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Fragile X syndrome (FXS), an inherited disorder characterized by mental retardation and autism-like behaviors, is caused by the failure to transcribe the gene for fragile X mental retardation protein (FMRP), a translational regulator and transporter of select mRNAs. FXS model mice (Fmr1 KO mice) exhibit impaired neuropeptide release. Release of biogenic amines does not differ between wild-type (WT) and Fmr1 KO mice. Rab3A, an mRNA cargo of FMRP involved in the recruitment of vesicles, is decreased by ~50% in synaptoneurosomes of Fmr1 KO mice; however, the number of dense-core vesicles (DCVs) does not differ between WT and Fmr1 KO mice. Therefore, deficits associated with FXS may reflect this aberrant vesicle release, specifically involving docking and fusion of peptidergic DCVs, and may lead to defective maturation and maintenance of synaptic connections.
Fragile X syndrome (FXS) is the most common cause of inherited mental retardation, with an incidence of 1 in 4000 males and 1 in 8000 females (1). The molecular mechanism underlying FXS involves the expansion of an unstable polymorphic CGG triplet repeat to >200 in the 5′ untranslated region of the Fmr1 gene (2,3). This results in hypermethylation of the Fmr1 promoter and silencing of the Fmr1 gene, with consequent absence of the protein product, fragile X mental retardation protein (FMRP) (3). FMRP is an mRNA-binding protein that is enriched at the synapse and known to regulate the transport and localized translation of mRNA in response to mGluR receptor activation (4). Many of the mRNA cargoes associated with FMRP encode proteins crucial for spine maturation and synaptic plasticity (5−8).
In the absence of FMRP, there is defective regulation of localized mRNA translation. This absence affects synaptic plasticity in FXS, with abnormalities in long-term potentiation (LTP) and long-term depression (LTD) (9,10) in Fmr1 knockout (KO) mice, which exhibit characteristics of FXS (11,12). The absence of FMRP should lead to dysregulated local protein levels in both axons and dendrites, but previous reports have focused largely on translational regulation deficits at the postsynaptic site in FXS. Studies by Hanson and Madison (13) and Lauterborn et al. (14) recently suggested possible presynaptic effects caused by the loss of FMRP, prompting us to examine neuropeptide release in FXS. The mRNA cargoes of FMRP include presynaptic proteins that participate in the secretory pathway, in particular, vesicle exocytosis (5,6). One such protein is Rab3A, a GTPase that cycles between a soluble Rab3A-GDP form and a vesicle membrane-bound Rab3A-GTP form and is involved in activity-dependent vesicle docking and fusion at the synapse (15,16). Changes in Rab3A levels would be expected to affect activity-dependent release of transmitters and modulators. Using Western blot analyses, we characterized the levels of this protein in wild-type (WT) and Fmr1 KO mice.
Next, we used matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to examine synaptoneurosomal preparations and probed the physiology of stimulus-evoked neuropeptide release in Fmr1 KO mice using live brain slices. Our results indicate that these mice are markedly deficient in neuropeptide release. In order to determine whether the neuropeptide release deficit in Fmr1 KO mice is a general deficit in dense-core vesicle (DCV) release, we used electrochemistry to examine the release of biogenic amines. We show that the release deficit is specific to peptides, because there is no significant difference in the release of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) from the striatum of WT and Fmr1 KO mice. Lastly, using electron microscopy to quantify the number of peptide-housing DCVs, we do not observe significant differences between WT and Fmr1 KO mice, again suggesting a specific release deficit in FXS.
We characterized the amount of Rab3A at the synapse using synaptoneurosomal preparations, which are enriched in intact pinched-off synaptic processes, from postnatal 10−14 day (P10−14) WT and Fmr1 KO mice. Using Western blot analysis, we found Rab3A expression to be reduced by ~50% in isolated cortical synapses of Fmr1 KO mice (n = 8) compared with that in WT mice (n = 8; p < 0.01) (Figure (Figure1a,b).1a,b). The amount of Rab3A in total cortical homogenates was also decreased in the Fmr1 KO mice, however, to a smaller extent, because total homogenates contain more somatic material (Figure (Figure1c,1c, n = 4, WT; n = 4, Fmr1 KO; p < 0.01).
The decrease in Rab3A may be accompanied by a decrease in Rab3 interacting proteins. Liao et al. (17) recently reported reduced protein levels of other Rab isoforms in Fmr1 KO mice, including several proteins involved in vesicle exocytosis. Rab3A KO mice have previously been shown to have altered activity-dependent vesicle release (15) and complete loss of LTP at the hippocampal CA3 mossy fiber synapses (18). Although the mechanism behind Rab3A function in FXS is currently unknown, we speculate that there may be an imbalance in Rab3A-GDP and Rab3A-GTP cycling. It has previously been shown that an accumulation of membrane-bound Rab3A-GTP leads to a reduction in docked DCVs (19). Van Weering et al. (20) have observed that neither Rab3A-GTP- nor Rab3A-GDP-locked mutant chromaffin cells exhibited any differences in the total amount of DCVs as compared with WT chromaffin cells.
To directly investigate whether there is a peptide release deficit associated with reduced Rab3A, we developed a MS-based protocol to examine stimulation-dependent release of peptides from WT and Fmr1 KO mouse cortical synaptoneurosomes. Previously reported methods primarily used electrophysiology to observe the effects of stimulation at Fmr1 KO mouse synapses (21). Synaptosomes have been used for many studies on neurotransmitter release (22). Here, we adapted small-volume (23,24) and neuropeptide release collection approaches (25−28) to enable MS-based peptide characterization of released peptides in Fmr1 KO mice. Briefly, synaptoneurosomal preparations from WT and Fmr1 KO mice (n = 4, WT; n = 5, Fmr1 KO) were stimulated with KCl (55 mM), and the releasates were concentrated and separated into fractions using reversed-phase liquid chromatography (LC). The fractions were analyzed using MALDI-MS. The resulting mass profiles of releasates from WT cortical synaptoneurosomes revealed stimulation-dependent release of several peptide signaling molecules as well as protein fragments (Figure (Figure2a).2a). Little or no stimulated peptide release was observed in cortical synaptoneurosomes from Fmr1 KO mice; the reduction in peptide release is at least 90% and perhaps greater (Figure (Figure2c),2c), with the large decrease making quantitative measures of decrease difficult. To consider whether the KCl stimulation data reflect presynaptic or postsynaptic peptide release events, (S)-3,5-dihydroxyphenylglycine (DHPG) was administered to stimulate postsynaptic group I metabotropic glutamate receptors in the WT synaptoneurosomes; this stimulation did not elicit synaptic peptide release. Thus, we suggest, but have not yet fully confirmed, that this deficit is presynaptic in nature. Several peptides and protein fragments observed in the releasates of stimulated WT synaptoneurosomes were present in sufficient amounts to confirm their identity with tandem MS (MS/MS) analysis. These peptides are primarily derived from structural and related proteins (such as actin, tubulin, and stathmin) and neuropeptide prohormones (Supporting Information, Table S1).
To confirm changes in the stimulation-induced release in a more intact preparation, we used brain slices obtained from P10−15 WT and Fmr1 KO mice, as described earlier (25). We chose two regions to study: (1) the cerebral cortex, because this was the source of our synaptoneurosomes, and (2) the suprachiasmatic nucleus (SCN), a well-defined nucleus that is highly peptidergic and for which we previously characterized robust peptide release (25). Using an on-tissue collection strategy, we collected releasates from the cortex and SCN of WT and Fmr1 KO mice using a micropipet tip packed with C18 solid-phase extraction beads. We then performed MALDI-MS analysis on releasates obtained before and after stimulation with 55 mM KCl. The MS profiles showed a significant increase in the number and intensity of peaks detected in releasates after stimulation of WT, but not KO slices, compared with the prestimulated collections (Figure (Figure3,3, n = 4, WT; n = 4, Fmr1 KO). In addition to arginine vasopressin, somatostatin, and proSAAS, which have been shown earlier to be released from the SCN (25), we identified several peptides from the cortex that were also identified from the synaptoneurosomal releasates.
Although we have shown that the impaired release mechanisms involve peptides, it is reasonable to speculate that other regulated release pathways might be affected, such as cytokine and growth factor release. For example, brain-derived neurotrophic factor (BDNF) release from CA3 neurons was shown to recruit a presynaptic component of plasticity (29). Lauterborn et al. (14) recently showed that the LTP deficiency observed in the hippocampal field CA1 of Fmr1 KO mice can be rescued by exogenous application of BDNF, even though there was no significant difference in the basal level of BDNF in homogenates of Fmr1 KO versus WT hippocampus; therefore, those results also imply a deficit in BDNF synaptic release in FXS.
To analyze the specificity of the observed release deficit, we used electrochemistry-based measurements to analyze the stimulus-evoked release of biogenic amines. Brain slices from P70 WT and Fmr1 KO mice containing the caudate putamen (CP), ventral bed nucleus of the stria terminalis (vBNST), or substantia nigra pars reticulata (SNr) were used to analyze the electrically stimulated release of DA, NE, and 5-HT, respectively, because these particular regions are enriched in these biogenic amines (30−32). DA release in striatal brain slices was evoked by a single-pulse electrical stimulation (Figure (Figure4a).4a). Stimulated release of 5-HT (20 pulses at 20 Hz) in slices of the SNr had a similar profile; however, the maximal release values and rate of uptake are both lower than those seen for DA (Figure (Figure4b).4b). NE release was evoked by 60 pulses at 60 Hz stimulation and demonstrated the slowest uptake of all three neurotransmitters (Figure (Figure4c).4c). Electrically stimulated DA release monitored by fast-scan cyclic voltammetry was unchanged in Fmr1 KO mice compared with WT mice (n = 6, WT; n = 7, Fmr1 KO; p > 0.05). Additionally, NE release in the vBNST stimulation was not different (n = 4, WT; n = 6, Fmr1 KO; p > 0.05) between Fmr1 KO and WT control animals. This trend was repeated in the SNr where stimulation evoked the same amount of 5-HT in both animals (n = 6, WT; n = 7, Fmr1 KO; p > 0.05). Similar results were found when monitoring exocytosis from chromaffin cells (Supporting Information, Figure S1 and Table S2). The lack of differences in release of biogenic amines between WT and Fmr1 KO mice shows that the presynaptic release deficit is not general but is transmitter specific.
It is known that both clear-core vesicles, which house many classical neurotransmitters, and large DCVs, which house peptides and perhaps the catecholamines, are mobilized and released in an activity-dependent manner (33,34). Because DCVs contain neuropeptides, we considered whether the decrease in the amount of peptides observed in Fmr1 KO mice was due to a decrease in the number of DCVs. We examined electron micrographs containing layer IV of the visual cortex from P15 and P25 WT and Fmr1 KO mice, utilizing unbiased stereological methods to look for differences in the density of DCVs (DCVs/μm3). While a decrease in DCVs/μm3 between WT and Fmr1 KO mice at both P15 and P25 was observed (Supporting Information, Figure S2), these differences did not reach significance. A significant decrease in DCVs/μm3 from P15 to P25 was observed for both genotypes. Specifically, our analysis revealed a main effect of age (p < 0.01) but no main effect of genotype (p = 0.220) and no genotype−age interaction (p = 0.996).
MS-based assays that are capable of analyzing release enable characterization of peptide secretion following chemical stimulation, without having to preselect the peptides (25,26,35). The ability to collect and characterize peptide release allows us to present the first report of impaired peptidergic release in the FXS mouse model. Although there is not a significant reduction of DCVs in FXS, there is a ~50% reduction in synaptic levels of Rab3A. These results suggest that some of the deficits in Fmr1 KO mice may be directly correlated with deficits in the peptide release machinery. Intriguingly, the release deficit observed in the Fmr1 KO mice involves the release of neuropeptides in the neocortex and does not involve biogenic amines in the striatum, suggesting a deficit in the release of peptidergic DCVs, at least in several brain regions of FXS mice. Because similar reductions in peptide release are observed in both functional synapses (synaptoneurosomes) and brain slices from the cerebral cortex and the SCN, this impairment appears to be fairly general and may be associated with synaptic machinery, perhaps aberrant DCV docking and fusion, in which Rab3A normally participates. Stimulated release of DA, NE, and 5-HT is unaffected in the striatum, and basal synaptic transmission in Fmr1 KO mice is similar to WT mice (36), results that point to a specific release defect in the brain.
In conclusion, there appears to be a role for cell-to-cell signaling in the etiology of FXS, possibly affecting one or more phases of the synaptogenesis and synaptic stabilization sequence (37,38). Peptides characterized as released from the synapses in an activity-dependent manner include fragments of structural proteins such as actin, tubulin, and stathmin, which are involved in exocytosis. Their dynamics play a role in the morphological plasticity of dendritic spines (39), and release of these peptides is reduced in Fmr1 KO mice. The resilience of this defect, as observed in synaptoneurosomes and slice preparations from several brain regions, suggests that this impaired peptide-specific release may be a fundamental biological deficit and may contribute to defective synaptic maturation and maintenance and the behavioral deficits known to be associated with FXS. The correlation between FXS and the neuropeptide release deficit suggests that this is a unique pharmacological target, which, if restored, may ameliorate some of the effects of FXS.
WT and sighted Fmr1 KO mice bred in the FVB.129P2-Fmr1tm1Cgr background were housed at the Beckman Institute at the University of Illinois at Urbana−Champaign (UIUC) or the University of North Carolina at Chapel Hill (UNC−CH) animal facility. Mice were maintained under controlled environmental conditions, with standard alternating periods of 12-h light and darkness. Rodent chow and water were provided ad libitum. Animal care and procedures in accordance with the National Institutes of Health regulations and protocols were approved by the UIUC and UNC−CH Institutional Animal Care and Use Committees.
Synaptoneurosomes were prepared from cortices of P10−14 WT and Fmr1 KO mice. For the MS measurements of synaptoneurosome release only, six experiments were performed using rat cortices to validate our ability to stimulate peptides and collect releasates (n = 6). Once the ability to collect peptide release was shown to be robust, we modified the procedures for smaller mouse samples. Briefly, mice were sacrificed by decapitation. Brains were removed and dissected, and several cerebral cortices were pooled to obtain adequate cellular material for each experiment (n = 4, WT; n = 5, Fmr1 KO with each sample consisting of two or three cortices). Cortices were homogenized in a glass−Teflon homogenizer in 1 mL of ice-cold homogenizing buffer (50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.5, 125 mM NaCl, 100 mM sucrose, 2 mM potassium acetate), filtered through a series of nylon mesh filters (149, 62, 30, and 10 μm; Small Parts, Inc., Miramar, FL) and finally filtered through a 10-μm polypropylene filter (Gelman Sciences, Ann Arbor, MI). Filters were washed at each step with the ice-cold homogenizing buffer. The final filtrate was spun briefly (4000 × g, 4 °C, 1 min) to remove cellular debris; final supernatant volume was ~1 mL. This synaptoneurosomal preparation was either used directly for stimulation and collection of releasates by MS or was lysed (for Western blot analysis) in a lysis buffer [final concentration 50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P-40, 20 nM okadaic acid, 20 μM sodium orthovanadate, phosphatase inhibitor mixture (Pierce, Rockford, IL), and protease inhibitor mixture (Roche, Indianapolis, IN)]. NP-40 soluble total cortical homogenate was prepared in the following way: cortices from a single mouse, P10−14, were homogenized in NP-40 lysis buffer [final concentration 50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P-40, 20 nM okadaic acid, 20 μM sodium orthovanadate, phosphatase inhibitor mixture (Pierce), and protease inhibitor mixture (Roche)] and spun (15000 × g, 4 °C, 15 min), and then the supernatant was collected.
For each synaptoneurosomal preparation and total cortical homogenate sample, 30 μg of lysed protein samples was run on 12% polyacrylamide gels, blotted to nitrocellulose membranes, and stained with rabbit polyclonal antibody specific for Rab3A (Abcam, Cambridge, MA). HRP-labeled secondary antirabbit antibody (Cell Signaling Technology, Inc., Danvers, MA) was detected by enhanced chemiluminescence (Pierce). To quantify and standardize protein levels, blots were stained for β-actin (Sigma Aldrich, St. Louis, MO) and with Amido Black (J. T. Baker, Phillipsburg, NJ) for total protein. Chemiluminescence was scanned in a FluorChem 8900 Imager (Alpha Innotech, San Leandro, CA) and relative optical densities were determined by using AlphaEaseFC software, version 4.0.1 (Alpha Innotech), normalized to β-actin level and total protein loaded.
The synaptoneurosomal preparations, obtained from two to three mouse cerebral cortices for each preparation, were divided into two equal aliquots used as a control (unstimulated) or as the stimulated sample. For the stimulated samples, 1 M KCl (55 mM final concentration) or 1 M DHPG (Tocris Cookson, Ellisville, MO; 5 mM final concentration), was added to one of the aliquots and incubated for 15 min. For the control samples, 50 μL of homogenizing buffer was added. After stimulation, the aliquots were centrifuged (4 min at 4000 × g at 4 °C), and the supernatant was collected. The peptides present in the supernatant were desalted and concentrated using a spin-column (Pierce) according to the manufacturer's protocols. These measurements were repeated for nine samples (from 21 animals). In two instances, after chemical stimulation, no peptides (including the normally ubiquitous β-thymosin) were detected via MS, indicating an issue with the preparation; in such cases, the data were excluded.
Brain slices were prepared from P10−P15 WT and Fmr1 KO mice. Briefly, mice were quickly decapitated, brains were removed, and a Vibratome (Vibratome 3000 Series; Ted Pella, Redding, CA) was used to obtain 400-μm sections. The sections were placed into a brain slice chamber (AutoMate Scientific, Inc., Berkeley, CA) equipped with a proportional temperature controller. Each slice was perfused with Earle's balanced salt solution (EBSS; without phenol red), supplemented with 24.6 mM glucose, 26.2 mM NaHCO3, and 2.5 mg/L gentamycin and saturated with 95% O2/5% CO2 at 37 °C, pH 7.4. Collection of releasates from each slice (n = 4, WT; n = 4, Fmr1 KO) was performed as described earlier (25) and analyzed by MALDI-MS.
The synaptoneurosomal releasates were separated using a capLC system (Micromass, U.K.) coupled to a robotic spotter (Proteineerfc, Bruker Daltonics, Billerica, MA). Briefly, the samples were initially loaded onto a 5-μL loop using a manual injector (VALCO Instruments Co. Inc., Houston, TX) and subsequently trapped onto a reversed-phase trap column (PepMap, C18, 5 μm, 100 Å, LC Packings, a Dionex Company, Sunnyvale, CA) using solvent C (90% water, 10% acetonitrile (ACN), 0.1% formic acid (FA), and 0.01% trifluoroacetic acid (TFA)) at a flow rate of 8 μL/min and washed for 5 min. Each sample was then eluted and separated on a reversed-phase column (LC Packings 300-μm i.d. × 15 cm, C18 PepMap100, 100 Å) using a gradient at 2 μL/min flow rate for 45 min. Gradients were generated using solvent A (95% water, 5% ACN, 0.1% TFA) and solvent B (95% ACN, 5% water, 0.1% FA, and 0.01% TFA). Gradients were changed linearly in three different steps; 5−30% solvent B in 15 min, 30−80% solvent B in 35 min, 80−5% solvent B in 40 min. The chromatographic profile was monitored using a UV detector at 210 nm. The capLC system was connected online to the Proteineerfc spotter to collect the fractions onto a MALDI target. Prior to spotting the fractions, the Proteineerfc was programmed to deposit 1 μL of α-cyano-4-hydroxy cinnamic acid MALDI matrix (10 mg/mL, 60% aqueous ACN, 0.1% TFA). For each sample, a total of 24 fractions were collected at 1 min intervals, corresponding to samples eluted from 18−42 min of the chromatographic run.
Mass spectrometric analysis of the samples was performed using a MALDI-TOF/TOF mass spectrometer (Ultraflex II, Bruker Daltonics). The instrument was externally calibrated using a mixture of standard peptides (angiotensin II, angiotensin I, substance P, bombesin, ACTH clip 1−17, ACTH clip 18−39, somatostatin 28, bradykinin fragment 1−7, renin substrate tetradecapeptide porcine) in the mass range 700−3200 Da (Bruker Daltonics). For each of the synaptoneurosomal releasate fractions, data was collected using 400 laser shots in 10 short steps by panning across the spot and combining the spectra. Following data collection, the mass spectra were smoothed and baseline corrected, and the peaks with high peak intensity and signal/noise ratio were tabulated. Releasates from the slice were directly analyzed using MALDI-MS.
MS/MS of selected molecular ion species was performed using the TOF/TOF feature of the Bruker Ultraflex II instrument. The MS/MS spectra were processed using FlexAnalysis 2.4 and Biotools 3.0 software packages (Bruker Daltonics). Partial de novo analysis was also performed on the MS/MS spectra using Peaks software (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) to assist in peptide identification. For the release of peptides from the SCN, previously sequenced peptides characterized in the SCN and supraoptic nucleus were used for peptide identification (25,40).
Eight P15 WT and ten P15 Fmr1 KO mice and eight P25 WT and ten P25 Fmr1 KO mice were used to analyze DCV quantity in layer IV of the visual cortex. Perfusion and tissue preparation procedures for transmission electron microscopy ultrastructural analyses of brain have been described in a previous report (41). Within layer IV of the visual cortex, a region of neuropil was selected based only on proximity to easily recognizable landmarks (capillaries, nuclei, etc.) and on the absence of folds in adjacent sections (aside from these two criteria, the region was selected randomly within layer IV). Micrographs from identical regions of consecutive (approximately 60-nm thick) serial sections were taken at a magnification of 8800× (final magnification 26,400×) by a Phillips CM-200 transmission electron microscope and analyzed by raters blind to the experimental group. The density of DCVs was estimated using the physical disector (42), in which disector pairs, composed of a "reference" section and a "look-up" section, are analyzed. For each disector pair, the fractionator (43) is used to establish one sampling site that is the area of the electron micrograph in layer IV of the visual cortex. Between 6 and 15 serial electron micrographs per tissue block were used for stereological analysis. DCVs were characterized by the presence of an electron-dense core and at least 50% of circumference surrounded by an intact identifiable double-membrane (44); clear-core vesicles were not quantified. The sections were analyzed for DCVs present in the reference section but not the look-up section. These DCVs were counted. DCVs present on both the reference section and look-up section were not counted. The formulas DCVv = Q/Vdis and Vdis = AframeTN were used to obtain DCV density, where DCVv is DCV density, Q is the number of DCVs present in the reference section but not the look-up section, Vdis is the disector volume of the tissue through which the DCVs are counted, Aframe is the area of the superimposed counting frame, T is section thickness, and N is the number of sections through which the counting is done.
The SPSS (SPSS Inc., Chicago, IL) statistical package (version 16.0) was used to conduct statistical analyses. Independent Student's t-tests were used to determine statistical significance. For DCV quantification, a two-way ANOVA was conducted to determine statistical significance for effects of age (P15, P25) and genotype (WT, Fmr1 KO) on 415 micrographs from 36 animals. For these analyses, p < 0.05 was considered statistically significant.
Chromaffin cells were prepared as previously described (45,46). Plates were maintained in a humidified, 5% CO2 atmosphere at 37 °C for at least 24 h prior to experimentation. Brain slices were prepared containing the CP (31), SNr (30), and BNST (32). Slices were superfused in artificial cerebrospinal fluid at 37 °C for 35−40 min prior to recording. Measurements were made with carbon-fiber microelectrodes prepared using T650 carbon fibers (47). At chromaffin cells, recordings were made by amperometry with disk electrodes (46). Measurements of DA, NE, and 5-HT release in brain slices were made using fast-scan cyclic voltammetry at cylindrical electrodes as previously described (30).
Exocytosis at chromaffin cells was triggered by 0.5 s pressure ejection of 60 mM K+ buffer from a pipet located 30 μm from the cell. Ten stimulations, separated by 30 s, were applied to each cell. In brain slices, release was electrically evoked (31). Amperometric spikes were analyzed with MiniAnalysis (Synaptosoft, Decatur, GA). Individual amperometric spike characteristics determined were the spike frequency, quantal size (Q), t1/2, and amplitude (48). In the slice experiments, the maximal neurotransmitter concentrations measured following stimulation are reported.
We thank Dr. G. Govindaiah for technical assistance with brain slice techniques. We also thank Dr. Deepa Venkitaramani and Shawn Kohler for assistance with statistical analysis of DCV quantification. We gratefully acknowledge Stephanie Baker for help with manuscript preparation.
National Institutes of Health, United States
S.P.A., S.H.K, J.V.S., B.M.K., K.T.T., R.M.W., W.T.G., and I.J.W. designed the research, S.P.A., A.E.L., S.H.K., S.R., B.M.K., K.T.T., and N.G.H. performed the research, S.P.A, B.M.K., K.T.T., and A.E.L. analyzed the data, and S.P.A., A.E.L., S.H.K., I.J.W., J.V.S., R.M.W., and W.T.G. wrote the paper.
The projected described was supported by the National Institute on Drug Abuse (NIDA) under Award Nos. P30DA018310 and DA017940, the National Institute of Neurological Disorders and Stroke (NINDS) under Award Nos. NS031609 and NS038879, and the National Institute of Mental Health (NIMH) under Award No. MH035321. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIDA, NINDS, NIMH, or the National Institutes of Health.
A list of the most prominent secreted peptides identified from WT synaptoneurosomes, vesicular release events at chromaffin cells from WT and Fmr1 KO mice, and representative EM images from visual cortex of WT and Fmr1 KO mice, together with quantification data. This material is available free of charge via the Internet at http://pubs.acs.org.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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7 Sep 2014, 14:46 by sangremuerto
Albums August 2014
2 Aug 2014, 13:24 by SuicideScrubhttp://userserve-ak.last.fm/serve/500/99153813/Future+Decay.jpg
Children of Technology - Future Decay
Dishammer - Under the Sign of the D-Beat Mark
Gama Bomb - Tales From the Grave in Space
Iron Maiden - The Number of the Beast
Machinae Supremacy - A View from the End of the World
Machinae Supremacy - Rise of a Digital Nation
Midnight - No Mercy For Mayhem
23 Feb 2014, 00:55 by nassuttaja2A list of all the shows that I have ever attended and all the artist that I have seen.
Ankkarock '09
AP-Fest Vol. 3
Aurinkotehtaan musiikkijuhlat '15
Flow '12
Hammer Open Air '10
Hammer Open Air '12
Hammer Open Air '13
Hellfest '12
Indie iltamat '12
Indie iltamat '13
Kuudes Aisti '13
Ruisrock '07
Ruisrock '08
Ruisrock '09
Ruisrock '10
Sonisphere '09
Turku Saatanalle II
Tuska '08
Tuska '09
Tuska '10
Tuska '11
Tuska '12
Tuska '13
Tuska '14
Unholy Alliance III
Åbo Massaker III
Epäkristus+Vomiting Skulls+Minoria @ Tillintupa, Paimio ?.?.2008
The Ocean+EF @ Klubi, Turku 27.05.2010
Rotten Sound+Trap Them+Gaza+Haust+The Kandidate @ Klubi, Turku 22.4.2011
Godflesh+Pedigree @ Tavastia, Helsinki 24.9.2011
Swallow the Sun+Oddland @ Klubi, Turku 3.2.2012
Sólstafir+Hexvessel @ Klubi, Turku 23.4.2012
Moonsorrow+Ghoul Patrol @ Virgin Oil, Helsinki 29.6.2012
Barathrum @ Darkside Club, Helsinki 30.6.2012
My Top 81 Albums of 2013
1 Jan 2014, 10:02 by wrathchild88Well hello there. Just to clarify what's going on here, I've listed all of the albums that I've listened to that were released this year in reverse order with some comments about the band and the album so that hopefully you'll have a good idea about what to expect from the album. Obviously I could write a lot more for each but I wouldn't be able to do that for every single album I've listen to this year, so I've put links up to youtube videos so that you can decide for yourself. Clearly there are some notable bands I've missed this year: Black Sabbath, Atlantean Kodex, Falkenbach, Revocation, Deicide, Motorhead, Hypocrisy, Satyricon, The Ruins of Beverast, Fleshgod Apocalypse to name but a few, but I think that I've got a good and wide selection of metal to recommend, or not as the case may be. There were plenty of really good albums this year and it was pretty hard to pick a favourite, but I managed it. If you just want a list of my top albums this year, scroll to the bottom. …
The Best, 2013
20 Dec 2013, 08:54 by asog666Airbourne - Black Dog Barking
Blood Tsunami - For Faen!
Carcass - Surgical Steel
Deeds of Flesh - Portals To Cannan
Deicide - In The Minds Of Evil
Entrails - Raging Death
Gama Bomb - The Terror Tapes
Hail of Bullets - III: The Rommel Chronicles
Hatriot - Heroes of Origin
Hell - Curse and Chapter
Immolation - Kingdom of Conspiracy
Inquisition - Obscure Verses for the Multiverse
Master - The Witchhunt
Morbosidad - Muerte de Cristo en Golgota
Onslaught - VI
Pestilence - Obsideo
Rotting Christ - Κατά τον δαίμονα εαυτού
Sodom - Epitome Of Torture
Suffocation - Pinnacle Of Bedlam
Temple of Baal - Verses Of Fire
Seen Live
28 Sep 2013, 14:24 by Unfurl_Bands and artists I've seen live - all of them, whether I like them or not :>
Agonija (3)
Aistė Smilgevičiūtė ir Skylė (2)
Ale va (2)
Alive Way (2)
Altar of Plagues
Andrius Kaniava
Angelus Apatrida
Anomaly (2)
Atalyja (3)
Atomine Elektrine
Aura Noir
Avenue Acoustic
Bad X Blend
Berserker (2)
Blood Axis (2)
Blue in Bossa
Body Cargo
Ceramic Hobs
Christian Galarreta
Crypts of Despair (3)
Cult of Fire
Darkened Nocturn Slaughtercult
Days of Eclipse (2)
Dead Congregation
Death in June
Deeper Upper (2)
Deströyer 666
Diktatūra (2)
Dirty Vegas
Downfall of Gaia
Driezhas (2)
Dust Bolt
The ridiculous list of gigs I've attended
18 Aug 2013, 14:42 by NecroZedThis doesn't include the classical and jazz concerts, for no reason other than that I forgot and can't be bothered to renumber everything. A * denotes a band I played in.
1 16/09/96 Jethro Tull -Sands Centre, Carlisle
2 14/02/97 Suede + Mansun + Raissa -Sands Centre, Carlisle
3 31/10/99 The Stranglers + Schindler - Sands Centre, Carlisle
4 28/05/00 The Australian Pink Floyd - Brewery Arts Centre, Kendal
5 15/07/00 Alice Cooper + 1 -City Hall, Newcastle
6 19/07/00 Unknown Canadian bands -A field, Port Colborne
7 08/09/00 Mel C + Tom Cat- Sands Centre, Carlisle
8 27/09/00 Space + Shed 7 + The Bluetones + Animalhouse - Millennium Dome,London
9 28/09/00 Bucks Fizz - The Venue, Canterbury
10 28/10/00 Some industrial band - Slimelight, London
11 09/01/01 Nearvana - The Venue, Canterbury
12 ??/01/01 Some UKC bands - Keynes JCR, Canterbury
13 18/01/01 The Eighties Experience - The Venue, Canterbury
14 20/02/01 Some death metal bands - Studio 41, Canterbury
Doomed to death
11 Aug 2013, 22:16 by uiterweerdSat 27 Jul – Stonehenge 2013
Of the 18 times this festival was organized previously, I have never been. From the moment I stepped onto the grounds, I knew this was going to be great. The atmosphere was incredibly relaxed. Due to the impending catastrophic weather, I went inside immediately and walked in on Funeral Whore. Solid death metal - a good start to the day. Obscenity were next, but they hardly made an impression on me I'm afraid. Outside, the recently reformed Dissect were playing and I managed to grab a few songs. They displayed a ferocious energy and I loved the enthousiasm of the lead singer. Many bands should take example. What happened afterwards is already a bit of a blur. Enthroned disappointed slightly, but this might be due to the fact that they were the only proper black metal band playing the festival. I think Exhumed were next, and I loved it, even though the live act is a bit silly, but at least they're setting themselves apart from all the other death/grind bands. …
Albums July 2013
8 Jul 2013, 13:01 by SuicideScrubBywar - Twelve Devil's Graveyards
D.R.I. - Thrash Zone
Dr. Living Dead - Dr. Living Dead
Dr. Living Dead - Radioactive Intervention
Fatal Embrace - Dark Pounding Steel
F.K.Ü. - 4: The Rise of the Mosh Mongers
F.K.Ü. - Where Moshers Dwell
Gama Bomb - The Terror Tapes
Havok - Time Is Up
Havok - Unnatural Selection
Motörhead - 1916
Motörhead - March ör Die
Ultra-Violence - Privilege To Overcome
Xentrix - For Whose Advantage?
Albums Owned...
2 Jun 2013, 05:56 by NLMenkeTotal: 613; CD: 499; CD-R: 30; tape: 77; vinyl: 4; DVD: 3
Me total: 366; CD: 329; CD-R: 28; tape: 2; vinyl: 4; DVD: 3
Parents total: 184; CD: 109; CD-R: n/a; tape: 75; vinyl: unknown
Sister total: 63; CD: 61; CD-R: 2; tape: n/a; vinyl: n/a
~Last updated: November 16, 2014; 12:15am~
*artists link to last.fm page - albums link to blog*
.38 Special
- 20th Century Masters: .38 Special (Me: CD)
- Special Forces (Parents: tape)
3 Inches of Blood
- Fire Up The Blades (Me: CD)
- Back In Black (Me: CD; Parents: tape)
- Dirty Deeds Done Dirty Cheap (Me: CD)
- Live (Sister: CD)
- The Razor's Edge (Sister: CD)
- Stiff Upper Lip (Me: CD)
- A Compilation Of The Best Of Balls To The Wall & Restless & Wild (Sister: CD)
- Balls To The Wall (Me: CD)
Adam Ant
- Friend Or Foe (Parents: tape)
- Aerosmith's Greatest Hits (Parents: CD)
- Big Ones (Me: CD)
- Just Push Play (Sister: CD)
- Toys In The Attic (Me: tape)

subset: commoncrawl


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Yuri Arcurs Launches PeopleImages.com
The world's top selling stock photographer Yuri Arcurs has had enough with low commissions and launches his own direct sales platform to pave the way for other creatives. http://www.PeopleImages.com
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Q: I am really into my wildlife photography but cannot afford a super zoom lens, I have a 300mm and am considering a teleconvertor. Has anybody had a experience with these and would you recommend them?
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The past two months has seen a ton of matches take place, some upsets caused and of course, big teams making their way to the top once again.
This year, the top 4 does look a bit different in both our titles and with the "Loser Bracket Semi Finals" kicking off tonight, we will see which of these teams can perform under pressure!
All eyes on Battlefield, as this title has once again broken our viewership barriers with our past records for viewership being beaten! Tonight will be no different where the highly anticipated match between 2 "underdog" teams will take place. ALG Vanguard and PuLse Elevate. Both of these teams have played some impressive matches. Both of these teams managed to beat a tournament favorite, PEW Battlefield. Both of these teams were seen as underdogs but have shown what incredible form they are in! Tonight will decide which of these two teams will make it through to the "Loser Bracket Final". Where they will face SvG iNFamous in the Loser bracket final. And of course, the winner of that match will then go on to face PuLse Evolve who have already made it to the Grand Final.
Let's take a closer look at these two teams competing tonight, look at the line ups, and have me add my personal opinion "Slang" to the pros and cons of each player and the role I believe each of these players will have in tonights match. Trust me, this will be a tight game so DON't miss this!
ALG Vanguard
Tte_Cadaver – More aggressive, famous for his "zou zou" and Ritalin fed rabbit like movements in game, making him really hard to shoot, very accurate shooter and plays right in front, Chances are he will be clashing with Fox and Elfie often in this game as they tend to play a similar lane in the game. EZ 30 bombs for Kyle EZ life EZ game
Tte_Nutt-sakk – Similar to Cadaver in aim, different in movement. This player has been the talk of the town lately for being a seriously good player with crazy reflexes. Also quite aggressive, Nuttsakk will join Cadaver in the middle most likely, being an aggressive player himself.
Tte_Giddyup – More defensive and laid back in playing style, I think the team relies on this player for the second line and even to hold spawn at times. Not very aggressive, but very hard to find if you are looking to wipe his squad.
Tte_RavenRM – This player flanks so much and plays so randomly with his movements that he can tilt a team really hard if they fail to pick him up early in the game. His flanks have proven to open up pushes and plants on previous streams. Pretty good shot and plays a calm and more sneaky role, can really cause some hurt.
Tte_Xectr – The captain, the rock, the more consistent player, Xectr will always be consistent in their matches and will lead his team by example. I think he has played a massive role in recent results and is heading the team in the right direction.
puLse-Joben – The beard that makes magical aim happen, some say that if you shave his beard he loses his ability to play…. The captain and the mind behind this team is Joben, someone more aggressive than most which seems to compliment this Pulse team and their aggressive play style.
puLse-eLf1e – Meme Machine, general nice guy, someone who plays the game so fast that sometimes he loads the next map while the first map is still being played. Elfie has been playing for a long time and has quite a bit of experience under his belt, a really good shot and a really good thinker in game. Elfiee will most likely meet with Cadaver and Nuttsakk in tonights match.
puLse-r0flc0pter – More calm, more defensive. Someone to balance out this team that runs on diesel and aggression. He is much needed to supply the team with a calmer mind and a backup plan and a safe area to play in. He usually plays a more supportive role and does it very well.
puLse-Fox – One of my favorite players at the moment, someone I think will be one of the big names in this game in the future. Raw talent and very young. This will be someone to keep your eye on tonight as this player is very accurate and very smart. He will be with Ellfie and will probably face off against Cadaver and Nuttsakk often. This engagement will be really interesting to see.
puLse-I-no-goud – This player is No Goud…..
Good luck to both these teams tonight! It will be streamed on our main channel. The match officially starts at 8:30


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Man Murdered in Broad Daylight in Venice
A man was gunned down in broad daylight in the Oakwood area of Venice today, authorities said.
The shooting at Brooks and Seventh avenues was reported about 4 p.m., Los Angeles Police Department Officer Tony Im said.
"Basically the suspect walked up to the victim and shot him," Im said.
The man, yet to be identified, was pronounced dead at the scene, Im said.
The shooter fled west on Brooks, he said. Earlier reports describing a suspect wearing a Star Wars mask turned out to be untrue, he said.
The shooter was described as a 5-foot, 8-inch to 5-foot, 9-inch African-American man in a black hooded sweatshirt, black jeans and black sunglasses, Im said. Witnesses said he appeared to be in his late 20s, according to the officer.
The area of million-dollar homes is a traditionally black community where two gangs, the African-American Venice Shoreline Crips and the Latino Venice 13 have long claimed turf.
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|Message view
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|Henry Story <henry.st...@bblfish.net>
|Re: [scala-user] Solution: taking context seriously was: A scala anti pattern -- looking for a name
|Sun, 24 Jul 2011 10:20:26 GMT
In a reply to Reto's post, which did not make it to the Clerezza list, but which is available here https://groups.google.com/d/msg/scala-user/IsJ1yXjd2lw/cdAsYsPc4-EJ Rex Kerr wrote > Is Clerezza intended to be used primarily on its console? If yes, then interactive usability is more important than maintainability of large blocks of code, since you don't typically have large blocks of code, but you have lots of interaction. Otherwise, you can take advantage of the fact that you have some control over the console, unlike raw Scala source code. You can have a modal console that wraps everything in a new context(x) { } line by line, or a context character like : that indicates that the following stuff is in the context of the current graph (which can be a signal for the wrapping). > > --Rex I agree with Rex there, there are other ways around the problem of the console. Henry On 22 Jul 2011, at 16:45, Reto Bachmann-Gmür wrote: > I think the issue boils down to a design recommendation for code using > the clerezza rdf library. > > Importing the members of an EzGraph as context makes perfectly sense > on the clerezza console. The "coding" there is imperative, one want to > see the results ideally after each line. Creating a block scoped to > graph is clearly much less user-friendly [1]. Also having the content > graph as default is very useful and shouldn't be sacrificed for > enforcing good design in another context. Opening and being in the > context you most often want to be is not only handy for rapid queries, > the shell is a triple sink where new triples can be added to the > clerezza instance simply by writing them down in a syntax similar to > the turtle or the n-triples rdf format. Not having to call a method on > some nodes on the graph makes a big difference in terms of usability. > In program code that as opposed to the commands on the shell is > deigned to last and which isn't executed while you type it is better > design practice to instantiate an anonymous subclass of EzGraph (which > could be renamed or aliased as context as in your examples) rather > than importing the members (methods and implicts) of an instance. > > Your anti-pattern example shows horrible code that you can write using > the library, you could very well write a similar example using the > java transaction api. The clerezza API however supports better coding > practices and the documentation shall focus on these, changing the api > to enforce these better practice would be at high costs for those > using the api interactively on the shell or in scripts. > > Reto > > > 1. the clerezza faq entry on how to reset the password shows an > example real-life usecase for switching graph-context on the console > > On Fri, Jul 22, 2011 at 10:12 AM, Henry Story <email@example.com> wrote: >> Great I think I have the solution now. >> This thread [1] has helped a lot to make clear the use of context, why >> imports that bring behaviour with them >> are confusing (they loose context) but also why it was tempting to use >> them. It turns out then that one has to take >> context seriously, and rewrite the library in order not to have to use the >> confusing implicit imports whilst making code >> as easy to read as if they were there (because they will be). Ie: we can >> use those imports but make them explicit, >> without ever writing them. If that sounds confusing, it turns out not to be >> in practice, as the following code shows. >> One can easily create two graph: >> val ez1 = new context { >> b_("reto") -- FOAF.name --> "Reto Bachman-Gmür".lang("rm") >> -- FOAF.homepage --> >> "http://bblfish.net/".uri >> }.graph >> val ez2 = new context { >> b_("hjs") -- FOAF.name --> "Henry Story" >> }.graph >> but one can then easily continue writing in each graph, in a layered way >> without confusion >> new context(ez1) { >> b_("reto") -- FOAF.interest --> "scala" >> } >> new context(ez2) { >> b_("hjs") -- FOAF.homepage --> "http://bblfish.net".uri >> } >> The context protects the blank nodes in the context, so they can't jump from >> one context to the other >> (well they can because the framework at the top is leaky - but that is >> something for another day), >> the subject of the context is very clear too, and one can even add methods >> to this context. >> The only thing that is odd from a technical perspective is that this will >> create a bunch of classes on >> each "new". >> So I must admit without the discussion I would not have walked down this >> path, which seems like a good one. >> Henry >> The commit for this is here: >> >> https://github.com/bblfish/clerezza/commit/6c95c934f15b96c753e3e8eb091482ccaf1b5e32 >> The files to look at are the new context class >> >> https://github.com/bblfish/clerezza/blob/6c95c934f15b96c753e3e8eb091482ccaf1b5e32/parent/rdf.scala.utils/src/main/scala/org/apache/clerezza/rdf/scala/utils/context.scala >> But especially the test class that has many examples of usage >> https://github.com/bblfish/clerezza/blob/6c95c934f15b96c753e3e8eb091482ccaf1b5e32/parent/rdf.scala.utils/src/test/scala/org/apache/clerezza/rdf/scala/utils/EzMGraphTest.scala >> [1] https://groups.google.com/d/topic/scala-user/IsJ1yXjd2lw/discussion >> On 21 Jul 2011, at 21:32, Henry Story wrote: >> >> On 21 Jul 2011, at 15:32, Rex Kerr wrote: >> >> But anyway, I am less concerned about the library than about standard use >> cases. Would anyone want to use different graphs? If yes, selecting them >> based on sort-of-hidden context is unwise. Henry's example made it look >> like using different graphs was a pretty reasonable thing to do. And note >> that a one-character variable is a lot more informative than a zero-variable >> character: >> >> graphContext(definitionsPreamble.currentGraph) { g => >> whatever -- g(edgelabel) --> soandso >> } >> >> Now you have, with a couple extra characters per call, documentation of what >> the relevant context is. >> >> Thanks Rex. This discussion has helped me also see how it could have seemed >> so >> attractive for Reto to want to use this import behaviour. >> We start from something very nice and simple we can all agree on (pretty >> much) >> val g = new EzMGraph { >> reto -- FOAF.knows --> ( bnode --FOAF.name --> "henry" ) >> -- FOAF.knows --> danny >> } >> This is both nice and readable, efficient and context bound. (And this was >> one of Reto's early ideas.) >> So bnode above is a method that returns a GraphNode (as defined in [1]) >> using the graph itself. Ie the above is equivalent >> to >> val g = new EzMGraph { >> node(reto) -- FOAF.knows --> ( this.bnode -- FOAF.name --> "henry" >> ) >> -- FOAF.knows --> danny >> } >> The good thing is that the result of ( this.bnode -- FOAF.name --> "henry" ) >> is a GraphNode with the same >> backing graph as EzMGraph, so there is no copying of information from one >> graph to another to do (which due >> to blank nodes in the semantic web, might end up being more complex than one >> may think) >> Now if one wanted to fill the graph with information from another context, >> one would want a notation >> more like >> val retoNd = g.node(reto) >> for (p <- people) { retoNode -- FOAF.knows --> ( p -- FOAF.knows --> me >> ) } >> ie for a list of people assert that Reto knows them and that they also know >> me . >> But here if p were say a URI for a person, then one would be creating in the >> code >> ( p -- FOAF.knows --> me ) >> an new GraphNode each time backed by a different empty graph, because of the >> implicit >> implicit def resource2graphNode(res: Resource) = new RichGraphNode(res, >> new SimpleMGraph()) >> This will feel wasteful, when compared to the import solution that Reto had >> proposed where all >> resources end up being transformed to GraphNodes with the same backing >> graph. >> With the code as it stands now in [1] one can still do that. Then one >> would have >> import g.node >> for (p <- people) { retoNode -- FOAF.knows --> ( p -- FOAF.knows --> me >> ) } >> So now each p will be transformed into a GraphNode using the same store >> as g. >> Of course, this is nothing more than one would have had one written >> for (p <- people) { retoNode -- FOAF.knows --> ( g.node(p) -- >> FOAF.knows --> me ) } >> It is more repetitive, and will be even more so the longer the code. But I >> still prefer it to the >> import above (for the moment). One feeds that something needs to be >> delimited more clearly. >> So perhaps the subclassing solution is the good one again for this >> new EzMGraph(g) { >> for (p <- people) { retoNode -- FOAF.knows --> ( p -- FOAF.knows >> --> me ) } >> } >> is not that much more verbose that then import version, but it does make >> it clear where the graph >> is delimited. With an type alias one could even make that look like this: >> new graph(g) { >> for (p <- people) { retoNode -- FOAF.knows --> ( p -- FOAF.knows >> --> me ) } >> } >> Is that perhaps good enough? >> Henry >> >> >> >> >> >> >> >> >> >> >> >> [1] https://github.com/bblfish/clerezza/blob/51be3b14a5488838d87094e81455ca806507b72b/parent/rdf.scala.utils/src/main/scala/org/apache/clerezza/rdf/scala/utils/EzMGraph.scala >> Social Web Architect >> http://bblfish.net/ >> >> Social Web Architect >> http://bblfish.net/ >> Social Web Architect http://bblfish.net/
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|By Marketwired .
Article Rating:
|September 15, 2017 08:59 PM EDT
VANCOUVER, BRITISH COLUMBIA -- (Marketwired) -- 09/15/17 -- Pure Gold Mining Inc. (TSX VENTURE: PGM) ("Pure Gold" or the "Company") is pleased to announce the results of voting at its Annual General Meeting of Shareholders ("AGM") held Friday, September 15, 2017 in Vancouver, Canada.
Shareholders voted in favour of all items put forward by the Board of Directors and management. All six of the individuals nominated for the board of directors were elected as set out below:
---------------------------------------------------------------------------- Votes For % Votes For Votes Withheld % Votes Withheld ---------------------------------------------------------------------------- Graeme Currie 69,431,916 99.99% 6,500 0.01% ---------------------------------------------------------------------------- Darin Labrenz 69,433,416 99.99% 5,000 0.01% ---------------------------------------------------------------------------- Mark O'Dea 69,438,416 100% 0 0.00% ---------------------------------------------------------------------------- Lenard Boggio 69,431,916 99.99% 6,500 0.01% ---------------------------------------------------------------------------- Rob Pease 65,754,916 94.69% 3,683,500 5.31% ---------------------------------------------------------------------------- Troy Fierro 69,438,416 100% 0 0.00% ----------------------------------------------------------------------------
Shareholders also voted in favour of (i) appointing PricewaterhouseCoopers LLP, Chartered Accountants, as auditors of the Company for the ensuing year and authorizing directors to fix their remuneration; and (ii) approving and ratifying the Company's Stock Option Plan. A total of 69,438,416 Pure Gold common shares were voted, representing 36.25% of total shares issued and outstanding as at the record date of the meeting.
Our mandate is pure and simple. To dream big. To colour outside the lines. To use smart science and creativity to unlock the next major discovery at the Madsen Gold Project in Red Lake, Ontario. And become Canada's next iconic gold company.
Additional information about the Company and its activities may be found on the Company's website at www.puregoldmining.ca and under the Company's profile at www.sedar.com.
Darin Labrenz, President & CEO
Neither TSX Venture Exchange nor its Regulation Services Provider (as that term is defined in the policies of the TSX Venture Exchange) accepts responsibility for the adequacy or accuracy of this release.
Investor inquiries:
[email protected]
SYS-CON Events announced today that SIGMA Corporation will exhibit at the Japan External Trade Organization (JETRO) Pavilion at SYS-CON's 21st International Cloud Expo®, which will take place on Oct 31 – Nov 2, 2017, at the Santa Clara Convention Center in Santa Clara, CA. uLaser flow inspection device from the Japanese top share to Global Standard! Then, make the best use of data to flip to next page. For more information, visit http://www.sigma-k.co.jp/en/.
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Most of the time there is a lot of work involved to move to the cloud, and most of that isn't really related to AWS or Azure or Google Cloud. Before we talk about public cloud vendors and DevOps tools, there are usually several technical and non-technical challenges that are connected to it and that every company needs to solve to move to the cloud. In his session at 21st Cloud Expo, Stefano Bellasio, CEO and founder of Cloud Academy Inc., will discuss what the tools, disciplines, and cultural...
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With the rise of DevOps, containers are at the brink of becoming a pervasive technology in Enterprise IT to accelerate application delivery for the business. When it comes to adopting containers in the enterprise, security is the highest adoption barrier. Is your organization ready to address the security risks with containers for your DevOps environment? In his session at @DevOpsSummit at 21st Cloud Expo, Chris Van Tuin, Chief Technologist, NA West at Red Hat, will discuss: The top security r...
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Sep. 20, 2017 02:30 PM EDT Reads: 450


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HPI Racing Roto Start Back Plate Nitro Star K4.6, Sure Fire .32, Axial .28 and .32
HPI Racing
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This is the HPI Roto Start System Shaft Drive Starter back plate from HPI Racing. This back plate works with HPI's K series of engines, such as the K4.6 found on the Savage SS .28 monster truck. This will also work with the new Racer's Edge Sure Fire .32 monster truck engine so you can drop it into your Savage and start it with a roto start instead of a pull start.
This product was added to our catalog on October 27, 2005.


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This year's National Conifer Week takes place from 30 September – 7 October 2017, encouraging garden centres around the country to embrace the beauty of conifers, and highlight them using eye-catching displays, to demonstrate the variety and choice of conifers available.
Spicer said younger customers had forgotten about bad publicity conifers got years ago because of sitka spruce plantations that cover huge areas of UK Forestry Commission land and leylandii high hedges, which have caused neighbour disputes because they block light.
He said newer, more colourful and compact conifers were leading sales.


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Cottontail knocked on the big front door, & was admitted to the Palace. There she stood in her funny country clothes but none of the other four Easter Bunnies laughed, for they were wise & kind & knew better.
Friday, April 28, 2017
Thursday, April 27, 2017
Monday, April 24, 2017
Saturday, April 22, 2017
Six Celebrities Who are Pro-Life on Abortion
Six Celebrities Who are Pro-Life on Abortion: 2015 will go down as monumental year for pro-life movement after a series of undercover videos exposed the nation's largest abortion chain, Planned Parenthood,
Thursday, April 20, 2017
Whatever is Lovely: An apple a day--oh wait, those are lemons
Whatever is Lovely: An apple a day--oh wait, those are lemons: The year 2014 was the year of the 365-day panic attack, and by panic attack I mean scared-to-leave-home-without-my-husband, day in, day out,...
Tuesday, April 18, 2017
Sunday, April 16, 2017
Saturday, April 15, 2017
Rachel Lamb and Joshua Brown's Wedding
Rachel Lamb and Joshua Brown's Wedding: We're thrilled to be sharing the full ceremony from Rachel and Josh's wedding with you, along with some of our favorite, never before seen moments from this special day. You can see it all right here On Demand!
Friday, April 14, 2017
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Undergraduate Degrees (Education)
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Information skills: bridging the gap (2007)This study involves a critical evaluation of the effectiveness of information skills training for final year dental undergraduate students at the School of Dentistry, Cardiff University. As a result of the introduction ...
Practitioners Perceptions of the effectiveness of Adult Education and Lifelong Learning in one specific town within the Blaenau Gwent area (2009)This dissertation examines the practitioner's perceptions of the effectiveness of Adult Education and Lifelong Learning in one specific town within the Blaenau Gwent area. lt explores the barriers in Adult Education and ...
Teachers' attitudes to the implementation of the Social and Emotional Aspects of Learning (SEAL) programme in a South Wales primary school (2009)The importance of healthy emotional development of young people has been recognised in a number of initiatives focussed on developing children's social, emotional and behavioural skills. The Social and Emotional Aspects ...
Teachers' perceptions of the importance of creativity in the early years classroom (2009)This dissertation explores creativity and provides an overall picture of the importance of the importance of creative development within education. The Welsh Assembly Government presents the chief guidelines on the ...
Professionals' perception of the implementation of the CHIPS peer support programme in a south Wales high school (2009)his research considered the extent to which a CHIPS (Childline in Partnership with Schools) peer mentoring programme, in place in a South Wales high school, is implemented to best benefit the pupils and staff of the school. ...
School staffs' perspectives of triggers for disruptive behaviour in a special educational needs environment (2008)This research reports upon school staff's perspective's of triggers for disruptive behaviour in a Special Educational Needs (SEN) environment. lt examines how a SEN impacts upon a child's holistic development and ...
Teachers' and educational professionals' perceptions of the factors that impact upon the educational attainment of 'looked after' children in a south east Wales local authority (2009)Backqround: The educational attainment of 'looked after' children has been the centre of political debate for decades. Studies and statistics suggest that'looked after' children significantly underachieve in comparison ...
"My mum's hair is green!" An exploratory study into the perceived effects of the Foundation Phase on development of children's creativity (2009)This dissertation focuses on the perce¡ved effects of the Foundation Phase on the development of children's creativity within a mainstream pr¡mary school sett¡ng. A qualitative approach to data collection was used. Semi ...
A comparative study of drug related education at key stage three and four in two south Wales secondary schools (2008)Using qualitative methodology (interviews, questionnaires and documented evidence) the study aimed to find out what policies and teaching süategies schools had in place for educating children at these key stages, I chose ...


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- Living room
- Bathroom
- Shower
- Bathtub
- Hairdryer
- Internet access
- Direct dial telephone
- Cable TV
- 220V power supply
- Fridge
- Mini fridge
- Individually adjustable air conditioning
- Safe
- Balcony/Terrace
- Smoke detector
Bijilo Beach
- Low Deposit Options Available
- Interest FREE Payment Plans
The hotel is located next to the sandy Bijilo Beach, which is only 500 meters away from the bustling tourist area of Senegambia, a long stretch of sandy beach. The hotel faces the ... Continue reading
The hotel is located next to the sandy Bijilo Beach, which is only 500 meters away from the bustling tourist area of Senegambia, a long stretch of sandy beach. The hotel faces the Atlantic Ocean and is only a 5-minute walk from the famous Bijilo forest park that features a range of exotic wild animals. There is also a selection of bars and restaurants located next to the hotel.With its convenient proximity to the Atlantic Ocean, its impeccably landscaped grounds, an intimately relaxed atmosphere and professional staff, the hotel is an ideal holiday choice. The hotel consists of a total of 55 elegant rooms and private bungalows. Guests can wine and dine in the ocean-view restaurant, café, bar or pub. Guests are welcomed in a lobby with 24-hour reception and check-out service. Facilities include air conditioning, a hotel safe, currency exchange, cloakroom, hairdressing salon, children's playground, room and laundry services, and bicycle hire. Business travellers are sure to appreciate the conference facilities and WLAN Internet access, whilst those arriving by car can leave their vehicle in the car park or garage.The standard rooms offer a perfect ambience and tranquillity. All rooms feature comfortable double beds, luxury duvets with extra pillows, and blackout curtains to ensure a good night's sleep. For the business traveller, the rooms have desks with telephone points and wireless high-speed Internet access. Each is en suite with a shower, bath and a hairdryer. All rooms have air conditioning, a fridge, direct line telephone, satellite TV, a safe, and a balcony or terrace.
- Year of construction: 2001
- Number of floors (main building): 2
- Disability-friendly rooms: 5
- Living room
- Bathroom
- Shower
- Bathtub
- Hairdryer
- Internet access
- Direct dial telephone
- Cable TV
- 220V power supply
- Fridge
- Mini fridge
- Individually adjustable air conditioning
- Safe
- Balcony
- Terrace
- Smoke detector
- 24-hour reception
- Wired Internet
- Wi-fi
- Room service
- Laundry service
- Launderette
- Bicycle hire service
- Air conditioning in public areas
- Smoke detector
- Hotel safe
- Currency exchange facilities
- Cloakroom
- Supermarket
- Shop
- Garden
- Bicycle storage
- Luggage room
- Café
- Bar
- Restaurant
- Non-smoking area
- Smoking area
- Highchairs
- Air conditioning in Restaurant
- Hairdressing salon
- Children playground
- Outdoor freshwater pool: 1
- Sun loungers (fees may apply)
- Parasols (fees may apply)
- Massage
- Pub
To make booking a holiday at Bijilo Beach in Bijilo easier for you, we provide a range of different payment options. To find out if you can book Bijilo Beach with a low deposit holiday option, please click the "Price and Availability" button above.
Essential Information
Time Zone
Plug TypeVoltage 220V 50Hz
This week's weather forecast
Hotel address : KOMBO COASTAL ROAD,, Bijilo, Gambia, 4061
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Questions and Answers about Bijilo Beach
If you can't find what you're looking for or if you have a question about this specific hotel, please get in touch via the Add a Question button below. Our knowledgeable team will aim to respond to your question within 72 hours. In the meantime, previously asked questions can be found below and answers to some of the most commonly asked questions can be found on our FAQs page.


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Boy's Fall/Winter Collection: Henleys and Bootcut Jeans
I made two Henleys for Henry, one out of woven fabric and one out of knit fabric. To make life easier on myself, instead of making more pants for Henry I tailored two pairs of jeans from wide leg to bootcut. This first Henley is the woven shirt. I upcycled a men's shirt to make, you can make one too with my
. I found the elephant shirt at the thrift store and cut it up for this shirt. It turned out really fun! And the jeans look baggy because they are still pretty long on Henry.
The knit Henry is great too. I did some blocking with different fabric to give it a really cool look. I used two men's t-shirts to make it. I also made the placket on the side so it gives it a cool asymmetrical look. I used snaps for the closure.
These pants are brown jeans that I tailored. It makes it look like Henry isn't drowning in them, but he still has plenty of room to move.
Our apartment complex makes huge piles of leaves and leaves them for awhile, so Henry gets lots of leaf pile time. He mostly loves stomping through the leaves, but I did catch some great leaf throwing pictures!
2013 Boy Collection: Fall/Winter
Boy Sewing Projects
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Artists Statement
Studied with Ian Fleming and R Henderson Blyth in Aberdeen and with J McIntosh Patrick at Hospitalfield. Met Kenzo Okada in Paris and developed a lifelong interest in oriental painting and zen philosophy, informed by the work of American colour field painters of the 1950s.
1953-1958 - Studied Grays School of Art, Aberdeen
1958-1959 - Hospitalfield College of Art, Arbroath and abroad
1960 - Member of The Scottish Society of Artists
1963-1968 - Education Officer, Council of Industrial Design, Scottish Committee
1968-1989 - Director, Aberdeen Art Gallery and Museums
1988-1998 - President, Royal Scottish Society of Painters in Watercolour
1989-1996 - Aberdeen City Arts and Recreation Officer
1995 - Member of The Royal Glasgow Institute of Fine Art
1998-Present - President, Royal Scottish Academy
1999-2007 - Trustee, National Galleries of Scotland
1980 - ESU Thynne Travelling Scholarship
- May Marshall Brown Award, RSW
- Sir William Gillies Award, RSA
1971 - Guthrie Award, RSA
1969 - Institute of Contemporary Prints Travel Award
Selected Exhibitions
Annual Exhibitions
RSW Annual Exhibitions
RGI Annual Exhibitions
RA Annual Exhibitions
RHA Annual Exhibitions
57 Gallery
Scottish Gallery
Demarco Gallery
Fruitmarket Gallery
Compass Gallery
Open Eye Gallery
Albemarle Gallery
Ingleby Gallery
Fine Art Society
Public Collections
National Gallery of Modern Art
Scottish Arts Council
Arts Council of Northern Ireland
Contemporary Art Society
Aberdeen Art Gallery and Museums
Glasgow Art Gallery and Museums
City Art Centre, Edinburgh
Perth Art Gallery and Museum
McManus Gallery, Dundee
Arburthnot Museum, Peterhead
Abbot Hall Art Gallery, Kendal
Hunterian Museum, University of Glasgow
Napier University
|Friends of Royal
Scottish Academy
Nuffield Foundation
Department of the Environment
Carnegie Dunfermline Trust
Grampian Hospitals
Strathclyde Education Authority
Pictures for Hospitals
Lothian Education Authority
Robert Fleming Holdings
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Deutsche Bank
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Residents on the Isle of Wight have started to receive either a new black wheelie bin or gull-proof sacks in preparation for the way household waste is collected from 2nd May.
Through the changes the Isle of Wight council are aiming to encourage more recycling, which brings money into the council and less waste going to landfill, which costs the council money and is less environmentally friendly.
Amey, the company with the 25 year waste disposal contract, have re-assessed all properties on the Island as to their suitability for wheelie bins (and size of).
Mass outrage
However, in the first week of the new bins being dispatched across the Island, an online petition has been created calling on the council to rethink their policy.
Set up by Julie King of Cowes, at time of publishing over 900 residents have signed the Change.org petition, angry at the allocation of 140 litre black wheelie bins.
New bins are "too small"
Many argue the bin is not going to be large enough to hold two week's black bag waste and say the council have been allocating the wrong size bins to any households of over three people.
The petition reads,
"The residents of the Isle of Wight have not been consulted about the changes to our waste collection, which we believe we should have been. Households up to 5 are only being given a 140 litre black bin which supposedly holds 4 black sacks of rubbish.
I've tested mine, it holds 2 full bags and almost half of another! A household of 3 upwards will make more rubbish than one of 1 to 2. You haven't taken into account pets who make rubbish, especially those who eat food pouches as they're not recyclable.
I want the policy changed so that either larger bins are provided to larger families (not just 6 or over) or it is agreed black sacks left at the kerbside will be collected.
If the whole Island is expected to take their own excess rubbish to the tip it will increase fly tipping (that will be nice for visitors), rubbish piled up in gardens and will increase car fumes (not good for the environment, contradicts why we're encouraged to recycle) and increase traffic, possibly causing congestion at the tips (one of which is closing soon for improvements).
If people choose to put their rubbish in neighbour's bins it could lead to conflict. Salford Council, Chichester Council and others use 140 litre bins but for single occupants only, the standard size they issue is 240 litres and 360 litres for a household of 6 or more. Please change the size of our bins Isle of Wight Council and Amey."
Public consultation
The Isle of Wight council did hold a public consultation in 2014 on waste collection and recycling prior to the selection of bidder for the 25 year contract.
More comments on the subject can be found on the online petition.
OnTheWight will get in touch with the IWC on Monday for a response to the petition and update once we hear back from them.
Saturday, 2nd April, 2016 6:33pm
By Sally Perry
ShortURL: http://wig.ht/2e60
Filed under: Island-wide, Isle of Wight Council, Isle of Wight News, Top story, Waste PFI Contract