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The vacuolar proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also regulates transmembrane proton (H+) secretion, which is necessary for an array of physiological functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells. Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite sex). We conclude that V-ATPase-mediated H+ secretion in the olfactory epithelium is required for optimal olfactory function.
Citation: Păunescu TG, Rodriguez S, Benz E, McKee M, Tyszkowski R, Albers MW, et al. (2012) Loss of the V-ATPase B1 Subunit Isoform Expressed in Non-Neuronal Cells of the Mouse Olfactory Epithelium Impairs Olfactory Function. PLoS ONE 7(9): e45395. https://doi.org/10.1371/journal.pone.0045395
Editor: Renping Zhou, Rutgers University, United States of America
Received: May 31, 2012; Accepted: August 22, 2012; Published: September 20, 2012
Copyright: © Paunescu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by the National Institutes of Health (NIH), www.nih.gov. T.G. Păunescu is supported by an NIH K01 grant DK-73266. Additional support was from NIH grants OD006662 (M.W. Albers) and DK-42956 (D. Brown). The Microscopy Core Facility of the Program in Membrane Biology receives additional support from the Boston Area Diabetes and Endocrinology Research Center (NIH grant DK-57521) and from the Center for the Study of Inflammatory Bowel Disease (NIH grant DK-43341). 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.
The vacuolar proton-translocating ATPase (vacuolar, or V-type, H+ATPase, or V-ATPase) plays an essential role in numerous vesicle trafficking processes. When expressed at the plasma membrane, the V-ATPase performs various cell type-specific functions: in renal collecting duct A-type intercalated cells, it is the main mediator of acid secretion by the kidney, thus maintaining body acid-base homeostasis [1], [2], [3], [4]; in epididymal clear cells, it is responsible for the acidification of the luminal fluid that is necessary for sperm storage and maturation [5], [6], [7]; in neutrophils and macrophages, it regulates cytosolic pH [8], [9]; in interdental cells of the inner ear, it is involved in regulating the pH of the endolymph [10], [11], [12]; and in osteoclasts, it is required for bone resorption [13], [14], [15], [16].
Given that the olfactory epithelium (OE) has been implicated in acid-base sensing and/or regulation [17] and that this tissue expresses carbonic anhydrase [17], [18], [19], [20], [21], an enzyme which is also highly expressed in H+-secreting cells in other tissues, we investigated whether the V-ATPase is expressed in this tissue. We detected various subunits of this enzyme in cells of the main OE (MOE) and of the vomeronasal organ (VNO) in the mouse [22]. Importantly, one of the V-ATPase subunits that we detected in the OE was the 56-kDa B1 subunit isoform, a marker of specialized H+-secreting cells in other organs, such as the kidney, lung, male reproductive tract, eye and ear [5], [12], [23], [24], [25], [26], [27]. We reported that the B1 subunit of the V-ATPase localizes to the apical microvilli of sustentacular cells (SCs) and to the lateral membrane in a subset of other epithelial cells of the mouse OE [22].
Based on the involvement of SCs in ion [28], [29], [30], [31], salt, and possibly water [28], [32] transport in the neuroepithelial mucous layer (NML), we hypothesized that V-ATPase-mediated H+ secretion may confer on SCs the role of acid-base regulators of the extracellular milieu in the OE. Consequently, the physiological significance of V-ATPase expression in the OE was assumed to involve acidifying the NML, which in turn could be important for sensitivity to odorants [22] - reminiscent of the way in which the mucosal concentration of various ions, including Na+, K+, and Ca2+, affects the sensitivity of odor detection [33]. It has been previously postulated not only that the V-ATPase is involved in regulating the pH of the NML but also that it may power other ion transport mechanisms and thus regulate the ionic balance of this layer [29]. Since in many mammals the NML contains odorant binding proteins [34], both odor discrimination and odor intensity could be dependent on pH, and hence on V-ATPase activity.
Absorption of various substances in the nasal mucosa depends on the pH of the formulation, exhibiting an increase in bioavailability at lower pH values – e.g. for azetirelin [35] and secretin [36] in rats and insulin in dogs [37]. For dairy products in which volatile fatty acids are important flavor compounds, the pH of the aqueous phase influences perception by affecting the dissociation equilibria of these acids [38]. Nasal toxicity varies depending on the thickness and pH of the NML, among other factors [39]. Vanilloid receptors in sensory neurons respond to capsaicin in a pH-dependent manner, i.e. the potency of capsaicin increases at lower pH values [40]. Moreover, V-ATPase alongside other putative membrane transporters is critical for maintaining intracellular pH [41], which may impact a wide array of physiologic and metabolic processes.
In the present study, we characterize the expression pattern of the V-ATPase in the MOE with greater resolution using immunohistochemical methods followed by fluorescence, confocal and electron microscopy. In addition, we investigate the involvement of this enzyme in olfactory function through behavioral studies in mice that are null for the critical B1 subunit of the V-ATPase.
To characterize V-ATPase expression in the mouse olfactory epithelium, we used previously described affinity-purified polyclonal antibodies raised in rabbit against mouse ATP6V1A (the V-ATPase 70-kDa "A" subunit) [42] and ATP6V1B1 (the V-ATPase 56-kDa "B1" subunit isoform) [22] and in chicken against mouse ATP6V1B1 [43].
We used the following commercially available antibodies as olfactory markers: a goat polyclonal antibody against olfactory marker protein (OMP) (WAKO Chemicals USA, Richmond, VA), a rabbit polyclonal antibody against cytokeratin-18 (CK-18) (Abcam, Cambridge, MA), a rabbit polyclonal antibody against cyclic nucleotide gated cation channel 2 (CNGA2) (Alomone Labs, Jerusalem, Israel), a mouse monoclonal anti-tubulin antibody and a rat monoclonal anti-L-CAM/E-cadherin antibody (Sigma-Aldrich, St. Louis, MO).
The secondary antibodies used for this study were indocarbocyanine (Cy3)- or fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit, Cy3-conjugated donkey anti-chicken and anti-rat, and Cy3- and FITC-conjugated donkey anti-goat antibodies purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Tissue Preparation and Immunohistochemistry
Olfactory epithelial tissues were prepared and subjected to immunohistochemistry protocols as previously reported [22]. Mouse pups (2-weeks) or adult mice, wild type (Atp6v1b1+/+) (C57BL6, Jackson Laboratory, Bar Harbor, ME) and B1 V-ATPase-deficient (Atp6v1b1−/−, B1-deficient) [44] were deeply anesthetized with 200 mg/kg body weight i.p. pentobarbital sodium (Nembutal, Abbott Laboratories, Abbott Park, IL). The head was dissected and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in phosphate-buffered saline (PBS) for 4 h at room temperature and subsequently overnight at 4°C. All animal studies were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care, in accordance with the NIH, Department of Agriculture, and AAALAC requirements.
Fixed tissues were extensively washed in PBS, and pup tissues were subsequently stored at 4°C in PBS containing 0.02% sodium azide until use. Adult tissues were stored in a similar way after being decalcified using Immunocal (Decal Chemical Corp., Tallman, NY) or Cal-Ex (Fisher Scientific, Pittsburgh, PA) per the manufacturer's recommendations. As discussed previously, cryosectioning of pup tissues did not require prior decalcification [22]. For cryosectioning, tissues were cryoprotected in PBS containing 0.9 M sucrose, embedded in Tissue-Tek OCT compound 4583 (Sakura Finetek USA, Inc., Torrance, CA), and frozen at −20°C as previously described [45], [46]. Sections were cut on a Leica CM3050 S cryostat (Leica Microsystems Inc., Buffalo Grove, IL) at 5 µm for regular immunohistochemistry and at 16 µm for 3-D image reconstruction. Tissue sections were collected onto Superfrost Plus microscope slides (Fisher Scientific), air-dried and stored at 4°C until use. For immunohistochemistry, sections were rehydrated in PBS, treated with sodium dodecyl sulfate (1% wt/vol. in PBS for 4 min) for antigen retrieval [47], washed in PBS, and incubated with 1% bovine serum albumin in PBS for 10 min followed by the primary antibody diluted in Dako antibody diluent (Dako, Carpinteria, CA) for 90 min at room temperature as previously described [22], [25], [46]. Slides were then washed and incubated for 1 h with the respective secondary antibody, rinsed in PBS, and mounted in Vectashield medium (Vector Laboratories, Burlingame, CA) containing 4,6-diamidino-2-phenylindole (DAPI). For dual immunofluorescence staining, the two primary antibodies were applied either simultaneously (followed by the secondary antibody mixture) or sequentially, with each primary antibody followed by the respective secondary antibody. We found these two approaches to yield similar results.
Digital images were acquired using a Nikon Eclipse 80i epifluorescence microscope (Nikon Instruments, Melville, NY) fitted with an Orca 100 CCD camera (Hamamatsu, Bridgewater, NJ) and analyzed using IPLab version 3.2.4 image processing software (Scanalytics, Inc., Fairfax, VA) [22], [25].
For confocal laser scanning microscopy, imaging was performed on a Zeiss Radiance 2000 confocal microscopy system (Carl Zeiss Microimaging, Thornwood, NY) using LaserSharp 2000 version 4.1 software. 3-D image reconstruction was performed using Volocity software (Improvision, Waltham, MA) on stacks of 60 images collected at 0.25 µm intervals along the z-axis as previously described [43]. Images were subsequently imported as 3-D projections into Adobe Photoshop version 6.0 image-editing software (Adobe Systems Inc., San Jose, CA). Final figures for epifluorescence and confocal microscopy were produced using the same software.
Immunogold Electron Microscopy
Tissues were processed and 50 µm thick sections were cut as described above. Tissue sections were floated off in PBS, dehydrated, embedded, and polymerized at 50°C in LR White resin (Electron Microscopy Sciences) as previously described [43], [48]. Thin (70–80 nm) sections were cut on a Leica EM UC7 ultramicrotome (Leica Microsystems) and incubated on drops of rabbit primary antibody against V-ATPase A-subunit diluted in Dako diluent for 2 h at room temperature, rinsed in PBS, and then incubated on drops of goat anti-rabbit IgG coupled to 15 nm gold particles (Ted Pella, Redding, CA) for 1 h at room temperature. After several rinses with distilled water, the grids were post-stained on drops of 2% uranyl acetate for 5 min, rinsed again in distilled water, and dried. For dual immunostaining, grids were incubated with a mixture of primary antibodies against V-ATPase A-subunit and tubulin. The secondary antibodies were also applied simultaneously, goat anti-rabbit IgG coupled to 15 nm gold particles and respectively anti-mouse IgG coupled to 10 nm gold particles (Ted Pella). Sections were examined in a JEM-1011 transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 80 kV, and images were acquired using an AMT XR60 digital imaging system (Advanced Microscopy Techniques, Danvers, MA). Final figures were produced using Adobe Photoshop as described above.
Home-cage Tests to Assess the Ability of Female Mice to Detect Male Urinary Odors
We collected urine from adult heterozygous (Atp6v1b1+/−) male mice and performed home-cage tests on sexually naive adult wild-type and B1-deficient females in vaginal proestrus or estrus, similar to previously described habituation-dishabituation tests [49], [50], [51]. Each wild-type or B1-deficient female received two 2-min. presentations of distilled water followed by two 2-min. presentations of male urine. These stimuli were presented at 4-min. intervals, by pipetting 10 µl of fluid on a 2 cm2 piece of filter paper taped to a plastic weigh boat [49], [50]. Stimulus investigation (sniffing) is defined as an inhalation directed at the filter paper with the tip of the nose within 1 cm of the filter paper. Animal behavior was followed in real time by visual observation, and stimulus investigation time was recorded using a programmable timer (Fisher Scientific). Unpaired t-tests were used to analyze the data.
Avoidance of a Predator Odor
We investigated the innate avoidance behavior that mice are known to display toward predator odors [52], [53] by using a test cage divided by a curtain into two compartments [54]. Specifically, trimethyl-thiazoline (TMT), a volatile odor isolated from fox feces, was used. Briefly, the experiment was performed in the dark during the nocturnal phase of the light cycle. The behavioral arena was placed in a chemical hood. Mice were habituated to the arena for 3×10 min with blank filter papers. After 7 min. of the third habituation, the filter papers were swapped with identical filter papers with 20 µL of water or 4% TMT dissolved in water, respectively. The remaining three minutes were video recorded and scored in a blinded fashion. Scoring included summing the accumulated time that the mouse spent freezing, defined as absence of movement save for thoracic oscillations consistent with breathing, and the number of sniffs within a 1 cm radius of each filter paper during the three minute epoch. Since the TMT concentration is suprathreshold, most avoidance and freezing behavior was initiated at distances much greater than 1 cm from the filter paper. While the mice had direct nasal access to the filter paper, we did not observe that any mouse directly contacted filter paper; thus, we interpret this phenotype as mediated by the main olfactory system and not the VNO. One-way analysis of variance and unpaired t-tests were used to analyze the data.
Mucosal pH Measurements
Whole olfactory epithelia from adult wild-type and B1-deficient female mice were dissected and cut with a razor blade into right, septal, and left parts. pH readings were taken from the luminal surface of each olfactory epithelial part using micro pH strips (pHydrion MicroFine, Micro Essential Laboratory, Brooklyn, NY) in the pH range of 6.0 to 8.0. Each micro pH strip was read by two different investigators unaware of the mouse genotype and averaged; the values from the three different regions were further averaged to yield the recorded mucosal pH value for the respective animal. For any given measurement point, a majority of the two investigators' readings were identical, and the largest difference between the two was 0.2 pH units. For any given animal, the differences between olfactory epithelial parts, if any, were within 0.2 pH units, with the exception of one mouse in which the differences ranged between 0.1 and 0.3 pH units. Unpaired t-tests were used for the statistical analysis of the data.
Results and Discussion
Localization of the V-ATPase in the Mouse Main Olfactory Epithelium (MOE)
The V-ATPase is a complex enzyme, composed of at least 14 subunits assembled into a transmembrane, proton- (H+-) translocating V0 sector and a cytosolic, catalytic V1 domain (Fig. 1) [55], [56], [57], [58]. In mammalian tissues the 56-kDa B subunit of the enzyme is expressed as two isoforms, B1 and B2. B1 is the B subunit isoform generally associated with regulated transmembrane H+ secretion in epithelia [5], [23], [24], [25], and localizes at high levels to the cell plasma membrane and to the submembrane domain. B2 is to a lesser extent associated with the cell plasma membrane, but predominantly with the membranes of intracellular organelles [22], [25].
(A) A schematic diagram showing the subunit composition of the V-ATPase. The cytosolic V1 domain is composed of subunits A through H (shown in white or light gray, marked with blue letters). The transmembrane V0 domain is composed of subunits a, c, c" (or b), d, e, and Ac45 (shown in blue, marked with white letters). Some of the subunit interactions are putative. (B) Section from a 3-D image reconstruction showing that B1 V-ATPase (red) localizes to the microvilli of olfactory sustentacular cells in a 2-week old female mouse pup. Apical cilia of olfactory sensory neurons are immunostained for CNGA2 (green). DAPI (blue) stains cell nuclei. Bar = 30 µm.
V-ATPase expression and localization in the MOE of adult and juvenile mice were investigated by immunohistochemistry. The results were similar in the two age groups [22]. The B1 subunit isoform localizes mostly to the apical microvilli of SCs, as we originally described [22]. To confirm and more thoroughly examine this localization pattern, we performed dual immunofluorescence staining for V-ATPase and the olfactory sensory neuron (OSN) markers, the cyclic nucleotide gated cation channel 2 (CNGA2) and the olfactory marker protein (OMP). Our results show that the V-ATPase B1 subunit does not colocalize with CNGA2, but instead is detected in a layer that does not extend into the lumen as far as the region occupied by this OSN marker. Similarly, the A subunit of the V-ATPase (Fig. 2A), which is the subunit that most closely associates with the B subunit within the complex V-ATPase structure (Fig. 1), does not colocalize with OMP (Fig. 2B). OMP-associated immunostaining is located more apically relative to the V-ATPase signal (Fig. 2C). These results are consistent with CNGA2 and OMP staining OSN cilia, whereas anti-V-ATPase antibodies stain SC microvilli.
The V-ATPase is expressed on the apical microvilli of SCs and on the basolateral membrane of a subpopulation of cells (arrows). Olfactory marker protein (OMP, B, green) localizes to the OSN cilia and to the basolateral membrane of OE cells (arrowheads) which, as revealed by the merge panel (C), do not coincide with the cells that express B1 in this membrane domain. DAPI (blue) stains cell nuclei. Bar = 10 µm.
Besides its SC localization, the V-ATPase was also detected in a subset of cells of the mouse MOE, where it localizes to the basolateral plasma membrane domain [22]. Given the relatively low density of these cells, their location within the MOE and their morphology, we determined that they are most likely not SCs. Dual immunostaining with the V-ATPase A (Fig. 2) and B1 subunit (data not shown) and OMP (Fig. 2) or CNGA2 (data not shown) also rules out the possibility that these cells may be OSNs, as previously presumed [22]. However, dual immunostaining with antibodies against the V-ATPase B1 subunit isoform and cytokeratin-18 (CK-18, Fig. 3) or L-CAM/E-cadherin (data not shown) reveals that the cells with basolateral membrane staining for V-ATPase also express these markers. Taken together, these data allow us to identify this epithelial cell subpopulation as microvillar cells, as previously described in the literature [59], [60].
As also shown in the previous figure for the A subunit, B1 localizes to the apical membrane microvilli of SCs and to the basolateral membrane of a subset of OE cells (A and D, arrows). Interestingly, these cells also exhibit basolateral membrane staining for CK-18 (B and E, arrows). The merge panels (C and F) confirm the co-expression of the V-ATPase B1 subunit and CK-18. Bar = 20 µm.
The cellular and subcellular localization of V-ATPase in the MOE was further investigated using immunogold electron microscopy (IEM). We found the A subunit of the V-ATPase to be expressed at high levels on the microvilli of olfactory SCs, confirming the immunofluorescence results presented above (Figs. 2–3). To a lesser extent, the A subunit also localized in the cytosolic domain of SCs and OSNs, consistent with the well known presence of A- (but not B1-) containing V-ATPases on the membrane of intracellular organelles in all cells [2], [3], [61]. As expected, tubulin localized exclusively to OSN cilia and not to SCs (Fig. 4, see also higher magnification inset).
The V-ATPase A subunit (panel A, arrowheads) is expressed at high levels in the microvilli of sustentacular cells (SC), but is minimally present in the olfactory sensory neurons (OSN), where it localizes to the cytosolic domain. Panel B shows the same expression pattern for the V-ATPase (arrowheads, 15 nm gold particles), whereas tubulin (arrows, 10 nm gold particles) localization is restricted to OSN cilia (shown at higher magnification in inset). Bar = 0.5 µm. Inset bar = 0.1 µm.
Consequently, the V-ATPase, including its B1 subunit isoform, which is a hallmark of regulated transmembrane H+ secretion, localizes to the apical membrane microvilli of olfactory sustentacular cells, but not to olfactory sensory neurons. The B1 subunit is also not expressed in the olfactory bulb at detectable levels by immunohistochemistry (T.G. Păunescu, unpublished observations) or in the brain in general, as previously determined by immunoblotting [24]. Brain V-ATPases typically contain the alternate B2 isoform and localize to the membranes of intracellular organelles.
Diminished Olfactory Function in B1-deficient Mice as Revealed by Home Cage Tests
As reviewed above, plasma membrane V-ATPases containing the B1 subunit isoform fulfill cell type-specific roles in all tissues and cells in which the enzyme has been detected. We, therefore, investigated the possible role of this enzyme in the OE. Home-cage tests performed on female wild type and B1-deficient mice with distilled water showed no significant difference between the two groups of animals or between presentations (all p values >0.05, n = 5 wild-type and n = 5 B1-deficient mice), as shown in Fig. 5. However, male urine presentations revealed that wild-type mice spent significantly more time investigating the urine of the opposite sex than B1-deficient mice (p = 0.001 for the first presentation and p = 0.009 for the second stimulus). Interestingly, while urine investigation time declined for the second exposure in wild-type mice (p<0.05), this parameter showed a slightly increasing trend in B1-deficient animals.
Water presentations revealed no significant difference between wild type and B1-deficient mice or between presentations. Both urine presentations showed that wild-type mice spent significantly more time investigating the urine of the opposite sex than Atp6v1b1−/− mice (n = 6). Data are shown as mean ± SEM (**, p<0.01).
The nature of this experiment does not preclude direct contact of urine with the nose and hence involvement of the VNO in generating the perception. However, we still interpret the observed deficit as arising from altered epithelial physiology since V-ATPases containing the B1 subunit isoform are also expressed in the VNO [22].
Diminished Olfactory Function in B1-deficient Mice as Revealed by Innate Avoidance Behavior Tests
The restricted expression pattern of the V-ATPase to the non-neuronal cells in the OE affords the opportunity to assess the functional role of this protein on olfactory function by surveying behavioral responses to odors isolated from predators with innate behavioral responses. The behavioral arena (Fig. 6A) consisted of a test cage divided by a curtain into two compartments, as described in "Methods". Adult B1-deficient male mice displayed a blunted response to trimethyl-thiazoline (TMT) compared to their wild type counterparts. The freezing behavior in the two groups of animals was similar in the absence of TMT (p = 0.12, data not shown), whereas the percentage of time spent freezing by B1-deficient mice in the presence of TMT was less than half the time recorded in wild-type animals (p = 0.02, n = 7 wild-type and n = 6 B1-deficient mice) (Fig. 6B). Also consistent with an olfactory deficit, the number of sniffs towards the TMT stimulus was higher in B1-deficient mice compared to wild type controls (p = 0.05) (Fig. 6C, Movie S1 and Movie S2).
(A) A schematic diagram of the behavioral arena is depicted. (B) The percentage of time freezing in adult male B1-deficient mice ("KO", n = 7) in the presence of the predator odor trimethyl-thiazoline (TMT) is less than half the time recorded in wild-type mice ("WT", n = 6). Data are shown as mean ± SEM (p = 0.02). (C) Increased investigation of TMT in the V-ATPase B1-deficient mice: The number of individual sniffs of TMT is increased in adult male B1-null mice ("KO", black bar) relative to wild-type mice ("WT", gray bar). Data is shown as mean ± SEM (p = 0.05) and confirm the blunted response to TMT in B1-deficient mice compared to their wild type counterparts.
In conclusion, our functional studies on mice deficient in the B1 subunit isoform of the V-ATPase [44], [45] revealed diminished innate avoidance behavior, measured as the number of TMT sniffs and time spent exhibiting freezing behavior in the presence of this predator odor, as well as the fact that they spent less time investigating the urine of the opposite sex in comparison to their wild-type counterparts. Female B1-deficient mice are less attracted to male specific urine and male B1-deficient mice avoid less and freeze less in response to a repulsive odorant. Both phenotypes could arise from either olfactory deficits and/or in central circuits that underlie assignment of valence to olfactory input. Since B1 is not expressed in the brain and is strongly expressed in the OE, we favor the olfactory hypothesis and not the central hypothesis.
Data from behavior tests point towards the involvement of V-ATPase in olfactory function. This finding is also consistent with the subcellular localization of the V-ATPase in wild-type vs. B1-deficient mice, as previously reported [22]. In intercalated cells of renal medullary collecting ducts from B1-deficient mice, V-ATPase activity is reduced to 28–40% of normal baseline values under circumstances where B2-containing V-ATPases are inserted maximally in the apical plasma membrane [45]. However, our preceding study showed that, unlike in these renal H+-secreting cells [44], [45], subapical V-ATPases containing the alternative B2 subunit isoform do not undergo a localization shift towards the apical microvilli of sustentacular cells in B1-deficient mice [22] and likely do not, therefore, compensate for the lack of the B1 isoform. Consequently, it should be expected that these animals have a defect in V-ATPase-mediated H+ secretion in the OE that negatively impacts their ability to detect urinary odors and odors with innate behavior responses. To investigate this possibility, we measured the pH of the luminal side of the olfactory epithelium directly and found a consistent and statistically significant (p = 0.0006) pH increase in the B1-deficient mice compared to wild-type animals. Mucosal pH averaged 6.758±0.036 in wild-type mice (mean ± SD, n = 4) compared to 7.010±0.012 in B1-deficient animals (mean ± SD, n = 4).
These data are consistent with our hypothesis that olfactory sustentacular cells may act through V-ATPases to regulate the pH of the neuroepithelial mucous layer (NML), which could in turn modulate the sensitivity to odorants [22]. This would not be unprecedented, since the NML concentration of various ions (e.g., Na+, K+, and Ca2+) was previously reported to affect the sensitivity of odor detection [33]. Moreover, the V-ATPase was previously postulated to power other ion transport mechanisms in the olfactory mucosa, thus regulating indirectly its ionic balance [29], as is known to happen in the inner ear [62].
We envisage that the pH of the NML could be important in various mechanisms included in classical models of olfaction, such as: 1) the absorption of odorant molecules from the air phase into the NML (mucus solubility and the equilibrium partition coefficient for the odorant at the air-fluid, i.e. mucus interface potentially being pH-dependent), 2) diffusion of odorants across the mucociliary layer (molecular diffusivity of the odorant presumably being pH-dependent), 3) odorant molecule binding to odorant binding protein, 4) odorant molecule-receptor binding on the OSN membrane and cilia (pH was shown to affect protein-ligand binding), and 5) odorant removal (the dissociation constant and the odorant uptake rate potentially being pH-dependent) [63], [64], [65], [66]. Moreover, some peptides and trace amines have been shown to mediate innate behavioral responses via olfaction [67], [68], [69], [70], [71], which could be modulated by pH.
It is of particular interest that the V-ATPase, and especially its B1 subunit isoform, is highly expressed in microvillar cells, where it localizes to the basolateral plasma membrane domain. As far as we know, there is no consensus on the specific physiological role fulfilled by microvillar cells. Intriguingly, we previously reported that cells exhibiting basolateral V-ATPase staining also express carbonic anhydrase type IV (CA IV) [22]. By analogy with CA II-rich olfactory sensory neurons [18], [20], [72] that were reported to be CO2 chemoreceptors [20], [73], we raised the question whether these cells may be involved in modulating the pH of the NML in response to CO2 [22]. While this idea is yet to be tested directly, its confirmation would provide a novel, unrecognized physiological role for this insufficiently characterized cell type.
The present study is the first to indicate the relevance of the V-ATPase, and presumably of V-ATPase-mediated proton secretion, in olfactory function. Undoubtedly, further functional and behavioral studies will allow a more comprehensive assessment of the physiological and clinical significance of V-ATPase expression in sustentacular and microvillar cells of the olfactory epithelium. At this point, we can only speculate on such possibilities. For example, modulating olfactory H+ secretion could offer the ability of up- or down-regulating the threshold of detection for certain odorants. Moreover, since the NML plays a role as a barrier against inhaled pathogens, and microbial and chemical toxins, regulating mucus pH may be relevant for protection against various specific diseases.
Supporting Information
Movie S1.
Representative video recording showing the behavior of a wild-type mouse three minutes after the TMT (left hand side) and water (right hand side) are introduced into the behavioral arena. A directed sniff is indicated by the "sniff" title that appears in the lower right hand corner after the sniff was scored (n = 1 sniff for the wild-type mouse shown).
Movie S2.
A similar video recording showing the behavior of a B1-deficient mouse three minutes after the TMT (left hand side) and water (right hand side) are introduced into the behavioral arena (n = 6 sniffs for the B1-deficient mouse shown). Freezing behavior was also quantified in these representative movies, but is not indicated by titles. The wild-type and B1-deficient mice were tested on the same day.
We thank Dr. Michael J. Baum (Boston University) for sharing his expertise on home-cage tests and for critically reviewing this manuscript, and Dr. James E. Schwob (Tufts University) for fruitful discussions and useful suggestions of antibodies used in this study as markers for olfactory epithelial cell types.
Author Contributions
Conceived and designed the experiments: TGP MWA DB. Performed the experiments: TGP RT EB SR MM. Analyzed the data: TGP SR MWA. Contributed reagents/materials/analysis tools: TGP MWA DB. Wrote the paper: TGP SR MWA DB.
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I have written various pieces about my quest to work with Ellen DeGeneres and how it is a journey that began years ago. (see "Dear Ellen: Will You Be My Disco Dance Partner" and "Are You There Ellen DeGeneres? It's Me, Amy" in previous blog entries) Now it is taking on a new form though. I am still 110% (eh, maybe 118%) behind my idea to come on as a correspondent for The Ellen Show doing sketches and interviews surrounding women's soccer but there is a bigger issue at hand now. There may not even be a professional league left next season. So there is a large group of fans who do not want to see this league fail doing everything they can do to help it succeed and continue on as a prosperous league. There is an old expression; the pen is mightier than the sword. Personally, if I owned a sword and knew how to use it I'd be charging in on a horse, Knight-style demanding a league. But I do not. I know how to write…kind of…so that's what I'll do.
The Women's Professional Soccer league (WPS) is in need of a 6th owner for a new team in order for the league to continue running. Many of us fans are reaching out to a certain celebrity, Ellen DeGeneres to help save the league and become an owner. I have a list of reasons why Ellen should get involved.
1- Ellen is the greatest human alive. WPS is the greatest league alive. Done deal.
2- Ellen showed support for the US Women's National Team this summer during their run at the World Cup.
3- Ellen would be an entrepreneurial owner; designing the uniforms with some Ellen flare, choreographing an opening number for the team to enter the field, hiring an English DJ to entertain during halftime AND she could instate an opening monologue into soccer. (Which I suppose I could fill in for her here and there on that one. Hint, hint. Wink, wink. Subtle, subtle)
4- Ellen would be able to turn a whole new viewing audience onto women's soccer. The viewers are out there, but without the proper marketing, exposure and air time they will never be given the opportunity to become true fans.
5- The world without WPS would be far less bright and would leave great sadness behind. This would cause Ellen to have to start working overtime in order to bring more laughter and light into people's lives where there is now a giant gaping black hole of WPS-less existence.
6- I used Arrested Development as an analogy in my last blog about saving the WPS. Portia De Rossi, Ellen's wife, brilliantly acted in Arrested Development and portrayed one of my favorite characters, Lindsay Bluth. Brilliant yet under rated show / brilliant yet under rated league. We saw what happened there; Ellen can't let it happen to the WPS too! Brilliance needs to be rewarded.
7- The WPS and Ellen are both all about good causes. The WPS has been a strong supporter of Breast Cancer Awareness through "Project Pink" games and also various other local causes amongst the communities they play in.
8- Piggybacking off #7…one of the teams, the Western New York Flash, held a dog day where they teamed up with the local ASPCA to find homes for animals and also encouraged dog owners to bring them out to the game. Ellen is a HUGE advocate for animal rights and an animal lover. The possibilities are endless in the amount of good Ellen could do through a team.
I could probably make a list to 100 if I had the time. (Or the attention span) But you all get the picture. Ellen DeGeneres and the WPS would be a perfect match and I hope we get to see it happen.
Ellen, if you're out there: Please consider this. If you need to make a pros and cons list I am more than happy to swing by your pad and speak with you. We can make a list, have some coffee, dance, me you and Portia can go shopping, maybe play a prank on Taylor Swift…we'll make a day of it. After thinking about it you will see what a great opportunity this could be. This league inspires young girls and shows them that they can succeed and live their dreams. It inspires adults to stay active and get into shape by seeing such strength and endurance displayed. It inspires parents to teach their kids to be healthy and active and it gives them role models for their kids to look up to. This is a league with talent, skill, entertainment, endurance, aggression, grace, poise and it's filled with athletes who possess all of those things plus a level head on their shoulders who are all smart, kind, and beautiful and they are idols to many. Please help save this league and give it hope for the future.
Call me up when you're ready to sit down and discuss. I'll be by the phone in my Ellen wig dancing over a coffee table anxiously waiting.
Also take a look....
And yet...another way to help! Another petition! SIGN!
Also take a look....
Check out this site for sure. Great organization helping to put the word out there and gives instructions on how you can help!
Another way to help...a petition you MUST sign started by an awesome dedicated fan!
And yet...another way to help! Another petition! SIGN!

subset: commoncrawl


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Or Login using BecomeGorgeous
Please fill the form below and follow the further instructions.
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Published on: 17 Jan 2009 by yunakona
Okayy, I have two pictures here.
I want my BANGS like this [NOT MY HAIR] and I was wondering if that would look okay on me?
I mean, I love hair like that, and my hair is a bit longer than my shoulders and it would be okay, right?
I'll accept any advice:)


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The Dilemma
The national average for employee disengagement is at 70% according to ongoing Gallup surveys. 18% of employees are actually actively disengaged, which means they are actively opposing progress. Brand promises are broken consistently when engagement is low. Gallup studies show that this leads to increased rates of accidents, errors, turnover, productivity, and lower sales and profits. The stakes are high.
If you cannot lead your own team, how can you lead others? How can your message and marketing find any traction if you don't have it in your own ranks?
The Solution
Studies show that employee engagement results in 37% less absenteeism and turnover, 48% fewer safety incidents, 41% fewer product defects, 21% higher productivity, 22% higher profitability, for those who implement engagement strategies fully.
The Notionfront Employee Engagement Consultation is a comprehensive data-driven approach to driving employee engagement. It is designed for businesses and organizations large or small. It is built on proven methods using tools from the Gallup organization. These techniques have been tested and implemented in fortune 500 companies and with millions of participants. So it is a tried and tested method. Our consultation has 4 modules. That can be taken independently or in its entirety. It is a multi-session engagement.
The 4 modules are: Asses, Spark, Inspire, and Sustain.
The Assess Module is an employee survey that uses the Gallup Q12® proprietary survey. This survey has been used with over 25 million employees. It has been distilled to 12 scientifically formulated questions. These questions were consistently answered affirmatively by top engaged employees. Notionfront will assist in the implementation of the survey and provide a report of the results.
The Spark Module is designed to establish foundational elements of labor engagement. It builds on the Q12® management principles for your organization. It also focuses on the creation of you corporate "why". Which is a core component of the Q12® culture and is often not properly formulated. Then it interactively educates to top level management on the Q12® cultural goals needed to establish the overall engagement strategy.
The Inspire Module is designed to inspire management with interactive educational sessions. It teaches the principles of the Q12® engagement strategy. Management is responsible for 70% of the engagement level within every organization. So we will also engage in interactive discussions about the challenges of implementation. The inspiration module can also be extended to training to the team and staff level.
The Sustain Module is designed to maintain focus on the Q12® engagement strategies. Like our brains, organization requires focusing to achieve growth. This module consists of internet based educational materials and optional in-person coaching.
Objectives of the Consultation
- Increase employee engagement
- Create an organizational "why"
- Create a plan to develop workforce engagement
- 37% less absenteeism and turnover, 48% fewer safety incidents, 41% fewer product defects, 21% higher productivity, 22% higher profitability, for those who implement Q12® engagement strategies fully.
It starts with the Notionfront Employee Engagement Consultation.
Note: Gallup®, Clifton StrengthsFinder®, StrengthsFinder®, the 34 Clifton StrengthsFinder® theme names, and the Q12® Survey are trademarks of the Gallup, Inc. Notionfront is using these materials as an independent entity and is not an official representative of the Gallup® organization.


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Project Noah is a tool to explore and document wildlife and a platform to harness the power of citizen scientists everywhere.
Cairina moschata
Found relaxing on the SCF campus bldg 25. I thonk it's a muscovy.
Lat: 27.44, Long: -82.59
Spotted on Mar 10, 2011 Submitted on Mar 10, 2011


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IBM unleashes 'big data' on wind, solar power management
Chinese megaplant adopts Big Blue's cloud tech – no, not that cloud, the ones in the sky
IBM has announced a weather-modeling and power-grid management system with the goal of increasing the effectiveness and efficiency of renewable energy sources such as wind and solar power.
"Applying analytics and harnessing big data will allow utilities to tackle the intermittent nature of renewable energy and forecast power production from solar and wind, in a way that has never been done before," IBM's global energy and utilities industry headman Brad Gammons said in a statement. "We have developed an intelligent system that combines weather and power forecasting to increase system availability and optimize power grid performance."
The system, called Hybrid Renewable Energy Forecasting, or HyRef, is part of the Smarter Energy component of IBM's Smarter Planet initiative. HyRef uses cloud-imaging tech and cameras that track cloud movement, and combines that data with info from sensors on wind turbines that keep track of wind direction, temperature, and speed. All of this data is fed into weather models and analyzed in such a way as to be able, IBM claims, to "produce accurate local weather forecasts within a wind farm as far as one month in advance, or in 15-minute increments."
By doing so, IBM says that HyRef can enable renewable-energy installations to better predict the power they can produce – wind and solar being variable sources – and thus to better anticipate the amount of power they will be able to provide to the grid to which they are connected. Such predictions enable managers to better accommodate the need to supplement renewables with such conventional power sources as coal and gas-fired power plants.
The better you can predict the weather,
the more efficient your renewables become
(click to enlarge)
HyRef builds on IBM's data analytics work with Denmark's Vestas Wind Systems, a leading provider of wind-power turbines with nearly 49,000 turbines installed in 73 countries. That effort focuses on turbine placement, and is based on what IBM calls "petabytes of data from weather reporters, tidal phases, sensors, satellite images, deforestation maps, and weather modeling research."
HyRef's weather-modeling data analytics are an outgrowth of its work on such projects as Big Blue's Deep Thunder high-resolution weather forecasting, which IBM says can help businesses and governments "tailor services, change routes and deploy equipment to minimize the effects of major weather events by reducing costs, improving service and even saving lives." [Interesting prioritization of benefits there, eh?—Ed.]
HyRef already has one major partner: China's Zhangbei National Energy Storage and Transmission Demonstration Project in the heavily industrialized Hebei Province, which combines wind and photovoltaic electricity generation with lithium-ion battery storage.
Zhangbei is currently rated at 216MW, and is scheduled to grow to 670MW as part of China's 5-year plan to cut back on its usage of fossil fuels. IBM says that adding HyRef to Zhangbei will increase its efficiency by around 10 per cent, which translates to an amount of power sufficient to provide electricity to an additional 14,000 homes – Hebei Province homes, in any case.
IBM China research analyst Meng Zhang notes that currently 20 to 40 per cent of energy from renewables can't be used because of its inherent variability. HyRef, he says, with its ability to better predict not only the output of the renewable sources but also the needs and conditions of the grid to which they are connected, can markedly increase that efficiency.
The market for wind power is expanding rapidly, IBM says, with wind now providing over 2.6 per cent of global electricity, and is projected to rise to 4.9 per cent by 2017. IBM clearly wants to be a major resource-management player in a market it projects to total $109.8bn by that latter date.
The Chinese government, faced with horrendous air-pollution problems, recently pledged $277bn toward an initiative aimed at improving air quality in and around Beijing and the adjacent Hebei Province – site of the Zhangbei complex that's implementing IBM's HyRef. ®


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Friend's Email:
Subject: I have found a CD that I think you would enjoy
|Groove Holmes
Night Glider
Genres: Jazz, Pop, Rock
CD Details
CD Reviews
Richard Groove Holmes at his Phattest and Phunkiest ! ! !
Eddie Landsberg | Tokyo, Japan | 11/12/2000
(4 out of 5 stars)
"During the '70s, Richard Groove Holmes got REAL funky. The irony is that most of it seems to have been recorded on smaller labels, and very little of it has seen the light of day. You'll hear lot's of stuff of his from the heavy cookin' '60s, and the Bluesy '80s, but the funkiest chapter of Groove's life, the '70s when he'd be kicking funk basslines on a nova string bass unit, screaming on a modified console with a Moog on top, and accompanied by funky wa wa guitar and sampler's wet dream beats - - seems to be lost in the vaults... until now... sort of...This album features hints of that sound. If you're a straight ahead Jazzer, it may not appeal much to you, but if you're a funk and groove-a-teer, its definitely damn smoking, possibly the funkier-est cuts of his in print. Its not Groove *at* his funkiest, but assuming you haven't heard Groove at his funkiest, The Night Glider may be one of the FUNKIEST Hammond organ albums you ever heard... and if you don't believe me take a quick listen to FLY JACK if you can.Speaking of FLY JACK, if this album tickles your funky check out Jack McDuff's funky Down Home Style... and keep your eyes out in the musty dusty vinyl bins ! ! !"
This is 70's Funk Lounge
furthur_57 | Belleville, Ontario, CANADA | 01/14/2003
(5 out of 5 stars)
"Imagine yourself sipping on a cocktail and smoking
a long cigarrette - dressed in a lime-green suit. This
is the music in the background. At times serious, at times
seriously silly, this is an excellent album of Lounge-Funk.
It doesn't get much funkier. Groove produced a number of albums with lesser known labels during his funkiest period, and most of those recordings were misplaced long ago. This is probably the closest we can get to the sound he was rattling out of his Hammond in those lost records. Although most of the tracks are recordings of Holmes with other bands, (he wasn't the front-liner), they are an excellent sample of the funkified organ groove that made him so famous, the Beastie Boys named a song after him.
This is an excellent album for midnight walks, lounging in a black-lit room with headphones, (with party favours), or dancing by yourself. Makes excellent background music for get-togethers as well - themed or not."


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It was the kind of look you get in passing. They sparsely last a moment and so you have to know what you're looking for before it happens. Knowing it was going on as it was happening was tough, but tougher still was acting on it. The excitement was electrifying. He had definitely given me the look.
Sometimes you think it's the look, and an unfortunate and unsubstantial glance is just that. When the guy takes off his sunglasses he's usually trying to tell you something. He wants it to be irrefutably clear without tinted lenses to obscure the meaning. I want him to want that anyway.
I looked back in that split second to give him an affirmative. I had to make sure it was slow and deliberate, but also delicately nonchalant; like I was trying to act surprised or sneak up on a grazing deer. I gave the slightest wink that might have only been me moving my eye, but he knew what I was doing. It was a sultry gesture that meant take me, and do it now.
Except that he didn't move. At first I doubted myself, I thought I had been so clear. Anything more and the rest of the people on the bus would look at me funny and tell me to put my clothes on. Maybe it wasn't the look? Maybe it was an accident or maybe he was just admiring my clothing; except that he wasn't looking at my clothes he was looking at my eyes. I could see it.
It dawned on me slowly. He was slow playing me. He wanted this to be foreplay. Otherwise we'd be on top of each other in a back alley by now and my inane inner-monologue would be replaced by wanton moans. Minx, he wanted to play THAT game. He was the predator. I was the prey. He was the captain of the rugby team. I was the guy who happened a glance in the shower.
This ought to be more fun than a quick jaunt on a bus.
I looked away, nervously. It was my role in our dance. I dredged up excess emotions to make me anxious and virginal. I controlled my breath, inhaling in a short staccatto fashion. The sweat was all real, it was a hot summer day and the bus was none too comfortable. Someone call a doctor because I'm feeling faint.
A response now wasn't a good thing. It meant that he didn't know what he was doing and was in fact unsure. It meant that he didn't want to fuck. It meant that, please, if you didn't mind… would you be so kind as to engage in lewd acts with me? Like a band nerd he'd use the formal terms for everything, intercourse, touching, and romance. I made the mistake of being the aggressor once to someone who happened to get lucky. He had no idea, he confessed the first time he jerked off he called it masturbation and he didn't come he ejaculated. He cried when it was done, I cried after he left.
I wanted no response at this stage. That means he knew how to play, that was safer. And there it was, nothing. No curt nod. No continued stare, just stoic avoidance. Hot.
I didn't need to check any playbook. I knew what to do. I'm not a slut, I just understand how things are done. Some people were born mechanics, or sportsfans; I was born with an intimate understanding of how to get some. How any situation could turn explosively sexual at any moment.
Coyly, I checked my watch. I was late for an appointment. I had a meeting with my boss. I needed to meet a friend for lunch. That's the after story. I just hate when someone has to rush off but says they'll call you. Be honest, it was a one shot deal don't bother with platitudes and empty promises. When we're finished he can linger in his conquest but I need to compose myself and rush off. I'll leave the 'Just Been Fucked' hair it'll be my victory. You can't buy that in a can. There was a holistic spontineity to the JBF hair that can't be replicated. It's nuclear waste that you don't need a geiger counter to detect.
Still nothing from him, I should be a little more tempting. I undid a button on my shirt and shook the collar. No response, he must be really good. I paused, trying to be casual before trying another tact. I stretched my leg out towards him into a more reclined position making the seat of the bus look inviting like the arm rest seat on a couch. I let this set in. He was sweating now. That was his game, role reversal. He was the confident jock turned bitch boy. The sergeant taken down a peg or two. I had to be the instigator now. I prowled around long enough, it was my turn to act.
Casually I scratched my knee. Then trapsed my hand up my inner thigh slowly like I was painting. He reclined in the way I had. He was enticed. I was a siren. I was a vampire. I'd appear to him as smoke wafting into his mind through some etheral miasma.
My other hand was extended outward across the back of the seats, so was his. The aisle separated our hands but I made him feel it. I made him yearn. Slowly his hand would crawl closer and closer.
I nodded my head to the tempo of a non-existant beat. I was in a trance. It was a compulsive hypnosis.
I was just thinking that I was the shit when the bus slammed to a stop and he got up.
Were we done? Was that it?
This is an old piece of writing that I did in 2009.


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The no-mount option is for when you want to glue letters directly to a flat surface using some kind of adhesive such as silicon caulk or liquid nails. The problem with this type of installation is that glue tends to squeeze out the edges and it can get very messy. Also, until your glue sets the letters can move around and for large letters that means you need to use long strips of masking tape to hold them in place.
Double sided tape can only be applied to letters up to 24" in height. If using this option then always use a liquid silicon caulk as well to protect the letters in case the tape should fail
This is the default stud mount. This option will have blocks glued to the backs of the letters that create a natural surface standoff of about 3/8" which can be enhanced by choosing a standoff length in the forms options. The blocks are drilled and tapped to accept studs(threaded rods) that get glued into holes drilled in the mounting surface. A drill mounting pattern is required.
Plastic pad extending beyond back of letter, typical with plastic letters, install product direct to wall without having to drill any holes. Installed with use of split stencil or spacing guide. Silicone is a common adhesive to hold such letters.
The combo or combination mount differs from the combination all mount in that it only has stud THROUGH pads on the tops of the letters and standard PAD mounts on the bottoms. This mount is useful where you want to drill as few holes as possible and still have mounting rods(studs) in the wall. Installed by drilling holes for the stud through pad and adhering non stud pads directly to mounting surface. A drill mounting pattern is required.
The combination all mount is stud through pad on all points. This gives you adjustability on every stud and is useful for irregular surfaces such as corrugated metal buildings, lap siding, stone walls or anywhere you want a standoff form the wall that is adjustable during installation. A drill mounting pattern is required
All other colors would be painted which adds 30% to the price.
Full size Drilling patterns are tracing of letters done by hand at the factory. The letters, with studs screwed into the back or into blocks, are then punched through the paper to show the exact location of the studs. Once taped onto a wall, the hole locations are then drilled and filled with adhesive for any installation using a threaded stud. $3.25 per letter.
A pounce pattern is an outline drawing of letters ordered, full size, made on a plotter with a pounce tool instead of a pen. This tool punches small holes into the paper, following the exact outline of the letters. The installer will then tape this drawing on the wall and "dust" the surface with chalk. The chalk will create a "dusting outline" of the letters on the wall that is then used as a guide for installing plain or pad mounted letters. $3.25 per letter.
Stencil is a full size cutout of smaller letters. This pattern cannot be ordered for Cast or Molded products. Guide for installing plain, pad or double face tape letters. Guide is taped to wall, letter set inside cutout, adhered to wall. $60 FOR UP TO 100 CHARACTERS. (We determine if Stencil or Split Stencil is best for your order.)
This pattern cannot be ordered for Cast or Molded products. Split Stencil is a partial cutout of bottom portion of the letters above the baseline, plus 2" below the baseline that provides a perfect way to install plain, pad or double face tape flat cut out letters. This split stencil is generated using the same file that we use to produce your letters. $60 FOR UP TO 100 CHARACTERS. We determine if Stencil or Split Stencil is best for your order.)
A guide for plain, pad or double face tape mounted letters. The letters in a specific copy are spelled out and properly spaced on a long pattern table or computer generated. A narrow (approximately 2") piece of paper contains small hash marks or plotted lower portions of the letters. This guide is then secured to the wall with tape by the installer and provides a visual guide for installing letters. $10 - ANY QUANTITY.
901 Polished Aluminum
902 Brushed Aluminum
903 Polished Brass Aluminum
904 Brushed Light Brass Aluminum
906 Brushed Copper Aluminum
908 Satin Black Aluminum
909 Satin Silver Aluminum
910 Satin Gold Aluminum
911 Polished Smoked Aluminum
912 Satin Bronze Aluminum
915 Brushed Brass Aluminum
916 Brushed Pewter Aluminum
917 Brushed Black Aluminum
924 Bronze Stainless Aluminum
925 Brushed Medium Bronze Aluminum
927 Light Stainless Aluminum
About Metal Faced Acrylic Letters, Numbers and Logos


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i have an Solar Dude (libra-energy.eu/en/product/inverter-mon ... j3KLfkveUk witch operates at the 868,45 MHZ frequency.
here are some pictures:
it's an device to measure the power of solar panels.
does any of you know how to connect this to an domotica system (Domoticz for example).
i have one for spare, so if someone is interested in making this work it would make my day,


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PSA opens two new branch offices
July 21, 2003
NORCROSS, Ga. - Pediatric Services of America (PSA) has opened new branch offices in San Antonio, Texas, and Chesapeake, Vir., to provide home medical equipment primarily for infants and children, the company announced last week.
These branch offices will let the company meet the demand of its growing medical equipment customer base, as well as better serve and support its already existing pediatric nursing operations in the areas.
PSA President and CEO Joseph D. Sansone stated, "The addition of these two new medical equipment offices are in response to our successful operation of pediatric nursing services in the same areas, and fits our strategy of building our market matrix of services in the communities we serve."
PSA operates a network of over 120 branch offices in 22 states. Through these offices, PSA provides a combination of services, including pediatric private-duty nursing, pediatric day-treatment centers, pharmacy services and home medical equipment.


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Alan V. Lowenstein, one of New Jersey's most prominent attorneys, passed away Tuesday, May 8 at the age of 93. Mr. Lowenstein has been recognized for his professional excellence, leadership, integrity, and passion for the underprivileged. He set a standard of "meritocracy" for Lowenstein Sandler, one that welcomes lawyers of character and ability regardless of race or creed.
"Alan Lowenstein's vision and leadership helped to create the firm that we are today: one that embraces the ideals of professional excellence, commitment to its clients' interests, civic duty and diversity," said managing director Michael L. Rodburg. "While the practice of law and the firm's leadership continue to evolve, the imprint of Alan's ideals will forever guide us. He will be deeply missed."
Mr. Lowenstein founded the firm in 1961 in Newark, NJ. Others in the founding group included Richard M. Sandler, Joseph LeVow Steinberg, Benedict M. Kohl and Arnold Fisher. For most of the past 46 years, Mr. Lowenstein oversaw the firm's expansion from five attorneys to more than 250, with offices in Roseland, NJ and New York City. Today, the firm has top-ranked corporate and litigation departments as well as several nationally ranked practices.
During his 60 years of experience in banking and business law, Mr. Lowenstein represented institutions and corporations. After graduating Harvard Law School in 1936, he went to Washington, DC to work with William O. Douglas, who was then organizing the Temporary National Economic Committee, the predecessor of the Securities & Exchange Commission. In 1940, he returned to his roots in Newark to begin what became a life-long practice of law. Among his notable professional achievements, Mr. Lowenstein was the principal draftsman of the Banking Act of 1948 and chair of the New Jersey Corporation Law Revision Commission (1963-1971). He was listed among The Best Lawyers in America, since its inception in 1983 until his retirement in 2001, as one of New Jersey's top corporate law attorneys.
Early on, Mr. Lowenstein demonstrated a social conscience. In 1930, at age 16, Mr. Lowenstein boarded a train to North Carolina to report for The Newark Evening News the plight of textile mill workers who were seeking to organize under a labor union. Later he was a leader in Newark's charter reform movement, helping to change the city's governmental structure. Recognizing the need for cultural programs in his community, Mr. Lowenstein joined the Board of the New Jersey Symphony Orchestra in 1960, and served as its president from 1970 to 1973. Under Mr. Lowenstein's stewardship, the New Jersey Symphony Orchestra was transformed from a suburban orchestra into one of the State's cultural icons. In 1999, the Alan V. and Amy Lowenstein Foundation founded the New Jersey Institute for Social Justice, an organization that works through the legislature and the courts to assure basic human needs, freedom from discrimination, equal educational opportunities, and equal access to all levels of the judicial system.
Guided by and in recognition of Mr. Lowenstein's vision and leadership, Lowenstein Sandler:
Founded the Alan V. Lowenstein Public Interest Fellowship at Essex-Newark Legal Services, reflecting the firm's enthusiasm for the program and commitment to this enduring relationship with Legal Services.
Established (in honor of his 80th birthday) the Alan V. Lowenstein Scholarship for graduates of schools of social work who intend to serve as professionals of Jewish federations in the United States and Canada.
Sustained a leadership role in providing pro bono legal services, and is the recognized leader among New Jersey law firms in providing these services to a wide array of institutions and individuals.
Mr. Lowenstein was also a published author. In May of 2001, he wrote Alan V. Lowenstein: New Jersey Lawyer And Community Leader, an autobiography that spans six decades of public and private service (published by Rutgers University Press and the New Jersey ICLE). He also completed the Rutgers Oral History Project: Recording The Life And Work of Alan Lowenstein: A History Graduate Project.


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Filmmaker Beth Harrington has been working on "The Winding Stream", the definitive Carter Family documentary, for well over a decade, having interviewed Johnny Cash, Mike Seeger, Rita Forrester, and many other luminaries, and recorded performances by some of country music's best artists (Roseanne Cash, George Jones, John Prine, and the Carolina Chocolate Drops among others)... And now, at last, the film is done and getting ready to be shown on the festival circuit! It will debut on March 15, 2014 at South X Southwest.
I'm so happy for Beth, and anxious to see this film myself! Beth, will there be a Seattle screening??
Watch the trailer here:
UPDATE: Rolling Stone gave "Winding Stream" a Best of SXSW mention!


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+1 800-971-2453
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See America
Cars, Planes, Trains….life moves us along at a quick pace. As you pedal your bike across the country there is time to see the sights, smell the flowers and take in the vast expanse of America's landscape.
Pick a Tour – Ride Your Bike
Feel the breeze in your face and ride the miles with new friends who share your passion for cycling. Ride coast-to-coast or choose a shorter tour that fits into your busy schedule. We are happy to help you make your plans.
Embrace the Journey
There are many reasons to ride your bike. The grand accomplishment of riding across the United States is only one of them. There's time to think, time to plan, time to reflect on where you've been and where you are going.
Take a look….join the fun!


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Asheville, NC (PRWEB) December 22, 2013
Supreme Skin, a prominent Asheville area skin care provider, is now revealing its methods for their signature anti-aging facial services. The firm desires to help people with services relating to a special noninvasive anti-aging facial in Asheville, NC to help people look younger and feel their best.
The anti-aging facial process is explained as first starting with a deep cleansing of the skin. The procedure starts by using a special system for a light exfoliation. Then they use microcurrent technology, which is from the bio-therapeutic family to deliver signal to the cellular level of body that produces something called a "Biological Chemical" effect. This specialized beauty service helps to improve numerous skin problems. It increases the production of ATP (adenosine triphosphate) which is the 'powerhouse of the cells, collagen and elastin. These are what we all lose as we age. This special facial is restorative to the skin at the cellular level, taking away wrinkles while firming and toning the skin.
Microcurrent is a low level form of electricity that mirrors the body's own natural electrical impulses. A microcurrent face lift is a non-surgical, non-invasive cosmetic technique that safely and effectively improves the health and appearance of the skin. It is also referred to as a BIO-ultimate face lift. These tiny microcurrent impulses encourage your body's currents at the cellular level. The signs of aging are greatly reduced, while skin tone and elasticity are dramatically improved. These relaxing treatments may be enjoyed as a one time, comprehensive facial, or in a series to reach optimal goals, as well as provide enhanced longevity of the results. This procedure is proven safe and effective, delivering noticeable results immediately.
A spokesperson for the company stated that, "this anti-aging facial in Asheville, NC helps people maintain younger looking skin without any side effects and without putting the skin at risk. We also make sure people can afford the services that we have because we want to make is available to everyone." They claim at Supreme Skin to get results even on the first treatment and dramatic results with a series and then minimal maintenance.
Read the full story at http://www.prweb.com/releases/2013/12/prweb11444219.htm.
|Source: PRWeb
Copyright©2012 Vocus, Inc.
All rights reserved


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All amendments to commence on 19 November 2016
Visa Condition 8107
As one of the few mandatory conditions imposed on the 457 visa, condition 8107 currently stipulates that a 457 visa holder cannot cease employment for more than 90 consecutive days. The amendment will reduce this 90 day time period to 60 days.
The practical effect of the change is that if a 457 visa holder does not find a new sponsor within 60 days of ceasing employment with their current sponsor, they will be in breach of their visa condition and their visa may be liable to cancellation.
Definition of 'Member of the Family Unit'
The 'member of the family unit' definition prescribes which family members a visa applicant is eligible to include in a visa application.
The changes will limit the definition to include the direct family members, that is, the spouse or de facto partner and the dependent children and step-children of the primary applicant or their spouse/de facto partner. It will also provide an age limit for eligible children of 23 years, or any age if that child is incapacitated to work, to be regarded as 'dependent' applicants.
Prior to these amendments, the definition operated to permit extended family members who were regarded as dependents of the primary applicant to be included on the application. For example, a widowed parent who was financially dependent on the primary visa applicant would have been regarded as a dependent member of the family unit and thereby included on the application. The restrictions brought on by the new definition will mean that these family members will have to seek alternative routes to migrate to Australia.
Tourist Visa - The Frequent Traveller Stream
The Government is trialling the introduction of a 10-year validity visitor visa for Chinese nationals.
The current Subclass 600 (Visitor) visa offers the following streams:
- Business Visitor
- Tourist Stream
- Sponsored Family
The 'Frequent Traveller Stream' will be introduced to this subclass and will permit visa holders to undertake both business visitor and tourism activities. The stream will facilitate the grant of a visa with up to 10 years validity, with multiple entries. The visa will allow a maximum three-month stay period after each entry, with no more than 12 months cumulative stay permitted in a 24 month period.
Applications lodged under this stream attract an application fee of AUD1,000 and is intended for high value frequent travellers from the People's Republic of China.
Commencement of the new Temporary Activity Visa Framework
Four visa subclasses will be available from 19 November:
- Subclass 400 Temporary Work (Short Stay Specialist) visa
- Subclass 403 Temporary Work (International Relations) visa
- Subclass 407 Training visa
- Subclass 408 Temporary Activity visa
While requirements for the subclass 400 visa remain largely unchanged on a practical level, the existing Training and Research (Subclass 402) visa will be repealed and replaced by the Subclass 407 Training visa (applicants wishing to undertake occupational training) and Subclass 408 Temporary Activity visa (applicants wishing to undertake research).
A uniform Visa Application Cost of $275 will be applicable to these visa subclasses.


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We deliver a diverse range of flexible and fully-accredited adult education, industry training and employment programs from our six training sites across Perth.
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We believe that education and training can help to break the cycle of adversity and create opportunities for a brighter future.
This is why we pride ourselves on providing our students with a safe and supportive learning environment in which they can develop the skills, confidence and independence necessary to broaden opportunities for work and a sustainable economic future.
I enjoyed the Certificate III in Business course a lot. The trainers were really helpful in helping me to get my Certificate - they did an awesome job. The atmosphere here is really, really nice. I would 100% recommend Centacare to other people. Centacare is awesome!
Centacare has been great and so supportive. Even though I'm not on Centrelink, I cannot afford to pay the whole thing but the payment is so flexible.
I had a personal crisis during work experience and I ended up in tears. I called my trainer on the way home and she stopped and talked to me and consoled me. Then I made an appointment with Andrea, Centacare's counsellor, and she has been very supportive. I highly recommend Centacare to everyone.
I would recommend Centacare to a lot of people; they give you the best experience and teach you a lot of knowledge that you'll have for the rest of your life. I'd tell anyone if they want to do the Certificate III in Individual Support (Ageing) course then join up, you'll have a good time. It was a very good experience.
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December 6, 2018
2018 LifeLink CHRISTMAS APPEAL: Centacare equips Diana with life skills for the workforce
After Diana Bonney left high school, she turned to Centacare Employment and Training to further her education.Centacare Employment and Training is an Archdiocesan social service agency receiving f...
October 30, 2018
Didn't Get the ATAR Score for Nursing? Here Are Some Options to Pursue Your Nursing Career
If you didn't get the right ATAR score for Nursing at university, don't stress! We've got you covered. There are other viable pathways available for you to get into the career in nursing that yo...
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text:
It's an interesting week to be a woman in New York, and in particular a black woman.
This week sees the UN General Assembly convene to consider the Millennium Development goals and its commitment to women, Newsweek publishes its 2011 global women's progress report (highlighting the fact that women hold the "highest political offices from Thailand to Brazil, Costa Rica and Australia", and there is the second annual Women: Inspiration and Enterprise (WIE) symposium in Soho, New York, hosted by Arianna Huffington, Sarah Brown and Donna Karan.
A panel on "What it means to be a woman now" struck a chord, as it shone a spotlight on successful black women, from co-founder of the conference Dee Poku (former head of international marketing at Paramount Pictures), to the model Iman and editor-at-large of Essence magazine, Mikki Taylor. As a black woman in the US media, there are now myriad opportunities. Call it the Michelle Obama effect, or the fact that there are simply more media channels and avenues open to black women in my industry, but it seems like it really is a great time to be a black woman in this country. I'm in awe of the number of black presenters on TV and in top editorial positions at mainstream magazines, such as newly appointed fashion features editor at Glamour magazine Rajni Jacques or Paper women's market editor Zandile Blay, not to mention Kenya Hunt, global fashion editor of the Metro newspaper in the US.
Then there is Soledad O'Brien, an anchor and special correspondent for CNN/US who produces whole segments highlighting the issues affecting people of colour, and Robin Givhan, headhunted from the Washington Post earlier this year by Newsweek because of her Pulitzer prize-winning fashion writing.
There is less of a feeling in the US that you have to wait for someone of colour to step aside to make room for you; there isn't just one Trevor McDonald (as wonderful as he is) presenting the news: a sentiment shared by a number of black British actors and entertainers, such as comedian Gina Yashere, who said to Hannah Pool in an interview with this newspaper: "I can't wait for Lenny Henry to die just so I can get a TV show". She is now selling out arenas in New York and LA.
Mikki Taylor at Essence also believes this is a good time to be a black woman in the US: "We are the women whose time has come. You hear it in their conversations, we are children of the dream, we're the outpouring of those who paved the way for us, who made it possible for us to own our lives now and be who we are. I think this has been coming a long time and I'm excited to see what we'll do with this," she says.
Like many women I've been helped by role models and mentors along the way. My mother cultivated my love of journalism in the first place by bringing home a poster of successful black journalist Claudia Jones [who is celebrated each year in the NUJ Claudia Jones lecture], and sticking it on the wall in my room. She then found a youth journalism club called Children's Express for me to attend. June Sarpong, who ran the WIE conference, also mentored me when I was a television runner on T4 [the youth show on Channel 4] back in the early 2000s by offering me real support and advice after she went from a receptionist at Channel 4 to presenting, with the help of mentors. Through June's success and my mother's belief in me, I never believed I couldn't have a career in the media because of the amount of melanin in my skin.
Of course it's not all good news: there still isn't gender equality in the workforce – recent figures from the US Department of Labour revealed that women in the US earn "81% of the median weekly earnings of their male counterparts" (a similar pattern of gender inequality in the boardroom is noticeable in the UK too). Also, even among women there are disparities across race in the US: "women's inflation-adjusted earnings grew significantly from 1979 to 2010. During this period inflation-adjusted earnings for white women rose by 33%, compared to 25% for black women," showing that "growth in real earnings for white women outpaced that of their black counterparts".
In addition to this, author and Stanford law professor Ralph Richard Banks has just launched a book called Is Marriage for White People? looking at dating and romance in black America. Banks looks at why successful black women are "half as likely as white women to be married, and more than three times as likely as white women never to marry". By conducting over 100 interviews with people of colour he focuses on arguments that are prolific in the community about black women significantly outperforming black men in school and in university, not to mention the high rates of incarceration for black men, which poses a challenge for women who want to marry within their race.
Globally, black women aren't fairing very well either, whether it be high rates of mortality in childbirth in Africa, women and children suffering the destruction and the slow rebuilding of Haiti, which has largely been ignored in recent months (once the celebrity angle disappeared), or an interesting fact I found out from a midwife and White Ribbon Alliance advocate from Tanzania at the WIE symposium, that pregnant women in her country have to walk up to 30km to get to health care facilities. So what do you think, is it a great time to be a woman now? Is it a great time to be a black woman? Do we need more mentors? Have we only begun to take the steps needed to create change and what is needed next?


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Thank you Sinnove, it's a real gold mine!
thank you very much for your kind words. The clip is amazing, I'll keep it for a future french project!
Yes, these we can use too, love those printies, they will look great on boxes and bags, mabe on fabric too.Thank you again!* marlies
Legg inn en kommentar


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Luxury Champagne Truffle Gift Box And Large Milk Chocolate Heart
Luxury Valentine gift box containing 12 Hamiltons handmade chocolate champagne truffles. Made with white chocolate the champagne truffles are rolled in milk chocolate flake and presented in a luxurious black box tied with a red satin ribbon.The box comes with a large milk chocolate Valentine heart
FREE UK delivery for orders over £20
- No reviews
These luxury Valentine gift boxes contain 12 handmade white chocolate Marc de champagne truffles coated in milk chocolate flake and make a perfect gift for Valentines day. The box is decorated with a satin red ribbon and comes with a Valentine chocolate heart. Treat your loved one this Valentines day
* 12 white chocolate champagne truffles
* Minimum net weight 140g
* Luxury gift box made from textured black board
* Hand tied with red satin ribbon
* Large milk chocolate heart piped with "I love You*
* Minimum net weight 130g
Milk chocolate contains: cocoa solids 35%min, milk solids 20% min. White chocolate contains: cocoa solids 28% min, milk solids 22% min.
Sugar, cocoa, milk, cream, vegeatable fat (palm, sunflower, rape, shea), whey, Marc de Champagne, alcohol, glucose, starch, soya lecithin, natural flavours.
Allergen Information:
Milk, Soya, May contain nuts.
Suitable for vegetarians
Nutrition per 100g
Calories (Kcal) 538, Energy (Kj) 2251, Fat (g) 33, Saturated fat (g) 20, Carbohydrate (g) 51, Sugar (g) 51, Protein (g) 6, Salt (g) 0.18


text: Hi, I am considering taking a teaching job in Bratislava. Is there someone who can offer advise about what it is like to live in Slovakia? The standard of living, etc.? I have done some basic research, but I figured that someone with personal experience can give me a better picture.