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Conceived and designed the experiments: DWS CSA RHE. Performed the experiments: DWS CSA. Analyzed the data: DWS CSA RHE. Wrote the paper: DWS RHE.
The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.
The physiological action of peptide hormones and neural peptides depends on their sorting to vesicles capable of regulated exocytosis in response to stimulation. Despite the diversity and importance of signals released by this pathway, surprisingly little is understood about the molecular mechanisms involved in sorting to and indeed formation of the large dense core vesicles (LDCVs) that mediate regulated secretion as opposed to secretory vesicles that undergo constitutive release. We recently used RNA interference in cell lines to identify a requirement for the adaptor protein AP-3 in sorting to the regulated secretory pathway, but the importance of this role in vivo has remained unknown. Using mutant mice lacking various subunits of the AP-3 complex, we now show that AP-3 is indeed required for appropriate, regulated secretion in multiple neuroendocrine cell types. Although AP-3 exists as both ubiquitous and neuronal forms, we also find that either form alone suffices to confer regulated secretion. The results show that AP-3 plays a novel and essential role in regulating the release of peptide hormones and neural peptides.
In contrast to most proteins which undergo immediate and unregulated secretion after biosynthesis, proteins destined for regulated release require sorting into LDCVs, but the mechanisms responsible for sorting to LDCVs and indeed LDCV formation remain poorly understood. LDCVs bud from the trans-Golgi network (TGN) [1], [2], [3], and previous work has suggested that lumenal interactions such as the aggregation of granulogenic proteins drive their formation [4], [5]. Indeed, sorting to LDCVs has been suggested to occur by default, with proteins destined for other organelles removed during the well-established process of LDCV maturation [6], [7]. However, direct analysis of budding from the TGN has demonstrated the sorting of regulated from constitutive cargo at this early step, before maturation [3]. In addition, LDCV membrane proteins such as carboxypeptidase E and sortilin have been proposed to serve as the receptors for soluble cargo [8], [9]. In contrast to these lumenal and membrane interactions, the cytosolic machinery involved in sorting to LDCVs and LDCV formation has remained poorly understood.
Several membrane proteins contain cytosolic sequences that direct them to LDCVs. For example, the neuronal vesicular monoamine transporter VMAT2, which fills neurosecretory vesicles with monoamine transmitter, depends on a conserved, C-terminal, cytoplasmic dileucine-like motif for sorting to LDCVs [10], [11], [12], and the LDCV membrane protein IA-2β (phogrin) relies on a remarkably similar sequence [13]. Since the requirement for a cytoplasmic motif suggested an interaction with cytosolic sorting machinery, we recently used VMAT as a reporter to screen by RNAi in Drosophila S2 cells for proteins involved in biogenesis of the regulated secretory pathway, identifying multiple subunits of the heterotetrameric adaptor protein AP-3 [14]. Loss of AP-3 results in mis-sorting of VMAT in both S2 and mammalian neuroendocrine PC12 cells, dysregulated secretion, a reduction in the number and alteration in the morphology of LDCVs [14]. Indeed, AP-3 RNAi disrupts sorting at the TGN and impairs the concentration of membrane proteins such as synaptotagmin that are required for regulated release [14]. However, most work in mammalian cells has focused on the role of AP-3 within the endolysosomal pathway, in trafficking from early endosome to lysosome.
Consistent with a role in the endolysosomal pathway, mocha mice (Mus musculus) lacking AP-3 show defects in lysosome-related organelles (LROs) such as melanosomes, platelet granules and synaptic vesicles [15], [16]. In addition to the abnormal coat color and a bleeding diathesis, however, the animals exhibit perinatal lethality, hyperactivity, head tilt, seizures and reduced fertility [17], [18], [19], and it has remained unclear whether a defect in the endolysosomal pathway can fully account for the severe phenotype. Importantly, previous work in S. cerevisiae has indicated a primary role for AP-3 in the biosynthetic pathway [20], [21], [22]. We have thus now used mocha mice to investigate the physiological role of mammalian AP-3 in regulated protein secretion.
To determine whether the loss of AP-3 in vivo affects regulated secretion, we cultured adrenal chromaffin cells from control and AP-3-deficient mocha mice, measuring the release of endogenous secretogranin II (SgII) in response to the nicotinic agonist DMPP [23]. Western blotting of the medium indicated that DMPP stimulates SgII secretion from control cells, but SgII was undetectable in the medium of mocha cells (Figure S1). However, the substantial reduction in cellular SgII content of mocha adrenal glands [14] and of cultured mocha adrenal chromaffin cells (Figure S1) made it difficult to determine whether the cells simply do not contain and release enough SgII to detect, or actually exhibit a defect in regulated release.
To assess regulated exocytosis by chromaffin granules, we used total internal reflection fluorescence (TIRF) microscopy to image neuropeptide and LDCV membrane protein reporters fused to the superecliptic pHluorin [24], [25]. The pHluorin is a modified form of green fluorescent protein (GFP) with increased sensitivity to protons that is quenched at the low internal pH of LDCVs and therefore increases in fluorescence with exposure to the higher external pH on exocytosis. Since neuropeptide Y (NPY)-pHluorin has been shown to undergo regulated exocytosis [26], we used lentiviral transduction to express this fusion protein and monitored individual exocytotic events at the plasma membrane of living chromaffin cells. In the absence of stimulation, control cells showed very few spontaneous fusion events over 90 s of imaging, but AP-3-deficient mocha cells exhibited substantially more (Figure 1A–1B). Both control and mocha cells showed a clear increase in exocytosis in response to stimulation by DMPP, but the extent of stimulation relative to baseline reveals an ~70% reduction in mocha cells compared to controls (Figure 1B). To extend these findings to an LDCV membrane protein, we transduced chromaffin cells with a virus encoding VMAT2-pHluorin, with the lumenal location of pHluorin enabling detection of release events [27], [28]. Similar to NPY-pHluorin, VMAT2-pHluorin also showed a clear increase in basal, unstimulated exocytosis in mocha relative to control cells, and this again resulted in an ~75% reduction in stimulated release (Figure 1C). The loss of AP-3 thus dysregulates the release of LDCVs as monitored using either soluble or membrane cargo.
The role of AP-3 in defining LDCV membrane protein composition has suggested that loss of the adaptor results in mixing of constitutive and regulated secretory pathways [14]. The resulting constitutive secretion (measured biochemically in the case of endogenous SgII [14]) could thus reflect either the spontaneous release of constitutive secretory vesicles which contain mis-sorted LDCV cargo but no dense core, or the dysregulated release of LDCVs. To distinguish between these possibilities, we again took advantage of TIRF microscopy. Release from constitutive vesicles without a dense core should yield events with reduced amplitude relative to controls, whereas dysregulated LDCV fusion should yield events with a size similar to controls. Analyzing individual exocytotic events, we observed that the basal as well as stimulated events observed in mocha cells have an amplitude similar to those observed in controls (Figure 1D). The heightened basal secretion observed in mocha cells thus apparently results from dysregulated exocytosis of LDCVs rather than the fusion of constitutive secretory vesicles without a dense core.
The increased basal exocytosis of NPY and VMAT2 from mocha chromaffin cells is consistent with earlier experiments using RNAi in PC12 cells [14], but does the loss of AP-3 also dysregulate release from other neuroendocrine tissues? Pancreatic β cells store insulin in LDCVs morphologically and biochemically similar to chromaffin granules [29], [30]. To assess the regulated release of insulin in vivo, we measured baseline serum insulin levels while fasting and stimulated levels 15–20 minutes after intraperitoneal injection of glucose (Figure 2A). Before glucose administration, we observed a slight reduction in the serum insulin levels of mocha mice relative to controls, but this did not reach significance. After glucose administration, the control animals showed a clear increase in serum insulin but the mocha mice did not (Figure 2A), suggesting a failure of regulated release.
To assess the consequences of dysregulated insulin release for carbohydrate metabolism, we also measured blood glucose. Surprisingly, we observed no clear difference in fasting blood glucose levels between control and mocha mice (Figure S2A). After glucose administration, mocha animals show an increase in glycemia but less than controls (Figure S2A), a surprising effect in light of the lower serum insulin levels which would have been expected to impair glucose tolerance (Figure 2A). The effects of the mocha mutation on blood glucose levels thus do not correlate with the effects on insulin. However, blood glucose levels reflect the combined action of multiple circulating hormones, many of which may be affected by the loss of AP-3. Indeed, the dysregulated release of other peptide hormones may indirectly affect the release of insulin.
To examine insulin release independent of systemic effects, we isolated pancreatic islets and acutely incubated them in medium containing either low or high concentrations of glucose. Figure 2B shows that in contrast to the clear stimulation of insulin release by high concentrations of glucose in control islet cells, mocha cells exhibit increased basal release with little if any stimulation by high glucose. As opposed to the reduced content of SgII in mocha chromaffin cells, cellular insulin levels show no difference between mocha and control islets (Figure 2C), indicating that the change in basal secretion is not secondary to altered expression of the hormone. In the case of insulin, the mocha mutation thus dramatically and selectively impairs regulated secretion.
Since the effects of the mocha mutation on release of other peptide hormones may complicate the observations in vivo, we also examined glucagon, a peptide released from pancreatic α cells that opposes the action of insulin: glucagon raises blood glucose levels in response to starvation. Although we observed no effect of the mocha mutation on fasting serum glucagon (Figure S2B), baseline glucagon secretion was increased in acutely isolated islets (Figure 2D). In addition, the glucagon content of mocha islet cells did not differ from controls (Figure 2E), and the morphology of the islets in situ appears unchanged (Figure S2C). Thus, mocha mice show dysregulated release of two peptide hormones with opposing actions, suggesting a global effect on peptide hormone release that makes it difficult to predict the net consequences for glucose homeostasis in vivo.
The heterotetrameric AP-3 complex is known to exist in two isoforms, one expressed by all tissues, and another expressed more specifically by neurons and endocrine tissue including the adrenal gland and pancreatic islets [16], [31], [32]. In metazoan cells, the ubiquitous isoform contributes to trafficking from early endosomes to the lysosome through a pathway that does not involve multivesicular bodies [15], [33]. In contrast, the neural isoform has been implicated in the formation of synaptic vesicles from an endosomal intermediate [34], [35], suggesting that this isoform may also contribute to the formation of LDCVs. To test this possibility, we used pearl mice lacking the ubiquitous isoform of the β3 subunit (β3A) and β3B knockouts lacking the neural isoform of β3. Since the δ subunit of AP-3 exists as only a single isoform, and the loss of one subunit usually destabilizes the complex [17], [36], we first stained cells in culture for δ to assess the effect of the mutations on the complex as a whole. We were surprised to observe no effect of losing either β3 isoform on the levels of immunoreactive δ in chromaffin cells (Figure 3A), particularly considering the abundance of the ubiquitous β3A subunit in most tissues. However, we did observe reduced expression of δ by β3A-deficient, non-chromaffin cells in the culture (Figure 3B), presumably because they do not express the neural isoform and therefore cannot exhibit redundancy. Consistent with the relative abundance of adrenal cortical cells [37] and of the ubiquitous AP-3 isoform, western analysis of adrenal homogenates showed low levels of AP-3 δ in β3A-deficient pearl animals (Figure 3C) but normal levels in β3B knockouts (Figure 3D).
To determine whether the loss of β3A or β3B influences the formation of LDCVs, we then examined the effects on SgII. We were surprised to observe that in contrast to mocha cells which showed the reduction previously reported [14], both β3 mutants had normal levels of SgII by immunofluorescence (Figure 3A). By western analysis of adrenal extracts, both β3A-deficient pearl and β3B knockouts also contained normal levels of immunoreactive SgII (Figure 3C–3D). However, loss of both isoforms in the adrenal gland of double mutant mice produced a reduction in SgII comparable to that observed in mocha mice (Figure 3E). With regard to the cellular content of SgII, the two isoforms thus exhibit redundancy.
The reduced expression of SgII in mice lacking AP-3 might reflect increased basal secretion or an entirely distinct process. LDCV contents have indeed been shown to undergo transcriptional regulation through a variety of mechanisms [30], [38]. We therefore measured adrenal SgII and chromogranin A (CgA) transcripts from control and mocha adrenals by quantitative reverse transcription (qRT)-PCR. Both SgII and CgA mRNA were substantially reduced (by ~50%) in mocha mice, although not to the same extent as the protein [14] (Figure 4A). PC12 cells showed a similar reduction in SgII mRNA after AP-3 RNAi (Figure 4B).
Since AP-3 influences trafficking within the endolysosomal pathway, loss of the adaptor may also influence SgII levels through increased degradation in the lysosome. To test this possibility, we inhibited lysosomal proteases after AP-3 knockdown in PC12 cells, but did not observe any increase in the levels of SgII (Figure 4C). On the other hand, the level of lysosomal hydrolase precursor procathepsin D dramatically increased in response to the inhibition of lysosomal degradation, indicating the effectiveness of the inhibitors (Figure 4C). The reduction in cellular SgII observed with loss of AP-3 thus reflects reduced expression as well as increased constitutive release, but not increased degradation.
AP-3 resembles AP-1 in terms of sequence, the ability to recognize similar trafficking motifs and subcellular location at endosomes and the Golgi apparatus [39], [40], [41]. In addition, AP-1 associates with immature LDCVs and promotes their maturation through the clathrin-dependent removal of proteins destined for other organelles [6], [42]. In mouse pituitary AtT-20 cells, maturation indeed contributes to regulated release by removing the inhibitory protein synaptotagmin 4 [43]. In PC12 and pancreatic islet cells, however, immature LDCVs can undergo regulated release [44], [45]. To determine whether silencing of AP-1 impairs regulated release from PC12 cells, we initially targeted AP-1 β-adaptin since this is the only mammalian AP-1 subunit without multiple isoforms and hence with reduced potential for redundancy. Despite highly efficient knockdown of the β1 subunit by RNAi, μ1A levels were not reduced (data not shown), raising the possibility that the β2 subunit (of AP-2) stabilized the complex by replacing β1 [46], [47]. We therefore targeted the γ subunit of AP-1, in particular the γ1 isoform which appears to show little redundancy with γ2 [48]. siRNA transfection reduced endogenous γ1 protein by ~80% (Figure 5A). It also reduced the stimulated secretion of SgII and the intracellular accumulation of SgII, very similar to AP-3 δ RNAi (Figure 5B,C). However, normalizing to the reduced cellular stores of SgII revealed more of an increase in basal SgII secretion than a reduction in stimulated release with AP-1 knockdown, in contrast to AP-3 RNAi (Figure 5D). AP-1 RNAi thus still reduces the extent of stimulation (stimulated/basal release) from 19-fold for control to 10-fold for AP-1 RNAi (p<0.005). AP-1 knockdown also potentiates the effect of AP-3 RNAi on stimulated secretion even after normalization to the reduced intracellular stores of SgII (Figure 5D). AP-1 thus contributes to regulated secretion, and its role appears independent at least in part from that of AP-3.
The results show that mocha mice have a major defect in the regulated secretion of peptide hormones. mocha animals exhibit hyperactivity, poor fertility, seizures and premature lethality [18], [19], but this has generally been attributed to a role for AP-3 in the endolysosomal pathway and the formation of LROs [15], [34]. We now find that mocha animals exhibit dysregulated exocytosis of adrenal chromaffin granules and both insulin- and glucagon-containing granules from pancreatic islet cells. All of the mocha cells examined show increased constitutive release relative to their cellular stores. In addition, they all show a reduced effect of stimulation, with a virtually complete loss of regulation in pancreatic β cells. Consistent with these findings in mocha mice, AP-3 RNAi increases constitutive and reduces stimulated LDCV release in PC12 cells [14]. Considering the parallel effects of AP-3 deficiency on LDCV behavior in chromaffin and pancreatic islet as well as PC12 cells, we infer that the dysregulated release observed in mocha mice reflects a common disturbance in the formation of LDCVs. The dysregulation of release by multiple neuroendocrine populations further suggests that a global defect in regulated protein secretion contributes to the phenotype of mocha mice, although the specific peptides contributing to individual features of the phenotype such as hyperactivity and seizures remain unknown.
In previous work, we found that the loss of AP-3 reduces the amount of SgII stored in PC12 cells and the adrenal gland [14]. This reflects the increased baseline exocytosis of LDCVs, but we now find that multiple granin mRNAs down-regulate as well, indicating unanticipated transcriptional effects of AP-3 deficiency. Indeed, the transcriptional down-regulation of certain LDCV cargo may account for the apparently different effects of AP-3 deficiency on different reporters and in different cells. The down-regulation of SgII mRNA in PC12 cells presumably makes it difficult to detect an increase in the absolute amount of SgII released constitutively, but transfection into PC12 cells of pHluorin-based reporters, which are not subject to this down-regulation, reveal the increased basal secretion [14]. We observe a similar increase in basal exocytosis of VMAT2- and NPY-pHluorin expressed in AP-3-deficient chromaffin cells using a lentivirus. In the case of pancreatic islets from mocha mice, cellular levels of insulin do not fall, presumably enabling us to detect the increase in basal insulin release.
Similar to AP-3 deficiency, loss of the major LDCV protein IA-2 reduces expression of multiple LDCV cargo [49], [50], suggesting that AP-3 may sort IA-2 to LDCVs. In the absence of AP-3, decreased LDCV IA-2 may thus result in reduced granin gene expression. Although AP-3 has a role in the endolysosomal pathway, we also find that the reduced granin content does not reflect increased degradation. The role for AP-3 in regulated secretion thus appears distinct from its well-established role in trafficking to the lysosome.
The analysis of isoform-specific knockouts indicates redundancy between the ubiquitous and neural isoforms of AP-3 with regard to LDCV formation. Using SgII as a reporter for a defect in the regulated secretory pathway, we find that only the loss of both ubiquitous β3A and neural β3B causes a reduction in adrenal SgII levels. However, the loss of these isoform-specific subunits has differential effects on other trafficking phenomena. In neurons, loss of β3B mimics the effect of the full mocha mutation, with reduced presynaptic expression of proteins such as the zinc transporter ZnT3 and the chloride carrier ClC-3 [35]. Loss of β3A, on the other hand, increases presynaptic expression of these proteins [35]. The redundancy of neural and ubiquitous AP-3 forms in LDCV formation thus contrasts with the opposing roles of the two isoforms in delivery of specific proteins to the nerve terminal.
How does AP-3 promote regulated secretion? In PC12 cells, the loss of AP-3 reduces the number of LDCVs and changes their morphology [14]. They appear less dense by gradient fractionation and larger by electron microscopy (EM). Consistent with this, previous work in mocha mice has shown enlarged chromaffin granules by amperometry and EM [31]. In addition, we found that AP-3 deficiency affects the membrane proteins required for regulated exocytosis: the calcium sensor synaptotagmin 1 shifts from LDCVs to lighter membranes [14]. An assay for budding from the TGN further shows that AP-3 deficiency impairs LDCV formation [14]. Despite the importance of AP-3 for LDCV formation, however, its role may be indirect, and previous work has indeed localized AP-3 primarily to endosomes [33], [41].
Several observations have suggested a role for AP-3 at the Golgi complex. In yeast, AP-3 contributes to a direct pathway from the Golgi to the vacuole [20], [22], [51]. Although this has been considered specific to yeast, work in mammalian cells has more recently supported a role for AP-3 in delivery of membrane proteins from the biosynthetic pathway to the lysosome [52], [53]. Biochemical studies have further demonstrated the specific binding of AP-3 to membranes derived from the Golgi or to artificial membranes containing the Golgi lipid phosphatidylinositol-4-phosphate (PI4P) [54], [55]. Ultrastructural studies with immunogold have also demonstrated a small pool of AP-3 at the Golgi complex [33], [41]. However, it remains possible that the role of AP-3 in LDCV formation is indirect, helping to recycle critical LDCV membrane proteins to the Golgi, or adding these proteins to LDCVs during their maturation [56].
Despite the complete loss of AP-3 and increased basal secretion, adrenal chromaffin cells from mocha mice still show residual stimulated release, raising the possibility that another system also contributes to LDCV formation. Interestingly, previous work has implicated the related adaptor AP-1 in the formation of secretory granules by other cell types, such as glue granules of the Drosophila exocrine salivary gland, and the Weibel-Palade bodies of mammalian endothelial cells [57], [58]. AP-1 also has a clear role in LDCV maturation [6], [42], but immature LDCVs can undergo release from PC12 cells [45]. We were therefore surprised to find that loss of AP-1 impairs regulated release by PC12 cells. It remains possible that maturation promotes regulated secretion even if it is not absolutely required. Alternatively, AP-1 may promote regulated release independent of LDCV maturation.
We also find that the loss of AP-1 exacerbates the dysregulation of release by AP-3 RNAi, suggesting that the two adaptors act through distinct mechanisms. If AP-1 promotes regulated secretion through its role in LDCV maturation, it may indeed act to remove proteins that interfere with regulated release, a process that occurs in AtT-20 cells [43]. We speculate that a proofreading role for AP-1 may become even more important in the absence of AP-3 to concentrate the membrane proteins required for regulated secretion.
All procedures involving animals were approved by the UCSF Institutional Animal Care and Use Committee.
The rabbit SgII antibody was obtained from Meridian Life Science, the mouse actin monoclonal antibody from Millipore, the mouse δ SA4 monoclonal antibody from the Developmental Studies Hybridoma Bank, the goat cathepsin D antibody from Santa Cruz, the mouse HA.11 monoclonal antibody from Covance, the mouse adaptin γ monoclonal antibody from BD Transduction Laboratories, the mouse insulin monoclonal antibody from Sigma, the guinea pig glucagon antibody from Linco, the mouse glucagon monoclonal antibody from Sigma and the rabbit somatostatin antibody from Thermo Scientific.
Silencer Select rat Ap3d1 (sense, 5′-CAUGGAUCAUGACCAAGAA-3′) and corresponding non-targeting control siRNAs were from Ambion. ON-TARGETplus rat Ap1g1 (sense, 5′-CAUAAAUAUUCUUGGUCGA-3′, 5′-GUGUGGAGAUGCACGCUUA-3′, 5′-UGUAACAGUGAUAACGAUA-3′, 5′-GGACUGGAAUUCACGGCAA-3′) and corresponding non-targeting pooled control siRNAs were from Dharmacon.
The sequences of NPY-pHluorin (a generous gift of R. Holz, U. Michigan) and VMAT2- pHluorin were amplified by PCR to add 5′ BamHI and 3′ EcoRI sites, then subcloned into the FUGW lentiviral expression vector, replacing the EGFP coding sequence.
PC12 cells were maintained in DMEH-21 medium supplemented with 10% horse serum (HS) and 5% cosmic calf serum (CCS; HyClone) in 5% CO2 at 37°C. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. HEK293T cells were maintained in DMEH-21 medium with 10% fetal bovine serum (FBS) in 5% CO2 at 37°C. Lentivirus was produced by transfecting HEK293T cells with FUGW, psPAX2 and pVSVG and Fugene HD (Roche) according to the manufacturer's instructions [59].
Mouse adrenal chromaffin cells were isolated and cultured as previously described [60]. Briefly, adrenal glands were dissected and placed in cold Ca++-, Mg++-free (CMF) Hank's balanced salt solution (HBSS). The surrounding fat and cortex were removed and the medullae transferred to tubes containing 300 U/ml Collagenase I (Worthington) in CMF-HBSS. Medullae were dissociated by shaking for 40 min at 37°C. Collagenase solution was then replaced by CMF-HBSS containing 200 µg/ml DNAse I (Sigma) and 1% heat-inactivated FBS (Gibco), the tissue triturated first with a P200 pipette tip, then with a 23 gauge needle. The cells were pelleted at 300 g for 8 min at room temperature and resuspended in pre-warmed culture medium. Cells were maintained in DMEH-21 medium supplemented with 10% FBS and antibiotics. For lentiviral transduction, freshly isolated chromaffin cells were plated in viral supernatant, and fresh medium was added the following morning.
For TIRF microscopy, control or mocha chromaffin cells were plated onto glass chambers coated with poly-L-lysine, immediately transduced with lentivirus encoding either NPY- or VMAT2-pHluorin and imaged live 4–7 days later. Images were typically collected for 40–50 ms at 10 Hz and room temperature using an inverted TIRF microscope (TE2000E; Nikon) with 100× Plan Apo 1.49 NA oil objective, a 1.5× tube lens and an electron-multiplying charge-coupled device camera (QuantEM; Photometrics). Basal exocytosis was measured in Tyrode's solution containing (in mM, 119 NaCl, 25 HEPES-NaOH, pH 7.4, 30 glucose, 2.5 KCl, 2 CaCl2, 2 MgCl2) over 90 s, and release stimulated for 60 s in Tyrode's containing 5 µM 1,1-Dimethyl-4-phenylpiperazinium (DMPP; Sigma). Individual exocytotic events were quantified manually using NIS-Elements software (Nikon). The amplitude of individual exocytotic events was measured by placing 2×2 pixel ROIs manually over the center of events, and the mean ROI intensity prior to an event subtracted from the maximum event intensity.
Glucose tolerance was assessed and serum insulin levels measured using 5–15 week-old mocha mice and age-matched controls. Mice were fasted overnight (~16 h), weighed the following morning, and blood samples collected for baseline glucose and insulin levels. Mice were then injected intraperitoneally with glucose at 2 mg/g body weight and blood samples collected from the tail vein at the time points indicated. Blood glucose levels were measured using the FreeStyle glucometer (Abbott). To measure serum insulin, the blood was allowed to clot, sedimented at 2000 g and 4°C for 20 min, and insulin levels determined using the Ultra Sensitive Mouse Insulin ELISA kit (Crystal Chem).
Islets were isolated as previously described from 9–16 week-old mocha mice and age-matched controls [61]. Briefly, islets were purified on a Ficoll gradient and allowed to recover for 1 h at 37°C. Five islets were aliquoted into tubes containing HEPES-buffered RPMI medium supplemented with either 2.8 mM glucose (basal) or 28 mM glucose (regulated) and incubated for 1 h at 37°C. The islets were then sedimented and the supernatants collected to measure insulin secretion. The pellets were resuspended and sonicated in 2 mM acetic acid, 0.25% RIA-grade BSA to extract intracellular insulin. Finally, the nuclei were lysed by additional sonication in 67 mM ammonium hydroxide, 0.2% Triton X-100. Secreted and cellular insulin were quantified by ELISA (Mercodia) according to the manufacturer's instructions, and islet DNA quantified to confirm that the amount of islets per tube was similar between conditions. Glucagon was measured by ELISA (R&D Systems).
Chromaffin cells were fixed by adding an equal volume of 4% formaldehyde in CMF-PBS to the culture medium and incubating for 20 min at room temperature. Cells were blocked and permeabilized in CMF-PBS containing 2% BSA, 1% fish skin gelatin and 0.02% saponin. Primary antibodies were diluted in blocking solution at 1500 (SgII) and 1100 (δ SA4). The following secondary antibodies conjugated to Alexa Fluor dyes (Invitrogen) were used at 11000 in blocking solution: goat anti-rabbit IgG 488 and goat anti-mouse IgG 594. Images were acquired using a Zeiss LSM 510 confocal microscope and 100× oil objective.
pearl and mocha mice were obtained from the Jackson Laboratory, and mocha animals were backcrossed to C57BL/6 to remove grizzled and Pde6brd1 alleles. Ap3b2 KO mice were obtained from V. Faundez (Emory) and S. Voglmaier (UCSF). Double mutant pe/pe; Ap3b2−/− mice were generated by crossing pe/pe; Ap3b2+/− males to pe/+; Ap3b2+/− females. Adrenal glands from 3–6 week-old mice were homogenized in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, and Complete protease inhibitors (Roche) with 1 mM EGTA and 1 mM PMSF. After sedimentation at 14,000 g to remove nuclei and cell debris, 20–40 µg protein was separated by electrophoresis through polyacrylamide, transferred to nitrocellulose, and the membranes immunoblotted for AP-3δ and SgII, with actin as loading control and the appropriate secondary antibodies conjugated to IRDye800 (Rockland Immunochemicals). The immunoreactivity was quantified by imaging with an Odyssey system (LI-COR Biosciences) and ImageJ (National Institutes of Health), and the signals normalized to actin. For western analysis of the SgII secreted from chromaffin cells, Tyrode's solution was collected, sedimented at 300 g for 3 min at 4°C, and the supernatant mixed with SDS-PAGE sample buffer before electrophoresis through polyacrylamide. Chromaffin cells were directly lysed by the addition of sample buffer. In this case, SgII and actin were detected using ECL plus (GE Healthcare).
Total RNA was isolated from PC12 cells and mouse adrenal glands using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. To improve the yield of adrenal RNA, ultrapure glycogen (Invitrogen) was added to the TRIzol as carrier. In addition, adrenal RNA was treated with RNAse-free DNAse I (NEB) to remove contaminating genomic DNA. cDNA was synthesized using oligo(dT) or gene-specific primers and a Transcriptor First Strand cDNA Synthesis kit (Roche). qPCR was performed with SYBR Green (Applied Biosystems) on a Stratagene Mx4000 machine. The following primers were used: rat SgII fwd: 5′-ACAATATAAGACAGAGGAAAATTTT-3′, rev: 5′-TGGATAAGAAGCAGAACTG-3′; rat β-actin fwd: 5′-CCGTGAAAAGATGACCCAGATC-3′, rev: 5′-CAGGGACAACACAGCCTG-3′; mouse CgA fwd: 5′-CCAACCGCAGAGCAGAG-3′, rev: 5′-AGCTGGTGGGCCACCTT-3′; mouse SgII fwd: 5′-AAGTGCTGGAGTACCTCAACC-3′, rev: 5′-TTACATGTTTTCCATGGCCCG-3′; mouse GAPDH fwd: 5′-ATGGTGAAGGTCGGTGTGAAC-3′, rev: 5′-TCCACTTTGCCACTGCAAATG-3′.
Two days after the second siRNA transfection, PC12 cells were incubated for ~24 h in complete medium supplemented with vehicle or a cocktail of lysosomal protease inhibitors (Sigma) including (in µM) 10 antipain, 10 leupeptin and 5 pepstatin A. Cells were washed on ice with cold PBS and lysed by the addition of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, and Complete protease inhibitors (Roche) plus 10 mM EDTA and 1 mM PMSF. Samples were analyzed by quantitative fluorescent immunoblotting.
Cells were transfected with siRNA (100 nM) on days 1 and 3 after plating, washed 2 days later and incubated in Tyrode's solution containing 2.5 mM K+ (basal) or 50 mM K+ (stimulated) for 30 min at 37°C. The supernatants were collected, cell lysates prepared as described above, and the samples analyzed by quantitative fluorescent immunoblotting.
Statistical analysis was performed using the Student's two-tailed t-test unless otherwise indicated.
mocha chromaffin cells display marked reductions in secreted and cellular SgII. Chromaffin cells were stimulated with 20 µM DMPP in Tyrode's solution, or left unstimulated in Tyrode's alone. After a 15 min incubation at 37°C, supernatants were collected and mixed with SDS-PAGE sample buffer, and cells lysed by the addition of sample buffer. Secreted and cellular SgII were detected by immunoblotting. DMPP clearly induces secretion of SgII from control cells, but not from mocha cells. Analysis of cellular SgII shows a marked reduction in mocha cells. Cellular actin was used as a loading control.
Glucose tolerance, serum glucagon and islet morphology in mocha mice. (A) Control and mocha mice were fasted overnight and challenged with glucose (2 mg/g body weight) delivered i.p. mocha mice show slightly improved glucose tolerance relative to controls. p<0.02 for the area under the glucose-time curve; n=8 control and n=5 mocha mice. (B) Control and mocha mice display comparable serum glucagon levels after an overnight fast. n=15 control and n=10 mocha mice. (C) mocha mice exhibit normal pancreatic islet morphology as determined by double staining for insulin/glucagon and glucagon/somatostatin. Scale bar indicates 30 µm.
We thank Kurt Thorn and the UCSF Nikon Imaging Center for their assistance with microscopy, Greg Szot and the UCSF Diabetes Center for their help with islet isolation, and members of the Edwards lab for helpful discussion.
DWS was funded by a predoctoral fellowship from the NIH (MH085406). CSA was supported by the Swiss National Science Foundation and the Feldman Family Foundation. RHE was supported by the NIH (MH096863). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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.


text:
By Ray Lilley, Associated Press
WELLINGTON, New Zealand A rare and threatened species of tiny frog has been found breeding in a New Zealand animal park, meaning its future may now be more secure, researchers said Monday.
The 13 finger nail-sized Maud Island froglets were discovered clinging to the backs of full-grown male frogs at the Karori Wildlife Sanctuary in the capital Wellington, said researcher Kerri Lukis. The frogs are normally found only on two islands in the Malborough Sounds region of New Zealand's South Island.
"Maud Island frogs have never been found breeding" before, even on their home island, said Lukis, a masters degree student at Victoria University in Wellington.
"It's wonderful timing for 2008 — International Year of the Frog and a Leap Year," she said.
The breeding suggests Maud Island frogs can be bred in other predator-free habitats — strengthening their prospects for survival, said Ben Bell, the biologist overseeing Lukis' studies.
The sanctuary's predator-proof fence gives the frogs a breeding environment like Maud Island that is safe from rats, Bell said.
Maud Island frogs are estimated to number up to 40,000 — most of them on the island from which they take their name and the rest on Motuara Island.
Don Newman, who is the threatened species science manager with the Conservation Department and was not involved in the frog program, said the breeding success adds a third location where the frogs have bred, a factor that "spreads the risk" and improves the species' chance of survival.
Maud Island frogs, one of four native New Zealand frogs, have evolved little over the last 70 million years, Lukis said, resulting in distinctive features and behaviors.
They do not croak, live in water or have webbed feet, she said.
Also unlike other frogs, these hatch from the egg as fully formed frogs without going through the tadpole stage.
Eggs are laid under rocks or logs and the male sits over the eggs until they hatch as well formed, tailed froglets.
In 2006, 60 Maud Island frogs were released in the frog enclosure at the wildlife sanctuary — a security-fenced area of some 620 acres set up to enable threatened native birds and other species to re-establish their numbers safe from introduced predators like rats, mice, stoats, ferrets and wild cats.
All four of New Zealand's surviving native frog species are threatened, with the rarest, Hamilton's frog, numbering less than 300.
Copyright 2008 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
Conversation guidelines: USA TODAY welcomes your thoughts, stories and information related to this article. Please stay on topic and be respectful of others. Keep the conversation appropriate for interested readers across the map.

subset: commoncrawl


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House Party Rate Special $150.00 for 3 hours or $200 for 4 hours and $45 for each additional hour of dj Service. Ask about wedding and company party rates. Wedding services 4 hours for $400.00 each additional hour is $85.00
My name is Joe and I have been Djing for over 12 years for a leading Dj company in LA. And I have lots of experience. Also I have M.C. Experience, along with doing interactives and games. I will dress for the occasion from causal to formal.
I have worked on all kinds of events from baby's 1st birthday, weddings, back yard, picnics, school dances, Carnivals, Store Grand Openings, USC Tailgate Party, retirement, anniversary, corporate events, Fund Raisers, Bank Openings, and KID PARTIES. And all types of holiday parties Halloween, Christmas, and New Year's. You name it I have probably worked it.
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I have a 2 speaker Set-up w/ a Dancing light.
References are available on request. Thank you for the opportunity to provide professional DJ services for your event.


text:
Zombie poster design. by Micky Betts by Mixta110Watch
©2012-2017 Mixta110
A poster design for all Zombie lovers. (see buy print for a copy)
pen and inks, and ditital coloring.
pen and inks, and ditital coloring.
Yea painting stuff like this take a lot of time. I'm looking forward to seeing that
Nice poster. Would make a nice relief.be great to paint.
Thanks! I'm considering a zombie relief plaque. Maybe just in bronze effect as if i sell a few i don't want to have to paint them all in detail. This is why i stopped selling my zombie bust sculpture.
Love it!


text:
- Description
Go natural with this earthy bedroom set, with simple lines and brown cherry finish. The contemporary lines of this bedroom set are accented with a unique flared headboard. The matching chest, dresser and night stand have the same understated design with a little flair, having silver pull handles.Includes
1 California King BedDimensions
California King Bed, 88 1/2" X 74 1/2" X 45 3/4"H
More from this collection: Midland
- Additional Information
Additional Information
SKU 13791306 MPN CM7600CK GTIN UPC 847289042808,847289042792,847289042815 Brand Furniture of America Collection Midland Assemble Length 88.5 Assemble Width 74.5 Assemble Height 45.75 Assembly Required Yes Material Wood Finish Other Sizes California King Bed Styles Platform Bed Styles No Shipping Time 1-2 Weeks Free Shipping No
- Reviews
Disclaimer: Pictures displayed may include optional products not included in the price. Computer monitors often display color variations. Color swatches are shown if alternative colors are available. Please call us or email us if you have any questions!


text:
Depth Chart
Kicker: Ryan Norton
Punter: Riley Dixon
Kickoffs: Riley Dixon
Holder: Darius Kelly
Returners: Prince-Tyson Gulley, Ervin Philips, George Morris, DeVante McFarlane, Brisly Estime, Ritchy Desir
2013 Statistics
Norton: 10-15 field goals, 44-yard long
Dixon: 75 punts, 3,161 yards, 42-yard average, 75-yard long, 21 inside the 20
Estime: 4 punt returns, 126-yards, 31-yard average, 70-yard long, 5 kickoff returns, 89-yards, 18-yard average, 21-yard long
Desir: 27 punt returns, 111-yards, 4-yard average, 23-yard long
Morris: 15 kickoff returns, 294-yards, 19-yard average, 35-yard long
McFarlane: 10 kickoff returns, 184-yards, 18-yard average, 24-yard long
Gulley: 6 kickoff returns, 154-yards, 25-yard average, 49-yard long
What We Know
With the loss of Ross Krautman before training camp started, Ryan Norton became the starting kicker. He was inconsistent last season but looked much better in training camp this year. Still, he is a question mark going into the season.
Riley Dixon emerged as a star from the punting position last season. He had 15-punts of over 50-yards last season including a school record 75-yarder. Dixon will be a strength this season.
The loss of Charley Loeb will go unnoticed by most, but he was the primary holder on field goal attempts. He often made up for bad snaps with his ability to catch the ball and place it quickly. Darius Kelly has experience holding in high school and junior college and will take over for Loeb this year. He has big shoes to fill.
The return game has some potentially dynamic players in Brisly Estime and Ervin Philips. Estime showed flashes of being a homerun threat as a punt returner last season. He set up the game-winning touchdown in the Texas Bowl with a 70-yard return late in the fourth quarter. He is dangerous in the open field and has elite speed.
Ritchy Desir will also return punts for Syracuse this season. Scott Shafer likes his steadiness as a guy who never drops the ball when fielding punts.
The kick return game was where Syracuse really struggled, ranking 94th nationally in that category. Shafer likes to use running backs as his primary returners, which means Gulley, Morris and McFarlane could all see time back there.
The intriguing player is the freshman Ervin Philips. He has more explosiveness than the other options and could give the Orange a dynamic threat back there. Look for Philips to get some opportunities.
Biggest Question
How consistent can Ryan Norton become? The loss of Ross Krautman hurt Syracuse last season. In fact, it may have cost them the game against Pittsburgh. His ability to consistently hit field goals this season, especially inside the 40-yard threshold, could be critical in a few games.
Keep an eye on how Norton responds early on. If he misses some, walk-ons Cole Murphy and Alex Hodgkinson could get an opportunity to show what they can do.
Season Outlook
For the first time in years, Syracuse has multiple dynamic return options. How much will Syracuse utilize Estime on special teams, however? The Orange do not want to get him hurt but know he is their best option for the big play. Dixon is a big weapon as a punter while Norton remains a question mark. A lot of games can be won or lost on special teams and Syracuse has a solid unit entering 2014 but still has questions to answer.


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Mr Gasket Blue Anodize Air Cleaner Nut
Part #: 5327
List Price: $8.18
Our Price: $4.09
You Save: $4.09
Air Cleaner Nut Blue Anodize, This clear aluminum air cleaner nut is made from billet aluminum with knurled sides for easy hand tightening. Measures 3/4" high x 1" dia. with 1/4"-20 thread.


text:
Old Scaly Mountain School House
circa 1900
Scaly Mountain
Women's Club
A small club . . . . accomplishing big things!
Club Events
2017 Charity Auction
Chocolate Fantasy
Events Summary
Club Images
Pancake Breakfasts
2016 Charity Auction Results
Merchant Supporters
Meet our Merchants
Merchants - How you can help!
Our Scholars
Meet our Scholars
Past Scholars
Scholarship Guidelines
Scholarship Application
About Us
Becoming a Member
Membership Application
Club Photos
How to Contact Us
Member Login
Member Login


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Denver police impersonator had mustache, Hawaiian shirt & possible Magnum P.I. fetish
Police impersonators tend to be delusional types. Remember the Colorado Springs man who tried to pull people over in a 1995 Cadillac equipped with a white strobe light? He'd likely identify with the mustached man who patted down and cuffed a bystander at a Colfax bus stop while clad in a Hawaiian shirt. Does this guy think he's Magnum P.I.? Maybe, even though he's in his fifties, has gray hair and is just five-eight. Dream on, pal. Get more details from the Denver Police Department release below:
Denver Police want the community to be aware of an incident involving a police impersonator. [Monday] at 12:30 p.m., the suspect approached a man while he stood at the bus stop at Colfax Avenue and Corona Street. The suspect told the victim he was under arrest and handcuffed him behind his back. The suspect patted the victim down and asked him where he lived. When the victim started questioning the suspect as to whom he was, the suspect showed a badge on a chain hanging around his neck for a second and told the victim he was a police officer. When the victim began to protest, the suspect took the handcuffs off the victim and walked away, last seen South bound in the 1400 block of Corona St. He did not have a radio and no vehicle was seen.
The suspect was described as a White male, in his 50's, 5'8", 170-pounds, Short Gray Hair and Mustache, Wearing a Hawaiian Type Multi-Color Shirt, Blue Jeans and had a Gray Bag on a Sling Over his Shoulder.
If you have any information regarding this suspect or this incident, please contact The Denver Police Department at 720-913-2000 or call Crime Stoppers at 720-913-STOP (7867). You can text to: CRIMES (274637) then title DMCS and enter your message or send an e-mail to metro-denvercrimestoppers.com. If the information you provide leads to the arrest and charging of a wanted individual, you can receive a cash award up to $2,000.
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Competitors Sponsorship Coors Light John Smiths Darts British Horse Racing The Grand National Heineken Budweiser World Cup 2016 FA Cup Carlsberg Arsenal FC Grass Roots Football FA Carlsberg Trophy The England Football Team FA Carlsberg Sunday Cup Carling The Scottish Cup Scottish Football Team The Scottish National Stadium Comedy Guinness Six Nations Rugby (Surfing, BMX, Skate)
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Content Carling Grolsch Static Content
Brand / Heritage Pub Finder Music News Gig Finder Festival Guide Daily Football News Competitions Guinness Static Content
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Brand / Range / Heritage TV Ads Pub Finder Football Fixtures Static Content
Brand / Range / Heritage Corona Static Content
Brand / Range / Heritage Video, Audio Text Rich Media Content High Quality
Lifestyle Content Food, Design,
Fashion, Brand UEFA Weekly Competition
(Now Closed) TV Ads James Bond
Experience Embedded Comedy Content Sharable Content
'Good Call Centre'
(Now Finished) Budlocator MOTM Match Pint Links To Bud TV Ticket Giveaways Movie/Game
Recommendations Gig Finder Football News,
updated 5 x a year Merchandise
& eCommerce eCommerce with range of
branded merch for customers and licencees Shop has a wide range of Murphys
branded merchandise Shop has a wide range of Guinness branded merchandise Sports Bar in Leicester Square Limited edition merchandise for prize giveaways YouTube Twitter Social
Media RSS Facebook Mobile
Apps CRM Registration Email Comms
eComms Questions 607,000 Budweiser 228,000 Tiger Beer John Smiths 137,000 Carlsberg Carling Zest App Carlsberg 3,268 Followers Peroni 1,250 Followers Fosters Funny 5000+ Followers Budweiser 289 Followers Tiger Beer 1,374 Followers John Smiths Racing News Corona 55,000 Likes Fosters 172,000 Likes Carlsberg Carlsberg For England Team 48,000 Likes That calls for a Carlsberg 988,000 Likes Carling 100,000 Likes Heineken 10,500,000 Likes Grolsch 15,000 Likes Peroni 49,000 likes Guinness 400,000 Likes Coors Light 162,000 Likes FB Playlist App powered by Spotify Carlsberg Drive aware third party App Heineken Rugby Fixtures App Budweiser MOTM Aurasma App Carling Zest Music Composer Grolsch Air Hockey Game Corona Beach VolleyBall Game Facebook
Log In Heineken Carling Budweiser Carling Budweiser Coors Light Signed up but no further comms Budweiser Emails promoting site content and poll questions John Smiths Newsletter with racing news content Grolsch Signed up but no further comms Carlsberg Signed up but no further comms Budweiser Coors Light John Smiths Carling Grolsch Name, Email, DOB, Postal Address, Mobile (Mandatory) Opt in email SMS Carling Welcome Email, no further comms Carlsberg Poll or Segmentation Guinness Guinness 1,500,000
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Recently a former ballet star dipped his toe in the world of gay porn calling it performance art. Many people scoffed at the idea of calling this type of entertainment as art.
But not everyone felt that way. Now comes Clayton Pettet, a 19 year-old art student at Central Saint Martins in London. In a performance piece called "Art School Stole My Virginity", he is set to pop his cherry for the first time in front of a live audience.
Clayton has been preparing for this 'performance art project' for three years. Abstaining from anal sex, all in preparation for this moment. He says, "The key thing about performance art is that it should only be performed once, and this is the ultimate once-in-a-lifetime performance." Isn't that the truth?
He continues, "Since culturally we do hold quite a lot of value to the idea of virginity I have decided to use mine and the loss of it to create a piece that I think will stimulate interesting debate and questions regarding the subject".
"I've held on to my virginity for 19 years, and I'm not throwing it away lightly. Basically it's like I am losing the stigma around virginity. I want the audience to see if anything has changed between me and my partner."
Not everyone sees this in the same light. The Lesbian and Gay Christian Movement have been critical of Clayton for cheapening sex and using this as a publicity stunt.
Spokesperson Rev Sharon Ferguson says, "My issue is around is this the right expression of someone's bodily sexuality? As an art project in front of an audience, where is the love, respect and mutuality in that? Stunts like this cheapens our own sexual relationships."
Nonetheless Clayton is currently having safe sex with his partner, and plans to debut this once in a lifetime performance on January 25, 2014.


text:
Audiences suddenly get younger
By Gary Levin, USA TODAY
On a single day this September, the ranks of 18-to-24 year
olds with TVs will suddenly swell by nearly 10%, or 2.4 million potential viewers.
Has there been a miraculous population boom? No. Nielsen
Media Research merely is adjusting its estimates of the viewing population,
as it does each fall, to coincide with the start of the new TV season.
Usually, Nielsen's annual tweaking is a non-event: Any
single age group grows at most 1% from year to year. But this year there's a
once-in-a-decade difference: Nielsen readjusts its TV-audience figures every
10 years to reflect U.S. Census data, and this fall's will take into account
changes tracked by the 2000 Census.
The adjustment reveals that Nielsen's annual projections
over the previous nine years severely underestimated the growth in the youngest
segment of viewers. Ages 12-17 will be adjusted up 4.8%, while ages 18-24 will
grow 9.6%. Those Nielsen figures are used to measure TV's most-watched shows,
and provide a yardstick to determine billions of dollars paid to the networks
for advertising time.
The discovery may not affect what we see in the new season,
but for networks courting younger viewers, such as WB, Fox, UPN and MTV, the
Nielsen corrections mean an instant boost in audience levels and in ad-dollar
Conversely, networks reaching older viewers may suffer
from Nielsen's reckoning of no growth among that segment despite an influx
of baby boomers because earlier estimates were overly generous.
"If you're WB, Fox or UPN, you're finally getting credit
for all of those viewers that were not accurately measured for many years,"
says Initiative Media analyst Stacey Lynn Koerner. "And for networks like CBS
... it's not necessarily working in their favor."
"We're all smiling here on this adjustment," says WB president
Jed Petrick. "We assume we've been undercounted for a long time. But we built
WB to cater to that group because we knew the older networks were ignoring it."
And some of those networks now confronted with startling
growth among the young viewers that many advertisers crave may now pursue
them even more aggressively. Shows including WB's Smallville and Fox's
The Simpsons, popular among teens and young adults, will see their audiences
grow even if ratings stay flat.
Nielsen ratings are expressed as a percentage of a population:
A show with a 6 rating among any age group, for instance, means 6% of those
potential viewers are watching.
But advertisers pay based on the number of viewers they
reach. "If the rating translates to more people, you can make more money," says
CBS research chief David Poltrack. "Sales departments wait for these numbers
anxiously and hope they can give them a more favorable basis to sell."
One factor driving a sudden increase among teens (up nearly
5% over last season) and the youngest adults is a startling change in the Hispanic
population as measured by Nielsen. This fall, they will represent 10% of U.S.
TV homes, up from 8% currently. In recent years, that percentage has been virtually
unchanged, even as the number of Hispanics an especially young group
has grown sharply.
The shift is one more example in networks' long-simmering
feud over discrepancies with Nielsen, the only authoritative source of ratings
And the timing was a further worry: The preliminary estimates
were delayed by Census-data revisions and weren't delivered to networks and
advertisers until late May, after they'd already begun selling ad time for the
new season.
"They were probably two to three weeks late," says ABC
research chief Larry Hyams. Some sponsors will have to adjust their already
completed ad plans to account for a jump in audience. "From an advertiser perspective,
things could be a little screwy; it just means double work."


text:
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Visa-Free St.Petersburg for Uppsala Students
- All ferry tickets (Stockholm-Helsinki-St.Petersburg-Tallinn-Stockholm)
- Russia entrance fee for visa-free travellers (25 Eur)
- 7 nights' accommodation in shared rooms/cabins, bedlinen included (4 nights in B- or C-class cabins without window on overnight ferries; 3 nights in the hostel).
- Transport to and from the ferry terminal in St Petersburg
- Guided bus tour in St Petersburg (museum fees not included, entrance is optional)
- Guided tour in the Hermitage Museum (free for students)
- Guided city walks in Helsinki and Tallinn
- Service from a Scanbalt tour manager and local guides
- Visit St Petersburg - one of the world's top destinations!
- Spend 4 crazy nights on the Baltic ferries
- Visit Helsinki and Tallinn
- Meals
- Optional activities
Watch how fun the tour is ;)
Tour Program
Day 3. St Petersburg
Day 4. St Petersburg
Optional Activities
The Catherine Palace was the Rococo summer residence of the Russian tsars, located in the town of Tsarskoye Selo (Pushkin), 25 km south-east of St. Petersburg. The compositional centre of the ensemble is the Catherine Palace – a splendid example of the Russian Baroque. Visitors are enraptured by the sumptuous décor of the Great Hall and the Golden Enfilade of state rooms that includes the world-famous Amber Room now returned to life. Today, as we enter the palace, we can sense the spirit of the times of Empresses Elizabeth and Catherine II and admire unique works of fine and applied art. More than a hundred historical monuments are scattered across the Catherine and Alexander Parks that have a joint area of 300 hectares; there are grand palaces and intimate pavilions, bridges and marble monuments, and also exotic structures imitating Gothic, Turkish and Chinese architecture that invest little corners of the parks with a romantic atmosphere.
Price includes: local guide escorting the group from St Petersburg, transport in tourist bus, stops on the way, entrance to the palace park, entrance to Catherine Palace.
Duration: 6h
*Takes place: March-April and all autumn departures
Peterhof Park, which is actually a series of palaces and gardens, located in Saint Petersburg, Russia, laid out on the orders of Peter the Great. These Palaces and gardens are sometimes referred as the "Russian Versailles". The palace-ensemble along with the city center is recognized as a UNESCO World Heritage Site. Peterhof is just as famous for the parks and its great fountains as for the palace buildings.
Includes: Entrance to the park area, transport in tourist bus, information from a local guide.
Duration: ~6h
*Takes place: May and June departures
This is definitely the best way to experience Russian culture – taste local cuisine and meanwhile watch professional artists perform traditional songs and dances!
Price: 30 Eur
Price includes: folk performance, dinner with Russian specialties - starter, main course, dessert, and of course, a vodka shot.
Glamorous and fun limousine ride, that takes you along the night streets of the city, regular stops are made by main city sights.
If you are interested in experiencing classical music and ballet the Marinsky and Hermitage Ballet are world class. We recommend you to visit their website where you also can book tickets. To get the tickets you like it's a good idea to book early. http://www.mariinsky.ru/en / http://www.hermitageballet.com/
There might be some good last-minute offers available in the ticket-booths on the streets.
Visa-Free Travel Info
To join this tour you don't need a VISA but you need to have travel Passport allowing crossing borders between non-Schengen countries.
! Please note, that ID card is not enough to travel to Russia
! Passport must be valid for at least 6 months after the tour finishes and you return to Schengen space. Those nationalities, that require a Schengen visa to stay in Scandinavia, need to check, that it is a multiple-entry visa. Single visa is valid only for one way trip!
The following info is valid only for those who are not citizens of the European Union:
! The citizens of countries that are not part of the Schengen union - all non-EU countries, except Norway, Switzerland, Liechtenstein and Iceland - please pay special attention to the following regulation:
If you have already stayed in European Union - for example, in Sweden, Norway, Denmark, Finland, etc. for more than 90 days - you need to have a valid residence permit in this country you currently live in (temporary one is also suitable). Those who will not be able to present their valid residence permit card when checking-in to the ferry to St.Petersburg, will not be allowed into Russia, and can be sent directly to their home country!
! We highly recommend you to make travel insurance valid in Russia for this trip!


text:
In a market that is glutted by designs that look identical, Lotus Romeo is like a breath of fresh air. Daring to be different and not another take on the standard Italian fare, Lotus Romeo is art and beauty applied to equestrian apparel. If you have not noticed already, there is no dearth of Italian […]
What's New
Presenting Miss Shield, Samshield's wide brim helmet
The much awaited Miss Shield Helmet by Samshield has just arrived into the United States. To be precise, it made its landing exactly 2 weeks ago at the end of week 3 of the Winter Equestrian Festival in Wellington.To say the helmet is spectacular and breathtaking in the same instant, would be an understatement. While […]
For Horses "FH" : A revolution in riding breeches
By now, you may have heard of the name, whether its "For Horses" or "FH". It is the small name in Italian equestrian apparel that is making waves across the United States. You may have spotted the iconic "4 horses" head logo surrounded by "F" and "H". Subtle and classy, "For Horses" inspires a return […]
TACKNRIDER – Wellington's Finest Tack Shop
Step into TACKNRIDER's Wellington store and you will feel like a kid in a candy store. The store is filled with equestrian goodness. From Freejump Stirrups, to Samshield Helmets to Equiline Breeches, we have it all. We have the biggest selection of Samshield Helmets, including the Samshield Shadow Matt Helmet family and the latest Crystal Medley helmet by Samshield.
We offer the latest in all things Equestrian, whether it is Shock Absorbing Stirrups from Flex-On or the Interchangeable Spurs by Mandtis. Check out the exciting new KASK Dogma Chrome Helmet that is making waves in Wellington. We promise your shopping experience will be anything but boring.
Our featured brands include Equiline, Samshield, Freejump, KASK, Tucci, Ogilvy.


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Call our experts: 888.808.FINE
Shop Personalized StationeryCarlson Craft Wedding Invitations Customize any Address Stamp to match your style!
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Whether you live in the warmth all year round or only see a few months of it, a comfortable pair of sandals is essential for supreme comfort when the sun is high. Footwear etc. has gathered an extraordinary array of supportive thong, slip-on, wedge, high heel and designer sandals fit for the most luxurious spring or summer day! If you struggle to find warm weather shoes that look as elegant as they feel, try one of the orthopedic sandals for women from Vionic sandals, Alegria sandals, Birkenstock, Taos sandals and more.
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|Property type
|Minimum Stay
|7 nights
Beautiful villa...Villa Marietta is a magnificent restored detached country house set in wonderful rustic grounds with a fantastic private swimming pool with panoramic views of the historic town of Cisternino. The town of Cisternino is under a 5 minute drive away and Ostuni, Martina Franca, Locorotondo and Alberobello only 15 to 30 minutes away. The whole region is widely considered to be the most beautiful and unspoilt in Puglia. You are only 15-20 minutes by car from excellent sandy beaches. The upper floor of the villa is available for rental, There are 3 large double bedrooms 1 with ensuite shower room all with comfortable mattresses and a family bathroom. Bright modern open plan Kitchen diner and lounge with the kitchen being equipped with all amenities including dishwasher, fridge freezer split level oven and hob. The outside boasts a fantastic modern sfioro swimming pool built to the highest standards, there is a fully equipped shower room, pool house with fridge/freezer and cooking facilities, there is also a stone built BBQ. The large terrazza offers dining facilities in the form of a large 10 seater cube rattan furniture. Why not do some sun bathing and relax on one of the modern sun beds that enjoys unobstructed sunshine from sunrise till dusk.
Private appartment on lower floor, consisting of 2 double bedrooms and kitchen/diner is occasionally used by owner but if more bedrooms are required please contact owner.
We are flexible with arrival and departure dates so that you can take advantage of cheaper mid week Ryanair flights.
Villa Marietta was just perfect for our family holiday this year. We have stayed in many villas across Europe over the years and I think this one topped them - it was so well equipped, excellent supply of linen and towels, super comfortable beds and Santa and Vito were so quick to respond if we had any questions and were really helpful - thank you so much.
We had such a great holiday, Italy is a beautiful country and we are seriously considering moving there in the future - I can certainly see me and my family there. It is such a lovely area as well, and we managed to visit lots of stunning places. We do intend on coming back to Cisternino again and that area as there was so much we didn't see and do.
The weather was surprisingly good for October - I had prepared for it not being too hot in the day so was pleasantly surprised when we had lots of lovely sunny days - the kids were in the pool everyday, which was crazy cause the water was cold due to the time of year - I did manage a few dips myself so it wasn't that bad! It did get a little chilly in the evenings which I hadn't prepped for, so wish I had taken a few more jumpers but all was good when in the Villa, as the heaters provided gave out a sufficient supply of heat.
Thanks for letting us rent your beautiful villa, I would certainly recommend it.
Really nice holiday retreat. Lovely views over the town in the distance. Pool area superb.
|Rate Period
|Weekend Night
My Standard Rate
7 night minimum stay
|No additional mandatory fees
|Refundable damage deposit


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Romania 50,000 Lei 2001 (2002)
Welcome back! Whilst you were away we added the functionality to view auctions and bid in pounds.
Romania - 2000-2004 Polymer Issue: Information on the Catawiki site about Romania 50,000 Lei 2001 (2002), 2001.
At its heart, Catawiki is an online catalogue with images of, and information on, collectibles. Our catalogue is compiled by users in a similar way to Wikipedia.


text: This article or section is a mob stub. You can help expand it by editing it. Mob boilerplate · Boilerplate help · Research: Wowhead WoWDB Use {{Stub/NPC}} for interactive non-player characters. This type of article is usually categorized under "Mobs", "Bosses" or "Creatures". Marshlight BleederLevel: 62 - 63Zangarmarsh Marshlight Bleeder can be found in Zangarmarsh. Objective of Edit [64] Stinging the Stingers Loot Edit [Marshlight Bleeder Venom] [Diaphanous Wing] External links Edit Wowhead Thottbot WoWDB Retrieved from "http://wowwiki.wikia.com/wiki/Marshlight_Bleeder?oldid=2607163" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Ferocity beasts Mob stubs Level 62 - 63 NPCs Wasps Zangarmarsh mobs Add category Cancel Save


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Sydney Roosters beat Newcastle Knights 29-12 in NRL clash at Sydney Football Stadium
A much-improved second-half performance helped the Sydney Roosters see off cellar-dwelling Newcastle 29-12 in a scrappy NRL clash on Saturday at the Sydney Football Stadium.
|at SFS, June 14 2014
|Chris Houston (NEW) - 50
|Sam Moa (SYD) - 19
|Sonny Bill Williams (SYD) - 2
|Run (m):
|Sam Moa (SYD) - 164
|Sydney Roosters: 29 (R Tuivasa-Sheck 2, S Kenny-Dowall, J Friend, I Liu tries; J Maloney 4 cons, field goal)
Newcastle: 12 (J Smith, J Mamo tries; T Roberts 2 cons)
Interview: Trent Robinson
Interview: Wayne Bennett
Interview: Willie Mason
View full stats sheet
Tries from Roger Tuivasa-Sheck, Shaun Kenny-Dowall, Jake Friend and Issac Liu after the restart sealed the two points in a scrappy affair played in front of a subdued crowd of 9,847.
It was a complete contrast to the premiers' opening 40 minutes when they looked disjointed in attack and lethargic in defence as the visitors belied their lowly position with a fully-committed showing.
The Knights came into the game on the back of six straight losses, but on the day the NRL announced it had taken ownership of the club from Nathan Tinkler's Hunter Sports Group, they appeared to have an extra spring in their step as they opened up a 12-6 lead at the break.
Veteran back-rower Jeremy Smith darted over from close range within eight minutes to open the scoring with Tyrone Roberts adding the extras.
A Tuivasa-Sheck four-pointer converted by James Maloney levelled the scores before Jake Mamo chalked up his first NRL try on the stroke of half-time with an excellent finish in the corner.
Former New South Wales halves pairing Mitchell Pearce and Maloney started to exert their influence in the second stanza as the Roosters woke up from the early slumber and, 11 minutes after half-time, Tuivasa-Sheck scored his second of the night.
The Kiwi winger took full advantage of James McManus' indecision under Maloney's towering bomb and touched down.
Kenny-Dowall then gave the hosts the lead for the first time with McManus again targeted, this time by Pearce, as the centre hauled in a perfectly weighted kick.
That try knocked the stuffing out of the Knights, who were camped in their own half for most of the second period and failed to add to the first-half tally, and Friend and Liu added some extra gloss to the scoreline late on.
Topics: rugby-league, sport, sydney-2000, nsw, australia, newcastle-2300
First posted


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Peach in Spanish
Does your child know what a peach seed looks like, or the flower before fruit appears? Check out the phases with this beautiful coloring page featuring el melocotón, that's "peach" in Spanish!
Follow the link for more coloring pages with fruits in Spanish.
Click to find similar content by grade or subject.


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During my last few years covering the Nationals, I never had any major travel problems. Not once.
This year, I've had a connecting flight to San Francisco delayed by two hours because President Obama was at the airport, causing me to miss most of the pregame access during the first day of the Nationals-Giants series, and now I've spent the morning in a mad scramble to rebook a flight to New York after my morning flight to LaGuardia was canceled by air traffic control (probably because of the storm that's moving up the East Coast). But I think it's because of the Curse of Roch Kubatko.
Anyway, I'm rebooked now on a 12:24 flight to JFK, which should get me to New York in plenty of time before tonight's 7:10 start at Citi Field. The forecast looks okay by game time, though I wouldn't expect players to be taking BP outside.
Jordan Zimmermann gets the start for the Nationals, pitching at Citi Field for the first time this year. Zimmermann faced the Mets on the road twice last year with mixed results. His first outing there, last April 26 in front of a crowd of 40,023, was an impressive statement for the rookie. Zimmermann got his second win in as many big-league starts, striking out five batters and allowing a run in 5 1/3 innings.
His next outing wasn't nearly as smooth. Zimmermann pitched five innings, giving up five runs on eight hits last May 27. Zimmermann struck out eight in that game, but allowed a big two-run homer to Daniel Murphy in the sixth inning (one of the Nationals' several adventures with home run replays at Citi Field).
Tonight, Zimmermann makes his final start of the year, having continued to oscillate between flashes of brilliance and outings short-circuited by homers. He's allowed seven of them in 25 innings this year, and though that's an extraordinarily high percentage, Zimmermann did give up 10 homers in 91 innings last year. His fastball is potent enough that when not located properly, he's probably going to surrender some bombs.
He'll get another crack at Citi Field tonight, against a Mets team that has all but packed it in for the year, and a shot to finish on a high note.


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Tourist Directory > Hotels
Summer Hotel
Contact:(+65) 6841 3229
Description: Enjoy comfort and privacy in the heart of Geylang by saving your dollars on lodging in Summer Hotel. Affordable and easy on the pocket, you will be glad you had that extra cash after exploring the streets filled with eateries serving a large variety of local cuisine. Most of the places open till past midnight so you will never be left hungry.
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Film Review: Ted's back and he's ballsier than ever
THE main star of Ted 2 may on the outset seem lovable and cuddly, but as we learned when he first burst onto our cinema screens three years ago, you won't get more lewd and crude than this roguish teddy-bear.
Though the novelty factor of the original has disappeared, the return of foul-mouthed Ted (voiced by Seth MacFarlane) and his best buddy John Bennett (Mark Wahlberg) is a welcome one.
This deliciously puerile comedy begins after our furry friend (who came to life after a childhood wish by John) and his new wife Tami-Lynn (Jessica Barth) are denied the chance to adopt a baby and Ted must prove in a court of law that he's an actual person.
But before they even think of adopting, Ted comes up with the idea of getting a sperm donor, seeing as he himself can't impregnate his wife due to not having a human penis.
Cue all sorts of mayhem in their hometown of Boston as Ted and John embark on a journey to find much-needed sperm to make the new baby. They decide to break into the home of one stunningly handsome man's mansion to retrieve some 'jizz' while he sleeps.
Most Irish people won't recognise the victim, but in fact he's one of America's most famous faces: American football's highest-paid player, Tom Brady of the New England Patriots, who's married to gorgeous supermodel Gisele Bundchen.
Let's just say that the gruesome twosome's attempts at masturbating the quarterback and reliving him of some precious protein shake don't go exactly as planned.
Generous John offers to donate his swimmers at a local sperm bank, which leads to the most hilarious scene in the film when he takes a wrong turn and ends up in a bit of a mess, which at this stage we'll leave to your imagination.
There are also a couple of funny cameos from the likes of Liam Neeson, Jay Leno and Amanda Seyfried as bungling, bong-loving lawyer Samantha.
After getting lost in a huge marijuana field, Ted, John and Samantha head to New York to convince Morgan Freeman's super-dooper lawyer Patrick Meighan to take on their case.
The storyline and humour begin to wane towards the end, though.
In an unnecessary and confusing subplot, creepy Donny (Giovanni Ribisi), who in the first film nearly killed Ted, is back. He is selling toy company Hasbro on an idea to kidnap Ted and carve him up to discover the secret to why he talks so they can duplicate him millions of times over for everyone who ever wanted the kind of friendship John and Ted have.
It all comes to a head at Comic-Con, where disaster strikes again.
There are also a couple of great musical numbers, including an opening wedding scene to Irving Berlin's Steppin Out With My Baby, while Seyfried — who is no stranger to singing given her history with Mamma Mia! — is tipped to win the film an Oscar for her track Mean Ol Moon.
Verdict: 3/5


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What is the policy on this on most enterprises?
3 Replies
Nov 6, 2008 at 2:33 UTC
Well Jack, I've never been in an office that has UPS connected to each workstation, but here's how I set up an accountancy firm, with HEAVY responsibilties via their Payroll Beauro.
UPS on all servers (that's a must) and I also have the network switches, and ADSL on UPS, and I have another UPS @ the Payroll computer.
This means when we get an outage, and they seem to becoming more frequent, the payroll dept keeps trading, and paying peoples wages. Power usually returns within 30mins, and we can keep trading till the batteries run out.
I have another client, who I'm trying to convince, to have "Bi-Wire" feed to the building that can be fed by a LARGE generator (no problems here, 'cos they actually hire these beasts) So when the power goes out, the generator kicks in.
Sounds drastic? Not really, as this is HQ, and if it goes down, none of the other six branches can access the systems. If you're looking at a multi million pound business, it makes sense.
Nov 6, 2008 at 9:47 UTC
The policy will vary based on the impact and frequency/duration of power outtages. The company I used to work for had a database application that absolutely could not have a client crash without corrupting indexes. And everyone used it. So we had UPSes on every station. Here, at a larger company, we only protect servers (of course), critical apps like Accounting and Payroll, the internet gateway, and stations that connect to resources that don't handle outtages very well, like legacy databases.
Nov 7, 2008 at 7:57 UTC
Big UPS's on everything in the server room...including all servers, switches and phone system. Smaller UPS's on all the machines deemed "critical" in the building including time clocks, payroll, and all other switches. We are a hospital so most of that, including the entire server room, is on generator...so it only has to hold for a minute at most. All of the other PC's do not have UPS's.
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


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Decisions, Decisions and Other Episodes is a fan-fiction VHS/DVD Release featuring one Maya and Miguel episode, one Pokemon episode, one All Grown Up episode, one SpongeBob SquarePants episode, one Thomas and Friends episode and one The New Adventures of Winnie The Pooh episode.
- Decisions, Decisions (Maya and Miguel)
- Tunnel Vision (Pokemon)
- Fools Rush In (All Grown Up)
- Fools In April (SpongeBob SquarePants)
- Thomas and The Circus (Thomas and Friends)
- Rabbit Takes A Holiday (The New Adventures of Winnie the Pooh)
- Distributed by USA Home Entertainment.
VHS/DVD Artwork
Front Cover
- Top: Maya and Miguel
- Bottom: Francine from "Fools Rush In"
Back Cover
- Snubbull from "Tunnel Vision"
- Thomas from "Thomas and The Circus"


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76 - 69 Final
Henderson, Duke easily overmatch St. John's once againFeb 20, 2009 - 6:12 AM NEW YORK (Ticker) -- A matchup with struggling St. John's was all Duke needed to get back on track.
Gerald Henderson scored 19 points as the ninth-ranked Blue Devils snapped a two-game skid with a 76-69 victory over the Red Storm in Thursday's non-conference matchup.
Jon Scheyer had 18 points and Kyle Singler added 15 for Duke (21-5), which beat St. John's for the sixth straight time, a series that has included a number of routs in recent years.
The Blue Devils, who were routed by North Carolina before losing at Boston College on Sunday, have beaten the Red Storm by an average of 16.5 points over the last six meetings despite a late charge by the hosts in this one.
"This has been a tough time for us, these last couple weeks," Duke coach Mike Krzyzewski told the Charlotte Observer. "We found out two new things today (with Elliot Williams and Miles Plumlee) which were good. We've got to get the other guys playing at the level we know we can play at."
For St. John's (12-14), the loss was its fifth straight and 13th in its last 16 games.
The Red Storm actually started the game well offensively, shooting 61 percent (11-of-18) in the first half. However, they found themselves down by 11 points at 37-26 due to 14 turnovers.
St. John's went on to finish with 18 giveaways while going 54 percent (27-of-50) from the floor.
Duke maintained a nice cushion through most of the second half, pulling ahead, 50-34, with 14:19 to play behind an 11-2 run.
D.J. Kennedy scored 20 points for St. John's.
Despite Duke's success in the series, Krzyzewski made a change to the starting lineup, inserting Williams as a guard. The freshman, who is more known for his defense, responded well, scoring a season-high 11 points on 5-of-6 shooting.
"It was nice," Williams told the Observer. "I was starting for Duke as a freshman at Madison Square Garden. I just tried to do the best I could. I put a lot of ball pressure on there and tried to come out with a win."
With normal point guard Greg Paulus pulled out, Scheyer moved over from shooting guard to handle the duties running the offense.
"It's not a thing where I'm just playing point guard for 40 minutes," Scheyer told the newspaper. "If someone is pressuring me, I can just hit Kyle. I hit Kyle a couple times. He brought it up. Or Gerald can. Or Elliot in that lineup. It's not really a big deal."
Although Paulus and other former starting guard Nolan Smith came off the bench, the Blue Devils ended up with only four points from their reserves. That is something that will need to improve as Duke hosts Wake Forest on Sunday.
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roots#1 Cards Fan Added 10 roots
Duke(9) 76, St. John's 69 FinalFeb 20 3:52 AM
- 5
rootsMattDOOM777 Added 5 roots
Duke(9) 76, St. John's 69 FinalFeb 19 10:26 PM
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Feb 19 9:17 PM
Feb 19 7:54 PM
Related News
- Krzyzewski has hernia surgery, expects to be healthy for Rio May 9
- Duke's Grayson Allen has hernia surgery Apr 22
- Duke's Jefferson receives medical hardship waiver Apr 22
- Duke freshman guard Derryck Thornton to transfer Apr 10
- Grayson Allen to return to Duke for junior season Apr 6
- Duke's Brandon Ingram declares for NBA Draft after 1 season Apr 4
- Duke: Coach K recovering from knee replacement surgery Apr 3
- Duke ends with longer-than-expected NCAA run, short bench Mar 25
- Duke's title defense ends with 82-68 loss to Oregon Mar 25
- Oregon takes the national stage against defending champ Duke Mar 23
- Defending champ Duke, upstart Yale ready for 2nd round clash Mar 18
- It's David vs. Goliath when UNC Wilmington meets Duke Mar 17


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Kataginu was a Rokugani sleeveless jacket with exaggerated shoulders. [1] [2]
- ↑ Scenes from the Empire 28, by Robert Denton, Seth Mason, and Shawn Carman
- ↑ Legend of the Five Rings; Fourth Edition, p. 207
|This page uses content from Wikipedia. The original article was at Kataginu. The list of authors can be seen in the page history. As with L5r: Legend of the Five Rings Wiki, the text of Wikipedia is available under the GNU Free Documentation License.
|This article is a stub. That means that it has been started, but is incomplete. You can help by adding to the information here.


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If you are suspected of driving while under the influence of alcohol or drugs, you may be arrested for DUI (driving under the influence). Depending on the state, the specific crime is also called DWI (driving while intoxicated), OUI (operating under the influence), or OWI (operating while intoxicated). A DUI conviction has serious consequences (for example, losing your driving privileges); but an experienced DUI attorney can often get the charges dropped or reduced, or may be able to negotiate lesser penalties depending on your circumstances and your past history.


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For years, we've provided full disclosure of our products' ingredients, including what ingredients are not contained in the product .Our "Contains No" statement lists gluten where applicable. For years we've gone one step further in assuring the gluten free status of our products by testing our finished products for gluten. We're also highlighting the gluten free status with a logo on the front panel of our labels. This helps your consumers quickly recognize the gluten free status without having to search for it elsewhere on the label
We phased this logo onto the labels of the products tested, beginning in the last quarter of 2009.
We confirm that none of the products we offer contain more than 10 ppm gluten, and use this logo on the front panel of the label.
An estimated 12 million people follow a gluten free diet. One in 133 has Celiac disease and more remain undiagnosed. Dietary supplements are important for those on a gluten free diet, as a gluten free diet often lacks certain vitamins and minerals, and many have nutrient malabsorption. Be prepared for National Celiac Awareness month in May and highlight our Double Verified Gluten Free Guaranteed Program logo on your labels!
Your customers will clearly see which of our dietary supplements are gluten free by the logo on the front panel of the label. They can also take comfort knowing that our products labeled with the logo have undergone in-house analytical testing for gluten (tested to contain no more than 10 ppm, or parts per million of gluten), to ensure they are gluten free. Our program provides a consistent, science-based set of standards that helps consumers easily choose our premium supplements.
In mid-2013 the FDA announced a decision defining Gluten Free as 20ppm, more than double what we allow in products.
All of Vitamer Labs finished products undergo gluten testing using an ELISA (Enzyme-linked immunosorbent assay) procedure (a machine that detects the presence of an antigen in a sample) to detect the presence of gluten


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I attend class today in the school time Mr./Mrs. Chen said that,"Today work is each schoolmate catches several snails." In my heartthought where can have the snail? I as soon as get the home, rushes to the vegetable garden in, liftsthe subtilis, I as soon as looked, happen to has several snails, thenexchanges the newspaper "the small cradle", again puts in a each onegreatly fat big snail "the cradle" in, brings them to go home. Gets in the home I to think that, "What does the snail eat?" Ithereupon have performed an experiment, I put them on a big disc, onall is putting various leaf, then I am waiting patiently, crossed awhile snail gradually to crawl to the cabbage leaf. Oh! The originalsnail likes eating the cabbage leaf! How many centimeters do I also want to measure its elongation? I put aruler on the table, I put the snail on a ruler head, puts the whitevegetable leaf on another, the snail continuously crawls to the whitevegetable leaf, I look at the ruler, is surprised, the snail length4.5 centimeters, this is shortest, long has 5 centimeters. I had alsoknown snail's length, thinks "the snail you or to be long quickly" I one all the white vegetable leaf which buys back from thefood market eat daily to them, sometimes also gives a water, slowlythe snail has grown up some, I also discovered the snail shell is adextrorotation. I am praying in the heart: "The snail, you must growup in a big hurry! Future you also will be able to give birth crowd ofyoung snails "
欢迎阅读《作文:Catchesthesnail》,"查字典"每日为您更新更多优秀的"三年级作文",请随时关注! Iattendclasstodayinthes