Berberine is a natural alkaloid derived from a traditional Chinese herbal medicine. It is known to modulate microRNA (miRNA) levels, although the mechanism for this action is unknown. Here, we previously demonstrate that the expression of 87 miRNAs is differentially affected by berberine in multiple myeloma cells. Among 49 miRNAs that are down-regulated, nine act as oncomirs, including miR-21. Integrative analysis showed that 28 of the down-regulated miRNAs participate in tumor protein p53 (TP53) signaling and other cancer pathways. miR-21 is involved in all these pathways, and is one of the most important oncomirs to be affected by berberine in multiple myeloma cells.
We confirmed that berberine down-regulated miRNA-21 expression and significantly up-regulated the expression of programmed cell death 4 (PDCD4), a predicted miR-21 target. Luciferase reporter assays confirmed that PDCD4 was directly regulated by miR-21. Bioinformatic analysis revealed that the miR-21 promoter can be targeted by signal transducer and activator of transcription 3 (STAT3). Down-regulation of interleukin 6 (IL6) by berberine might lead to inhibition of miR-21 transcription through STAT3 down-regulation in multiple myeloma. Furthermore, both berberine and seed-targeting anti-miR-21 oligonucleotide induced apoptosis, G2-phase cell cycle arrest and colony inhibition in multiple myeloma cell lines. Depletion of PDCD4 by short interfering RNA could rescue berberine-induced cytotoxicity in multiple myeloma cells.
Our results suggest that berberine suppresses multiple myeloma cell growth, at least in part, by down-regulating miR-21 levels possibly through IL6/STAT3. This led to increased PDCD4 expression, which is likely to result in suppression of the p53 signaling pathway. These findings may also provide new mechanistic insight into the anti-cancer effects of certain compounds in traditional Chinese herbal medicines.
Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of plasma cells (PCs) within the bone marrow (BM). Globally, its incidence varies from 1 per 100,000 people in China to about 4 per 100,000 people in most developed countries [1, 2]. MM is characterized by profound genomic instability involving both numerical and structural chromosomal aberrations of potential prognostic relevance [1, 2]. Nearly half of MM tumors are hyperdiploid (HD) with multiple trisomies of non-random odd-numbered chromosomes, a low prevalence of chromosomal translocations involving the immunoglobulin heavy chain (IgH) locus at 14q32 and chromosome 13 deletion . It has been suggested that chromosomal abnormalities and other types of genetic or epigenetic alterations might contribute to miRNA deregulation in cancer [4–6].
miRNAs are a newly discovered class of endogenous non-coding small RNAs that regulate gene expression through degrading target mRNAs and/or suppressing their translation by binding to the 3′-untranslated region (3′-UTR) of target genes. Bioinformatic predictions indicate that 30% of all human genes are regulated by miRNAs. Thus, miRNAs are involved in a variety of biological processes, from development and differentiation to survival, apoptosis, and senescence [7–9].
Accumulating evidence suggests that miRNAs that are significantly over-expressed in tumors may be a novel class of oncogene. Termed "oncomirs", these oncogene miRNAs usually promote tumor development by negatively regulating tumor suppressor genes that control various biological processes. Therefore, altering oncomir expression might be a valuable strategy for cancer treatment [10, 11].
Differential miRNA expression and high levels of oncomirs, including miR-21, miR-155, miR-17-92, and miR-125b, have been reported in MM. miR-21 is frequently over-expressed in MM and is involved in proliferation, apoptosis, cell cycle, drug-resistance, and pathogenesis [6, 12–15]. Loffler et al. demonstrated that interleukin-6 (IL6) regulates miR-21 transcription in IL6-dependent human myeloma cell lines (HMCLs) through a signal transducer and activator of transcription 3 (STAT3)-related mechanism. Importantly, ectopic expression of miR-21 was sufficient to sustain growth of IL6-dependent MM cells in the absence of IL6 . This evidence indicates that miR-21 is an important oncomir in MM.
Berberine (BB), an alkaloid that was initially isolated from Chinese herbs, is currently used as a traditional medicine to treat diarrhea caused by bacteria, although the mechanism for this action is unknown. Accumulating evidence suggests that BB also elicits anti-cancer effects by inhibiting cell growth and inducing apoptosis in a variety of cancer cell lines [17–20]. Animal studies have shown that BB can inhibit chemical-induced carcinogenesis, tumor promotion, and tumor invasion [21, 22]. Recent studies also show that BB exerts anti-cancer effects by inhibiting proliferation and reproduction of certain tumorigenic microorganisms and viruses, such as Helicobacter pylori and hepatitis B. BB can also regulate the transcription of some oncogenes and carcinogenesis-related genes via interactions with DNA and RNA. Furthermore, BB is a broad-spectrum enzyme inhibitor that affects N-acetyltransferase, cyclooxygenase-2, and topoisomerase activities, as well as gene expression and protein synthesis . Thus, BB can regulate many oncogenic mRNAs and proteins. However, whether BB can regulate miRNAs remains unknown.
miRNAs in animals have a highly conserved 5′-end sequence consisting of 7–8 nt called the seed sequence. The seed sequence binds with 100% complementarity to the target mRNA and is a key feature in the recognition between a miRNA and its target mRNA . Inhibition of the seed sequence leads to a loss of mature miRNA function, and is the target of anti-miRNA oligonucleotides (AMOs) [25, 26].
In this research, we performed microarray analysis to explore the possibility that BB regulates miRNA expression. Our results show that BB differentially regulates the expression of a number of miRNAs. Forty-nine miRNAs were down-regulated, of which 28 were shown by KEGG analysis to be involved in p53 signaling, the cell cycle and other cancer pathways. Of the 49 miRNAs, miR-21 had the most target genes and participates in all the signaling pathways and can, therefore, be considered as one of the most important oncomirs. The role of miR-21 in MM was further investigated with the use of AMO-miR-21. Our findings provide new insight into anti-cancer mechanisms of traditional Chinese herbal medicines and provide evidence that they are effective in treating cancer.
Based on our preliminary study, the MM cell line, RPMI-8266, was treated with 75 μM BB for 48 h. Total miRNA from 1 × 108 cells was isolated and labeled using an mirVANA™ miRNA Isolation kit and mirVANA™ miRNA labeling kit (Ambion, Austin, TX, USA).. Samples (4 μg) labeled with Cy3/Cy5 were hybridized on miRNA microarrays (CSC-GE-3, chipscreen biosciences, Shenzhen, China). After air drying, each chip was scanned with a Generation III array scanner (Amersham Pharmacia). Data analyses were performed using Imagequant 5.0 (Array Vision 6.0).
miRFocus software (http://mirfocus.org), developed by LC Science USA, was used to analyze miRNA-target gene pathways and to determine related miRNA annotations (Additional file 1: Figure S1).
An anti-miR-21 oligonucleotide (AMO-miR-21) was designed according to sequence complementary to mature miRNA-21: AMO-miR-21, 5′-ATAAGCTA-3′ (8 bp). A control scramble AMO (SCR) 5′ -TCATACTA-3′ (8 bp) was also synthesized (Additional file 1: Figure S2). All oligodeoxynucleotides were chemically synthesized and modified with phosphorothioate and/or fluorescein isothiocyanate (FITC) by the Shanghai Sangon Bio-engineering Company, China. The siRNA sequence of PDCD4 (siPDCD4) was 5′-AAGGUGGCUGGAACAUCUAUU-3′. The RNA duplexes were synthesized and purified by Shanghai GenePharma Company, China.
MM cell lines (RPMI-8266 and U226) were obtained from the Shanghai Institute of Cell Biology, China. The cells were cultured in RPMI containing 25 mM HEPES, 10% fetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, and 50 U/mL streptomycin. The cells were grown in RPMI-1640 medium containing 10% fetal calf serum (FCS) at 37°C in a 5% CO2 humidified atmosphere (Thermo FORMA 3110).
BB was purchased from Sigma-Aldrich. RPMI-8266 and U226 cells in the exponential phase of growth were seeded in 96- or 24-well plates (Costar) and transfected with 0.5 μM AMO-miR-21 using Lipofectamine 2000 (Invitrogen) in serum-free RPMI-1640. PDCD4 siRNA and control SCR (100 nM) were transfected into RPMI-8266 and U226 cells using Lipofectamine 2000 according to the manufacturer's instructions.
The full-length human PDCD4 3′-UTR (1917 bp) was PCR-amplified from cDNA with the following primers: 5′-ccgctcgag ATATAAGAACTCTTGCAGTCT-3′ and 5′-ataagaatgcggccgc ACAGAGGATCTTTACATGTTTA-3′ containing NotI and XhoI restriction site overhangs, respectively (indicated in italic type). The amplified product, which contains one putative miR-21 binding site, was cloned into psiCHECK-2 (Promega) immediately downstream of the Renilla luciferase reporter gene and was named PDCD4 3′-UTR. Site-directed mutagenesis was performed using the QuikChange II XL site-directed mutagenesis kit (Stratagene) to change three nucleotides in the seed sequence, in which ATAAGCTA was substituted by TAGCTACT. The mutant plasmid was named PDCD4-mut- 3′-UTR.
RPMI-8266 cells were cotransfected with 100 nM miR-21 or 0.5 μM AMO-miR-21 together with PDCD4 3′-UTR or PDCD4- mut-UTR and assayed for luciferase activity 24 h post-transfection using the Dual-Luciferase Reporter Assay System (E1910, Promega). For each sample, firefly luciferase activity was normalized against Renilla luciferase activity.
RPMI-8266 and U226 cells were treated with 75 μM and 120 μM BB, respectively. Total RNA was extracted in TRIzol (Invitrogen). The levels of miR-21 and U6 small nuclear RNA (snRNA) were determined using a miRNA RT-PCR Quantitation Kit (Shang Hai Gene Pharma Company). U6 snRNA was used as the internal control, and the fold-change in miR-21 expression was calculated using the 2−ΔΔCT method.
RPMI-8266 cells were transfected with 0.5 μM AMO-miR-21 using Lipofectamine 2000 and cultured for 48 h. Levels of PDCD4 mRNA were determined using SYBR-Green real-time PCR assays. PDCD4 primers used were 5′-CCAAAGAAAGGTGGTGCA-3′ and 5′-TGAGGTACTTCCAGTTCC-3′ and GAPDH primers were 5′-CAACGGATTTGGTCGTATT-3′ and 5′-CACAGTCTTCTGG GTGGC-3′. PDCD4 mRNA levels were normalized to those of GAPDH.
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer in the presence of proteinase inhibitor (Biocolor BioScience & Technology Company, Shanghai, China). Cell lysates (30 μg) were denatured in Laemmli sample buffer (Bio-Rad) for 5 min at 95.1°C, separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% (w/v) fat-free milk in phosphate-buffered saline (PBS) and 0.5% (v/v) Tween-20 for 1 h, and then incubated with anti-PDCD4 antibody (Cell Signaling Technology) at room temperature for 2 h. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibody. Signals were visualized with enhanced chemiluminescence (ECL) (BeyoECL Plus, Beyotime), and analyzed using a BI-2000 system (Beyotime, Haimen, Jiangsu province, China),.
RPMI-8266 and U226 cells were treated with 75 μM and 120 μM BB, respectively.
IL6 protein levels in supernatants were determined using a human IL6 ELISA kit (R&D Systems).
RPMI-8266 cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,4- diphenyl-tetrazolium bromide (MTT) assays. Briefly, cells were seeded at a density of 1 × 105 cells/ml in 96-well plates (100 μl/well). The cells were treated with BB (75 μM) or AMO-miR-21 (0.5 μM). At 48 h post-treatment, 20 μl MTT stock solution (5 mg/ml) was added to each well, and the plate was incubated for 4 h at 37°C. The media was then removed, and dimethyl sulfoxide (DMSO) (150 μl) was added to dissolve the blue formazan crystals produced by viable cells. Cell viability was assessed by measuring the absorbance at 570 nm on a Bio-Rad microtiter plate reader.
The colony assay for dispersed single cells was performed to measure the capacity of cells to form colonies. Cells treated with BB (75 μM) or AMO-miR-21 (0.5 μM) were seeded onto a 24-well plate (2 × 103 cells per well) and mixed thoroughly with 0.9% methylcellulose solution in RPMI-1640 containing 20% FBS. Single cells were randomly and evenly distributed throughout each well. Colonies were formed during incubation for 1–2 weeks at 37°C in a 5% CO2 humidified atmosphere. Light microscopy was used to observe and count colonies containing more than 50 cells.
Flow cytometry (Coulter Elite; Fullerton, CA, USA) was performed to analyze cell cycle profiles and levels of apoptosis. For cell cycle analysis, cells were collected, rinsed twice with PBS, fixed in 70% ethanol for 1 h at 4°C and stained with propidium iodide (PI) solution (50 μg/ml) containing RNAse A (200 ug/ml). Cell cycle was analyzed using flow cytometry according to DNA content. To analyze apoptosis, cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and PI. For each sample, data from approximately 10,000 cells were recorded in the list mode on logarithmic scales. Apoptotic and necrotic cells were analyzed by performing quadrant statistics on PI-negative/annexin V-positive cells and PI/annexin V double-positive cells, respectively.
Of the 1152 miRNAs represented on the microarray, 87 were differentially expressed between BB-treated and control cells (Additional file 1: Figure S3); 49 were down-regulated, and 38 were up-regulated compared to control. Further analysis revealed that nine of the 49 down-regulated miRNAs were potential oncomirs, and three of the 38 up-regulated miRNAs are considered tumor suppressor genes (Additional file 2: Table S1 and Additional file 2: Table S2). Among the 49 down-regulated miRNAs, mirFocus software identified 28 that are involved in p53 signaling, the cell cycle and other cancer pathways (Figure 1). Of these, miR-21, has the most identified target genes and participates in all the above signaling pathways. Thus mir-21 can be considered as one of the most important oncomirs. These results suggest that BB suppression of MM might involve miRNA-mediated gene expression.
Ingenuity analysis of miRNA predictive pathways. Among the 49 down-regulated miRNAs, 28 were shown by mirFocus to be involved in p53 signaling, cell cycle and other cancer pathways. miR-21 participates in all the above signaling pathways, and can be considered as one of the most important oncomirs in the effects of BB on MM.
To validate down-regulation of miR-21 by BB and to investigate the effect of exogenous IL6 on BB-modulated miRNA-21 expression in MM, RPMI-8266 and U226 cells were treated without or with 75 μM or 120 μM BB, respectively, in the absence or presence of IL6 (0.5 ng/ml). Total RNA and/or protein was isolated and analyzed for the expression of miR-21 and PDCD4 using real-time PCR and western blot analyses. As shown in Figure 1, BB treatment significantly down-regulated miR-21 levels in a dose-dependent manner, and exogenous IL6 somewhat rescued BB-mediated down-regulation of miR-21 (Figure 2A). Interestingly, BB also reduced the level of IL6 in supernatant (Figure 2B). IL6 is an important oncogene in MM and is involved in miR-21 transcription . These results confirmed that BB down-regulates miR-21 and IL6 and consequently the miRNA target gene, PDCD4 was up-regulated (Figure 2C, 2D). Meanwhile, exogenous IL6 could rescue BB-induced miRNA-21 down-regulation in MM cells.
Effect of BB on levels of miRNA-21, IL6, and PDCD4 and the effect of exogenous IL6 on BB-induced miRNA-21 expression. RPMI-8266 and U226 cells were treated with 75 μM and 120 μM BB, respectively, in the absence or presence of IL6 (0.5 ng/ml) and harvested 48 h after treatment. Total RNA and protein were isolated and analyzed for levels of miRNA-21 and PDCD4. (A) miR-21 and U6 snRNA levels were determined by quantitative real-time PCR in the absence or presence of IL6. (B) The IL6 protein levels in supernatants were determined using an ELISA kit. (C) Relative PDCD4 mRNA levels were measured with SYBR-Green real-time PCR. (D) PDCD4 protein levels were assessed by western blot analysis. *p < 0.01 vs. control. The data show that BB significantly down-regulated miR-21 and IL6 levels in RPMI-8266 and U226 cells and up-regulated PDCD4 mRNA and protein levels in 8266 cells. Notably, exogenous IL6 could attenuate the reduction in miRNA-21 expression induced by BB.
Bioinformatic analysis showed that sequence upstream of the miR-21 gene contained two putative STAT3 binding sites (Additional file 1: Figure S4A). To investigate whether BB affects STAT3 mRNA levels, RPMI-8266 and U226 cells were treated with 75 μM and 120 μM BB, respectively. STAT3 mRNA levels were significantly decreased (Additional file 1: Figure S4B). STAT3 is recruited to the miR-21 regulatory region in response to IL6 ; therefore, BB-mediated down-regulation of IL6 might cause transcriptional inhibition of miR-21 via reduced STAT3 action.
Because BB treatment reduced miRNA-21 levels in MM cells, we next examined whether BB also regulated the expression of genes targeted by miRNA-21. We first focused on the PDCD4 gene, which is known to be targeted by miR-21. AMO-miR-21 was transfected into RPMI-8266 cells to knock down endogenous mir-21. As shown in Additional file 1: Figure S5A and S5B, PDCD4 mRNA and protein levels were significantly increased in AMO-miR-21-transfected cells compared with control-transfected cells, suggesting that PDCD4 is regulated by miR-21.
We performed luciferase reporter assays to determine whether PDCD4 is a direct target of miR-21. The luciferase reporter plasmid containing the 3′-UTR of PDCD4 (PDCD4-3′UTR) or a mutant PDCD4 3′-UTR containing a mutation in the putative miR-21 binding site (PDCD4-mut-3′UTR) were co-transfected with AMO-miR-21 or a miRNA-21 mimic. The firefly luciferase plasmid was used as an internal control. As shown in Additional file 1: Figure S5C, over-expression of the miR-21 mimic significantly suppressed luciferase activity, whereas transfection of AMO-miR-21 significantly increased luciferase activity in cells transfected with PDCD4-3′UTR. In contrast, luciferase activities in cells transfected with PDCD4-mut-3′UTR were not significantly changed by either over-expression or knockdown of miR-21 (Additional file 1: Figure S5C,S5D). These results indicate that PDCD4 might be a direct target of miR-21.
AMO-mir-21 was modified with FITC, and transfected into RPMI-8266 cells using Lipofectamine 2000. High levels of AMO-mir-21-FITC were detected, mainly in the cytoplasm (Additional file 1: Figure S6A). Flow cytometry showed that at 24 h and 48 h post-transfection 95.27% and 85.8% of cells, respectively, were FITC-positive (Additional file 1: Figure S6B).
To determine the effects of BB on MM cell growth via miR-21, RPMI-8266 cells were treated with BB (75 μM) or AMO-miR-21 (0.5 μM) as described in Materials and Methods. Cell viability was assessed by triplicate MTT assays. As shown in Figure 3A, treatment with BB at 50 μM or higher significantly inhibited cell proliferation. Similar results were also observed when miR-21 was knocked down by AMO-miR-21 (Figure 3A), and these effects were dose-dependent. Thus, both BB and AMO-miR-21 inhibited MM cell viability, indicating that the BB-mediated cell death might involve miR-21 down-regulation.
BB and AMO-miR-21 inhibit cell viability and induce apoptosis and G2 phase cell cycle arrest. RPMI-8266 cells were treated with various concentrations of BB and AMO-miR-21 then plated in 96-well plates in medium containing 10% FCS and cultured for another 48 h. Cell viability was assessed by triplicate MTT assays. (A) BB and AMO-miR-21. *p < 0.01 vs. blank and Scramble controls. Both BB and AMO-miR-21 inhibited cell viability in a dose-dependent manner. RPMI-8266 cells were treated with 75 μM BB or 0.5 μM AMO-miR-21. Cells were then stained with FITC-conjugated annexin V and PI for 15 min., and then analyzed by flow cytometry. (B) Both BB and AMO-miR-21 promoted apoptosis and IL6 could reduce BB-induced apoptosis. *p < 0.01 vs. control. (C) Both BB and AMO-miR-21 significantly induced G2-phase cell cycle arrest. *p < 0.01 vs. blank or SCR controls.
We next investigated whether BB treatment or miR-21 inhibition could induce apoptosis in MM cells. To this end, RPMI-8266 cells were treated with BB or transfected with AMO-miR-21. The cells were then stained with annexin V and PI and analyzed by flow cytometry. As shown in Figure 3B and Additional file 1: Figure S7, both treatments significantly increased the number of apoptotic cells, indicating that BB treatment and miR-21 inhibition induce apoptosis.
To investigate the effect of BB treatment or miR-21 inhibition on the cell cycle, RPMI-8266 cells were treated with BB or transfected with AMO-miR-21 and then subjected to cell cycle analysis by flow cytometry. As shown in Figure 3C and Additional file 1: Figure S8, BB treatment resulted in an accumulation of cells in the G2/M phase. Interestingly, miR-21 inhibition by AMO-miR-21 produced almost identical effects on the cell cycle to those observed following BB treatment. These results indicate that BB-induced G2/M arrest in MM cells is at least partially mediated via miR-21 down-regulation.
Colony growth is closely related to neoplastic capacity. To investigate the effect of BB and AMO-miR-21 on colony formation in RPMI-8266 cells, colony growth assays were performed. One week post-treatment, we found that RPMI-8266 cells treated with AMO-miR-21 or BB showed fewer colonies, compared to the control groups (Figure 4). These results indicate that both BB and miR-21 knockdown had an inhibitory effect on the malignant growth capacity of MM cells.
BB and AMO-miR-21 suppress colony formation. Colony growth capacity was assessed by methylcellulose colony formation assays. (A) Images of representative colony formation. (B) After incubation for 1 week, RPMI-8266 cells treated with 75 μM BB or 0.5 μM AMO-miR-21 were observed under a light microscope and the number of colonies counted. Three independent experiments were carried out.
To examine whether miR-21 down-regulation is associated with BB-induced cytotoxicity, we further studied the effect of PDCD4 knockdown in MM cells. We designed three siRNAs against the PDCD4 gene and evaluated their knockdown efficiency using real-time PCR analysis. We found that one siRNA efficiently knocked down PDCD4 mRNA and down-regulated PDCD4 protein levels in RPMI-8266 cells (Figure 5A,B).
siRNA down-regulation of PDCD4 expression and rescue of BB-induced growth inhibition. RPMI-8266 cells were treated with 100 nM PDCD4 siRNA for 6 h, and harvested 48 h after treatment. Total RNA and protein were isolated and analyzed for PDCD4 expression levels. (A) PDCD4 mRNA levels were determined by SYBR-Green real-time PCR. (B) PDCD4 protein levels were determined by western blot analysis. PDCD4 siRNA significantly down-regulated PDCD4 levels in RPMI-8266 cells. *p < 0.01, vs. control. RPMI-8266 and U226 cells were pre-treated as above and then plated in 96-well plates in medium containing 10% FCS for a further 72 h in the presence of BB (75 μM). Cell viability was assessed by triplicate MTT assays. The results indicate that PDCD4 siRNA can rescue BB-induced growth inhibition. (C) RPMI-8266 cells. (D) U226 cells. *p < 0.01 vs. control.
Next, we examined whether PDCD4 knockdown could rescue the cytotoxic effect of BB treatment. RPMI-8266 and U226 cells transfected with PDCD4 or control siRNA were treated with BB for 72 h, and cell viability was assessed by MTT assays. As shown in Figure 5C,D, PDCD4 siRNA transfection alone did not affect cell viability. However, PDCD4 depletion significantly ameliorated BB-induced growth inhibition, indicating that PDCD4 could be a downstream effector protein in the BB-induced cell growth inhibition pathway, and that it might be a tumor suppressor in MM cells.
Traditional Chinese medicine is an inexpensive treatment strategy. However, for most treatments neither the underlying therapeutic mechanisms nor the molecular targets of active compounds are well defined. Here, we describe a mechanism in MM cells by which BB inhibits cell proliferation and induces apoptosis and cell cycle arrest (Figure 3) via the regulation of miR-21 (Figure 2A).
miR-21 is over-expressed in the vast majority of cancer types analyzed so far and is thus recognized as an important oncomir [27, 28]. Inhibition of miR-21 suppressed cell growth in culture and tumor growth in a xenograft mouse model . Recently, Medina et al. demonstrated that miR-21 over-expression leads to a pre-B malignant lymphoid-like phenotype and that inhibiting miR-21 alone induces complete tumor regression in a few days, suggesting that miR-21 is a central oncomiR in tumor formation . Interestingly, MM is also a B cell neoplasm of clonal malignant cells in the bone marrow. Another group has also suggested that miR-21 as a key oncomiR in MM .Our results show that BB down-regulates miR-21 levels in MM cell lines (RPMI-8266 and U226) (Figure 2A), but does not affect miR-21 levels in IL6-independent human myeloid leukemia cells (HL60, NB4, and K562 cell lines; data not shown).
Bioinformatic analysis identified two putative STAT3 binding sites upstream of the miR-21 gene (Additional file 1: Figure S4A), and STAT3 mRNA levels were significantly decreased by BB (Additional file 1: Figure S4B). STAT3 has been validated to be recruited to the miR-21 regulatory region in response to IL6 . Our data indicate that IL6 levels in cell supernatants were significantly reduced by BB. Exogenous IL6 could partly ameliorate BB-mediated miR-21 down-regulation (Figure 2A). Thus, in MM, down-regulation of IL6 by BB might lead to the inhibition of miR-21 transcription through STAT3 down-regulation.
Antisense oligonucleotides targeting mRNAs have been successfully used to identify miRNA functions and for the development of therapeutic agents [31–33]. As miRNAs are small nucleic acids (19–24 nt), antisense inhibition is considered to be the best and possibly the only practical approach for specific pharmacological inhibition of their function . Recently, an 8-mer nucleic acid complementary to the seed region of an miRNA, called a tiny anti-miRNA, was used to antagonize miRNA function [25, 26]. In this study, high levels of AMO-mir-21-FITC were detected by confocal microscopy, mainly in the cytoplasm (Additional file 1: Figure S6A), and 95.27% of cells were FITC positive at 24 h post-transfection (Additional file 1: Figure S6B). Here, we have investigated the anti-MM effects of AMO-miR-21 and compared them with those of BB.
We also verified that PDCD4 is a direct target of miR-21 (Additional file 1: Figure S5). Like AMO-miR-21, BB treatment down-regulated miR-21 and up-regulated PDCD4. Intriguingly, BB and AMO-miR-21 produced almost identical effects on cell proliferation, apoptosis, and cell cycle profile (Figure 3). Therefore, we conclude that the anti-MM activity of BB might be achieved, at least in part, through the inhibition of miR-21/PDCD4 signaling.
The identification of miRNA target genes is necessary to assess the roles of aberrantly expressed miRNAs in human cancer and to subsequently develop miRNA-based gene therapies. miR-21 is strongly up-regulated in a variety of human neoplastic disorders. It can target and, therefore, can potentially regulate a number of important tumor suppressor genes (e.g., PDCD4, PTEN, TPM1, SPRY, RECK, and NFIB), which has attracted the attention of researchers in various fields [34, 35]. PDCD4, a 64 kDa protein, is an important, recently identified tumor suppressor that inhibits cell transformation, translation and invasion. PDCD4 is down-regulated in several types of human cancer [36–38], and is an independent predictor of poor prognosis in renal cell carcinoma patients . Consistent with this notion, we show here that both BB and AMO-miR-21 up-regulate PDCD4 by suppressing miR-21 (Additional file 1: Figure S5A-B, Figure 2C-D). Silencing PDCD4 with siRNA rescued BB-induced cytotoxicity (Figure 5). Furthermore, PDCD4 modulates the expression of other genes on two levels. PDCD4 affects transcription of certain genes by inhibiting the activity of specific transcription factors, such as c-Jun [40, 41], Sp1  and p53 . In addition, PDCD4 is thought to act as a suppressor of translation. A substantial body of evidence also suggests that PDCD4 is associated with p53 mRNA and suppresses its translation, thereby maintaining a low level of p53 in unstressed cells. This suppression is abrogated due to low levels of PDCD4 after DNA damage . These observations are consistent with our predictions from the bioinformatic analysis (Figure 1).
Mechanisms of drug-insensitivity or resistance are involved in the apoptotic capacity of cancer cells . Almost all cytotoxic anti-tumor drugs used clinically exert their effects by inducing apoptosis . It is known that tumors escape apoptotic signals by expressing anti-apoptotic proteins, such as Bcl-2 family proteins [45, 46]. Our study indicated that, similar to the effect caused by AMO-miR-21 inhibition of miR-21, BB treatment significantly promoted apoptosis, demonstrating the potential of BB and of targeting miR-21 for the treatment of MM (Figure 3). Hu et al. also show that miR-21 mediates berberine-induced apoptosis in human multiple myeloma cells .
To the best of our knowledge, our study is the first to show that the traditional Chinese medicine, BB, modulates the expression profile of many miRNAs in MM cells by down-regulating oncomirs and/or up-regulating tumor suppressor miRNAs. Our data show that BB treatment of MM cells down-regulated miR-21, probably via inhibition of IL6/STAT3, and led to the up-regulation of PDCD4, which was likely to result in suppression of the p53 signaling pathway. Therefore, BB is a therapeutic candidate for the treatment of MM (Additional file 1: Figure S10). Our work also provides new insight into the mechanisms underlying the anti-cancer effect of a traditional Chinese herbal medicine.
Xiaochuang Luo, Jingyi Gu, Rongxuan Zhu contributed equally to this work.
This work was supported by grants from the National Natural Science Foundation of China (81170496), the Fundamental Research Funds for the Central Universities (no. 21609406), the Science and Technology Plan Projects of Guangdong Province, and the Key Discipline Construction Foundation of Jinan University. We are grateful to Chipscreening Inc. (Shenzhen, Guangdong, China) for microarray services and to Land biology Inc. (Guangzhou, China) for providing luciferase reporter services.
JF conceived and designed the experiments. XL, JG, RZ and MF performed the experiments. JF and YL analyzed the data. XZ and JF contributed reagents, materials and analytical tools. JF, YL and XZ wrote the paper. All authors read and approved the final manuscript.


text:
Manchester Arena attack
What happened in Manchester? What we know so far about the attack
Key facts in the aftermath of the explosion that killed at least 22 people and left 59 injured
Manchester attack – latest updates
Kevin Rawlinson and Alice Ross
Tue 23 May 2017 19.22 EDT First published on Mon 22 May 2017 21.14 EDT
How the Manchester attack unfolded
A terrorist attack hit a concert in Manchester on Monday evening. Here is what we know so far:
Police have confirmed that at least 22 people were killed in the explosion at an Ariana Grande concert at the Manchester Arena. Some of them were children.
Why are troops being deployed on the streets of the UK?
Under operation Temperer the British army is to deploy about 900 troops around the UK to support police and free up officers to focus on other duties, including making arrests.
The troops will be stationed from Wednesday, replacing police guards at No 10 Downing Street, parliament and Buckingham Palace, and elsewhere around London. Theresa May said the deployment "would help police keep the public safe."
Police officers in Manchester and elsewhere will discuss specific deployments with their military regional counterparts. The soldiers will be in uniform but under the command of the police. Ewen MacAskill
Theresa May, the prime minister, declared that the threat level has been raised from severe to critical, meaning authorities believe a further attack is imminent.
Almost 1,000 troops will be deployed to Britain streets after the threat level was raised. About 984 troops will be stationed from Wednesday, replacing police guards at No 10 Downing Street, parliament and Buckingham Palace, and elsewhere around London. The changing of the guard ceremony at the palace has been cancelled.
Police named the suspect as Salman Ramadan Abedi, 22, who they said was born in Britain and was from a Libyan background. He has not been formally identified by the coroner. A 23-year-old man arrested on Tuesday is believed to be Ismail Abedi, the brother of the suspected bomber. He is still being questioned.
Three men have been arrested in south Manchester in connection with the investigation into the attack, Manchester police have announced.
Officers said 59 people were hospitalised and 60 treated by paramedics at the scene following the explosion, which hit the venue shortly after the concert finished. Twelve of those taken to hospital were aged under 16, North West ambulance service said.
May confirmed the incident was a terrorist attack as she addressed the media outside Downing Street.
It is the deadliest terror attack to hit the UK since the 7/7 London bombings in July 2005.
Who were the victims?
Saffie Rousos and Georgina Callander Composite: PA/Instagram
Around 21,000 people are reported to have been at the concert at the time of the explosion.
Twelve victims have been named: Georgina Callander, 18; Saffie Rose Roussos, eight; Olivia Campbell, 15; Kelly Brewster, 32; John Atkinson, 26; Alison Howe, 45; Lisa Lees, 47; Martin Hett, 29; Angelika Klis, 40; Marcin Klis, 42; Nell Jones, 14 and Jane Tweddle.
Police say their priority is to establish whether Abedi was acting alone or part of a network.
In messages posted online, Islamic State claimed responsibility for the attack.
The police say they believe the attacker was carrying an improvised explosive device, which he detonated.
Multiple witnesses said they heard an explosion, with one telling the Guardian the blast shook the building, before "everyone screamed and tried to get out".
A 23-year-old man was arrested on Tuesday morning in south Manchester, the police said. The Arndale shopping centre was evacuated and a man was arrested there but this was unconnected to the attacks, the police said.
Armed police carried out a controlled explosion at a house in Fallowfield and raided a block of flats in Whalley Range, both in south Manchester, on Tuesday morning as part of the investigation into the attacks.
Manchester map
Where did it happen?
Manchester Arena said the incident took place "outside the venue in a public space".
The blast was reported to have hit the foyer of the building at about 10.30pm, British Transport police said.
Victims have been taken to eight hospitals across the Manchester area.
Large parts of the city around the arena have been sealed off. Victoria station has been closed and is expected to be closed throughout Tuesday.
Police have asked the public to avoid Manchester city centre on Tuesday as they continued to work in the area. Some roads in the city centre have been closed for much of the day and public transport has faced disruption.
Police carried out a controlled explosion in the Cathedral Gardens area but the item destroyed was not suspicious.
Where it happened
How did Theresa May react?
Theresa May has said her thoughts were with the victims and families of those affected in "what is being treated by the police as an appalling terrorist attack". In a televised appearance outside 10 Downing Street, she attacked the "appalling, sickening cowardice" of the bombing.
Theresa May decries 'warped and twisted mind' of Manchester attacker
Other reactions
The home secretary, Amber Rudd, described the bombing as a "barbaric attack" that targeted "young people, children out at a pop concert".
May chaired two meetings of the Cobra government emergency committee. She also travelled to Manchester to meet the chief constable, mayor and emergency services, she said, and visited Manchester children's hospital.
Manchester's new mayor, Andy Burnham, attended the Cobra meeting via video-link and attended a vigil in Albert Square at 6pm on Tuesday.
The Queen issued a statement condemning the "act of barbarity", offering her sympathies to those affected and thanking the emergency services. "The whole nation has been shocked" by the attacks, she said.
The Labour leader, Jeremy Corbyn, and the Liberal Democrat leader, Tim Farron, have expressed their sympathies for the victims.
General election campaigning has been suspended.
The US president, Donald Trump, gave a statement from his trip to Israel in which he called the attackers "evil losers". Trump spoke to the prime minister to offer US assistance in the investigation into the attack and assuring her that "Americans stand with the people of the United Kingdom", according to a read-out of the call.
The London mayor, Sadiq Khan, condemned "barbaric acts of terrorism".
Police reaction
The Metropolitan police said it asked for soldiers to be sent on to the streets in order to free up armed officers currently deployed on guard duties.
The force's commissioner, Cressida Dick, said additional police would be on duty in London throughout the weekend.
Police urged those who are concerned about loved ones who were in the area to call the National Casualty Bureau on 0800 096 0095 (+448000960095).
Police have issued an urgent appeal for anyone who was in the city centre between 8pm and 11pm on Monday night and has dashcam footage to submit it to the National Police Chiefs' Council image appeal site.

redpajama_set_name: RedPajamaCommonCrawl


I've used your website a ton, all the way back to your early days. I've never had one of your recipes fail. Until today. I followed this one to the letter and got a curdled, soup mess. I'm at a loss as to how this happened. I did use russets and I did use half n half. I think I'm going to go back to the old bechamel method.
Hi Karen, oh that's weird! No idea why that happened.
So if you parboil to cut baking time, what is the method? Do you bake 20 min covered then uncover and add cheese and bake 25 uncovered? It doesn't help to make one modification and not explain full change. Thank you.
Hi, Denise — The parboiling method is described at the end of the recipe. And yes, you would just divide the covered and uncovered time similar to the full recipe. I've modified the instructions slightly to make this a little more clear. Thank you!
Made this tonight for my husband and me — delicious. We both loved it. Didn't have any bacon and didn't miss it. Used cream and 2% milk and it was certainly rich enough. For cheese, used swiss, parmesan and gruyere. Can't wait for left-overs tomorrow and will make again for company. THANKS!
I love this recipe, it's my go to scalloped potato recipe. Although I leave off the bacon, in my opinion it's better without bacon, but that's just my personal opinion. I do advise pre boiling the potatoes. But it's a great recipe, I've been using it for years now.
My experience with parboiling is that it makes the dish watery. Think it must have something to do with leaching the starch. So I just plan on cooking for the whole long time. They're totally worth it.
I tried carmalizing red onions and adding that as a layer. It's fantastic.
Daaaang… this was good. Used russets and a white onion, and the full 3 cups of half and half. Instead of Swiss or Gruyere I used a sharp local farmer's cheese, which has a nice strong flavor and melts well. Omitted the bacon, used the chives. Loved it. 90 minutes was enough.
a golden brown top when cooked & takes about 45 mins.
I think this recipe turned out just the way it was supposed to, so I was pleased with that aspect. The texture was just right: not soupy at all and the potatoes were perfectly cooked. However, I think the onion overpowered the other flavors. It should taste mostly of comforting potatoes and cheese, in my opinion, but instead I was left with that bitter onion aftertaste. Next time, I will halve or maybe even third the onion. Also, I made the mistake of not peeling potatoes thoroughly as I like the chunkiness of potatoes with a little skin. But in this dish, it didn't work; we got a bit of "dirty" flavor from the skins. Next time, will peel all the way. Finally, I did add a little finely chopped broccoli to the mix, and it turned out great! The whole dish was much tastier as leftovers the next day, and even better the third!
Made this last Sunday as we hosted a little get together for most of my wife's family (she has two sisters and three brothers plus grown kids). We served it with a honey-smoked ham from Costco and chicken legs I grilled on my Big Green Egg in the rain. I used red potatoes instead of russets as my wife had just bought a big bag at Costco. Used our food processor to slice the potatos which made the prep go pretty quick. Everyone loved it! Will definetely make this again.
We parboiled at first, as suggested and used a shallow pan, as suggested.
It turned out so delicious my husband ate 50% of the total pan!!! Whatever's leftover, he has claimed them for lunch tomorrow in the office!!!
Thank you Elise!!! It's another winner!
Well,i tried this recipe, and since the layers were not very shallow – maybe 4″ deep – i raised the temp to 400. Also didnt have cream so i used 1%. the 1% didnt thicken well, so i left it in longer, to see if more would evaporate. at the end, i just had a big mess – the milk curdled and the cheese separated into oil… uck. DONT raise the temp on this!
I don't have any cream, can I just use 2% milk instead of half and half?
I just made this for Easter and i had to throw it down the drain!!! I thought i was a great cook but this makes me scared to try new recipes!! I made it one day ahead and put in the fridge overnight and when i took it out the potatoes looked black. I thought it might change once i baked it, but i tasted it and it had gotten mushy. It smelled great while baking but it was too embarassing to present to my guests. Thankfully my mother in-law made mashed potatoes for back up.
I am glad I saw this review. I was going to prepare the day before cooking. I will not do that now. Thank you!
I made this for Christmas and it was good… even better the next day (and the next)!!! I goofed and had too many things that needed to go in the oven, and not enough time, so I had to bake it at 425 for the first 45 minutes or so which curdled the cream – but it was still tasty.
If it's as good reheated, and apparently shouldn't be prepared a day ahead, was it the thought on cooking the day before serving and then reheating? Help, because I want it to be as excellent as it sounds but need to plan ahead. Thanks.
These potatoes were delicious, I made them for a side dish for a holiday dinner. I doubled the recipe and used a large 10×15 dish, making sure the potatoes were sliced VERY thin. No problems, the potatoes were tender, the cheese was brown and crispy, and I used the oven times listed. Thanks, this is a great side that I will definitely make again!
Everyone raved about these. I have to say they did bake probably 2 hrs because I did not parboil the potatoes. And, although I have not made scalloped potatoes in years, I had a recollection of shaking a bit of (Wondra)flour over each layer, which I did and I think that did keep it from being runny. The flavour was wonderful and as I said, all raved. Thank you, it was an Easter success!
You can leave potatoes soaking in water (or water and milk) covered in the fridge for 2 or 3 days with no adverse affects. Make sure the potatoes are completely submerged as any out of the water will turn gray. Soaking for long periods will 'leach' some of the starch out of the potatoes and that starch ends up in the bottom of the bowl. Be aware of this if you are counting on the starch from the potatoes thickening your sauce. Scoop the potatoes out of the bowl rather than dumping them into a strainer as whatever is on the bottom of your bowl will end up on the top of your potatoes.


text:
Q: Firefox addon: Window.open() vs CTRL-N I created a simple firefox add on using addon-builder that installs & successfully appears in the add on toolbar at the bottom of the browser.
If I press ctrl-n, open new tab, open new window, or open private browsing window in firefox, then I see and can use my addon. However, if another site programmatically opens a window using window.open(), then my addon doesn't appear.
Is this by design? Or is there a setting that I can include in my addon so that it always appears, even if the window was opened through window.open() instead of ctrl-n?

A: When sites open with window.open, they specify which parts of the browser UI will show. You may be able to place it somewhere less likely to be removed, like the navigation toolbar. The add-ons toolbar sounds like it's going away soon, anyway.

redpajama_set_name: RedPajamaStackExchange


Soft sponge circles designed to protect your clients' ears from heat discomfort while under your hood dryer.
Matty Foam Ear Shields are washable, so you can rinse and reuse.
£2.88 (inc. VAT) Why no VAT? We're a trade wholesaler and quote our prices without VAT to help our VAT-registered customers understand the true cost of a purchase.
Pack of 100 bonnet-style disposable heating caps for use in perming.


text:
New York Mets Will Need Surprises in the Bullpen: Don't Expect Addison Reed Dominance Again
Jul 10, 2016; New York City, NY, USA; New York Mets relief pitcher Addison Reed (43) delivers a pitch during the ninth inning against the Washington Nationals at Citi Field. Mandatory Credit: Anthony Gruppuso-USA TODAY Sports
Nobody should expect Addison Reed to duplicate his 2016 season. This is why the New York Mets will need a few bullpen surprises in 2017.
The guy was absolutely magnificent a season ago. In fact, he's been brilliant since arrival on the scene late in 2015, coming from Arizona, a previous house of horrors for New York Mets setup man Addison Reed.
This doesn't automatically mean Jeurys Familia's right-hand man out of New York's bullpen will showcase extraordinary innings in 2017.
It's nothing personal. This is simply how bullpens in Major League Baseball operate. Very rarely can bullpen arms be depended upon year-after-year. The position is one of, if not the most unreliable spot in all of baseball (closer aside).
In 2016, the team who finished first in all the land in bullpen ERA were the Los Angeles Dodgers. They sported a sparkling 3.35 ERA. In 2015, the very same team finished 19th with a 3.91 clip. While closer Kanley Jansen has remained as steady as a rock, the club's middle relief stepped up big time over the course of one season.
Last year, Addison Reed was downright filthy. His 1.97 ERA and 91 strikeouts over 77.2 innings only tells a fraction of the story. His stuff, his attitude, his demeanor — it fit brilliantly as the Mets 8th-inning man leading up to Familia.
To rely on the man, however, is another story.
Reed, 28, hasn't been failproof throughout his six-year MLB career. Once thought of as the franchise closer in Chicago with the White Sox, Reed struggled to the tune of a 4.75 ERA and 1.364 WHIP in 2012. Two seasons later in Arizona didn't solve any of Reed's issues. His first full season as closer with the Diamondbacks yielded a 4.25 ERA with blown saves left and right. The worst part about his line was the 1.7 HR/9 innings mark.
Twenty-fifteen was the year that saw Reed take a backseat in the bullpen. He shifted to setup man and was eventually scooped up by a keen Sandy Alderson.
He's been brilliant ever since.
But this is where things get interesting.
Can Reed do it again? Of course, he can. At the same time, he can struggle, mightily, especially considering he'll likely start the season as closer in Familia's absence (pending his domestic violence troubles). His past, extremely shaky past provides reason for concern and Terry Collins will need to pray a few of his other bullpen arms step up.
Behind Reed is lefty Jerry Blevins, another guy coming off a stellar campaign. At 33-years of age, the Mets lefty who missed all of seven games in 2015, pitched to a 2.77 ERA while striking out 52 in 42 innings pitched. Again, he'll be another guy the Mets are depending on in that 'pen.
Alongside Blevins is a mashup of arms — Fernando Salas, Hansel Robles, Josh Smoker, Josh Edgin, Erik Goeddel and Sean Gilmartin. One or two of these guys will need to step up if these Mets have any serious designs of contending in 2017.
Sure, Reed and Familia can come out and be just as dominant as they were a season ago. But when it comes to the inconsistencies of bullpens from year to year, contending teams always need a guy or two to surprise.
It's just the nature of the beast.
Reed is coming off a year that saw him destroy his past highs in games (80) and innings (77.2) pitched (previous high of 68 games and 59.1 innings pitched). Until he proves he can get it done at a high level in back-to-back workload seasons, a backup plan must be firmly in place.
The only question now is, "Who is the backup plan in the bullpen for the New York Mets in 2017?"
What they'll need is the next version of Jeurys Familia circa 2015.
NEXT: Any Mets Lineup Without Jose Reyes is Non-Authentic
Terry Collins


NeNe Leakes is pleading her case. The RHOA star knows people think she was acting especially shady during part 1 of the RHOA reunion but according to her, it's for good reason. NeNe recently sat down with E! News' Daily Pop to talk her role in "How High 2" and Sunday's reunion.
NeNe told the hosts that she STILL considers all the housewives her "coworkers" especially Cynthia Bailey who NeNe says is "acting like she's bleeding." NeNe's adamant that she's being portrayed as a villain when it's clearly someone who's not as "nice" as they seem.
NeNe also added that the audio clip that aired on the RHOA finale of Cynthia and Kandi seemingly trying to cover up the fact that they invited Kenya Moore to a party was only released because NeNe pressed for it herself.
NeNe apparently had insider insight that Cynthia was "backstabbing her" and she begged producers to release the audio.
SIGHHHHH. All this drama over Kenya Moore, huh?
Watch NeNe talk her ex-friend Cynthia below.
Do YOU think she's being dramatic or does she have a point?
If your friend invites your former friend to a party and doesn't tell you does it mean that you should sever ties???
While NeNe claims that Cynthia's bashing her in the press it looks like Cynthia thinks it the other way around. She told our sister site MadameNoire that she and NeNe are not friends and she finds it "disgusting" to tear another woman down.


text:
Three more state attorneys endorse Ashley Moody for Attorney General
Peter SchorschFebruary 6, 20184min
2018 Attorney General's Race
Ashley Moody
Bernie McCabe
Brad King
Brian Haas
Bruce Colton
Dennis Ward
Ed Brodsky
Glenn Hess
Jay Fant
Pam bondi
Phi Archer
political endorsement
political endorsements
R.J. Larizza
Ross Spano
Ryan Torrens
Sean Shaw
William Cervone
Republican Attorney General candidate Ashley Moody announced Tuesday that three state attorneys endorsed her bid to succeed Pam Bondi in 2018.
"As a former circuit court judge, the endorsement of these State Attorneys, many of whom have spent their lifetime dedicated to justice and the pursuit of justice for victims and their communities, is tremendous. I am honored to have their support and as part of our team. Together we will work to ensure the rule of law prevails and victims have a voice in the process," Moody said.
The endorsements came in from 19th Circuit State Attorney Bruce Colton, 14th Circuit State Attorney Glenn Hess and 8th Circuit State Attorney William Cervone.
The trio join previous state attorney backers Phi Archer, Ed Brodsky, Brian Haas, Brad King, R.J. Larizza, Bernie McCabe and Dennis Ward, giving Moody the backing of half of Florida's 20 state attorneys.
Moody has also picked up endorsements from Bondi and 33 Republican county sheriffs.
"I know firsthand how important it is we have an Attorney General who stands firm in support of the rule of law and knows what it takes to put criminals behind bars. To ensure we have criminals brought to justice, we must have an Attorney General who understands the law, has practiced in a courtroom, and has a strong record of defending our communities. Ashley Moody is the only candidate prepared to be our next Attorney General," said Colton.
Hess added that the state needs "an experienced and tough Attorney General" and that Moody "has proven throughout her career that she is ready to lead our state as Florida's Top Cop."
Cervone echoed his colleague's calls for an Attorney General with tough-on-crime experience.
"When it comes to the safety and security of Floridians we need an Attorney General who knows what it takes to combat crime and lock up violent criminals. Ashley Moody not only has a demonstrated history of accomplishing just that, but also has the temperament and tenacity to continue that fight in Tallahassee. I'm proud to endorse her as our next Attorney General," he said.
Moody is running against state Reps. Jay Fant, Ross Spano and Frank White in the Republican Primary race. Competing for the Democratic nomination are Tampa Rep. Sean Shaw and Ryan Torrens.
At the end of 2017, White led in fundraising with $1.95 million on hand between his campaign and committee accounts, followed by Moody with $1.2 million in the bank, Fant with just shy of $1 million and Spano with about $50,000.
White's total includes $1.5 million of his own money, while Fant has put $750,000 of his own cash into his campaign.
Peter Schorsch
Peter Schorsch is the President of Extensive Enterprises and is the publisher of some of Florida's most influential new media websites, including Florida Politics and Sunburn, the morning read of what's hot in Florida politics. Schorsch is also the publisher of INFLUENCE Magazine. For several years, Peter's blog was ranked by the Washington Post as the best state-based blog in Florida. In addition to his publishing efforts, Peter is a political consultant to several of the state's largest governmental affairs and public relations firms. Peter lives in St. Petersburg with his wife, Michelle, and their daughter, Ella.
previousCounty Commissioner Dave Eggers backing Nick DiCeglie in HD 66 race
nextSenators lash out at insurance industry during hearing on AOB reform


Attorney Leonard W. Foy III with Foy Law Office, PLLC practices in the area of real estate with a concentration in residential and commercial real estate transactions. Mr. Foy has represented clients in thousands of commercial and residential real estate transactions during his 23 years as a practicing attorney. He has offices in Nashua, NH and Lawrence, MA.


text:
Comedy Birthdays for June 4
Happy Birthday today to British actor and comic Russell Brand (born 1975) and American actress and comedienne Rosalind Russell (1907 – Nov 28, 1976).
Rosalind Russell had a forty-year career on both stage and screen, winning a Tony Award and all five Golden Globes for which she was nominated. Her best known film roles include the fast-talking girl reporter in His Girl Friday and the larger-than-life Mame Dennis in Auntie Mame.
A character actress of the first order, Russell was just as hilarious in screwball comedies as she was playing wealthy heiresses and even professional women such as judges and psychiatrists. Russell attributed her atypically long Hollywood career to the fact that she never became a sex symbol, playing on her looks as a basis for career.
Russell Brand first acheived mainstream attention in the UK in 2004 as host of Big Brother's Big Mouth, a spin-off of the popular "reality" show. His first major film role was St Trinian's in 2007, followed by roles in Forgetting Sarah Marshall and a starring role in 2010's Get Him to the Greek and 2011's remake of Arthur.
During his six-year career, Brand has had more than his share of controversy, including a reputation for drug use, alcoholism, and promiscuity; on-air prank calls that led to his resignation from the BBC, and various controversial appearances on various award shows.
Posted in Comedy Birthdays
← Happy Birthday, Chuck BarrisPeople You Wish You Knew →
Crock rocker S. Irwin
Crocodile Hunter Steve Irwin was most well-known for being a wildlife expert and television personal[...]
2/16 - Happy Birthday, Edgar Bergen
Here's the pitch... "We are going to put a ventriloquist on the radio!" "Wait, the audience ca[...]
2/5 - Happy Birthday, Christopher Guest
When the gods of music and comedy merge, Christopher Guest becomes their mavin. This performer gave [...]
Mr Brenner, you're looking older
Davie Brenner was a favorite comic when Johnny Carson ruled the television airwaves. This master of [...]
2/03 - Happy Birthday, Shelley Berman
Shelley Berman There are names that get lost in the bane of history, people who were successful in [...]
Tommy Smothers' Mom liked him best....
Tommy Smothers is half of one of greatest comedy duo's of all time--The Smothers Brothers. They wou[...]
Pauly Shore is having a birthday
Pauly "The Weasel" Shore was destined to become a comic. His mother owned a famous comedy club in L[...]


Professional Concrete Cutters & Core Drilling Experts. Servicing areas from The Tweed, Gold Coast to Brisbane and Surrounds. Searching for reliable tradies? Call us for all your concrete sawing & core drilling requirements.
We at Cut It Out Concrete Sawing & Core Drilling pride ourselves on our reliable, professional and clean services. Providing obligation free quotes and inspections of the work to be required, no job is too small for us! Cut It Out Concrete Sawing & Core Drilling provides services to the Domestic and Commercial Building & Construction Industries in the Gold Coast, Northern New South Wales (Byron Bay, Lennox Heads, Tweed) and Brisbane areas.
Offering multiple concrete core drilling services, both hand held and drill rig mounted systems, drilling through floors and walls starting from 14mm up to 400 mm + in diameter. We can drill almost to any depth starting from 5 mm.
Out Concrete Cutters use small road saws that can fit into tighter spaces e.g. smaller entrances & doorways. And a large powerful road saw suitable for jobs with a lot of metres to cut. We offer multiple concrete cutting services using Petrol, Diesel, Electric or High-Frequency equipment.
We are highly experienced in the installation of fixed, surface mounted and removable bollards. If you are looking for added security around your shop/carpark or complex then call us for a quote to install your bollards.
We provide obligation free quotes and inspections. No job is too small or too large for us. We service the domestic market as well as private businesses and government organisations.
High quality service. They were very mindful of not cutting into areas where there maybe electric or plumbing services. Very efficient, precise and well priced. Highly recommended!
Professional and reliable, Cut it Out always respond quickly and endeavour to complete all projects in a timely manner. A pleasure to work with.
Have dealt with Kris & Joe for over 5 years on a variety of projects and scope of works. Absolutely a pleasure to deal with, outstanding workmanship and top people to boot!!! Cant recommend them enough!!!
Very happy with Chris & Joe went over and above to try and accomodate our situation and time frame, very professional service, great people to deal with. Would not hesitate to recommend this company.
Servicing Gold Coast, Brisbane and Tweed Heads Areas!
Cut It Out Concrete Sawing & Core Drilling are proud supporters of this amazing children's charity.


Daniel Chester French's summer home and studio is located in Stockbridge, Massachusetts. "Chesterwood" (named after French's grandfather's house in Chester, New Hampshire) is a 122 acre estate where many of French's best known works were designed. The house, studio and gardens are open to the public (an admission fee is charged) and there is a modest gift shop on the property. Chesterwood also has exhibits of modern sculpture.
Different things about Chesterwood appeal to different people. For me, French's studio is the high point. This large structure has many of French's original models and what many consider to be his most stunning work, "Andromeda." The studio, designed by Henry Bacon and completed in 1898 raises question upon question for any guest who is confronted with a dizzying array of images. Access to the house and studio are by guided tour only. During the many times I have been to Chesterwood I have found the guides to be helpful and informative, although I have often wished guides had more detailed information about specific pieces in the studio as I find myself constantly wondering, "What is that?" only to be told, sadly, "I don't know..." Chesterwood seems to be organized for the tourist interested in a casual look at French and his creative life; those wanting an in depth study of his work will need to look elsewhere although the visitor center (where the gift shop is located) has a good exhibit about several of French's works (most recently updated in 2001).
Chesterwood is open from May 1 - October 31. Photography is not allowed inside the studio or house but the photos below were taken on the Chesterwood grounds and give an idea of some of the things that await the visitor. Anyone interested in French's work will not be disappointed in visiting Chesterwood. While in the area, visitors can also take in the Norman Rockwell Museum and Tanglewood, the summer home of the Boston Symphony Orchestra. For more information about Chesterwood, visit the Chesterwood website.
This is a view of French's studio. The entrance (for tours) is from the porch to the left. The original entrance leading to the reception room is on the right side (where tours exit). To the right, out of view, are beautiful formal gardens.
This closer view of the studio shows the reception room on the right. Note the large number of windows that flood the studio with light, and how tall the studio is, built to accommodate large models.
When tour groups are taken to the studio, they first congregate on the porch. To the left of the porch you can see a interesting contraption French had built into the studio. Consideration of the effect of lighting is an important part of creating sculpture. So he could see his works in progress in natural light (since most were to be displayed outdoors), French had a short railroad track constructed. French would move a sculpture onto a part of the floor of the inside of the studio that was actually on wheels. When the large door was opened, the statue could be rolled outside into natural light.
This view shows the full outdoor extension of the track.
The doorway leading from the porch to the studio is seen on the right. The bench built into the outside wall is flanked with two rejected models of Indians for the Francis Parkman Memorial (Jamaica Plain, MA). If one was to turn around and look over the porch, you would be greeted with a magnificent view of Monument Mountain in Great Barrington, Massachusetts.
The rejected Parkman Memorial model set into the wall to the left of the bench.
The rejected Parkman Memorial model set into the wall to the right of the bench.
Photography at Chesterwood is not allowed inside French's studio, his house or in the museum attached to the gift shop. On a recent visit to Chesterwood, I took the last tour of the day and upon leaving French's studio and standing outside, snapped this photo through a window in the closed door. It gives but a taste of the wonders awaiting a visitor to Chesterwood. Covered in a white sheet for the night is French's stunning "Andromeda." Behind "Andromeda" with outstretched arms is a model for French's "The Genius of Creation" and Memory is seen at far right in clay. Studies for the Clark Memorial are on the wall. This and much more awaits the visitor to Chesterwood. Do not miss an opportunity to visit Chesterwood and immerse yourself more deeply in the work of Daniel Chester French.
The tour of Chesterwood begins at the gift shop which then leads to a small garden for an orientation session. In the garden is a bronze casting of French's 1912 statue of a standing Abraham Lincoln, the original of which stands at the State House in Lincoln, Nebraska.
A closeup view of French's standing Lincoln.


text:
Свен Якобссон (,  17 квітня 1914 —  9 липня 1983) — шведський футболіст, що грав на позиції півзахисника.

Виступав за клуб ГАІС, а також національну збірну Швеції.

Клубна кар'єра
У дорослому футболі дебютував 1934 року виступами за команду клубу ГАІС, кольори якої і захищав протягом усієї своєї кар'єри гравця, що тривала вісімнадцять років.

Помер 9 липня 1983 року на 70-му році життя.

Виступи за збірну
1937 року дебютував в офіційних матчах у складі національної збірної Швеції. Протягом кар'єри у національній команді, яка тривала 11 років, провів у формі головної команди країни лише 7 матчів, забивши 1 гол.

У складі збірної був учасником чемпіонату світу 1938 року у Франції, де взяв участь у двох матчах і відзначився автоголом у програному з рахунком 1:5 півфінальному матчі проти збірної Угорщини, ставши таким чином лише третім гравцем в історії, який забивав у власні ворота у фінальних частинах чемпіонатів світу.

Посилання

шведські футболісти
Гравці збірної Швеції з футболу
Футболісти ГАІСа

redpajama_set_name: RedPajamaWikipedia


text:
Oil jumps most since June as Canada cuts after Saudi-Russia pact
A worker passes stores of oil drums and gas flares while working aboard an offshore oil platform in the Persian Gulf's Salman Oil Field, operated by the National Iranian Offshore Oil Co., near Lavan island, Iran, on Friday, Jan. 6. 2017. Nov. 5 is the day when sweeping U.S. sanctions on Iran's energy and banking sectors go back into effect after Trump's decision in May to walk away from the six-nation deal with Iran that suspended them. Photographer: Ali Mohammadi/Bloomberg
Oil was jolted higher by efforts across the globe to support prices as Saudi Arabia and Russia extended their pact to manage the market and Canada's largest producing province ordered unprecedented output curbs. Prices retreated slightly after Qatar said it was leaving OPEC.
After their worst month in a decade, futures in New York and London advanced more than 5 percent on Monday. Although Moscow and Riyadh have yet to confirm any fresh cuts, the agreement by the Russian and Saudi leaders over the weekend opens the door for a deal at the OPEC meeting this week in Vienna. Alberta's decision to curtail production by 325,000 barrels a day put a rocket under oil's rally before Qatar's surprise announcement that it was leaving the producer group to focus on gas.
Bullish investors are taking heart that the world's biggest producers are ready to take action after crude collapsed into a bear market on fears of a glut. Speculation over whether the Organisation of Petroleum Exporting Countries and its partners will curb output has gripped the market in recent weeks, causing volatility to spike. While OPEC delegates said the Saudi and Russian leaders have given political blessing for an agreement, questions remain, including the size of any potential cut.
"We may see prices recovering to $60 a barrel this month and depending on how much and until when OPEC's output curbs will continue, we might be heading toward $70," Sungchil Will Yun, Seoul-based commodity analyst at HI Investment & Futures, said by phone. "Canada's production cuts are adding to the bullish sentiment in the oil market, removing concerns of oversupply that has so far been dampening prices."
West Texas Intermediate for January delivery climbed as much as $2.92, or 5.7 percent, to $53.85 a barrel on the New York Mercantile Exchange, its biggest intraday gain since June. The contract traded at $53.51 at 3:37 p.m. in Seoul, after losing 22 percent last month. Total volume traded was more than triple the 100-day average.
Brent for February settlement rose as much as 5.3 percent to $62.60 a barrel on London's ICE Futures Europe exchange. The global benchmark crude was at an $8.59 premium to WTI for the same month. The January contract expired on Friday after declining 1.3 percent.
OPEC's President and the United Arab Emirates Energy Minister Suhail Al Mazrouei, said he was optimistic OPEC+ will reach an agreement in Vienna over a cut in production for 2019. Technical teams are working on the level of the cuts necessary and the reference baseline for the reduction, he said.
Alberta's unprecedented curbs, announced Sunday, are an effort to ease a crisis in the nation's energy industry. The province will reduce production of raw crude and bitumen by 8.7 percent starting in January until the levels of excess oil in storage are drawn down. The reduction would then drop to 95,000 barrels a day until the end of next year at the latest.
The amount being cut is more than the total production of each of OPEC's three smallest members: Equatorial Guinea, Gabon and the Republic of Congo.
"It's always more impactful when everyone joins the fray," said Stephen Innes, head of Asia Pacific trading at Oanda Corp. "It definitely highlights just how critical a supply cut at this juncture is needed to stop oil prices for plummeting. It's a bullish set up all around."
Alberta's producers have been particularly battered by crude's slump amid surging oil-sands output, a shortage of pipeline space and heavy U.S. refinery maintenance. The province is following the advice of companies like Cenovus Energy Inc. and Canadian Natural Resources Ltd., which have been hammered by record low prices for heavy Canadian crude that at one point were $50 a barrel less than U.S. grades.
Meanwhile, Qatar's energy minister said the country, which accounted for less than 2 percent of OPEC's production in October, would leave from January and thereafter would not be committed to any agreements by the group.
Prices got a further boost as Donald Trump and his Chinese counterpart Xi Jinping called a truce in their trade dispute, with the U.S. president agreeing to postpone a planned tariff hike on Chinese goods for three months in return for greater purchases of American products. The pact prompted a rally in riskier assets, including oil, as fears over the economic fallout of the trade war ease.
Other oil-market news: Money managers cut total positions on WTI crude to the lowest level in five years, according to U.S. data released Friday. Oil explorers expanded drilling in American fields as OPEC and allied crude exporters prepared to discuss production controls and cratering prices.
Hydrasun rising in the east
Oil gets boost from trade deals, but demand concerns persist
Oil holds biggest weekly drop since July on easing Gulf tension
Oil prices up after Iran attacks reignite supply fears
Updated: Oil flirts with $70 as Middle East crisis deepens fear of supply disruption


Are you looking for Lawrence Frames 755523 Lawrence Frames 2x3 Black Wood Picture Frame - Gallery Collection from Lawrence Frames? We found best price for you. Click on button below to buy Lawrence Frames 755523 Lawrence Frames 2x3 Black Wood Picture Frame - Gallery Collection for the lowest price.


You can choose from an assortment of electives in addition to -- or independent of -- the core program. Because every child has different gifts and interests, we want you to be able to choose from a wide variety of add-on classes to round out your student's education. Most elective courses meet once a week for 75 minute classes for 28 weeks; some courses are longer, some shorter. Elective homework varies by course. Students come and go throughout the day; and study hall is available in the library for those who have a dormant period between classes.


Based in North East England, D. C. PAT Testing is an accomplished company offering portable appliance testing to Businesses, Schools, Landlords and the Public sector throughout the UK. Formed in 2007 the company has many years experience in the PAT testing industry. DC Pat pride themselves on the level of service provided to all customers throughout the UK. They always work to the highest standards laid down by the Institution of Electrical Engineers. DC Pat are fully qualified to both City and Guilds 2377 Part 001 and City and Guilds 2377 Part 002. D. C. PAT Engineers have a wealth of experience in the electrical Testing industry for 240V, 110V and 415V appliances. This experience includes appliance testing at industrial, commercial and domestic premises. Work carried out will fulfill all obligations under the Health and Safety at Work Regulations and the Electricity at Work Regulations. Staff are fully trained to BS7671 16th Edition Wiring Regulations.
J L Technical Services offers reliable professional portable appliance testing (PAT) in Bristol, North Somerset and surround area. Working with our customers to ensure they are in compliance with the current regulations and "Codes of Practise" regarding the maintenance of electrical equipment at work.
Carrying out testing to standards laid down in the latest 4th Edition "Codes of Practise for In-service and Testing of Electrical Equipment" by The Institution of Engineering and Technology.
All our engineers are qualified to City and Guilds " Electrical Testing and Inspection" (2377), and all of our test equipment is annually calibrated.
We are able to carry out the testing at a time that will minimise disruption to your business.
PAT Testing or P.A.T Testing from 65p per appliance and based in Cardiff. We cover all of South Wales (Including Newport, Cwmbran, Caerphilly, Bridgend, Treforest, Pontypridd, Rhondda, Merthyr, Swansea) and the Bristol area. We offer portable appliance testing to a diverse range of companies and individuals, including schools and colleges, hospitals, offices, restaurants, retail shops, letting agencies and landlords to name but a few.
Low cost professional PAT Testing - portable appliance testing service, fully qualified engineers, fully insured, CRB checked, no call out charges, free minor repairs, free replacement plugs and fuses, friendly reliable service. We cover all areas!! For more information on our services including prices, please visit our website. Alternatively telephone or e.mail us!
A local and competitive PAT testing service. I also offer a microwave leakage test service, in conjunction with my PAT test service. For small business''s which require the use of their electrical equipment, during business hours. I can conduct my testing out of hours.
Westcourt Facilities Management is a provider of Hard and Soft Service to SME and Domestic customers. We are a one stop shop for needs such as Cleaning, Window Cleaning, Portable Appliance and Socket Testing to vending machines & water stations to stationery. Westcourt also offers Fire and Safety Training, Health and Safety, 24 Hours Emergency Call out which may be advisable for glazing, alarm call outs. Harder Services will include Air Conditioning. Graffiti Removal. We offer tailor packages that provide for all your and your staff needs and necsseties, thus making us an asset to all.


CBS won the first night of the November sweep in viewers and adults 25-54 and placed a competitive second in adults 18-49, according to preliminary Nielsen live plus same day ratings for Thursday, Nov. 2.
The eighth episode of SURVIVOR: COOK ISLANDS won its time slot in viewers (15.56m), adults 25-54 (6.9/16), adults 18-49 (5.5/14).
Compared to the last first run episode (10/19/06), SURVIVOR was up +1% in households (9.0/14 from 8.9/14), +8% in adults 25-54 (from 6.4/16), +4% in adults 18-49 (from 5.3/14) and added +540,000 viewers (from 15.02m, +4%).
SURVIVOR: COOK ISLANDS topped "Ugly Betty" in adults 18-49 (vs. 4.4/11, +25%), adults 25-54 (vs. 5.1/12, +35%), viewers (vs. 13.73m, +1.83m viewers or +13%). This is SURVIVOR's largest adults 25-54 advantage over "Ugly Betty" (+1.8 rating points) and matches the largest adults 18-49 advantage (+1.1 rating points).
CSI was a solid second in households (12.8/19), viewers (20.53m, -0.43m behind ABC), adults 25-54 (8.5/18) and adults 18-49 (6.9/16). Compared to the last first run episode (10/19/06), CSI was up +1% in households (from 12.7/19), +2% in adults 25-54 (from 8.3/18), even in adults 18-49 (from 6.9/16) and up slightly in viewers (from 20.49m).
At 10:00 PM, SHARK was second in households (8.8/15, -0.1 behind NBC), viewers (13.17m, -0.60m behind NBC), adults 25-54 (4.9/12) and adults 18-49 (3.7/10).
For the night, CBS was first in households (10.2/16), viewers (16.42m), adults 25-54 (6.8/15) and second in adults 18-49 (5.3/13, -0.3 behind ABC).
CBS won the first night of the November sweep in viewers and adults 25-54 and placed a competitive second in adults 18-49, according to Nielsen live plus same day ratings for Thursday, Nov. 2.
The eighth episode of SURVIVOR: COOK ISLANDS won its time slot in viewers (15.30m), adults 25-54 (6.8/16) and adults 18-49 (5.4/14).
Compared to the last first run episode (10/19/06), SURVIVOR was up +6% in adults 25-54 (from 6.4/16), +2% in adults 18-49 (from 5.3/14) and added +280,000 viewers (from 15.02m, +2%).
SURVIVOR: COOK ISLANDS topped "Ugly Betty" in adults 18-49 (vs. 4.4/11, +23%), adults 25-54 (vs. 5.1/12, +33%), viewers (vs. 13.62m, +1.68m viewers or +12%). This matches SURVIVOR's largest adults 25-54 advantage over "Ugly Betty" (+1.7 rating points) and is the largest adults 18-49 advantage (+1.0 rating points) since October 12th.
CSI was a strong second in households (12.9/19), viewers (20.77m, -0.26m behind ABC), adults 25-54 (8.6/18) and adults 18-49 (7.0/16). Compared to the last first run episode (10/19/06), CSI was up +2% in households (from 12.7/19), +4% in adults 25-54 (from 8.3/18), +1% in adults 18-49 (from 6.9/16) and added +280,000 viewers (from 20.49m, +1%).
At 10:00 PM, SHARK was tied for first in households (8.9/15, with NBC), second in viewers (13.32m, -0.53m behind NBC), adults 25-54 (4.9/12) and adults 18-49 (3.7/10).
For the night, CBS was first in households (10.2/16), viewers (16.50m), adults 25-54 (6.8/15) and second in adults 18-49 (5.4/13, -0.1 behind ABC).


text:
Mike Carlson, NASM- CPT, CFL1
No doubt you have seen someone riding one by now, maybe from a distance so it appears as if he or she is walking on water. Up close, it looks like something an ancient Polynesian fisherman would have used. Honestly, that's not far off. Stand-up paddle boarding is the throwback sport that's starting to make waves.
"Prone paddle boarding has been around since the 1930s," says Christian Shubin, owner of Poseidon Stand-Up Paddle Surfing in Santa Monica, Calif., (poseidonstandup.com). "All over the world, stand-up paddle boarders have been entering traditional paddle-board events. Now there are more stand-up boarders than prone boarders."
Stand-up paddle boarding (often referred to as "SUP'ing") can be traced back to ancient Hawaiian paddle surfing. Modern SUP'ing also began in Hawaii, in the 1960s, as a way for surfing instructors to teach whole classes of gremmies. In recent years, pro surfers like Laird Hamilton have helped push the SUP renaissance, a surge in popularity that is likely because of the sport's versatility. Thrill seekers can ride waves just like traditional surfers. Endurance athletes love putting in competitive miles, such as in Southern California's Battle of the Paddle event, which attracts 1,000 paddle-board racers every year. And those attracted to SUP's leisurely touring qualities find a laid-back noninvasive way to commune with the sea, river or lake.
And no matter why you paddle, you'll get a great workout. "You end up engaging all your core muscles as well as your upper and lower body," Shubin says. "Every time you paddle, it's like doing a little crunch." It's also highly accessible to beginners. You might hit the drink a few times, but even the first try will be enjoyable. Still, Shubin recommends staying out of the waves on a maiden paddle-board voyage, getting your feet wet in a marina or lake instead. He offers these other useful tips for newbie paddlers.
The Stance: Unlike surfing, both feet should be pointed toward the nose of the board. In the very middle of most boards is a handle. Stand over that spot with your feet about shoulder-width apart. "You can stagger your feet a little bit. Sometime that gives you more balance. But for the most part, you want to keep them pretty even," Shubin says.
The Grip: Grab the paddle with two hands, one on top of the grip and one halfway down the shaft. The paddles have a bend in the shaft just above the blade. Hold the paddle so the blade is angled away from you rather than scooping toward you.
The Stroke: Keep the arm that's holding the top of the paddle extended the whole time. Reach out as far as you can, fully submerge the blade in the water and stroke. Pull back with your bottom hand at the same time, bending at the waist and digging into the water. It's a gross motion made up of many smaller moves. "You are bending everything just a little bit," Shubin advises. "Your arms are moving a little, you're bending at the waist a little bit and twisting your body just slightly with your shoulders."
New paddlers should expect legs and shoulders to tire out the quickest, but you don't have to be in ridiculous shape to enjoy your first time on a paddle board. And broad sports skills can make the learning curve even shorter. "Surfers tend to do well because of their ocean experience and confidence in the water, and skiers do well because of their balance and leg strength," Shubin says. "Women who do yoga pick it up very quickly, too, because of their balance and flexibility."
Round 'Em Up
Get Your Lebowski On
Get Your Kicks
Aloha, Mr. Muscle
You Got Served
Get Energized