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Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types
© Guduric-Fuchs et al.; licensee BioMed Central Ltd. 2012
Received: 30 April 2012
Accepted: 16 July 2012
Published: 1 August 2012
MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported.
Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p).
The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.
microRNA Deep sequencing Extracellular vesicles ExosomesBackground
Membrane-bound extracellular vesicles (EVs), are referred to by various terms in the literature, including microvesicles or microparticles. However, they may be divided into two main types formed either by budding of the plasma membrane (shedding vesicles, ectosomes; approximately 100 nm −1 μm in diameter) or by exocytosis of multivesicular bodies (MVB) (exosomes; approximately 50–100 nm) [1–3]. While it is important to recognize that these two main populations of EVs have distinct biogenesis pathways, it remains technically challenging to isolate their respective pure populations due to their overlapping size distributions and most preparations are therefore heterogeneous [3]. EVs are targeted to specific recipient cells by surface receptors which mediate their internalisation and uptake of their content, comprising lipids, proteins, mRNAs and microRNAs (miRNAs) [2, 4]. A significant physiological role for EVs is supported by their implication in cancer progression, angiogenesis, cell reprogramming and differentiation [5–9].
MiRNAs are a class of small RNA molecules that regulate gene expression by binding to specific mRNA targets and triggering their degradation or inhibiting their translation. Primary miRNAs (pri-miRNAs), transcribed either as independent genes or as introns of coding genes, are processed to form precursor hairpin loops (pre-miRNAs) and then mature miRNAs by the RNAse III enzymes Drosha and Dicer respectively. One strand of the mature miRNA is loaded into an RNA induced silencing complex (RISC), which mediates the interaction between the miRNA and its target mRNA molecules. Argonaute (Ago) proteins are part of the RISC and mediate cleavage of target mRNAs [10, 11].
Recently, miRNAs have been found extracellularly, being encapsulated within vesicles (EVs), or associated with Ago2 or NPM1 proteins or high-density lipoproteins (HDL) [12–19]. EV preparations isolated by ultracentrifugation are heterogeneous and include both exosomes and other vesicles. Extracellular miRNAs have emerged as prognostic markers for various diseases, such as cancer and cardiovascular disorders [13, 20–23]. Recent studies have demonstrated that miRNAs can be shuttled between cells in EVs and modulate gene expression [6, 24–26] and control different physiological functions in the recipients [27–30].
It has been shown that miRNA export into exosomes occurs via the ceramide-dependent secretory pathway and independently of the endosomal sorting complex required for transport (ESCRT) [24]. Although the mechanism of release of shedding vesicles is less well known, the role of P4-ATPase and ESCRT has been recognized recently [31]. Several studies have suggested that the miRNA content of EVs does not simply reflect the miRNA repertoire of the cells of origin and that some miRNAs are selectively exported or retained within the cell [25, 27, 32–34]. However, the mechanism whereby specific miRNAs are selectively exported is unknown.
The potential for exploiting extracellular vesicles as vehicles for delivery of therapeutic molecules has been appreciated [35–38]. For example, EVs prepared from a patient's own cells could potentially be enriched with specific siRNAs and/or miRNAs and used as a tool for therapeutic delivery of these regulatory RNA molecules. Alternatively, the therapeutic effect could be achieved by transplanting cells engineered to over-express critical miRNAs, which are delivered to the target cells via EVs[33, 36]. To most effectively harness EVs as therapeutic tools, it will be crucial to understand and control how they are loaded with regulatory RNAs.
As a step towards understanding the mechanism of selective export of miRNAs to EVs we have applied deep sequencing to characterize the global expression pattern of small RNAs in HEK293T cells and the EVs that they release. Although the enrichment of overexpressed miRNA in EVs has been shown by RT-qPCR in HEK293T cells, mesenchymal stem cells, macrophages and immune cells [24, 25, 27, 28, 37], it is not known to what extent overexpressed miRNAs are exported into EVs and how they affect the export of endogenous miRNAs. To address these questions, small RNAs from cells transfected with miR-146a expressing plasmid and their EVs were also subjected to deep sequencing. Ectopic overexpression of miR-146a in HEK293T exosomes was previously shown to exert a functional effect upon recipient cells, including downregulation of target gene expression [24].
Comparison of our data with other publicly available data sets of miRNA expression in cells and their EVs revealed a subset of miRNAs that tend to be preferentially exported in various cell types, suggesting a common function in intercellular communication.
To compare the global miRNA profile of non-transfected and pSM30-miR-146a transfected cells and their EVs, small RNA libraries were prepared and sequenced on an Illumina Genome Analyzer. Raw reads were subjected to adaptor removal and aligned to miRBase (Release 17, http://www.mirbase.org/) [39]. Both raw and processed data has been deposited in Gene Expression Omnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/), with accession number GSE38916. The total numbers of reads >15 bp matching each miRNA were calculated. These values were then normalized to number of reads per million mapped (RPMM) in each library, to enable direct comparison of expression levels. As a measure of the relative levels of expression of individual miRNAs in cells and EVs, the log (base two) of the ratio of the read number in EVs over that in cells was calculated (log2 (EV/cell)). The miRNA expression data for cells and EVs is presented in Additional file 1: Table S1.
miRNAs most preferentially released into EVs
miRNA | Non-transfected cells & EVs | miR-146a - Transfected cells & EVs | ||||
RPMM cells | RPMM EV | Log2 (EV/Cells) | RPMM cells | RPMM EV | Log2 (EV/Cells) | |
mir-451 | 0.4 | 3945.0 | 13.3 | 1.7 | 7982.5 | 12.2 |
mir-150 | 0.4 | 2475.0 | 12.6 | 0.3 | 663.0 | 10.9 |
mir-146a* | 10.4 | 1321.2 | 7.0 | 317863.3* | 440496.3* | 0.5 |
mir-320c | 24.3 | 2347.5 | 6.6 | 57.8 | 1803.4 | 5.0 |
mir-143 | 39.9 | 3232.5 | 6.3 | 8327.3 | 354.4 | 8.5 |
mir-720 | 65.4 | 3157.5 | 5.6 | 3208.9 | 46.2 | 5.5 |
mir-125a-5p | 214.7 | 9360.0 | 5.4 | 439.0 | 2121.6 | 2.3 |
mir-486 | 105.7 | 3900.0 | 5.2 | 72.8 | 7637.8 | 6.7 |
mir-149 | 89.0 | 3142.5 | 5.1 | 85.3 | 928.2 | 3.4 |
mir-2110 | 43.5 | 1380.0 | 5.0 | 175.0 | 928.2 | 2.4 |
mir-320a | 762.1 | 23190.0 | 4.9 | 1250.0 | 11483.2 | 3.2 |
mir-197 | 472.4 | 8347.5 | 4.1 | 724.0 | 6046.6 | 3.1 |
mir-339 | 91.4 | 1515.0 | 4.1 | 178.0 | 610.0 | 1.8 |
mir-99b | 2065.2 | 27420.0 | 3.7 | 4900.0 | 8353.8 | 0.8 |
mir-4286 | 108.5 | 1245.0 | 3.5 | 72.1 | 1617.7 | 4.5 |
mir-92b | 515.1 | 5542.5 | 3.4 | 484.0 | 4800.1 | 3.3 |
mir-222 | 774.1 | 7882.5 | 3.3 | 1200.0 | 10634.5 | 3.1 |
mir-423-5p | 5737.2 | 55762.5 | 3.3 | 5290.0 | 22435.9 | 2.1 |
mir-877 | 467.2 | 4425.0 | 3.2 | 2000.0 | 2121.6 | 0.1 |
mir-191 | 9501.8 | 88717.5 | 3.2 | 15000.0 | 40893.8 | 1.4 |
mir-1292 | 140.0 | 1222.5 | 3.1 | 256.0 | 397.8 | 0.6 |
mir-1246 | 782.8 | 6570.0 | 3.1 | 156.0 | 11456.6 | 6.2 |
miRNAs most preferentially retained within cells
miRNA | Non-transfected cells & EVs | miR-146a - Transfected cells & EVs | ||||
RPMM cells | RPMM EV | Log2 (EV/Cells) | RPMM cells | RPMM EV | Log2 (EV/Cells) | |
mir-218 | 1342.24 | 135.00 | −3.31 | 1460.00 | 132.60 | −3.46 |
mir-15a | 2411.56 | 435.00 | −2.47 | 2120.00 | 371.28 | −2.51 |
mir-140 | 1181.04 | 277.50 | −2.09 | 1130.00 | 185.64 | −2.61 |
mir-148b | 18756.99 | 5047.50 | −1.89 | 17300.00 | 5383.56 | −1.68 |
mir-378 | 397404.00 | 109560.00 | −1.86 | 192000.00 | 117695.76 | −0.71 |
mir-16 | 7225.49 | 2100.00 | −1.78 | 7190.00 | 2625.48 | −1.45 |
mir-20a | 45087.00 | 14460.00 | −1.64 | 52700.00 | 14957.28 | −1.82 |
mir-424 | 1230.92 | 397.50 | −1.63 | 878.00 | 477.36 | −0.88 |
mir-18a | 4535.03 | 1477.50 | −1.62 | 4520.00 | 1405.56 | −1.69 |
let-7f | 16253.27 | 5520.00 | −1.56 | 31964.69 | 11055.00 | −1.53 |
let-7 g | 4999.47 | 1950.00 | −1.36 | 5580.00 | 4826.64 | −0.21 |
mir-130b | 2194.90 | 877.50 | −1.32 | 2180.00 | 769.08 | −1.50 |
mir-15b | 2820.93 | 1147.50 | −1.30 | 2780.00 | 1087.32 | −1.35 |
mir-598 | 1066.13 | 435.00 | −1.29 | 1400.00 | 397.80 | −1.82 |
mir-26b | 1594.80 | 660.00 | −1.27 | 1440.00 | 689.52 | −1.06 |
mir-340 | 4503.91 | 2137.50 | −1.08 | 1870.00 | 2280.72 | 0.29 |
The most highly exported miRNA in EVs in our data, miR-451, was also reported previously to be highly exported from breast cancer cells lines and mesenchymal stem cells [25, 33]. This was intriguing, and it prompted us to speculate that the same miRNAs may be preferentially exported or retained in various cell types. We therefore tested two other cell types, namely a human microvascular endothelial cell line, (HMEC-1) and primary outgrowth endothelial progenitor cells (OECs) with the same panel of primers used to confirm sequencing data in HEK293T cells. For the 12 miRNAs expressed in both endothelial cells and EVs the proportions retained or released followed a similar pattern to that observed in HEK293T cells (Figure 5B). Two miRNAs, miR-1246 and miR-9 were not detected and miR-7 was not detected in endothelial EVs.
MiR-451 is the only reported miRNA that is not processed by Dicer, but instead uses an Ago2-mediated pathway for its maturation [40–42]. It is tempting to speculate that this unique processing pathway might be connected with the high extracellular export of miR-451. In this case one might expect the most exported miRNAs to also be the most affected by loss of Ago2 activity. We therefore ranked miRNAs according to the reduction in their expression in bone marrow of Ago2 ko mice compared to the wild type [43]. Remarkably, in addition to miR-451, several other miRNAs among the top 6 affected by the Ago2 deletion (miR150, miR-486, and miR-144) were also found to be highly exported in several data sets (see column (K) in Figure 6).
MiRNAs are involved in regulation of a wide array of biological processes, from development to immunity. Extracellular and circulating miRNAs have been found both in the non-vesicular fraction and encapsulated in EVs. They have been shown to associate with lipids (HDL) and proteins (Ago2 and NPM1). While the role of EV miRNAs in cell-to-cell communication has now been widely accepted [3] and HDL-mediated miRNA transfer has been shown to regulate recipient cell gene expression [19], there is still no direct evidence that miRNAs associated with proteins act in a paracrine fashion [16–18].
Realisation of the potential importance of miRNAs in cell-to-cell communication has triggered research into the mechanisms of miRNA release and their effects upon recipient cells [4]. Evidence is accumulating that miRNA export into EVs is selective, but it is not known how this export is regulated. Several studies have compared the miRNA content of cells and EVs using microarrays or RT-qPCR. Deep sequencing is an alternative approach for measuring miRNA expression which has the advantage of providing a digital measure that does not require an internal standard for normalisation. This is particularly important when comparing miRNA levels between cells and EVs, where no common internal standards have been identified. Although several previous studies have applied deep sequencing in similar scenarios, they involved fewer reads and the expression of only selected miRNAs was reported [48, 49]. Here we report a global comparison of the miRNA pools in HEK293T cells and their EVs. Our analysis confirmed previous observations that some miRNAs are preferentially exported and found to be enriched in EVs.
Enrichment of a miRNA in EVs after its overexpression in cells has been measured previously by RT-qPCR. However, it is important to reveal how such overexpression compares with and affects expression and export of endogenous miRNAs. We found overexpressed miR-146a to contribute almost a third of all reads in cells and even more in EVs, representing almost half of their cargo. It is very important for the miRNA therapeutic field to understand the consequence of miRNA overexpression. Our data suggest that if a miRNA is overexpressed in a specific cell type the possibility that a significant amount of this miRNA will be exported and potentially affect other cells should be considered. Although we cannot assess the effect of miR-146a overexpression upon the absolute expression of other miRNAs, this effect is likely to be similar in both cells and EVs. However, our data demonstrates that the relative expression levels of endogenous miRNAs (and their EV:cell miRNA ratios) remain very stable and are largely unaffected by the miRNA overexpression. The fact that selective miRNA export is tightly regulated suggests that it plays an important role in cell physiology. This will be an important consideration not only for further studies into the mechanism of miRNA export but also for the development of methods for enrichment of EVs with miRNA for therapies. It is possible that the overexpressed miRNA and endogenous miRNAs use different export routes and therefore do not affect each other. Since our EV preparation does not distinguish between exosomes and shedding vesicles we cannot exclude that one of these is preferentially used to carry overexpressed miRNA. Fractionation of EVs will be necessary to further examine this possibility. Nevertheless, there may be an overlap between the two pathways, as a recent study has suggested that shedding vesicles and exosomes require the same GTPase (RAB-11) and ESCRT proteins for their biogenesis and therefore may be regulated by the same mechanisms, regardless of their distinct cellular localization [31].
Comparison of our data with publicly available expression sets from other cell types revealed that some miRNAs tend to be universally exported. It is possible that this reflects release of the same type of vesicles (eg exosome) by all the cell types analysed. The commonly exported miRNAs possibly fulfill a common function in intercellular communication. MiR-451 was found to be the miRNA with the highest ratio between expression levels in EVs and cells. In relation to its maturation miR-451 is a non-canonical miRNA, processed by the Ago2 protein, in contrast to other miRNAs that commonly require Dicer for this process [40, 41, 43]. Ago2 has been shown previously to be associated with both EV and non-vesicular miRNAs [17, 18, 49]. Comparison of miRNA expression between cells and their EV counterparts from other published data sets reveals miR-451 to be one of the most highly exported miRNAs in multiple cell types. Deletion of Ago2 in bone marrow leads to reduced expression of several other miRNAs in addition to miR-451 [43], notably some of the miRNAs that are most preferentially exported from various different cell types. These data suggest that there may be a connection between Ago2 processing and the selection of miRNAs for export. Recent studies have demonstrated the physical colocalisation of RISC and MVBs and their functional interactions in regulating miRNA loading and RNA silencing [49, 50]. Being a key component of RISC, Ago2 may potentially be involved in directing miRNA towards exosomal secretion. However it has not been investigated whether Ago2 mediated miR-451 processing also occurs within RISC and in colocalisation with MVB membrane, or in another subcellular compartment. Future studies are required to characterize miRNA export into specific EV populations and must also address the challenge of dissecting the function of Ago2 in miRNA processing from its potential role in miRNA export.
Specific endogenous miRNAs, most notably miR-451 are selectively released from HEK293T cells in EVs by a process that may involve Ago2. Conservation amongst the miRNAs exported by different cells suggests that this process may be widely used to regulate similar target cell functions. Enrichment in genes involved in endocytosis amongst the targets of the most selectively exported miRNAs suggests that these functions may include intercellular vesicular transport itself. The demonstration that EV miRNA content can be manipulated by cellular overexpression of a specific miRNA bodes well for future therapeutic use of this system to deliver miRNAs/siRNAs to target cells.
Cell culture
HEK293T cells from the American Type Culture Collection (ATCC) were cultured in DMEM (Life technologies, Paisley, UK) supplemented with 10% fetal calf serum (FCS) and 100 μg/ml Primocin™ (InvivoGen). Human microvascular endothelial cells (HMEC-1) [51] were cultured in MCDB 131 medium (Life technologies) containing 10% FCS, L-glutamine (2 mM), epidermal growth factor (10 ng/ml, Life technologies), hydrocortisone (1 μg/ml, Life technologies), and 100 μg/ml Primocin. As described previously [52], OECs were obtained from the mononuclear cell fraction of umbilical cord blood, obtained under full ethical approval from The Office for Research Ethics Committees Northern Ireland (ORECNI). Cells were resuspended in EGM-2 medium (Lonza) supplemented with 10% FCS and seeded on 24 well culture plates pre-coated with rat tail collagen type 1 (BD Biosciences) at a density of 1×107/ml All cells were cultured under standard conditions at 5% CO2, 37°C, and 95% humidity.
MiRNA cloning
The mature miRNA sequences for miR-146a, were inserted into pSM30 vector according to the published protocol [53]. Briefly, the oligos containing the miR-146a mature sequence and the miR-30 loop sequence were designed as follows: top strand miR-146a oligo: 5'- AGCGGACCCATGGAATTCAGTTCTCATAGTGAAGCCACAGATGTATGAGAACTGAATTCCATGGGTT-3' and the bottom strand miR-146a oligo: 5'-GGCAAACCCATGGAATTCAGTTCTCATACATCTGTGGCTTCACTATGAGAACTGAATTCCATGGGTC-3'). Oligos were synthesized and HPLC purified by Eurogentec (Seraing, Belgium). Five μl of each oligo (100 μM stock solution) were mixed with 8 μl water and 2 μl of 10 x restriction buffer 3 (New England Biolabs), heated at 95°C for 4 minutes and left at room temperature for 15 minutes to anneal. Annealed oligos were diluted 2500 x to get 10 nM for ligation. Annealed oligos were ligated into pSM30 vector using Rapid Ligation Kit (Roche) with 15:1 insert:vector ratio. pSM30 empty vector was digested with BSMBI restriction enzyme and linearized plasmid was gel purified. DH5α competent cells were transformed with the ligation reaction and positive colonies were selected on agar plates supplemented with kanamycin.
HEK293T cells were seeded at 3×104 cells/ml into T75 flasks and transfected after 24 hours with Turbofect in vitro transfection reagent (Fermentas, York, UK) according to the manufacturer's instructions. After 16 hours media were replaced with media containing 10% serum depleted of EVs by centrifugation for 2 hours at 100000 g. EVs were collected from the conditioned media after 48 h hours.
Isolation and characterisation of EVs
EVs were isolated as described previously [25, 54]. Cell debris was removed by centrifugation at 2000 g for 30 minutes and vesicles were isolated by ultracentrifugation at 100000 g for 1 hour.
For Electron microscopy HEK293T EVs pellets were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C overnight. After post-fixation in 1% osmium tetroxide, pellets were dehydrated with increasing alcohol concentrations and embedded in Spurr resin. Ultrathin sections were prepared using an ultramicrotome, placed on copper grids (Agar Scientific Ltd, UK), stained with uranyl acetate and lead citrate, and examined using a JEOL 100 CX transmission electron microscope.
RNA extraction
RNA was extracted using a miRNeasy kit (Qiagen, Crawley, UK) or High pure miRNA isolation kit (Roche, Burgess Hill, UK) according to the manufacturers' protocols. RNA purity and quantity was determined using a Nanodrop spectrophotometer (Thermo Scientific) or Qubit fluorimeter (Life Technologies). RNA integrity was assessed using RNA 2000 and small RNA chips on a Bioanalyzer (Agilent).
Deep sequencing
Small RNA libraries were prepared using a sample prep kit v1.5 kit (Illumina) following the manufacturer's protocol. Cluster generation and sequencing on a Genome Analyzer II was performed at the Trinity Genome Sequencing Laboratory, Dublin (http://www.medicine.tcd.ie/sequencing). Sequencing data were analyzed using Genomics workbench software (CLCbio, Aarhus, Denmark). After removal of adapter sequences reads >15 bp were aligned to miRBase (Release 17) for annotation, allowing no more than 2 mismatches. Reads were normalized to reads per million mapped (RPMM) to enable comparison between different libraries. If a miRNA was found in both EV data sets but was detected in only one data set from cells (due to its low expression value) it was assigned one read in the other cell data set for the purpose of calculating an EV:cells ratio.
Target genes were predicted for miRNAs of interest using the microT-CDS algorithm available at http://www.microrna.gr/microT-CDS with a strict miTG Score Threshold of 0.8 [44]. Lists were integrated using Microsoft Access and pathway analysis of the KEGG database[55] performed using the The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7[56] available at http://david.abcc.ncifcrf.gov/. Predicted target sites supported by CLIP-Seq reads were downloaded from Starbase (http://starbase.sysu.edu.cn/) [47].
Specific miRNAs were amplified using a modified version of the method described by Shi and Chiang [57]. One microgram of RNA was polyadenylated using poly(A) polymerase (PAP; Ambion, Foster City, CA) at 37°C for 1 hour in a 25 μl reaction mixture. RNA was then reverse transcribed with 200 U of SuperScript III reverse transcriptase (Life technologies, Paisley, UK) and 0.5 μg poly (T) adapter. Primers for specific miRNAs were designed from miRNA sequences obtained from miRBase and our sequencing data and synthesized by Eurogentec (Seraing, Liège, Belgium) (Additional file 5: Table S5). The reverse primer was the 3' adapter primer (3' RACE outer primer in the FirstChoice RLM-RACE kit; Ambion).
RT-qPCR was performed using Maxima SYBR Green qPCR mastermix (Fermentas) in 10 μl reactions containing 2 μl of 1:15 cDNA dilution. Reactions were performed on a LightCycler 480 (Roche), with the initial preincubation at 50°C for 2 min and activation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 60 s, with fluorescence acquired after 15 s of the 60°C step. Primer efficiencies were determined using serial dilutions of pooled cDNA and the reactions were optimized to achieve efficiencies of ~2 for all primers used. The RNA from three independent experiments was analyzed and all PCR reactions were performed in triplicates. Gene expression data were normalized to miR-26a. The relative expression was determined as 2−ΔCt, where ΔCt = Ct(miRNA)-Ct(miR-26a).
Analysis of publicly available miRNA expression data
Publicly available data were included in analysis if they had expression data for EVs (exosomes, microvesicles) and corresponding cells. Microarray data were downloaded from GEO database [27, 28, 33] or retrieved from supplementary data [58, 59]. Difference between expression in cells and EVs was determined as the ratio of expression EV:cells for microarrays or as delta Ct for RT-qPCR arrays and miRNAs were ranked accordingly. Using Microsoft Access miRNAs that are present in our data were pulled out from all the other data sets with their own ranks kept. Colours were assigned to miRNAs according to their ranks using Microsoft Excel (conditional formatting).
- CLIP-Seq:
Crosslinking immunoprecipitation sequencing
- EVs:
Extracellular vesicles
Human microvascular endothelial cell line
Multivesicular bodies
- OECs:
Outgrowth endothelial progenitor cells
RNA-Induced Silencing Complex
Reads per million mapped.
This work was supported by grant number BB/H005498/1from the Biotechnology and Biological Sciences Research Council (BBSRC). The authors thank Gerry Mahon and Liza Colhoun for assistance with electron microscopy and Dr. G. Du (University of Texas Health Science Center at Houston, TX, USA) for kindly providing pSM30. The Trinity Genome Sequencing Laboratory is a core facility funded by Science Foundation Ireland and supported by the Trinity Centre for High Performance Computing.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

subset: commoncrawl


text:
Beauty Tip
H2-Oh! Water for Your Skin
Thermal water spray is a summer beauty essential
-Paige Herman Axel
The idea of buying water in a can may seem a bit wasteful at first, but there's more to spritzable H20 than meets the eye. Most importantly, they aren't just tap water, these are mineral-rich thermal waters sourced from deep in the Earth, so they're packed with minerals and other natural good-for-your-skin ingredients. A few of the most popular and easiest to find are from Eau Thermale Avene. Evian, La Roche-Posay and Vichy, and they're relatively inexpensive.
Here are 8 reasons why you should have a can in your bag at all times.
1. The fine mist is refreshing on a hot summer day.
2. They can set your makeup and leave you with a youthful, dewy look.
3. The minerals are soothing to a sunburn.
4. Doctors often recommend them to help skin recover after in-office treatments like microdermbrasion and chemical peels.
5. They help hydrate the skin, especially on airplanes (but make sure you have a small, travel-approved can).
6. If you're in a bind and can't wash your face, you can spray on and wipe off with a cotton ball.
7. The minerals act as antioxidants, protecting your skin from damage.
8. Thermal waters can be used to soothe even the most sensitive skin, and help relieve rosacea symptoms.
Go ahead – take the plunge and try out a can of thermal water for your skin. Your face will thank you for it.


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She's got herpes of the lip, too.
Im not smitten, dude. She wasn't born when she met Mr Alexander. What evidence has been presented at this trial, that shes capable of such violence, to the point of planning it out. The prosecution hasn't even presented any evidence of incidents of rage, born out of jealousy.A 28 year old capable of planning that kind of violence, a monster as you said, would most certainly showed plenty of signs of it in her life.
Dude, she lied about it - twice already.
Besides people who commit 'crimes of passion' - usually don't exhibit signs of past violence, they don't have a 'record'. Premeditation is another story.
The prosecution has plenty of evidence. So much so to the fact that their last-ditch effort was to put her on the stand in the first place. Any defense team with even a chance at a prayer wouldn't allow their defendant to get slaughtered under cross-examination, but they did it anyway.
In a self defense case, the defendant almost always has to take the stand.The only motive would be his Cancun trip with another women.She knew about that for a year.The bottom line to me is, if she planned on killing him,stole a gun to do it, why stay a day and a half at his house, have sex with him, and then kill him with a knife???In the day and a half she was there, she probably had 100 chances to take out the gun and pull the trigger five times to the back of his head.There was a pic she took of him with his back facing her,perfect chance.A planned killing, no way
The point is if she planned this, and based on her lies after his death, she took the riskiest option. What was it,allowing him to get out of the shower and face her to do it.what if he wrestles the gun out of her hand and kills her.If she planned this, and then lied, she didn't want to get caught. Why stay there a day and a half, makes no sense.
I wonder if the attorney is tappin dat ass.
In this photo, she has big breasts.
Looks like a whole truckload of "crazy eye" to me.
They interviewed a couple that was friends with the victim. The wife said that none of the women in their circle of friends trusted her...and that she herself would not leave her husband alone in the same room with her for fear she would put the moves on.
She said words to the effect of "she exuded sexual energy."


text:
Marist (25-21-1) | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 6 | 16 | 1 |
(22) Virginia Tech (33-19) | 2 | 0 | 2 | 0 | 2 | 0 | 0 | 0 | 0 | 1 | 7 | 9 | 0 |
BLACKSBURG, Va. – Tyler Horan, again, led the Hokies offense, but it was Mark Zagunis' second home run of the day that sent No. 22 Virginia Tech off the field with 7-6 victory over visiting Marist in 10 frames at English Field in non-conference action Sunday afternoon.
In all, the Hokies hit five home runs that accounted for all of its runs, and three of them were hit with two outs that produced five RBI. Tech improved to 33-19 with the victory as Marist fell to 25-21-1 with the loss.
Sean Keselica and Zagunis each hit solo home runs in the first inning to open the scoring for Tech, and in the third, Horan hit a two-out, two-run shot that put the Hokies up 4-0.
The Red Foxes plated solo runs in both the fourth and fifth frame to cut their deficit in half only to see Horan go deep again with another two-out, two-run home run. It was his fourth long ball of the weekend and his team-leading 10th of the season, which made it 6-2.
Brad Markey started the game on the mound and went six frames with four strikeouts. He allowed two runs on nine hits and walked one, but was helped by the Hokie defense that turned three double plays behind him.
However, Marist, who had 16 hits in the game, but just one for extra bases, got to the Tech bullpen, combining for seven hits in three innings and scored two runs in both the eighth and ninth to knot the game and send it to extra innings.
After Clark Labitan worked a scoreless 10th – the team's seventh extra-inning game of the year – Zagunis came to the plate with one out and ended the game on a 2-2 pitch that gave him his first career multi-home run game and his eighth long ball of the season.
Labitan earned his first win of the year and is now 1-5, while Jordan Eich dropped to 1-2 with the loss.
Tech, who over the weekend secured a spot in the upcoming ACC Baseball Championship in Durham, N.C., will play its final ACC series of the year when it welcomes Wake Forest to English Field for a three-game set. The series starts on Thursday at 5:30 p.m.
Additional Notes: Tech has now won 11 of its last 13 games. The five home runs hit by the Hokies were the most hit by the team since February 25, 2011 when they hit seven versus Niagara. It was also the last time two players each hit two home runs in a single game – Tim Smalling and Andrew Rash.Zagunis scored a career-high four runs in the game – two on his own home runs and one each on Horan's home runs. Horan's three-game week saw him hit .538 with three doubles and four home runs (1.692 slugging percentage) and drive in nine RBI. He now leads the team with a .332 batting average and .599 slugging percentage. Horan becomes the first Hokie to hit 10 home runs in back-to-back seasons since Brad Bauder did it in 2002 and 2003. This was Tech's 14th walk-off win under Coach Hughes. Zagunis joins Horan, Rash and Austin Wates as the only men under Hughes to hit a walk-off home run. The seven extra-inning games is a school record as is the five wins, as they improved to 5-2 with the victory.
For updates on Virginia Tech baseball, follow the Hokies on Twitter (@VT_Baseball).


text:
10 Grassy Pond Road Harwich Port, MA 02646
10 Grassy Pond Road is located in Harwich Port, MA. At this time, the property has an estimated value of $456,000 and yearly taxes of $3,385. This home has 3 bedrooms, 2 bathrooms, and approximately 1,550 sqft of living space. The property was built in 1960. Similar homes nearby have 3 bedrooms, 2 baths and sell for around $450,000.
Check out Harwich Port homes for sale, read the Harwich Port real estate news or review the Harwich Port real estate trends. Here is a summary of local market conditions over the past 30 days:
17 Homes for Sale
$750,000 Median List Price
113 Days on Market
8.5 Months Inventory
3 Bed, 2 Bath Detached
4 Bed, 2 Bath Detached
2 Bed, 1 Bath Detached
3 Bed, 1 Bath Detached
3 Bed, 2½ Bath Detached
3 Bed, 3 Bath Detached
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More sellers put their homes up for sale in the Harwich Port market over the last 30 days and the number of new listings shot up this week to five. Home sales remained unchanged from the prior period... Read More »
Over the last 30 days, the Harwich Port market picked up with two homes sold. Within the same time frame, South Yarmouth had 13 sales, the most of all cities in the area. The median sales price droppe... Read More »
The number of homes coming into the Harwich Port market tumbled to one listing over the last 30 days, while sales remained right where they were as once again one home was sold over the same period. T... Read More »
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A clean revolution could enhance U.S. energy security and create a stronger economy, yet arguments for it are unlikely to find their way into the presidential campaign, argues John Elkington.
Whichever way you run the numbers about climate change, 2012 looks set to go into the record-books. In America alone, this year's heat wave broke over 40,000 temperature records. In the 1980s, the US weather-related insurance cost was around $3bn a year, compared to $20bn (and rising) today. Hurricane Katrina alone cost more than £200bn, representing over 1% of GDP. And then there are little things like West Nile virus, unknown in the US before 1988, but now spreading as a result of rising temperatures – resulting in a record 2,000 cases by August 2012, and 87 deaths.
But how many of us expect climate change to be central to the debates between Obama and Romney in the final stages of the US presidential elections? I, for one, am not holding my breath. And yet the degree to which this rising challenge is debated should be seen by the wider world as a key indicator of whether America is fit for purpose – and fit to lead – in the 21st century.


text:
quark is a valid word in this word list. For a definition, see the external dictionary links below.
The word "quark" uses 5 letters: A K Q R U.
No direct anagrams for quark found in this word list.
Words formed by adding one letter before or after quark (in bold), or to akqru in any order:
e - quaker s - quarks
Shorter words found within quark:
ar ark auk ka qua
List shorter words within quark, sorted by length
Words formed from any letters in quark, plus an optional blank or existing letter
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text:
Here's a creamy comforting potato casserole to make when you need a side dish sure to please a large group. It's so simple to assemble, and everyone likes the flavor.
-Merrill Powers
Spearville, Kansas
Top 10 Potato Salad Recipes
- 5 cans (12 ounces each) evaporated milk
- 7-1/2 cups milk
- 5 cans (10-3/4 ounces each) condensed cream of chicken soup, undiluted
- 5 cans (10-3/4 ounces each) condensed cheddar cheese soup, undiluted
- 1 pound butter, melted
- 1 package (12 ounces) cornflakes, crushed
- 3 medium onions, finely chopped
- 10 packages (2 pounds each) frozen cubed hash brown potatoes, thawed
- In several large bowls, combine all ingredients. Transfer to 10 greased 11-in. x 7-in. baking dishes. Bake, uncovered, at 350° for 45-55 minutes or until potatoes are tender. Yield: 10 casseroles (about 10 servings each).
Originally published as Potatoes for a Crowd in Taste of Home
October/November 2004, p40
Reviews for Potatoes for a Crowd
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Reviewed May. 3, 2009
"Breaking it down is easy; just divide each ingredient by 10 (the number of casseroles it makes). You will have half-used cans of things, but you can use them for later dishes."
Reviewed Apr. 7, 2009
"Can someone break this recipe down for like one casserole? It sounds good but not that much!"
Reviewed Jan. 22, 2009
"Potatoes for a Crowd: I was wondering if this recipe can be made in a NESCO 18 quart roaster? I would love to be able to make this for a family reunion but, not sure if this whole recipe will work in the roaster. Can anyone help?


text:
It seems that even before the Kindle Fire has been announced, we have yet another challenger to the Nook Color's Reading Tablet crown. It's the 7″ Kobo Vox eReader Tablet with a LCD screen.
All information on the Kobo Vox eReader Tablet is courtesy the FCC. Engadget had the scoop but didn't make the effort to actually read the FCC documents and see the picture very helpfully included:
Kobo has filed papers at the FCC for the 'Kobo Vox eReader Tablet'. Engadget has the scoop on the Kobo Vox eReader Tablet and makes a mess trying to guess what it is. Engadget uses delightful phrases like –
shrouded in bookish mystery
What's this mystery e-reader?
Is it a reworked version of the relatively recently released Touch Edition?
Well … The fact that it's named 'Kobo Vox eReader Tablet' doesn't leave much room for speculation, does it? Or that picture included above with 'LCD' written right on the screen.
Here's what we roughly know –
- Kobo EReader Reading Tablet with model number K080.
- There's a 7″ LCD screen.
- Probably manufactured by Wistron Corporation as all the documents at the FCC are from Wistron. Wistron is located in Taiwan (Taipei to be precise).
- It supports 802.11 b, g, and n.
- It has a WLAN antenna (chip antenna). Its located at the top left of the device (right above the LCD screen).
- Battery has a rating of 3.7V 4000mAh, 14.8 Wh. Model Number is D1-11-04. The Power adapter used supplies 5 Volts at 2.0A. It takes input in the 100 to 240 Volts range – so it might be a worldwide release.
- There's mention of rotating 90 degrees, 180 degrees, and 270 degrees. Suggests either an accelerometer or a choice between Portrait and Landscape mode.
- The dimensions are 19 centimeters by 13 centimeters. That's roughly 7.48″ by 5.11″. Quite compact for a 7″ screen device. The bezel seems to be a bit thick at 1.5 centimeters on the side (0.59″).
- There's a LCD screen. The screen dimensions are 15.5 centimeters (6.1″) and 9 centimeters (3.54″). Which would make the diagonal around 7.05″. So we have a 7″ LCD screen.
- Testing took place roughly between August 30th to September 14th. Suggests a release should be imminent. Testing took place at least in USA, Zürich (perhaps, not sure), Canada, and Taiwan. Again, suggests a possible worldwide release.
- Support for MicroSD cards. Support for a headset. USB Support.
- There were some internal modifications made after initial submission.
Will add more when more news about the Kobo Vox Reading Tablet becomes available. For the moment it seems that just like Kindle vs Nook vs Kobo, we will see a very interesting eReader tablet battle – Kindle Fire vs Nook Color vs Kobo Vox.


text:
Tesla Motors (NASDAQ:TSLA) CEO Elon Musk took center stage at the D: All Things Digital technology conference on Thursday and unveiled his plans to triple the automaker's Supercharger coverage area this month. Musk also promised that by the end of 2013, Tesla car owners will be able to drive from Los Angeles to New York using only Superchargers.
It sounds like a good deal to just about any driver, and especially to overly mobile ones. But as with all cases, consumers want to know the facts before signing their names on the line. Here is all you need to know about the Tesla Supercharger:
1. What is a Supercharger?
Let's start with the basics. A Supercharger performs the same function that a Tesla owner's ordinary electric current at home does, but it serves to ameliorate the stress users may face when driving far distances across cities, fearing that their vehicle will run out of power. Superchargers provide a full charge in a little under an hour, and are strategically placed between cities and well-traveled highways to allow owners to drive from station to station with minimal stops and little anxiety.


text:
What does AUTO mean in Military?
This page is about the meanings of the acronym/abbreviation/shorthand AUTO in the Governmental field in general and in the Military terminology in particular.
Find a translation for AUTO in other languages:
Select another language:
What does AUTO mean?
- car, auto, automobile, machine, motorcar(noun)
- a motor vehicle with four wheels; usually propelled by an internal combustion engine
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Hi all, Zombie_X here with another review! This won't be as in depth as the T1's review but I will give you all the needed info.
The Beyerdynamic DT880 sound can be classified as being treble oriented with slightly recessed mids (will get tot hat later) and having slow or muddy bass (will get to that as well).
- Source: PC via Pop-Pulse SPDIF-II (to Optical), Marantz VC6001 CD Player
- Amp: Woo Audio WA3+, Audio-GD ROC
- DAC: Music Hall DAC25.2 (with NOS Mullard E88CC tube)
- IC's : ZXAC custom made RCA and XLR cables
I find the treble on the DT880/600Ohm to be slightly smoother than the 250Ohm and 32Ohm models out there. It's very extended and crystalline in it's presentation. I find it to be harsh on poorly mastered or recorded material but when used with reference grade material the treble is to die for. Very articulate and grain free.
Many say the DT880 is leaner in the midrange but I have to say they are not. The treble shines over the mids and makes them seem recessed or muted. The mids are very transparent with lots of detail and also have great resolving capabilities. Vocals sound very nice and realistic and guitars have an edge to them which is very nice.
Yet again I hear this talk of the slow and muddy DT880 bass. The 250Ohm has this as well as the 32Ohm, but not the 600Ohm model. The bass is very punchy when needed and is very extended with great texturing and layering. It's is also not sluggish in anyway as long as the recordings bass is not that way. The bass is very tight and well controlled without being to dominant of the sound. Many on [...] have said that the DT880 also has bloated bass which is simply not true.
The soundstage is very well layered and offers a lot of detail and separation of the various instruments. I find it to be quite accurate and spacious with good air. In comparison to the HD600 it is a bit bigger in terms of width and depth of the presentation. It's not the biggest out there but it is better than others.
These 600Ohm headphones need strong amping. I would recommend a nice OTL tube amp for them like my Woo Audio WA3+. They need a significant voltage swing to function properly.
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The Alaska SeaLife Center's entryway is getting a facelift thanks to the generosity of the Rasmuson Foundation!
Its new ticketing counter will have a more modern design, more ticketing stations, more storage, and flat screen TV displays.
The new look will create a more friendly, educational, and informative experience for our guests. The SeaLife Center hopes visitors will be able to visit this summer and check it out!
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Johan Molina – Business
Full disclosure: Johan Molina did not have a great first year at the University of Rhode Island. His grades were slipping, and he wasn't involved in any student activities. He sat himself down and had a little talk. Coasting through college was not an option. Growing up in one of the toughest neighborhoods in Providence, he knew he could do anything if he set his mind to it. And he did.


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Credits & Description:Advertising Agency: McCann Erickson, Argentina The Radio ad titled Italy was done by McCann Buenos Aires advertising agency for product: Madison Rent a Car (brand: Madison) in Argentina. It was released in the Mar 2012. |
Latest Madison Ads & Commercials
© 2004-2017 Lixil Graphics Ltd. All trademarks, logos and copyrights are the property of their respective owners.


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Chatting With Chapey
Chatting With Chapey
February is a month that has many events that will be of interest to
Since everyone loves a parade, let me take this opportunity to invite you to our annual fundraiser at Antun's on Saturday, February 17, 2001, at 7 p.m. to benefit the Queens St. Patrick's Day Parade. As you are well aware, funds raised at this annual event will help us to defray part of the costs of our Queens parade. As you have noted from Beverly Baxter's column this year we are celebrating men and women of distinction who have served our community and deserve recognition for their efforts.
Please join us at Antun's for our
dinner dance. You will have a great time as we have every year and help a worthy cause. For tickets please call Mike Benn (318-4749) or Peggy or William Neville. We will look forward to seeing you there.
February is also National Snack Food month. This is very good news to many people who like myself love to eat snacks. Now, we even have a reason to celebrate our "snaking". When Hershey did a survey they discovered that about 90 percent of those who participated considered themselves to be part of the "in group" of snackers. If this doesn't surprise you, the next result of the survey will. More than 60 percent of the survey responders considered chocolate to be their favorite snack.
I could have saved the people at Hershey all the money that they spent on the survey. If they called me I could have told them that I love to snack and I love chocolate. One interesting item on the chocolate front - when I was taking my doctoral studies at Rutgers in New Jersey I stopped at a shop in New Brunswick to buy my husband a chocolate Valentine. After I paid for this very nice novelty chocolate item I discovered that it was made at Madeline Chocolates in the Rockaways. It's nice to know that we have excellent chocolate made right here at Madelines. Be sure to go and buy them.
February is also the 30th anniversary of the NASDAQ Stock Market. It is a market that built itself on electronic trading. NASDAQ, when it was created, was viewed as a technological wonder. Surprisingly, one area where the NASDAQ hasn't been competitive is in converting to decimal pricing. In this area the New York Stock Exchange is ahead. NASDAQ doesn't agree with this criticism because it feels that the NASDAQ is dealing with more stocks
and smaller companies, which are not similar to the NYSE and they are
expanding to new markets in Europe and Japan. However, regardless of your viewpoint on this NASDAQ issue, the NASDAQ is to be congratulated for starting on February 8, 1971 with what was considered "junk" issues and moving to becoming a very close second to the New York Stock Exchange.
Parades, chocolates and the NASDAQ have one thing in common. They are all being celebrated in February.
Special Note: Best wishes to Mary Dever on her retirement. Mary is an
outstanding professional who has contributed to the lives of the children in our district. She and her late husband Jerry Dever have devoted so many valuable years in our school system. Good luck to Mary in her future endeavors.


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As inauguration draws near, thousands head to Washington
January 19, 2013 10:00 AMWASHINGTON
Camille Carlos got on a bus in the middle of the night, spent hours pushing her way through crowds, waited in long lines to use smelly portable toilets and tried to stay warm by taking turns using hand warmers shared among friends.
After all that, she didn't even get to see what she'd come 268 miles for: the 2008 swearing-in of President Barack Obama. Her first view of the new president came late that night when she returned to her North Fayette home and flipped on CNN.
The day was cold, it was exhausting, it gave her sore feet -- and she can't wait to do it again.
Ms. Carlos, 52, is among an estimated 800,000 expected to trek to Washington Monday for Mr. Obama's second swearing-in. Most, like Ms. Carlos, will have no tickets and will find themselves in a massive crowd on the National Mall. If they're lucky enough to find good spots they'll be able to watch the swearing-in on 21-foot screens towering overhead.
Three thousand constituents requested 10,000 tickets from Sen. Bob Casey, D-Pa., but he had only 400 to distribute.
House members each got 198 tickets, many of which went to state officials and campaign volunteers. Most offices implemented lottery systems to distribute the rest.
"Some people had rather compelling stories of how hard they worked. Some of these people did hundreds of hours of phone banking" for the president, said Rep. Mike Doyle, D-Forest Hills. "These are volunteers. They don't get paid. They do it out of a sense of wanting to make their country better, and I feel like that person should at least get to see the president sworn in."
Lawmakers also made it a priority to provide tickets to constituents whose efforts to attend the 2009 inaugural were thwarted by a logistical nightmare that became known as the "tunnel of doom." Thousands of ticket-holders were directed into an underground traffic tunnel where they were trapped for hours and missed the inauguration. Officials blamed the incident on unanticipated crowd-control problems.
Other ticketed attendees in 2009 were directed to line up at a security gate that was never opened to provide access. Carol and Martin McWilliams of the Bucks County borough of Langhorne were among them.
"We were so excited. We drove down the night before, stayed in a hotel and awoke at the crack of dawn to get in line and they never opened the gate. You could see this whole open space inside the gate where we were supposed to be but nobody ever let us in," said Ms. McWilliams, 67.
"It was a great disappointment but we were still thrilled to be anywhere near the event, to be part of that day," she said.
Her colleagues at the New Jersey Education Association, where she is a secretary, can't believe she's going back.
"One co-worker said, 'I can't understand why you'd put yourself through that again,' and I said I'm just so excited to go back,'" said Ms. McWilliams, who scored her tickets through Mr. Casey's office.
Those without tickets will have a better shot of viewing the president later Monday when he participates in a parade from the Capitol to the White House.
More than 2,800 groups applied to participate in the parade. About 60 have been selected to participate, including Boy Scout Troop 358 in Philadelphia.
Pennsylvania is among eight states that will be represented by custom-designed floats. Pennsylvania is being highlighted because it is the birthplace of Vice President Joe Biden. The float will depict the Liberty Bell and the state seal.
Ms. Carlos had a good view of the 2009 inaugural parade, but it wasn't easily attained.
"We went there with determination, determination, determination. It took us all day to squeeze through there but we got all the way up to the barricades. Let me tell you, it was hard," she said. "But it was worth it and that's why I'm going again."
Arthur Keys has attended so many inaugurations he's lost count. The first was Jimmy Carter's in 1976 and what he remembers most about it was the chill. At 28 degrees, it was among the four coldest inaugurals on record.
"I'm used to the cold. I wear my Pennsylvania long johns," said Mr. Keys, 67, who has homes in both Amity, where he is a farmer, and Washington, D.C., where he serves as president of International Relief and Development, a nonprofit humanitarian organization.
With each inaugural he has attended it has become more frustrating to navigate crowds and get through ever-increasing security but, he says, it's worth it to see history in the making.
"It's really a bipartisan thing. You see Republicans and Democrats there, and there are a lot of things to participate in," said Mr. Keys, who planned to attend the swearing-in, view the parade and then head to the official inaugural ball at the Washington Convention Center.
There are hundreds of thousands more like him traveling from all across the country and from other parts of the world to celebrate Mr. Obama's second inauguration.
"We are a nation of ritual, and any time there's a public demonstration of ritual people want to be there," said Joseph Valenzano, assistant professor of communication at the University of Dayton. "Inauguration Day is a real moment in history. It only happens once every four years and this is President Obama's last one."
He compared Inauguration Day in D.C. to New Year's Eve in Times Square.
"There's celebration. It's a feel-good time, and people want to be part of that moment," he said.
Ms. Carlos -- who dressed her best, wore a mink coat and carried a designer handbag to the 2008 inaugural -- said she has "a little obsession" with presidential pomp and circumstance.
"I like seeing it. I like the dignitary part. I like the whole process," she said.
She finds herself a little disappointed in Mr. Obama, who didn't meet her expectations during his first term in office.
She said she attended the 2009 inauguration thinking that this new president would do more for the economy. Instead, she found herself laid off from a job she loved after 20 years with FedEx.
"I just thought Americans would be doing better off [under Mr. Obama] but it just seems like we're stuck in the mud," she said.
Still, she remains a supporter. She hopes he'll do more in his second term.
The theme of this inauguration is "Faith in America's Future."
While the public swearing-in will be held Monday, the president will already have been officially sworn in during a private ceremony Sunday, the actual start of his second term.
nation - regionBureau Chief Tracie Mauriello: 1-703-996-9292 or email@example.com. First Published January 19, 2013 5:00 AM


text: That's Nutsdodai1/16/08 9:45amFiled to: Men at WorkBallsNutsnutztrailer testiclestruck nutzVirginia151EditPromoteShare to KinjaToggle Conversation toolsGo to permalinkPerhaps you've seen trucks or trailers with large rubber testicles attached to the back? Virginia state delegate Lionel Spruill has introduced a bill to ban "displaying replicas of human genitalia." He claims the big balls are a safety issue, because drivers can get distracted. Spruill was prompted to take action when a constituent's young daughter asked her father to explain the nutsacks. Spruill also had the stones to say he'll bring a set of the $24.95 trailer testicles with him for legislative show-and-tell. Frankly, it's unbelievable that these things haven't been outlawed already. Think they'd let women drive around with labia majora on their cars? Bollocks! [CBS News]Reply151 repliesLeave a reply


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"Sessions with Ramon" sounds like a show on public radio highlighting latin jazz fusion. That sounds like it may be interesting in the sense that if you're driving in your car and you turn to the station (presuming you don't have satellite radio or some sweet ass Pandora setup), you can expose yourself to some "world music" and consider yourself slightly more cultured.
Please, blog, may I have some more?


text: The house by Thomas Bendel is made of prefab concrete and then built together on site. The use of glass is reserved for the front and the back of the house using aluminum as a frame material. The placement of this big windows takes the views and displays it as a big wallpaper. I would agree that the house needs a little bit more of decoration as it looks like a nuclear silo… maybe a wall or two in dark charcoal and bright yellow and you are done.


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"I have to give this class one of my extremely rare 10s.
Take this if you are serious about getting in shape. There is no minute wasted. However, because it's outside and feels like playing, the class leaves you feeling rejuvenated and eager for the next day." Aimee Heckel, Editor - Daily Camera Other fitness programs lack the progressive improvement that a camp series can offer. Boot Camp's multi-week program was designed to systematically work through all your muscle groups, target cardiovascular performance & endurance while incrementally increasing the intensity of the effort. Why is Boot Camp so Effective?
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Discover for yourself why 70% of Boot Campers are repeat or referrals. "I would recommend Boot Camp because I had such good results: by the end of my first session, I cut 53 seconds from my timed mile, I was able to do twice as many push-ups, 20 more sit-ups, and lost about 6 pounds!" |
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HYPE Arduino Bot Robotics
Main Learn about the Arduino open-source electronic prototyping platform, and join the community of Arduino makers.
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Today: March 30, 2017
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Live from Chattanooga, TN
Turnstone SOTO Collection
The State of the Office Collection is a range of desktop organizers and convenience oriented accessories for one who wants a premium quality, productive workspace. Designed by Turnstone, the SOTO Collection is based around the SOTO Rail which fits firmly onto the back of your desktop. A large collection of add ons can be purchased a la carte that attach to the SOTO Rail, including storage boxes, display shelves, and file organizers. You can tell a huge difference between a desk that utilizes an effective system like the Turnstone SOTO Collection and one without. Everything shown helps knockout clutter and inefficiency. Get it right here at Smart Furniture.
The SOTO State of the Office Collection


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Cal-UCLA Football Game to be Replayed on TV
Oct. 16, 2000
Berkeley - Fox Sports Bay Area will re-air the dramatic Cal-UCLA football game twice this week. A slightly edited three-hour version of the game will be shown on Fox Sports Bay Area on Tuesday (October 17) at 7 p.m. and again on Wednesday (October 18) at 12 noon.
The Cal-UCLA football telecast was produced by Fox Sports West 2 and aired in southern California late Sunday afternoon. Bill McDonald and Mike Sherrard called the game for the network.
Cal delivered an impressive 46-38 victory over the 13th ranked Bruins on Saturday, breaking a four-game losing streak.
The weekly half-hour Cal football highlight show will air this week on Wednesday at 9:00 p.m. after a San Jose Sharks hockey match.
Another great Cal victory, the Cal women's soccer 2-0 victory over Stanford this past Sunday will also be shown this week on Fox Sports Bay Area. That match will air at 1:00 p.m. on Tuesday (October 17).


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18 items found |
You can send Gifts For Mother to Dominican Republic in the following cities.
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As we previously reported, a Chihuahua was rescued from a roadway median on Interstate 680 in Walnut Creek during rush hour Friday evening.
A CHP officer spotted the small dog on the highway's concrete center divider around 6 p.m. while driving south on I-680 near the North Main Street overpass, CHP Officer John Fransen said.
The officer exited his car, used a protein bar to coax the Chihuahua toward him and was able to pick up the dog, Fransen said.
The dog was uninjured and was taken by Contra Costa Animal Services personnel to the animal shelter in Martinez.
Although Fransen said he didn't know how the dog ended up on the freeway, he said, it's likely that someone left the animal there. "As sad as it sounds, it actually happens pretty often," he said.
Since Friday night, there have been several offers from the public to adopt the dog, the officer said.
RELATED STORY: PICTURE: Look! It's a Chihuahua ON the Center Median of I-680 in Walnut Creek


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Jennifer Steinhauer in the Times reports that some Republicans are running away from the Ryan Plan (you know, the one that changes Medicare from a health insurance plan to an underfunded subsidy), while others are trying to figure out if they should support in order to gain Tea Party votes. As policy, of course, it never had a chance to pass the Senate or of being signed by President Obama (and every Republican staffer Politico could find agrees), so it was pure political theater from the start. As Paul Krugman points out, the goal may have been to win over the pundits — a group that is vastly more concerned with the deficit than ordinary voters — but even that failed. (They got Jacob Weisberg, but he backpedaled furiously, and they got David Brooks, which was mainly amusing because then we got to watch Krugman trying to observe intra-Times decorum by not going after Brooks by name). Now Republicans are wondering if the loss of a Congressional seat in a conservative New York district was Ryan's fault.
But while I'd like to think that the nation is recovering its senses, at least on what Republicans mean for Medicare, I'm not optimistic. Brad DeLong put it well:
"the political lesson of the past two years is now that you win elections by denouncing the other party's plans to control Medicare spending in the long run — whether those plans are smart like the Affordable Care Act or profoundly stupid like the replacement of Medicare by RyanCare for the aged — sitting back, and waiting for the voters to reward you."
Remember that at the same time they were plotting the destruction of Medicare as we know it,* the Republicans were also attacking the Obama administration for planning to reduce Medicare spending (from its projected levels, not its actual levels). Remember when Mitch McConnell's office released press releases, on consecutive days, saying, "Cutting Medicare Is Not What Americans Want" and "Expanding Medicare 'A Plan for Financial Ruin'"? Politically, the only mistake they made — if they made one at all — was putting out those press releases on consecutive days from the same office. I'm certain that a majority of Americans would agree with both of those positions, standing on their own.
Most likely, the Republicans will reposition the Ryan Plan as an attempt to "save Medicare" (wait, they're already doing that). They will try to eliminate the provisions in the Affordable Care Act that attempt to reduce Medicare costs by making Medicare spending more efficient, like the Independent Payment Authorization Board, demonizing them as government intrusion into medical treatment decisions. The answer, they will say, is a "Medicare" system free of government involvement. And if they can create enough gridlock to make sure the Affordable Care Act fails, then five or ten years from now, when the debt projections are even worse, they will take another shot at privatizing Medicare. Remember, this wasn't the first one: Newt Gingrich took a shot back in 1995. As long as the Republicans can use voters' fears of "big government" to block real reforms to Medicare, time is on their side. At some point, even if they have to wait twenty years, the fiscal imbalance will be so big that they will be able to eliminate Medicare.
The Obama administration knows this game. They knew what they were doing when they put cost-control measures into the Affordable Care Act, and I'm sure Obama will veto anything that prevents those measures from working. But it's still an uphill battle.
* No, I don't think this is an overstatement. The Ryan Plan would change Medicare from a guaranteed level of health insurance coverage to a voucher that may or may not buy you health insurance at all.


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text: Oddish (Emerald 5) 704pages on this wiki Add New Page Edit Classic editor History Comments0 Share ODDISH Nickname(s) N/A Pokémon Species Oddish Level xx Status Active Acquired xd xh xm Attacks XXXX Gender ♀ Another Oddish that A caught. S/he has a xxx nature. Contents[show] HistoryEdit NameEdit Personality/LoreEdit TriviaEdit Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Generation 3 Gen 3 Characters Safari Zone Pokemon Great Cabbage Harvest Add category Cancel Save