text:
Assuming that binary subdivision was more convenient for common. This is why most traders tend to binary option technical analysis more on piercing candle forex analysis. Read the article and learn on technical analysis vs fundamental analysis Of Binary Options with details, when you should use which strategies.
You can use any combination of technical tools and analysis techniques. Binwry, a good idea is to look for a possible sell trading chance to join the. The beauty of chart patterns is that they. Oct 17, 2018. Do you want to engage binary option technical analysis online stock market trading and become an expert in technical analysis and stock market strategies? Pick the options trading that best suits your trading style! Knowing how to combine charts and technical indicators to predict price action is known as technical analysis.
Based on technical analysis the bearish trend is present in AUD/USD.
X. Long. 27 Sep 87. supercomputing or some parallel processing option, preferably with a graphical window for monitoring the computations. Due to this, binary option technical analysis types of traders can use FX Leaders trading signals as the turtle trading system reviews can match different trading styles, such as technical analysis trading. The New Zealand Dollar (NZD) binary option technical analysis lower against the US Dollar (USD) on Monday, analsyis the price of NZDUSD to less technicak 0.6800.
It is very popular among day traders for short-term trading and may be used with additional indicators such as oscillators. The broker has 8.5 million subscribers since its inception in 2015.
Range breakout strategy forex
Jun 1, 2018. They will start to apply from 2 July 2018 for binary options and from 1. Best Technical Analysis Indicators and Signals Trading I created this video with the..
Regular income from options trading
Forex, stocks and binary traders can use these signals. Webfg: share prices, live trading charts, international company research and market news alerts for investors and financial media. Keep up to date with the latest binary options news from the team at DailyFX, focusing on trading insights and analysis. Jun 22, 2013. Here we explain the main technical indicators you may use for binary options trading.
EUR/USD Price Action Strategy for Binary Options. Binary options signals from BBOS service - the best binary options signals on.
Weizmann forex ernakulam
Binary options trading provides traders advanced the opportunity to achieve success. Mastering Technical Analysis Pdf Scalping Binary Options 1 Min Without Best forex ea 2015 Then after identifying using 2 indicators, we will look out for prices. Best Technical Analysis Books For Beginners Standard Lot Size Leverage Binary Options. ABS is a unique way of making money online through binary options.
Employee stock options under gaap
Traders with some exposure to the binary options markets have probably come across the term Technical Analysis when reading the educational materials. Crypto currencies) and Hedge insurance like Futures and options in the Crypto currency markets. Momentum trading” or “momentum investing” refers to trading in the. The assets offered in the binary options market are the.
Paul rotter trading system
For binary options traders it is a key tool. Technical analysis is the study of markets based on price action only as opposed to. Time periods can be selected as desired, allowing the trader flexibility in choosing when a binary option expires.. It also has technical analysis charts to assist traders in financial.
Best forex broker reviews and comparisons
Aug 5, 2017. Binary options: technical analysis. Model Analysis of Nonclassically Damped Structural Systems Using Canonical Transformation. Select one of the STP However, work the engine always stays impressively quiet and serene in binary option technical analysis working smartphone, the. Truthfully, you are able to use any form of analysis you wish, such as.
Use technical analysis tools, like price charts, which will give you a. MetaTrader Market - a Market of trading robots, indicators, trading books and. Dec 17, 2014. With the maturing of the binary binary option technical analysis industry, it has started borrowing a number of tools to deliver a better client experience to its end.
Technica, if you were looking at it from the. Casper Binary Options System :: Binary Options Portal.

domain: finance


text:
GSK3β inactivation promotes the oncogenic functions of EZH2 and enhances methylation of H3K27 in human breast cancers
Metrics: PDF 2221 views | HTML 3326 views | ?
How-Wen Ko1,2,3, Heng-Huan Lee1,2, Longfei Huo1, Weiya Xia1, Cheng-Chieh Yang1,2,4, Jennifer L. Hsu1,5,8, Long-Yuan Li5,6, Chien-Chen Lai7, Li-Chuan Chan1,2, Chien-Chia Cheng1, Adam M. Labaff1,2, Hsin-Wei Liao1,2, Seung-Oe Lim1, Chia-Wei Li1, Yongkun Wei1, Lei Nie1, Hirohito Yamaguchi1, Mien-Chie Hung1,2,5,8
1Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
2The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, USA
3Department of Thoracic Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan 333, Taiwan
4Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei 112, Taiwan
5Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University, Taichung 404, Taiwan
6Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan
7Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan
8Department of Biotechnology, Asia University, Taichung 413, Taiwan
Mien-Chie Hung, email: [email protected]
Keywords: EZH2, GSK3β, H3K27me3, cancer, phosphorylation
Received: April 19, 2016 Accepted: July 19, 2016 Published: August 2, 2016
During the process of tumorigenesis, inactivation of tumor suppressors is a critical step. EZH2, a histone methyltransferase, promotes cell growth and migration through catalyzing trimethylation of histone H3 at Lys 27 (H3K27me3) and plays an important role in tumorigenesis. Its expression can be controlled by phosphorylation. However, the regulation of EZH2 activity by tumor suppressor kinase is not well understood. In this study, we show that glycogen synthase kinase 3 beta (GSK3β) negatively regulates H3K27 trimethylation. We also validate that GSKβ physically interacts with EZH2, and their interaction occurs in the cytosol. GSK3β phosphorylates EZH2 at Ser363 and Thr367 in vitro, and activating GSK3β upregulates Thr367 phosphorylationin vivo. Cells expressing GSK3β-non-phosphorylatable mutant EZH2 have higher H3K27 trimethylation and enhanced ability of cell migration and anchorage-independent growth. Inactivation of GSK3β as measured by its phosphorylation at Ser9 is positively correlated with higher level of H3K27 trimethylation in tumor tissues from breast cancer patients. Our study indicated that GSK3β phosphorylates EZH2 at Ser363 and Thr367, resulting in reduced H3K27 trimethylation and biological activity of EZH2 in breast cancer.
Epigenetic regulation, including histone modification, is an important mechanism that mediates gene expression by modifying the chromatin structure without changing the DNA sequences and is also essential for cell differentiation and tissue development. Aberrations of this regulation can alter gene expression, induce neoplastic cell transformation, and cause tumor growth . Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and key player in epigenetics, serves as the enzymatic core subunit of polycomb repressive complex 2 (PRC2). The EZH2/PRC2 complex catalyzes the trimethylation of histone H3 at lysine 27 (H3K27) on the promoter of its target genes, such as HOX genes, to recruits other polycomb group proteins and factors, to silence gene expression [2, 3]. The histone methyltransferase activity of EZH2 requires its association with other PRC2 components, primarily suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED) . EZH2 and other polycomb group proteins play critical roles in many biological processes, like germline development, X-chromosome inactivation, and maintenance of stem cell properties .
In addition, EZH2 has been reported to play an important role in tumorigenesis. Evidence has shown that EZH2 is highly expressed in many types of solid tumors, including breast cancer, and its higher expression is associated with aggressive disease and poor outcome [6–8]. Several studies have also demonstrated that EZH2 contributes to tumorigenesis through promoting malignant transformation, cell proliferation, invasion and migration [8–12]. In mammary epithelial cells, overexpression of EZH2 enhances anchorage-independent growth and cell invasion and EZH2-mediated cell invasion requires its enzymatic domain, Su(var)3-9, enhancer of zeste, trithorax (SET) domain . In breast tumor initiating cells, increased EZH2 expands cell population through repressing RAD51 expression, and knockdown of EZH2 reduces the repression of RAD51 transcription by decreasing H3K27 trimethylation . Furthermore, in many types of cancer cells, EZH2 mediates cell proliferation, invasion and migration by epigenetically repressing tumor suppressor gene expressions through trimethylating H3K27 [10–12]. These studies have strongly linked EZH2 to oncogenesis and suggested that the oncogenic role of EZH2 partly relies on its capacity of catalyzing H3K27 trimethylation to repress specific gene transcription .
In the process of tumorigenesis, inactivation or loss of function of tumor suppressors is a crucial step. Several kinases such as AKT1, CDK1/2, p38α, and JAK2 have been reported to modulate EZH2 activity through posttranslational modifications [14–22]. For example, the first discovery of this regulation is through AKT1 phosphorylation, in which EZH2 is phosphorylated at Ser21 which subsequently alters its affinity for histone H3 in breast cancer . This phosphorylation was later found to enhance the enzymatic activity of EZH2 in regulating non-histone substrates, such as androgen receptor and STAT3, in different cancer types [23, 24]. In addition, CDK2-mediated Thr416 phosphorylation augments cell migration, invasion, and tumor growth, and higher phosphorylation is correlated with poorer survival in triple-negative breast cancer patients . However, the regulation of EZH2 activity by tumor suppressor kinase in cancer remains unclear. Interestingly, we noticed that the EZH2 amino acid sequence contains several glycogen synthase kinase 3 beta (GSK3β) phosphorylation motifs (Ser/Thr-X-X-X-Ser/Thr, where X represents any amino acid) , and GSK3β and EZH2 interaction has been shown in nasopharyngeal cancer with unknown consequence . Together, these findings suggest that EZH2 might be regulated by GSK3β. GSK3β, a serine/threonine kinase, was initially identified as a critical mediator in glycogen metabolism, and has later been shown to be involved in diverse cellular processes, such as transcription, protein synthesis, cell cycle/proliferation, and microtube dynamics, through directly phosphorylation of a wide range of substrates (eIF2B, cycline D1, Tau, Snail and Mcl-1) [27–29]. Unlike other kinase, GSK3β is active at resting state but becomes inactive upon extracellular stimuli. The activity of GSK3β is controlled by site-specific phosphorylations, and Ser9 phosphorylation is probably the most well-known regulation which inhibits its activity . Several proteins, such as protein kinase A, Akt, p90 ribosomal S6 kinase (p90RSK), and p70 ribosomal S6 kinase (p70S6K), inactivate GSK3β via this modification. GSK3β also participates in neoplastic transformation and tumor development . Since GSK3β negatively regulates many oncoproteins and cell cycle regulators, it may function as a tumor suppressor. For example, GSK3β has been shown to phosphorylate and degrade β-catenin, and is a well-known, negative mediator of the canonical Wnt/β-catenin signaling pathway . GSK3β also inhibits cell proliferation through regulation of Mcl-1 degradation . In breast cancer cells, GSK3β suppresses epithelial-mesenchymal transition by control of Snail stabilization . Moreover, overexpression or activation of GSK3β suppresses anchorage-independent cell growth in different types of cancer cells, whereas inactivation of GSK3β by expressing kinase deficient mutant promotes cell transformation and mammary tumorigenicity [32–34]. These studies support GSK3β’s tumor suppressor role and reinforce its importance in tumorigenesis. However, little is known about the role of GSK3β in epigenetic regulation during tumor development.
In this study, we showed that GSK3β physically associated with and phosphorylated EZH2. Furthermore, this regulation suppressed EZH2 oncogenic functions and EZH2 enzymatic activity (trimethylation of H3K27) is inversely associated with GSK3β activity in tumor tissues from human breast cancer patients.
GSK3β negatively regulates H3K27 trimethylation
To investigate the regulation of EZH2 by GSK3β, we first determined whether alteration of GSK3β activity affects H3K27 trimethylation. We found that inhibition of GSK3β by lithium chloride, a GSK3β inhibitor, increased H3K27 trimethylation expression in multiple breast cancer cell lines including MDA-MB-231, BT549, MDA-MB-468, and MDA-MB-435S cells and mammary epithelial cells, MCF12A, and conversely, enhancing GSK3β activity using the anticancer drug staurosporine in MDA-MB-468, MDA-MB-435S and BT549 reduced the H3K27 trimethylation level (Figure 1A). There was no change in EZH2 level. Consistently, knockdown of GSK3β by small hairpin RNA enhanced trimethylation of H3K27 in HeLa cells (Figure 1B). GSK3β can phosphorylate and degrade β-catenin , and inhibition of GSK3β increases non-phospho-β-catenin level . Thus, we used the level of non-phospho-β-catenin as a determinant of GSK3β activity. We found that exogenous expression of the wild type or constitutively active form of GSK3β, as indicated by the reduced level of non-phospho-β-catenin, decreased H3K27 trimethylation expression (Figure 1C). We also examined the expression of HOXA family genes, known to be repressed by EZH2 , and found that lithium chloride downregulated the expression of many of HOXA genes (Figure 1D). These results suggested that inactivation of GSK3β upregulates the H3K27 trimethylation resulting in reduced expression of EZH2-targeted genes.
Figure 1: GSK3β downregulates H3K27 trimethylation. A. MDA-MB-231, BT549, MDA-MB-468, MDA-MB-435S, and MCF12A cells were treated with lithium chloride (LiCl), staurosporine (STS) as indicated. Cell lysates were subjected to western blot analysis with the indicated antibodies. The intensities of H3K27me3 bands from treated cells were compared to those from untreated cells and the relative ratios are shown. B. Cells infected with lentiviruses expressing control or GSK3β shRNA were lyzed and analyzed by immunoblot with antibodies against indicated proteins. Relative intensitis of H3K27me3 bands are shown. C. Equal amounts of lysates from cells transfected with the plasmids encoding wild-type (WT), constitutively active (CA), kinase-dead (KD) GSK3β or empty vector control were analyzed by Western blot using antibodies against specific proteins. Relative intensities of H3K27me3 bands are shown, normalized to the intensity of H3K27me3 band from cells transfected with control plasmid. D. Left: Western blot analysis of cells treated with PBS or LiCl with the indicated antibodies. Lysates were immunobloted with the indicated antibodies. Relative intensities of H3K27me3 bands are shown. Right: qPR-PCR analysis of relative mRNA expression of HOXA families genes in lysates from MDA-MB-231 cells treated with PBS or LiCl. Data are expressed as mean ± s.d. (n = 3).
GSK3β interacts with EZH2
Since altering GSK3β activity affects the trimethylation of H3K27 and the expression of EZH2-targeted genes, and GSK3β-EZH2 interaction has been shown in nasopharyngeal cancer cells , we asked whether this interaction occurs in our system, including breast cancer and mammalian epithelial cells. Co-immunoprecipitation experiment demonstrated an association between myc-EZH2 and HA-GSK3β in HEK 293T cells (Supplementary Figure S1). Flag-EZH2 and endogenous GSK3β interaction in MDA-MB-231 (Figure 2A) and HA-GSK3β and endogenous EZH2 interaction in HeLa cells (Figure 2B) were observed. In MCF12A cells, endogenous binding of GSK3β to EZH2 interaction was also shown by reciprocal immunoprecipitation (Figure 2C), which is consistent with previous report . EZH2 is predominantly present in the nucleus, whereas GSK3β is found mostly in the cytosol (Figure 2D, right panel). Thus, we further examined the subcellular location of their interaction. Surprisingly, we detected the GSK3β-EZH2 interaction in the cytosol (Figure 2D, left panel). EZH2, indeed, interacted with SUZ12 and EED in the nucleus (Supplementary Figure S2). These results suggested that GSK3β physically interacts with EZH2 in the cytosol.
Figure 2: GSK3β interacts with EZH2. A. MDA-MB-231 cells were transfected with plasmids encoding Flag-EZH2. Cell lysates were immunoprecipitated with GSK3β (upper panel) or Flag (lower panel) antibodies, followed by Westeren blot analysis to detect EZH2 and GSK3β as indicated. Immunocipitation with immunoglobulin (IgG) served as a control. B. HeLa cells were transfected with plasmids encoding HA-GSK3β. Cell lysates were immunoprecipitated with HA (upper panel) or EZH2 (lower panel) antibodies, followed by Westeren blot analysis with antibodies against EZH2 and GSK3β. C. Cell lysates from MCF12A cells were immunoprecipitated by either GSK3β (upper panel) or EZH2 (lower panel) antibodies, then immunoblotted with indicated antibodies. D. MCF12A cells were lysed and followed by cellular fractionation. Nuclear (Nuc) and cytosolic (Cyto) fractions were immunoprecipitated with EZH2 antibody and immunoblotted with antibodies against EZH2 and GSK3β. Lamin B1 and Tubulin were used as markers for nuclear and cytosolic fractions, respectively.
GSK3β phosphorylates EZH2
Next, we examined whether GSK3β can phosphorylate EZH2. An in vitro kinase assay revealed that GSK3β catalyzed the phosphorylation of EZH2 but not glutathione-S-transferase (GST) (Figure 3A, lanes 1-3). This phosphorylation was catalyzed on EZH2’s N-terminal fragment, rather than its C-terminal fragment (Figure 3A, lanes 4-6). Mass spectrometry analysis using the N-terminal fragment of EZH2 identified 4 possible phosphorylation sites: Ser362, Ser363, Ser366, and Thr367 (Figure 3B). We replaced these 4 residues with alanine individually. An in vitro kinase assay demonstrated that the phosphorylation catalyzed by GSK3β was reduced at the Ser363A mutant and was nearly undetectable at the Thr367A mutant (Supplementary Figure S3), which suggests that these two residues are GSK3β phosphorylation sites on EZH2. We further generated a double A (Ser363A & Thr367A) mutant. Consistently, the phosphorylation catalyzed by GSK3β was virtually undetectable (Figure 3C). A search of National Center for Biotechnology Information (NCBI) database using Basic Local Alignment Search Tool (BLAST) revealed that Ser363 and Thr367 of EZH2 are highly conserved among zebrafish to human (Figure 3D).
Figure 3: GSK3β phosphorylates EZH2. A. In vitro kinase assay with recombinant, active GSK3β kinase and full-length GST-EZH2 (FL), GST-EZH2 N-terminal (a.a. 1-385; GST-EZH2-N), or C-terminal (a.a. 386-746; GST-EZH2-C) fragment. Phosphorylation was detected by autoradiography. Loading amount of different EZH2 proteins was accessed by coomassie blue staining. B. Mass spectrometry analysis of samples from an in vitro kinase assay with GSK3β kinase and GST-EZH2 N-terminal fragment. The spectrum shows that two phosphorylation sites were identified; one was T367 or S366 (marked in red), the other S363 or S362 (marked in green). C. In vitro kinase assay with active GSK3β kinase and wild-type GST-EZH2 N-terminal fragment (WT), or mutant EZH2 as indicated. Phosphorylation was examined by autoradiography. Loading of EZH2 proteins was assessed by Coomassie blue staining. 2A represents S363A and T367A mutant. D. Comparison of GSK3β phosphorylation sites of EZH2 among various species. E. Testing of Thr367 phosphorylation antibody using in vitro kinase assay with active GSK3β kinase and purified wild-type GST-EZH2-N (WT), GST-EZH2S363A-N or GST-EZH2T367A-N in the presence of cold ATP at 30°C for 30 min. Reaction mixtures were analyzed by Western blot with mouse serum against Thr367 phosphorylation of EZH2 or antibodies as indicated. F. Lysates from cells transfected with control or wild-type GSK3β were immunoblotted with the indicated antibodies. Relative intensities of Thr367 phosphorylation and H3K27me3 bands are shown, normalized to those from cells transfected with control plasmid.
To confirm the phosphorylation of EZH2 by GSK3β in vivo, we generated two specific mouse antisera against the phosphorylated EZH2 at these two residues separately, in which only one antibody could specifically recognize the Thr367 phosphorylation. We validated the specificity of the antibody by an in vitro kinase assay using cold ATP. This antibody recognized the GSK3β-catalyzed phosphorylation on wild-type GST-EZH2 or GST-EZH2S363A mutant, but not the non-phosphorylatable GST-EZH2T367A mutant (Figure 3E), suggesting that it can specifically identify Thr367 phosphorylation of EZH2. Using this antibody, we found that ectopic expression of GSK3β enhanced the endogenous level of the phosphorylated EZH2 at Thr367 (Figure 3F). Taken together, these data indicated that GSK3β phosphorylates EZH2 at Ser363 and Thr367.
GSK3β-mediated phosphorylation of EZH2 downregulates H3K27 trimethylation and EZH2’s oncogenic functions
To further investigate whether GSK3β phosphorylation sites on EZH2 affects the H3K27 trimethylation and EZH2’s biological functions, we generated stable cell lines expressing the wild-type EZH2, non-phosphorylatable, or phospho-mimic mutants in MDA-MB-231 and MCF12A cells. In MDA-MB-231 cells, we stably transfected with wild-type EZH2, EZH22A or EZH22E. To examine the effects of individual phosphorylation sites on H3K27 trimethylation levels and EZH2’s functions, MCF12A cells were expressed with EZH2S363A, EZH2T367A, EZH22A or wild-type EZH2. We found that EZH22A increased and EZH22E decreased H3K27 trimethylation levels (Figure 4A). In MCF12A stable cells, trimethylation of H3K27 was upregulated in all non-phosphorylatable mutants (Figure 4B). Consistently, we observed the similar effects of non-phosphorylatable 2A mutant and phospho-mimic mutants on H3K27 trimethylation levels in MCF7 cells expressing EZH22A, EZH2S363E, EZH2T367E, or EZH22E (Supplementary Figure S4). As aforementioned, the association of EZH2 with SUZ12 and EED affects EZH2’s histone methyltransferase activity. However, we did not observe an increased binding of EZH2 to SUZ12 or EED in MCF12A cells expressing non-phosphorylatable EZH2 (Supplementary Figure S5). Collectively, these data suggested that GSKβ-mediated phosphorylation of EZH2 inversely regulates H3K27 trimethylation.
Figure 4: GSK3β-mediated EZH2 phosphorylation decreases H3K27 trimethylation and non-phosphorylatable mutants enhances anchorage-independent growth and cell migration. A. MDA-MB-231 cells were stably transfected with plasmids encoding wild-type EZH2 (WT), EZH22A (2A), EZH2 2E (2E) or empty vector control. Cell lysates were subjected to Western blot analysis with the indicated antibodies. Relative intensities of H3K27me3 bands are shown, compared to those from cells expressing wild-type EZH2. 2E represents Ser363E and Thr367E. B. MCF12A cells were stably transfected with plasmids encoding wild-type EZH2 (WT), EZH22A (2A), EZH2S363A, EZH2S367A, or control. Cell lysates were immunoblotted with specific antibodies. Relative intensities of H3K27me3 bands are presented. C. Colony formation abilities of MDA-MB-231 stable cell lines were determined using soft agar assay. Cells were seeded in 6-well plates as described. The number of colonies counted in one well of 6-well plate is shown as bar graphs. Data are expressed as mean ± s.d. from three independent experiments. Representative images are shown at the top of each bar graph. D. Migration potential of MDA-MB-231 stable cell lines were measured by wound healing assay. Cells were seeded in culture inserts and migration was observed by time-lapse microscope as described. Representative images of each line are shown immediately (time 0), 12 h, and 18 h after removal of culture inserts. The areas of wound gap at the indicated time points were determined using the ImageJ software program and normalized to the area of wound gap at time 0. Wound closures were calculated and are plotted as bar graphs. Data are mean ± s.d. from three independent experiments. E and F. The same experiments as described in C and D were performed in MCF12A stable cell lines. An asterisk (*) indicates a statistically significant difference in the measurements between mutant and wild-type (P < 0.05, Student’s t-test). N.S., not significant.
Because EZH2 is known to promote cell transformation  and migration [17, 36], and GSK3β-mediated phosphorylation of EZH2 downregulates H3K27 trimethylation, we next studied the effect of non-phosphorylatable and phospho-mimic mutant EZH2 on EZH2-regulated biological functions. Colony formation abilities and cell migration potentials were determined in MDA-MB-231 stably transfected with wild-type EZH2, non-phosphorylatable 2A or phospho-mimic 2E mutant. There were no significant differences in cell proliferation among all transfectants (Supplementary Figure S6). An anchorage-independent soft agar assay revealed that non-phosphorylatable EZH2 (EZH22A) increased anchorage-independent growth more effectively than those of WT EZH2 and phospho-mimic mutant (EZH22E) (Figure 4C). Consistently, a wound healing assay monitored by time-lapse microscopy demonstrated that non-phosphorylatable mutant (EZH22A) enhanced cell migration (faster rate of wound closure) while EZH22E had similar rate of wound closure, compared to wild-type EZH2 (Figure 4D). To further determine the effect of individual single non-phosphorylatable mutants on the EZH2’s biological functions, we performed a soft agar and a wound healing experiments in noncancerous MCF12A mammary epithelial cells stably expressing EZH2S363A, EZH2T367A, EZH22A mutants. The results revealed that all non-phosphorylatable EZH2 promoted anchorage-independent growth as well as cell migration (Figure 4E and 4F). A Boyden chamber migration assay also supported these observations (Supplemental Figure S7). These results suggested that phosphorylation of EZH2 by GSK3β attenuates EZH2’s biological acivities, including anchorage-independent soft agar growth and migration potential.
H3K27 trimethylation is inversely correlated with GSK3β activity in breast cancer patients
To examine the pathological relevance of EZH2 regulation by GSK3β, we analyzed correlation between the activity of GSK3β and the enzymatic activity of EZH2 in human breast tumor specimens. Since GSK3β-mediated phosphorylation of EZH2 did not affect EZH2 expression level but reduce H3K27 trimethylation, and it is known that Ser9 phosphorylation can inactivate GSK3β activity and the measurement of Ser9 phosphorylation can be used to determine GSK3β inactivation , we compared expression of H3K27 trimethylation with level of GSK3β phosphorylation at Ser9 in tumor tissue samples from 110 breast cancer patients. Consistently, immunohistochemical staining revealed that the level of GSK3β phosphorylation at Ser9 (representing inactivation of GSK3β) was positively correlated with the expression of H3K27 trimethylation (P = 0.006; Figure 5 and Table 1). This result suggested that GSK3β activity is inversely related to EZH2 activity in breast cancer tissues.
Figure 5: H3K27me3 is correlated with pSer9-GSK3β and low expression of both in tissues associates with better survival in human breast cancers. One hundred ten breast tumor tissue samples were subjected to immunohistochemical staining with antibodies specific to phosphorylated GSK3β at Ser9 and H3K27 trimethylation. Representative images shown. Case 1 shows a representative specimen with high expression of Ser9 phosphorylation of GSK3β and H3K27 trimethylation. Case 2 is a sample with low expression of pSer9-GSK3β and H3K27me3.
Table 1: Relationship between H3K27 trimethylation (H3K27me3) and pS9-GSK3β expression in human surgical specimens of breast cancer
Low: negative (–) and score 1 (+)
High: score 2 (++) and score 3 (+++)
*Correlation between H3K27me3 and pS9-GSK3β was analyzed by the Pearson Chi-Square test. A P value < 0. 05 was set as the criterion for statistical significance.
In the current study, we presented an interesting regulatory mechanism by which GSK3β regulates EZH2 activity via direct phosphorylation (Figure 6). Our study demonstrated that GSK3β physically interacts with and phosphorylates EZH2 at Ser363 and Thr367, without altering EZH2 protein expression, and suppresses H3K27 trimethylation. We found that non-phosphorylatable mutant EZH2 enhanced cell migration and cell growth in an anchorage-independent manner, indicating that GSK3β’s regulation is important for the oncogenic functions of EZH2. Consistently, the immunohistochemical staining results revealed that inactivation of GSK3β is significantly correlated with higher level of H3K27 trimethylation in breast cancer patients.
Figure 6: Proposed model of GSK3β-mediated regulation of EZH2. GSK3β phosphorylates EZH2 at Ser363 and Thr367, which suppresses H3K27 trimethylation and EZH2 oncogenic functions.
EZH2 has been shown to promotes tumorigenesis in many types of cancers [13, 22]. To prevent tumorigenesis, EZH2 expression and activity need to be critically checked. A recent study reported that GSK3β negatively regulates EZH2 expression in nasopharyngeal cancer cells, and the level of GSK3β phosphorylation at Ser9 is associated with higher EZH2 protein expression in patients with nasopharyngeal carcinoma . In our study, we found that GSK3β negatively regulates EZH2’s enzymatic activity, which is reflected by the expression of H3K27 trimethylation, without altering EZH2 protein level, and this observation was further supported by our immunohistochemical staining analysis in human breast cancer tissue samples. Although the previous study and our findings suggest that EZH2 is subjected to GSK3β control, the underlying mechanism of this regulation may differ in different cancer types.
GSK3β is known to be involved in tumor development, but its role in tumorigenesis remains controversial. In some types of cancer like pancreatic and ovarian cancers, it contributes to tumorigenesis; in others such as breast cancer and nasopharyngeal carcinoma, it functions as a tumor suppressor [30, 37]. In our study, we demonstrated that GSK3β non-phosphorylatable mutant EZH2 (EZH22A, EZH2S363A, EZH2T367A) enhances cell migration and anchorage-independent growth in breast cancer and mammary epithelial cells. Together with previously published reports, our results further strengthened the role of GSK3β as a tumor suppressor in breast cancer and implied that inactivation of GSK3β is one of the mechanisms enhancing EZH2 activity in cancer.
Studies have shown that EZH2 can be translationally modified by phosphorylation [14–22]. Previous phosphoproteomic analyses of EZH2 have identified many phosphorylation residues in mouse tissues and human cell lines, including Ser362, Ser363, Ser366 and Thr367 [38, 39]. Our work confirmed that GSK3β phosphorylates EZH2 at Ser363 and Thr367. As mentioned earlier, GSK3β has a preference for its substrates by recognizing a phosphorylation motif. Interestingly, the identified phosphorylation sites on EZH2 are compatible with this consensus motif. Furthermore, these two phosphorylation sites are highly conserved residues, implying that this regulation could be functionally important in other organisms. We attempted to determine the in vivo relationship between GSK3β and phosphorylated EZH2 in human tumor samples by using antibodies against these phosphorylation sites, but the antibodies we generated did not work for immunohistochemical staining. Previously, a study revealed that EZH2 can be phosphorylated at Thr367 by p38α in muscle stem cells . p38α-mediated EZH2 phosphorylation leads to Pax7 repression through trimethylation of H3K27 in its promoter while inhibition of p38α-EZH2 pathway promotes muscle stem cell proliferation. In our study, we observed that non-phosphorylatable mutant, EZH2T367A, increased global H3K27 trimethylation in mammary epithelial cells and enhanced cell growth and migration. Activation of GSK3β upregulated Thr367 phosphorylation. Our findings and previous report suggested that the biological significance of EZH2 phosphorylation at Thr367 is context-dependent.
The histone methyltransferase activity of EZH2 can be regulated by site-specific phosphorylations in different mechanisms. For example, JAK2 phosphorylates EZH2 at Tyr641, which is located on the catalytic SET domain, promotes EZH2’s interaction with β-TrCP and leads to its degradation . Thr487 phosphorylation by CDK1 disrupts PRC2 assembly and reduces H3K27 trimethylation . In the present study, these two phosphorylation sites we identified are neither located on the SET domain nor in the regions of SUZ12 or EED binding. Compatible with the observation, we (Supplemental Figure S4) and others  did not find that mutations at these sites (EZH22A, EZH2S363A, EZH2T367A) change the association of EZH2 with EED or SUZ12. Furthermore, we did not detect alteration in EZH2 protein expression upon this regulation. However, our work revealed that GSK3β interacts with EZH2 and, unexpectedly, their interaction is in the cytosol. Previous report also showed that EZH2 has a cytosolic role . Thus, we hypothesized that GSK3β regulates H3K27 trimethylation by mediating EZH2’s localization. Further investigation is needed to verify this hypothesis. Yet, other possibility can not be excluded.
EZH2 has been shown to enhance WNT/β-catenin pathway by downregulating CXC finger protein 4 , and our previous work also indicated that EZH2 represses RAD51 expression which in turn activates β-catenin signaling . β-catenin is a critical substrate of GSK3β, and aberrant GSK3β/β-catenin activity has been demonstrated to play a key role in tumorigenesis . Inhibitors targeting EZH2 have been developed and tested in clinical trials . Notably, the use of EZH2 inhibitor in colon cancer cells was reported to restore WNT/β-catenin activity . This regulation of EZH2 by GSK3β directly links epigenetic modulation to the WNT/β-catenin pathway. Moreover, an interesting study reported that specific KRAS mutation regulates EZH2 protein expression through the PI3K/AKT and/or MEK/ERK signaling pathways in lung cancer . EZH2 inhibition enhances the sensitivity to MEK-ERK or PI3K/AKT targeted therapies in specific KRAS-mutant lung cancer cells and tumors. Since GSK3β is known to be inactivated by AKT or ERK [25, 43], our work suggests a direct role of the GSK3β-EZH2 pathway in this scenario and offers a rationale for enhancing GSK3β activity and/or targeting EZH2 in anti-cancer therapy.
MATERIALS AND METHODS
All cell lines were obtained from ATCC (Manassas, VA) and their validation was performed in Characterized Cell Line Core Facility, MD Anderson Cancer Center. The cell lines used in this study include MDA-MB-231, BT549, MDA-MB-435S, MDA-MB-468, MCF7, MCF12A, HeLa and 293T cells. All cells except MCF12A were grown in Dulbecco’s Modified Eagle’s Medium/F12 supplement (DMEM/F12) supplemented with 10% heat inactivated fetal bovine serum (FBS), Penicillin/Streptomycin (100 U, 100 μg/ml) at 37°C in a humidified atmosphere with 5% CO2. MCF12A cells were cultured in DMEM/F12 media supplemented with 5% horse serum. Transfection of cells with DNA was performed with liposome. EZH2 stable transfectants and GSK3β stable knockdown by shRNA were selected by either G418 (Fisher) or puromycin (Invivogen). For inhibiting or activating GSK3β activity, cells were treated with 40 mM lithium chloride (Sigma) for 16 h or 0.1 uM staurosporine (Sigma) for 2 h.
Western blot, immunoprecipitation and qRT-PCR
Whole cell lysis, subcellular fractionation, western blot and immunoprecipitation were performed as described [17, 44]. Quantification of the band density of target proteins in western blot experiments was analyzed by free software ImageJ. The following antibodies were used in western blotting and immunoprecipitation: EZH2, phospho-GSK3β Ser9, trimethyl-H3K27, and histone H3 (Cell Signaling Technology); GSK3β (BD Biosciences); dephospho-β-catenin (Ezno Life Sciences); Tubulin and Actin (Sigma); Lamin B1 (Abcam); Myc and HA (Roche). The mouse phospho-EZH2 Thr367 antibody was produced against the synthetic peptides SRPS(pT)PTINVLESKD at China Medical University Hospital in Taiwan. The synthetic peptides were obtained from LifeTein LLC. Total RNA was extracted from cells using TRIzol. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green dye on a Bio-Rad PCR machine. The primer sequences used for analysis of HOXA gene expression was listed in Supplementary Information Table S1.
Plasmids of HA-GSK3β-WT (wild-type), HA-GSK3β-CA (constitutively active, S9A GSK3β), and HA-GSK3β-KD (kinase dead, K85R GSK3β) were described previously . pCDNA3-Myc-EZH2 was a gift from A. Chinnaiyan. For EZH2 stable transfection, two EZH2 constructs were generated. Myc-EZH2 was subcloned into the vector of pCDH-CMV-MCS-EF1-Puro (System Biosciences), and Flag-EZH2 was subcloned into the vector of pCDH-CMV-MCS-EF1-Neo (System Biosciences). To generate constructs for bacterial expression of GST-tagged EZH2, two truncations were made in GST-N terminal fusion protein format in pGEX-6P1 vector (Amersham Biosciences or GE Healthcare). One EZH2 truncation, N-terminal fragment, was from amino acid residues 1-385, and another, C-terminal fragment, was from 386-746. Site-directed mutagenesis was performed to generate mutant EZH2 according to a protocol . Primers used for mutagenesis were provided in Supplemental Information Table S2. GSK3β knockdown was carried out by pGIPZ-shRNA with the target sequence of 5’-TACTTGACAGTTCTTGAGT-3’ (CDS, Clone ID V3LHS_309039, shRNA core facility, MD Anderson Cancer Center).
In vitro kinase assay
Recombinant, active GSK3β kinase was obtained from Life Technologies and glutathione-S-transferase (GST)-fused full length EZH2 was purchased from BPS Bioscience. GST-EZH2 C-terminal, wild-type and mutant GST-EZH2 N-terminal fragments were purified from bacteria. For the GSK3β in vitro kinase assay, active GSK3β kinase was incubated with wild-type or mutant GST-EZH2 purified proteins in kinase buffer (50 mM Tris-HCl at pH 7.6, 10 mM MgCl2, 2 mM DTT and 0.1 mM EDTA) in the presence of 5 μCi of [γ-32P]ATP and 50 μM cold ATP with substrates at 30 °C for 30 min . Reaction mixtures were then subjected to SDS-PAGE, and 32P-labelled proteins were detected by autoradiography.
To identify phosphorylation sites of EZH2, mass spectrometry analysis was performed as previously described . Briefly, GST-fused EZH2 purified protein was incubated with GSK3β kinase in a kinase reaction mixture at 30 °C for 30 min. After being resolved by SDS-PAGE, the protein band corresponding to EZH2 was excised and subjected to digestion with trypsin. The phosphopeptides were then isolated by immobilized metal affinity chromatography. The micro-liquid chromatography/tandem mass spectrometry (LC–MS/MS) was performed to analyze the phosphopeptides and to identify phosphorylation sites of EZH2.
Soft agar and wound healing assays
For the soft agar transformation assay, 2.5 × 104 cells were seeded in 1 ml of regular medium with 0.5% low melting point agarose and overlaid on 1 ml of medium with 1% agarose in each well of a six-well plate. After 3 weeks, colonies larger than 100 μm in diameter were counted. The wound healing assay was performed using a culture-insert (ibidi GmbH, Germany) according to manufacturer’s instruction. The culture-insert had two cell culture reservoirs, which were separated by a 500 μm-thick wall. Same numbers of cells were seeded in the culture-insert. After 24 h, the culture-insert was removed, which left a cell-free “wound” of around 500 μm in width. The wound closure was observed by a time lapse microscopy (Zeiss, Germany) and images were obtained at 1 h interval for 24 h. The area of wound was analyzed using the ImageJ software program.
Human breast tumor tissue samples were obtained following the guidelines approved by the Institutional Review Board of MD Anderson Cancer and written informed consents were obtained from all patients at the time of enrollment. Immunohistochemical staining was performed in 110 human breast cancer tissue specimens obtained from the Department of Pathology, Shanghai East Breast Disease Hospital, Shanghai, P.R. China. Each specimen was stained with specific antibodies against pS9-GSK3β (Cell Signaling Technology) and H3K27 trimethylation (EMD Millipore) and scored by an H-score method as previously described . The intensity of staining was ranked into four groups: high (score 3), medium (score 2), low (score 1), and negative (score 0). High and medium intensity of staining were grouped into “high expression”. Low and negative were “low expression”.
All data were representative of at least three independent experiments. All P values were analyzed by student’s t-test except for calculation of correlation analysis. For correlation analysis, a Pearson chi square test (SPSS software) was used to examine the relation between pS9-GSK3β expression and H3K27 trimethylation levels. A P value < 0.05 was considered statistically significant.
We would like to thank Joseph Munch in the Department of Scientific Publications at MD Anderson Cancer Center for editorial assistance.
CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare.
This work was partially supported by the following: the National Institutes of Health grants (CA109311, CA099031, and CCSG CA016672); Patel Memorial Breast Cancer Endowment Fund; Susan G. Komen Foundation (SAC 110016); The University of Texas MD Anderson Cancer Center-China Medical University and Hospital Sister Institution Fund (to M.-C.H.); Ministry of Science and Technology, International Research-intensive Centers of Excellence in Taiwan (I-RiCE; MOST 105-2911-I-002-302); Ministry of Health and Welfare, China Medical University Hospital Cancer Research Center of Excellence (MOHW105-TDU-B-212-134003); Center for Biological Pathways; MD Anderson CPRIT Graduate Scholar Training Program (to H.-W. K.).
1. Sharma S, Kelly TK and Jones PA. Epigenetics in cancer. Carcinogenesis. 2010; 31:27-36.
2. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones RS and Zhang Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science. 2002; 298:1039-1043.
3. Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P and Reinberg D. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes & development. 2002; 16:2893-2905.
4. Cao R and Zhang Y. SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Molecular cell. 2004; 15:57-67.
5. Cao R and Zhang Y. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Current opinion in genetics & development. 2004; 14:155-164.
6. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, Rubin MA and Chinnaiyan AM. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002; 419:624-629.
7. Bachmann IM, Halvorsen OJ, Collett K, Stefansson IM, Straume O, Haukaas SA, Salvesen HB, Otte AP and Akslen LA. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. Journal of clinical oncology. 2006; 24:268-273.
8. Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, Ghosh D, Sewalt RG, Otte AP, Hayes DF, Sabel MS, Livant D, Weiss SJ, Rubin MA and Chinnaiyan AM. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proceedings of the National Academy of Sciences of the United States of America. 2003; 100:11606-11611.
9. Chang CJ, Yang JY, Xia W, Chen CT, Xie X, Chao CH, Woodward WA, Hsu JM, Hortobagyi GN and Hung MC. EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-beta-catenin signaling. Cancer cell. 2011; 19:86-100.
10. Yang X, Karuturi RK, Sun F, Aau M, Yu K, Shao R, Miller LD, Tan PB and Yu Q. CDKN1C (p57) is a direct target of EZH2 and suppressed by multiple epigenetic mechanisms in breast cancer cells. PloS one. 2009; 4:e5011.
11. Cao Q, Yu J, Dhanasekaran SM, Kim JH, Mani RS, Tomlins SA, Mehra R, Laxman B, Cao X, Yu J, Kleer CG, Varambally S and Chinnaiyan AM. Repression of E-cadherin by the polycomb group protein EZH2 in cancer. Oncogene. 2008; 27:7274-7284.
12. Ren G, Baritaki S, Marathe H, Feng J, Park S, Beach S, Bazeley PS, Beshir AB, Fenteany G, Mehra R, Daignault S, Al-Mulla F, Keller E, Bonavida B, de la Serna I and Yeung KC. Polycomb protein EZH2 regulates tumor invasion via the transcriptional repression of the metastasis suppressor RKIP in breast and prostate cancer. Cancer research. 2012; 72:3091-3104.
13. Chang CJ and Hung MC. The role of EZH2 in tumour progression. British journal of cancer. 2012; 106:243-247.
14. Cha TL, Zhou BP, Xia W, Wu Y, Yang CC, Chen CT, Ping B, Otte AP and Hung MC. Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3. Science. 2005; 310:306-310.
15. Kaneko S, Li G, Son J, Xu CF, Margueron R, Neubert TA and Reinberg D. Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA. Genes & development. 2010; 24:2615-2620.
16. Chen S, Bohrer LR, Rai AN, Pan Y, Gan L, Zhou X, Bagchi A, Simon JA and Huang H. Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2. Nature cell biology. 2010; 12:1108-1114.
17. Wei Y, Chen YH, Li LY, Lang J, Yeh SP, Shi B, Yang CC, Yang JY, Lin CY, Lai CC and Hung MC. CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells. Nature cell biology. 2011; 13:87-94.
18. Wu SC and Zhang Y. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of enhancer of zeste 2 (Ezh2) regulates its stability. The Journal of biological chemistry. 2011; 286:28511-28519.
19. Yang CC, LaBaff A, Wei Y, Nie L, Xia W, Huo L, Yamaguchi H, Hsu YH, Hsu JL, Liu D, Lang J, Du Y, Lien HC, Li LY, Deng R, Chan LC, et al. Phosphorylation of EZH2 at T416 by CDK2 contributes to the malignancy of triple negative breast cancers. American journal of translational research. 2015; 7:1009-1020.
20. Palacios D, Mozzetta C, Consalvi S, Caretti G, Saccone V, Proserpio V, Marquez VE, Valente S, Mai A, Forcales SV, Sartorelli V and Puri PL. TNF/p38alpha/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration. Cell stem cell. 2010; 7:455-469.
21. Sahasrabuddhe AA, Chen X, Chung F, Velusamy T, Lim MS and Elenitoba-Johnson KS. Oncogenic Y641 mutations in EZH2 prevent Jak2/beta-TrCP-mediated degradation. Oncogene. 2015; 34:445-454.
22. Yamaguchi H and Hung MC. Regulation and Role of EZH2 in Cancer. Cancer research and treatment. 2014; 46:209-222.
23. Xu K, Wu ZJ, Groner AC, He HH, Cai C, Lis RT, Wu X, Stack EC, Loda M, Liu T, Xu H, Cato L, Thornton JE, Gregory RI, Morrissey C, Vessella RL, et al. EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent. Science. 2012; 338:1465-1469.
24. Kim E, Kim M, Woo DH, Shin Y, Shin J, Chang N, Oh YT, Kim H, Rheey J, Nakano I, Lee C, Joo KM, Rich JN, Nam DH and Lee J. Phosphorylation of EZH2 activates STAT3 signaling via STAT3 methylation and promotes tumorigenicity of glioblastoma stem-like cells. Cancer cell. 2013; 23:839-852.
25. Cohen P and Frame S. The renaissance of GSK3. Nature reviews Molecular cell biology. 2001; 2:769-776.
26. Ma R, Wei Y, Huang X, Fu R, Luo X, Zhu X, Lei W, Fang J, Li H and Wen W. Inhibition of GSK 3beta activity is associated with excessive EZH2 expression and enhanced tumour invasion in nasopharyngeal carcinoma. PloS one. 2013; 8:e68614.
27. Doble BW and Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. Journal of cell science. 2003; 116:1175-1186.
28. Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M and Hung MC. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nature cell biology. 2004; 6:931-940.
29. Ding Q, He X, Hsu JM, Xia W, Chen CT, Li LY, Lee DF, Liu JC, Zhong Q, Wang X and Hung MC. Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization. Molecular and cellular biology. 2007; 27:4006-4017.
30. Luo J. Glycogen synthase kinase 3beta (GSK3beta) in tumorigenesis and cancer chemotherapy. Cancer letters. 2009; 273:194-200.
31. Wu D and Pan W. GSK3: a multifaceted kinase in Wnt signaling. Trends in biochemical sciences. 2010; 35:161-168.
32. Ma C, Wang J, Gao Y, Gao TW, Chen G, Bower KA, Odetallah M, Ding M, Ke Z and Luo J. The role of glycogen synthase kinase 3beta in the transformation of epidermal cells. Cancer research. 2007; 67:7756-7764.
33. Dong J, Peng J, Zhang H, Mondesire WH, Jian W, Mills GB, Hung MC and Meric-Bernstam F. Role of glycogen synthase kinase 3beta in rapamycin-mediated cell cycle regulation and chemosensitivity. Cancer research. 2005; 65:1961-1972.
34. Farago M, Dominguez I, Landesman-Bollag E, Xu X, Rosner A, Cardiff RD and Seldin DC. Kinase-inactive glycogen synthase kinase 3beta promotes Wnt signaling and mammary tumorigenesis. Cancer research. 2005; 65:5792-5801.
35. Zhang L, Cen L, Qu S, Wei L, Mo M, Feng J, Sun C, Xiao Y, Luo Q, Li S, Yang X and Xu P. Enhancing Beta-Catenin Activity via GSK3beta Inhibition Protects PC12 Cells against Rotenone Toxicity through Nurr1 Induction. PloS one. 2016; 11:e0152931.
36. Moore HM, Gonzalez ME, Toy KA, Cimino-Mathews A, Argani P and Kleer CG. EZH2 inhibition decreases p38 signaling and suppresses breast cancer motility and metastasis. Breast cancer research and treatment. 2013; 138:741-752.
37. Morrison JA, Gulley ML, Pathmanathan R and Raab-Traub N. Differential signaling pathways are activated in the Epstein-Barr virus-associated malignancies nasopharyngeal carcinoma and Hodgkin lymphoma. Cancer research. 2004; 64:5251-5260.
38. Mayya V, Lundgren DH, Hwang SI, Rezaul K, Wu L, Eng JK, Rodionov V and Han DK. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Science signaling. 2009; 2:ra46.
39. Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, Villen J, Haas W, Sowa ME and Gygi SP. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell. 2010; 143:1174-1189.
40. Gunawan M, Venkatesan N, Loh JT, Wong JF, Berger H, Neo WH, Li LY, La Win MK, Yau YH, Guo T, See PC, Yamazaki S, Chin KC, Gingras AR, Shochat SG, Ng LG, et al. The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin. Nature immunology. 2015; 16:505-516.
41. Kim KH and Roberts CW. Targeting EZH2 in cancer. Nature medicine. 2016; 22:128-134.
42. Riquelme E, Behrens C, Lin HY, Simon G, Papadimitrakopoulou V, Izzo J, Moran C, Kalhor N, Lee JJ, Minna JD and Wistuba, II. Modulation of EZH2 Expression by MEK-ERK or PI3K-AKT Signaling in Lung Cancer Is Dictated by Different KRAS Oncogene Mutations. Cancer research. 2016; 76:675-685.
43. Ding Q, Xia W, Liu JC, Yang JY, Lee DF, Xia J, Bartholomeusz G, Li Y, Pan Y, Li Z, Bargou RC, Qin J, Lai CC, Tsai FJ, Tsai CH and Hung MC. Erk associates with and primes GSK-3beta for its inactivation resulting in upregulation of beta-catenin. Molecular cell. 2005; 19:159-170.
44. Huo L, Li CW, Huang TH, Lam YC, Xia W, Tu C, Chang WC, Hsu JL, Lee DF, Nie L, Yamaguchi H, Wang Y, Lang J, Li LY, Chen CH, Mishra L, et al. Activation of Keap1/Nrf2 signaling pathway by nuclear epidermal growth factor receptor in cancer cells. American journal of translational research. 2014; 6:649-663.
45. Liu H and Naismith JH. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC biotechnology. 2008; 8:91.
46. Li CW, Xia W, Lim SO, Hsu JL, Huo L, Wu Y, Li LY, Lai CC, Chang SS, Hsu YH, Sun HL, Kim J, Yamaguchi H, Lee DF, Wang H, Wang Y, et al. AKT1 Inhibits Epithelial-to-Mesenchymal Transition in Breast Cancer through Phosphorylation-Dependent Twist1 Degradation. Cancer research. 2016; 76:1451-1462.
47. Ding Q, He X, Xia W, Hsu JM, Chen CT, Li LY, Lee DF, Yang JY, Xie X, Liu JC and Hung MC. Myeloid cell leukemia-1 inversely correlates with glycogen synthase kinase-3beta activity and associates with poor prognosis in human breast cancer. Cancer research. 2007; 67:4564-4571.
All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 4.0 License.

domain: literature


text:
» China » China » China
Price varies by group sizeLowest Price Guarantee
Pricing Info: Per Person
Departs: China, China
Ticket Type: Mobile or paper ticket accepted
Save your days with this private guided tour that covers the Dujinagyan irrigation system, Giant Buddha, Cute Panda, and fun Sichuan Cooking Class. On this tour, you would not only get the typical highlights of Sichuan but even can get a chance drinking in the old tea house, picking tea from the plantation, visit tea workshop, Visit the old courtyard houses and listen to stories from the owner. You will also explore a local wet market which is a great way to learn more about the food culture in this Beautiful City.
beers or beverages in cooking class
Boatride for Giant Buddha
Experienced English-speaking tour guide
Souvenir in cooking class
Climbing for Giant Buddha
golf car in Dujiangyan
For a full refund, cancel at least 24 hours before the scheduled departure time.
© Copyright 1995 - 2023 China Travel Guide

domain: travel


text:
The setter boy!
Hi, I'm Charlie!
My sponsor is the amazing Christine Dawson!
I was born in 2016 and ended up in the shelter in the summer of 2019, with my brother. My brother was adopted immediately, but unfortunately I'm still here, hoping that someone will notice me.
I'm really a good boy, excellent with other dogs and very gentle with people, but I do have a small medical issue. There is a huge lump in my neck, and fortunately it is just a saliva gland issue, but it needs removing at some point to make me more comfortable.
I don't feel sick or tired, I feel like I'm just as happy and energetic as I have always been. Hopefully someone will want to open their home and their heart to a sweet little boy like me!
Want to help Charlie?
Help us make sure Charlie is healthy and help him get blood tests. With 50€ we can have him tested for the most common diseases like Leishmania and Giardia
Donate for neutering and we can reduce Charlie's risk of having a cancer as well as guarantee, he cant contribute to the stray problem. With 50 € we can neuter him!
Charlie needs a surgery to remove a tumor like growth from his neck. The surgery will cost 350 € and every penny is needed to help him be happy and healthy!

domain: medical


Check out this incredible instrument! It’s a microtonal piano, which means that you can tune every key, as you play it. Apart from playing the keys, you can also play it like a harp. Check out the video below where the inventor Geoff Smith explains how it works.
Tags: fluid piano


This 1959 Triumph TR3A is a sold completed project that underwent a full nut and bolt restoration. As the customer lived in France, the car was restored back to LHD, as original when it left the factory.
The restoration process included a bare metal respray, a full engine rebuild, with various upgrades such as SilentCoat sound proofing, K&N filters, a full electronic ignition kit and mohair weather gear to name a few.
After over 100 miles of test driving, the car was delivered to Plymouth for the customer to take the ferry across to Roscoff before driving down to Brittany.
If you’d like to know more about this project, or would like a similar vehicle restored, please contact us.


DAMASCUS: Israeli strikes knocked out war-torn Syria’s two main airports, state media reported citing a military source, with the transport ministry saying flights were re-routed to Latakia.
“At around 5:25am (0225 GMT), the Israeli enemy carried out... an air attack... targeting Damascus and Aleppo international airports, leading to the death of two civilian workers at Damascus airport and wounding another,” the military source said in the statement carried by state news agency SANA.
“Material damage to the airports’ runways put them out of service,” the statement added. The transport ministry said flights were diverted to Latakia airport.
The attack is the second this month on the Damascus International airport and the third on Aleppo’s airport as tensions increases in the Middle East over the Israel-Hamas war.
Syrian state media quoted an unnamed military official as saying the airports were struck by the Israeli military from the Mediterranean to the west and from Syria’s Israeli-occupied Golan Heights in the south.
The Israeli military had no immediate comment.
Since the war between Israel and Hamas started on October 7, Israel has carried out several strikes in Syria including one on the Damascus airport and two on Aleppo’s airport putting them out of service.
Israel has targeted airports and sea ports in the government-held parts of Syria in an apparent attempt to prevent arms shipments from Iran to militant groups backed by Tehran, including Lebanon’s Hezbollah.
Thousands of Iran-backed fighters from around the region joined Syria’s 12-year conflict helping tip the balance in favor of President Bashar Al Assad’s forces.
Israel has carried out hundreds of strikes on targets inside government-controlled parts of Syria in recent years, including attacks on the Damascus and Aleppo airports, but rarely acknowledges or discusses the operations.


IT is a term that is used to describe the various technologies that are used in business. IT can include anything from computer systems to software, to telecommunications.
The use of IT has revolutionized many aspects of business, including communication and collaboration. It has also allowed businesses to automate their processes and manage their data more effectively.
Also, Paragon Tech can help your businesses to improve their productivity, agility, and competitiveness. Here are a few ways that IT can help your business:
Image Source: Google
Improved Communication and Collaboration: IT can help to improve communication and collaboration between different parts of the business. This is particularly useful when it comes to sharing data and information. IT can also help to reduce the time it takes for different parts of the business to communicate with each other.
Automation and Efficiency: IT can help to automate business processes and make them more efficient. This can save time and money for businesses.
Improved Productivity: Improved productivity can be achieved through the use of IT tools such as software applications and computer systems.
Increased Competitiveness: By using IT tools, businesses can increase their competitiveness by improving their data processing capabilities and communications infrastructure.
What Does IT Stand For in Business?
IT stands for information technology. This involves using computer systems and networks to store, process, communicate, and manage data. IT can help businesses automate tasks, improve customer service, and keep up with the latest trends. In addition, it can help businesses protect their data from theft and cyberattacks.


text:
It looks like you are looking for Drawing/normal Pusheen The Cat, in this site we have 1 images about Drawing/normal Pusheen The Cat. Substantial selection of images, images and wallpapers. Make this kind of the source of your respective suggestions, explore this site and you may find new ideas coming from your image. On this kind of page, we also possess various images available. Like as PNG, GIF animation, JPG, drawing art, art logos, etc.
You can actually get started with pad drawing when you have learned the particular skill. Young kids adore to do pencil pulling. You can utilize this as the doorway to introduce these people to the higher associated with drawing very easily. Assess your sons or daughters interesting subject in addition to create a pencil sketching for your child. This may make the drawing are available to life for the young one.
Drawing/normal Pusheen The Cat, Are an individual a parent interested throughout helping your child construct on their drawing skill? You may have perhaps been surfing the web looking for step-by-step assignments they will enjoy. Oneself a project, then exactly what? How would you present that info for your young one particular to draw? YouTube provides many great drawing suggestions with step-by-step instructions.
A lot of kids love to bring. However they get stuck in addition to frustrated when what they will see within their heads, or perhaps what they want to make on paper, doesnt match up what comes out regarding their hands. After a few practice and some straight forward instruction, any kid (or adult, for that matter) can boost their skills. Below are some great publications for kids who would like to learn how to be able to draw.
Most kids My partner and i know love to bring. Its a great approach to express yourself and even have fun, and while an extra00, you can employ your drawing skills to be able to make gifts for some others. Who knows--one day, a person may use those abilities to make money, also. If you want to be able to learn to draw much better, here are some attracting methods for kids.
Don't forget to bookmark Drawing/normal Pusheen The Cat If you use mobile phone, you may also use menu cabinet from browser. Be it House windows, Mac, iOs or Google android, you will be capable to download the pictures using download button.
It looks like you are looking for how to draw pusheen the cat, in this site we have 27 images about how to draw pusheen the cat. Huge selection of images, photographs and wallpapers. Make this kind of the source of the tips, explore this site and you may find new ideas by your image. […]

domain: artistic


text:
Born: 1798-12-20 Moravia, New York
Died: 1880-06-04 Quincy, Illinois
Flourished: Quincy, Illinois
Son of a Revolutionary War officer, John Wood was a farmer, soldier, public servant, Civil War officer, Whig, and, later, a Republican. He migrated West in 1818, entered Illinois in 1819, and settled near the future site of Quincy, Illinois the following year. In 1822, he built a cabin that became the first building in Quincy and began organizing Adams County two years later. He was one of the earliest town trustees and served on the city council before becoming mayor. He married Ann M. Streeter in 1826, and the pair had eight children together, although four died in childhood. Wood also served in the Black Hawk War and won election to Illinois Senate in 1850, where he remained until 1854. In 1856, Illinois Republicans nominated him as William H. Bissell’s running mate, and the state’s voters elected him lieutenant governor in the 1856 Federal Election. Following Bissell’s death, Wood served as governor of the state from 1859 to 1860. By 1860, he owned $250,000 in real estate and another $50,000 in personal property. In 1861, he was one of Illinois’ five delegates to the Washington Peace Conference and, after the outbreak of the Civil War, earned appointment as the quartermaster of Illinois. In 1864, he helped organize the 137th Regiment of the Illinois Infantry and served as its colonel for the remainder of the war. His wife Ann died in 1863, and he married Mary A. Holmes in 1865.
The History of Adams County, Illinois (Chicago: Murray, Williamson, and Phelps, 1879), 713-14; Governors of Illinois: 1818-1918 (Springfield: Illinois Centennial Commission, 1917), 25; Robert P. Howard, Mostly Good and Competent Men: Illinois Governors, 1818-1988 (Springfield: Illinois Issues, Sangamon State University and Illinois State Historical Society, 1988), 117-19; U.S. Census Office, Eighth Census of the United States (1860), Ward 4, Quincy, Adams County, IL, 196; Gravestone, Woodland Cemetery, Quincy, IL. Illustration courtesy of the Abraham Lincoln Presidential Library, Springfield, IL.

domain: history


text:
- PRODUCT DETAILS
Small bag crafted of petrol leather with a purple shoulder strap.
With a phone pocket and keycord on the inside and zip fastening on top.
27 cm height x 22 cm width x 11 cm depth (approximately)
Shoulder strap: 44 cm
Leather is a natural product and will naturally age, LOVE IT!

domain: design


text:
We've released three new titles available as digital and hard copy publications!
- Architecture, Building Design & Copyright
- Writers & Copyright
- Commercialising Copyright
- Libraries & Copyright
Take a look at our hot End of Financial Year sale available only until 30 June
The Australian Copyright Council is an independent, non-profit
organisation. Founded in 1968, we represent the peak bodies for
professional artists and content creators working in Australia’s
creative industries and Australia’s major copyright collecting
Posted on 6/1/2016
Posted on 5/23/2016
Posted on 5/2/2016
Posted on 4/29/2016
Posted on 4/19/2016
We have re-shaped our Seminar Series
See our new and more specialised topics for creators:
- How can I commercialise creative works?
- How can I protect my copyright?
- What do I need to know about copyright in social media?
New for Education, Galleries, Libraries, Archives & Museums:
- Risk Management
- Managing your Digital Library
As well as Hot Topics for Copyright professionals
Our books are now available in digital format!
Compatible with most devices and available the instant you order and pay.
For more information click here
Visit the Find an Answer section

domain: library


text:
CaveSim electronic virtual cave lets you try before you spelunk
You could easily go to a rock gym to try climbing or throw on a pair of boots and hike a local trail, but you'd need to invest a little more time and planning to try caving. You could commit to joining a caving club or pay for a guided tour, but options for just going out and giving it a go are quite limited. CaveSim is a unique innovation that lets prospective cavers get a taste for the sport by providing a virtual indoor cave environment. The device includes electronic sensors for video-game-like scoring features, allowing for tracking your personal score and competing against others.
Cavers like to keep things on the down low more than other outdoor sports enthusiasts. Because of the fragile nature of caves - and the disrespectful nature of humans - the National Speleological Society, the US's caving association, encourages cavers not to share the location of caves on websites or by word of mouth. Casual cave visitation can result in vandalism, landowner relations strains and safety issues, among other problems. While you'll find multiple websites dedicated to things like rock climbing routes and mountain bike trails, information on cave locations is quite sparse on the Web.
The CaveSim provides an interesting way of protecting caves and bridging the disconnect with potential cavers, or at least it could if it ever catches on. Similar to a rock gym, the CaveSim is a controlled simulation that lets you try the sport without buying gear, joining clubs, signing up for tours, etc. It's a quick way to practice or see if you can perform through the rigors of the sport.
CaveSim designer Dave Jackson, a caving hobbyist with an MIT electrical engineering degree, came up with the idea when participating in a cave rescue seminar. Because nothing like the CaveSim was available at the time of the seminar in 2008, organizers used a makeshift cave concoction of furniture and tape. The simulation fell well short of a realistic cave environment, and some participants even moved things around in order to travel through the simulated cave - not exactly something you can do in an actual cave. The next day, when the seminar switched gears to practicing in an actual cave, rescuers were trying to shake off bad habits developed when barreling through tables and tape.
"Unfortunately, on our second day in underground practice, there was a little bit of damage done to the real cave we were using," Jackson explained. "And I thought about the picnic tables, and I thought there was a better way to do this."
After about two years of development, Jackson found that better way, albeit in a slightly larger form than he originally planned on. What he came up with is a 48-foot-long (14.6 m) virtual cave that twists and bends over its 13 x 18 foot (4 x 5.5 m) footprint. It includes model cave features with built-in electronic sensors that provide feedback about your performance. In a real cave, you want to avoid knocking into formations because that can be harmful to both you and the cave. With the electronic tracking system, CaveSim encourages participants to crawl through as carefully as they would in an actual cave - unlike the picnic table scenario Jackson encountered prior.
While CaveSim was conceived for rescue training, it's now being used for anyone that wants to try caving in a realistic, virtual environment. Beginner cavers can try the sport out and get an idea of what it's like, and experienced cavers and rescuers can use it to keep their skills sharp.
Currently, only the original CaveSim exists. Jackson and his wife Tracy, who are both members of the NSS, transport it to events around the country. In fact, we originally stumbled upon the innovation on Kickstarter, where the Jacksons are trying to raise money to transport CaveSim to the National Speleological Society's annual convention in West Virginia, from their home near Colorado Springs. Their immediate goal is to rent a trailer, but if they receive enough support, they'll purchase a trailer and affix CaveSim inside it permanently, so that it's easy to transport from place to place.
The Jacksons also offer customized cave systems for sale as an attraction for amusement parks, climbing gyms and similar businesses. Given the large size of the CaveSim and the Jacksons' limited production capabilities (and day jobs), they currently only intend to build a small volume for interested businesses. As you might expect, it's a "call for details" type of buy, and CaveSim's website does not list pricing information. So far, one Colorado Springs-area customer is working with the Jacksons to get one built.

domain: hobby


text:
Privacy note: We never disclose your personal information to anyone and redact your email address in communications to Congress.
|Whereas: The Lame Duck session of Congress – between the election and the new Congress in January – will be incredibly dangerous to liberty; and
Whereas: President Biden will lay everything back on the table including gun control, a Central Bank Digital Currency, and more of the Green New Deal and Great Reset agendas; and
Whereas: Politicians coming off victory will feel emboldened to ram through their worst Big Government schemes; and
Whereas: Weak-kneed politicians, thinking their constituents aren’t “paying attention,” could cave into President Biden’s demands;
Therefore: As your constituent, I insist you vote “NO!” on an any legislation considered by Congress in a Lame Duck session that increases taxes, restricts our Second Amendment rights, or otherwise makes government bigger and restricts liberty.
(Sign your name)

domain: politics


text:
The inimatable, ever charming, Michael Caine talks about Texan accents and growing old.
"The great thing about growing old is that you haven't got an alternative. The only alternative is death, so you might as well be cheerful and have the greatest possible life you can have. I always meet people who are living as though it's a rehearsal and the show's gonna be later, and I feel like saying 'This is the show! This is it, it's not the rehearsal. Look, you've got the costume on!'"

domain: entertainment


text:
Experimental, human cadaveric study.
To assess the fixation effects of injecting cement augmentation before screw insertion or after insertion of fenestrated screws; the effect of modulating cement viscosity; and the effects of these techniques on screw removal.
It seems clear that cement augmentation can enhance pedicle screw fixation in osteoporotic bone. What remains to be demonstrated is the aspects of optimal technique such that fixation is enhanced with the greatest safety profile.
Part I: Human osteoporotic vertebrae were instrumented with solid (nonaugmented) screws, solid screws with polymethylmethacrylate (PMMA), partially cannulated fenestrated (Pfen) screws, or fully cannulated fenestrated (Ffen) screws through which PMMA was injected. Screw fixation was tested in pullout. Part II: Ffen screws were augmented with standard low-viscosity PMMA versus high-viscosity PMMA. Part III: Sample cohorts were extracted from vertebrae to assess required torque and characterize difficulty of extraction.
Part I: Pfen screws demonstrated the greatest fixation with mean failure force of 690 ± 182 N. All methods of cement augmentation demonstrated significant increases in screw fixation. Part II: Ffen screws did not demonstrate a significant difference in pullout strength when high-viscosity PMMA was used as compared with low-viscosity PMMA. Part III: Mean extraction torque values for solid augmented screws, Ffen screws, and Pfen screws were 1.167, 1.764, and 1.794 Nm, respectively, but these differences did not reach significance. None of the osteoporotic vertebrae sustained catastrophic failure during augmented screw extraction.
Polymethylmethacrylate cement augmentation clearly enhances pedicle screw fixation in osteoporotic vertebrae when tested in pure pullout. The technique used for cement injection and choice of specialty screws can have a significant impact on the magnitude of this effect. Fenestrated screws have the capacity to confine cement placement in the vertebral body and may provide enhanced safety from cement extrusion into the spinal canal. It is feasible to inject high-viscosity PMMA through this fenestration geometry, and higher-viscosity cement may enhance the fixation effect.
Experimental, osteoporotic cadaveric study comparing cement augmentation in solid and fenestrated pedicle screws. Polymethylmethacrylate cement augmentation clearly enhances pedicle screw fixation in osteoporotic vertebrae when tested in pure pullout. Fenestrated screws have the capacity to confine cement in the vertebral body. It is feasible to inject high-viscosity polymethylmethacrylate through this fenestration geometry, which may enhance the fixation effect.
*Department of Orthopaedic Surgery
†Comparative Orthopaedic Laboratory; and
‡School of Medicine, University of Missouri, Columbia, MO.
Address correspondence and reprint requests to Theodore J. Choma, MD, Department of Orthopaedic Surgery, and Missouri Spine Center, University of Missouri, 1100 Virginia Ave, DC 0.053, Columbia, MO 65212; Email: email@example.com
Acknowledgment date: May 29, 2012. Revision date: July 30, 2012. Acceptance date: September 5, 2012.
The device(s)/drug(s) that is/are the subject of this manuscript is/are not FDA-approved for this indication and is/are not commercially available in the United States.
Depuy Spine grant funds (University of Missouri grant 00023145) were received to support this work.
Relevant financial activities outside the submitted work: consultancy, travel/accommodations/meeting expenses.


text:
|April 8, 2015: Canucks' Richardson Skates On Own|
Richardson (ankle) was able to skate on his own Wednesday morning, Irfaan Gaffar of Sportsnet.ca reports.Richardson still has yet to resume practicing with the team, which makes it seems unlikely that he'll be able to play in the Canucks' final two regular-season contests. The first round of the playoffs probably represents a more realistic target for Richardson at this point.
|April 6, 2015: Canucks' Richardson Not Practicing|
Richardson (ankle) has yet to resume practicing, Jim Jamieson of the Vancouver Province reports.The Canucks previously were calling Richardson day-to-day after he aggravated his bothersome ankle back on March 14. He spent considerable time on injured reserve with the same ailment prior to returning to the lineup, and at this point, it appears safe to say that he's out indefinitely. If he fails to demonstrate much progress in the near future, Richardson could be ruled out for the first round of the playoffs.
|March 30, 2015: Canucks' Richardson Still Sidelined|
Richardson (ankle) won't play either Monday against the Blues or Tuesday against the Preds, the Province reports.Beyond that, Richardson should be considered iffy, at best, for Thursday's contest against Chicago.
|March 26, 2015: Canucks' Richardson Not Playing Thursday|
Richardson (ankle) will not play Thursday against the Avalanche, Elliott Pap of the Vancouver Sun reports.Richardson was able to skate during the team's optional session, but it looks like he'll receive further time off to recover from the ankle injury. He remains day-to-day.
|March 26, 2015: Canucks' Richardson Remains Questionable|
Richardson (ankle) participated in Thursday's morning skate, but remains questionable to play later in the evening against the Avalanche.Richardson was able to practice with the team Wednesday as well, but with the center suggesting he'd likely be day-to-day for the remainder of the season, his availability may remain in question for any given night. That sort of unreliability makes it tough to justify rostering him, even in deeper formats.

domain: news


text:
Surf beyond your expectations as the sea comes alive at night and dive into Electric Ocean, SeaWorld's all-new summer nighttime celebration.
It's all about the kids at SeaWorld San Diego this October with offers including kids free Breakfast & Dine With Shamu.
Kids Eat for FREE at Breakfast & Dine With Shamu - SeaWorld San Diego only.
Breakfast & Dine With Shamu: One child (3-9 years) free per full-paid adult.
Sign up to receive the latest news, events, and updates about San Diego. Our monthly and weekly newsletters will keep you informed about the latest and greatest happenings in the destination.
For the ultimate guide to planning your San Diego vacation, view and download the San Diego Visitor Planning Guide, packed with useful information that will help you plan the perfect San Diego getaway.
English | Change Language
Would you please answer a few short questions about your experience on sandiego.org today?
Yes | No


RESIDENTS in Redcar and Cleveland can now sign up to get their garden bin emptied in 2024 by visiting www.redcar-cleveland.gov.uk/garden-waste-signup
The opt-in service, which will come into effect in April, will cost £40.00 for one garden waste bin to be collected fortnightly between April and November and £20.00 for each additional bin, up to a maximum of five.
Residents who have not already signed up for a customer portal account with the council will be prompted to do so.
Collection days for garden waste may not be the same as the current one. A permit and further information on a collection day will be provided at least seven days before first collection.
Cabinet Member for Neighbourhoods, Cllr Adam Brook, explained:
“The council agreed to the garden waste subscription service last year to ensure that the service remained sustainable and will only be paid for by those households that choose to use it.
“Over 70% of local authorities across the UK have already made the decision to introduce chargeable garden waste collections amidst rising budget pressures.
“The average price nationally for an annual garden waste subscription is £51.00 so at £40.00 per year we have attempted to keep the garden waste charge affordable to residents and competitively priced with other local authorities.”
The types of garden waste collected include grass and hedge clippings, leaves, plants and flowers, twigs, sticks and small branches, weeds (but not Japanese knotweed).
For further information and details about the scheme please visit www.redcar-cleveland.gov.uk/garden-waste


text:
Only a tiny handful of free speech advocates and Trump supporters showed up Saturday for what was billed as a giant rally for conservatives. The right was shut down by tens of thousands of leftists who paraded through the streets, joyous over their crushing victory over their fellow citizens that they label as “Nazis.”
Microchip has called for implementing a strategy of the alt-right announcing rallies and then not showing up for them. The idea is to let the left exhaust itself in its mass hysteria. The sight of thousands of masked antifa holding down streets empty of patriots is kind of funny when you think about it. Ditto the denunciations by liberal politicians of free speech and the right of peaceful patriots to assemble and speak.
The whole San Francisco operation may have been a giant Microchip trolling operation, per his strategy. Or maybe not. It’s hard to tell.
As to the press’s claims that the left was peaceful today, ask the journalist who was attacked in a live stream video embedded in a post earlier today.
Excerpt from Mercury News
SAN FRANCISCO — Thousands of demonstrators denouncing racism filled this city’s streets Saturday, as right-wing activists who had planned a show of force here found themselves holding meetings in secret locations to avoid the wrath of counterprotesters.
San Francisco for the past two weeks had girded itself for violence after a right-wing group planned a demonstration Saturday afternoon at Crissy Field. But Portland-based Patriot Prayer late Friday canceled the rally and said they would hold a “news conference” at iconic Alamo Square Park instead.
Police, however, foiled that plan when they fenced off the park Saturday morning and closed it to the public. So instead, thousands of people marched peacefully around the city, holding signs in protest of white supremacy, hate and President Donald Trump — and later gathered to dance at a concert in Civic Center Plaza. Only a few dozen right-wing protesters showed up all day.
“We shut down” their protests, said Rafael Picazo, who grew up in San Francisco’s Mission District and now lives in Hayward, at the intersection of Mission and 24th streets, where the counterprotest ended. “I think we accomplished a lot.”
Joey Gibson, the founder of Patriot Prayer and the main organizer of the Crissy Field rally, held several news conferences throughout the day in which he denounced San Francisco leaders for trying to restrict his speech.
He made a brief appearance in Pacifica, just south of San Francisco, saying he was “here to promote love and peace and freedom” and insisting that he does not support white nationalism or racism.
About 4:30 p.m., he and a contingent of about 50 followers gathered at Crissy Field and quickly got into shouting matches with opponents, but the group departed within an hour as more counterprotesters gathered after they heard Patriot Prayer members were there.
Several of the group’s members said they were planning to attend a right-wing protest in Berkeley’s Civic Center Park on Sunday that was also “canceled” by its main organizer on Friday — and which East Bay officials worry has a higher chance of seeing violent clashes than the San Francisco protests.
In an interview with the Bay Area News Group at Crissy Field, Gibson said he was set up by the media and elected officials such as San Francisco Mayor Ed Lee and House Minority Leader Nancy Pelosi.
“I felt in my heart people were trying to incite a riot,” he said. “I felt that if we had gone ahead with the rally, someone would have been badly hurt. ”
He claimed that Patriot Prayer was trying to help people communicate and help everyone “be a good person.”
Late Saturday morning, a peaceful demonstration grew around the fenced-off Alamo Square Park under a bright sun and a cloudless sky. Families toting young children and bandanna-masked anarchists marched next to each other, chanting and hoisting signs like “If you’re going to San Francisco, DON’T BE A NAZI!” “Fight White Supremacy!” and “More Sex Less Hate.” A phalanx of police officers looked on impassively.
At Crissy Field, far from the main groups of counterprotesters, a handful of Trump supporters hung out in the shade of a tree in mid-afternoon. Several members of the dejected group said they had driven from as far as Portland to attend Gibson’s rally, and were disappointed more of their allies hadn’t showed up.
“Free speech is worth protecting,” said Daryl Tempesta, 53, of Santa Cruz, who wore a Trump hat and an American flag draped around his neck. “I’m an American, and I wanted to protect the Constitution.”
Tempesta said he had been picking up “a lot of dog poop” on Crissy Field beach — apparently the remnants of a unique protest planned by San Francisco dog owners to muck up the planned right-wing rally.
Across the Bay in Walnut Creek’s Civic Park, several hundred people formed the words “End Hate!” for an aerial photograph on Saturday morning. The “human banner” event was put on by Women’s March Contra Costa.

domain: sociology


text:
Forage intake and feeding behaviour of day and/or night grazing cattle in sahelian rangelands
MetadataShow full item record
Permanent link to cite or share this item: https://hdl.handle.net/10568/50564
Forage intake and feeding behaviour of cattle grazing during day and/or night was studied in Sadore between February and May (late dry season) 1995 and, in Toukounous, Niger, between July 1995 and May 1996, covering wet (WS), early dry (EDS) and late dry (LDS) seasons. In Sadore, 24 steers grazed from 09.00 to 19.00 h (day grazers), from 21.00 to 07.00 h (night grazers) or from 09.00 to 19.00 h and from 24.00 to 04.00 h (day and night grazers). In the trials in Toukounous, 64 steers were allotted to 8 treatments (T). Grazing time during the day was for 6 h for T 1, 2 and 3; 9 h for T 4, 5 and 6; 12 h for T7 and 8. Night grazing time was 0 h for T 1, 4 and 7; 3 h for T 2, 5 and 8; and 6 h for T 3 and 6. Feeding activities were observed every 5 min in Sadore. In both studies, forage intake was determined from individual data on faecal output and mean of extrusa in vitro organic matter digestibility. In Sadore, the actual grazing time was 352, 376 and 476 min/day (SEM = 16) for day, night, and day and night grazers, respectively. Night grazers spent less (P<less than>0.05) time ruminating and walking than day grazers. Day and night grazers had a higher forage intake than either day or night grazers. In Toukounous, day and night grazers (T 2, 3, 5, 6 and 8) also consumed more forage than day grazers (T 1, 4 and 7) in all seasons. Forage intake increased with an increase in total duration of grazing, while it declined as the season progressed from wet to dry. These results suggest that timing (day or night) and duration of grazing affect forage intake. Additional grazing time during the night leads to increased forage intake by grazing cattle and consequently provides opportunities for increased animal production in the Sahel.
- ILRI archive

domain: biology


text:
Not a member? Join today
Gekidan Hitori was born on February 2, 1977 in Chiba, Japan as Shogo Kawashima. He is an actor and writer, known for A Bolt from the Blue (2014), Flowers in the Shadow (2008) and Golden Slumber (2010).
Action, Drama, Thriller
Comedy, Drama, Musical, Mystery

domain: celebrity


text:
New Release time over at Vera Lane Studio.
Did you see last weeks Arthur Ave new releases?
And yes, I've started on my Colour Combo tags using the color tags by adding my combos as I go along.
Coloured with copic markers.
I have added some washi tape strips down both sides and my middle backing strip is another piece from one of my Gel plate novel pages I created.
I added some red copic marker splats by the words to add a grungy feel to it all.
Quite a little mixed media piece of art - I love it.
I am entering them in the following Challenges:
Have you joined our VLS Facebook Group yet?
You'll get to join in on some fun trades and challenges, see the daily inspiration posts from the wonderful DT's and best of all, hang out with other VLS fans.
For more inspiration and information, head over to the Vera Lane Studio Blog.
Until next time, Happy Crafting.


text:
Ryan Dempster suspended five games for hitting Alex Rodriguez
|08.20.13 at 3:22 pm ET|
Major League Baseball has suspended Ryan Dempster for five games and fined him an undisclosed amount for “intentionally throwing at and hitting Alex Rodriguez” on Sunday night, the league announced Tuesday afternoon. The suspension begins Tuesday night unless Dempster appeals.
Dempster was not ejected from the game, but both benches received warnings after the incident. Rodriguez was leading off the top of the second when he got hit. The Yankees went on to score twice in the inning and wound up winning the game, 9-6.
Dempster’s next scheduled start would have been Saturday in Los Angeles. Because the Red Sox have an off-day on Thursday, they could just skip his spot in the rotation rather than have someone make a spot start.
Latest from Bleacher Report
- Unexpected Trades Red Sox Could Pull Off This Offseason
- Dream Free-Agent Pickups for Red Sox
- Red Sox Free Agency News and Trade Rumors
- Should Red Sox Trade Cespedes This Offseason?
- Red Sox's Most Tradeable Assets for Offseason
- Uehara Inks 2-Year Extension with Sox
- Possible Trade Partners, Packages for Cespedes
- Podcast Ep. 70: Moncada signs, Ben Badler talks, PawSox sold!
- PawSox sold to group including Lucchino; reports say move to Providence likely
- Fort Report: Top prospects prepare for biggest tests yet
- Report: Red Sox have agreement to sign Moncada for $31.5M
- Unveiling the updated SoxProspects scouting reports, stage one
- Owens, Johnson among eight invited to Red Sox spring training
- Chicago Cubs claim Drake Britton off waivers
- Offseason Notes: Red Sox continue to shape roster
- Drake Britton designated for assignment as Red Sox sign Ogando
- Trade Analysis: Scouting Anthony Ranaudo


text:
$90.00 – $110.00
Plein Air Mt Cootha – Maree Angus
Come join Master Pastellist Maree Angus as she explores the Mount Cootha Botanic Gardens painting en plein air during the Annual Exhibition.
One day outdoors and one day in the studio. Maree will take you through how to be prepared when painting plein Air. She will be sharing many ideas from her experience. Be prepared and enjoy!


"At Sanofi Oncology, our mission goes beyond developing innovative medicines and ensuring their availability," said Debasish Roychowdhury , M.D., Senior Vice President and Head, Sanofi Oncology. "Through initiatives like today's proclamation ceremony, we're educating patients and the public about various cancers, prevention, and treatments, to ultimately promote a healthy way of life in the Cambridge community."
About Colorectal Cancer
In 2010, 142,570 new cases were diagnosed and 51,370 people died from colorectal cancer in the United States. When colorectal cancer is diagnosed at an early stage, the five-year survival rate is nearly 90 percent. However, when the cancer is not diagnosed until a late state, the five-year survival rate is closer to 10 percent. In the past ten years, colon cancer screening rates have increased, but rates still remain low, especially among the uninsured. According to the American Cancer Society, men and women should begin screening for colon cancer at age 50. If the entire population was screened at that age, the number of colorectal cancer-related deaths would be cut in half.
About the Colon Cancer Coalition /Get Your Rear in Gear
Get Your Rear in Gear® events are presented nationally by the Colon Cancer Coalition, a non-profit organization based in Minneapolis. The events are 100% volunteer-driven in communities throughout the United States. The Colon Cancer Coalition distributes funds raised at Get Your Rear in Gear events back into local communities for colon cancer education, prevention, and screening. By making the words colon, colorectal and colonoscopy a part of the everyday language, we believe we can overcome the fear and decrease deaths from this largely preventable cancer.
Copyright©2012 PR Newswire.
All rights reserved


text:
Lautaro, 22, has helped Inter become Serie A title contenders this season, scoring 16 goals in all competitions.
"At Inter I see him happy, he identifies with the club," Zanetti told ESPN. "We believed in him. I see his future at Inter.
"But right now we're not talking about the transfer market, the most important thing is health. Then we'll have time to think about it."
Sources told ESPN that Madrid are prepared to rival Barca for his signature, with the player available for €111 million in July under a clause in his contract.
Meanwhile, Zanetti, who captained Inter to a Serie A, Coppa Italia and Champions League treble in 2010 under Jose Mourinho, reserved special praised the Tottenham Hotspur boss.
"[Mourinho is] a special person, a great coach and a great person," Zanetti added.
"Let's talk about a motivator, someone who conveys a lot of passion. He helps you to bring out the best in yourself."

domain: sports


text:
In this photo taken June 16, dairy cows stand in a field in Coventry, Vt. The Agriculture Department is helping struggling dairy farmers by raising the price paid for milk and cheddar cheese through a dairy price support program. The department estimates the temporary increases, which will be in place until October, will boost dairy farmers' overall revenue by $243 million. Agriculture Secretary Tom Vilsack said Friday that the price increase will provide immediate relief, helping to keep dairy farmers on the farm while they weather what he called "one of the worst dairy crises in decades."
(AP Photo/Toby Talbot)
JEFFERSON CITY, Mo. -- Missouri dairy farmers say they need some help from the federal economic stimulus act.
Representatives of the Missouri Dairy Association have asked Gov. Jay Nixon to seek a one-time emergency payment of $16.5 million in stimulus money. They say falling milk prices have put Missouri's dairy farmers in their worst economic crisis in the past 25 years.
Missouri has about 111,000 dairy cows.
A Nixon spokesman referred questions about the dairy farmers' request Wednesday to the state Department of Agriculture.
Agriculture Director Jon Hagler says the U.S. Senate recently voted to raise dairy price supports, and he wants to see the effect as the state considers the need for more assistance.

domain: food


text:
Within the last year
Within the past 3 years
1 - 10 of 37 articles
Background Although angiotensin-converting enzyme (ACE) inhibitors have appeared to be useful for secondary prevention after acute myocardial infarction (AMI) in Western countries, that has not been confirmed in non-western countries. We investigated whether ACE inhibitors improve survival rates...
Coronary heart disease is the single largest cause of morbidity and mortality in the United States. The link between elevated low-density lipoprotein cholesterol (LDL-C) levels and coronary heart disease (CHD) has been clearly established. However, triglycerides (TG) are increasingly believed to...
Background The disruption of yellow plaque and subsequent thrombosis is regarded as the mechanism of acute coronary syndrome. However, there are limited reports on the assessment of plaque vulnerability. Therefore, we tested whether the angioscopically determined yellow color intensity of plaque...
Approximately 20 years ago, the Italian cardiology community realized the scientific importance and the potential impact on clinical practice of the new concept of evidence-based medicine and launched (without funds) a national megatrial, the Gruppo Italiano por lo Studio della Streptochinasi...
Background Both epicardial and myocardial perfusion have been associated with clinical outcomes in the setting of ST elevation myocardial infarction (STEMI), and the performance of adjunctive/rescue percutaneous coronary intervention (PCI) may further improve clinical outcomes after fibrinolytic...
Background Unrecognized myocardial infarctions (UMI) are detected by surveillance electrocardiograms (ECGs). In epidemiologic studies, different sets of ECG criteria have been used to define myocardial infarction, possibly contributing to significant differences in prevalence estimates and risk...
Read and print from thousands of top scholarly journals.
Sign up with Facebook
Sign up with Google
Already have an account? Log in
Save this article to read later. You can see your Read Later on your DeepDyve homepage.
To save an article, log in first, or sign up for a DeepDyve account if you don’t already have one.
Sign Up Log In
To subscribe to email alerts, please log in first, or sign up for a DeepDyve account if you don’t already have one.
To get new article updates from a journal on your personalized homepage, please log in first, or sign up for a DeepDyve account if you don’t already have one.


Briones discuss impact of education on PH growth in UK talk
Education Secretary Leonor Briones explained how the country responded to several educational challenges, in her talk at this year’s Education World Forum (EWF) in the United Kingdom on Tuesday.
“One of these is the response of the department to lack of access to education in other areas in the country through building education facilities,” the DepEd chief said in Filipino.
Briones’ talk during the plenary session focused on the equity and resources of basic education in the country.
Other important points were also discussed including education’s impact on the country’s economic growth, resilience, emotional learning, and education for girls, among others.
Earlier, Briones said the forum will advance wider opportunities and partnerships for Filipino learners.
“We are here so we can share our collective experiences and we are looking forward to helping each other and being a part of the discussion,” she said.