text:
Small RNA sequencing reveals miR-642a-3p as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis
DOI: 10.1186/gb-2011-12-7-r64
© Zaragosi et al.; licensee BioMed Central Ltd. 2011
Received: 3 January 2011
Accepted: 18 July 2011
Published: 18 July 2011
In severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation.
We used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis.
Overall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells.
Obesity, by itself or associated with ancillary disorders such as diabetes and cardiovascular pathologies, represents a major public health issue in developed countries. In severe obesity, as well as in normal development, the growth of adipose tissue is the result of adipocyte hypertrophy and hyperplasia. It is now well established that a pool of multipotent progenitor cells persists in adipose tissue throughout life and is able to differentiate to give rise to adipocytes [1–3]. Certain key events controlling the terminal differentiation of progenitors into adipocytes have been identified. Transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs) are known to play a critical role in this process [4]. However, the molecular mechanisms controlling the early steps of adipocyte progenitor commitment towards adipocyte differentiation remain poorly understood. Several lines of evidence suggest that osteoblasts and adipocytes share the same precursor cell type. Mesenchymal stem cells isolated from different tissues can differentiate into both lineages at a clonal level [2, 5, 6]. A reciprocal and inverse relationship exists between adipogenesis and osteogenesis [7–9]. Pathophysiological conditions such as ageing or osteoporosis, for instance, involve a concomitant decrease in trabecular bone volume and an increase in bone-marrow adipocyte numbers [10]. Moreover, molecular mechanisms that activate differentiation towards one lineage often inhibit differentiation towards the opposite fate. Several signaling pathways, including the bone morphogenetic protein, Wnt, Hedgehog and insulin-like growth factor pathways, as well as transcription factors such as PPARγ and RUNX2 (runt-related transcription factor 2), have already been shown to modulate the balance between adipogenesis and osteogenesis (reviewed in [11]).
MicroRNAs (miRNAs) are a subclass of regulatory, non-coding RNAs that regulate gene expression at a post-transcriptional level by affecting mRNA translation and stability [12]. Up to 30% of human genes could potentially be regulated by miRNAs [13]. The ability of a miRNA to interact with many targets, together with the possibility for several miRNAs to share the same target, represent powerful regulatory mechanisms that tremendously increase the complexity of biological networks. Over the past few years, miRNAs have been shown to regulate many cellular processes, including adipogenesis and osteogenesis. miR-103, miR-143, miR-17~92, miR-21, and miR-204/211 have been reported to promote adipogenesis [14–18], while the miR-27 family inhibits this process [19]. Similarly, osteogenesis is regulated positively by miR-29b, and negatively by miR-133, miR-135 and miR-125b [20].
Our present work aims to clarify the role of miRNAs in the regulation of adipogenesis. We have characterized small RNAs that are modulated by adipogenic differentiation in human adipose tissue-derived stem (hMADS) cells by a deep-sequencing approach. Among the RNA species we sequenced, miRNAs were the most abundant class of annotated small RNAs. However, we also found significant variations in expression levels of non-annotated small RNAs during adipogenic differentiation. A current bioinformatics challenge in small RNA research is the prediction of RNA targets and how their regulation is integrated into already existing biological networks. We performed such a study in the specific context of the miR-30 family, in order to evaluate the capacities of these miRNAs to regulate adipogenesis. Our investigations focused on the transcription factor RUNX2, a major regulator of osteogenesis, which we established as a bona fide target of miR-30a and miR-30d.
Global analysis of miRNAs by high-throughput sequencing during adipogenesis of hMADS cells
To identify small RNAs that are differentially expressed during human adipogenesis, hMADS cells were differentiated into adipocytes in vitro. RNA was extracted from confluent undifferentiated (day 0) cells and from cells that were differentiated for 3 or 8 days. Differentiation efficiency was checked by expression profiling of specific genes, such as those encoding adiponectin and PPARγ (Additional file 1).
Top 26 regulated miRNAs during adipogenesis
P-value AD8/ND
Maximal read number across all samples (per million miRNA reads)
3.64 ± 0.04
7.32 ± 0.08
0.72 ± 0.42
4.32 ± 0.11
0.28 ± 0.02
4.24 ± 0.05
1.68 ± 0.10
2.53 ± 0.13
1.49 ± 0.32
4.51 ± 0.49
0.89 ± 0.10
1.46 ± 0.08
0.43 ± 0.01
2.41 ± 0.2
4.10 ± 1.34
4.51 ± 1.28
-0.85 ± 0.02
-1.48 ± 0.03
1.09 ± 0.18
2.49 ± 0.33
1.41 ± 0.01
1.98 ± 0.01
0.56 ± 0.15
1.25 ± 0.31
3.70 ± 0.12
3.73 ± 0.11
1.54 ± 0.18
3.05 ± 0.27
0.36 ± 0.03
2.15 ± 0.46
-0.25 ± 0.02
1.53 ± 0.33
0.53 ± 0.42
1.82 ± 0.46
-0.47 ± 0.18
-1.57 ± 0.60
0.41 ± 0.06
1.27 ± 0.31
0.52 ± 0.13
1.41 ± 0.08
1.66 ± 0.24
2.82 ± 0.64
-1.64 ± 0.03
-2.09 ± 0.52
2.14 ± 0.83
3.14 ± 0.73
-0.58 ± 0.07
-1.3 ± 0.33
-0.62 ± 0.07
-1.29 ± 0.29
The expression patterns of miRNAs that were previously reported in adipocytes or their precursors are in agreement with published data, as summarized in Additional file 3. However, the adipogenesis-dependent regulation of many of the differentially expressed miRNAs we identified has never been described before; these include miR-642a-3p, miR-345, miR-193b, miR-29c, miR-664, miR-10b, miR-136, miR-22*, miR-181a, miR-154*, let-7a, let-7b and let-7c.
Up-regulation of miR-642a-3p, miR-378/378* and miR-30 miRNAs suggests their contribution to adipogenesis
The expression profile of the miRNAs that were strongly up-regulated during adipogenesis (miR-642a-3p, miR-378, miR-30a, miR-30b, miR-30c, miR-30d, miR-30e, and miR-193b) was validated by quantitative PCR (qPCR; Additional file 4). Although some of the fold changes obtained by this technique were not strictly equal to those obtained by deep sequencing, this approach confirmed qualitatively the stimulation of the expression for all of these miRNAs.
Incidentally, miR-378 microRNAs, also highly regulated in our model, have a genomic location in intron 1 of PPARGC1B (Additional file 5) and miR-378 has already been described as positively regulated in adipogenesis (Additional file 3). In addition to miR-378, our data confirmed that the miR-30 family was up-regulated in adipogenesis (Table 1). Interestingly, the relative abundance of the miR-30 family varies from 1.1% in undifferentiated cells to 4.9% in adipocyte-differentiated cells (Figure 2b). In particular, miR-30a and miR-30d accounted for 3.7% of all sequenced miRNAs in adipocyte-differentiated hMADS cells. Even though none of the miR-30 family members are encoded within introns of pro-adipogenic sites, their increased abundance is likely to reflect a major role in differentiation.
Gain and loss of function studies reveal that the miR-30 family favors adipogenesis
Given their up-regulation after induction of adipogenesis and their high abundance in adipocytes, we focused on the role of miR-30 family members in adipogenesis. We altered their expression by transfecting synthetic miR-30 miRNAs or the corresponding antagomirs. Inhibition of the miR-30 family was achieved with the transfection of a combination of three oligonucleotides that can target and inhibit activity of the whole miR-30 family. Over-expression was obtained with transfection of pre-miRNAs for miR-30a and miR-30d. In both cases, sub-confluent hMADS cells were transfected and then submitted to adipogenic differentiation three days later, once cells had reached confluency. Adipogenesis was scored after 10 days (miRNA knock-down) or 4 days (miRNA over-expression) in differentiating medium. At each analyzed time point, inactivation and over-expression were efficient, as shown by qPCR (Additional file 6).
miR-30 miRNAs stimulate adipogenesis via inhibition of the osteogenesis transcription factor RUNX2
To identify molecular mechanisms that would regulate the effects of miR-30 miRNAs on adipogenesis, bioinformatics prediction of their targets was performed with TargetScan. It revealed that RUNX2 bears several conserved binding sites for these miRNAs (Additional file 7). RUNX2, also known as CBFA1, is a key regulator of osteogenesis and its expression is detected at the undifferentiated state. It increases during osteogenesis and decreases during adipogenesis [24] (Additional file 1).
HEK-293T cells were co-transfected with either construct together with the following synthetic pre-miRNAs: negative control, miR-30a, miR-30d or miR-378 (as RUNX2 does not bear any putative binding site for this miRNA, miR-378 was used here as an additional control). When cells were transfected with pSi-CHECK™-2 bearing the first putative binding site, none of the tested miRNAs had any effect on luciferase activity. In contrast, with pSi-CHECK™-2 bearing the last two binding sites, miR-30a and miR-30d triggered a more than two-fold decrease in luciferase activity compared to the control miRNA (Figure 5c). As expected, miR-378 had no effect on luciferase activity. Importantly, this effect was confirmed at the protein level for endogenous RUNX2. Transfection of sub-confluent hMADS cells with pre-miR-30a or pre-miR-30d induced a 0.61-fold or 0.48-fold decrease in RUNX2 protein levels, respectively (Figure 5d). Thus, these results demonstrate that RUNX2 is a bona fide target of miR-30a and miR-30d.
Finally, we sought to establish a direct link between miR-30 effects on adipogenesis and RUNX2 targeting. We used the target site blocker (TSB) strategy to mask miR-30 binding sites 2 and 3 in the RUNX2 3' UTR. Transfection with RUNX2 miR-30-specific TSB, but not a control TSB, significantly decreased miR-30a stimulation of adipogenesis (Figure 5E). In conclusion, RUNX2 targeting is, at least in part, responsible for miR-30 positive effects on adipocyte differentiation.
Adipocyte differentiation is a complex process combining several levels of regulation. Signaling pathways, such as cAMP and insulin signaling pathways, as well as key transcription factors, such as PPARγ, C/EBPβ and Krüppel-like transcription factors (KLFs), have been extensively studied [4, 25].
Our results suggest a direct role of miRNA-mediated post-transcriptional regulation in adipogenesis. In particular, we show that the miR-30 family is a positive, key regulator of adipocyte differentiation in a human adipose tissue-derived stem cell model. The up-regulation of miR-30 expression is triggered at early stages of adipocyte differentiation (day 3) and increases until terminal differentiation. Of note, all miR-30 miRNAs do not belong to the same genomic cluster (Additional file 8). In particular, miR-30a and miR-30d are encoded by genes located on distinct chromosomes, suggesting coordinated regulation of distinct genomic regions. Factors that are responsible for this coordinated regulation have not yet been elucidated.
In order to dissect the molecular mechanisms involved in the effects of miR-30 on adipogenesis, we searched for predicted target genes. We focused on RUNX2, which is a well-established regulator of osteogenesis. Indeed, an inverse relationship is known to regulate the balance between adipogenesis and osteogenesis. Thus, identifying miRNAs that are up-regulated during adipogenesis and that negatively regulate a key osteogenesis transcription factor is of major importance. In fact, the RUNX2 pathway has been reported as a potent inhibitor of the expression of the master gene for adipogenesis, PPARγ [26]. Thus, it is tempting to speculate that RUNX2 inhibition is required for adipocyte differentiation and that miR-30 miRNAs play a critical role in this process.
We show here for the first time that miR-30 miRNAs target RUNX2. Huang and co-workers [18] recently demonstrated that miR-204 and miR-211, which were up-regulated during adipogenesis of human bone marrow stem cells, also target RUNX2. However, we found that miR-204 and miR-211 were expressed at extremely low levels - for example, below our 0.03% threshold - while miR-30 represented 4.9% of the miRNA reads in adipocytes. This is probably not due to a deep sequencing cloning bias, as miR-204 detection was above average and better than that of miR-30 in a synthetic equimolar miRNA panel that we sequenced in similar conditions (data not shown). Thus, in our system, this very low abundance of miR-204 and miR-211 suggests that their impact on RUNX2 and differentiation is minor when compared with the highly expressed miR-30 family. Importantly, we also showed that miR-30 stimulation of adipogenesis was impaired by masking miR-30 binding sites in the 3' UTR of RUNX2, and preliminary data suggest that miR-30 inhibition might stimulate osteogenesis. Altogether, these data strongly support a direct and functional link between RUNX2 and miR-30, but does not exclude the contribution of additional miR-30 targets. In an attempt to identify the ones that were regulated at the RNA level, we performed a transcriptome analysis of hMADS cells that were transfected with pre-miR-30a or pre-miR-30d and then submitted to adipocyte differentiation for 4 days. Using miRonTop [27], we verified that predicted miR-30 targets were correctly enriched in these experiments. Statistical scores were highest for the miR-30 family (P-value = 5.32.10-10), showing its strong overall impact in these cells. In the list of predicted miR-30 targets, we noticed the presence of CBFB (core binding factor beta), a co-transcription factor that forms a heterodimer with RUNX proteins [28]. CBFB was down-regulated after over-expression of miR-30a and -miR30d, and slightly up-regulated in the antimiR-30 condition. Since CBFB was shown to be essential for functions of RUNX1 and RUNX2 [28], these additional data may explain the drastic effect of miR-30 on adipogenesis.
In addition to miR-30 miRNAs, we identified potent up-regulation of other miRNA families, such as miR-378 (35.7-fold), during adipogenic differentiation. A role of decreased miR-378 expression in osteogenesis in the osteoblastic cell line MC3T3-E1 has been suggested recently [29]. Indeed, miR-378 appears to target nephronectin, which is a positive regulator of osteoblastic differentiation. Very recently, Gerin and co-workers [30] identified miR-378/378* as positive regulators of lipogenesis.
Although expressed at lower levels than the highly abundant miR-30 family, two members of the miR-642 family were the most highly up-regulated miRNA in our adipogenesis model. The function of these miRNAs has not been reported before. Of interest, in a recent study identifying the association of miR-519b with human obesity, Martinelli and co-workers [31] also detected that miR-642a was up-regulated in 19 out of 20 fat depots of obese subjects. In our data, no reads corresponding to miR-642a were detected for undifferentiated cells, indicating highly adipogenic-restricted expression. Amongst both miR-642a isoforms, only miR-642a-3p was above the 0.03% threshold in our model. Yet, until recently (September 2010), only miR-642a-5p was present in mirBase release 15 (named miR-642 in release 15) and, thus, detectable on commercial microarrays. The current mirBase release (release 17) includes two miR-642 entries: miR-642a (miR-642a-5p), which was detected at one copy in a unique, high-throughput sequencing experiment; and miR-642b, which is backed by an unknown number of reads.
As shown in Additional file 8, miR-642b is, in fact, located on the opposite strand to miR-642a. The mature sequence annotated in mirBase for miR-642b is the 3p arm of the pre-miRNA. While we also detected miR-642b, this sequence was much less (14-fold) abundant than miR-642a-3p. miR-642a-3p and miR-642b sequences are, in fact, quite similar and only diverge by one base in their 3' end. This observation raises doubts about the bona fide existence of miR-642b. In our dataset, the few reads that were attributed to miR-642b could, in fact, correspond to miR-642a-3p reads bearing sequencing errors. To support this hypothesis, we counted the reads attributed to each miR-642 species within the raw read files. This approach requires conversion of each miRNA sequence into the corresponding color-space sequence, and a perfect match search for these sequences in the read files. This method confirmed that miR-642b was detected at very low levels compared with miR-642a-3p (Table S4 in Additional file 9). We also verified the quality of miR-642a-3p sequencing. Figure S6 in Additional file 9 shows that the positions allowing discrimination of miR-642-3p from miR-642b correspond to high quality values. These values suggest that the corresponding reads were correctly assigned to miR-642-3p.
More generally, this raises questions about the quality of some mirBase annotations. In particular, for miRNAs with highly tissue-specific expression, such as miR-642a, the low numbers of reads backing the mirBase entries might lead to incorrect annotations.
Even though our study focused on miRNAs, we also noted that 34.2% of reads that were mapped to the reference genome did not correspond to any annotated small RNA. Our small RNA cloning strategy only captures small RNAs that are, as miRNAs, 5'-phosphorylated and, thus, eliminates RNA degradation products generated by the major cellular ribonucleases, which generate fragments that are not 5'-phosphorylated.
Some of those un-annotated, small RNAs were significantly regulated during adipogenesis (not shown). Most of the regulated sequences are located within the introns of annotated genes. For instance, we identified an adipocyte-enriched, 21-bp sequence within the fourth intron of NCOA2 (nuclear receptor coactivator 2, or transcriptional intermediary factor 2 (TIF2); Additional file 10). It is noteworthy that NCOA2 is associated with obesity. In fact, TIF2-/- mice are resistant to diet-induced obesity and TIF-/- mouse embryonic fibroblasts store lipids with a much lower efficiency than TIF2+/+ mouse embryonic fibroblasts [32].
We also found that 2.6 to 6.3% of small RNA reads mapped to tRNA sequences. Recently, Lee and co-workers [33] described a new class of tRNA-derived small RNAs, termed tRFs, that are not products of random degradation or biogenesis. In our data, we found abundant reads matching the 5' end of mature tRNA (Additional file 10). No function for this class of small RNA has yet been suggested.
We identified several annotated, but also previously unknown, small RNAs that are regulated during adipogenesis, such as miR-642a-3p. Deep sequencing also allowed the relative abundance of each miRNA to be estimated, revealing miRNAs that reach relatively high expression levels and are, thus, potentially relevant in adipogenesis. Amongst the adipogenesis-induced miRNAs, miR-30 reached the highest levels during differentiation. We show that this miRNA family plays an important role in adipogenesis via the targeting of RUNX2, a major regulator of osteogenesis.
Materials and methods
Cell culture
hMADS cells were obtained from the stroma of human adipose tissue as described previously [34]. Briefly, we used the stroma-vascular fraction of white adipose tissue from young donors (1 month old to 7 years old). Adipose tissue was collected, with the informed consent of the parents, as surgical scraps from surgical specimens from various surgeries, as approved by the Centre Hospitalier Universitaire Nice Review Board. Approximately 200 mg of adipose tissue were dissociated with type A collagenase and the stroma-vascular fraction was separated from the adipocyte fraction by centrifugation. The crude stroma-vascular fraction was plated on uncoated culture dishes; 12 hours after plating, non-adherent cells were removed by a medium change and adherent cells (termed CA by Rodriguez et al. [34]) were maintained in the proliferation medium, which is composed of DMEM (low glucose) containing 10% fetal calf serum, 0.01 M HEPES, 100 U/ml penicillin and streptomycin. The hMADS cell populations included in this study were isolated from a 4-month-old (hMADS) male [34]. HEK 293 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in monolayer culture in DMEM supplemented with 10% fetal calf serum.
In vitrohMADS cell differentiation
Adipocyte differentiation was induced on the day hMADS cells reached confluency. Adipogenic medium was composed of DMEM/Ham's F12 media supplemented with 10 μg/ml transferrin, 0.86 μM insulin, 0.2 nM triiodothyronine, 1 μM dexamethasone, 100 μM isobutyl-methylxanthine and 1 μM rosiglitazone. Three days later, the medium was changed (dexamethasone and isobutyl-methylxanthine were omitted).
Evaluation of hMADS cell adipocyte differentiation
Neutral lipid accumulation was evaluated by Oil red O staining, as previously described [35]. GPDH activity was performed in triplicate wells, using the method described previously [36] (GPDH is an enzyme that is required for the formation of triglycerides). Expression of the adipogenesis-induced markers PPARγ2, FABP4, adiponectin and C/EBPβ was also evaluated by real-time qPCR.
RNA extraction
hMADS cells were lysed by addition of TRIZOL reagent (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) on the cell layers. Total RNAs containing the small RNA fraction were then purified on a RNeasy kit column (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Purity and concentration of total RNA samples were first evaluated using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA samples were run in a RNA nano-chip into a 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA) to verify the integrity of the RNA samples.
Gene expression analysis by real-time qPCR and DNA microarray
RNAs were retro-transcribed with the Mirscript RT kit (Qiagen). Quantitative PCR was performed using LightCycler® 480 SYBR Green I Master mix and Light Cycler 480 real-time PCR machine (Roche Applied Science, Indianapolis, IN, USA). Expression levels of transcripts were evaluated using the comparative CT method (2-deltaCT). Transcript levels of POLR2A and RPL13 were used for sample normalization. Results are log2-transformed fold changes of normalized 2-deltaCT. Data were obtained from three independent experiments and are represented as average ± standard error. Primer sequences are detailed in Additional file 11.
DNA microarrays experiments were performed on Agilent Sureprint G3 Human GE 8x60K microarrays according to the manufacturer's instructions. The experimental data and microarray design have been deposited in the NCBI Gene Expression Omnibus [37] under series GSE29207.
Small RNA cloning and sequencing
Total RNA containing the small RNA fraction were isolated from hMADS cells as described above. The SOLiD™ Small RNA Expression Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) was used to build a library of double-stranded DNA molecules from the population of small RNAs present in the different samples, which were then read using the Applied Biosystems SOLiD™ System sequencing according to the manufacturer's instructions. Briefly, total RNAs containing the small RNA fraction were hybridized (at 65°C for 10 minutes, then at 16°C for 5 minutes) and ligated (at 16°C for 16 hours) to adapters that are provided by the Small RNA Expression Kit. Adaptor mix A (AdA) and adaptor mix B (AdB) were used to produce templates for sequencing small RNAs from the 5' ends and from the 3' ends, respectively. As described in the Small RNA Expression Kit, samples were then reverse transcribed (at 42°C for 30 minutes) to synthesize cDNA and treated with RNAse H (37°C for 30 minutes). Small RNA libraries were amplified by PCR (17 cycles) and size selected on 8% polyacrylamide gels. The 105- to 150-bp material (corresponding to 15- to 50-bp small RNAs) was excised from the gel and eluted in nuclease-free water (70°C for 3 hours). DNA concentrations of all samples were measured by qPCR.
Libraries were amplified by emulsion PCR and sequenced on SOLiD according to the manufacturer's instructions. Read length was 35 bp. The experimental data have been deposited in the NCBI Gene Expression Omnibus under series GSE25715.
Small RNA deep sequencing data analysis
Color-space reads were matched against annotated databases using the Small RNA Analysis Pipeline Tool v5.0 (RNA2MAP), provided by Applied Biosystems, using the following parameters: one color-space mismatch within the first 18 bases of the reads, called the 'seed sequence' and two color-space mismatches on the following positions of the reads. First, small RNA reads were matched against the human genome (hg19), then versus miRBase release 16 to identify matches with non-human miRNA, and finally versus non-coding RNA sequences from fRNAdb, a database of ncRNA.org. For each annotated miRNA that was sequenced, the number of sequences for miRNAs was normalized to a total of 106 miRNA sequences. The amount of each miRNA was determined following a linear model. Only miRNAs with at least 300 counts per million in at least one of the experimental conditions were conserved for differential expression analysis. The significance of the difference between the experimental and control groups was estimated by an empirical Bayes method using the limma package from Bioconductor [38].
Inactivation and over-expression of miRNAs
All transfections were performed with HighPerfect transfection reagent (Qiagen). For inactivation of miRNA expression, sub-confluent hMADS cells were transfected with a combination of three DNA/LNA mixmers with a phosphorothioate backbone (Exiqon, Vedbaek, Denmark), at a final concentration of 40 nM for each. The sequences of these three DNA/LNA mixmers were 5'-CAGTCGGGGATGTTTAC-3', 5'-CAGTCGAGGATGTTTAC-3', and 5'-GAGTGTAGGATGTTTAC-3'. The simultaneous use of these three oligonucleotides successfully inhibited all miRNAs from the miR-30 family (Exiqon, personal communication; Additional file 6). The sequence for the mismatch control oligonucleotide was CAGTCGAAGCTGTTTAC.
For over-expression, sub-confluent hMADS cells were transfected with Pre-miR™ miRNA precursor molecules (Ambion, Life Technologies Corporation, Carlsbad, CA, USA), at a final concentration of 40 nM. The negative control was the 'Pre-miR™ miRNA Precursor Molecules - Negative Control #1'.
For both over-expression and inhibition studies, hMADS cells were submitted to adipogenic medium 3 days after transfection. For the target protection experiment, sub-confluent hMADS cells were transfected with TSBs, which are custom designed LNA oligonucleotides with a phosphorothioate backbone (Exiqon); the sequences were 5'-ACATGAAGTAAACACACA-3' for miR-30-TSB and 5'-CAGTCGAAGCTGTTTAC-3' for TSB-neg (mismatch control). TSBs were used at a concentration of 20 nM. The day after this first transfection, hMADS cells were co-transfected with miR-30 Pre-miR™ miRNA precursor molecules (Ambion) at a final concentration of 40 nM, together with the miR-30-TSB again, or the mismatch control TSB. hMADS cells were then submitted to adipogenic medium the day after the second transfection.
Cloning of RUNX23' UTR in pSi-CHECK™-2
Partial sequences (positions 32 to 332 and positions 3,102 to 3,421) from the 3' UTR of RUNX2 (ENST00000465038) were amplified by PCR and cloned at the XhoI and NotI sites of pSi-CHECK™-2 (Promega, Madison, WI, USA). Synthetic miRNAs (miR-30a, miR-30d and miR-378) as well as negative control (miR-Neg) were purchased from Ambion. HEK 293T cells (20,000 per well) were reverse transfected in 96-well white plates with 100 ng of pSi-CHECK™-2 plasmid and 5 nmol of synthetic miRNAs using 1 μl of lipofectamine 2000 (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). The, 48 hours after transfection, renilla and firefly luciferase activities were assayed with the Dual Glo Luciferase Assay System (Promega) and measured with a luminometer (Luminoskan Ascent, Thermo Scientific, Waltham, MA, USA).
Preparation of cell extracts and western blot analysis
Cells were rinsed with phosphate-buffered saline and solubilized in stop buffer containing 50 mmol/l HEPES, pH 7.2, 150 mmol/l NaCl, 10 mmol/l EDTA, 10 mmol/l Na4P2O7, 2 mmol/l Na3VO4, and 1% Triton X-100 supplemented with Protease Inhibitor Cocktail (Roche). RUNX2 antibody (MBL, Woburn, MA, USA) was used at a final concentration of 0.5 ng/μl. Secondary horseradish peroxidase-conjugated antibody was purchased from Promega.
- bp:
base pair
core binding factor beta
CCAAT/enhancer-binding protein
Dulbecco's modified Eagle's medium
fatty acid binding protein 4
glucose-dependent insulinotropic polypeptide receptor
glycerol-3-phosphate dehydrogenase
- hMADS:
human multipotent adipose-derived stem
- miRNA:
nuclear receptor coactivator 2
- piRNA:
piwi-interacting RNA
peroxisome proliferator-activated receptor
- qPCR:
quantitative polymerase chain reaction
runt-related transcription factor 2
- snoRNA:
small nucleolar RNA
transcriptional intermediary factor 2
target site blocker
untranslated region.
We acknowledge the excellent support from the Nice Sophia-Antipolis Functional Genomics Platform and from Niels Frandsen from Exiqon. This work was supported by CNRS, the Association pour la Recherche sur le Cancer (ARC post-doctoral Fellowship to LEZ and grant number 4983) and INCa (PL0079, PB).
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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.


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Boxham tree
One recent rare sunny day here in the UK I made sure I got out and about in Edenbridge to have a walk on the fields and snap some shots. This is a shot of a tree with Broxham Farm in the background.
And thanks for everyone putting the HDR version to #11 for Feb 22, 2008 on Explore!
This photo is part of my .photography collection on flickr.
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Taken on February 10, 2008

subset: commoncrawl


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"...France's leading nutritionist Dr. Jean-Michel Cohen shares 150 easy-to-prepare recipes, practical checklists, and addresses the physical, psychological, and cultural factors that impact our ability to control our relationship with food. Through his progressive, holistic three-step weight loss plan you'll get the best-practices of the French way of eating, a new approach to food and a way to celebrate life, helping you look and feel your best." ~San Francisco Guardian
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The Zagat Review
Chef-owner Robert Wiedmaier upholds "haute-cuisine" traditions at his West End flagship offering "exceptional" French-Belgian "classics with a twist" served with "panache" in an "elegant but not pretentious" atmosphere; the package includes a "luxurious" contemporary space, and while its expense-account pricing leads some to reserve it as a "special treat", there's an "excellent" pre-theater deal that includes transport to and from Kennedy Center.
Zagat reviews are compiled from individual user reviews. Write your review.Write your review.
What To Order
Insider Tip
Sommelier Moez Ben Achour presides over an award-winning wine program that begs for exploration.
Marcel's by Robert Wiedmaier
2401 Pennsylvania Ave NW
Washington, District of Columbia 20037
2401 Pennsylvania Ave NW | Map
Washington, District of Columbia 20037


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text: OverviewBack to top Jarrow Formulas - Amino Surge - 250 Tablets Item# :63715UPC# :790011150039 Brand:Jarrow Formulas Size/Form:250 Tablets Dosage:1 Tablet(s) AMINO SURGE™ is a rich source of various forms of amino acids. AMINO SURGE™ is especially rich in Branched Chain Amino Acids (223 mg per tablet), which are essential for formation of muscle mass and positive nitrogen balance.AMINO SURGE™ is a rich source of Amino peptides from whey proteins which is the richest source of Branched Chain Amino Acids, resulting in an ideal amino acid ratio and superior absorption.AMINO SURGE™ is formulated with synergists that enhance the absorption, metabolism and efficacy of amino acids. Amino acids are relatively simple molecules with a molecular weight of about 100. All body proteins are produced from amino acids. Amino acids play a role in energy production, as the carbon backbone can also be shunted into the Tri-Carboxylic Acid (TCA), or Krebs cycle. Structural proteins, such as collagen, cartilage and elastin are rich in amino acids. Amino acids are also modified to produce hormones, enzymes, neurotransmitters and other substances.Keep out of the reach of children.


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- Properties
- Agents
- Offices
- About the Area
- News
- Videos
- About Us About Us
- Preferences
- Back
- Change Currency:
With professional experience spanning both the east and west coasts of the U.S., Christine Krenos has developed sensitivity to the diverse lifestyles and cultural distinctions of her often bi-coastal and international clientele. Her patience, understanding, diplomacy and desire to truly listen to her clients' needs and goals combine in communication skill unsurpassed among her peers. Christine's clients and colleagues alike also know her as highly organized, proficient at win-win negotiation and possessing boundless energy.
A Pennsylvania native, Christine earned a Bachelor's Degree in Education, followed by a Certificate in Strategic Hospitality Management from Cornell. Her subsequent professional positions took her to Florida, Maryland, Washington, D.C. and Los Angeles during her years spent as an educator and well over a decade in management in the hospitality industry. Her major supervisory role on the opening team of The Ritz-Carlton, Los Angeles and JW Marriott Los Angeles L.A. LIVE, for example, contributed immensely to her business, communication and organizational acumen. Overall, those invaluable experiences allowed her to develop and build on the organization, negotiation and people skills vital in her role as a Realtor.
Today, Christine works with other highly successful real estate agents and represents the finest Napa Valley and Sonoma Valley properties and the most discriminating buyers and sellers. Dedicated to providing a demystified and fulfilling real estate experience for her clients, she is proud of her ability to truly listen to and understand people and their unique situation. That ability, combined with her myriad talents and skills, places her in an elite group of real estate professionals. It is an echelon requiring constant development in the areas of contracts, market study, state-of-the-art marketing technology and more. Even her personal interests such as tennis and Bikram Yoga are challenging on many levels. In fact, accepting and excelling at both professional and personal challenges are what ultimately sets Christine apart from her peers and spell success on behalf of her clients.
m: (707) 227-8661o: (707) 227-8661f: (707) 935-1784
25 East Napa Street
Sonoma, CA 95476
Office: (707) 227-8661Fax: (707) 935-1784


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Just a quick update to our supporters!
VIDA and our Chilean partner FEDES are planning to establish 12 health clinics that will be supported by a Chilean physician group who will staff the clinics on a rotating basis.
In April 2011, a VIDA corporate partner visited FEDES projects in Chile to assess various program options.
The project is currently identifying facilities to house the clinics and also exploring various mobile clinic options.
The program is also working with Chilean vintners to create a sustainable project. Viva la vino!
There is much more work to do - but with your help we can do it!
Project Reports on GlobalGiving are posted directly to globalgiving.org by Project Leaders as they are completed, generally every 3-4 months. To protect the integrity of these documents, GlobalGiving does not alter them; therefore you may find some language or formatting issues.
If you donate to this project or have donated to this project, you will get an e-mail when this project posts a report. You can also subscribe for reports via e-mail without donating.
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Assistance with data analysis - repost
- Status Closed
- Budget €30 - €250 EUR
- Total Bids 6
Project Description
I require assistance analysing data in R for a small project. I have done much of the work myself but have encountered a few errors that I need help with. I also need some aspects explained.
Freelancers should be:
1. Willing to work with me until I am 100% satisfied. Only when all errors are resolved and I am 100% happy will payment be released.
2. Fluent English and an ability to explain things clearly is required.
3. Bidders should be experts in R, particular CLUSTER ANALYSIS
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Scallops With Lemon Garlic And Parsley
|Fresh chopped parsley
|1⁄2 Cup (8 tbs)
|2 Clove (10 gm), minced
|2 Tablespoon
|Olive oil
|2 Tablespoon
|Sea scallops
|1⁄2 Pound, trimmed
|Dry vermouth
|1⁄2 Cup (8 tbs)
|1⁄4 Teaspoon
|1⁄8 Teaspoon
1. Grate yellow zest from lemons, being careful not to include any bitter white pith. In a small bowl, combine lemon zest, parsley, and garlic. Set gremokta aside.
2. In a nonreactive large frying pan over medium-high heat, melt butter with olive oil. Add scallops to pan and cook 2 minutes on each side, or until opaque but still tender and juicy. Add vermouth and heat, 1 to 2 minutes. Remove from heat. Season with salt and pepper. Add half the gremolata to pan and toss together. Pass remaining gremokta on the side.
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 783 Calories from Fat 499
% Daily Value*
Total Fat 57 g87.1%
Saturated Fat 19.8 g99%
Trans Fat 0 g
Cholesterol 139.4 mg46.5%
Sodium 881 mg36.7%
Total Carbohydrates 24 g7.8%
Dietary Fiber 6.2 g24.7%
Sugars 1.6 g
Protein 41 g81.8%
Vitamin A 43.3% Vitamin C 198.7%
Calcium 18.3% Iron 17.9%
*Based on a 2000 Calorie diet


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yes limo taxis
Chauffeur Service, Limousine Service, Airport Transfers
Our chauffeurs
Our clean Luxury Limousines and Taxis for chauffeur services are driven by Professionally Experienced Drivers who with their profound skill ensure that your trip is Enjoyable and that you arrive at your destination on time, in style and with comfort.
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Our Cost effective chauffeur services can be used for
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Airport Transfers
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Sightseeing and day tours
Wedding cars& ceremonial transports
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Limousine Hire Services
Special parcel Delivery Services
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text: Pinch to zoom Personalised Silver Bar Necklace by Soremi £65$111.55€77.90$83.29 121 customer reviews(121) Favourite Favourite View your favourites chain chain (required)16 inch trace chain18 inch trace chain (+ £3.50)20 inch trace chain (+ £4.50)16 inch curb chain (+ £7.50)18 inch curb chain (+ £8.50)20 inch curb chain (+ £12)24 inch curb chain (+ £15)chain (required)16 inch trace chain18 inch trace chain (+ $6)20 inch trace chain (+ $7.72)16 inch curb chain (+ $12.87)18 inch curb chain (+ $14.58)20 inch curb chain (+ $20.59)24 inch curb chain (+ $25.74)chain (required)16 inch trace chain18 inch trace chain (+ €4.19)20 inch trace chain (+ €5.39)16 inch curb chain (+ €8.98)18 inch curb chain (+ €10.18)20 inch curb chain (+ €14.38)24 inch curb chain (+ €17.97)chain (required)16 inch trace chain18 inch trace chain (+ $4.48)20 inch trace chain (+ $5.76)16 inch curb chain (+ $9.61)18 inch curb chain (+ $10.89)20 inch curb chain (+ $15.37)24 inch curb chain (+ $19.22) number of silver barsnumber of silver bars (required)OneTwo (+ £28)Three (+ £56)Four (+ £84)Five (+ £112)number of silver bars (required)OneTwo (+ $48.05)Three (+ $96.10)Four (+ $144.16)Five (+ $192.21)number of silver bars (required)OneTwo (+ €33.55)Three (+ €67.11)Four (+ €100.67)Five (+ €134.23)number of silver bars (required)OneTwo (+ $35.87)Three (+ $71.75)Four (+ $107.63)Five (+ $143.51) personalisation (required) personalisation personalisation personalisation personalisation text on two sides of bartext on two sides of barYes please (+ £10)text on two sides of barYes please (+ $17.16)text on two sides of barYes please (+ €11.98)text on two sides of barYes please (+ $12.81) guaranteed day after dispatch deliveryguaranteed day after dispatch deliverySpecial delivery please (+ £6.90)guaranteed day after dispatch deliverySpecial delivery please (+ $11.84)guaranteed day after dispatch deliverySpecial delivery please (+ €8.26)guaranteed day after dispatch deliverySpecial delivery please (+ $8.84) quantity personalise & pay by Soremi Location: W. Yorks, GB read seller's profile see seller's complete range more items from this seller Personalised Hidden Initial Necklace by Soremi £60$102.97€71.91$76.88 Personalised Solid Gold Bar Necklace by Soremi £356$610.96€426.66$456.17 Silver Personalised Bar Charm Pendant by Soremi £85$145.87€101.87$108.91 Silver Personalised Satin Men's Necklace by Soremi £69$118.41€82.69$88.41 Personalised Scroll Necklace by Soremi £98$168.18€117.45$125.57 Personalised Mens Hoop Bracelet by Soremi £65$111.55€77.90$83.29 view all you may also like PRODUCT DETAILS CUSTOMER REVIEWS DELIVERY RETURNS Made in Britain can be personalised exclusive ask seller a question save for later Personalised sterling silver hand punched bar pendant, hand made by Ghazal and her lovely team of jewellers in their Yorkshire Studios. This timeless pendant is the perfect way to carry text of your choice on your jewellery. One of our very first designs, it has become one of our classics. Often copied but never bettered! Each bar has our trademark 'perfectly imperfect' hand punched text of your choice which we carefully apply to the sides of each bar. The cost is £65 for the first bar and necklace and then a further £28 per bar thereafter. Each bar varies in size from 2.5cm to 5cm long. The pendants come on a choice of necklaces: 40.5cm (16"), 45.5cm (18") and 51cm (20") trace chains (as photographed) which have a more delicate look, and 45.5cm (18") and 51cm (20") curb chains which are a heavier/thicker chain. Simply let us know which names/words you would like and their spelling. Characters available : All letters in upper case only, numbers 0 -9, : . , &. Where coordinates are required we will use a . in the appropriate place to denote 'degrees' . We also offer smaller letters too (1mm) eg for McRALANE. Please note that these pendants are made entirely by hand with hand punched letters and are therefore not uniform – any irregularities are part of the design and charm of the necklace. Bars lengths and their order are chosen by Ghazal in order to best achieve the maximum aesthetical effect unless otherwise requested. Characters available : All letters of the alphabet in capitals, 1-10 . , ' " ? ! # & made from: 925 Sterling Silver dimensions: Each bar varies in size from 2.5cm to 5cm long. product code: 43101 121 customer reviews see more customer reviews read about our product rating system ratings guide trusted independent customer feedbackforum excellent bad good poor This product is dispatched to you by Soremi. Personalised items will take 1-2 weeks to dispatch form time of order. You have 28 days, from receipt of cancellable goods, to notify the seller if you wish to cancel your order or exchange an item. Please note: goods that are personalised, bespoke or made-to-order to your specific requirements, perishable products and personal items sold with a hygiene seal (cosmetics, underwear) in instances where the seal is broken are non-refundable, unless faulty. Read more about our returns policy.Should you choose to return or exchange your order you will need to deliver the item(s) to the UK, where this seller is based. Soremi's return policy Please see notonthehighstreet.com's returns policy.


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Riverton FamilySearch Library/Youth Conference
Search and Serve Youth Conference
The Riverton FamilySearch Library offers a one day Family History youth conference activity for ward and multi-wards.
The library youth conference activities are designed to help youth and their leaders feel the Spirit of Elijah and learn how its influence can bless the lives of both the living and the dead through Family History and Temple Ordinance work.
Workshops provide instruction on the key elements of genealogical research and offer hands-on experience to help participants with their individual family history responsibilities. Young Men and Women will also be encouraged to use their technical skills to help their own family, ward members, friends and others with their family history efforts.
Elder David A. Bednar's Invitation and Promise to Youth
"The skills and aptitude evident among many young people today are a preparation to contribute to the work of salvation.
"I invite the young people of the Church to learn about and experience the Spirit of Elijah. I encourage you to study, to search out your ancestors, and to prepare yourselves to perform proxy baptisms in the house of the Lord for your kindred dead. And I urge you to help other people identify their family histories.
"As you respond in faith to this invitation, your hearts shall turn to the fathers. …Your love and gratitude for your ancestors will increase. Your testimony of and conversion to the Savior will become deep and abiding. And I promise you will be protected against the intensifying influence of the adversary. As you participate and love this holy work, you will be safeguarded in your youth and throughout your lives."
2013 Schedule
This activity will be available every Wednesday, Thursday and Friday; June 5 through August 23, 2013; except for June 7, July 3, 4, 5, 24, and August 2.
The deadline for registration is May 1, 2013. Reservations will be taken on a first-come, first-served basis. Book early to secure a date.
Click Here for the Online Registration Form
Group Sizes
The library can accommodate ward or multi-ward groups with a maximum of 100 youth and leaders.
- A FamilySearch lanyard and all training material for the workshops will be furnished to participants at no cost. There will be a charge for the printing of optional pedigree fan charts.
- The cost of lunch at the Riverton Office Building cafeteria will be approximately $5.50 for a hamburger, fries and a large drink.
Detailed instructions for participants will be sent to leaders following registration. However, it is important to note that all participants must register for an LDS account. Youth and leaders need to have access to their username and password to participate fully in the conference activities.
Conference Workshop Outline
10:00 a.m. - Welcome and Introduction
The conference will begin with a short overview of the library's resources and layout, an outline of the day's activities, and some important guidelines.
= =


text: Located on Ao Nang's main esplanade is this highly acclaimed 140 seat seafood restaurant that attracts rave reviews on Trip Advisor. Glorious views and sunsets over the Andaman set the scene for an idyllic dining experience. Menu pricing is moderate and attracts the customers but the business still maintains an excellent income far in excess of the asking price. The restaurant has two levels with indoor and outdoor seating, upstairs lounge and an ocean-terrace. Approximate floor space of 430sqm. Three extra rooms are available for staff accommodation. The restaurant has been in operation for over 11 years and the owners now want to take a back seat from running such a busy restaurant. Ao Nang's tourism is ever increasing and businesses in such a prime location and at this price are in short supply as no one wants to sell due to the large revenues. A turn-key business that will break even in around one year of operation and some serious money can be made by a committed buyer.


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Healthy communities — including those that attract active, health-conscious retirees — begin with healthy mothers and babies.
That is why it's imperative to invest in initiatives designed to provide all pregnant women and newborns, regardless of race or income, with adequate care and counseling to promote good health and well-being.
The public and private sectors have made such investments in Sarasota County since 2008 — creating and operating the "Save My Life" program, which targets mothers and babies at the greatest risk of health problems.
But as reporter Dale White and Dr. Washington Hill recently wrote in the Herald-Tribune, funding for the program will expire at the end of February.
A modestly priced fund-raising event will be held tonight; if $5,000 is raised, an anonymous donor will provide a matching donation. Those contributions would enable the program to continue operating in the short term.
The event organizers and donor deserve credit for recognizing the need for, and value of, Save My Life.
Yet this program is so vital — to at-risk mothers and their children, and to the entire community — that it warrants even greater investments, providing the potential for long-term sustainability.
A community-based program
Save My Life was not initiated by distant bureaucrats seeking to spend money on generic programs. It originated after student researchers at the University of South Florida noticed a spike in fetal deaths among African-Americans in Sarasota County.
The Healthy Start Coalition — whose goal is to improve the health and well-being of all Sarasota County's pregnant women, infants and young children — worked with a small group of doctors and social-service advocates to begin providing prenatal and post-natal services to women and children in Newtown, the longtime center of Sarasota's African-American community.
The Community Foundation of Sarasota County provided grants to fund start-up and operational costs; some state Health Department dollars and private donations supplemented the grants.
As a result, Save My Life has worked to address racial disparities in what Dr. Hill, a highly regarded specialist in maternal and fetal medicine, calls "birth outcomes."
The initiative is tailored to the African-American community, which experiences significantly higher rates of fetal or infant mortality, premature births and low-birth weights than the rest of the population.
Classes and outreach
Hill described the essence of the program in a guest column Monday:
"This program offers small group and individual classes on the importance of health during childbearing years and during pregnancy, stress reduction, childbirth education, breast-feeding education, in-home support for breast-feeding and safe sleep practices for infants called 'Back to Back for a Safe Sleep.' Outreach efforts, education and classes are offered by the only African-American childbirth educator in Sarasota County, who is trusted and well known in the community."
These services have been provided for three years at a nominal cost of $89,000; the childbirth educator is a contract employee who pays her own taxes and health-insurance premiums. An additional $6,000 in grants have subsidized the purchase of safe bassinets for newborns.
Investments in Save My Life provide benefits not only to individual mothers and children but to the community.
The care of premature and low birth-weight babies is extraordinarily expensive; infants and children who receive inadequate care and attention are more likely to have difficulty learning and becoming productive citizens.
Ideally, the state Health Department would help fund this program and similar initiatives in every county — at least partially.
But the fate of Save My Life, and the health and well-being of the entire county, is in this community's hands.
* For more information about Save My Life, call (941) 373-7070.
Saving 'Save My Life'
Healthy communities — including those that attract active, health-conscious retirees — begin with healthy mothers and babies.


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Serious wine enthusiasts need a serious wine glass. A wine glass that works to capture every nuance of every wine. This fantastic all-rounder does just that. Set of two.
Thank you for visiting Dartington Crystal Wine Masters Chefs Taster Glasses, Set of 2 on shoppingfind.co.uk
We bring the products from many websites to one website for you to search, save and compare.


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Currently, the Highlander is offered in both naturally aspirated and hybrid versions, with front- and all-wheel drive. We don't expect the formula to change too much, since sales of the Japanese CUV are still steady – well in line with newer players in the segment like the Ford Explorer, Nissan Pathfinder and GM's Lambda triplets (Chevrolet Traverse, GMC Acadia and Buick Enclave).
We'll have the full skinny on the new Highlander in the coming weeks. For now, scroll down to read Toyota's incredibly brief press blast.
TORRANCE, Calif., March 13, 2013 – Toyota will stage the world debut of the all-new 2014 Highlander mid-size SUV at a press conference at the 2013 New York Auto Show.
The press conference will be held on Wednesday, March 27, at 9:10 a.m. Eastern Time in the Galleria Level Four at the Jacob Javits Center.


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Hotel Ristorante Vittoria
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The Middle of Nowhere
It has happened my friends. We are almost incommunicado. Satellite coverage has dropped to its lowest levels. So, it would be almost impossible for me to upload photos.Therefore, I will finish my blog after I get home and have a normal internet connection. Sorry.
Many activities on the ship; talent shows, galley tours, movies, and the usual relaxation pursuits such as reading. I am wading through the biography of Andrew Carnegie at ...
To Florence
... to move and much consternation - police are there too. It seems that two cars are parked on the corner and around the corner making it impossible for the bus to negotiate the very tight corner. We watch the drama unfold, as one of the drivers, an elderly woman, who seems to pretend to have a bad hip and plead sympathy, returns to her car. She seems to be arguing with the 2 young women police officers and being very dramatic about her hip. Finally she moves her car ...
Last day of Tour
... honey flavours & fresh goats cheese. Our last lunch of the tour was a degustation lunch, a 9 course feast, from which we are still recovering! Lol absolutely amazing & a great way to finish a wonderful week . 4 antipastos. 2 pastas, 2 meats & dolce . They sure know how to eat over ...
Torino (Turin) Grand City
... but I have yet to try a gelato better than Hunter Belle's ? This afternoon we start our official Food & wine tour with aperitifs & dinner......... & what a lovely night it was. Our tour guide Rob, a winemaker from Margaret River WA seems like a great guy & full of local knowledge. Our other member of our tour is Susan from Ballina. Think it's going to be a fun week.Had a beautiful dinner at a very special restaurant. Great ...
Chapter 6 - I don't like Mondays
... up the atmosphere of central Turin. Exhausted, we returned to the hotel and, to be honest, didn't feel up to facing the outside world just at that moment. And so we treated ourselves to a night in watching the only channel that was in English: BBC News. The Greek crisis was at it's peak and we lay in the cool of the room watching live coverage of the potential Grexit from the EU. Have just about never had a better night in my life.
The morning ...


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Pharmacology is all around us - discover more
19 Nov 2015
The Royal Institution of Great Britain, London
Sir Salvador Moncada was invited to share his considerable experience and insights of "Pharmacology and the understanding of vascular biology: the last 45 years and looking to the future"
07 Oct 2015
The Victoria Gallery & Museum, Liverpool
Encouraging Fellows and Honorary Fellows to meet and network as a group, this will also be an opportunity to congratulate the Society's newest Fellows.
19 Jun 2015
Sheffield Motorpoint Arena, Sheffield, UK
Led by the Society of Biology Careers Committee.
15 Jun 2015
Glassboat Restaurant, Welsh Back, Bristol, UK
Encouraging all members, from undergraduates to honorary fellows, to meet and network.
05 – 06 Jun 2015
Cheltenham, UK
The British Pharmacological Society and the Biochemical Society led this activity to help visitors to have a go at designing their own medicine.
04 Jun 2015
A discussion of the current state and future of prescribing in the NHS and and how individuals can engage in future prescribing decisions taken on their behalf by their doctors.
14 Apr 2015
National Museum of Scotland, Edinburgh, UK
A discussion of the future of prescribing in the NHS and and how individuals can engage in future prescribing decisions taken on their behalf by their doctors.
13 – 14 Apr 2015
UWE, Bristol, UK
07 Apr 2015
Presented by The Physiological Society and the British Pharmacological Society
29 Mar 2015
Westin Boston Waterfront, Boston, USA
Hosted by the British Pharmacological Society.


text: OverviewBack to top Dolisos - EluSun - 60 Capsules Item# :60397UPC# :050860144611 Brand:Dolisos Size/Form:60 Capsules Servings:30 Dosage:2 Capsule(s) Just because summer has faded doesn't mean your tan has to. Elusun self-tanning supplements help you maintain a healthy golden glow without exposing your skin to the harsh rays of the sun. Using the richest source of natural carotenes, Elusun works from the inside out to advance the skin's natural pigmentation process, promoting an even head-to-toe tan with no messy bronzing gels or creams.This unique three-in-one formula also contains Vitamins C and E - valuable antioxidants known to reverse the signs of aging, as well as Borage oil to help your skin maintain proper moisture levels.


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French Connection jersey print dress is a versatile option for daytime. Pair with pumps and a chunky knit cardi for the weekend.
Dandelion Jersey Dress has a V-neck and button-fastening placket with contrast trim, short sleeves and attached fabric tie.
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I have mixed feelings about TED talks. In the beginning, I was addicted…Dan Gilbert's 2004 presentation about Happiness might have been the first one I watched more than once …and who can forget Brene' Brown sharing her research on vulnerability? Goosebumps! Of course, the popularity of TED made everyone want to learn how to do "that." Its about great storytelling, right? Yes… if the stories are true. Particularly when it comes to scientific studies. But that's not what happened.
A bit more research and I discovered my beloved Dan Gilbert was telling stories as well. Gilbert issued corrections to two of the stories he told in that first video. No wonder I've been so uncomfortable with the way storytelling is being taught. Making a truth more vivid and engaging is one thing, but recent leaps of inference or stories that make a correlation sound like cause/effect? that just keeps us in the dark. Specifically videos about recent neurological studies that purport to explain storytelling. I think we need to stop "telling stories" that oxytocin or the magic of a "narrative arc" explain storytelling. It's much bigger than science can explain. Storytelling is an art – subjective, emotional, and as variable as humanity is diverse.


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Fashioning London
Clothing and the Modern Metropolis
By (author) Christopher Breward
Format: Hardback
Normal Price: $130.00
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Fashioning London by Christopher Breward
Book DescriptionOver the past three centuries, London has established itself as one of the worlds most inventive fashion capitals. City life and fashion have always been intertwined, but nowhere has this relationship been more excitingly expressed than on the streets of London. Fashioning London looks at the manner in which particular styles of dress became associated with this leading international city, ultimately challenging the dominance of Paris, Milan and New York.From the ballrooms and boxing rings of the eighteenth century, through Victorian extremes of poverty and conspicuous consumption, to the flamboyant explosions of subcultural taste that define the capital today, Londoners have constantly offered an idiosyncratic reading of fashionability that has profoundly influenced the nature of style elsewhere. Breward constructs an original history of clothing in London its manufacture, promotion and cultural meaning while showing how issues of space, architecture and performance impinge on notions of fashionability. It highlights the importance of such outfits as the dandy's suit, the dolly bird's mini-skirt and the second-hand ensemble of the punk in forming our understanding of the capital's distinctive character. Drawing on a range of sources, including paintings, street photography, maps, tourist guides, literature, stage and press representations, Fashioning London paints a vivid and definitive portrait of Londons iconoclastic style.
Buy Fashioning London book by Christopher Breward from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9781859737873
(234mm x 156mm x 14mm)
Pages: 224
Imprint: Berg Publishers
Publisher: Bloomsbury Publishing PLC
Publish Date: 4-Mar-2004
Country of Publication: United Kingdom
Other Editions...
Books By Author Christopher Breward
Hot Metal, Hardback (October 2016)
Hot Metal focuses on the experience of Australian print-workers between the 1960s and 1980s, concentrating on labour, production, design and culture in the context of deindustrialisation.
Suit, Hardback (April 2016)
Beautifully illustrated and written with authority, Suit offers new perspectives on this most mundane, and at the same time poetic and beautiful, product of modern culture.
Fashion Studies, Hardback (January 2016)
Through case studies focussing on the work of an international range of scholars, Fashion Studies examines how ethnographic and material culture methodologies can be applied effectively in fashion research practice.
Material Relations, Paperback (December 2015)» View all books by Christopher Breward
Material relations tells the story of nineteenth and early twentieth century middle-class families by exploring the domestic spaces they inhabited and the material goods they prized. By opening the doors of the house, the book sheds new light on aspects of family life including love, marriage, sex, childhood and death.
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Author Biography - Christopher Breward
Christopher Breward is Professor in Historical and Cultural Studies, London College of Fashion.
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Who Works for Startups? The Relation between Firm Age, Employee Age, and Growth
Paige Parker Ouimet
University of North Carolina at Chapel Hill
Rebecca Zarutskie
Federal Reserve Board
October 1, 2011
US Census Bureau Center for Economic Studies Paper No. CES-WP-11-31
We present evidence that young employees are an important ingredient in the creation and growth of firms. Our results suggest that young employees possess attributes or skills, such as willingness to take risk or innovativeness, which make them relatively more valuable in young, high growth, firms. Young firms disproportionately hire young employees, controlling for firm size, industry, geography and time. Young employees in young firms command higher wages than young employees in older firms and earn wages that are relatively more equal to older employees within the same firm. Moreover, young employees disproportionately join young firms that subsequently exhibit higher growth and raise venture capital financing. Finally, we show that an increase in the regional supply of young workers increases the rate of new firm creation. Our results are relevant for investors and executives in young, high growth, firms, as well as policymakers interested in fostering entrepreneurship.
Number of Pages in PDF File: 43
Date posted: October 20, 2011
Suggested Citation
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Welcome to The Mystic Sea! Welcome to the realm of Mara Gimel. Mara Gimel means Camel Of The Mystic Sea. It is my role to lead you through this world of the occult, the unseen, the unexplained .... enter this realm with an open mind, and remember there is more to this world than we are being told .... it's time to remember what you have forgotten.
Tuesday, 2 July 2013
Quote for the Day
"If you see your path laid out in front of you -- Step one, Step two, Step three -- you only know one thing... it is not your path. Your path is created in the moment of action. If you can see it laid out in front of you, you can be sure it is someone else's path. That is why you see it so clearly." -- Joseph Campbell


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(based on 14 reviews)
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Beautiful and Affordable
By Tammy
from Overland Park, KS
About Me Prhris Administrator
Comments about Baudville Acrylic Glacier Trophy - Small:
Bottom Line Yes, I would recommend this to a friend
Was this review helpful? Yes / No
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Impressive and affordable
By Manager
from Fort Lauderdale, Florida
About Me Healthcare Industry
We presented this acrylic glacier trophy to our employees to recognize the great work they have contributed year after year upon winning a prestigious award. The waterfall edges appeared 3-D. Precise laser engraving personalized are pre-written award verse
Service and delivery comments:
The delivery was prompt. The product arrived in excellent condition and was securely packaged. The customer service representative and recognition specialist were very helpful. We would definitely use Baudville again!
Great Quality
By Bridget
from Toledo, OH
Turned out great, quick delivery.
Might be small, but has BIG impact!
By For the Bank
from Williamsport, PA
About Me Dept Manager
I needed an award that wasn't extravagent, but well stated in appearance. This did it! It might be on the smaller side, but the design and affects make this a great piece.
Excellent product
By Stacey
from San Dimas, CA
We purchased these trophies for our annual awards for "best performers." Our budget is limited, so we chose the small acrylic glacier trophy. I was concerned that it might be too small, but it ended up larger than I expected, and perfect for our needs. As always, the personalization came out beautifully.


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Married couple charged with murdering Heather Elvis after husband's affair as house search 'reveals evidence of killing'
- Sidney and Tammy Moorer were arrested on Friday and charged with indecent exposure and obstructing justice in the case of Heather Elvis
- On Monday they appeared in court and the charges of kidnapping and murder were added
- Authorities said they had probable cause to charge them for murder after a search of their home on Friday
- Elvis, 20, vanished in December after driving her car to a boat landing
- Cell phone records show Sidney Moorer - who authorities say had had an affair with Elvis - was in contact with her on the day she went missing
A South Carolina couple suspected in the mysterious disappearance of the husband's former lover have now been charged with murdering her.
Sidney and Tammy Moorer kept their heads down as they appeared in court in Conway, South Carolina on Monday - three days after authorities searched the couple's home and found evidence giving them probable cause to charge the Moorers with killing 20-year-old student Heather Elvis.
Horry County Police Chief Saundra Rhodes said that authorities have not yet found Elvis' body but continue to carry out investigations. Elvis' family have spoken of their hope she will be found alive.
The couple, who have three children, were arrested on Friday on indecent exposure and obstructing justice charges. Kidnapping and murder charges were added at Monday's court appearance.
Scroll down for video
Charged: Sidney Moorer, left, and his wife Tammy, right, appeared in a South Carolina court on Monday charged with the murder and kidnapping of Heather Elvis, 20, more than two months after she was last seen
New charges: The Elvis family, left, sits at a table at a hearing for Sidney Moorer on Monday
Elvis, a cosmetology student, vanished on December 18 after she drove her car - willingly, authorities believe - to a boat landing building in Myrtle Beach. It was found empty on December 19.
Cell phone records show Sidney Moorer, 38, was in contact with Miss Elvis at 6am on the day she disappeared.
In the original police report, seen by WMBF News, Miss Elvis was said to have been in a relationship with Moorer, who she had met while working in a restaurant.
Her roommate had told police the affair had ended about a month before Miss Elvis disappeared, and that on the night she went missing, the pair were back in contact.
The roommate allegedly said Miss Elvis told her Moorer had called to say he was leaving his wife so he could be with her.
A December 19 police report included a statement from Sidney Moorer admitting he had been in contact with Miss Elvis, to tell her to 'quit calling him and that was it'.
Mystery: Heather, 20, disappeared in December after driving herself to a boat landing in South Carolina
Where is she? Elvis drove to Peachtree Landing, pictured, on December 18 and her empty car was found there the next day. Authorities have not found her body but said they believe she has been murdered
The couple's home was searched Friday morning by police who, Lieutenant Robert Kegler said, had a search warrant after analyzing video surveillance.
the night Miss Elvis disappeared, she had been on a date with a man who
dropped her off at the apartment at about 2am. That man has been ruled
out of the investigation.
believe Miss Elvis later drove herself to the boat landing where her
car was found, although the reason she would go there is not known.
Sidney Moorer's attorney T. Kirk Truslow said the case is a rush to judgment.
'Sidney has no connection to, nor knowledge of, any facts surrounding the disappearance of Heather Elvis,' Truslow said in a statement. 'I firmly believe Sidney will be completely exonerated.'
Tammy Moorer's attorney added that she will be cleared.
the best of my knowledge, Tammy does not know Heather Elvis at all,'
defense attorney Greg McCollum said. 'I don't think they've ever met.'
Search: After searching the couple's South Carolina home on Friday (pictured), authorities said they had probable cause to charge the couple with murder
Evidence: Police search for evidence at the couple's South Carolina home on Friday after they were arrested
Elvis' family said they didn't care about the new charges - only what had happened to their daughter.
'If Heather is safe and unharmed, I don't care what they do to them,' Terry Elvis, her father, told ABC News. 'They can turn [the Moorers] loose. I really don't care.'
As details about the arrest became
known, members of the 300-strong search party who have been looking for
Miss Elvis lined the street outside the Moorer property.
group has been tirelessly searching for the young woman, combing the
surrounding woodland and boat landing, since she disappeared.
On a Facebook page dedicated to the search, the Elvis family posted a statement on Friday, calling on residents to keep searching.
'Please be aware that there are rumors flying everywhere that Heather has been found. This is not the case,' the post said. 'Please keep your flyers and posters up and on display till Heather is home.'
Charged: Tammy and Sidney Moorer, who had an affair with student Heather Elvis before she vanished in December, were arrested on Friday and on Sunday, new charges were added
Arrested: Sidney and Tammy Moorer have been charged with obstructing justice and indecent exposure
Trial: Sidney and Tammy Moorer are being held on $20,000 bond each
Terry Elvis, Heather's father, had previously released the last picture he had received of his daughter, which showed her learning to drive a stick shift on her date.
Mr Elvis explained in an essay on xoJane that he has a manual transmission truck he had been saving to give to one of his daughters, but that neither of them had ever learned how to drive a stick shift.
At the
end of her date the night before she disappeared, the man she was
meeting offered to teach her how to drive his manual truck in a mall
parking lot and she excitingly sent the picture of her driving to her
father to share the accomplishment.
'It is the last communication I have had with my daughter, the last picture I have of her on my phone,' Mr Elvis said.
'I didn't talk to Heather about it, but she had a good time,' Mr Elvis added about the date. 'I didn't think much of it.'
Mr Elvis has worked tirelessly in the police investigation and search for his daughter.
Still searching: More than 300 volunteers are helping with the hunt to find Heather Elvis
Last picture: Elvis sent this picture showing her driving stick shift to
her father the night before she vanished
Search: Terry Elvis prints missing person posters for his daughter as the family continue their search
In his essay, Mr Elvis wrote that his daughter's disappearance had taught him not to take his loved ones for granted since you never know when you might lose them.
'Hold your loved ones close, tell them how much you love them every day. Never let them doubt how much love you have for them so that if you are ever in the position I am in, you will be able to look in the mirror with less regret,' Mr Elvis said.
The family still hope Miss Elvis will be found alive.
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Lake Montezuma Arizona Golf Courses
|Beaver Creek Golf Resort
Additional Golf Course Reviews:
|Ken McDonald Golf Course -- Solid muni course
|Review by: Craig
|This course has forgiving fairways, but not easy fairways. By that I mean that you get away with a small hook or slice, but a large one causes problems. With the mature landscaping on this course, those problems frequently include trees. As I found out 3 times in one hole. :)) The greens were OK. They had just been sanded at the beginning of the week, and it showed, but they were still very playable. It was nice to see a muni course taking care of maintenance even during the summer. There practice area, including putting green, is large and very well kept. The pro shop was also decent. There is a restaurant on site, but I didn't visit it. The only negative was the typical one for muni courses - pace of play. Even on a Sunday afternoon in the summer heat, the course was full. The price was very good - summer rates were $18.50 for 18 holes and a cart. Actually, this was better than 'very good'. Overall, this course provides an excellent value - good course for a great price. I heartily recommend this course for all levels of players.
|From Scottsdale, AZ, USA


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- 1536 CUDA Cores
- 2GB 256-bit GDDR5
- PCI Express 3.0
This review is from: EVGA SuperClocked w/ ACX Cooling 02G-P4-2774-KR G-SYNC Support GeForce GTX 770 2GB 256-bit GDDR5 PCI Express 3.0 SLI Support Video Card
Looks nice, stays cool, and runs very well.
No 'cons', but a bit of an issue that is completely my fault and not EVGA's/Nvidia's (See other thoughts).
Other Thoughts:
I get a bit of whine, but I blame my PSU for it.
If you can't decide whether to go with a HD7970 or this GTX 770, go with this one. This was one of the only times I have not used AMD, and it has been great so far. The speeds are similar, but the User Interfaces of Nvidia's software and GeForce experience are fantastic. I am tired of AMD's poorly created UI.
My biggest complaint is that after I bought it, I decided I wanted to set up a WC loop. The problem is that this card has non-reference PCB and it is extremely hard to find a suitable water block. The ACX fan(s) are awesome, but I like the aesthetics of a WC loop. This incompatibility is my fault, and not a CON at all. If I had thought of WC earlier, I would have gone with a reference GTX 770.
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