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Pharmaceuticals 2011, 4(3), 457-469; https://doi.org/10.3390/ph4030457
Abstract: G protein-coupled receptors (GPCRs) are crucial elements in mammalian signal transduction, and are considered to represent potent drug targets. We have previously developed a GPCR assay system in cultured cells based on complementation of split fragments of click beetle (Pyrearinus termitilluminans) luciferase. The interaction of GPCRs with its target, β-arrestin, resulted in strong emission of bioluminescence upon stimulation with its specific ligand. In this study, we improved precision of the GPCR assay system by using railroad worm (Phrixothrix hirtus) luciferase as an internal control. We generated stable cell lines harboring the railroad worm luciferase and quantitatively evaluate the extent of GPCR-β-arrestin interactions. We showed concentration-dependent bioluminescence responses for four GPCRs: β2-adrenoceptor, endothelin receptor type A, α2-adrenoceptor and human μ-opioid receptor. We also demonstrated that the variation of responses was reduced significantly by normalizing the data with bioluminescence from railroad worm luciferase. This assay system represents a simple and reliable approach for screening drug candidates in a high throughput manner.
1. Introduction
G-protein coupled receptors (GPCRs) form a large and diverse multigene superfamily of integral membrane proteins that are involved in many important physiological functions [1]. GPCRs control key physiological functions, such as neurotransmission, hormone and enzyme release from endocrine and exocrine glands, immune responses, cardiac and smooth muscle contraction, and blood pressure regulation. These receptors are not only crucial targets for basic biology but also prominent components for drug discovery [2,3]. A large part of efforts towards drug discovery and development is focused on finding chemicals that affect the ability of ligands to bind to GPCRs, either to inhibit or accelerate certain cellular processes. However, as our knowledge of the complexity of GPCRs' function increases, particularly when studied in a cellular environment, it has become clear that novel approaches are needed to monitor the interaction of GPCRs with their targets. β-arrestins are multifunctional adaptors that mediate the desensitization, internalization, and some signaling functions of GPCRs [3].
In order to assay and detect the GPCR-β-arrestin interactions, powerful technologies based on protein fragment complementation have been used for screening chemicals and drug candidates [4-8]. The principle of protein fragment complementation is that a reporter protein, which is an enzyme or a fluorescent protein, is divided into two fragments so that they cannot function alone. These fragments are fused to potentially-interacting protein partners and complementation upon interaction leads to restore its function. The restored enzyme activity or fluorescence can be detected as an indicator of protein-protein interactions [7-11]. In recent years, GFP-fragment complementation has been used for the technique of visualizing GPCR-β-arrestin interactions [12]. Localization of the interactions in living cells was visualized with the technique of GFP-fragment complementation. However, the technique has some limitations such that it often requires for high-energy excitation, which results in high background signals. In contrast, bioluminescent proteins such as firefly, Renilla, Gaussia and click beetle emit light by a biochemical reaction without any excitation sources. Taking such strong advantage of the bioluminescent enzyme, we have developed novel green luciferase fragments from click beetle (Brazilian Pyrearinus termitilluminans; Emerald Luc; ELuc) for the study of GPCR-β-arrestin interactions in live cells (Figure 1) [13,14]. The use of click beetle luciferase has a strong advantage of brightness; the photon count of click beetle luciferase is estimated as being over 10-fold higher than those of firefly luciferase. In addition, the spectrum of firefly luciferase is known to change in a pH-dependent manner, whereas click beetle luciferases have the property of pH independence of the spectra [15,16]. However, in spite of some advantages, the split fragments from the click beetle failed to produce sufficient levels of precision. The activity of luciferase is changed in proportion to the concentrations of d-luciferin and ATP. In addition, the number of the cells in 96-well microtiter plates is different in each well, which makes the precision worse in the data handling.
In this study, we show a method of precision-improved bioluminescence analysis for monitoring GPCR-β-arrestin interactions in living cells based on ELuc fragment complementation. To reduce fluctuation of bioluminescence between different wells, we used a red color luciferase from the railroad worm (Phrixothrix hirtus; RWLuc) as an internal control. The RWLuc is the best candidate as an internal control because the substrate of RWLuc is the same as that of ELuc, d-luciferin.
In addition, the emission maximum of RWLuc is 625 nm, which is distant from that of ELuc (emission maximum: 535 nm) [17,18]. We generated four kinds of stable cell lines of HEK293 cells transfected with β-arrestin (ARRB2) and different GPCRs, which were connected with a pair of ELuc fragments. Moreover, RWLuc was transfected into the HEK293 cells, and then new stable cell lines were generated. To evaluate quantitatively the effect of chemicals that act on GPCRs, bioluminescence intensity from the ELuc was divided by that from RWLuc. By this procedure, standard deviations of the bioluminescence responses were improved and quantitative evaluation became possible on 96-well microtiter plate formats.
2. Experimental
2.1. Materials
The cDNA of click beetle (Brazilian Pyrearinus termitilluminans) luciferase (Emerald Luc; ELuc) and Emerald Luciferase Assay Reagent were obtained from Toyobo Co. Ltd. (Japan). The cDNA of railroad worm (Phrixotrix hirtus) luciferase (RWLuc) was a gift from Dr. Y. Ohmiya (AIST, Japan). The human cDNAs of α2-adrenoceptor (ADRA2A; NM_000681.3) and μ-opioid receptor (OPRM1; NM_001008503.1) were obtained from GeneCopoeia Inc. (Rockville, MD). The cDNA library of human brain, DNA polymerase and restriction, modification enzymes were obtained from Takara Bio Inc. (Japan). Damgo, endothelin 1 (ET-1), and epinephrine were purchased from Wako Pure Chemical Industries Ltd. (Japan). DRAQ5™ dye was obtained from Biostatus Ltd. (Leicestershire, UK). Mouse anti-V5 antibody and donkey anti-mouse IgG antibody tagged with AlexaFluoro 488, Hoechst 33342, and expression vectors of pcDNA3.1/myc-His(B) and pcDNA4/V5-His(B) were purchased from Invitrogen Corp. (Carlsbad, CA).
2.2. Construction of Plasmids
The cDNAs used for the plasmid construction were generated using the standard polymerase chain reaction (PCR) with gene specific primer and Pyrobest DNA polymerase. The cDNAs of β-arrestin (ARRB2; NM_004313.3), β2-adrenoceptor (ADRB2; NM_000024.5) and endothelin receptor type A (EDNRA; NM_001957.3) were amplified from the human brain cDNA library. The PCR fragments were subcloned into restriction enzyme sites of mammalian expression vectors, pcDNA3.1/myc-His(B) or pcDNA4/V5-His(B) (Figure 2). All PCR fragments were sequenced using a genetic analyzer (ABI310; Applied Biosystems). For transfection of the cells, the expression vectors were purified using a DNA purification system (Purelink HiPure Plasmid Filter, Invitrogen Corp.).
2.3. Transfection, Cell Culture and Generation of Stable Cell Lines
The HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose) supplemented with 10% fetal bovine serum (FBS), 100 unit/mL penicillin, and 100 μg/mL streptomycin at 37 °C in an incubator with 5% CO2. Transfection of plasmids into the cells was performed with a reagent (TransIT Transfection; TransIT®-LT1; Mirus Co., TX). In order to obtain cell lines which stably expressed GPCR and β-arrestin, the transfected cells were screened with both 0.8 mg/mL geneticin (G418) and 0.04 mg/mL zeocin. To generate stable cell lines which expressed RWLuc, the HEK293 cells that have already expressed the GPCR and β-arrestin were further transfected with the plasmid of RWLuc, and then the cells were screened with 0.05 mg/mL blasticidin. The expression of proteins in each stable cell line was confirmed by the existence of both red and green bioluminescence.
2.4. Measurement of Bioluminescence Spectra
The stable cell lines were cultured on 96-well microtiter plates and transferred into a small vial, measurement of spectrum, for 1 min for each sample. Where necessary, the cells are stimulated with 1× 10−6 M isoproterenol for 12 min. Bioluminescence spectra were measured using an AB-1850 LumiFL-spectrocapture (ATTO Corp., Tokyo, Japan). Bioluminescence spectra were collected for 1 min in each sample.
2.5. Bioluminescence Assays of GPCR-β-Arrestin Interactions
The cells were cultured with a phenol red free DMEM supplemented with 1% FBS on 96-well microtiter plates, and incubated at 37 °C in 5% CO2 overnight. The cells were stimulated with different concentrations of ligands and incubated for a given time at 37 °C in 5% CO2. Before measurement of luciferase activities, 100 μL/well of the Emerald Luciferase Assay reagent (Toyobo Co. Ltd., Japan) was added in each well. The ELuc and RWLuc activities were measured using a 96-well microtitier plate reader (TriStar LB941; Berthold Technologies GmbH and Co. KG, Germany) with one band-pass filter of 525 ± 25 nm for bioluminescence from ELuc and the other band-pass filter of 630 ± 30 nm for bioluminescence from RWLuc. All measurements were performed five or six times with different wells of culture plates. The values were averaged and presented with standard deviations.
3. Results and Discussion
3.1. Generation and Characterization of Cell Lines Stably Expressing RWLuc
The dissecting sites of ELuc are very important to obtain maximum responses and to avoid background signals. We have previously identified new dissecting sites from the ELuc with a significant improvement of the signal-to-background ratio and absolute photon counts [14]. Based on the previous study, the combination of N-terminal ELuc (1-415 aa) and C-terminal ELuc (394-542 aa) has been applied to monitoring the interaction between GPCRs and β-arrestin (Figure 1). Among various kinds of GPCRs, β2-adrenoceptor (ADRB2) was chosen for the initial experiments. The ADRB2 was connected with the C-terminal ELuc fragments, whereas β-arrestin was connected with the N-terminal one. Both fusion proteins were stably expressed in the HEK293 cells, named HEK293-ARRB2-ADRB2 cells. The use of HEK293 cells has a few advantages; it is possible to express exogenous proteins in higher level and growth rate of the cells is relatively fast. The cells were seeded on 96-well microtiter plates and then stimulated with the ligand, isopreternol. Bioluminescence increased concomitantly with increasing concentrations of isopreterenol and the maximum responses obtained was 15–40-fold higher than the background bioluminescence (data not shown). However, absolute photon counts of the bioluminescence changed in each well of the microtiter plates because of a little difference in d-luciferin, ATP and the number of cells. In order to minimize such unwanted fluctuation, we further constructed a cell line that stably expressed RWLuc in the HEK293-ARRB2-ADRB2 cell line. Of more than 100 subcloned cells, we obtained a cell line that stably expressed the RWLuc, which was named HEK293-ARRB2-ADRB2-RWLuc.
In order to characterize the stable cell line, the bioluminescence emission spectra were examined using a lumino-spectral meter (Figure 3A). The bioluminescence spectrum from the cell line in the absence of ligands showed a single emission peak at the wavelength of 625 nm. The emission maximum is consistent with that of RWLuc, indicating that the RWLuc was stably expressed in the cells. In the presence of 1.0 μM isoproterenol, we observed an enhanced emission peak at the wavelength of 535 nm, attributed to complementation of split ELuc fragments. Absolute photon count at 535 nm from ELuc was almost the same as that at the 625 nm from RWLuc, indicating that RWLuc is useful for internal control of ELuc fragment complementation.
Next, to examine characteristics of the newly generated HEK293-ARRB2-ADRB2-RWLuc stable cell line, we investigated the quantitative assessment of the isoproterenol-induced ADRB2–β-arrestin interaction in the cells. The HEK293-ARRB2-ADRB2-RWLuc stable cells were grown on 96-well microtiter plates, and then stimulated with each concentration of isoproterenol for 12 min. The bioluminescence through a band-pass filter of 525 ± 25 nm was separated from the bioluminescence through a band-pass filter of 630 ± 30 nm. Figure 3B shows raw data of bioluminescence through the filter of 525 ± 25 nm with different concentrations of isoproterenol. The photon counts of bioluminescence increased concomitantly with increasing concentrations of isoproterenol from 1.0 × 10−9 to 1.0 × 10−5 M. On the other hand, a little change in the photon counts from bioluminescence through 630 ± 30 nm was found in the various concentrations of isoproterenol. Background luminescence in the absence of isoproterenol was 924 (525 ± 25 nm) and 123 (630 ± 30 nm) and mechanical noise of plate readers in the absence of cells was 16 (525 ± 25 nm) and 11 (630 ± 30 nm). The maximum photon counts produced were about 35-fold higher than the background luminescence. Absolute photon counts through 630 ± 30 nm were lower than those through 525 ± 25 nm although the maximal response was obtained at 625 nm in the spectral analysis (Figure 3a). This may be due to the fact that the sensitivity of the plate reader over 600 nm was lower than that of the spectrometer and, therefore, the photon counts through 525 ± 25 nm became higher than those through 630 ± 30 nm.
To evaluate quantitatively the effect of isoproterenol, bioluminescence intensities from ELuc were normalized against those from RWLuc. The normalization was performed such that the bioluminescence through the filter of 525 ± 25 nm was divided by the bioluminescence through the filter of 630 ± 30 nm, which was defined as a relative luminescence unit (RLU). Figure 3C shows the bioluminescence changes in the RLU, which was evaluated from photon counts in the presence of different concentrations of isoproterenol (Figure 3b). The RLU values reached a maximum in the presence of 1 × 10−6 M isoproterenol, which was 13-fold higher than that in the absence of isoproterenol. The EC50 value obtained for isoproterenol was 1.6 × 10−10 M. The value was lower than that reported previously by the β-galactosidase fragment complementation assay (2.7 × 10−8 M) [4].
To compare the standard deviations between the raw data of bioluminescence intensity and its normalized values of RLU, coefficient of variation (CV) was calculated. The CV values were 0.38 ± 0.14 for the raw data of bioluminescence and 0.25 ± 0.15 for the RLU. This result demonstrated that the standard deviation of the bioluminescence responses was significantly improved by normalization with the bioluminescence of RWLuc.
Finally, the effect of cell number in wells and concentrations of the substrate on the luminescence response of the system was systematically investigated. Upon 80–90% cell populations, no difference in the maximal response was obtained (data not shown). However, when cells were confluent about 100% of well, the photon counts decreased because of the cell death. In addition, when concentration of d-luciferin was changed from 0.5 mM to 10 mM, the maximum photon counts were almost the same (Figure 4), indicating that the present dual-color assay is not influenced by cell population or substrate concentrations.
3.2. Application to Different GPCRs
Next, we have applied this assay system to different kinds of GPCRs. We have constructed three new cell lines that stably expressed GPCRs of endothelin receptor type A (EDNRA), α2-adrenoceptor (ADRA2A) and human μ-opioid receptor (OPRM1), each of which was connected with the C-terminal fragment of ELuc. The cell lines were named HEK293-ARRB2-EDNRA, HEK293-ARRB2-ADRA2A and HEK293-ARRB2-OPRM1, respectively. The localization of each receptor was examined by immunostaining with Alexa-Fluoro 488 under a fluorescence microscope. All GPCRs localized on the plasma membrane although some portions of the receptors existed in the endoplasmic reticulum (Figure 5A). We next examined responses of bioluminescence for each specific ligand, endothelin-1 for EDNRA, epinephrine for ADRA2A and damgo for OPRM1. The bioluminescence responses depended on the concentration of each ligand and the maximum responses were 15-30-fold higher than the background bioluminescence (Figure 5B). Interestingly, time-dependant analysis of photon counts upon ligand stimulation exhibited different response time for the bioluminescence (Figure 5C). In the case of EDNRA, the maximum bioluminescence reached at 5–7 min after addition of endothelin-1. Decreases in the bioluminescence after maximum response were swift, and the response at 60 min was almost half of the maximum response. On the other hand, time-dependent responses of the bioluminescence for ADRA2A and OPRM1 were slower than the response for EDNRA. The maximum responses for ADRA2A and OPRM1 were obtained at 10 min and 20 min after stimulation of the corresponding ligands, respectively. The differences of the time-dependent changes in the bioluminescence between GPCRs were also observed in the previous study, which might be originated from dissociation of GPCR-β-arrestin interactions and/or decomposition of GPCRs [14].
Finally, using the cell lines of HEK293-ARRB2-EDNRA, HEK293-ARRB2-ADRA2A and HEK293-ARRB2-OPRM1, we generated stable cell lines which stably expressed RWLuc, named HEK293-ARRB2-EDNRA-RWLuc, HEK293-ARRB2-ADRA2A-RWLuc and HEK293-ARRB2-OPRM1-RWLuc, respectively. The bioluminescence intensities with the filter of 525 ± 25 nm increased with increasing in each ligand, and the maximum responses were approximately 25-fold for EDNRA, 15-fold for ADRA2A and 30-fold for OPRM1 higher than the background bioluminescence (Figure 6A).
On the other hand, no remarkable change in the bioluminescence through the filter of 630 ± 30 nm was obtained. When we calculate RLU, we found increases in the RLU values; 10-fold for endothelin receptor type A (EDNRA), 6-fold for α2-adrenoceptor (ADRA2A) and 8-fold for human μ-opioid receptor (OPRM1), respectively (Figure 6B). The EC50 values were 6.9 × 10−9 M for EDNRA, 3.4 × 10−10 M for ADRA2A and 1.0 × 10−9 M for OPRM1. The CV values calculated from the raw data of bioluminescence and those of RLU were 0.33 ± 0.21 and 0.21 ± 0.10 for EDNRA, 0.37 ± 0.14 and 0.21 ± 0.10 for ADRA2A, and 0.20 ± 0.07 and 0.20 ± 0.07 for OPRM1, respectively. In any cases, standard deviations were even or improved by introducing the process of normalization of the raw data with RWLuc.
The normalized curves show different maximal signal amplitudes (RLU) from one GPCR to another. One of the reasons is the difference of maximal bioluminescence responses for the GPCRs in raw data: When the higher maximal response of photon counts was obtained after ligand stimulation, the maximal RLU value became higher number. The other reason is the differences of expression levels of RWLuc. We could not control the expression level or RWLuc or GPCRs between the four kinds of stable cell lines, which resulted in the differences of the RLU values. These facts indicate that it is impossible to compare RLU values between the different GPCRs.
A more widely used technology is the bioluminescence resonance energy transfer (BRET) assay, of which basic concept is identical to the present method. However, because of spectral overlaps between bioluminescence and fluorescence, rigorous data analyses are needed to distinguish non-specific interaction from true oligomeric protein interactions. In comparison to such existing method, the present method presents strong advantages in terms of the detection period—less than 10 min, a high signal-to-background ratio of the bioluminescence, and high sensitivity of absolute photon counts.
Dual color assay reporters are also commonly used to improve experimental accuracy and precision [19]. The activity of one reporter gene generally represents molecular events in a target cell, while the activity of the second reporter gene provides an internal control. Thus, dual reporter assays often allow for more reliable interpretation of the experimental data by reducing extraneous influences. Dual-reporter applications utilizing firefly luciferase in combination with either chloramphenicol acetyltransferase (CAT), β-galactosidase (β-Gal) or β-glucuronidase (GUS) have been used [20,21]. However, those co-reporter combinations diminish advantages of luciferase. For example, while the luciferase assay can be performed and quantitated in seconds, the CAT, β-Gal and GUS assays are endpoint assays requiring lengthy incubation periods prior to quantitation. In addition, these other reporters are limited in their sensitivity and in the range of their linear response; care must be taken not to exceed these ranges. In this context, a GPCR assay method with dual-luciferases has recently been developed with a built-in control using Renilla luciferase [22]. Normalization of firefly luciferase activity with Renilla luciferase activity assists in compensating for potential variability in cell density and nonspecific effects caused by the compounds. However, the reporter assay system is not so accurate because the Renilla luciferase activity has to be measured separately with a different substrate, coelenterazine. This assay system can be affected from the differences in the d-luciferin and ATP concentrations. In comparison to such previous methods, the present assay system provides simple assay procedure and allows us to monitor the effects of ligands on GPCR quantitatively. There is no need to consider unwanted differences in d-luciferin and ATP concentrations inside the cells, which is unique and quite useful for constructing a high-throughput screening system.
In the design of this assay system, the GPCRs were connected with C-terminal fragment of ELuc. Effects of the fusion construct on the original function of GPCRs were not evaluated extensively in the present study. However, the C-terminal fragment of ELuc is composed of only 149 amino acids, which is much smaller than the β-galactosidase fragment and GFP. In the previous studies, it has been reported that GPCRs fused with GFP or β-galactosidase fragment bound to their agonists and antagonists in a normal concentration ranges and their internalization events resulted [4,12]. Considering these data, there may be no effect of connection of the ELuc fragment with GPCR on their specific functions.
4. Conclusions
In this work, we have applied split fragments from click beetle luciferase to monitoring new GPCR-β-arrestin interactions. To justify variables of data originated from the cell number, ATP concentrations, the amount of d-luciferin and transfection efficiency, we generated the stable cell lines that expressed full length of RWLuc as an internal control. We normalized the obtained data with the bioluminescence of RWLuc, and precision of the normalized values was significantly improved in comparison to simple evaluation from absolute photon counts. Usefulness of this new system was demonstrated with four GPCRs; β2-adrenoceptor, endothelin receptor type A, α2-adrenoceptor and human μ-opioid receptor, and the present method will be further applicable for any kinds of GPCRs if their interacting proteins are present. Because the assay procedure is quite simple and the obtained data is reliable, the methods become a powerful new approach for the study of GPCR analysis and for screening of drug candidates.
This is work was supported by the Japan Society for the Promotion of Science (JSPS), New Energy Industrial Technology Development Organization (NEDO), and in part by the Global COE Program and grants (S0801035), MEXT, Japan.
- Dorsam, R.T.; Gutkind, J.S. G-protein-coupled receptors and cancer. Nat. Rev. Cancer 2007, 7, 79–94. [Google Scholar]
- Klabunde, T.; Hessler, G. Drug design strategies for targeting G-protein-coupled receptors. ChemBioChem 2002, 3, 928–944. [Google Scholar]
- Schmid, C.L.; Bohn, L.M. Physiological and pharmacological implications of β-arrestin regulation. Pharmacol. Ther. 2009, 121, 285–293. [Google Scholar]
- Yan, Y.X.; Boldt-Houle, D.M.; Tillotson, B.P.; Gee, M.A.; D'Eon, B.J.; Chang, X.J.; Olesen, C.E.; Palmer, M.A. Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using β-galactosidase enzyme complementation technology. J. Biomol. Screen. 2002, 7, 451–459. [Google Scholar]
- Yu, H.; West, M.; Keon, B.H.; Bilter, G.K.; Owens, S.; Lamerdin, J.; Westwick, J.K. Measuring drug action in the cellular context using protein-fragment complementation assays. Assay Drug Dev. Technol. 2003, 1, 811–822. [Google Scholar]
- von Degenfeld, G.; Wehrman, T.S.; Hammer, M.M.; Blau, H.M. A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals. FASEB J. 2007, 21, 3819–3826. [Google Scholar]
- Michnick, S.W.; Ear, P.H.; Manderson, E.N.; Remy, I.; Stefan, E. Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat. Rev. Drug Discov. 2007, 6, 569–582. [Google Scholar]
- Luker, K.E.; Gupta, M.; Luker, G.D. Imaging CXCR4 signaling with firefly luciferase complementation. Anal. Chem. 2008, 80, 5565–5573. [Google Scholar]
- Ozawa, T.; Kaihara, A.; Sato, M.; Tachihara, K.; Umezawa, Y. Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal. Chem. 2001, 73, 2516–2521. [Google Scholar]
- Kaihara, A.; Kawai, Y.; Sato, M.; Ozawa, T.; Umezawa, Y. Locating a protein-protein interaction in living cells via split Renilla luciferase complementation. Anal. Chem. 2003, 75, 4176–4181. [Google Scholar]
- Hu, C.D.; Kerppola, T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat. Biotechnol. 2003, 21, 539–545. [Google Scholar]
- Hammer, M.M.; Wehrman, T.S.; Blau, H.M. A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization. FASEB J. 2007, 21, 3827–3834. [Google Scholar]
- Hida, N.; Awais, M.; Takeuchi, M.; Ueno, N.; Tashiro, M.; Takagi, C.; Singh, T.; Hayashi, M.; Ohmiya, Y.; Ozawa, T. High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases. PLoS One 2009, 4, e5868. [Google Scholar]
- Misawa, N.; Kafi, A.K.M.; Hattori, M.; Miura, K.; Masuda, K.; Ozawa, T. Rapid and high-sensitivity cell-based assays of protein-protein interactions using split click beetle luciferase complementation: an approach to the study of G-protein-coupled receptors. Anal. Chem. 2010, 82, 2552–2560. [Google Scholar]
- Viviani, V.R.; Uchida, A.; Viviani, W.; Ohmiya, Y. The influence of Ala243 (Gly247), Arg215 and Thr226 (Asn230) on the bioluminescence spectra and pH-sensitivity of railroad worm, click beetle and firefly luciferases. Photochem. Photobiol. 2002, 76, 538–544. [Google Scholar]
- Viviani, V.R.; Arnoldi, F.G.; Neto, A.J.; Oehlmeyer, T.L.; Bechara, E.J.; Ohmiya, Y. The structural origin and biological function of pH-sensitivity in firefly luciferases. Photochem. Photobiol. Sci. 2008, 7, 159–169. [Google Scholar]
- Viviani, V.R.; Bechara, E.J.; Ohmiya, Y. Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures. Biochemistry 1999, 38, 8271–8279. [Google Scholar]
- Viviani, V.R.; Arnoldi, F.G.C.; Venkatesh, B.; Neto, A.J.S.; Ogawa, F.G.T.; Oehlmeyer, A.T.L.; Ohmiya, Y. Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases. J. Biochem. 2006, 140, 467–474. [Google Scholar]
- Nakajima, Y.; Kimura, T.; Sugata, K.; Enomoto, T.; Asakawa, A.; Kubota, H.; Ikeda, M.; Ohmiya, Y. Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. Biotechniques 2005, 38, 891–894. [Google Scholar]
- Sugihara, K.; Park, H.M.; Muramatsu, T. In vivo gene electroporation confers strong transient expression of foreign genes in the chicken testis. Poult. Sci. 2000, 79, 1116–1119. [Google Scholar]
- Deroo, B.J.; Archer, T.K. Proteasome inhibitors reduce luciferase and β-galactosidase activity in tissue culture cells. J. Biol. Chem. 2002, 277, 20120–20123. [Google Scholar]
- Paguio, A.; Garvin, D.; Swanson, B.; Worzella, T.; Wood, K.; Fan, F. Using Luciferase reporter assays to screen for GPCR modulators. Cell Notes 2006, 16, 22–25. [Google Scholar]
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


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New Investors Nokia Growth Partners & Qualcomm Join Series B Lead BlueRun Ventures
New York, NY, February 13th, 2013 – Verve Mobile, the leader in location-powered mobile advertising, announced today it has closed its Series C financing led by Nokia Growth Partners, a global growth stage venture firm focused on mobile technology, services and media with participation from new investor Qualcomm Incorporated, acting through its venture investment group, Qualcomm Ventures (QCOM), and Series B lead investor BlueRun Ventures. The capital will be used to further develop and expand Verve's proprietary mobile location-based advertising and publishing products and to grow its marketing and sales capabilities.
"Verve's focus is combining big data, location-based services (LBS) and ad technologies to make mobile advertising work better for advertisers and publishers," says Tom MacIsaac, Verve Mobile CEO. "Nokia and Qualcomm are global leaders in mobile technology innovation and have important insights, assets, initiatives and relationships that can help Verve maintain its lead in location-powered mobile advertising."
John Gardner of Nokia Growth Partners has joined Verve's Board in connection with the financing, and Quinn Li of Qualcomm Ventures has become a Board Observer.
About Verve
Verve Mobile is the pioneering leader in location-based mobile advertising. Our customers are national brand advertisers who want to engage consumers on their mobile devices with location-aware, data-driven and highly targeted marketing. We also serve premium publishers across the nation, delivering the platforms and services they need to power and monetize their mobile properties. The Verve location-powered technology platform and the proprietary data it generates are revolutionizing mobile media and advertising. It enables consumer and content brands to reach their desired audiences on the go, out and about, and closer to the buying decision than ever before. Verve has offices in New York, NY, Washington D.C. and San Diego, CA. For more information, visit www.vervemobile.com
About Nokia Growth Partners
Nokia Growth Partners invests in companies that are changing the face of mobility, communications and the internet. NGP offers industry expertise, capital and an extensive network, enabling entrepreneurs to build disruptive, industry-changing companies and take them to the global market. With offices in the US, Europe, India and China, NGP extends the reach of companies making their products and services local everywhere. Visit www.nokiagrowthpartners.com for more information.
The post Verve Mobile Secures $14M in Series C Funding appeared first on Nokia Growth Partners.


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We visited Wayanad recently and one of the places we visited during our trip is Uravu. It is a non-governmental, non-profit organization which strives to provide employment to the underprivileged people by making handicrafts from bamboo. It is tucked away in a small town Thrikkaipetta in Wayanad District. I had read about this on someone else's blog and had it on my must-see list when I visited Wayanad last week.
Uravu is around half an hour drive away from Enteveedu, the home-stay where we stayed. We reached this place at around 9 and we could see people already busy working. We first visited the stores where they sell handicraft items. The store was a sight to see. Bamboo everywhere, in all possible colors and variety and shapes. I was mesmerized with the amount of innovation and creativity around me.
The first sight I caught was this corner where bamboo vases in all possible sizes were neatly arranged. There were many more stocked away, which I couldn't see. Apparently, bamboo vases sell like hot cake.
Then there is this corner which had beautiful lamp shades. I am not sure how much heat bamboo can withstand, but lamp shades look so pretty. Don't you agree?
Look at the far right one. The design is so intricate! Makes me want to buy all those.
Why stop at lamp shades? Look at this hanging piece which looks so elegant.
Candle stands, pen stands, files, pens – lots of useful and beautiful things.
How about some jewelery? Who would have thought bamboo can be worn around your neck? And such pretty necklaces too!
These women are making thin strips of bamboo which will be used to make curtains and mats. They cut strips first and then file them to get a smooth finish.
A worker making door curtains and floor mats using the loom. He was working on it methodically – there was a rhythm to his work which was so mesmerizing to watch.
Another worker making a lamp shade.
A few women polishing (applying varnish) to finished items.
They do screen printing on order. You select the design and they make any item you want. Look at these freshly printed key chains.
I was so impressed with this little factory that I had half the mind to buy all the items there. I controlled my urge and picked a few things – a serving tray, a few pens, a fruit basket and a very interesting musical instrument which makes the sound of raindrops. You have to hear it to believe it.
Some may label these items as expensive, but as someone who hand knits sweaters, I know how much time and effort goes into making something hand-made. A machine knit sweater will always cost less than a hand-knit one, but where is the love and the human touch in it, I say? I did spend a big amount on buying these bamboo items which I might probably get for cheaper here in Bangalore, but this is my way of supporting this local seller.


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Fear And Loathing in the Tropics Frogs Adapt to Survive
Written by Murray Carpenter and originally published in The Boston Globe, November 24, 2008. Used with permission.
GAMBOA, Panama — The eyes of a caiman, an alligator-like reptile, reflect the beam of a flashlight shining on the pond, as, nearby, a snake slips through the shore undergrowth. Large spiders patrol the margins, and predacious water bugs skim the water's surface. It's a tough place to be a frog. But it's a great place to study fear and death.
That is exactly what Boston University associate professor Karen Warkentin—standing at water's edge wearing rubber boots and a headlamp—is doing. Along the way, she and her colleagues are challenging basic biological assumptions by illuminating the many different ways frogs develop in response to their environments.
It rained buckets earlier, and red-eyed tree frogs are calling enthusiastically. These slender frogs are to the tropics what loons are to New England—photogenic emblems of the wild. They are also fruitful research subjects. Warkentin has been studying them since 1991, when she staked out a pond in Costa Rica . The pond attracted many frogs and snakes, and she noticed two things: Frog eggs were not hatching at the same age, and snakes were eating a lot of the eggs.
After hundreds of hours of observation in the field and in the lab, she found that when a snake attacks a cluster of frog eggs, the vibrations prompt the embryos to hatch early. The eggs are mature at seven or eight days, but will hatch as early as five days in response to an attack. By hatching early, the embryos trade certain death by snake for the risk of being slightly underdeveloped and more vulnerable to predators when they splash down into the pond.
When Warkentin first presented her observations, many herpetologists harrumphed. But they came around after watching her videos of the attacks.
Warkentin says her research is part of a growing body of work showing how environmental influences affect development and survival. "It's not just what genes you have, but how you use them," said Warkentin. "Genes matter, the environment matters, and what you get in the end is dependent on both of them."
Since her initial discovery, she says, "I've kind of followed where these frogs have led me." That included a fellowship with the Smithsonian Tropical Research Institute, where she is a research associate. She spends at least two months a year at a Smithsonian field lab in an old school building in this Panama Canal town. Not only are there a number of well-studied frog ponds nearby, the institute excavated this pond for Warkentin four years ago, sculpted to her specs among trees at the edge of the jungle. The amphibians colonized it as soon as it held water.
Warkentin and her students have had some surprises.
Warkentin and Justin Touchon, a doctoral candidate at BU, found that tadpoles raised in tanks with fish develop clear, slender tails, while those in tanks with dragonfly larvae develop big red tails. The adaptations allow them to better escape the different predators.
"What it shows is that these animals are much more complex than we think," said Touchon.
Touchon also found that hourglass tree frogs, which usually lay eggs on leaves over water, instead laying their eggs in the water—marking the first time that a vertebrate has been known to lay eggs both on land and in the water, a sort of evolutionary bridge species.
In one experiment, Touchon moved frogs that were about to lay eggs to enclosures with water-filled kiddie pools and overhanging plants. Half the enclosures were in an open field, where eggs laid on leaves would be at risk of drying up in the hot daytime temperatures, the other half were in the shade nearby. Although neither group of frogs had ever seen their new habitat in daylight, the vast majority of each group laid their eggs in the place best suited to the daytime conditions—in the cooling water for those in the sun, on the leaves for those in the shade.
"The idea that frogs make these really smart decisions about where to lay their eggs surprises most people," said Touchon. His photo of these frogs made the cover of the Proceedings of the National Academies of Science, which published his paper in May.
Warkentin has found complexity at every turn. Not only can red-eyed tree frogs hatch early to escape predators, they can also complete their metamorphosis and leave ponds early to escape tadpole predators such as water bugs, or delay metamorphosis when there's an abundance of pond-side predators such as fishing spiders. Fear of predators, it turns out, strongly influences not just frog behavior but also physical development.
Warkentin is also investigating early development, growth within the egg, and how eggs hatch.
But her biggest project is nicknamed "fear and death." It occupies a large field near the experimental pond. Each of the 200 tanks that hold 100 gallons is stocked with tadpoles. Some tanks have no predators, some have dragonfly larvae, and some have water bugs. In some tanks the predators are loose among the prey, in others they are suspended in nets where they are fed tadpoles but can't prey on the general population. The tadpoles detect the predators' presence through chemical signals.
Warkentin hopes this project will provide answers to a big question: In a frog's world, "what's the relative importance of fear and death?"


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Grey Lynn Community Centre – Hirer's Information
Getting in You do not need a key. The Centre will be open.
Payment : To secure your booking payment must be received
at least 10 days prior to the event. We accept cash or cheque only.
Setting Up : All setting up and after event cleaning must be conducted within the time booked…
Smoking : The Centre is smoke free, a designated smoking area is located in
front of the building. An ashtray is located on the wall.
Cleaning : to be completed by Hirer
Tables wiped down and stacked face to face.
Plastic chairs stacked 10 high in the storage cupboards.
Fabric chairs stacked five high within the Oval room.
Grounds : remove all litter from garden, decks and street frontage.
Kitchen Users : are responsible for the provision of tea towels, scourers and detergent etc.
When using the stove please turn on the extractor fan above.
Wipe inside and out of stove, dishwasher and fridge.
Sweep and mop the floors.
Check toilets are clean and tidy.
All rubbish must be removed from the premises by the Hirer.
Fire Alarm
Smoking, candles, incense, smoke machines or any other smoke emitting devices are not permitted to be used at the Centre. Prior permission is required from the Centre Coordinator for use of barbeques or spits in the grounds.
Should actions of a Hirer result in a fire callout by the Fire Department the Hirer shall be held responsible for the callout fee of $1125.00
After Hours Assistance
Evening/Weekend Caretaker 027 2717210
Auckland Council 24hours 379 2020


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Looking to play some softball? We currently have 7 leagues to choose from. Please click on the league name for more information.
West Chester Wildfire - Youth Girl's Fast Pitch
Chester County Men's Slowpitch - Adult Men's 6-12' Arc Pitch Softball
Chester County Coed - Adult Coed 6-12' Arc Pitch Softball
West Chester Modified - Adult Men's Modified Pitch Softball
County Church Modified - Adult Men's Modified Pitch Softball
Brandywine Valley Senior League - Adult Men's 60 & over Softball
Chester County Women's Slowpitch - Adult Women's 6-12' Arc Pitch Softball


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MADISON, Wis. (8/25/15)--It's not too early to start thinking about the holiday season, especially if it concerns the year-end bottom line. This year's holiday retail season is expected to be strong in the United States, according to the latest eMarketer forecast.
U.S. retail sales in the months of November and December will increase 5.7% year over year, reaching $885.7 billion, eMarketer predicted. That's an upward adjustment from the 3.2% growth rate predicted earlier this year--and the highest since the 6.3% rise in 2011.
"Retail sales in November and December 2015 are expected to show a healthy increase over what was experienced during the same period in 2014," said eMarketer analyst Monica Peart. "This expectation is driven primarily by the fact that gas station sales, which make up roughly 12% of overall retail sales, dropped rather dramatically in late 2014.
"Increases in real income from wages, further decreases in unemployment and an increased willingness to spend in traditional retail categories that missed out on the windfall in gas prices earlier on in the year should also drive growth," Peart added.
Retail ecommerce holiday season sales are expected to decline slightly this year to 13.9% compared with 14.4% in 2014. However, the market share of ecommerce sales will increase to 9% of total retail sales, or $70.4 billion, up from 8.3% last year.
Similarly, mobile will play an increased role in holiday sales this year. eMarketer expects U.S. retail mobile commerce sales to rise 32.2% in the full year of 2015--more than double the 14.2% increase forecast for retail ecommerce sales as a whole. By the end of 2016, 25% of all retail ecommerce sales in the United States will take place via mobile devices.


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Bernie Sanders' stepdaughter is considering a run for mayor of Burlington, Vermont, the same office that was the starting point for the independent U.S. senator's political career.
Carina Driscoll is a former city council member and progressive state legislator. She said Monday that she will make a decision by the end of the month.
Democratic Mayor Miro Weinberger is expected to seek re-election in March 2018.
The latest news from around the state
Driscoll says she believes it's time to consider a new direction and a new vision for Burlington. She says she'd like city hall to provide more leadership of some of the city's public assets, such as its telecom utility.
Her stepfather was elected Burlington's mayor in 1981, upsetting the status quo of city politics and launching his political career.


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Toronto's culinary landscape is as diverse as it is delicious. Constantly expanding, the city welcomes many new eateries every year. Whether you're a local foodie or a tourist interested in broadening your horizons, the downtown area is perfect for finding new cuisine and interesting atmospheres in which to indulge. The following are 4 Toronto restaurants set to open in 2014.
Pai is a brand new Thai restaurant that will be opening on Duncan Street. This eatery is being established by Nuit and Jeff Regular, the geniuses behind Khao San Road (who they recently severed ties with), Sabai Sabai and Sukhothai - a few of the city's most popular Thai locations. Pai plans to bring the complex flavours and dishes unique to Northern Thailand to Toronto.
Apiecalypse Now!
Mentioned in our Guide to Vegan Bakeries, Apiecalypse Now! offers an incredible range of baked goods that are 100% free of animal cruelty. They've been successfully selling their goods at city farmer's markets for a number of years and 2014 will see the opening of the bakery's very first retail storefront. It is set to be located on Markham Street – stop by for a treat that won't leave you feeling guilty!
Barsa Taberna
Barsa Taberna is set to be Market Street's newest addition in early 2014. Located in the heart of downtown, across from St. Lawrence Market, this Barcelona-inspired restaurant will offer an incredible menu of Spanish tapas and small plates. It will be run by talented chef Michael Smith (who worked previously at the Gladstone Hotel), who plans to devise brunch, lunch, dinner and late-night menus.
Red Sauce


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Last modified: 2011-12-31 by german editorial team
Keywords: mecklenburg-west pomerania | stralsund | hanseatic | arrowhead (white) | cross (white) |
Links: FOTW homepage | search | disclaimer and copyright | write us | mirrors
3:5 image by Stefan Schwoon
See also:
Stralsund, the old hanseatic city opposite of Rügen Island had a long tradition. In the newer history she was part of Rostock District in times of the DDR, became a county free city after 1990 and finally became part of bew county of Vorpommern-Rügen and the county's capital on 4 September 2011.
Klaus-Michael Schneider, 2 Nov 2011
Stralsund is a north German city, in the Land Mecklenburg-Westpomerania, population about 76,000. The flag they used [for the Hansa festival in Bergen, Norway], was red with a white cross and arrowhead.
Jan Oskar Engene, 14 May 1996
A flag version of the arms, canting for German Strahl (=ray), Slavic stral (=arrow). Sources: Staack 1997 and Günther 1999a. br>Stefan Schwoon, 13 Feb 2001
From Ralf Hartemink's International Civic Arms website:
Stralsund was founded as Stralow and the arrow (Stral in German) is a canting symbol. (...) The arrow as a symbol already appears on the first known seal of the city, which is known from 1265 and 1274. It shows a small boat under the arrow, as well as two swimming fish. (...) Until the 18th century (...) the arrow is (...) used as arms of the city, but the colours are uncertain. Similarly, the arrow, sometimes accompanied by a cross, is used on the coins of the city.
Literature: O. Hupp, Königreich Preussen, Wappen der Städte, Flecken und Dörfer, reprint from 1896 and 1989, Kulturstiftung der deutschen Vertriebenen, Bonn, 1993; H. Ewe, Schiffe auf Siegeln, Hinstorff, Rostock, 1972.


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ROCK - Hard Rock/Metal
Within the Grey is a hard rock band from Moline, Illinois, not far from the Chicagoland area. Our sound can be described by combining what moves us about the elements of underground hardcores fearless heavy side, the driving riffs of modern and classic metal, and the vocal-melody fueled style of todays radio rock. The band is influenced by such artists as: Metallica, Korn, Three Days Grace, Beartooth and Underoath to name a few. In a way we are taking what we love about these artist and their respective genres and blending it to cultivate our own style and brand. Within the Grey has just completed recording three songs with a local studio in Geneseo, Illinois. Within the Grey has been playing in and around the Midwest area for just over a years time. We have had the pleasure of sharing the stage with such notable bands as: Gemini Syndrome, Metal Church, Red Sun Rising, Otherwise, Shallow Side and 3 Years Hollow. We are Within the Grey, and we believe that is how the world must operate. There is no such thing as any facet of life being either black or white, it is all a matter of perspective.
Sorry, no MP3s available


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D5-BAV14 od, 8055967927145
New product
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By buying this product you can collect up to 150 loyalty points. Your cart will total 150 points that can be converted into a voucher of 1.65 €.
DEFCON 5 ENDURANCE VEST CARRIER Coyote
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Tasca utility di grandi dimensioni. misura 21 x...


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May 6, 2006
We will, over time, take a hammer to all manner of weird monetary ideologies that have grown up over the years. They are practically unlimited in number. One of these ideologies is the notion expressed in the equation:
M is the "amount of money" (left conveniently undefined)
V is a mystery variable commonly termed "velocity"
P is "prices", as if "prices" could be accurately reduced to a single number. For now, we can consider this the GDP deflator.
T is "transactions," as if anybody really knows what transactions are going on. For now, we can consider this "real" GDP. P*T can be generalized as nominal GDP.
This notion is some sort of holdover from the bad old days when people had a goal (utterly mistaken) of trying to make the study of economies into something like the study of thermodynamics, i.e. the construction of steam engines. Even today, gullible young people are encouraged to learn higher math in preparation for graduate work in economics, which is a joke that became unfunny a long time ago. I used to say that one can be a great economist with 8th grade math, but today I suspect even that may be an overstatement.
Now, up above, I said that the equation MV=PT is a "notion" expressed in an equation. The notion expressed, of course, is that one can manipulate an economy via monetary manipulation. Which is of course true; the problem is that you can't manipulate an economy to any good. The effects are all bad.
One of the many problems here is that V is a meaningless fudge ratio. Let me show you what I mean. Instead of M="money," let's say that M="number of hits for Lindsay Lohan on Google". It is a mathematical truism that:[Lindsay Lohan hits on Google]*[some number]=[nominal GDP]
Or, to use the example in the title of this brief,["money" arbitrarily defined]*[some number]=[my butt]
Both of these are a mathematical certainty! Both are utterly meaningless! This "MV=PT" nonsense has been around a very long time, as it was also a favorite notion of the Mercantilist thinkers of Britain in the 1600-1750 period, who were also eager to manipulate the monetary conditions of that area, despite that over a century of such manipulation never did much good for anybody. This is what Adam Smith had to say about these issues:
"What is the proportion which the circulating money of any country bears to the whole value of the annual produce circulated by means of it, it is, perhaps, impossible to determine. It had been computed by different authors at a fifth, at a tenth, at a twentieth, and at a thirtieth part of that value."
Today, "money" is typically defined as predominantly short-term lending, and the dollar is in use worldwide, with 80%+ of banknotes now apparently circulating overseas, making the assertion of any relationships of a worldwide currency with the nominal GDP of one country doubly preposterous.
The 1970s and 1980s were the heyday of using "MV=PT" directly. In time we will examine the damage wrought. I will point out, by way of example, that in 1970, the Nixon economists decided that they wanted a 9.0% increase in nominal GDP over the coming year, to get the economy out of the 1970 recession. Didn't "MV=PT" indicate that this could be directly accomplished by simply increasing "M"? Of course, "M" can't be increased directly, as it is typically taken as a measure of short-term credit, so the question became: how much would the Fed increase the base money supply to increase "M" enough to produce a 9.0% increase in nominal GDP? This calculation fell to Art Laffer, then head of the Office of Management and Budget, who did as he was told. Arthur Burns, Nixon's old buddy installed at the Fed in 1970, was given his marching orders, with the result that the Bretton Woods gold standard was blown up in 1971 and the world entered a decade of phenomenally painful inflation.
Of course, the Nixonites just wanted to play games, but the notion of "MV=PT" — utterly meaningless in itself — gave them a convenient justification or veneer of respectability with which they could delude themselves and others that what they were about to was in the best interests of all involved.
Then, in the early 1980s, the Fed attempted to control inflation by application of "MV=PT," which was a big mess but ultimately succeeded, because, by that point, what they really waned was a convenient justification and veneer of respectability by which they could undertake a very crude, destructive and sloppy monetary contraction, to end the inflationary period of the 1970s. This enabled them to live out their fantasy that "fighting inflation" required "recession," when, of course, solving an inflation problem should lead to economic boom!
In the 1990s, the Bank of Japan and other economic types (including virtually all foreign commentators) totally failed to understand monetary deflation in Japan as the result of a rising value of the yen since 1985 (the "yen going to Y2" although it actually went to more like three yen!). They thought that a low BOJ target interest rate would encourage banks to increase short-term lending/deposits, thus increasing "M", and hopefully thus increasing "P*T" or nominal GDP. When that plan didn't work, they claimed that the "transmission mechanism was broken," when all that was broken was their fallacious theories. The real solution was to lower the value of the yen by a) announcing such a policy, and b) increasing the supply of base money. They never really got a) right, but finally implemented b) in 2001, in a shy and barely effectual sort of way, after half a decade of pressure mostly from a few foreign thinkers (including me) who got it mostly right. Result? Reflation and recovery!
(By the way, the Japanese government has overshot its mark and already has an inflation problem, which will likely produce some big surprises in the future for them!)
So you see, getting these principles right is very important, and can mean the difference between economic disaster and success.


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Archive.org recently added a gigantic collection of the influential science fiction magazine Galaxy Science Fiction to its site that is free for anyone to read, worldwide. While it's not a complete collection of the magazine's run, there are many pieces of sci-fi history in the collection. Get the details after the jump.
$1 a month:
You'll get:
- My thanks & gratitude
- Your name listed in a special "Thank You" post on The Code every month
- Access to Patron-only posts and updates
- One free Patron only exclusive download each month


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Honestly can just get a completely fresh/new client at this point?
####Title Fix: Honestly can we just get a completely fresh/new client at this point? * One that doesn't use your entire RAM? * One that doesn't lag for no reasons even if you have a decent PC RIG? * One that has an option to disable certain animations and features to make it run even more smoothly? * Or you know, one that actually functions properly? The amount of bugs/issues/lag that the current Client has is unbelievable. And there are even more issues outside Client. So it would be nice to just get a completely revamped Client that has better UX (User Experience) because the UX in it's current form is literally a joke in comparison to a lot of game launchers out there. If they could resolve the Client Issues then they could move on to resolve the In-Game issues (like the recent Mouse related issues, alt tabbing out of game, massive fps drops, really high ping and so on...). Instead of making League from ground up in a mobile platform they should of done it to PC first since that is their main Platform where most players play the game.
- Citação
- Conecte-se para comentar
- Citação
- Editar
- Excluir Excluído
- Tem certeza
- Sim
- Não
- Desfazer exclusão
- Exibir na ferramenta de moderação


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Prison Riot Sparks Calls For Transparency
Here's an excerpt from Erin Rosa's article, click at the bottom to read the entire thing.
The federal government needs to be more transparent in providing local communities with information about the violent incidents that occur in Colorado prisons. That's the message from public officials reacting to recent news that a prison in the state has been on lockdown status since August, just months after a violent yard riot in April left two inmates dead.
In a series of articles published last week, The Colorado Independent reported that the high-security United States Penitentiary in the southern city of Florence — operated by the federal Bureau of Prisons — had been in lockdown status since early August following a violent inmate-on-inmate assault.
Lockdown status means that inmates are generally confined to their housing units or cells.
The incident comes just months after a deadly yard riot in April that ended with two inmates being killed by guard gunfire, and on top of that, the prison's warden Sara Revell was just given the annual Excellence in Prison Management award from the bureau for overall management of staff and inmates.
During the riot, guards used a heavy arsenal of weaponry, including more than 200 M-16 rifle rounds, more than 300 pepper balls and nearly 12 long-range CS gas canisters, according to documentation obtained from sources inside the prison.
Rep. Buffie McFadyen, a Pueblo Democrat whose district includes the penitentiary, said the news showed how necessary it was for the bureau to release key details about the riot to the public.
"There's a problem with the secrecy the bureau functions in to begin with overall. They're not a very transparent organization," McFadyen said, noting that shes talks regularly with prison workers and officials with the correctional officers union. "I have to cultivate people to talk to. I don't get answers back from the bureau."
Colorado Independent


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Grandville Christian Preschools are committed to early childhood education. We offer many options for you and your child. Our caring staff have been chosen for their dedicated love of the Lord and their desire to educate young children. We believe that each child is a precious gift from God and we strive to meet each child's needs spiritually, emotionally and academically. Within our nurturing environment, your child is able to explore God's world and develop their individual gifts and abilities.
Focus at Spanish Immersion Preschool
Our Spanish language immersion program offers children the opportunity to fully experience a second language and culture.
Immersion is a method of educating children by instructing them in a "target" language so they become bilingual. The classroom environment becomes the setting for second language acquisition through core subject content instruction, educational discourse, and social interaction.
About Our Preschool Program
Research revels the following benefits to learning a second language:
- A positive effect on intellectual growth.
- Flexibility in thinking, greater sensitivity to language, and a better ear for listening.
- Enhancement in understanding native language.
- Greater ability to learn additional languages.
- Appreciation of other cultures and global perspective.
- Increased career opportunities.
Other things you should know:
- Our bilingual preschool teachers use a variety of ways to get your child excited about learning the Spanish language. These include pictures, movement, activities, music, puppets and games.
- Spanish Immersion preschool strives to keep low teacher-to-student ratios in an effort to provide the best immersion education experience possible.
- Our staff is CPR trained and First-Aid certified.
- Toilet training is not a requirement to begin preschool.
- Open mid- September through May.
Spanish Immersion Preschool Program Choices
- Tuesday and Thursday morning preschool
for 3-year-olds: 8:45 – 11:15 AM
- Tuesday and Thursday afternoon preschool
for 3-year-olds: 12:45 – 3:15 PM
- Child must be three by September 1 to enroll in three-school
- Monday, Wednesday and Friday morning preschool
for 4-year-olds: 8:45 – 11:15 AM
- Monday, Wednesday and Friday afternoon preschool
for 4-year-olds: 12:45 – 3:15 PM
- Child must be four by September 1 to enroll in preschool


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A Federated Congregation of the American Baptist Churches USA & the Presbyterian Church USA
| Home | About Us | Services | Ministries | Resources | Contact Us |
Some Resources
Visit Us On Facebook
Click here for : Worldview Quiz
Scroll Down for Useful Links and Information for Spiritual Growth.
Crosswalk-Website with family topics and daily devotions.
Heartlight-Daily devotionals and articles.
Family Life Network- Radio ministries.
American Baptist Churches USA
Presbyterian Church USA
211 2-1-1 provides free and confidential information and referral.
rightnow- the mission of the church matters
Village of Nunda -Official Website of the Village of Nunda
The Layman Online-Presbyterian Lay Committee


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Welcome to the first of the Book Witch Blogs!
Been pondering mailing lists and such for a while now but a) didn't have anything to report regularly enough, and, b) didn't want to start something only to find I couldn't sustain it! A bit further down the road now, particularly with the Events side now in place, so giving this a try and if it works out can use it as the basis for a (monthly) newsletter or similar (or that's the thought process anyway!)
Thanks to much help from my ever-so-glamorous assistant the website has been flooded with new additions this past week – both "new new" and "new pre-loved" including some very amazing rare and limited editions as part of the ongoing cataloguing process (for more see Special Finds). Brief glitch on day one when we hit some memory issues (the website's not ours!) but fortunately resolved quite quickly.
June's workshop will be a re-run of Runes (try saying that ten times fast) and will be on the 15th – as usual you can book online or in the shop, see Events for further info. I've been absolutely loving doing them, and looking forward to some of the new topics I'm hoping to cover in the coming months.
I also managed to de-Americanise the website (apologies to Americans, but some of the terminology was driving me crazy) so no more 'Cart' and 'Newness' nonsense, it should be Baskets and Date Added all round now!
Another new addition is 'Book Prescriptions' – more designed for 'distance customers' since (hopefully) anyone who pops in the shop knows they can always have a blether about suggestions and recommendations, but this gives everyone the opportunity the chance to fill in a wee form to do the same (or even just for days like this when it's chucking it down and you don't want to venture forth…)
Seems like most of the past week involved minor skirmishes with the website – I think it largely came out a draw but time will tell whether it's all working properly and it might take me a while to get it as 'pretty' as I would like (if any WP gurus want to volunteer their help I'd be more than willing to accept, done a not-bad job at bodging through myself so far but know my limitations!)


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While Miami is best known for palm trees swaying among neon-lit avenues, Texas is probably most famous for cattle, oil, lots of excess...and old fashioned duels to the death.
Read Also:
- Haven's Isaac Grillo Wins Inaugural Ultimate Miami Bartender Award at Magic City Casino
That vision apparently has made two of Miami's hottest young chefs homesick for the lone star state. You see, Haven's chef Todd Erickson came to Miami from the Big-D (Dallas for you tenderfoots), and The Federal's Cesar Zapata graduated from the Art Institute Houston before working under James Beard-nominated chef Timothy Keating in Houston.
So, to pay tribute to their long-lost former homes, both chefs decided to hold one of those duels -- only knives and pans will be used in place of rifles and pistols (thank heavens for progress).
The duel will be held at The Federal on Tuesday, October 30, at 7:30 p.m. and you're invited to bear witness to this epic battle, eating your way through the evening...and having a say in the chefs' fates.
The five-course menu. consisting of five Texan themes, will be divided up between the two chefs. The dishes will be paired with Spoetzl Brewery beers out of Austin, Texas. The menu is:
Chopped beef, roasted jalapeño, fried egg, Texas toast
Hatch roasted chicken, melted leek tamale
charred sweet corn salsa, queso de cabra
Smoked short rib, Texas chili, sweet potatoes, spicy pickles
Game Duo
Battle quail
Hummingbird cake and cream cheese mousse
melted pecan praline, caramelized pineapple
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At the end of the meal, diners will be handed a score card and will be asked to rate the chefs on taste, plating, and originality. Score cards will also be entered into a raffle for a dinner for two at the restaurant of their choice -- The Federal or Haven.
The dinner, served family style, is $75 per person, excluding tax and gratuity. Tickets can be purchased in advance here.
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Vacuum packaging extends shelf-life, ensures quality, prevents products from drying out, protects it from outside influences and improves hygienic handling.
Low Cost
Vacuum packing is a very cost effective procedure for preserving perishable food products. Very low packing material costs, short process times and a relatively low investment for long-lasting equipment makes vacuum packing by far the most economical and most efficient way of packaging perishable food products. But also for other applications, such as protecting goods and space saving logistics or storage, vacuum packing is an ideal solution.
Vacuum bags and film
For vacuum packaging special vacuum bags are required. The bags are coextruded with mostly a combination of polyethelene (PE) for strength, but also for sealability, and nylon (PA) as a barrier to prevent air from penetrating the bag over time. Good quality bags will maintain vacuum in the bag over a long period. The bags are often treaded so that the don't stick together and processed in a clean environment. Special cook bags will allow the product to be heated in a water bath.


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St. Paul the Apostle Catholic Church is a growing and diverse community with a small parish feel. United in prayer and worship, we reach out to others, leading and serving as one with Christ.
Effective February 25, 2011, Cardinal DiNardo appointed Fr. Wencil Pavlovsky as our new Pastor. Fr. Wencil served in the Archdiocese as Pastor at St. John Fisher in Richmond and St. Augustine in Southeast Houston. He was formally installed as the new Pastor of St. Paul the Apostle Catholic Church in a Mass celebrated by Msgr. Leo Wleczyk, our Episcopal Vicar, on Sunday, March 27, 2011. Father Wencil guides our spiritual diverse and multi-cultural family. He has imprinted the St. Paul community with his own unique and nurturing spirit, leading us in giving Glory to God.


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After lots of talking, the proposal is written.
We've begun to negotiate the unique territory which emerges from each new collaboration, and found some shared space from which to begin. It's exciting and full of potential.
We've told a few other people about what we're planning to do, and we've been encouraged by their responses.
Now, we've just got to cross our fingers…


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Edge of Black (A Samantha Owens Novel)
CategoriesAll | Mystery | Suspense / Thriller
Edge of Black (A Samantha Owens Novel)
by: J.T. Ellison
Dr. Samantha Owens is starting over: new city, new job, new man, new life. She's trying to put some distance between herself and the devastating loss of her husband and children—but old hurts leave scars.
Before she's even unpacked her office at Georgetown University's forensic pathology department, she's called to consult on a case that's rocked the capital and the country. An unknown pathogen released into the Washington Metro has caused nationwide panic. Three people died—just three.
A miracle and a puzzle…
Amid the media frenzy and Homeland Security alarm bells, Sam painstakingly dissects the lives of those three victims and makes an unsettling conclusion. This is no textbook terrorist causing mayhem with broad strokes, but an artist wielding a much finer, more pointed instrument of destruction. An assassin, whose motive is deeply personal and far from understandable.
Xander Whitfield, a former army ranger and Sam's new boyfriend, knows about seeing the world in shades of gray. About feeling compelled to do the wrong thing for the right reasons. Only his disturbing kinship with a killer can lead Sam to the truth…and once more into the line of fire.
Book Details
File Size: 462 KB
Print Length: 362 pages
Page Numbers Source ISBN: 0778313727
Publisher: MIRA (December 1, 2012)
Sold by: Amazon Digital Services, Inc.
About the Author
J.T. Ellison is the bestselling author of the critically acclaimed Taylor Jackson series, including All The Pretty Girls, 14, Judas Kiss and The Cold Room. She is the bi-monthly Friday columnist at the Anthony Award nominated blog Murderati, and was named "Best Mystery/Thriller Writer of 2008" by the Nashville Scene.


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- Home
- Property
- United States of America
- North Dakota
- Burke
- Home with Acreage-Recreational-Vacant Land
- Pheasant Hunting Retreat - 280 acres & Home
Pheasant Hunting Retreat - 280 acres & Home, Portal, North Dakota, USA
Pheasant Hunting Retreat - 280 acres & Home, Portal, North Dakota, USA, 280.00 Acres
Main Info
- Street Address : 201 2nd Ave. East
- Postal / Zip Code : 58772
- State / Province : North Dakota
- County : Burke
- Closest City : Portal
- Lot Size Acres : 280.00 Acres
- Dwelling : Yes
Tax Year : 2017
Estimated Taxes per year are $2,300.00
Broker Info
Joe Bender
Four Seasons Realty(P:) 701-389-8934 (M:)701-389-8934 [email protected] fourseasonsrealtynd.com
Joe's knowledge in residential, commercial, ranch and agriculture lands, along with experience in selling lake properties, and a true appreciation for North Dakota, makes working with Joe right choice. Since moving to Bottineau, North Dakota in 2005 where he calls home today with his wife Angela and two kids Alex and Abby, Joe has been selling real estate for the past 9 years. Joe now owns Four Seasons Realty in Bottineau and with his background in business management, residential and land development; it has made him a top successful Broker in the area. Joe's willingness to go the distance for his clients and his ability to understand the Dakota diversity of lands has created a great working relationship with his clients. When he is not listing and selling, Joe is an avid hunter, from siting in his tree stand to flushing upland game. Joe's love for the outdoors began with growing up in Western Montana and being lucky enough to have a wonderful father who introduced him to the great outdoors. Joe continues to pass along that same love to his kids on the great plains of the Dakota's. Joe is eager to share his passion and work ethic with his clients in the great state of North Dakota.
Contact us for more information on this property
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You wake up in the morning to the sun rising across Willow Lake and to the sound of the ducks and geese on there way south and to the bugle of the elk off in the distance, and you realize this isnt a dream it is reality on this great Turtle Mountain property. This property is a dream that rarely comes true let alone on the open market in the turtle mountains and it can be yours! This property is one that I could write a book on, but let the pictures do the talking. You really need to see it....Read More
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$609800 242.00 Acres
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CRP & Hunting Paradise, 640 acres, Berthold, North Dakota, USA
Here is an amazing piece of property for sale south west of Berthold, ND. This land is all in CRP and the current contract expires in 2027. This is one of the most beautiful upland game and waterfowl hunting parcels in the area. The terrain is very rolling and there are many large bodies of water found thru out the parcel. There are pheasants, sharptail grouse and many ducks and geese. There are also numerous whitetail deer found on the property. There is a large reservior located a few mil....Read More
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$989000 640.00 Acres
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CRP & Farm Land - Great Hunting, 582 acres, Columbus, North Dakota, USA
Here is an opportunity to purchase 582 acres located in Burke county, ND along highway 40 between Columbus and Powers Lake. There are 5 parcels totaling 582 all continuous acres. The owners of the land currently have about 301 acres in CRP. There are two parcels under CRP contract on the farm. All contracts expire on Nov. 30, 2022. Here's the numbers: The 1st contract is $30.85/acre 250.8 acres, the 2nd contract $32.37/acre 50.3 acres and the remaining acreage is native cattle g....Read More
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$699900 582.00 Acres
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Bar & Restaurant - Lake Sakakawea & Missouri River, Pick City, North Dakota, USA
Great Location - Great Reputation! Well-established bar and restaurant in a prime recreation area in central ND. Located on the headwaters of Lake Sakakawea and the Missouri River. This is a rare opportunity to own a small-town business while taking advantage of the abundant tourist traffic throughout the area. This 2000 sq ft property offers seating for up to 100 people, including an outdoor deck plus room for expansion. Currently this establishment offers a full menu and full liquor licen....Read More


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© 2019 RSM US LLP. All rights reserved.
Our Commitment to Audit Quality and Professional Excellence
At RSM US LLP, it's all about our clients. That's why we have demanded audit excellence for more than 90 years.
As professionals who provide opinions as to the fairness of financial statements, we define our clients as the users of those financial statements. Our clients are the shareholders of a public company, the owners of a private company, and other stakeholders. For auditors, "serving clients" means providing services with objectivity, competence and due care for the benefit of the stakeholders who demand audit excellence.
Our commitment to audit quality and professional excellence provides information for our clients and others on matters directly related to audit quality, such as governance, leadership, independence, audit performance, and monitoring. It also includes summaries of certain actions we have undertaken and plan to take to improve our system of quality control.


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Self-proclaimed "Ambassador of Rap for the Capital," Washington, D.C
As early as 2006 Wale has had a string of hits in D.C.-Maryland-Virginia area. Much of his work is inspired by 80's go-go music. It is my understanding, this artists has produced about 10 mixtapes in his time. I came across the title of this track via a twitter page, looked it up and have been playing it every since. The following track also features Gucci Mane (I know he is everywhere). You can really feel the go-go era in this song. Wale has an undeniable flow that is metaphoric yet enticing. This beat makes you want to "beat your feet", even if you're from down south. It also makes all the "Pretty Girls" want to "clap clap, like this".
If you are looking for a new track to get you moving this week, I recommend this download. Click the player below to hear the track.
Wale's debut album "Attention Deficit" is set to hit stores November 10, 2009.
Enjoy this track or artists? Share your thoughts.
Have a track or artists you enjoy? Contact me.


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4-Week Trip Planning Timeline (as of 10/29/12)
Trip Duty Descriptions Overview Grid (as of 9/28/12)
TRIP DUTY DESCRIPTIONS: (Click link for detailed list of duties)
- Outdoor Program Coordinator (Adult) – pre-trip research & maps, guides Trip Leader in his job, makes reservations & deposits, requests payments as necessary, submits tour permits
- Patrol Advisor (Adult) – guides patrol through trip planning & preparation process during meetings & on-line meetings to ensure that everything gets done, supervises patrol during trip, oversees patrol clean up & follow up upon return from trip.
- Trip Leader (Scout &/or Adult) – Responsible for making Trip Flier & for overall trip planning – location, departure & return times, routes, activities, etc.
- Trip Patrol Leader – Assigns trip duties & coordinates/oversees the work of patrol members in carrying out their assigned trip responsibilities
- Trip Cook/Asst Cook – Plans the menu, makes shopping list, cooks while in camp.
- Trip Grubmaster – Buys & stores food until trip, prepares/pre-packages food for trip, buys ice for cooler.
- Trip Quartermaster -inventories patrol chuck box before & after trip, checks out needed equipment for patrol & its members, ensures all equipment is returned clean, dry, and in good working order
- Trip Firemaster – plans trip campfire program prior to trip, oversees gathering of wood in camp, sets fire lay, lights & puts out camp fires, enforces fire safety rules
- Trip Waterman – make Patrol Gear list for Trip Plan, (re-) fills water jugs in camp &/or sets up water purification station during hiking/backpacking trips
- Trip Cleanup Captain -makes Personal Gear list for Trip Plan, sets up dish washing station in camp, washes patrol cooking dishes, responsible for trash & recycling, follows Leave No Trace guidelines
- Trip Skill Instructor – responsible for teaching assigned rank &/or merit badge skills during trip, prepares teaching plan & brings needed materials, signs off when scout has completed/mastered requirements taught.


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From the trailhead in Lions Bay, the trail plunges into your typical North Shore forest and climbs steadily along an old logging road. The trail is marked with flagging tape and with stone arrows on the ground. Keep right. The trail passes a waterfall on your left and offers some peek-a-boo views of Howe Sound on your right. The logging road narrows to a trail and after about an hour and a half to two hours you should reach the turn off to Harvey Creek. It is well signed. The trail descends steeply to the bridge crossing. (The old Harvey Creek crossing is closed now and this new bridge crossing replaces it) There are some nice views of Howe Sound from the bridge.
The trail gets trickier now. For the next two hours or so you switch back through thick forest, scrambling over fallen logs and clambering up the trail, which consists mainly of rocks and roots in many places. This is the steepest part of the trail except for the climb up to the summit of course. Finally, the trail deposits you out onto the Howe Sound Crest where you are rewarded for your efforts with views of the clear-cut slopes of Mt. Harvey to your left and in front, and the West Lion to your right.
Continue along the well marked trail up towards the West Lion. The grade eases but its still not easy going as you hop from rock to rock along the heather covered slope. In half an hour or so you will reach the Howe Sound Crest. Most people stop here. It's a great place for lunch with views of Capilano Lake, the city, and many of the North Shore Mountains.
If you choose to continue to the summit, descend into the gap between the Lions using the fixed rope. The worst part of the ascent is the first part where you must traverse a dangerously exposed section. It's not terribly difficult except for the fact that falling is NOT an option. Keep right and look for the orange markers. The ?trail? is well marked with good handholds almost all the way up. That being said, this is NOT for inexperienced climbers. The climb is dangerously exposed in MANY places. Go slow and test your holds, many of which are roots and branches. Do not attempt the summit of the West Lion without proper footwear.
The views from the summit are phenomenal. We could see Black Tusk, Garibaldi, Cathedral, Coliseum, Needles, Crown and many more of the North Shore peaks. It took us 4.5 hours from the trailhead to the summit. Helicopters regularly circled the peak bringing tourists in close to watch the climbers. This was kind of fun the first time but after 4 times it was getting distracting and annoying.
I expected the climb down from the summit to be worse than the climb up but was pleasantly surprised to find that it wasn't as bad as I expected. It was much more enjoyable than the rest of the descent. My knees took a hell of a beating on the long way down. The trail through the trees and then down the logging road seemed to go on forever. We made it back to the car at 5:30. We'd spent an hour on the summit with two more short breaks on the way up and down so all told we hiked for about 7.5 hours.
This is not a hike for the inexperienced or unfit but is a rewarding challenge for experienced, fit, prepared hikers.
One word of warning, do not leave anything lying around. It's a sad reality that people are stinkers. Last week a friend of mine had his trekking poles swiped after he left them at the base while he climbed to the summit. I had someone pick up my camera when I had taken off my pack and was resting on the way down.
Take Hwy 1 to Lions Bay. Take Oceanview Road, and go left on Cross Creek Road, and then right on Centre Road. Go left on Bayview Road, left on Mountain Drive and left on Sunset Drive. There is parking at the trailhead but unless you get there early you'll have to park 1 km down the road at the school on Mountain Drive.


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Army bomb disposal experts who regularly put their lives at risk to foil dissident republican attacks should be awarded a General Service Medal.
That is the call from the Ulster Unionist Party's Doug Beattie, a former Royal Irish Regiment Captain whose bravery in driving the Taleban out of the Afghan town of Gashmir earned him the Military Cross in 2007.
The war hero turned politician spoke out in praise of the army bomb disposal team based at Aldergrove after an attack on a police officer in Londonderry on Wednesday.
Mr Beattie said the army's Ammunition Technical Officers (ATO) kept Northern Ireland safe from "dissident republican psychopaths".
His party colleague, Tom Elliott MP, has written to Secretary of State for Defence, Michael Fallon MP, to call for the bomb disposal squad to be awarded the General Service Medal.
Mr Beattie described the attack in Londonderry on Wednesday as "vile and indiscriminate".
He continued: "It is worth noting that when such incidents occur, it is 321 Explosive Ordnance Disposal (EOD) Squadron that deploys to deal with them. Based out of Aldergrove they attend incidents like this approximately once a week. Each incident they are called to attend potentially involves deadly devices that could kill or seriously maim.
"It is a high risk role and a specialised skill that helps keep the people of Northern Ireland safe from these dissident republican psychopaths.
"However although they are on this high risk task, as part of Op HELVETIC, they do not receive the medal recognition their predecessors received as part of Op BANNER. The General Service Medal (GSM) is not routinely awarded to those from the military who undertake Military Aid to Civil Power known as MACP."
He added: "When your time in the military is served and civilian life becomes your normality, it is the medals you were awarded that reflect the hardship and sacrifice you have suffered during your time in the military. They are something to cherish and pass onto your family in later years."
Mr Beattie added: "We owe these men and women a great debt of gratitude"
An Army spokesperson said: "Our explosives ordnance disposal personnel provide vital support, often in testing circumstances, and operate at the very highest levels of professionalism in order to keep the public safe.
"As with all of our personnel, we carefully consider every request to recognise their extraordinary accomplishments."


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Yesterday was crazy.
I went to the house to pick up a few things. It's hard being there. I look around and see the life I will no longer have. Then I see him and I miss him. I mean, I miss the us. The good times when he would want to be fun and would allow it. I let my mood be dictated on how his day was going or how he treated me. I do miss the times when we would laugh. I use to do that a lot more.
Work went quick because I had to leave at noon to pick up my niece from the airport and then I had a dentist appointment.
I got back an hour before we closed and did all the close out invoicing for the day.
Sez and I went to dinner and to the casino. It was so much fun. We both walked out winners. I can't remember the last time I was even in that casino!
I got a great text during dinner letting me know that things had gone well, but not as well as expected. This is going to sound fucked up, but I am so glad it didn't go TOO well.
This gives me an opportunity to put my foot and the door and show what I can do.
I got a phone call later that night from my old friend and I laughed for what felt like an hour straight. It's been like that a lot with me. People have been coming into my life and I can feel that part of me coming back. That very social, sweet, caring and FUNNY person.
We talked up until 11pm about stupid things and people we use to know. For the most part, I thought my memory was pretty good, but it's not until you're talking to people you knew when you were a kid that you start remembering the small details.
He mentioned a friend that I forgot we even had and I was shocked that my memory could completely erase someone I had known for that long, completely out. I was reminded of the long summers on the dock of Woodridge/Truetts lake.
We promised each other that we would go back to Shingletown and visit the old haunts. How strange of a trip will that be? He wants to see his father's memorial bridge. That one will be hard for both of us. I can't remember crying so hard for another person's pain. That funeral was the hardest one to go to. A police officer. I saw him there after years of radio silence and he hugged me with such intensity. I wouldn't have guessed I would see him almost a year later when his 23 year old sister died. Things like that shouldn't happen to a kid.
I'm glad we talk again though.
One Comment
That bridge. That funeral. That dock. Those times. Those memories.
Your transitions in life right now. Your discoveries. Your triumphs of self.
It's almost like two distinct individuals are wandering around with so much the same life.
Here's to you, Chica. And here's to me. And here's to the next basket of great things life brings for each of us. <3


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While the EU tends to allow a long lead-in period for some of its legislation, this is not always a total success, as the General Data Protection Regulation (GDPR) has proved.
Sometimes businesses only start to take notice when the implementation period has almost run out.
This could be the case with amendments to the Industrial Emissions Directive (IED), according to a leading water and waste water management company which has warned that thousands of British companies face potential fines and disciplinary action, including the closing of facilities, due to the far-reaching effects of new tightened rules under the IED.
These are set out in "best available techniques" (BATs) or BREF (BAT reference) document requirements, set to come into force later this year.
Declan Maguire, operations director at Alpheus Environmental, has suggested that new obligations are only now becoming apparent as sectoral guidelines come into place.
"Companies that were previously IED compliant will suddenly become non-compliant as they fail to achieve the new standards. If companies are not proactively establishing baseline reports of emissions and addressing deficiencies it will lead to penalties and ultimately facility closures, and no business can sustain this."
Under the IED, companies are already obliged to reduce harmful industrial emissions, including emissions of waste water and generation of waste, and these rules will become more stringent once the new BREFs are in force.
It is expected that guidance documents currently under preparation by the European Commission will include increased responsibilities in the design, construction, and operation of industrial facilities, specifically including water treatment.
According to Mr Maguire, companies in water-intensive industries such as food and beverages, electronics, leisure, alcohol, manufacturing and pharmaceuticals will be most affected by the new responsibilities.
And despite Brexit, he concluded, industry experts believe these rules will still be maintained in UK law.
See app.croneri.co.uk for in-depth guidance on the IED.


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New York Daily News Vol.93 -Number 333
Wednesday May 23, 2012
Marvel's big announcement yesterday on The View that Northstar and his boyfriend Kyle were to be married in a nigh-unprecedented same-sex interracial wedding was picked up by various international news periodicals, the largest of which in New York City is the venerable New York Daily News, which has been continually published since 1919. It's actually the fourth-largest newspaper by circulation in New York City, but none of the other large New York City papers picked up on the news, including USA Today, The New York Times, Wall Street Journal, The New York Post, nor Newsday. The New York Daily News gave nearly a full page of coverage, the most extensive of any of the national or international papers covering this event. Guardian (Mac), Sasquatch, Snowbird, Shaman, Aurora, Puck and Marrina appear in preview art to the cover of Astonishing X-Men #51, and of course Northstar and Kyle appear as well, including images from Astonishing X-Men #50, released on the same day as this issue.
The article, titled "Gay wedding for 'X-Men'" is nearly identical to the on-line article published the day before, with a few minor wording changes. The amazing wrap-around cover to Astonishing X-Men #51 by Dustin Weaver and colored by Rachelle Rosenberg is the main illustration modified with an overlaid caption that unfortunately obscures part of Snowbird but otherwise reproduced well in newsprint. Other images in the article are a modified version of the soon-to-be famous proposal splash page by Mike Perkins which has the speech bubble thankfully enlarged, and in the lower left corner, a great image of Northstar snipped from the cover art of Astonshing X-Men #50.
It should be noted that both Reuters and AP picked up the story, so in addition to Ethan Sacks' story above, dozens and dozens of other newspapers (in more than one language!) published an article, some with an accompanying image, some in color! Here is an abridged list of major newspapers from around the world who I have confirm to have joined The New York Daily News in correctly identifying the newsworthiness of Northstar's wedding (all in English except where noted) by publishing the story in their print version:
Cape Breton Post, Canada
Cebu Daily, Philippines
Daily Telegraph, UK
Edmonton Journal, Canada
El Universal, Mexico (Spanish)
Hindustan Times (Delhi), India
Hindustan Times (Kolkata), India
Hindustan Times (Mumbai), India
Il Giornale, Italy (Italian)
Journal Pioneer, Canada
Kingston Whig-Standard, Canada
Los Angeles Times
Nanaimo Daily News, Canada
National Post, Canada
Northern Territory News, Australia
Pittsburgh Post-Gazette
San Jose Mercury News
South China Morning Post, Hong Kong
The Amherst Daily News, Canada
The Atlanta Journal-Constitution
The Dominion Post, New Zealand
The News, New Glasgow, Canada
The Press, New Zealand
The Southland Times, New Zealand
The Sun Times, Canada
The Timaru Herald, New Zealand
The Trentonian
Times Colonist, Canada
Toronto Star, Canada
Toronto Sun, Canada
Truro Daily News, Canada
Washington Times Daily
Winnipeg Free Press, Canada
… and more!
Nearly all of these contained at least one image, so globally the number of people who saw this Alpha Flight appearance today was likely in the tens of millions. The circulation for the New York Daily News alone is around 700,000 and is easily eclipsed by the Los Angeles Times (800,000), Daily Telegraph (900,000) and Hindustan Times (1MM+), so I'm fairly certain of this estimation.
Note: Yes, that's Israeli supermodel Bar Refaeli nude on the cover of the New York Daily News today. Sigh. Something about this blog just can't keep the sexy away…


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In an exclusive interview with CNBC's Becky Quick last night(Wednesday) in Omaha, Warren Buffett says the current market chaos and turmoil will probably create buying opportunities for him and Berkshire Hathaway:
"You get more excited when there's a lot going on, you can't help it. And frankly, it will probably present more opportunity to us because when dislocations occur things get more mispriced and that sort of thing...
"So it can be a time of opportunity. It won't be for sure, but generally speaking, when there's a certain amount of chaos in certain sections, the fallout, and its unpredictable where the fallout will be, but the fallout sometimes offers some real opportunites."
Video clips of Becky's entire interview with Warren Buffett have been posted on Warren Buffett Watch.