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Distribution of Y-Receptors in Murine Lingual Epithelia
Maria D. Hurtado,
Affiliation Department of Pediatrics, University of Florida, Gainesville, Florida, United States of America
Andres Acosta,
Affiliation Department of Medicine, University of Florida, Gainesville, Florida, United States of America
Paola P. Riveros,
Affiliation Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health (NIH), Bethesda, Maryland, United States of America
Bruce J. Baum,
Kirill Ukhanov,
Affiliation Departments of Neuroscience and Psychiatry, Center for Smell and Taste, University of Florida, Gainesville, Florida, United States of America
Alicia R. Brown,
Cedrick D. Dotson,
Herbert Herzog,
Affiliation Neuroscience Program, Garvan Institute of Medical Research, Sydney, Australia
Sergei Zolotukhin
* E-mail: szlt@ufl.edu
Maria D. Hurtado Andres Acosta ... Sergei Zolotukhin
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Peptide hormones and their cognate receptors belonging to neuropeptide Y (NPY) family mediate diverse biological functions in a number of tissues. Recently, we discovered the presence of the gut satiation peptide YY (PYY) in saliva of mice and humans and defined its role in the regulation of food intake and body weight maintenance. Here we report the systematic analysis of expression patterns of all NPY receptors (Rs), Y1R, Y2R, Y4R, and Y5R in lingual epithelia in mice. Using four independent assays, immunohistochemistry, in situ hybridization, immunocytochemistry and RT PCR, we show that the morphologically different layers of the keratinized stratified epithelium of the dorsal layer of the tongue express Y receptors in a very distinctive yet overlapping pattern. In particular, the monolayer of basal progenitor cells expresses both Y1 and Y2 receptors. Y1Rs are present in the parabasal prickle cell layer and the granular layer, while differentiated keratinocytes display abundant Y5Rs. Y4Rs are expressed substantially in the neuronal fibers innervating the lamina propria and mechanoreceptors. Basal epithelial cells positive for Y2Rs respond robustly to PYY3–36 by increasing intracellular Ca2+ suggesting their possible functional interaction with salivary PYY. In taste buds of the circumvallate papillae, some taste receptor cells (TRCs) express YRs localized primarily at the apical domain, indicative of their potential role in taste perception. Some of the YR-positive TRCs are co-localized with neuronal cell adhesion molecule (NCAM), suggesting that these TRCs may have synaptic contacts with nerve terminals. In summary, we show that all YRs are abundantly expressed in multiple lingual cell types, including epithelial progenitors, keratinocytes, neuronal dendrites and TRCs. These results suggest that these receptors may be involved in the mediation of a wide variety of functions, including proliferation, differentiation, motility, taste perception and satiation.
Citation: Hurtado MD, Acosta A, Riveros PP, Baum BJ, Ukhanov K, Brown AR, et al. (2012) Distribution of Y-Receptors in Murine Lingual Epithelia. PLoS ONE 7(9): e46358. https://doi.org/10.1371/journal.pone.0046358
Editor: Wolfgang Meyerhof, German Institute for Human Nutrition, Germany
Received: June 22, 2012; Accepted: August 29, 2012; Published: September 26, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by 1R01DK62302-01 (National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases) to SZ; the Division of Intramural Research of the National Institute of Dental and Craniofacial Research in part supported this research (BB). HH is funded by an NHMRC of Australia Senior Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Neuropeptide Y (NPY), Peptide YY (PYY), and Pancreatic Polypeptide (PP) belong to a family of peptides sharing similar hairpin-like PP-fold structural homology and evolutionary history [1]. NPY is widely expressed in the central as well as in the peripheral nervous system; PYY is released mostly by L-endocrine cells in the distal gut epithelia, while PP is produced by specialized cell in the pancreas. These peptides mediate various complementary and often opposing metabolic functions such as appetite and satiation, energy intake and expenditure; cell proliferation, migration, and differentiation; neuromodulation, angiogenesis, osteogenesis, and many other biological processes. This diversity of functions is mediated through the extensive redundancy of PP-fold peptides binding to five known receptors (Rs), Npy1r, Npy2r, Npy4r, Npy5r, and Npy6r (hereafter referred to as Y1R, Y2R, Y4R, Y5R, and y6R). The YRs belong to the rhodopsin-like superfamily of metabotropic G Protein-Coupled Receptors (GPCRs). All YRs act through Gi/o signaling pathway inhibiting cAMP synthesis, activating Protein Kinase C (PKC), Mitogen-Activated Protein Kinase (MAPK), or Phospholipase C (PLC), thus inducing release of intracellular Ca2+. In addition, YR downstream signaling modulates the conductance of membrane Ca2+ and inwardly rectifying K+ (GIRK) channels. The pharmacological redundancy of NPY family receptors is further increased by the action of dipeptidyl-peptidase-IV (DPPIV), a serine exopeptidase that truncates NPY and PYY at their N termini producing peptides NPY3–36 and PYY3–36 and thereby changing their binding specificity.
Adding more complexity to the physiological role of PP-fold peptides, we have recently documented that PYY3–36 is present in saliva and showed the expression of its preferred receptor, Y2R, in the basal layer of the progenitor cells of the tongue epithelia and von Ebner's gland [2]. Although the innate physiological functions of salivary PYY3–36 are yet to be fully determined, we have presented data that support a role of salivary PYY in the modulation of food intake (FI) and in the accumulation of body weight. This anorexigenic effect is apparently mediated through the activation of Y2 receptors in a subpopulation of cells in the oral mucosa [2]. Other groups have shown the presence of NPY in human saliva [3] and the expression of the NPY gene in the taste receptor cells (TRCs) in the rodent [4]. Given the widespread pattern of expression of PP-fold peptides and cognate YRs in other tissues, and taking into account their pleiotropic functions and the redundancy of their interactions, it was important to determine whether other members of the NPY gene family are also expressed in the oral cavity. The purpose of the current investigation, therefore, was to identify the expression of genes coding for most studied members of the YR family (Y1R, Y2R, Y4R, Y5R) in tongue epithelia cells.
In vitro YR antibody validation
HEK 293 cells were transfected with plasmids expressing murine Y1R, Y2R, Y4R, Y5R, or GFP cDNAs under the control of the strong constitutive Cytomegalovirus-Chicken b-actin (CBA) promoter. Two days after transfection, cells were fixed on cover slips and subjected to immunocytochemistry (ICC) analysis using the respective antibodies and conditions employed for YR detection in tissue samples (see Immunostaining section, below). The source of all antibodies, dilutions, and controls is listed in Table 1.
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Table 1. Antibodies used for immunolocalization studies.
https://doi.org/10.1371/journal.pone.0046358.t001
Ethics Statement.
This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida (UF, Permit Number: 03059 "Modulation of taste sensitivity by PYY signaling"). All procedures were done in accordance with the principles of the National Research Council's guide for the Care and Use of Laboratory Animals. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Tissues were collected from sacrificed animals and immediately frozen in O.C.T. Compound. Mice were housed at 22–24°C in a 12 hours dark/light cycle and had access to water and food ad libitum unless indicated otherwise.
Tongues and brains were harvested from wild type C57Bl/6 male mice from Charles River, as well as from homozygous Y1R KO [5] and Y2R KO [6] mice. Both KO strains are maintained at the UF animal facility. Genotype was confirmed using the following genotyping primer sets: Y1R KO: primer mY1-H 5′-TGGCAAAACAGGTCCCTG-3′; and primer mY1-P5 5′- CTAGCCAGTTGGTAATGG-3′; Y2R KO: primer NHE-3A 5′-TTAACATCAGCTGGCCTAGC-3′, and primer NHE-6 5′-GGAAGTCACCAACTAGAATGG-3′.
Immunostaining
YR immunoreactivity staining.
For specific information on the source of all antibodies, dilutions, and controls please see Table 1. Tissues were harvested from overnight fasted animals and immediately frozen in O.C.T. Compound. Four µm thick coronal sections were cut using a cryostat (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany), mounted onto Fisher Superfrost Plus slides and post-fixed in 4% paraformaldehyde for ten minutes. YR immunolocalization was conducted utilizing the tyramide signaling amplification (TSA) kit (Perkin Elmer). Tissues were blocked in 0.3% H2O2 in Tris-buffered saline (TBS) for 30 minutes at room temperature (RT) to eliminate endogenous peroxidase activity, followed by blocking with 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl and 0.5% Blocking Reagent from Perkin Elmer; for 60 minutes at room temperature to reduce nonspecific antibody binding. Sections were then incubated with primary rabbit anti-YR in TNT (0.1 M Tris HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) overnight at 4°C, followed by secondary goat anti-rabbit IgG (Fab')2 conjugated to horseradish peroxidase (Abcam; 1∶1000 for 60 min at RT). Staining was detected using Fluorescein provided in the TSA kit (1∶300 for 7 min at RT). Negative controls were run concomitantly. All sections were counterstained with DAPI (4′,6-diaminidino-2-phenylindole).
Mirror section staining.
To overcome the technical limitations imposed by the origins of antibodies (all four a-YRs were raised in rabbits), we utilized a mirror section staining method. In brief, the first section is mounted with its inner surface turned upwards on one slide, whereas the subsequent adjacent section is mounted on the next slide with its outer surface upwards. In this way, the complementary faces could be hybridized to two different antibodies without concern about the secondary antibody cross-reactivity. Although not entirely identical, the characteristic structures of the epithelial layers on the two separate slides provide sufficient visual guidance to identify distinctive cell layers or even particular cells.
YRs/NCAM (neural cell adhesion molecule) double staining.
Because YR and NCAM primary antibodies were raised in rabbits, double immune-labeling was performed using a modified indirect immunofluorescence (IF) protocol and the TSA kit allowing for the localization of two antigens in the same tissue specimen when both primary antibodies produced in the same host [4], [7]. The use of TSA permits control for type I and II interference [4]. A first primary antibody is used at very low concentrations that can only be detected with amplification kits such as TSA and not with a conventional protocol (interference I). A first secondary antibody is then used to detect this first primary, however only the Fab' fragment can be used in order to avoid binding of a second primary antibody to the first secondary antibody (interference II). This protocol has been fully described by Kaya et al. [7] and has been followed without modifications. Specifically, immediately after detection with the TSA Fluorescein, sections were extensively washed in TNT and then blocked with 10% donkey serum in TNT for 60 min at RT. Tissues were subsequently incubated with the second primary antibody rabbit anti-NCAM (Millipore; 1∶250 in 10% donkey serum overnight at 4°C), and visualized, using standard methods, with AF555 donkey anti-rabbit IgG (Invitrogen, 1∶1000 in TNT for 60 min at RT). All double-labeled sections were counterstained with DAPI.
In situ hybridization (ISH) was performed using QuantiGene® ViewRNA ISH Tissue Assay (Affymetrix) following the manufacturer's protocol. Gene-specific probe sets for murine Y1R, Y2R, Y4R, and Y5R mRNAs consisting, on average, of 20 different probe pairs were custom-designed and synthesized by Affymetrix. Where available, the respective tissues from the germline KO mice (Y1R, Y2R) were used as negative control samples. For Y4R and Y5R hybridizations, a no-probe sample was utilized as a negative control per the Affymetrix manual's recommendations. Hybridized target mRNAs were visualized using confocal fluorescent microscopy, or by bright field microscopy.
Ca2+ imaging
Adult C57Bl/6 mice were used in this study. Following sacrifice, the tongue was quickly dissected in the ice-cold modified artificial cerebrospinal fluid (ACSF) saturated with 95% O2 and 5% CO2 that contained: 120 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 1.25 mM Na2HPO4, 1 mM MgSO4, 1 mM CaCl2, 10 mM glucose, 305 mOsm. Small tissue blocks were embedded in 4% low-melting agarose and sectioned using Leica V1000 vibratome. Lingual slices were placed in a perfusion chamber (RC-26, Warner Instruments). Slices were incubated with 10 µM fura-2/AM for 30 min at 37°C. The chamber was then mounted on the stage of the upright microscope (Zeiss Axioskop FS2) equipped with a water-immersion 40× objective lens (Neofluar, NA1.4). Solutions were applied locally using a fine bore capillary attached to a 8-channel Teflon manifold (BioLogic, France) under the control of the perfusion system (VC-6, Warner Instruments) controlled by pClamp 9.2 (Molecular Devices). Images were acquired with a cooled CCD camera (ORCA R2, Hamamatsu) under the control of Imaging Workbench 6 software (INDEC Systems). The illumination system (Lambda DG-4, Sutter Instruments) was coupled to the microscope with a liquid light guide. Ratiometric imaging was performed using a Fura-2 filter set (excitation at 340 nm or 380 nm, emission at 510 nm, dichroic mirror 435 nm). Before calculating the ratio at 340/380 nm all images were corrected for background fluorescence. Off-line analysis and image processing was performed using Imaging Workbench 6, ImageJ 1.46 (available at http://rsbweb.nih.gov/ij/index.html) and ClampFit 9.2 (Molecular Devices).
RNA extraction and RT-PCR analysis
Approximately 1×2 mm section of the non-gustatory tongue epithelium anterior to the circumvallate papillae (CV) was cut with microscissors under a dissecting microscope; the muscle layer was removed. A 1 mm punch (biopsy punch- Miltex, Inc, York, PA) was used to collect the CV. Although the majority of the proximal epithelia tissues and the ventral muscle were removed, the samples likely included some non-gustatory tissue. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA); DNA was digested with RNase free DNase (Qiagen Inc, Valencia, CA) followed by RNA cleanup with the RNeasy Micro kit (Qiagen). One microgram of RNA was reverse-transcribed with Superscript III (Invitrogen). Products were amplified for 35 cycles with gene-specific primers (Table 2) utilizing RedTaq Ready Mix (Sigma-Aldrich, Saint Louis, MO). "No cDNA" samples were prepared for each primer set. DNA contamination was tested with control, intron-spanning primers. Products were resolved in a 2% agarose gel.
Table 2. NPY family RT-PCR primers.
Previously, we have demonstrated the expression of Y2Rs in basal lingual epithelial cells [2]. The dorsal surface of the tongue is covered by a specialized mucosa consisting of keratinized stratified epithelium [8]. Stratified epithelium is characterized by the high turnover rate of cells in response to mechanical and chemical contacts. Because of the known functions of YRs in cell proliferation [9], it was of interest to explore whether epithelial cells express these receptors.
RT-PCR analysis
To characterize the expression of other members of YR family, we performed RT-PCR analysis using RNA isolated from the non-gustatory lingual epithelium, as well from the taste buds. All members of the NPY family, including four cognate receptors, Y1R, Y2R, Y4R, and Y5R, as well as their respective agonists NPY and PYY were expressed in taste buds (Fig. 1) and in non-gustatory epithelium (data not shown). The identities of PCR fragments were confirmed by sequence analysis (data not shown). In addition several pairs of primers were utilized for each member of the NPY family, including those designed to amplify intron-spanning fragment of the respective gene. PCR amplification was mRNA-specific and no chromosomal DNA contamination was detected (data not shown).
Figure 1. RT-PCR analysis of the expression of NPY family members in the lingual epithelia.
The names of the genes and the expected PCR fragments sizes (in bp) are shown above the respective lanes. NC – negative (no cDNA) control.
https://doi.org/10.1371/journal.pone.0046358.g001
ISH analysis
To validate the RT-PCR results using an independent method, we analyzed the expression of the Y1R, Y2R, Y4R, and Y5R genes using an ISH assay in coronal sections of lingual tissues. Each custom-designed probe set included, on average, 20 oligonucleotides pairs complementary to the target mRNA in the regions divergent from other family members. As such, each probe set represented a highly specific reagent hybridizing to the respective target at high selectivity. The protocol was validated in brain sections, while tissues from KO mice, when available, were used as negative controls (Fig. 2A, C). Alternatively, no-probe control tissues from C57Bl/6 mice were utilized. All control tissues exhibited no non-specific binding (data not shown). The positive control hybridization with the brain tissues resulted in a pattern consistent with the data described in several publications [10], [11], [12], [13], [14], [15]. These latter results clearly demonstrated the specificity of the YR probe sets.
Figure 2. Validation of in situ hybridization (ISH) probe sets by ISH analysis of Y-receptor (YR) gene expression in the dentate gyrus of the mouse brain.
A and C – negative control samples, Y1R KO and Y2R KO, respectively; B, D, E, and F – positive control samples, WT C57Bl/6 mice. Representative fields outlined by the dashed rectangles in panels A through F are shown as close-up images either below the respective panels (panels A, B, C, and D), or to the right of the panel (panels E and F). Red dots show fluorescently visualized YR mRNAs. DAPI-stained nuclear DNA is shown in pseudo-colored green hue for better viewing.
In the dorsal epithelial layer of the tongue, the pattern for YR mRNAs expression, appeared to be different for different subtypes: Y1R expression was more prevalent in the superficial layers, including keratinocytes (Fig. 3B), while Y2R was more pronounced in, but not restricted to, the basal epithelial layer (Fig. 3D). Interestingly, we detected neuronal fibers positive for Y4R-encoding mRNA in the subepithelial region close to the basal laminae (Fig. 3E, arrows). Y5R mRNA staining was faint but clearly detectable in all layers of the epithelia, including keratinocytes (Fig. 3F). Similar to the brain tissue negative controls, the nonspecific staining in the negative control KO sections was negligible (Fig. 3A, C), as well as in the no-probe control sections with tissue from WT mice (data not shown).
Figure 3. In situ hybridization analysis of Y-receptor (YR) gene expression in the dorsal lingual epithelium.
A and C - (−) control samples, Y1R KO and Y2R KO, respectively; B, D, E, and F – wt C57Bl/6 samples. Bidirectional arrows indicate dorsal/ventral (D/V) coordinates of the section. One-direction arrows in panel E point at Y4R-positive neuronal fiber/s. Red dots show fluorescently visualized YR mRNAs. DAPI-stained nuclear DNA is shown in pseudo-colored green hue for better viewing.
In the gustatory epithelium from the CV, the expression of the respective Y1R, Y2R, Y4R, and Y5R mRNAs also showed an absence of non-specific binding from both KO control mice (Fig. 4A, D) and no-probe control sections from WT mice (data not shown). The distribution of YR expression was not restricted to any particular cell population within the taste bud, but rather appeared in many cells localized at the apical and basolateral portions of the CV (Fig. 4B, C E, F, G, H, I).
Figure 4. In situ hybridization analysis of Y-receptor (YR) gene expression in the circumvallate papillae.
A and D – negative control samples, Y1R KO and Y2R KO, respectively; B, E, F, and G - samples from wt C57Bl/6 hybridized to Y1R, Y2R, Y5R, and Y4R probe sets, respectively; C, H, and I – close up images of the representative areas from the respective panels (B, G and F, respectively) outlined by white dashed rectangles. The borders of several taste buds are outlined with yellow dashed lines. Red dots show fluorescently visualized Y-receptor mRNAs. DAPI-stained nuclear DNA is shown in pseudo-colored green hue for better viewing.
Validation of YR antibodies
To characterize the expression of YR subtypes in a cell-specific manner, we next conducted an IHC analysis. YRs belong to GPCR family of receptors and, as such, are homologous to each other as well as to other GPCRs [16]. It is recognized that a significant fraction of commercially available GPCR antibodies lack specificity and selectivity resulting in binding to other subtypes within the family [17], [18]. It was therefore essential to validate the antibodies used herein prior to conducting an IHC experiment. Utilizing ICC analysis in HEK 293 cells expressing Y-receptors subtypes with the conditions employed for YR detection in tissue samples, we clearly demonstrated that each antibody reagent interacts exclusively with its respective antigen, i.e. Y1R, Y2R, Y4R, or Y5R with no detectable cross hybridization to any other YR subtypes (Fig. 5A).
Figure 5. Validation of Y-receptor (YR) antibodies.
A - Immunofluorescence (IF) analysis of 293 HEK cells expressing murine YR cDNAs. Columns – cells transfected with Y1R, Y2R, Y4R, Y5R-expressing plasmids, respectively. Rows – cells on cover slips incubated to a-Y1R, a-Y2R, a-Y4R, or a-Y5R antibodies, respectively. Please note peripheral (membrane-associated) localization of YRs. B - IF analysis of YRs in the dentate gyrus (coronal sectioned planes) of mouse brain. The diffuse staining for Y1R reflects the distribution of Y1R positive neuronal fibers rather than cell bodies.
We selected the hippocampal dentate gyrus region as a positive control tissue because of its robust expression of YRs in a very specific and well-characterized fashion. Staining of brain sections revealed patterns of YR-positive neuronal cell bodies and fibers that are comparable to those previously reported (Fig. 5B) [10], [11], [13], [14], [15]. In particular, expression patterns of Y1R and Y2R appeared to be complementary, i.e. high levels of Y1R expression in one region corresponded to the low levels of Y2R, and vice versa [11].
Immunohistochemical (IHC) analysis
Using a mirror section staining protocol, we next determined that morphologically different layers of lingual epithelia expressed YRs in a distinctive yet overlapping pattern. Three separate complementary pairs of mirror sections were analyzed by hybridization to YR antibodies: Y1R and Y2R (Fig. 6A and B, respectively); Y2R and Y5R (Fig. 6C and D, respectively); Y1R and Y5R (Fig. 6E and F, respectively). The basal epithelial cell monolayer expressed both Y1Rs (Fig. 6A, E) and Y2Rs (Fig. 6B, C). Y2R expression appeared to be restricted only to this monolayer (Fig. 6C, right panel), while Y1R, on the other hand, was present in the parabasal prickle cell layer, and the granular layer (Fig. 6A, right panel). Differentiated keratinocytes displayed very low levels of Y1R protein, however, they displayed considerable expression of Y5Rs (Fig. 6F, right panel).
Figure 6. Immunolocalization of Y1, Y2, Y4, and Y5 receptors (Rs) in the dorsal epithelium of murine tongue.
Mirror section pairs (Panels A and B, C and D, E and F) were hybridized to the respective YR antibody (green), followed by DAPI counterstain (blue), as indicated in the upper left corner of each panel. For better viewing, the confocal images in B, D, and F were reflected horizontally. Representative areas of the epithelium, positive for either YR (dashed rectangles in the left-sided panels), are shown as close-up images on the right next to each respective panel. The irregular columned structures at the epithelial surface are transversely sectioned filiform papillae. G - Y4R-positive neuronal fibers (green) are located in the subepithelial region underlying the basal laminae. H – co-localization of Y4R and NCAM (red) immunoreactivity within mechanoreceptors of Meissner corpuscles (MC). As a morpho-histological reference of the dorsal lingual epithelium structure, an hematoxylin and eosin stained section is shown in panel I. Panel J shows a hypothetical diagram of a lingual dorsal epithelium layer and the differentiation/migration lineage of cell types expressing respective YR subtypes. K5 – cytokeratin-5 [2].
Unlike Y1R, Y2R, or Y5R, the expression of Y4R was not detected in the basal, or keratinized epithelial cells. Instead, it was restricted to the neuronal fibers extending within the subepithelial region close to the basal laminae of the lingual epithelia (Fig. 6G). Very few YR4-positive fibers were also positive for NCAM (data not shown) suggesting that the majority of these projections represented intraepithelial axons with free nerve endings. In addition, Y4R-positive neuronal fibers were also abundant in some areas of the lamina propria, in particular, in the fibers innervating mechanoreceptors (Meissner corpuscles) that were also positive for NCAM (Fig. 6H).
Functional assay for YR-positive epithelial cells
Previously, it has been shown that YRs mediate their responses through the Gi/o signaling pathway, reviewed in [1]. Several second messengers produced downstream of activated Y1, Y2, and Y4Rs include also inositol triphosphate, which is responsible for the increase in intracellular Ca2+ concentration [19], [20], [21]. To assess whether YR-positive cells in the dorsal epithelial layer could respond functionally to salivary PYY3–36, we conducted ex vivo Ca2+-imaging experiments on lingual sections loaded with fura-2/AM, a ratiometric fluorescent dye that binds to free intracellular Ca2+. Indeed, incubation of sections with PYY3–36 (40 µg/ml; applied locally) induced a clear elevation in intracellular Ca2+ concentration in the basal cellular monolayer (Fig. 7A, B). The PYY3–36 activation was rather uniform and led to a slow intracellular Ca2+ elevation in a majority of cells in the basal cellular layer (Fig. 6C). Qualitatively, a very similar pattern of response was observed in different slices loaded with a non-ratiometric Ca2+ indicator fluo-3/AM (data not shown). As a positive control to account for the functional integrity of cells we used ATP (20 µM) to activate strong Ca2+ signaling through its binding to purinergic receptors that are ubiquitously expressed in most cellular types (Fig. 7C) [22]. Application of ATP evoked Ca2+ signaling with a significant delay and variable in amplitude compared to the PYY-induced signal, i.e., shorter delay and more uniform in shape (Fig. 7C).
Figure 7. PYY3–36 induces intracellular Ca2+ in the lingual basal cell epithelium layer.
A – differential interference contrast micrograph of the agarose embedded acutely-obtained mouse lingual slice. The basal layer of epithelial Y1R/Y2R/K5-positive cells is outlined by a yellow dashed line. B – The same slice was loaded with a Ca2+ sensitive indicator dye, fura-2/AM. A static fluorescence image was captured at 380 nm excitation showing strong fura-2 loading in the basal cellular layer. C – Representative traces recorded from 13 individual cells selected in the slice shown in B. PYY3–36 (40 µg/ml) was applied by a local microperfusion for 30 sec indicated with the shaded box. ATP (20 µM) was used as a positive functional control. The data were collected from 38 representative cells individually analyzed in 3 slice preparations from two different animals.
Expression of YRs in taste bud cells
To characterize whether TRCs in the CV expressed YRs, we used an IHC assay in tongue sections harvested from mice sacrificed after a 24-hour fast. In agreement with data gathered from the dorsal lingual epithelial layer (Fig. 6A, B), epithelial cells forming the CV's outer edges also expressed Y1R, Y2R, and Y5Rs in a selective manner (Fig. 8A, B, and D, block arrows). In addition, cell monolayers lining the ducts of von Ebner's glands were also positive for Y1R and Y2Rs (Fig. 8A, B, indicated by VEG).
Figure 8. Immunolocalization of Y receptors (YRs) in taste receptor cells (TRCs).
Panels A, B, C, and D demonstrate Y1R-, Y2R-, Y4R-, and Y5R-positive TRCs, respectively. Areas within the dashed rectangle are shown as a series of magnified images to the right, stained for YRs (green), NCAM (red), DAPI (blue), respectively, with the merged images shown between YRs and NCAM. Open block arrows in the panels A, B, and D point at the YR-positive epithelial cells; filled arrowheads show apical parts of the YR-positive TRCs; open arrowheads – basolateral parts; VEG – von Ebner's gland. The majority of NCAM-positive intragemmal fibers are also Y4R-immunopositive (Panel 8C), but do not appear to express Y1Rs, Y2Rs, or Y5Rs.
A subpopulation of TRCs was positive for Y1Rs (Fig. 8A), Y2Rs (Fig. 8B), Y4Rs (Fig. 8C), or Y5Rs (Fig. 8D). In contrast to the epithelial cells, the staining pattern did not delineate the entire contour of cells, showing instead preferential accumulation of YRs within the apical part of the cells (filled arrowheads in Fig. 8A–C, respective magnified images in the panels on the right). To begin to understand the putative functions of YRs, we co-stained YRs with a molecular marker, NCAM that is expressed in intragemmal nerve fibers. It appears that many Y1R, Y2R, Y4R, and Y5R-positive cells also express NCAM (Panels A through C, magnified images to the right). Only Y4R expression was detected in the taste bud intragemmal nerve fibers.
Recently, we showed that a member of the PP-fold family, the satiation gut peptide PYY3–36, is present in murine and human saliva [2]. In mice, salivary PYY3–36 primarily derives from plasma. It is also synthesized in the taste receptor cells in taste buds of the tongue. We have demonstrated that augmentation of salivary PYY3–36 induces stronger satiation and that this effect is mediated through the activation of the preferred Y2Rs expressed in the lingual epithelial cells [2]. Taking into account the presence of other PP-fold peptides in the saliva [3], the redundancy of their interactions with YRs, and the multitude of their physiological functions, it was reasonable to hypothesize that the other YRs were expressed in the oral cavity tissues. To explore the mechanisms of salivary PYY3–36 action and to identify peptide's molecular target substrates, we set out to characterize the expression pattern of all major members of the YR family in lingual epithelia.
The dorsal surface of the tongue is covered by a specialized mucosa consisting of keratinized stratified epithelium (for review, please see [8]). In addition to its primary function protecting the underlying tissues during mastication, it also incorporates structures with gustatory functions (CV, fungiform, and foliate papillae), mechanical structures (filiform papillae), and mechanoreceptors (Meissner corpuscles). In addition, a glandular component of the lingual epithelium includes specialized salivary glands such as von Ebner's glands. In the current report, we focus on the dorsal lingual epithelia and the CV.
Using IHC analysis and a set of validated primary antibodies for Y1Rs, Y2Rs, Y4Rs, and Y5Rs, we determined that morphologically different layers [8] of the lingual epithelia expressed YRs in a very distinctive yet overlapping pattern (Fig. 6H). The basal epithelial cell monolayer expressed both Y1Rs and Y2Rs; Y1Rs were present in the parabasal prickle cell layer and the granular layer, while differentiated keratinocytes displayed an abundance of Y5Rs. ISH analysis essentially corroborated this pattern, although the Y5R-specific probe set appeared to hybridize to the target mRNA poorly, even in control brain tissue, where Y5Rs are known to be robustly expressed [15].
The Y2R-positive basal epithelial layer, which is also cytokeratin-5 (K5)-positive [2], responded robustly to PYY3–36 by increasing intracellular Ca2+ concentration indicating the receptors' physiological activity. To identify precisely the activated Y receptor subtype, specific receptor antagonists will be needed. Meanwhile, it seems reasonable to postulate that PYY3–36/Y2R/Gi signaling in K5-positive epithelial cells plays a proliferative role inducing mitotic activity of the progenitor cells in response to the mastication-related loss of keratinized lingual epithelial cells. In this regard, the postprandial increase in salivary PYY3–36 [2] could provide a feedback signal inducing such proliferation. Moreover, since in oral epithelia, proliferation also occurs in parabasal cells [23], the Y1R-positive prickle cell layer could be responsive to salivary NPY as well. Gi signaling in K5-progenitor cells can also mediate their motility, polarity and migration towards the dorsal layer of the keratinized filiform papillae. Such YR-dependent proliferation, migration, and differentiation has been previously described for the neurons in the adult mouse brain [24], olfactory neuronal precursors [25], and human endothelial cells [26]. There is also strong evidence supporting the NPY system's angiogenic and mitogenic functions in vascular smooth muscle cells and its potential role in endothelial cells wound healing [27]. A hypothetical diagram of a lingual dorsal epithelium layer and the differentiation/migration lineage of cell types expressing respective Y receptors subtypes is shown in Fig. 6J.
Assuming that the Y1R/Y2R-positive basal epithelial cells are modulated by salivary NPY and PYY, one has to postulate a mechanism of trans-cellular transport of these hormones through the superficial layers of keratinocytes forming hard-to-penetrate tight junctions. Interestingly, one of the outcomes of the PP-fold peptides interacting with their cognate GPCR YRs is the internalization and recycling of the respective agonist/receptor complex inside the cell [28], [29], [30]. It is, therefore conceivable that salivary PP-fold agonists are transported through cell layers akin to a relay race baton where directional cell-to-cell handoff is mediated by the increased affinity towards receptors, e.g., Y5R<Y1R<Y2R. The other possible endogenous source of PP-fold peptides are oral mucosal neuroendocrine cells, also known as Merkel cells, which are distributed along the basal layer of oral keratinized epithelium (for a review, please see [31]). We also have observed multiple PYY-positive cells in the lamina propria in close vicinity to the basal epithelial layer (data not shown).
The Y4R appears to be markedly expressed in most of the neuronal fibers innervating the lamina propria and taste buds. Some of these projections, especially fibers in mechanoreceptors of Meissner corpuscles and gustatory fibers in the CV, also expressed NCAM, indicating possible synaptic connections with receptor sensory cells, while most of the subepithelial fibers were NCAM negative, indicating free nerve endings. We also detected Y4R mRNA-positive fibers using an ISH protocol (Fig. 3E), suggestive of an anterograde trafficking of the Y4R mRNA along dendrites at least in some neurons.
Gustatory papillae are distinctive structures on the dorsal tongue epithelia incorporating several types of cells including basal epithelial cells, keratinized cells, TRCs organized in tight clusters (taste buds), and gustatory neuronal fibers innervating TRCs. Epithelial cells and TRCs derive from the lingual embryonic undifferentiated epithelium and are constantly turned over in the adult animal. All tested YR subtypes were prominently expressed in murine TRCs showing preferential apical distribution within cells. This distribution would make YRs easily accessible to paracrine salivary PP-fold peptides, suggesting their possible roles in modulating taste perception [32], although some taste receptors do not display such preferential apical distribution [33]. Conversely, some YRs-positive TRCs accumulated YRs in their basolateral portion (open arrowheads in Fig. 8A–C), which makes these cells accessible to the paracrine, or endocrine PYY, or NPY, synthesized inside taste buds [2], [4].
Within each taste bud, TRCs fall into three morphological subtypes, Types I through III, which seem to correspond to functional classes (reviewed in [34]). In order to understand the functional role of YRs in TRCs, one needs to assign YR-positive TRCs to a particular cell subtype co-staining YRs with a known cell type marker. Here we show that a subpopulation of YR-positive TRCs are co-localized with NCAM, a neuronal marker expressed in some Type III cells as well as in intragemmal nerve fibers. Because of technical limitations, we were unable to quantify the exact ratio of YR/NCAM-positive TRCs due to the specific staining contours. Experiments are currently under way to characterize the precise expression pattern of YRs in Type I–III TCRs and to identify taste qualities modulated by YR subtypes.
In summary, we have conducted a systematic analysis of the expression of genes encoding members of NPY family peptides and YRs in the dorsal lingual epithelia of the mouse. We have utilized independent assays (IHC, ISH, ICC and RT-PCR) using validated primary antibodies, custom-designed target-specific probe sets, and intron-spanning PCR primers to identify cells expressing YR subtypes. Herein, we show for the first time that all members of the NPY family are abundantly expressed in specific and selective fashion in multiple lingual cell types, including epithelial progenitors, keratinocytes, neuronal dendrites, and TRCs. Because of this remarkable diversity, and because of the redundancy of agonist/receptor interactions, NPY family peptides and their cognate receptors in the oral cavity may mediate a wide variety of functions, including proliferation, differentiation, motility, taste perception, as well as satiation. All of these multiple functions and their respective molecular mechanisms are subjects of the ongoing investigations.
Conceived and designed the experiments: MDH AA CDD HH SZ. Performed the experiments: MDH PPR AB KU SZ. Analyzed the data: MDH AA BB HH CDD SZ. Contributed reagents/materials/analysis tools: BB HH. Wrote the paper: MDH AA BB KU HH.
1. Zhang L, Bijker MS, Herzog H (2011) The neuropeptide Y system: pathophysiological and therapeutic implications in obesity and cancer. Pharmacol Ther 131: 91–113.
2. Acosta A, Hurtado MD, Gorbatyuk O, La Sala M, Duncan D, et al. (2011) Salivary PYY: A Putative Bypass to Satiety. PLoS One 6: e26137.
3. Dawidson I, Blom M, Lundeberg T, Theodorsson E, Angmar-Mansson B (1997) Neuropeptides in the saliva of healthy subjects. Life Sci 60: 269–278.
4. Zhao FL, Shen T, Kaya N, Lu SG, Cao Y, et al. (2005) Expression, physiological action, and coexpression patterns of neuropeptide Y in rat taste-bud cells. Proc Natl Acad Sci U S A 102: 11100–11105.
5. Pedrazzini T, Seydoux J, Kunstner P, Aubert JF, Grouzmann E, et al. (1998) Cardiovascular response, feeding behavior and locomotor activity in mice lacking the NPY Y1 receptor. Nat Med 4: 722–726.
6. Tschenett A, Singewald N, Carli M, Balducci C, Salchner P, et al. (2003) Reduced anxiety and improved stress coping ability in mice lacking NPY-Y2 receptors. Eur J Neurosci 18: 143–148.
7. Kaya N, Shen T, Lu SG, Zhao FL, Herness S (2004) A paracrine signaling role for serotonin in rat taste buds: expression and localization of serotonin receptor subtypes. Am J Physiol Regul Integr Comp Physiol 286: R649–658.
8. Squier CA, Kremer MJ (2001) Biology of oral mucosa and esophagus. J Natl Cancer Inst Monogr 7–15.
9. Mannon PJ, Mele JM (2000) Peptide YY Y1 receptor activates mitogen-activated protein kinase and proliferation in gut epithelial cells via the epidermal growth factor receptor. Biochem J 350 Pt 3: 655–661.
10. Stanic D, Brumovsky P, Fetissov S, Shuster S, Herzog H, et al. (2006) Characterization of neuropeptide Y2 receptor protein expression in the mouse brain. I. Distribution in cell bodies and nerve terminals. J Comp Neurol 499: 357–390.
11. Stanic D, Mulder J, Watanabe M, Hokfelt T (2011) Characterization of NPY Y2 receptor protein expression in the mouse brain. II. Coexistence with NPY, the Y1 receptor, and other neurotransmitter-related molecules. J Comp Neurol 519: 1219–1257.
12. Fetissov SO, Byrne LC, Hassani H, Ernfors P, Hokfelt T (2004) Characterization of neuropeptide Y Y2 and Y5 receptor expression in the mouse hypothalamus. J Comp Neurol 470: 256–265.
13. Kopp J, Xu ZQ, Zhang X, Pedrazzini T, Herzog H, et al. (2002) Expression of the neuropeptide Y Y1 receptor in the CNS of rat and of wild-type and Y1 receptor knock-out mice. Focus on immunohistochemical localization. Neuroscience 111: 443–532.
14. Parker RM, Herzog H (1999) Regional distribution of Y-receptor subtype mRNAs in rat brain. Eur J Neurosci 11: 1431–1448.
15. Wolak ML, DeJoseph MR, Cator AD, Mokashi AS, Brownfield MS, et al. (2003) Comparative distribution of neuropeptide Y Y1 and Y5 receptors in the rat brain by using immunohistochemistry. J Comp Neurol 464: 285–311.
16. Dumont Y, Redrobe JP, Quirion R (2002) Neuropeptide Y receptors; Pangalos MN, Davies CH, editors. Oxford, UK: Oxford University Press.
17. Saper C (2005) An Open Letter to Our Readers on the Use of Antibodies. The Journal of Comparative Neurology 493: 477–478.
18. Michel M, Wieland T, Tsujimoto G (2009) How reliable are G-protein-coupled receptor antibodies. Naunyn-Schmied Arch Pharmacol 379: 385–388.
19. Herzog H, Hort YJ, Ball HJ, Hayes G, Shine J, et al. (1992) Cloned human neuropeptide Y receptor couples to two different second messenger systems. Proc Natl Acad Sci U S A 89: 5794–5798.
20. Bard JA, Walker MW, Branchek TA, Weinshank RL (1995) Cloning and functional expression of a human Y4 subtype receptor for pancreatic polypeptide, neuropeptide Y, and peptide YY. J Biol Chem 270: 26762–26765.
21. Gerald C, Walker MW, Vaysse PJ, He C, Branchek TA, et al. (1995) Expression cloning and pharmacological characterization of a human hippocampal neuropeptide Y/peptide YY Y2 receptor subtype. J Biol Chem 270: 26758–26761.
22. Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50: 413–492.
23. Tipoe GL, Jin Y, White FH (1996) The relationship between vascularity and cell proliferation in human normal and pathological lesions of the oral cheek epithelium. Eur J Cancer B Oral Oncol 32B: 24–31.
24. Stanic D, Paratcha G, Ledda F, Herzog H, Kopin AS, et al. (2008) Peptidergic influences on proliferation, migration, and placement of neural progenitors in the adult mouse forebrain. Proc Natl Acad Sci U S A 105: 3610–3615.
25. Hansel DE, Eipper BA, Ronnett GV (2001) Neuropeptide Y functions as a neuroproliferative factor. Nature 410: 940–944.
26. Movafagh S, Hobson JP, Spiegel S, Kleinman HK, Zukowska Z (2006) Neuropeptide Y induces migration, proliferation, and tube formation of endothelial cells bimodally via Y1, Y2, and Y5 receptors. FASEB J 20: 1924–1926.
27. Ghersi G, Chen W, Lee EW, Zukowska Z (2001) Critical role of dipeptidyl peptidase IV in neuropeptide Y-mediated endothelial cell migration in response to wounding. Peptides 22: 453–458.
28. Parker SL, Kane JK, Parker MS, Berglund MM, Lundell IA, et al. (2001) Cloned neuropeptide Y (NPY) Y1 and pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells show considerable agonist-driven internalization, in contrast to the NPY Y2 receptor. Eur J Biochem 268: 877–886.
29. Fabry M, Langer M, Rothen-Rutishauser B, Wunderli-Allenspach H, Hocker H, et al. (2000) Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK-N-MC. Eur J Biochem 267: 5631–5637.
30. Gicquiaux H, Lecat S, Gaire M, Dieterlen A, Mely Y, et al. (2002) Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor. J Biol Chem 277: 6645–6655.
31. Mahomed F (2010) Neuroendocrine cells and associated malignancies of the oral mucosa: a review. J Oral Pathol Med 39: 121–127.
32. Hoon MA, Adler E, Lindemeier J, Battey JF, Ryba NJ, et al. (1999) Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell 96: 541–551.
33. Ohmoto M, Matsumoto I, Yasuoka A, Yoshihara Y, Abe K (2008) Genetic tracing of the gustatory and trigeminal neural pathways originating from T1R3-expressing taste receptor cells and solitary chemoreceptor cells. Mol Cell Neurosci 38: 505–517.
34. Yoshida R, Ninomiya Y (2010) New insights into the signal transmission from taste cells to gustatory nerve fibers. Int Rev Cell Mol Biol 279: 101–134.
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Whats with the new '24 style' shooting of The Bill?!
Discussion in 'TV Show Forum' started by MitsiTurbo, Jan 24, 2008.
Its really starting to annoy me now! The zooming into faces, quick panning and all that other stuff that reminds me of 24. Just doesnt suit the Bill! I dont watch it all the time, just when theres nothing else on, so I dont know how long its been like this.
But anyway... its annoying me.
This is ITV's attempt to bring "The Bill" into the 20th Century, let alone the 21st!
Seriously, it looks awful doesn't it?, and it's now a soap - sorry, I mean "serial drama" - and in all honesty, it should be put to rest now, because compared with every other serious police series, it's awful. Nowhere near as good as the heady days of the late-1980's and early-90's!
Anyone own a new style XC90?


text:
Atlantic News | Local Breaking | CTV News Atlantic
Prince Edward Island is reporting an increase in hospitalizations on Monday, with seven in hospital being treated for COVID-19, one of whom is in intensive care.
A new study shows, nearly two years into the COVID-19 pandemic, more than a third of working Canadians say they feel a sense of burnout.
Nova Scotia has expanded eligibility for COVID-19 booster doses to include anyone 18 years of age and older.
Tributes continue to pour in for former federal and Nova Scotia NDP leader Alexa McDonough, who died on Saturday after a struggle with Alzheimer's disease.
The New Brunswick government announced it will provide a wage top-up for workers who provide services to vulnerable residents.
Nova Scotia to open three new regional African Nova Scotian Affairs offices
Nova Scotia announced the addition of three new regional African Nova Scotian Affairs offices Monday.
Sunday was a day months in the making, as volunteers with the Out of the Cold Community Association put the finishing touches inside the emergency modular units that will eventually house 26 people.
Report on 'defunding' Halifax police recommends reforms, no specific budget cuts
A committee tasked with defining how to defund Halifax-area police has instead produced a wide-ranging report recommending a shift away from a "reactive" enforcement-based policing model -- but it includes no specific budget cuts.
As 400 public schools in Nova Scotia prepare to resume in-person classes on Monday, the president of the province's teachers union says he has "severe doubts" as to whether they can stay open until the end of the week.
Police in Halifax have charged an 18-year-old woman in connection with a robbery and five thefts that happened in the city over the last few months.
An RCMP officer based in rural Nova Scotia has been charged with sexual assault.
Cosmetology association calls for lockdown restrictions in N.B. to be lifed for its industry
Estheticians, hair stylists and nail technicians are calling on the New Brunswick government to lift lockdown restrictions on their industry.
Several factors impact empty shelves in Maritimes
Tusket, N.S. woman joins her husband in the centenarian club
January 13, 2022 was a special day for Annie Muise of Tusket, N.S., as she marked a major milestone.
Caper Bretoner cashes in: Bet paid out almost 30 years after it was made
A Cape Breton man is cashing in on a bet he made with his friend in 1994.
Scientist to recreate Mabel Bell's 100-year old gardens in Cape Breton
There's no doubt Alexander Graham Bell has left an indelible mark on history, but his wife Mabel was also a trailblazer. Now, almost 100 years after her death, a Nova Scotia scientist is recreating her ecologically significant gardens.
Cape Breton man who made a name for himself with beer cap mosaics incorporates tabs in his artwork
Teddy Hogan's hobby requires patience and a lot of time, which is why most days you will find him in his backyard garage from morning to night.
Maritime medical experts share advice on how to treat COVID-19 symptoms at home, when to seek help
Medical professionals share advice on how to care for yourself at home if you've tested positive for COVID-19.
'It's her lifelong dream': Cape Breton woman is 'princess for hire' in Halifax
It looks less like work and more like play, but being a real-life princess is Miranda Pike's job. At 23 years old, Miranda is the owner of Little Princess Parties Halifax.
Son of former N.S. 'fiddling premier' now head of new Cape Breton radio station that plays all local music
The son of a man known to some in Cape Breton as the former "fiddling premier" is picking up on some of his father's passion for Celtic music.
Despite many health care professionals' warnings, there are some who are talking about catching Omicron to 'get it over with,' and idea one doctor calls 'playing with dynamite.'
New program to provide menstrual hygiene products to New Brunswick schools
Students in New Brunswick will now have access to free menstrual hygiene products at school.
CTV Morning Live Atlantic for Monday, January 17, 2022
News update: Monday, Jan. 17, 2022
Weather update: Monday, Jan. 17, 2022
The latest on the potato ban
Staying safe during the omicron outbreak
The Betty White challenge
Remembering Alexa McDonough
CTV Morning Live Atlantic for Friday, January 14, 2022
News update: Friday, Jan. 14, 2022
Drake Batherson becomes latest Nova Scotian picked for NHL All-Star Game
When you think about NHL All-Stars from Nova Scotia the names Crosby, MacKinnon and Marchand usually come to mind.
Dad and daughter duo conquer Mount Kilimanjaro for a good cause
A father and daughter duo has just returned home to Fredericton after experiencing an adventure of a lifetime.
N.B. nursing home hoping to brighten community during uncertain times
Pine Grove Nursing Home in Fredericton is trying to bring a little extra light during uncertain times.
Mask standards shift as Omicron variant spreads
Given the high contagion level of Omicron, experts say the virus can slip through single-layer cloth masks. When schools re-open Monday, Nova Scotia Premier Tim Houston wants to provide the best quality mask for everyone.
CTV Morning Live 7 a.m. CTV News at Noon Noon CTV News at Five 5 p.m. CTV News at Six 6 p.m. CTV News at 11:30 11:30 p.m.
N.S. Potential COVID Exposures
N.B. Potential Public Exposures
N.S. Public School COVID-19 Exposures
Mount Kilimanjaro Fundraiser
The Measha Series
Van Gough 360
Ouka the fluffy flying dog
Nova Scotia Test Kits
New Brunswick Test Kits
P.E.I. Test Kits
Bird Tape
Honour Candy
NYE Kitchen Party
HRM NYE
Highlights from Across Canada
Concern and anger grows for hundreds of birds trapped inside old Cape Breton church
It's been several years since St. Anthony's Church on Dominion Street in Glace Bay closed its doors to the public, and since that time the vacant facility has attracted hundreds of birds, both inside and out.
Before he was known as Barto Built, sharing his backcountry 4x4 adventures on YouTube, Bart Sutherland was known as a 'rock-crawler,' driving his truck over seemingly insurmountable obstacles.
B.C. son helps mom replace cherished memories after toonie collection stolen
After a fire destroyed most of the pictures showing her children's past, Mandy Lamarche discovered she could recall their priceless moments together through coin collecting. When her collection was stolen, her son
A 30-year-old Ontario woman whose cancer surgery was postponed multiple times because of COVID-19 has secured a new date for her procedure.
B.C. couple explain why they haven't removed hostage-taking cats from blender box
Like all epic stories, this one starts quite simply, beginning with the delivery of a blender in a box.
'Highly improbable' Omicron spread to China via mail: Sharma
Feds, province agree to help fund search for unmarked graves at Sault former residential school
Calls for B.C. to take action to help internationally-educated nurses get to work
Nishnawbe Aski Nation calling on Ottawa, Ontario to better fund fire services

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: How do I get GreenSock draggable and onClick working simultaneously? I am trying to enable the user to navigate through different content on the site by either, dragging the clock-button to rotate it (created using the greensock animation library) to different points specified to change the content or, clicking on the clock-button which also rotates to change the content.  At the moment I can do one or the other, but as soon as I enable both and trigger a click event and then a drag event (or vice versa), the clock-button snaps to positions in the wrong order (but still the defined snaps).
The code is below.

var clockButton = document.querySelector('#clock-button');
var snaps = [0, 45, 135, 180, 225, 315, 360];
if ((window.screen.width > 1024) && (detectTouchscreen() === false)){
    var draggable = Draggable.create([clockButton], {
            type: "rotation",
            allowEventDefault: true,
            throwProps: true,
            onDrag: onRotate,
            liveSnap: function(value) {
            position = getClosestIndex((value + 360 * 99999999) % 360, snaps); //adding 360 * 999999999 in order to ensure it's always a positive rotational value
            return snaps[position];
            },
            onClick: function(){
                draggable[0].disable();
                if (snaps[position] === 0){
                    snaps[position] = 45;
                    draggable[0].enable();
                } else if (snaps[position] === 45){
                    snaps[position] = 135;
                    draggable[0].enable();
                } else if (snaps[position] === 135){
                    snaps[position] = 180;
                    draggable[0].enable();
                } else if (snaps[position] === 180){
                    snaps[position] = 225;
                    draggable[0].enable();
                } else if etc....

                }
            }

        });
}
<img id="clock-button" src="assets/img.png"
   data-deg="0" data-toggle="tab">

The content is changed using the onRotate function which is just hiding and showing elements dependant on the position.

A: This is working code:
var draggable = Draggable.create(clockButton, {
            type: "rotation",
            dragResistance: .01,
            dragClickables: true,
            liveSnap: onLiveSnap,
            allowContextMenu:true,
            onClick:
                function(e){
                adjusting = true;
                this.update();
                if (snaps[position] < 360){
                    snaps[position] = snaps[position ++];
                } else {
                    position = [1];
                }
                // console.log(snaps[position]);
                TweenLite.set(this.target, {
                    rotation: snaps[position]
                });
                this.endDrag();
                onRotate();

                adjusting = false;
            },

        onRelease: function() {
            this.update();
            if(this.tween && (adjusting || this.timeSinceDrag() > 0.02)){
                this.tween.kill();
            }

        }
    });

redpajama_set_name: RedPajamaStackExchange


Should We Go to Disney World?: 2017 Edition.
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text:
What to Expect After Saying 'I Love You' to All 12 Zodiac Signs?
Goran Nikolov
I am an astrology writer fueled by wide-eyed interest in all things otherwordlish, laser beam phrases and style most simple, yet enigmatic.
If you're horoscope obsessed, you've probably let the zodiac make some big and tough decisions for you. You've also probably judged people based on their zodiac sign – admit it. And I would bet my bottom dollar that you've checked a girl's/boy's zodiac sign to see if you're compatible after just meeting her/him. But I'm not here to judge you, because I, just like you, am a big zodiac enthusiast and I do believe that the stars and the universe have something to do with our lives.
Your zodiac sign says a lot about you as a person, but it can also tell you which horoscope signs you are most compatible with. Saying those three little words is a major step in a relationship and is usually the starting point of a strong and healthy relationship. The interesting part is that each zodiac sign says "I love you" differently. Some add too much drama into it and fail epicly, while others spit it out completely accidentally as they cannot keep that kind of an enormous feeling to themselves. On the receiving end, zodiac signs are all different too. Find out the proper way you should reply to "I love you" or what to expect to hear from your crush when you say the words.
Aries: "Awww! I Love You Moooore!"
People born under this sign are not very good with expressing feelings and they need some time to get to the phase of saying "I love you", but once they do, they are the most committed partners from the zodiac. In relationships, they are honest and will very likely put your needs before theirs. When you get to the stage to admit how you feel about an Aries, you can expect nothing less than "I love you more".
Taurus: "Umm... so soon? Are you serious?"
The only thing Taurus loves more than food is love. People born under Taurus tend to be careful, deliberate and passionate by nature when it comes to love. It's not easy to get to the heart of these people, which is why Tauruses are known for dismissing signs of affection until they are 100% sure that it is exactly what they want. If you rush the three magical words, be sure that you will be asked if you are serious with your feelings.
Gemini: "Really? I mean, it's awesome, but... reallyyy?!"
As you probably know, people whose zodiac sign is Gemini are dual-natured and inconsistent. They are also known for their tendency to avoid talking about feelings and emotions, as they can awkwardly change the subject whenever they feel uncomfortable. Having a dual personality makes this zodiac sign nearly impossible to predict how will they handle those three words and it could be something between an enthusiastic "Reallyyy?" and a cold-hearted "Wait. Really?".
Cancer: "You love me? Are you sure?"
You should expect nothing less than asking how sure you are about your feelings towards people who are characterized as committed, protective, caring, and dependable. Cancers are passionate lovers and they love deeply, but not until they feel completely certain that they're safe with you and you feel the same way they do. They may not be very good at saying or responding to "I love you", but they're excellent in making sure you know they love you via sweet gestures.
Leo: "Uum, why wouldn't you?"
Driven by desire to love and be loved, Leos are the most loyal and the sincerest of all astrology signs. You cannot shock a Leo by telling them "I love you", because they're so into themselves that they basically expect you to love them. Although these people are highly social and always tend to meet and hang out with new people, they are not afraid to settle down when they find their true love. Thanks to their confidence, they know how to handle this situation better than any other sign.
Virgo: "Thanks."
Virgo is known for being practical and emotionally strong, but they are not very good at talking about feelings as they are conservative. Instead of admitting feelings, Virgo would just hope you will figure out how they feel about you. When on the receiving end, they will need some time to think about it and see if they can see a future with you. However, these analytical individuals do recognize your effort and bravery and appreciate it, so you may get a "thanks".
Libra: *confused staring, followed by nervous laughter*
Wait, what? You tell a Libra that you love her/him and you threaten her/his perfectly balanced life, expecting to get those words back at you? If you haven't heard, Libras are incredibly indecisive and that kind of commitment proposal would easily confuse them. They are also very picky when it comes to choosing a partner, but once they find it, they are devoted, loving and caring partners and will try their best to make your life together harmonious.
Scorpio: "Okay."
There may be no zodiac sign that is more passionate than the Scorpio, but there surely are many that respond better. If you ask a Scorpio, they really do believe that this is the perfect reply. As mysterious and emotionally cold as they seem at first, many people are surprised by the fact that these people actually long for finding the one true love. They are capable of fierce loyalty, but not with just anyone. Scorpios are hesitant to say "I love you" too soon and wouldn't like to hear it too soon either.
Sagittarius: "Noo. I love YOUUU!"
People born under this zodiac sign are adventurous and fun-loving and when they are in love, they shower their partners with affection. Sagittariuses tend to get bored quickly, so their partners must be adventurers and free-spirited in order to make it work. Once they find a perfect match, they will spare no means to show how they feel. If you tell a Sagittarius that you love her/him, they will jump into your arms and start a competition of "but I love you more".
Capricorn: "I know."
Capricorn is the Han Solo of the zodiac. Ambitious, career-driven and compassionate, people whose zodiac sign is Capricorn are very likely to be looking for a best friend in their partner as the single most important criteria. They also prefer someone who shares the same life goals as they do and they are very picky when it comes to relationships. So when you admit your feelings to a Capricorn, he/she will probably go full-Han-Solo mode and tell you that he/she already knows. And he/she really does, because otherwise, you wouldn't reach that stage in your relationship.
Aquarius: "Love?"
Aquarius may be the most eccentric and most innovative sign of all astrological signs, but when it comes to love, they are best described as awkward. It's hard to tell how would an Aquarius respond, as these people are more logical than emotional and would like to rationalize what you're saying before they reply. The philosopher Aquarius is also likely to question the concept of love and evaluate if that's something he/she would benefit from. But when they know they are with the right person, they will selflessly give all of their love.
Pisces: "Huh?"
Pisces is the most sensitive and most compassionate sign of the zodiac and people born under this sign tend to be incredibly emotional. These romantics will do anything for love, even forgive almost anything to anyone, including cheating. Interestingly, though, they never lose faith in love. Pisces find a lot of ways to show they care about you and love you, but if you tell them that you love them when they don't expect it, they will ask you to repeat what you've just said to make sure they've heard you correctly. They hate rejections, so they will make sure it is the real deal before they rush into anything.
Did you find these reactions and replies correct?
YES! Absolutely! That's totally how I respond!
Seriously? This must be a joke.
Not entirely, but it's a fun read!
All the credits for this Hub belong to Culture Astrology.
Everything You Need to Know About a Taurus and Aries Romance
by Andrea Lawrence3
Everything You Need to Know About a Sagittarius and Pisces Romance
Signs Your Crush Likes You Through Texting: Zodiac Version
by Andrea Lawrence10
When Your Boyfriend Will Propose According to His Zodiac
The Capricorn Man and Love
by Kodama Network35
Aquarius and Virgo Compatibility: Why Is This a Powerful Match?
Pisces and Leo Relationship Compatibility (A Love Match Made in Heaven)
Why Pisces and Pisces Make for a Great Relationship
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Proline is one of the best fishing boat brands on the market. This 2008 Dual Console has been well taken care of in dry storage. She boasts a Mercury 225 Verado engine with only 340 hours! Her helm is equipped with a Garmin 541s GPS, stereo, compass, and depth-finder. Her enclosed porta-potti and the helm are shaded by her bimini top. A full boat cover, lockers, live-wells, and Fusion speakers can be found within the boat. There is no trailer included.


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COR-NTD
Research & outcomes
NTDConnectOR
What are Neglected Tropical Diseases
NTD Toolbox
Secretariat (NTD-SC)
Charles Mackenzie
Associate Director, Technical Development
Charles Mackenzie, Associate Director, Technical Development and a pathologist with expertise in tropical diseases, focuses on laboratory support and country program activities on neglected tropical diseases (NTDs) for the NTD Support Center in conjunction with the Mectizan Donation Program. He has over 30 years experience of working closely with a number of countries in Africa and Latin America on onchocerciasis and lymphatic filariasis, as well as a long career at the London School of Hygiene and Tropical Medicine and Michigan State University researching the pathogenesis of filarial and other infectious diseases. His main areas of activity at the Task Force for Global Health involve the development of a laboratory support system to aid countries as they move to the elimination and control of NTDs, as well as catalyzing the global efforts to eliminate onchocerciasis. Charles is also the current Chair of the Global Alliance to Eliminate of Lymphatic Filariasis (GAELF), as well as other committees that focus on patient care for NTDs.
EMAIL:CMACKENZIE@TASKFORCE.ORG
Coalition for Operational Research
on Neglected Tropical Diseases
Secretariat, Neglected Tropical Diseases Support Center
This website for the Coalition for Operational Research on NTDs (COR-NTD) received financial support from the Bill & Melinda Gates Foundation and by the United States Agency for International Development through its Neglected Tropical Disease Program. This information provided on the website is not official government information and does not represent the views or positions of the U.S. Agency for International Development or the U.S. Government or the U.K. Foreign, Commonwealth and Development Office or the U.K. Government.
A Remora website by Manta Ray Media


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Last Updated: Monday, 26 September 2005, 18:38 GMT 19:38 UK
US anti-war mother held at rally
Protesters held placards with the names of US soldiers killed in Iraq
Cindy Sheehan, the anti-war protester and mother of a US soldier killed in Iraq, has been arrested during a demonstration outside the White House.
Ms Sheehan and several dozen other protesters were held by police after failing to move on from the pavement in Washington DC.
She first attracted worldwide attention when she held a 26-day vigil outside President George Bush's Texas ranch.
Her arrest follows a massive anti-war march in the capital over the weekend.
Ms Sheehan was among a group of about 500 people who staged what they called a "civil disobedience" demonstration to bring more attention to the US military presence in Iraq.
The pavement outside the White House is a restricted area, where people by law must keep moving.
The mother's Texas vigil, prompted by her eldest son's death in action in Iraq in 2004, has made her a figurehead for the anti-war movement in the US.
See US police arrest Cindy Sheehan
Protest mother back at Bush ranch
Anti-war Sheehan quits Texas camp
Vigils to support Sheehan protest
Why mothers push for peace
Bush rejects mother's Iraq plea
US anti-war movement gains steam


HomeRubber Bracelet WristbandsStars and Stripes American Flag Rubber Bracelet Wristband - Adult 8"
Buy our Stars and Stripes American Flag Rubber Bracelet Wristband and display All American stars and red stripes. Our Stars and Stripes Rubber Bracelet Wristband is made from 100% Silicone Rubber. This seamless Rubber Bracelet Wristbands comes packaged in an individual polybag.
Printed with white on 2 colors of rubber for a patriotic message - the two colors of rubber mix together at the edges - each one has a unique look. Some others are all blue rubber with white and red ink to achieve a similiar look - we'll pick which ones you get.
These patriotic wristbands look great and have been a fantastic way to fundraise at my elementary school in support of a local 9/11 Memorial Stair Climb. I would highly recommend purchasing the "youth" size for smaller children like kindergarteners/first graders, yet all other students did well with the "adult" size. I will continue to purchase from the Support Store in the future!
Best UsesFundraising for patriotic events.
I really like the way they are made. One piece, no seam, I hope as you said it is. Bought 100 of these to give as I SEE fit. I feel they are better made than others (ones representing cares). Those have split on me telling me they were not a single/one piece. I like so far and others are gratefull.
Lasts 3-6 months of continuous wear before stars and stripes start to fade. Well worth it for $2.50.
Bought several of these bands to give to my veteran friends in celebration of our American ideals.
Wrist band was a gift for my husband. He wears it all of the time. I would say he really likes it! Great quality and service!
This is a high quality item for a reasonable price!
We looked everywhere for these and couldn't find them locally. My husband went online and found that you have an assortment of them, so we ordered enough to last us a long time. They are just what we were looking for!
These bracelets are really nice. The quality is much better than I expected. Each bracelet comes individually wrapped so no sticking together like some I've seen. They fit adults comfortably too.
i love these things. a simple way to show support for the men and women doing what must be done, at great personal cost. I give these stars and stripes away, the more the merrier!
Best Usesgive them to others - show our support to our own!
Fabulous! 2nd time buying, LOVE 'EM!


Sound Proof Systems BV, founded in the year 2000, is specialized in canopies.
To provide our worldwide customer base with a compact, easy to transport product, SPS has developed a range of self-assembly "FLAT PACK" canopies. The unique concept creates enormous advantages for the transport of a complete canopy to OEM customers. A Flat-Pack kit consists of a base brame, fuel tank, fuel gauge, engine/alternator supports, anti vibration mounts, battery holder and a canopy.
All Sound Proof Systems canopies are powder coated to withstand arduous weather conditions and are supplied with assembly manuals showing component identification drawings, for ease of construction.
Besides the standard products Sound Proof Systems developes and produces also bespoke products with very low noise levels.
Sound Proof Systems has a standard range of canopies for different type of generators. These canopies are designed in such a way that the whole canopy is packed as a kit; the so called "Flat-Pack canopies". This to minimize transport and storage costs.
This is almost a 1-2-3 system to quickly and easily built a generator set. The "kit" contains all the items, such as cross member, silencer, tank, exhaust system, that are needed to built a set. The construction manual give also clear guide lines to built the canopy.


Have you ever wondered how some people seem to make all the effortless income and live the dream, while others, even when they have the skills, (and maybe even work harder), still seem to hustle around town and struggle to reach the "next level" in their Network Marketing business?
Yeah I did too. And it used to make me mad when I would see all of the Facebook ads… you know the ones… "Are you struggling in your MLM?" and "Stop chasing your friends and family", from some nerdy internet guru..
I really thought these "internet marketers" were just bottom-feeding and trying to use their tactics to steal my distributors! (Or maybe just "sell them something"!
Well, long story short, I ended up in Las Vegas at their seminar, and what they were saying was making way too much sense. Undeniable.
The funny this was, it worked almost immediately. The first day I put out a blog post, I got a lead from Facebook.
Another network marketer that I was connected with. He wanted to know what I was doing, and if it was working.
I told him the truth: "I don't know, but I can send you a helpful link if you'd like." I told him what I was doing, and he bought the course that same night.
As I dug in and got super sidetracked into my blog, I started to lose all focus on actually building my network marketing business.
I was afraid of my Network Marketing income going down, so I put the "internet stuff" on the shelf for a while. I would put up a blog post here and there, but no real clear direction.
Then my company started to "fizzle out" and lose momentum. I didn't want to see it, because I had invested so much time and energy into it, but my checks were dwindling.
I was tired of chasing everyone, and I felt like none of my team was working as hard as I was. I was running around, state to state, and even country to country, like a freaking maniac.
This time, however, I was ready. By then, I had met too many friends that have made 6, 7, and even 8 figure incomes, leveraging and marketing their "freedom businesses" online.
I knew I needed change for the better, so I dropped in without much hesitation this time. I also immediately upgraded to "VIP" so I could get more coaching and better commissions if I sold the system/courses to other networkers.
I wasn't gonna mess around this time. I knew this was the way, and I knew this system was "the one"! I watched the modules and implemented.
I paused 'em and followed the directions, step by step, and pressed play again, all the way 'til I was dialed… I built wrote up a super valuable blog post, set up my targeted Facebook Ad, and clicked the button… "Process Order".
Butterflies in my stomach. Finally fell asleep. What did I wake up to? Leads. … and when I checked my email later that night? … SALES!!
Holy sh*t! A few months later, messages from a ton of other network marketers, thanking me for helping them discover a better way of building, so they don't have to feel like the slimy salesman at family outings.
Now they only talk to people who are interested and ready to buy… Freaking genius!
It's crazy to know that you can be picky about who you want to attract and work with! There are so many millions of people out there on Facebook, that you can choose and laser target exactly who you want to work with, and there's an endless sea of them!
Many times your target market will be someone exactly like you.
Think about it: Whatever your pain/frustration was, it made you open to looking at something else, right?
Now, what are your target market's worst fears? What is their worst frustration? What is important to them? What do they wake up thinking about, that stays on their mind constantly?
Why do people watch movies? It's because they just want to shut their brain off and relax after work, and be entranced into a storyline.
So if you learn to slip into your story, and talk about your pain or frustrations, (WHY you're doing the business), then you'll never really have to schpeel anyone.
They don't really care about "what" it is that you have. They care about WHY you're doing it.
And if your story, (why you're doing what you're doing), resinates with what's important in their lives, they will attract toward your solution, no matter which package it comes in.
Your story will help bring down their "sales filter", so you can actually get them to be curious enough to show up for themselves.
Do you have the solution to their problem/problems? If you do, awesome! Give it to them!
If you don't, then go figure it out, and then give it to them! This makes you ultra valuable to them!
Maybe you still have the same exact problem. Even better! Now you have the chance to become bigger than your obstacle that has been holding you back!
Google it. YouTube it. Find the solution and overcome it yourself! Only then can you help others!
The amount of money you make in this world is directly proportioned to how valuable you are to others. The catch is, building yourself into a valuable person takes time, so in order to have the success you want to have, you must become the person you needed to be.
It took me years learn what NOT to do, and I have become extremely skilled in Network Marketing.
So in my post, I knew I could help them, with no strings attached, and go above and beyond, to make sure they don't make the mistakes I made.
Learning and having a business is hard, because you have to think, so naturally, people don't like to learn as much as they like to be entertained.
Most of the time, the information sticks better when it's part of a story too. If you can somewhat entertain them while you're teaching them, they'll have a great experience and probably connect you/your site with "great experiences"!
This will also create trust, and "people only buy from people they know, like, and trust"!
After I give them what they need, and it was super valuable and game-changing for them, they know that I am trustworthy.
Then I can invite them to learn even more, and who doesn't want even MORE great training that changes your business?
Honestly, selling used to be a weakness of mine. I never liked being pushy like a "sales guy" or anything.
Also, I'm not the strongest with follow up, so having a system to do this stuff for me was KEY, and in this case, EMP (Elite Marketing Pro), gave me an email autoresponder follow up sequence that sells FOR me, with good sales letters and a whole turn key process. That changed everything for our business.
And for follow up: They say the average person needs 5-8 follow ups to make their decision to buy, and when you have kids, a business, wife, etc., that's just about impossible!
I've just never had time for that, so this is why it was a weakness of mine! Well when you have emails going out every day to laser targeted entrepreneurs who are already looking for solutions, you don't have ANY rejection, and the emails are doing the selling FOR you, even while you're SLEEPING!
I can't tell you how satisfying it feels to wake up to emails that say "you've made commissions" every morning, (but I'm sure you'll know soon enough).
Yes you have to come out of your comfort zone and learn this stuff initially, but if you're dead serious about taking your business where it needs to be, it's literally the ONLY way with today's new-school internet business models.
FEWFF! Crazy right? Are you crazy enough to know that it works?
I hope that was eye opening and valuable for you… Leave me a comment or share if you liked it!
P.S. – You can click here if you have any questions. I can't wait to hear your story!


Superior Oldsmobile cars yearn for superior parts... clearly.
If you're tired of the high prices from your mechanic and need some Oldsmobile replacement parts, then we've got what you are looking for. All Oldsmobile drivers will know that nothing but top quality replacement Wheel Hub & Bearing parts are the only ones that belong on their vehicle. Since you drive a Oldsmobile, you should recognize that maintaining it running well is important. Wheel Hub & Bearing failure Wheel Hub & Bearing is faulty it can result in an unquestionably annoying experience for even the most seasoned drivers.
Finally... you are changing your ride's Wheel Hub & Bearing.
Though the wheel was invented about 10,000 years ago, it required a special flanged mounting surface so it could be attached for an axle. A wheel hub is just that part, and your Oldsmobile needs one for each wheel so that it can roll smoothly as you drive down the road. There will be a noticeable amount of wobble, noise, and vibration if yours is bent or broken. Not only that, but if the wheel hub in your Oldsmobile has an integrated wheel bearing that has gone bad, you'll have to replace the entire assembly at once.
Because our live sales, customer service, and technical support staff are available via phone and email, CarPartsDiscount.com thrives to be your last stop online for the best replacement Wheel Hub & Bearing parts. We at Car Parts Discount would consider it our greatest honor to be your preferred choice for Oldsmobile parts online. Being an enthusiast of a vehicle that's famous for its a dash of luxury means you shouldn't trust it to the cheapest parts you can find. To find the applicable Wheel Hub & Bearing parts for Oldsmobile 88, 98, Achieva, Alero, Aurora, Bravada, Cutlass, Cutlass Ciera, Cutlass Cruiser, Cutlass Supreme, Intrigue, LSS, Regency, Silhouette and more, choose your model and checkout with a smile.


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Witness: Drivers drag racing before Avon crash
One seriously injured in Route 24 crash
Updated: 5:16 PM EDT May 1, 2014
Pam Cross
A violent car crash on Route 24 in Avon is under investigation after witnesses said the two drivers involved were drag racing.Witnesses said two cars were racing and one lost control, crashing into a grassy area.Watch report"I saw a couple of cars pass me, and I thought, 'Wow those guys are going pretty fast.' And as I came up to Harrison Boulevard, I saw one of the cars, looks like he went across the grass and crashed," witness Dennis Bunting said.Bunting stopped and saw one man partly ejected and another nearby."I stopped to see if I can help the people out," he said. "By then, another tow truck stopped, and an off-duty EMT came up and we did what we could do on scene until State Police arrived," he said.State police haven't said whether drag racing was the issue.The injured man was rushed to a Boston hospital."The guy that was half ejected, he was in pretty bad shape. There was a lot of blood on the scene. He was pretty bad," Bunting said.
AVON, Mass. —
A violent car crash on Route 24 in Avon is under investigation after witnesses said the two drivers involved were drag racing.
Witnesses said two cars were racing and one lost control, crashing into a grassy area.
Watch report
"I saw a couple of cars pass me, and I thought, 'Wow those guys are going pretty fast.' And as I came up to Harrison Boulevard, I saw one of the cars, looks like he went across the grass and crashed," witness Dennis Bunting said.
Bunting stopped and saw one man partly ejected and another nearby.
"I stopped to see if I can help the people out," he said. "By then, another tow truck stopped, and an off-duty EMT came up and we did what we could do on scene until State Police arrived," he said.
State police haven't said whether drag racing was the issue.
The injured man was rushed to a Boston hospital.
"The guy that was half ejected, he was in pretty bad shape. There was a lot of blood on the scene. He was pretty bad," Bunting said.


If you're looking for the best Chimney Sweep in Westwood, KS then look no further, you've found the right team to help with all your fireplace and chimney needs!
The homes of Westwood and the chimneys that serve them are over fifty years old. The character of these older structures is second to none. However, just like the homes, the hearths and flues have unique needs. With many of the homes, the availability of modern flue liners was hit and miss. The parging of smoke chambers and the sizing of flues are common issues we see with local chimneys.
The homes were frequently built by non-commercial builders, so consequently, chimneys and fireplaces are one of a kind. Sometimes the flues are too small for the fireplace and too large for the utility flues. These issues found by Full Service Chimney are easily repaired. We have been servicing the homes here for thirty years. By staying abreast of the current technologies in the field of hearths and flues, we offer homeowners options to keep their home safe and their hearth enjoyable.
Homeowners that have us out for an annual chimney sweep and inspection makes chimney upkeep far less expensive and easily handled. If it has been a while since you have had us out, contact one of our certified technicians today. We will take the time to discover what is going on with your system and listen to your plans for the hearth and fireplace. We would love to help you keep your hearth fires burning for years to come.
Westwood may not be well known to the rest of Johnson County's inhabitants, but chances are that they have driven through it on Rainbow Boulevard and just didn't realize it. The town, incorporated in 1949, covers one half square mile in the area tucked into the northeast corner of the county between Roeland Park, KCK, and KCMO. It also borders Westwood Hills, which voted not to merge in 1973. Although the city is small in comparison to its neighboring cities, it still offers its 1,700 residents retail and corporate options, an elementary school, a park, a community center, and city police. The city is proud of the pleasant and accessible community it has to offer residents and visitors.
Full Service Chimney inspects, cleans, and repairs Westwood chimney's daily… as we've done for over 30 years!


text:
Q: Package for magento connect 1.0 and 2.0 When submitting my package to magento connect I'm getting a rejection with the following message:

Extension Packaging: Repackage your extension correctly so that
  compatible Community Editions and/or Enterprise Editions correspond
  with correct channels. (Magento Connect 1.0 and Magento Connect 2.0).
  DO NOT resubmit as a new extension. It will be rejected and deleted
  from your account. Repackage and resubmit as instructed in the
  guideline

I packed my extension using the built-in extension packager on magento 1.7 and I marked  "1.5 and later" and  "Pre 1.5" both.
What am I missing here?

A: Roy, packager creates 2 different packages in var/connect/ and var/pear/. The first package is   Magento Connect 2.0 version, the second one - 1.0 version.
The trick is that you should upload version for magento connect 1.0 from var/pear/ and it will be automatically converted to the 2.0 version

A: Magento has a set of guides on how to write, package extension and design your profile http://www.magentocommerce.com/magento-connect/create_your_extension/
The link to Magento Connect Packing Extension Guidelines file is below:
http://info.magento.com/rs/magentocommerce/images/packagingmagentoconnectextensions6%200.pdf


Yea! I am so excited to hear that you are enjoying the Moms Panel! We love knowing that our guests are finding us very helpful when it comes to planning for upcoming Walt Disney World vacations. You are very lucky living in Florida! If I lived there, I would be grinning from ear-to -ear knowing that WDW is in the very state in which I reside.
You are more than welcome to set dining reservations at any of the WDW Resorts. Just have your reservation number handy as you approach the resort entry.


text:
John Arthur ist der Name folgender Personen:

* John Arthur (Politiker) (1875–1914), australischer Politiker
 John Arthur (Boxer) (1929–2005), südafrikanischer Boxer
 John R. Arthur (* 1931), US-amerikanischer Chemiker

redpajama_set_name: RedPajamaWikipedia


text:
Wellesley Public School Libraries
K-12 Research Guides
Wellesley High School Research Guides
Cold War Research Guide
Cold War Research Guide: Home
Databases for Background Info
Books for Background Info
Cold War Reference Library
Click the picture or link to access.
Encyclopedia of Cold War Politics by Brandon Toropov
Call Number: REF 973.92 TOR
Cold War by James R. Arnold (Editor); Roberta Wiener (Editor)
Call Number: REF 909.82 COL
Search the WHS Library Catalog
Anywhere Title Author
Suggested Books in Library
The Cold War by Meredith Day (Editor); Britannica Educational Publishing Staff (Contribution by)
Call Number: 909.82 COL
Cold War by Carole K. Fink
Call Number: 909.82 FIN
Berlin on the Brink by Daniel F. Harrington
Call Number: 943 HAR
The War in Vietnam by Anthony O. Edmonds
Call Number: 959.704 EDM
The Berlin Wall by Frederick Taylor
Call Number: 943 TAY
The Korean Conflict by Burton I. Kaufman
Call Number: 951.9042 KAU
Next: Primary Sources >>
Last Updated: Sep 23, 2019 10:19 AM
URL: https://libguides.wellesleyps.org/hs/coldwar
The Wellesley Public School System does not discriminate on the basis of race, color, religion, national origin, age, gender, gender identity, sexual orientation, or disability in admission to, access to, employment in, or treatment in its programs and activities. The Wellesley Public School System is committed to maintaining a school environment free of harassment based on race, color, religion, national origin, age, gender, gender identity, sexual orientation, or disability. All district academic and nonacademic programs and events are offered regardless of race, color, religion, national origin, age, gender, gender identity, sexual orientation, or disability.Harassment by administrators, certified and support personnel, students, vendors and other individuals at school or at school-sponsored events is unlawful and is strictly prohibited. The Wellesley Public School System requires all employees and students to conduct themselves in an appropriate manner with respect to their fellow employees, students and all members of the school community.The Director of Human Resources, 40 Kingsbury Street, Wellesley, MA 02481, 781-446-6200, has been designated as the employee responsible for coordinating the Wellesley Public School district's efforts to implement this nondiscriminatory policy.
School Committee Policies • Legal Notices • Accessibility


+(0-4) to radiusPlace into an item socket of the right colour to gain this skill. Right click to remove from a socket. , or Minion Instability.
No lightning damage will be added to damage that is not part of a "hit", such as damage over time effects. The lightning damage will however be added to the weapon hits from Puncture, Caustic Arrow, Viper Strike, and any other skill that hits.
Added Cold Support and Added Lightning Support have been buffed, bringing them on par with the Added Chaos Support gem. Added Cold Support now does 35.1% more damage at gem level 20. Added Lightning Support now does 17.5% more damage at gem level 20.
Increased the added damage at all levels. Now grants 0.5% increased lightning damage per 1% quality instead of increased chance to shock and shock duration.
The mana multiplier has been reduced from 140% to 130%.
Added Lightning Damage has been increased by 15% at level 1, up to 100% at level 15.
Added Lightning Damage has been moved from level 15 to level 12.
This page was last edited on 23 February 2019, at 12:48.


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The Chicks
How to See Kacey Musgraves, the Chicks Perform at ACL Fest
If you're not in Austin, Texas to catch the 2022 ACL Fest this weekend, you can still watch the show from the comfort of home.
The Chicks' Divorces Taught Them to Write From Real Life
Plus, Natalie Maines and Martie Maguire explain how going through divorces at the same time helped them feel less alone.
The Chicks Will Work For Social Causes on Upcoming Tour
The trio is partnering with environmental nonprofit Reverb on their upcoming amphitheater tour.
Cody Johnson Covers the Chicks' 'Travelin' Soldier'
It's a really powerful performance.
Jeremy Chua