROXUS has a contoured gamer's seat and comes with a custom PS3 controller. Or if you prefer the Xbox 360, there is a GYROXUS gaming chair that supports the Xbox too.
Link via DVICE | Photo: Boys' Stuff
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I have both an Xbox 360 and a PS3, so that means I need 2 of these chairs? Ouch! Is there one for the Wii too? Is there one for PC games too?
Is there any padding in it?
Is there any padding in it?
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text: 21-year-old Winnipeg man has been sentenced to 12 years in prison for two gang-related shootings in the city's West End, including one that unintentionally injured a 10-year-old girl. Paramedics take the girl, who was 10 years old at the time, to hospital following the May 26, 2010, shooting on Victor Street. (CBC) The man pleaded guilty on Monday to two counts of discharging a firearm in connection with the May 2010 incidents, including the May 26 shooting of a house on Victor Street. Court heard that the man, who was then a 19-year-old member of the Indian Posse street gang, fired three shots at the home as an act of retaliation for a drive-by shooting that killed a fellow gang member the day before. Inside the house was the girl, who was hit in the knee by a bullet that went through the front window. Her sister, who was eight at the time, was superficially injured by flying glass and debris. Neighbours and the man's own younger brother told police they saw him with a rifle at the time of the incident. The man was arrested two days later at The Forks, with a machete hidden in his pants. A teenage co-accused pleaded guilty in September 2010 to aggravated assault and assault with a weapon in connection to the attack.


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We've moved to www.cupcakesandcutlery.com - you'll be redirected to this post in a moment.
It pays to have friends who like to entertain. This weekend, the other two cupcakes, A & T, threw me my baby sprinkle (baby shower for 2nd or more child). It was AMAZING!!! I hope my words and photos can do it justice. I know it must have taken forever to put together and all that hard work truly made me feel extra special. So let's jump right in, shall we? The party was held at T's new house which we affectionately call The Resort and Spa based on it's size and ambiance. Without any decorations at all the location would have been magical, but with a canopy and some paper lanterns, the courtyard was turned in to an amazing outdoor dining room. Everything coordinated with the original invitation design (which I don't have a photo of! sorry!) from the linens, to the flowers and napkin rings. To welcome my guests, T pulled out her chocolate fountain (how lucky am I to know someone who has this!!) and laid out an impressive spread of cookies and fruits. So good!!! It was hard to part with the chocolate, but finally it was time to move outside for lunch. You may remember us mentioning T's friend Eve the caterer (here and here). She did all the food and as always, it was decadent and perfect. She made a tossed green salad with fruit and nuts in a light, yet flavorful vinaigrette. There was steamed asparagus washed in butter, and caramelized Italian sausage with a squeeze of lemon. But the real star of the show...the mac and cheese!!! It is the BEST I have ever tasted. I believe there was a bit of blue cheese throughout the cream sauce and then it was topped with toasty breadcrumbs and bacon. Heaven in a ramekin. Eve never disappoints. When we had finished eating, we each took a card from a bowl on the table and answered the question that it posed. They were a great way to get everyone to interact with each other and wasn't really a game. They were mainly things that had to do with being a parent, since everyone in attendance was a mom (except for my 6 year old niece:) Like what was your most embarrassing moment as a mom or guessing when my new baby would be born. After we had digested enough to get up from the table, we went inside to open gifts. Baby #2 got a lot of nice gifts and we just have a few things left to pick up before his arrival. But let's move on to the next part - dessert. There was more chocolate fountain to be had but T also pulled out some pastries made by Eve. She had made individual strawberry shortcakes, some sort of caramel apple gallete type dessert on puffed pastry and THE BEST LEMON BARS I HAVE EVER HAD IN MY ENTIRE LIFE. And trust me, there have been a few lemon bars in my days. With everyone pleasantly stuffed and the party slowly winding down, T made sure that I took a Polaroid with each guest to put in a small guest book she had put together. I always try to make sure that I get a photo of each guest at a party but when it is your own party it is that much harder to tote around your camera. I was grateful that she had thought of this and figured out a nice way to take care of it. It was also a great way to make sure that I got a moment with each guest which is also sometimes hard to do. And last but not least, as guests were leaving, they were sent off with a Sprinkles cupcake!!!! Oh how I had been craving a good cupcake!!! What a sweet ending to an amazing sprinkle. Thank you guys so much for everything you did. It's not about how much the party cost, or even how the overall party turned out. It means so much to me to know that you took time out of your busy lives to put this all together to make me feel like a queen for a day. I have a HUGE act to follow on T's shower in a few weeks. I hope I can deliver. A and T if I have left anything out that you want to ad, please feel free!!!!


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I've got a couple of days break at the end of reading week so I've headed up north to do a bit more of the coast to coast walk that Eleanor and I started last October. Today was a drive up to Liverpool, where she's been scouring archives maritime archives for her research, and a chance of an afternoon explore.
I don't really know the city very well at all, but it seems to me quite unique. The sloping rope walks down from the University and Cathedrals to the Mersey offering a faded memento of the mighty enterprises on which the docks were built - coffee, sugar, slaves. The sound of seagulls, the ever present mystery of the Liver bird gracing the tall buildings, motif ed on the street signs and painted into the stained glass of Victorian windows. The absurd taken for real, the ludic for lyric and everywhere a sharp sense of being on the edge of things. One moment laughter, the next tears. Does anywhere wear its sense of good humoured resilience more proudly than Liverpool?
We headed off to the weird and wonderful subterranean Williamson tunnels in Edge Hill and were given the last guided tour of the day by John, an eccentric mountain climber and retired geography teacher who's been clearing out debris and rubbish from the folly for the last twenty five years. The tunnels stretch for miles and only a tiny section has up to now been reclaimed.
Nobody's quite sure why they were initially built. The most persuasive theory is that Joseph Williamson who made his money in tobacco, believed in the dignity of labour and philantrophically set up the project to provide employment for soldiers returning from the Napoleonic wars. Another theory is that he was part of a fanatical religious set and was preparing a vast underground city for the people of Liverpool to retreat into come Armageddon. Whatever the reason when he died in 1840 work immediately stopped.
What will become of them? Hard to tell. John leads a tiny band of volunteers who fund raise to keep the clearance going through the tours, an occasional jumble sale and the running of a small kitchen selling waggon wheels and cups of tea to the police station on the other side of the street. Whatever Williamson's reasons for starting to dig into the soft sandstone I'm sure he'd be proud of the persistent determination of the local enthusiasts who are keeping alive his mysterious legacy. They've clearly inherited his spirit of exploration.


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This blog is my 101 things to do in 1001 days project. It was started and never finished back in March but my goal is to restart it and actually complete the list this time around.
Monday, May 16, 2016
101 in 1001 an Update
So it has been a while since I updated this blog and I am very sorry about that. So here is what is going to happen, I am going to do a quick update of things on this list.
So one thing that needs to be issued my failures:
6. Work out 4 times a week - maybe on this one I should have specified an amount of time like I did with the 10,000 steps one but I did not. So overall I think this one is a failure since most of February I was sick and then I never really got back into my working out.
34. Blog at least once a week during this 1001 days project - Well since you have not seen an update since week 2, this one is going to be incomplete as well. I am sorry about failing on this one but a part of me knew this was going to be hard.
98. Keep up with the items on this list weekly...well I think that this falls into the same failure category as #34.
Well lets see if I have completed anything on this list:
20. Go back to school - Well not that it was a hard one to get back into. But as of now I am registered to go back to school in the fall (I was going to take a summer class but it was already filled).
Posted by Samantha Miks at 8:00 AM No comments:
Labels: 101 in 1001
Wednesday, January 20, 2016
101 in 1001: Week 2 in Review
With another week done on my 101 things in 1001 days it is time for another status update. This week has been a little bit easier and harder at the same time but lets see what we are on our way to completing on our list now.
1. Lose 100 pounds - I know that this one is a big one on my list of things but I have made it to my first milestone of my 100 pounds lost and took off my first 20 pounds.
Update on my weight loss story
A photo posted by Samantha Jo (@amethystprincss) on
4. Give up Sodas for 1 Month - Yep, I have now completed 20 days without a soda. Though the first few days of this goal was pretty hard this last week was pretty easy. I just have to survive the next few days to make the 31 day goal.
6. Workout 4 times a week - This one continued really well this last week, I was able to find time to get in a workout everyday even though 1 of them was only a quick 20 minute walk before having dinner and game night with friends. But overall I did good and got in 6, 30 minute or longer walk.
37. Complete the Zombies Run 5K - Well I am happy to say that I am on my way to completion on this one with week 2 being complete.
Week 2 of #zombiesrun5k complete. Glad for a day or two before I start week 3.
A photo posted by Samantha Jo (@amethystprincss) on
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Wednesday, January 13, 2016
101 in 1001: Week 1 in Review
This first week of my New Year, New Me has been amazing. Though I have not completed anything on the list I have made progress to a few of them.
4. Give up Soda's for 1 month - Though I started this one a little bit before the start of my challenge to myself (Jan 1) I am still going strong. I have replaced the Soda's with tea in the morning which has been great. Check out some of my good morning tea posts on Instagram
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37. Complete the Zombies Run 5K training - As you can see in the above statement I have started the Zombies Run 5K training and have already started week 2 of the 8 week training.
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Though I have not really started everything on this list in full, I have gotten my feet in the waters and looking into making sure I get everything done. I can not wait to get to some of the other items on the list and look forward to sharing the journey with all of you.
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Monday, January 11, 2016
Thoughts during Zombies, Run 5K training
So Zombies, Run 5K is more of an introduction into the Zombies, Run Universe. The first mission I have dubbed Zombies 5K 0.0 is the exact same first mission as you see in the main Zombies Run program. This is your arrival into Abel Township.
Now that you have done your introduction as Runner 5 it is time to get into shape and ready for the Zombies. In this story the Doctor and Sam Yao are teaching you to be a faster runner which is something that the town needs as it seems all their runners have been victims of smoke inhalation.
The first week you are going pretty easy getting into the work out with:
10 min walk
(repeating 10 times)
1 min walk
15 seconds slow run
10 min free form run (I was not sure what this was, but it is a run/walk at your own pace)
Now let me admit, before really starting this program I have not really been a walker/runner. I would rather read a book or just about anything else than do this, but with my goal for the year being to make healthier choices I need to get up and move some more. Why not make that movement be a story mode workout. But in order to get the the story I need to get some stamina on workout going. Like every person who starts a new workout you have some thoughts that go through your head when you first start out and here are some of the thoughts that went through my head as I did my first Zombies, Run 5K workout.
- I can do this
- 5 minutes in and I am still going strong
- Ok, 10 minute walk done my legs burn, but I can do this.
- First 15 second run wasn't so bad.
- 4 of my 15 second runs done now I hurt and I really want to quit.
- OMG, I love this song, I can go a little longer.
- Only 2 more 15 second runs to go, yep I can do this.
- OK, last 15 second run, lets make it count.
- Last 2 minute Walk/Run and I can see my car, lets go.
- FINISHED, see I knew I could do this.
Though there was a lot of questionable moments on surviving this workout you will see that I did in fact complete the workout and live to tell the tale. I would also love to tell you that I did make it through the other 2 workouts for the week. There may have been a little mud a few times but I got the workouts in and was very proud of my 3 completed workouts last week. I can not wait to get on to week 2's workouts this week.
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Wednesday, January 6, 2016
New year....new me
So I have started this many of times: my goal to lose weight and be a better me. So hopefully 2016 is the year of success.
When I was in high school, I was able to have some success in weight loss with the Atkins diet so I am attempting it all over again. So here is to a new year with healthier choices from eating salad at work instead of a burger and fries and even walking after work at the park instead of going home and binge watching shows on Netflix.
With almost 200 pounds to lose to reach a healthy weight for me, I really do have a long ways to go on this journey. I am really excited to get started on it. So excited that during my day off yesterday I went and got somethings to make some healthy grab and go lunches to take to work.
So with a few days into 2016, I am starting my journey of hard work and hopefully great payoffs.
This was a picture I took of myself just a few days ago, so lets see how well I can take some of this weight off and get to a healthier, happier me.
Posted by Samantha Miks at 2:00 PM No comments:
Labels: 101in1001, diet, fitness, weight loss
101 Things in 1001 Days
Start date - TODAY January 6, 2016
Goal Date - October 3, 2018
But without further ado my list of 101 things
1. loose 100 lbs
2. Reach my Goal Weight (145)
3. give up fast food for 1 month
4. Give up sodas for one month
5. Get 10,000 Steps on my fit bit 4 times a week for 1 month
6. Work out 4 Times a week (Failed)
7. Run in a Disney Princess Run
8. Make Christmas cards like my mom did a few years ago and send them out to friends and family with pictures and updates
9. Send a secret to postsecrets
10. Complete a Photo-a-Day project on instagram
11. Learn to Knit
12. Complete at least 1 365 Project or 2
13. Make my own outfit for a Disney Princess Run
14. Make a quilt
15. Learn to crochet
16. Do a Photo-Scavenger Hunt
17. Read 50 most challenged/banned books
18. Read 100 books in 1 years time
19. Read 1 non-fiction book
21. Become fluent in Spanish
22. finish college with a BA Degree
23. Apply to start my Masters degree
24. Watch 30 movies newly released in theaters
25. Write a Novel/Book
26. Complete NaNoWriMo at least 1 time
27. Participate in VEDA (Vlog Every Day in April)
28. Host a Movie Night (Girls Night with Friends)
29. See something on Broadway
30. See the nutcracker
31. Throw myself a GREAT Birthday Party
32. Go to 1 concert
33. Listen to 1 artist I have never heard of before
34. Blog at least once a week during this 1001 Days (Failed)
35. Play any Video Game all the way through
36. Complete the Zombies, Run
37. Complete the Zombies, Run 5K
38. Go Ice Skating during the winter
39. See 200 Movies never before seen movies
40. Get my Passport
41. Go to Universal Studios
42. Go on an European Cruise
43. Fly 1st Class
44. Visit at least 2 Never before Visited Countries
45. Visit at least 5 states never been to before
46. Swim in the Pacific Ocean
47. Go on a cruise leaving from Galveston
48. Go to a Book Festival
49. Go to Mardi Gras
50. Go to A Book Conference
51. Visit Ben & Jerry's Ice Cream Factory/Blue Bell Ice Cream Factory
52. Go to Stone Mt. and walk to the top.
53. Visit all three Schlitterbahn parks in one summer
54. Visit the Riverwalk in San Antonio
55. Visit Colorado again
56. Go to Comic Con once
57. Go to VidCon
58. Visit Disney Land
59. Go to Disney World again
60. Get my own Domain and Hosting for this site/Book Blog
61. once I move out get a small dog
62. Buy a New Computer (Desktop)
63. Get a MacBook
64. Get a Little Black Dress
65. Go to a Wine Tasting and Find a Favorite Wine
66. The Items on THIS LIST http://familyshare.com/money/10-things-every-girl-needs-in-her-closet
67. Take a #Selfie a day on instagram for 1 Month
68. Make a friend and become penpals
69. Have at least 500 twitter followers
70. Have at least 50 e-mail followers/and 5,000 page views on the blog in one month
71. Have 500 Instagram Followers
72. Meet 10 new people
73. Tell a month of my life in photos
74. Learn to Speak Spanish
75. Send a Message in a Bottle
76. Ask 20 people to suggest a book and read them all
77. Don't say anything negative for 1 day
78. Send a secret to Post Secret
79. Fall in Love
80. Move out of my parents house
81. Make a list of 101 meaningful quotes to me
82. Try 5 drinks I have never tried before
83. Organize my Closet
84. Complete 10 items on the 75 skills every Woman Should Master list
85. Save $1251.25 which is the price of 1 coke a day from the vending machine at work
86. Make a Dress from Scratch
87. Answer the 5000 Question Survey
88. Redecorate bedroom and bathroom
89. Learn to apply makeup correctly and make it look nice.
90. Pay off my Car
91. Get my Tires Replaced
92. Keep my car clean and well cared for (this means learning to take care of my car myself)
93. Learn to Crochet and donate a blanket to a woman's shelter
94. Do one Project 4 Awesome Video
95. Give away old clothes never worn anymore
96. For every Failed ITEM donate $1 to a charity of Followers Choice
97. Find a Charity of MY CHOICE and donate $100
98. Keep up with the information on this list. Not everyday thing but at least weekly if not twice a week. (Failed)
99. Save all change for the duration of 1001 days and cash in for something fun to do.
100. design a t-shirt to sell
101. Write a new list like this one
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Labels: 101 in 1001
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These have been a favourite of mine for a long time and I have imported them several times before from ebay. Billed simply as 'white chocolate flavoured cups with a peanut butter filling' these came in a pack of two which weighed 42.0g.
One thing I always love about Reese's products is the way the nutty smells always make themselves known when you open the packet. As usual they made themselves immediatedly apparent when I slit open the foil wrapper.
The white chocolate I have to admit wasn't the best quality ever but the cream based, sweet milk flavours were nicely forthcoming and led for a smooth flowing dairy taste. What I loved about these white cups so much was the way the outer chocolate and the peanut butter combined to produce one of the most sinfully enjoyable creamy, salty, sweet nutty tastes going. The contrast in the sweet creamy flavours of the chocolate and the nutty inner filling was nothing other than delicious - I savoured every last bite.
Overall these white cups have to go down as my favourite Reese's product I have tried yet. I happen to think that Reese's milk chocolate is pretty poor, so I was pleased that the white chocolate made for such a phenomenal synergy with the inner peanut butter. Granted, this wasn't white chocolate in it's grandest form, but it was still delicious nonetheless. This is one of the best things I have reviewed on chocolate mission thus far.
9.0 out of 10


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I have been asked to share the following event information.
On Thursday October 18th at 6:00pm in the Quad infront of Main Building at the University of Prince Edward Island (Rain location is McMillan Hall in the WA Murphy Student Centre) the UPEI Rainbow Alliance will be holding their 3rd annual Candlelight Vigil Against Homophobia and Bullying. This event is to educate people, and remember those who have taken their lives, or had their lives taken, due to homophobia and other kinds of bullying. There will be guest speakers, musical performances, and the Candlelight ceremony.
This is a great chance to support the Alliance. They are also looking for more youth to talk briefly about any experiences they have of being bullied for any, or no reason.
If you want more information, contact Elyse at email@example.com.


text:
Today's consumers are more knowledgeable, networked, and vocal. For them consumption is not merely an act of buying products and services, but an expression of their creative potential. Consequently, they are demanding a say and a voice in how companies conceive, develop, and deliver value to them.
It is not surprising, therefore, to hear that a large number of companies are transforming how they innovate―not only in terms of developing new products and services, but in how they are created, delivered, and supported to customers.
Open Innovation thinking, where companies collaborate with suppliers, distributors, and customers to co-create unique value, is fast replacing traditional thinking that viewed innovation as a proprietary activity and marketing as a static, one-way broadcast.
However, while there is significant advocacy and buy-in for collaborating with customers, there is little guidance for companies on how to undertake the journey from applause and appreciation to execution. Only reading about others' success stories – Nike, Hallmark, P&G, Mozilla, etc. – is not sufficient for helping a company develop a blueprint for themselves.
Strategy and branding consultant Gaurav Bhalla presents a concrete framework to help companies systematically and effectively design and implement collaborative innovation programs with their customers that can be applied in both business-to-business and business-to-consumer contexts.
The author describes how today's technologies allow companies to create dynamic dialogues with their customers through shared networks, virtual marketing techniques, and blogs to develop deeper relationships that reinforce brand loyalty and ultimately drive growth.
They challenge traditional approaches to market research that measure "customer satisfaction" from a rear-window perspective, and help companies and their customers look forward instead.
"In today's customer-empowered world, collaboration and co-creation competencies are critical to the future growth of a company…Gaurav Bhalla offers a concrete framework and specific examples that managers can use to implement value co-creation programs with their customers. A must-read for companies not wishing to get left behind!"
Vijay Govindarajan, Earl C. Daum 1924 Professor of International Business, Tuck School of Business, Dartmouth College
"Collaboration and co-creation is the sweet spot for rethinking how companies should practice marketing and innovation…Gaurav Bhalla's book is very timely, and offers readers an effective way for building businesses around customers."
Nicolas Mirzayantz, Group President, Fragrances, International Flavors and Fragrances
"Companies can't afford to fake it. Customer-driven innovation has moved from the edge to become a core business practice. Gaurav Bhalla helps you understand what it takes to make this shift, and not a moment too soon."
John Hagel III, Co-Chairman, Deloitte Center for the Edge, and co-author, The Power of Pull
"If running your business seems more difficult lately, reading this book will help you understand why. It will also provide insights into how collaboration and co-creation can improve your company's performance."
Vince Barabba, Author, Meeting of the Minds, and Founder and Chairman, Market Insight Corporation
"Gaurav Bhalla is after big game: how the world's leading institutions are connecting and collaborating with their most important asset – their customers – in novel and important ways…This book is an important one to read for any marketing, market research, or product innovation professional."
Steve Howe, CEO, Passenger
"Based on my research it is clear that user co-creation is a strong driver for transforming the marketing and innovation programs of those companies that dare to listen and respond. Gaurav Bhalla provides a solid framework to guide this process and a rich set of case stories to explain why and how."
Jacob Buur, Professor of Participatory Innovation, Research Director of SPIRE, University of Southern Denmark


text:
...persistent deficits and continually mounting debt would have several negative economic consequences for the United States. Some of those consequences would arise gradually: A growing portion of people's savings would go to purchase government debt rather than toward investments in productive capital goods such as factories and computers; that "crowding out" of investment would lead to lower output and incomes than would otherwise occur. ...a growing level of federal debt would also increase the probability of a sudden fiscal crisis, during which investors would lose confidence in the government's ability to manage its budget, and the government would thereby lose its ability to borrow at affordable rates. ...If the United States encountered a fiscal crisis, the abrupt rise in interest rates would reflect investors' fears that the government would renege on the terms of its existing debt or that it would increase the supply of money to finance its activities or pay creditors and thereby boost inflation.
At some point, even Republicans should be smart enough to figure out that this game is rigged. Then again, the GOP controlled Congress for a dozen years and failed to reform either CBO or its counterpart on the revenue side, the Joint Committee on Taxation (which is infamous for its assumption that tax policy has no impact on overall economic performance).