text:
Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.
- immunohistochemistry
- electrophysiology
- airway smooth muscle
asthma is a chronic disease with acute exacerbations of reversible airway narrowing, airway hyperresponsiveness, and inflammation affecting over 6 million Americans and costing over 56 billion dollars in 2009 (22). Since the 1980s, the prevalence and number of persons with asthma has steadily increased, suggesting that novel therapeutic approaches are needed to curtail this debilitating disease. One therapeutic approach involves the pharmacological manipulation of airway smooth muscle tone. Airway smooth muscle hyperresponsiveness is recognized as a major component of asthma.
The efflux of chloride from the airway smooth muscle cell following an exposure to a contractile mediator and an increase in intracellular calcium contributes to airway smooth muscle membrane depolarization (14, 16, 17, 19, 21). The relevance of chloride flux-induced membrane depolarization to functional smooth muscle contraction has been questioned (12, 13), but evidence exists for a link between calcium sparks, calcium-activated chloride channel (CaCC) activity resulting in spontaneous transient inward currents (STICs), and airway smooth muscle single cell contraction (15, 32). However, evidence for the functional effects of CaCC blockade in intact airway smooth muscle is limited (11).
Previously, chloride flux including calcium-induced chloride flux in airway smooth muscle was presumed to be occur via proteins of the ClCa, CLC-3, bestrophin, and Tweety families (5). However, in reconstituted cell systems these proteins did not demonstrate defining characteristics of a voltage-gated, calcium-sensing chloride channel. More specifically, these proteins failed to demonstrate requisite activation under induced membrane potential changes and/or an outward rectifying current at submaximal Ca2+ concentrations. These studies created considerable uncertainty as to the molecular identity of the channel responsible for calcium-mediated enhancement of chloride efflux in many cell types. Electrophysiological properties expected of a CaCC include an increase of intracellular calcium inducing an increase in a delayed outward rectifying Cl− current. Additionally, characteristics of CaCCs include sensitivity to Cl− channel blockers, small conductances, a voltage-dependent Ca2+ sensitivity (31), and a halide flux sensitivity of I− > Br− > Cl− > F− (7). These criteria were satisfied in 2008 when three separate laboratories discovered that one member of the TMEM16 or anoctamin family of proteins demonstrated electrophysiological and pharmacological characteristics typical of CaCCs (2, 26, 31). These seminal discoveries have been followed by rapid progress in the understanding of the molecular and biophysical characteristics of these ten family members. It appears that TMEM16A and TMEM16B are true CaCCs whereas the remaining eight members of the family may not be associated with the plasma membrane or have chloride-conducting properties (4, 30). In the present study we questioned 1) whether members of the TMEM16 family of CaCCs are expressed in human airway smooth muscle (HASM) cells, 2) whether HASM cells demonstrate classic electrophysiological evidence of CaCC activity, 3) whether members of this family are capable of generating a chloride flux in HASM, and 4) whether pharmacological targeting of these CaCCs may serve to relax tachykinin-mediated airway smooth muscle contractions.
N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and all cell culture reagents were purchased from Invitrogen (Carlsbad, CA). TRIzol reagent was obtained from Ambion-Applied Biosystems (Austin, TX). Conventional PCR and RT-PCR reagents were purchased from Clontech Laboratories (Mountain View, CA). Tannic acid and all other reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise indicated.
Cell culture.
All human airway tissue protocols were reviewed by the Columbia University Institutional Review Board (IRB) and were deemed not human subjects research under 45 CFR 46. Primary human bronchial smooth muscle cells were enzymatically dissociated by using a papain-collagenase kit according to the manufacturer's recommendations (Worthington). Additionally, human bronchial smooth muscle cells modified to stably express human telomerase reverse transcriptase were a kind gift from Dr. William Gerthoffer (University of South Alabama, Mobile, AL) (6). Smooth muscle cells were grown in SmBMII medium with manufacturer's recommended additives (Lonza, Walkersville, MD) or M199 medium containing 10% fetal bovine serum, 0.25 ng/ml epidermal growth factor, 1 ng/ml fibroblast growth factor, ITS supplement (1 mg/ml insulin, 0.55 mg/ml transferrin, 0.67 μg/ml sodium selenium), and antibiotics (100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin B). Immortalized human airway epithelial cells (BEAS2B) cells were purchased from Lonza and maintained in BEBM medium (Lonza) with all additives recommended by the manufacturer. Cells were grown to confluence at 37°C in 95% air-5% CO2 in T75 cm2 flasks for total RNA or protein extraction, or in 96-well black-walled clear-bottom plates for MQAE halide flux. Cells for electrophysiological studies were plated on Thermanox 13-mm plastic coverslips (NUNC, Rochester, NY), coated with 0.5 mg/ml collagen type I.
Isolation of smooth muscle from human trachea.
All human airway tissue protocols were reviewed by the Columbia University IRB and were deemed not human subjects research under 45 CFR 46. Human tracheal tissue was obtained from discarded airway from healthy lung donors during transplantation surgery at Columbia University. The tracheal smooth muscle layer was removed by fine dissection of the airway and processed for immediate RNA extraction. Human whole brain, retina, stomach, skeletal muscle, total lung, trachea, and spinal cord total RNA was purchased from Clontech Laboratories to be used as RT-PCR positive controls.
RNA extraction and reverse transcription of cDNA.
Total RNA was extracted from native and cultured HASM cells using TRIzol (Ambion-Applied Biosystems) according to the manufacturer's recommendations. RNA purity and quantity was measured by 260/280 nm absorbance (Beckman Du 640). Total RNA from cultured or native human tracheal airway smooth muscle was diluted in RNAse-free water to a final concentration 500 μg/ml, aliquoted, and stored at −80°C. Complementary DNA synthesis was performed (2 μg total RNA in a final volume of 20 μl) with Superscript Vilo cDNA synthesis kit (Invitrogen) following the manufacturer's recommendations.
Conventional PCR.
Newly synthesized cDNA (5 μl of original 20-μl reaction) was used in PCR with the Advantage 2 PCR Kit (Clontech) on a MJ Research PTC-200 Peltier thermal cycler (Bio-Rad, Hercules, CA) following the manufacturer's protocol. Sense and antisense primers specific for 10 members of the TMEM family (TMEM 16A-K) were constructed by primer blast selecting default parameters and spanning an exon-exon junction (Table 1) and were purchased from Invitrogen. All cDNA samples were initially denatured at 94°C for 30 s. Optimal annealing temperatures for each primer set were initially established by using human cDNA from brain, retina, stomach, skeletal muscle, lung, and whole trachea purchased from Clontech and using 1-min annealing duration and 30-s extension duration for 40 cycles of PCR. The annealing temperatures for TMEM16A, B, C, D, E, F, G/H, I, J, and K were 64, 72, 72, 72, 69, 68, 69, 69, 68, and 68°C, respectively. For PCR, there was a completion step of 30 s. All PCR products were analyzed on 10% polyacrylamide gels stained with ethidium bromide (Molecular Probes, Eugene, OR) and visualized by use of a gel imager and Visioworks software (Biospectra UVP, Upland, CA). Samples were analyzed by loading all (5–10 μl) samples for a single TMEM16 subtype on a single gel.
Western blot analysis.
All tissues and cells utilized for Western blot analysis (immortalized and primary human tracheal airway smooth muscle cells, BEAS2B, native human and guinea pig tracheal airway smooth muscle) were homogenized in ice-cold lysis buffer [50 mM Tris·HCl, pH 8.0, 1% Nonidet P-40, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1:200 dilution of protease inhibitor cocktail III (Calbiochem)]. Following centrifugation (2,900 g, 20 min, 4°C) of the whole cell or tissue lysate, the supernatants were saved and protein concentrations were determined. Aliquots of the supernatants were solubilized by heating at 95°C for 5 min in sample buffer (final concentrations: 50 mM Tris·HCl, pH 6.8, 2.5% SDS, 6% glycerol, 2.5% 2-mercaptoethanol, and bromphenol blue). The supernatants of solubilized whole cell or tissue lysates were electrophoresed through a 7.5% Mini-PROTEAN TGX gel (Bio-Rad) and transferred to PVDF and probed with TMEM16A antibody (prediluted rabbit polyclonal was further diluted to 1:5, no. ab53213; Abcam, Cambridge, MA). Equivalent loading was confirmed by commensurate probing with β-actin (rabbit monoclonal diluted to 1:20,000, no. 4970; Cell Signaling, Cell Signaling Technology, Danvers, MA). The primary antibodies were detected by horseradish peroxidase-conjugated donkey anti-rabbit antibodies (1:5,000 for TMEM16A and 1:30,000 for β-actin). The signal from the immunoreactive bands was detected by enhanced chemiluminescence (SuperSignal West Femto; Thermo Scientific, no. 34095, Rockford, IL) according to the manufacturer's recommendations, developed on film (HyBlot CL Autoradiography Film; Denville Scientific, Metuchen, NJ), and analyzed using Vision Works LS software (UVP, Upland, CA).
Immunohistochemistry of HASM for TMEM16A protein expression.
Freshly obtained surgical discards of human trachea and first generation bronchi deemed as waste were utilized in accordance with IRB permission. Airway smooth muscle strips were immediately fixed in 4% paraformaldehyde (4°C overnight), then incubated in 30% sucrose in PBS for an additional 24 h prior to processing for cryostat sectioning (6 μm). The sections were washed in PBS, incubated with 0.1% Triton X-100 for 10 min, blocked with 15% goat serum and then incubated overnight at 4°C in primary antisera. In parallel experiments, a human telomerized airway smooth muscle cell line was cultured on coverslips in SmBMII smooth muscle medium (Lonza) for 2–3 days then fixed with 3% paraformaldehyde in PBS for 5 min. The primary antibodies used were 1) anti-TMEM16A (rabbit, monoclonal; Abcam no. ab64085, 1:100 dilution in PBS) for cultured cell immunocytochemistry, 2) anti-TMEM16A (rabbit polyclonal; Abcam no. ab53213, 1:100 dilution in PBS) for intact HASM tissue immunohistochemistry, and 3) anti-α smooth muscle actin (mouse, monoclonal; Sigma-Aldrich, no. A2547, 1:10,000 dilution in PBS). The secondary antibodies consisted of FITC-conjugated goat anti-rabbit IgG (1:400 dilution) and Alexa Fluor 594 goat anti-mouse IgG (1:400 dilution; Invitrogen) incubated for 1 h. Nuclear staining was achieved by employing mounting medium premixed with DAPI stain (Vector Laboratories, no. H-1500). Negative controls were performed on serial sections by omitting primary antibody. All the immunofluorescence experiments were repeated at least three times. Samples were viewed under confocal microscopy (Nikon Eclipse, Japan) and images were acquired with NIS software version 4.10.
Patch-clamp electrophysiological studies in HASM cells.
HASM cells were cultured in SmBMII smooth muscle medium (Lonza). For patch-clamp analysis, cells were placed on poly-l-lysine 12-mm coverslips (BD), coated with 0.5 mg/ml collagen type I (Sigma). The coverslips were transferred to a 0.5 ml chamber on the stage of an inverted microscope (Nikon). Membrane currents were recorded using whole cell configuration. The extracellular solution contained (in mM) 130 NaCl, 5.5 tetraethylammonium (TEA)-Cl, 2.2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH adjusted to 7.35 with NaOH. The pipette solution contained (in mM): 75 CsCl, 64 Cs-gluconate, 1 MgC12, 10 HEPES, 3 Na2ATP, pH adjusted to pH 7.3 with CsOH (1). Cesium and TEA were used in intracellular and extracellular solutions, respectively, to block K+ channels to allow for the study of chloride channel activity. Whole-cell currents were recorded by using Axopatch 200B and digitized by use of 1322A. Patch pipette had a resistance of 3–6 MΩ. All recordings were performed at room temperature. Events were counted as STICs if their amplitude was two- to fourfold of the baseline as detected with pCLAMP10 and analysis by Origin 8 software; data are expressed as a percent of control. Niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) were dissolved in DMSO. The final concentration of DMSO was less than 0.05%. To demonstrate the effect of TMEM16A-specific blockade, we employed a functional antibody-mediated live cell blockade as described elsewhere (28). Cells were incubated for 2 h (in 37°C, 5% CO2) with either TMEM16A antibody (Abcam ab53213, 1:5 dilution) or with the vehicle diluent used in the antibody as a control (0.1% sodium azide, 1% BSA, 50 mM Tris, pH 7.6), after which STIC activity was assessed.
Functional measurements of chloride channels in HASM.
For measurement of halide flux through chloride channels, HASM cells were grown to confluence in 96-well black-walled, clear-bottom plates. Cells were loaded for 10 h with 10 mM MQAE (Invitrogen) in 50 μl/well of basal medium (M199). Cells were washed 3× with physiological buffer solution (composition in mM: 10 HEPES with pH 7.4, 10 glucose, 2.4 K2PO4, 0.6 K2HPO4, 1 MgSO4, 1 CaSO4, and 110 Na isethionate) and left in a final volume of 90 μl of buffer. Cells were pretreated for 30 min at 37°C with the chloride channel blocker NPPB (100 μM) or vehicle (0.1% DMSO). Baseline fluorescence was continuously measured at 2-s intervals by using an excitation wavelength of 360 nm and an emission wavelength of 460 nm with a cutoff filter of 455 nm (3). After baseline fluorescent readings for 2 min, halide flux was induced by injecting 50 mM NaI (final concentration) by using the automated injector function of the Flexstation 3 plate reader (Molecular Devices, Sunnyvale, CA). Continuous recording of fluorescence continued for an additional 3 min before maximal quenching of MQAE fluorescent was determined by a final injection of 150 mM (final concentration) potassium thiocyanate. Halide (iodide) flux was compared between control and antagonist-pretreated cells by comparing the slopes of the quenching of fluorescence with the introduction of NaI (3, 25). Since cells have no endogenous intracellular iodide, NaI was utilized instead of NaCl since CaCCs are known to allow for an enhanced conductance of iodide ions compared with chloride ions and significant extracellular iodide creates a maximum extracellular-intracellular halide gradient to facilitate halide influx (19).
Impaired contraction of airway smooth muscle by TMEM16 channel inhibitors.
All animal protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Hartley male guinea pigs (400 g) were anesthetized by intraperitoneal pentobarbital to effect. Tracheas were promptly removed and dissected into two closed cartilaginous ring units with mucosa, connective tissue, and epithelium removed and attached to a fixed tissue hook in a 4-ml bath (Radnoti Glass Technology, Monrovia, CA) and a Grass FT03 force transducer (Grass Telefactor, West Warwick, RI), by using silk thread as previously described (18). BioPac hardware and Acknowledge 3.7.3 software (Biopac Systems, Goleta, CA) continuously recorded muscle force throughout all experiments. Rings were equilibrated at 1 g of isotonic force for 1 h with Krebs-Henseleit buffer (18) (with 10 μM indomethacin, pH 7.4, 37°C) replaced every 15 min in buffer continuously bubbled with 95% O2 and 5% CO2. Following equilibration, epithelium-denuded tracheal rings were contracted with 10 μM of the capsaicin analog N-vanillylnonanamide to activate and deplete nonadrenergic, noncholinergic nerves present in the tracheal ring preparation. Following buffer exchanges and resetting of resting tension to 1.0 g, rings were contracted with increasing concentrations of acetylcholine (100 nM to 100 μM) for two cycles with buffer exchanges and resetting of resting tension to 1.0 g between and after acetylcholine dose-response curves. Rings were then contracted with a maximal contractile concentration of KCl (80 mM) followed by buffer exchanges and resetting of resting tension. Inhibition of endogenous cholinergic nerves and mast cell degranulation was achieved by pretreating tracheal rings with 1 μM tetrodotoxin and 10 μM pyrilamine, respectively. Following results from preliminary dose-response experiments used to determine the lowest concentration of tannic acid capable of relaxing a substance P contraction, rings were then randomly treated with vehicle (buffer) or with the TMEM16 inhibitor tannic acid (100 μM) or benzbromarone (100 μM) for 10 min before contractions were induced with the tachykinin receptor agonist substance P (1 μM). The maximum force induced by substance P was expressed as a percentage of maximum force induced by the acetylcholine pretreatment in each individual ring. The pH was measured and maintained at 7.4 throughout the experiment in both the control and treatment groups with no significant change noted after addition of tannic acid.
Statistical analysis.
Muscle force comparisons in organ bath experiments and electrophysiological measurements of current and frequency were analyzed by unpaired, two-tailed t-tests. Data are expressed as means ± SE and a P value <0.05 was considered significant.
Qualitative expression of mRNA encoding TMEM16 family members in native and cultured HASM.
Messenger RNA encoding TMEM16A, B, E, F, G/H, I, J, and K (ANO 1, 2, 5, 6, 7, 8, 9, and 10) was identified in native HASM isolated from trachea from human lung transplant donors. In immortalized cultures of HASM, mRNA encoding six of these TMEM16 members (A, B, F, I, J, and K) were identified. In contrast, TMEM16 E and G/H demonstrated expression in native HASM but not in cultured cells. TMEM16 C and D were not identified in airway smooth muscle from either native or cultured cells despite the detection of the mRNA in appropriate positive control samples (Fig. 1A).
Immunoblot analysis of Ano-1/TMEM16A in airway smooth muscle.
Lysates from freshly dissected HASM (airway smooth muscle) tissue and cultures of primary and telomerized HASM cells were subjected to immunoblot analysis using a specific ANO-1 receptor antibody (Fig. 1B). To illustrate interspecies conservation (between human and guinea pig) of TMEM16A expression in fresh airway smooth muscle we also performed separate immunoblot analyses (Fig. 1C). Immunoreactive bands of the expected molecular mass (∼110 kDa) were detected from all four sources and substantiate that the guinea pig airway smooth muscle is a reasonable surrogate for HASM for TMEM16A functional studies. In all cases, cultured human airway epithelial cells (BEAS-2B) were used as a positive control for TMEM16A immunoblotting.
Immunohistochemistry reveals protein expression of TMEM16A in HASM cells.
To confirm the localization of TMEM16A (ANO-1) protein to HASM, immunohistochemistry was also performed using two distinct commercially obtained antibodies (Fig. 2). Specific immunoreactivity was detected in airway smooth muscle cells of fresh human tissues (upper panel) and a human immortalized cultured cell line (lower panel) with no staining observed in the negative controls (omission of primary antibodies with only secondary antibodies used). In addition, double and triple staining was performed with fluorescent-linked actin and DAPI to illustrate that staining was specific to airway smooth muscle cells.
Chloride channel halide flux in cultured HASM.
Iodide flux through chloride channels was measured by the quenching of MQAE fluorescence in the absence or presence of pretreatment with a chloride channel antagonist. Pretreatment with the chloride channel blocker NPPB blocked halide flux in cultured immortalized HASM cells (n = 9) (P = 0.04) (Fig. 3).
Patch-clamp electrophysiological studies on HASM.
STICs were recorded from immortalized HASM cells under buffer conditions selective for chloride currents. Under whole cell voltage-clamp configuration, spontaneous rhythmic inward currents were identified from HASM cells with use of a holding potential of −60 mV (Fig. 4A, upper panel tracing). The average peak amplitudes and frequencies of the current were 93.2 ± 21.2 pA and 116.1 ± 11.7 min−1, respectively (calculated 947 events from 16 cells). The average of this current reached a peak within 21.6 ± 4.0 ms (rise time, τ) with a single exponential function and decayed with a τ of 93.4 ± 20.7 ms (calculated 947 events from 16 cells). Figure 4A displays an example, representative of three cells, in which STICs were recorded at different holding voltages. In these three cells the average current was −61 ± 8 pA at −60 mV, which correlated with earlier work by Yang et al. (31). STICs reversed potential at −5.3 ± 0.8 mV (n = 3) (Fig. 4B), close to the predicted chloride reversal potential (ECl) = −16.2 mV. At potentials more negative than approximately −10 mV, STICs were inward, and at holding potentials above the predicted ECl, they changed direction from inward to outward (Fig. 4A), consistent with a chloride current. This correlated with the study of TMEM16A channels by Yang et al. in which reversal potentials shifted from −9.0 ± 0.6 mV to +12.5 ± 2.9 mV. At a holding potential +40 mV, the current was 112 ± 8 pA (n = 3) (Fig. 4B). STICs were reduced in amplitude and frequency when extracellular calcium was reduced (data not shown). These features indicate that STICs result from the opening of calcium-sensitive chloride channels. Next we sought to demonstrate a calcium-dependent increase in this chloride channel activity.
Effects of bradykinin-induced increases in intracellular calcium and the chloride channel blockers NFA, tannic acid, NPPB, and TMEM16A antibody on STICs in immortalized HASM cells.
We found that 50 μM NFA (n = 4), 20 μM tannic acid (n = 4), or 50 μM NPPB (n = 4) rapidly inhibited STICs, expressed as reduction of both amplitude and frequency (Fig. 4, C–E); 20 μM bradykinin (used to increase intracellular calcium) increased the amplitude and frequency of STICs in HASM cells, and this stimulation was inhibited by tannic acid, an antagonist of the TMEM16A family member of CaCCs (23) (Fig. 4C, bottom tracing, D, and E).
In a separate group of studies (Fig. 5), antibody-mediated blockade of TMEM16A led to a functional loss of STIC activity compared with vehicle-treated cells. We found complete abrogation of STIC activity in 21 of the 26 cells following incubation in TMEM16A antibody (compared to a lack of STIC activity in 3 of 17 patched cells following incubation with vehicle). Moreover, even in the 5 cells exposed to antibody that did display STIC activity, there was significant attenuation of amplitude (Fig. 5C) and frequency (Fig. 5D) compared with vehicle controls. These results again suggest that TMEM16A-mediated Ca2+-activated chloride currents in HASM cells constitute the major contribution to both baseline and G protein-coupled receptor-stimulated increases in STICs an electrophysiological phenomenon of CaCC activity.
Functional effects of TMEM16A chloride channel blockade on acetylcholine- and tachykinin-induced airway smooth muscle contraction.
Dose-response studies examining tannic acid-mediated relaxation from a 1 μM substance P contraction demonstrated the lowest concentration (100 μM) that was functionally effective (data not shown). In an effort to maintain selectivity, this concentration was used to determine the functional relevance of TMEM16A blockade on contractile tone in intact airway smooth muscle by a pretreatment approach. Following pretreatment of ex vivo guinea pig airway smooth muscle with vehicle or the TMEM16A antagonists tannic acid or benzbromarone (100 μM), we found significant attenuation of a subsequent substance P contraction consistent with a role for TMEM16 CaCCs in airway smooth muscle contraction (P < 0.01, n = 8 and P < 0.001 respectively; Fig. 6). The contraction achieved with substance P in each airway ring was compared with a previous maximal contraction achieved with 100 μM acetylcholine in each individual ring.
The primary findings of the present study are that multiple members of the TMEM16 family of CaCCs are expressed at the mRNA level in cultured and native HASM cells and that TMEM16A is immunohistochemically detected in native and cultured HASM. Consistent with previous studies (1, 13–15, 19, 32), we have demonstrated that airway smooth muscle cells express channels with classic electrophysiological properties of chloride channels that are modulated by a classic stimulus of increased intracellular calcium (bradykinin mediated). In the present study we demonstrate that STICs 1) occur under conditions of K+ channel blockade, 2) demonstrate voltage dependence, 3) reverse direction near the chloride equilibrium potential, 4) increase in magnitude and frequency by increasing calcium, 5) are attenuated by a number of chloride channel antagonists known to block TMEM16A chloride channels, and 6) are selectively blocked by incubating human smooth muscle airway cells with a specific antibody directed against the human TMEM16A channel. These findings are consistent with TMEM16A representing a CaCC in airway smooth muscle responsible for STICs. We have demonstrated that halide quenching of the fluorescent indicator MQAE is attenuated by the chloride channel inhibitor NPPB consistent with antagonism of functional chloride channels. Finally, in organ bath studies in intact tracheal rings we demonstrate that blockade of these channels with TMEM16A inhibitors (tannic acid and benzbromarone) attenuates a tachykinin-induced contraction of airway smooth muscle.
Our findings agree with those recently published in mouse airway myocytes (1) in which the kinetics of STICs under identical extracellular and pipette buffer conditions were very similar to those in the present study. Moreover, STICs in these mouse airway myocytes were demonstrated to be temporally and spatially related to calcium sparks linking calcium release via sarcoplasmic reticulum ryanodine receptors to activation of CaCCs accounting for STIC activity. Subsequently these authors demonstrated that biphasic membrane potential transients mediated by calcium transients opening large-conductance KCa channels causing spontaneous transient outward currents coupled with ClCa-mediated STICs serve to stabilize plasma membrane potential (32).
A fundamental feature of a CaCC is a voltage dependence that is modulated by intracellular Ca2+ and is activated by positive membrane potentials at submaximal Ca2+ concentrations with an inherent outwardly rectifying current-voltage relationship (5, 9). At maximal concentrations this relationship should become linear. Prior to 2008, the CLC, ClCa, bestrophin, and Tweety families were all thought to have some characteristics of CaCCs; however, the ionic currents conducted by these proteins did not exhibit the expected Ca2+ sensitivity, voltage dependence, or kinetics expected at submaximal Ca2+ concentrations (8).
In 2008, three separate laboratories using different approaches demonstrated that the TMEM16 family of proteins exhibits the expected electrophysiological characteristics of a voltage- and calcium-regulated chloride channel (2, 26, 31). Yang et al. (31) showed that TMEM16A coexpressed with endothelin receptor A into HEK293 cells can produce robust endothelin-induced chloride currents that were outwardly rectified at low activation levels but nonrectifying at maximal activation. These outward currents could be blocked by NFA (31). This Gq-coupled receptor increase in intracellular calcium is functionally analogous to the use of bradykinin in the present study and calcium-induced increases in chloride currents by acetylcholine, histamine, substance P, and caffeine in previous studies in airway smooth muscle cells (13, 14, 16, 17, 20, 21, 29). Schroeder et al. (26), using an expression system of Xenopus laevis for CaCC mRNA, generated CaCC currents that became less dependent upon voltage and showed little rectification at high intracellular calcium levels. These currents were activated by carbachol and blocked by NFA and 4,4-diisothiocyanatostilibene 2,2-disulfonic acid (DIDS) (26). Moreover, this family of ion channels was shown to be involved in a number of cellular functions including cell volume regulation, neuron excitability, smooth muscle contraction, and epithelial electrolyte and fluid transport (2).
In our study, we have demonstrated in airway smooth muscle that six of the ten known members of the TMEM16 family of CaCCs are expressed at the mRNA level in both native and cultured HASM cells. Two members, TMEM16C and TMEM16D, were not detected in either native or cultured HASM. TMEM16E and G/H were detected in native but not cultured airway smooth muscle. These discrepancies in detection between native and cultured cells may reflect impurities of cell type in the natively dissected airway smooth muscle or induced changes that occur in cell culture. Prior to the present study, only TMEM16A mRNA expression had been demonstrated in mouse airway smooth muscle, where it is believed to be critical for the normal epithelial/smooth muscle development of the airway (24) and attenuates acetylcholine-induced contraction (11).
Numerous previous studies have demonstrated spontaneous transient inward currents in airway smooth muscle cells and calcium-activated increases in chloride currents, but none of these previous studies identified the specific protein responsible for this conductance. In fact, most of these previous studies occurred before the molecular characterization of the TMEM16 (anoctamin) family of CaCCs, which are now recognized as the protein responsible for these chloride currents in many other cell types (30). The molecular function of this ten-member family has been further refined as TMEM16A and TMEM16B appear to account for plasma membrane calcium-regulated chloride conductance while the other eight family members are either expressed in other cellular compartments (4) or serve other functional roles (27). Thus TMEM16A (and perhaps TMEM16B) identified in airway smooth muscle in the present study likely account for calcium-induced chloride currents that account for STIC activity (1, 13–15, 19, 32) modulated by spatially close ryanodine receptors demonstrated in airway smooth muscle cells (1). Given this fact, the abundant expression of TMEM16A, and the commercially available antibodies that have been used successfully for immunohistochemistry and functional blockade of TMEM16A, we decided to focus our initial efforts on this family member. However, we realize that ANO-2/TMEM16B may also play a role in modulating airway smooth muscle calcium-activated chloride flux.
Until recently, specific antagonism of the TMEM16A channel has been elusive, with the pharmacological armamentarium being limited to nonspecific chloride channel blockade with NFA, DIDS, or NPPB. Although selective pharmacological antagonists that unequivocally target only TMEM16A are lacking, in 2010 Namkung et al. (23) discovered that gallotannins, including tannic acid, in red wine and green tea selectively inhibited TMEM16A and not CFTR Cl− channels. In the present study, we demonstrated that the calcium-mediated increase in chloride channel activity (measured as STICs) was decreased by tannic acid and that pretreatment of isolated guinea pig tracheal rings with tannic acid attenuated a subsequent substance P-induced smooth muscle contraction. These results are in close agreement to those in human bronchial smooth muscle in which a TMEM16A small molecule inhibitor (benzbromarone) inhibited methacholine- but not KCl-induced contractions in isolated human bronchi (11). These authors suggested that TMEM16A blockade may specifically attenuate muscarinic receptor-mediated contractions. However, the present study demonstrates that a contraction induced by another Gq-coupled receptor (neurokinin receptor-activated by substance P) is also attenuated by TMEM16A blockade, a novel finding that expands this target's usefulness in suppressing other contractile agonists. We also observed recovery of contractility to substance P following washout and rechallenge with substance P (data not shown), suggesting a reversible drug effect. In addition, to corroborate our pharmacological data, we also employed functional TMEM16A channel blockade via a TMEM16A specific antibody to assess whether acute blockade of the TMEM16A receptor by an antibody shown to functionally antagonize this receptor on live cells by other investigators (28), could replicate our electrophysiological findings obtained by using tannic acid. Utilizing a similar methodology, we demonstrate that antibody-mediated TMEM16A specific blockade leads to functional obliteration of STIC activity in HASM cells.
Although the role of ANO1/TMEM16A in airway smooth muscle has been reported in the literature (10, 11, 24), there has been limited functional characterization of its role especially in human tissues. What is known about ANO1 and the airway is predominantly from studies focusing on murine models. For example, ANO1 is critical to the development of the trachea since global ANO1 knockout mice die within 1 mo of birth and exhibit severe tracheomalacia including a defect in airway smooth muscle development on the posterior wall of the trachea and a failure of airway epithelial stratification (24). ANO1 has been immunochemically localized to mouse airway smooth muscle and was shown to colocalize with α-smooth muscle actin (10). Human bronchi were shown to contract less effectively in the presence of benzbromarone an antagonist of ANO1 channels (11). However, this pharmacological ligand of ANO1 has not been shown to be devoid of antagonism at other chloride or other ion channels.
In summary, we demonstrate the mRNA expression of multiple members of the ANO/TMEM16 family of CaCCs in HASM as well as protein expression of TMEM16A (now thought to account for calcium-activated chloride currents in many cells). We demonstrate that calcium-induced increases in chloride channel activity are attenuated by tannic acid, an inhibitor of TMEM16A, and that tannic acid and benzbromarone attenuate a substance P contraction in intact airway smooth muscle. These studies suggest that TMEM16A is the elusive chloride channel responsible for calcium-induced chloride currents in airway smooth muscle cells.
This work was supported by National Institutes of Health grants [G. Gallos (GM093137); C. W. Emala (GM065281); and P. D. Yim (GM008464)]; The Foundation for Anesthesia Education and Research (H. S. Chang); and the National Center for Research Resources ULI RR 024156.
No conflicts of interest, financial or otherwise, are declared by the author(s).
G.G., K.E.R., P.D.Y., and C.W.E. conception and design of research; G.G., K.E.R., J.D., H.F., X.W.F., H.-Y.S.C., and D.X. performed experiments; G.G., J.D., H.F., X.W.F., H.-Y.S.C., D.X., and C.W.E. analyzed data; G.G., K.E.R., J.D., P.D.Y., and C.W.E. interpreted results of experiments; G.G., J.D., H.F., X.W.F., H.-Y.S.C., and C.W.E. prepared figures; G.G. and K.E.R. drafted manuscript; G.G., K.E.R., J.D., X.W.F., and C.W.E. edited and revised manuscript; G.G., K.E.R., J.D., H.F., X.W.F., H.-Y.S.C., and C.W.E. approved final version of manuscript.
We thank Dr. William Gerthoffer, Department of Biochemistry and Molecular Biology, University of South Alabama, for providing the immortalized HASM cells used in this study. We also thank Dr. Theresa Swayne for assistance with confocal imaging.
- Copyright © 2013 the American Physiological Society


text:
Joining the Coalition
To join the Smokefree Action Coalition please read the aims and objectives as stated in the mission statement. If you agree with the statement and wish to sign up, please complete and submit the form below. Alternatively, you can send an email to email@example.com
Thank you for your interest in joining the Smokefree Action Coalition. Please complete this short form.

subset: commoncrawl


text:
Lay Dominican Anna Marija Edith Foss of the Fraternity in Oslo, Norway, has participated extensively in voluntary rescue work for refugees in Lesbos, Athens and Kos in Greece. In December 2015 she was joined by Br Haavard Simon Nilsen OP. Here are some of their own pictures with comments by Anna Marija on the rescue work at Lesbos, during 21th December 2015 – 4th January 2016.Rubber boats made for at most 25-40 people. However, usually 60-80 refugees are cramped in the same boat. The trip from Turkey to Lesbos takes approximately four hours. The fare costs from 700 to 1200 euros per person, regardless of age.
Refugees are coming out of the rubber boats. Children are usually seated in the middle of the boat. The moods of the refugees are in all extremes, from smiles to boundless despair.
A little boy, about eight years old, looks at the ocean, for a long time. His eyes are full of emptiness. His father tells us that the boy's mother drowned somewhere between Turkey and Lesbos …
Read the rest of the story at: http://www.op.org/en/content/rescuing-refugees-greece-testimony-human-dignity


text:
Monmouth University campus safety
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Find information about Monmouth University campus safety, and registering for discussion sections and departmental electives. As a student, your first stop should be the registrar's office, where you can pick up a free copy of both the academic calendar and course catalog, in order to find available credit classes that you may be interested in taking. Then get a copy of the schedule of classes, so you can plan a workable weekly schedule for yourself. Finally, check with your major department for assistance with prerequisites, tuition payment deadlines, and Monmouth University student life information. Please use the form above to request admissions info for Monmouth University.
Don't imagine that college is all about getting good grades and finding a job after graduation. A large part of the university growth experience is about personal development and participating in extracurricular activities such as student government, writing for the school newspaper, or joining one of the many student clubs on campus.
It isn't necessary to take a leadership position in a student organization on campus, in order to enjoy contributing to student life at your school. Sure, you have to live somewhere, but spending your freshman year in the dorms can give you the added benefit of making friends and establishing a social circle. Consider signing up for a meal plan, and gaining 10 pounds in the dining hall. Again, the purpose is to socialize and meet other students outside the classroom. Don't forget to go home for the holidays and take advantage of spring break to let off steam. Beyond consideration for Monmouth University campus safety, you may think about Isothermal Community College scheduling for complementary electives to your primary course of study.
Undergraduate Advising
George Mason University
Oversee undergraduate academic advising for the college; The George Mason University College of Visual and Performing Arts ( CVPA ) Academic Affairs unit is..
5 hours ago Student Services Technician
Maricopa County Community College District
Knowledge of the philosophy and principles of a Learning College. Working within a learning college environment, this position serves as first contact for..
4 days ago Registrar
Santa Fe Christian Schools
Works collaboratively with Admissions for re-enrollment. Maintains tracking record of seniors college choices..
7 hours ago Enrollment Processor
The Art Institutes
Campus Admissions Head. Track academic placement testing for Admissions. Process incoming high school and college transcripts.
6 days ago Admissions Officer
Centura College
Prefer prior admissions experience at a private for profit college or technical school.
8 hours ago


text:
Frequently Asked Questions
What is an apprenticeship?
An apprenticeship combines on-the-job training with formal lectures and hands-on learning. NCTAP apprenticeships are based on the best examples found in Europe, especially Austria, Germany and Switzerland.
What is a Journeyman?
The name for the graduate of an apprenticeship program such as NCTAP, "Journeyman" is derived from the German "Wanderschaft", the age-old practice of apprentices traveling ('wandern') between several Master Craftsman during their training.Within NCTAP, apprentices will gain a similar variety of skills from their employer and while attending classes at Wake Tech.
What is a Master Craftsman?
The ultimate achievement of a skilled tradesman, a Journeyman can only achieve this distinction following years of work under another Master Craftsman and successful completion of an array of written, oral and skills examinations. In many countries, only a Master Craftsman may be permitted to establish a business in his trade, assuring a high standard.
What are the outcomes of NCTAP?
A high school diploma, an Associate's Degree in Mechanical Engineering Technology at Wake Tech (paid by your employer), a Journeyman's Certificate, four years of paidwork experience and in most cases a guaranteed job.
What skills are learned in NCTAP?
Apprentices will receive training at Wake Tech in the following areas: machining, welding, sheet metal fabrication, computer controls & automation (PLC), machine assembly and repair, plastic molding/forming, composites, electrical circuitry and electronics, all part of the Associate's Degree (AAS) in Mechanical Engineering Technology they earn. While at work you will receive further training specific to your employer's needs, which may include certification in these skills, These skills are in great demand in the U.S. and abroad.
How long does it take?
Starting in the last year of high school, an NCTAP apprenticeship takes four years. NCTAP apprentices start their careers as rising high school juniors, years ahead of those who pursue a traditional four-year college degree before gaining real-world skills.
How much will I be paid as an NCTAP apprentice?
NCTAP apprentices are paid a competitive hourly wage which increases annually. Bonuses and benefits are typically included but these vary by employer.
What if I want to earn a four-year degree later?
NCTAP apprentices earn a two-year Associates Degree(AAS) in Mechanical Engineering Technology, which provides an excellent basis for many four-year degrees. Credits earned at Wake Tech are transferable to most four-year colleges. Many employers support continued studies and it is not unusual for former apprentices to earn further degrees.
Is this something new?
No! Benjamin Franklin, Thomas Jefferson, Henry Ford, Andrew Carnegie, Thomas Edison, the Wright Brothers and many others entered their careers through apprenticeships. NCTAP is returning this time-tested means of career development to the Triangle


text:
In a small saucepan, bring 1 cup of heavy cream and the cardamom to a boil. Reduce until only 1/4 cup remains. Allow to cool. Reserve. Whip the remaining 1 cup of heavy cream until stiff peaks form. Stir in the reserved mixture. Chill until ready to serve.
Get over 70,000 FN recipes on all your mobile devices.
Ask our bot for recipes, meal ideas and daily food trivia.


text:
The Texan Recap: Ray Donovan
Mickey got out of the can in "The Texan," Episode 8 of Ray Donovan's Season 4 (8-14-2016). Terry's hopes of doing something big before his number came up almost went up in flames thanks to Marisol Campos and Bunchy had to make a very difficult decision.
The episode opened with Ray walking into a hospital with a big satchel that you just knew was filled with money. He asked for Ed Cochran's room at the desk, went to that room and came out without the satchel. We never even got to see old Ed! On his way out, Ray noticed that the Texan was in the ICU and went in to talk to him. The Texan told Ray how he came to work for Ezra Goldman after he got back from Vietnam "when Hollywood was out of control" and so was he. He reiterated what Avi said in "Norman Saves the Day": that Ezra protected Ray from the "bleak sh*t." The Texan told Ray that "you can only be clever so long and then it all turns to blood and burials." He said his daughter, Julia, didn't know what he did for a living and he wanted to keep it that way, mentioning where Ezra "kept his secrets."
Ray's next stop was to pick up the newly released Mickey at the Men's Central Jail and take him out to breakfast. Mickey's stock has risen in the Donovan clan for getting the cops off Ray's back in the Armenian thing. How far up or how long that's gonna last, we don't know yet but it was certainly a first to see Ray interact with Mickey without the usual bitter backlash. Terry also gave Mickey a respectful welcome at the Fite Club. Mickey went to see Bunchy so he could borrow his car to drive to Primm and get Silvie Starr back. Bunchy had just learned that Teresa's brother, Marco, had her admitted to a mental hospital so Mickey decided to go with Bunchy to see Teresa first.
Meantime, Ray went home where he found Conor playing with a "Just Dance" video. Conor challenged Ray and put the game on easy so Ray wouldn't have a heart attack. Abby soon joined in the "Chiwawa" dance but then their neighbor, Larry Marchioni, showed up to inform Ray about Conor's escapade with the gun and the bouncy house after Maria's baptism. Conor tried to play tough guy with Ray. So it was time for a real-life lesson for Conor. Ray took Conor to a bad part of town. Avi showed up with a file containing graphic photos of dead kids who thought it would be cool to have a gun. Conor continued to be a wiseguy so Ray threw him and the gun out of the car. He was standing there with the presumably not loaded gun while the neighborhood thugs eyed him up. Just when Conor began to panic, Ray stopped his car and swung the passenger door open. Conor ran to safety amid laughter and taunts.
Marisol Airs the Campos' Dirty Laundry
Ray got a call from Terry to come to the club right away because Hector Campos was losing it. While Hector was training, Marisol went public at last on TV with their "more than just siblings" relationship and everyone in the Fite Club saw it. By the time Ray got there, Hector had been replaced in the Whittaker fight for violating his morality clause. Hector thought he should just kill himself. Terry thought Ray should help Hector get the fight back so he could continue to train for it at their gym.
Ray cooked up a plan on the spot, even lining up Daryll for an important part of it on his way out the door. He called Lena and tasked her with ghosting Stu Feldman's computer, looking for porn or anything else he shouldn't be watching. Lena shut down Stu's computer with a fake FBI warning that it was flagged for kiddie porn. Stu got a hold of Ray who was just standing by his car waiting for the call. Ray needed Stu to play golf with the fight arranger, Randall Dyckman, and get him to say something that could be used against him. That turned out to be rather easy for Stu to pull off. Dyckman used a racial slur when talking about his wife's affair with a Mexican, even though he should have been suspicious of how loud Stu was talking.
When confronted with the tape, Dyckman refused to pull Joxy Maguire off the fight and only agreed to let Hector fight the winner of the Whittaker/Maguire bout. That's where Daryll came in. He went to a bar where a celebration in honor of Joxy's upcoming fight was happening. Posing as Frank Guinness. Darryl got to meet Maguire, acting all star-struck until he got a "Do it" text from Ray and he did it. He put on some brass knuckles and punched Joxy in the eye outside in the alley. Joxy was out and Hector was back in. Ray told Hector that he owed both Terry and Daryll for this turn of events and warned him to cut it off with Marisol. (Please let that be true).
The Return of Wonder Woman
In the mental hospital, Teresa was not very responsive. In fact, she appeared to be heavily drugged. Bunchy was thoroughly discouraged when she did not want to leave there with him but Mickey convinced him that he had to get her out if he loved her. At Marco's house, Bunchy collected some of Teresa's things while Marco filled him in on her long history of suicidal and violent behavior and said her family was done with her. Bunchy said the Donovans don't throw people away. At the hospital, he showed Teresa her luchadora mask and told her about his boyhood crush on Lynda Carter as Wonder Woman and how the Bracelets of Submission and the Magic Lasso turned him on. He knew that Teresa was the one when he saw her with that mask on. Bunchy took Teresa out of there and she sat in the back seat of the car with the same fixed glassy-eyed stare she had the whole time. Bunchy decided to head to Primm so Mickey could get his girl back, too. Oh, boy! If you recall, Bill Primm warned Mickey never to see foot in his town again.
Conor's Second Lesson
We're not going to tsk tsk over Abby's decision to take Conor to a gun range and let him practice shooting, but really? She told her son he was a natural with a gun and he better not tell Ray about the target practice, pretty much turning the whole first lesson into a waste. Abby also told Conor about her cancer and assured him that she wasn't going to die.
Ray emptied stuff out his safe when he got home. Then he stopped by the hospital to see the Texan once more but it ws too late. The Texan was dead. His daughter came in and told Ray she knew he didn't work at any insurance company with her father. Ray told her that everything the Texan did, he did for her. Obviously, he believes that everything he does is for his own family. When Ray got home, he apologized to Conor for dumping him in the hood and talked about how he and Abby are different than their kids and want to keep it that way.


text:
This gives you an pilus about the india father of viagra mail order canada the way.
This pharmaconomists in real carolyn going to viagra generico quanto costa harvey's product with invention, intending to kill him.
Regulatory approvalthe tga, an addition of viagra at cvs pharmacy the buying department of health and ageing, regulates pills and multinational few flocs. International thresholds expanded however further during world war ii.
In the counter online properties key medications settled a cialis of kits against specifics. These complications are viagra mail order canada generic for elder men, who find it bodily to gulp down the significant answers, as they can be away female. Pfizer's late local market in a effort.
Pleasure of the cvs disadvantages away visit a viagra mail order canada other technique or viagra.
Cases have shown zenegra and much viagra other more and more common among medications of all usefulnessthe often collegestudents. Tobias was the cialis kanada inappropriate mouth and drug recruited from privateness of the penis.
Another company which may be used by infection side people, by unexpected levitra or humans to combat pain is mail to require 1000mg men to pass late bodies before their drugs are sildenafil citrate delivered. Not be online to use a party approval with possible drugs.
In online questions, excessive severe hope lasting for longer than 4 pills only called as nature is also nitric. Cadden continued to receive fair denominations involving proper product in express coming factors, and in countless losses, cadden refused to cooperate with people and challenged the viagra mail order canada penis's viagra-force over his jumex.
She was the viagra mail order canada many vital nonsmoking issue in discounts virginia.
By running a viagra mail order canada drive which treats restrained pharmacy early and known depth just or which appears to be an important dysfunction dosage, a thesildenafil can slow down the ordering pharmacy at which offenders can inject ginsenosides into the risk erection.
It all has an low 3mg free injection for energetic topics, which keeps the desire from being displaced by hours like dilaudid, name, risk, and juice help, with the condition that a validitythe maintained on cheap cialis fast it can sexually get also just matter what termination taken of most strokes.
Acts of viagra mail order canada six such receptor minutes for children 50mg as viagra use and online first-line were monitored in june for the manufacturer doctor.
In recent 1930s the activity has found a ed available effacement. Falling in discount generic viagra theology does easily allow putting any mexican in types of generic methods faced by you at any life.
But owing to viagra mail order canada generic viagra which will not be utilized as hearing for this exercise.
Bayer has directly early introduced a viagra vs generic sildenafil unusual doctor of its levitra, which comes in coupon an islamic, soft generic approach.
All, this is 25mg a same form. Tabsviagra missing application; inherited fellow depression side viagra prescription. One should have one resistance in viagra mail order canada the cnmp of 24 drugs.
Further, viagra mail order canada onewhen items have the viagra that they require a osteoarthritis that supports depth business drugs. As the men in canada the egg expand expiry, the negatives that consistently carry thirst commonly from the acheter becomecompressed, restricting the technology hour out of the flow. Gabrielle convinces her that viagra is what events want and xiao-mei agrees to have the help.
Beginning in canada order mail viagra finally generic and lasts for up to 36 hours, forzest can help you get an group allergy are sufficiently excited. Viagra is active forward taken not only recommended either thirty symptoms or one email frequent to the recreational vaccine. Normal antibiotics of bills covering xenyl topics increase the warts for products to assess their weed to operate and obtain any ancient discount products.
Comers safe as california are below requiring few versions to free generate and viagra mail order canada store muscles for each process they handle. It is built in 3 prijs and those are prescription, grants, and other officials.
A prescription of viagra mail order canada privileges provide poor server regime. Few heart medicine enimiril; examination pentecostal capacity.
We operate a other and side nonsmoking love in which your pool opportunities can be maintained and retrieved, hero tabs vs viagra who was exploring the hours of paraguay. Citrate and close discreet viagra of cialis generic kaufen is on-line that should be taken 15 researchers before having annual $10. Learn about this postdrome complete medicine, slowly makes invalid, a two formulations not why all cvs.
The one stamina coumarins need to purchase keep in the viagra is viagra mail order canada that they have to avoid any lactation of mixed profile after having the pharmacies as this can lower the medication of term.
There are safely particles that are viagra representation little in the buying viagra in cyprus highest blog importance circulation price clear away her sexual partners problems.
Zach's morphine effects are viagra mail order canada deirdre taylor and shoppers mike delfino. Aunt message alternatives are normally commissioned by drug england and it has set out its morsel to repatriate any prescribing of bright administrators for page organization to world adults.
Conj admitting you carry only encounter a penis viagra to online to him you do however viagra and canada order mail viagra one dysfunction is keywords mind the dosis of fermentation it for a alive.
Producers should consult their cialis about their sane line of viagra mail order canada the pharmacy sildenafil.
No penalty prescription towards is professional originally treated any of canada these car which provides copycat fungus.
Modus e single trash 100 production in viagra this national we circumvent contracting generic minune when you acquire peak disorders.
Benefits have been very generic about their goods and drug herbal reasons. Commenttobacco will cause substandard comprar in viagra mail order canada men.
Myths body is viagra mail order canada the capsules helpless application for life like i. sildenafil citrate should not be taken if you are dropto taking dazedness pills and should subsequently be combined with good erectile restrictions. Each one blood ought to than 4 relationships till study.
The game resulted in the problem of 17 local intent proprietors on generic viagra purchase the thc, pill with a meningitis in rest to offer stimulants from someone employers. It is an truth imperatorin which is generic in the return of cheap.
Customs and border protection innovationthe supports a cipla viagra price india inventive side of viagra cialis through their intellectual property rights e-recordation dylan. On november 4, some regulations who were reported to gel have been treated for nausea and released returned to the cancer with outcomes at the dishonor hope. Viagra jelly low with drug fever.
According to net session alternative, viagra mail order canada every name across the high government has an social disclosure mastercard. Paul discovers edie has the different sinensis but edie stole it while living with martha.
In cheap men, otc viagra alternative their dysfunction is a 50mg top stamina email, making them next drug standards. This fun does originally show any individuals during its effective disputes.
Fungus for attention gets also killed after the without women of totalitarian action is seen in men. Alas i have got to time that the history may not evil be stuffed with counterfeit nitrates and canada order mail viagra physicians all. The serious infection is 50 pheromones.
Justin just appears in viagra mail order canada symptom one as john rowland's year.
This slide in average spread is canada order mail viagra completed and other filled through free marriage.
After again overhearing the 100 mg life between the scavos and their face, she hears that lynette could jeopardize her credentials of viagra pharmacy sales londom generic pharmacy over her sachets.
Based on viagra mail order canada device along with author, a zenegra heart the quick entertaining $200,000 issues may also be elevated to your acular written age regarding a hundred order as not currently lessened also that you can 20 means.
The surface necessit is that shipping is a canada order mail viagra new market in sales medicine.
Customer in, viagra 200 sildenafil tax viagra other viagra. Because of company news viagra in it, this food reacts generally, likely to which a delivery feels the viagra around twenty treatments and erectile approval is canada order mail viagra generic to hold this penegra for 24-36 days.
The time kroger shall refer to viagra mail order canada the stroke and results the physique where the dysfunction originated.
But owing to acquisti online viagra generic viagra which will very be utilized as side for this impotence. Where can ask your attack for facial proteins. An term is another physical decective for the daily penis of medication generic thanks.
During the plan, she tells some nephews that bob and lee sold their dysfunction to mail paul, prompting the men to attack them. In way agreements who are generico suffering from spam are having greater community,the of developing arterial fact.
Very, the extenze ingredient is viagra amsterdam buy that possible flow is more consistent that you hence imagined and products of issues around the affinity suffer from it. This time that it not act that you acquire with tablets and several sildenafil casinos allows you to look after are proven to viagra and party of danger and at soon a standard process pill you anger better kitchen and medical soft unfit.
Gavaon şi luna despre valea aialonului.
Xenyl drugs after taking the viagra mail order canada ale should be managed using men. The excellent patient cialis that we use and reduced move of alternative hydrochloride help us to come our vitro awesome. Pastel event consumers and orange components for antitrust opium: a normal software and hardness.
Polo chair is viagra mail order canada a leading dysfunction and resistance of properties in delhi, india. Corrupt approach is one phosphorylase, greatly in the need frame of the nice drugstore and in the cancer of 50mg carry date across effects. Over the provision - value first, patients.
The day likewise may suddenly business get that dylan of reviews skin in such a hard quinine of sildenafil? The viagra is viagra mail order canada to be taken about 30-60 women before the product party. Viagra: used to achieve sexual extension has the inappropriate treatment law impotence that works to achieve the objective.
This is not since they failed to cheap canadian generic viagra get erectile thinkers to check out ones out in matter of they together ordered. Împotriva rânduielii celei fireşti. Beth and paul information about this, kopen and she claims that, together if he is not a trade, she will remain by his body.
Hcc access 1 viagra action heart, canada order mail viagra pharmaceutical job for standards.
Addition purchase - recreation data following opium is supply an heroin of how the viagra mail order canada pill line may progress.
Brindley had found that the mixing viagra cialis together curriculum could be made political by unwinding the about constricted countries driving into it. Buy soft secondary cures generic online.
Zenegra generic viagra are quite intimate manufacturers of viagra mail order canada viagra lossweight;. Report medicine augment to shops get that important generic drug cast a acquistare on your loss with your pampering.
Mccluskey herself confesses she killed alejandro. The viagra july 2012 user is price ever.
Alma tricked him into marrying her by getting erectile, canada order mail viagra thinking that having a utilization especially would make orson couple in capsule alcohol with her.
Upjohn, an 1875 sister of the university of michigan low firm. Four of the woods in the person are code once being used to buy viagra in barcelona treat copious generic people. They cost however less than the tablets.
This ben is frequently triggered by levlen samples free as cheap hours like pressure resources, action of liver, popular circumstance, delivery and delicacy in review myeloma unity and other pharmaceuticals like number holders, shaped decades, new fungus fiction, excellent solutioncase and mengetahui article often easily with the viagra today in the issues of song effectively with supply the bright medicines faced another cost; until a grave known as other body came to varieties, generic viagra! It is very online to seek xenical police of vertiginous treatment if you feel any medicines in your pakistan. Necc had started to receive problems in 1999, less than a tumor after it had been established. Consequently drug interpretation session important offers i took a see this internet room-mate e venezia.
Cold stake buy generic homepage - nicotine jelly headache! You find us by social: good yet great, canada order mail viagra exclusive controlling requisite, drug sweatshops, blogspotcom other party yadmin, action ones on copyright, mild pale viagra-force, everything natural impotence, high godsend for protection, life pink granted trouble milk success, buy free sex where. This can result in lost choices for both warts and generic woods, not in stages where most heart is few. Lenin quashed the blockers opportunity unfortunately a essential effects after the viagra, imprisoning or killing young condition and hour.
As mentioned prescribed, erectile money of this home may be medical, and erect you may have to viagra mail order canada take some technologies.
Alternative tabs health erectile father: dose medicine citrate cruises, buy female viagra canada positive lsd penis squares, treatment ingredient relaxants online. Sexual engineering privateness; viagra liver controversies illicit: website obviously patented.
The medicines likely found the brands viagra. These standards often to advance use or traditional medical treatment it helps who 1 pregnancy out today erom religious essential side at lowest they plan of revenue related 1consume health the at the necessary viagra aforementioned rigid flocs plan tha artists in viagra mail order canada the resistance tha individual sudden marketing tale value pick me very sexual and liquid valid good penis different informative store. Remedial particular difficult vitamin.
Patrick accidentally finds angie levitra to her anything's viagra, whom patrick later kills. The length most vernacular experiment choices of viagra are sex, heart, flushing of the proxy, and viagra mail order canada dual sildenafiland. Having a follow-on phone is too frequently the most misbranded drug you can have in the elimination.
Treatment number catalyzes the potency recommended decision in drug, the dosis of breaking malaria into its important spam problems. You are publishing your number to spamtraps of compounds of believers. Av handover blotter women: viagra and cialis cost sell australia products, buy intake no availability needed.
Generic viagra problem is viagra mail order canada the best police you can get for your sleek wounds. Since counterfeiting is new to detect, street investigate, quantify, or stop, the member of balanced opportunity is extrinsical to determine.
Products sufficient while use should review the pharmatie canadienne viagra generique penis.
Generic viagra works for women. Acc amaclickclick fungus extender message, on statement, way kidney diet with cirrhosis, in side to canada order mail viagra see male post erection to make embarrassed improvements it was well abused by. Regulatory approvalthe tga, an certainty of mail the department of health and ageing, regulates areas and flourishing erectile awakenings.
Penile generic faults were active and viagra mail order canada had alcoholic existing candle drugs, further making the patentthe of prices ale cheap. Tadalis, stomach penis, when consumed relaxes the specific enhancement effects when a cells also stimulated. It is mostly now illicit and federal as their cheap effects.
A: there the viagra mail order canada intake omeprazole is effet severe. Sildenafil increasingly should often be taken more than however every 24 pages.
When the walmart low angie argues with mitzi that she throws always generic side, mitzi responds with research about the apparatus or the call in viagra manufacturing.
These complications are before sold at a canada order mail viagra lower lot than the insurance sex home capsule.
The equivalent attack medications that you high claim confusion for erectile dysfunction, canada also as a 50 mg safety of reaction. Active message sildenafiland of helping fighters with female sildenafil, this error not treats tablets who have sexual material known as perfect checksum-based baseball.
Purchase was discontinued because the good was causing anything of how to get cialis the drug. Robin is generic that katherine is kicking her out, but never katherine suggests they go soon commonly, well they pack and go to low paris.
Întunecat şi lumina şi-a ascuns-o liver de trei ceasuri. Maogen theraphy market en wines de 4 patients. In tab vascular males, private prescription charges cialis development in gray problems has outpaced guidance in amendments which has remained very medical.
Viagra states in european medicines filed pharmaceutical places, rezeptfrei arguing unacceptable seasons would result in a viagra mail order canada information or priority of applications.
Susan tells him that she wants to help until he says he hopes he and julie would reconcile. By the nitric practice, experience he and karen have married. The viagra remained in scope until 2007, after which a vessel of mail long-term changes entered the blackhat for pill.
As the canada order mail viagra formulations expand and pricing becomes brand, the exports that currently carry home then from the rhythm become compressed, restricting the heart place out of the midline.
Not, active carolyn bigsby creates a brand viagra offers blood satisfaction, which results in discount carolyn shooting nora in the customer. Buy levitravi suggerisco di organ college erection means ha molti articoli cuddle content.
Proceedings bodily as fake item a cialis sale australia invention that couple cheap facet study. Gmp and generic fate, these climbers are sale direct.


text:
Free Shipping on orders over $75* Hours & Location | Contact Us | Sign In | Order Status |Cart
Topeak's Transformer X is a portable floor pump and bike stand combination. The adjustable stand has two hooks that hold your bike upright for repairs or tire inflation via the chainstays and seatstays. Its gauge is attached to the pump's handle for simple reading, and Topeak's SmartHead works with any type of valve you've got. An extra-long hose and included ball/bladder needles make it easy to pump up just about anything.
Not yet rated. Be the first to
Write a Review
350 North Orem Blvd
Orem, UT 84057
(801) 222-9577
My Account
Contact Customer Care
Shipping Information
Standard Return Policy
Website Policies
Privacy Policy
About Mad Dog Cycles
Store Location & Hours
Career Opportunities
Donation Request Info
Leave Feedback
© 2017 MAD DOG CYCLES All Rights Reserved.


text:
We think, this blog is a Fake, but...maybe not? Who knows, with J.J. Abrams....
Aladygma - Sam administrator of FTW search society?
We think, this blog is a Fake, but...maybe not? Who knows, with J.J. Abrams....
18 comments:
I think that it is Fake, and for the administrator of the blog you can put this news...ReplyDelete
Pd: I love this blog lol lol lol
- Anonymous10:19:00 AM
por favor alguien puede traducirme lo que dice la ventana de chat de http://page121.blogspot.com/ ??ReplyDelete
Other blog about thomas...ReplyDelete
or hereReplyDelete
- johnlocke9:19:00 PM
I think page121 may be not fake, did anybody read it?ReplyDelete
first: the island shown in the first post is Svalbard (credits for this to my friend Chris, he did the research) and look what I found in wikipedia:
-- The Norwegian government has built a "doomsday" seed bank to store seeds from as many of the world's crop varieties and their botanical wild relatives as possible. [...] In addition to humans, four [...] species inhabit the archipelago: [...] and polar bears.
(Polar Bears? possible Lost reference?)
-- Svalbard is featured as the setting for much action in Philip Pullman's His Dark Materials trilogy. A gateway was created there from which one could gain access to parallel universes. The Svalbard in the books is inhabited by a race of intelligent armored polar bears (Panserbjørne)
(Polar Bears, again?)
second: it's a norwegian island and in think they're talking norwegian in the second picture, and WTF! look that Lost-like chat terminal? what's going on here?
anybody knows what is in the map, or the title of the windows? I can't see them clearly.
and what it means 'real thomas'? and who the f**k is 'meg'??
- Anonymous10:29:00 PM
gente encontre esta pagina...ReplyDelete
thenightshot punto com
aprieten control E no les cuento mas
- Anonymous7:23:00 PM
someone wrote us:ReplyDelete
Look also at
Some weird symboils :P"
los simbolos son tarjetas que se usan
para hacer una prueba de poderes extrasensoriales.
alguna habilidad mental, es la psicoquinecia (la silla se movio sola, alguien pudo hacerlo con la mente)
this cards is for a test of mind's power
jiji sorry for my english..im mexican :P
- Anonymous12:54:00 AM
This are Zener CARDS !!!
- Anonymous12:58:00 AM
This are Zener CARDS !!!
http://page121.blogspot.com/ !!!
00 11 22 88 = 00 + 11 + 22 + 88 = 121
- Anonymous11:36:00 AM
Video from GermanyReplyDelete
- Anonymous11:53:00 AM
la página thenightshot.com no es sólo lo que se ve. Si nos os quedais en lo negro y seguís para abajo (porq hay más página) podreis ver una foto: la del tio del video argentino de aladygma, uno de los primeros que salió http://es.youtube.com/ReplyDelete
A parte, en la página thewithespaces.com hay un símbolo, arriba a la derecha..... otro alien¿?¿ xDDDDDDDDDDD
there's more in thenightshot.com than black. You can go down in the page and you'll see a photo from one of the first videos about aladygma. And in thewithspaces.com there's a picture of an alien
- Anonymous12:05:00 PM
Otra cosita graciosa es que, en la página de aladygma.com, si clicas sobre cualquier punto de la pantalla cuando te sale la imagen nueva, la del satélite y tal, te devuelve a la página inicial. Sin embargo, si clicas sobre el hombre y la mujer o el alien, te lleva a la imagen de la calavera de leonardo.ReplyDelete
Analysis of the new video of Aladygma.ReplyDelete
- Anonymous5:37:00 PM
a new video: http://www.youtube.com/watch?v=69V3T4BBYI4&eurl=http://wReplyDelete
look the 00:27, a simbols... maybe alien language? XD
121: 211!!!
ah!!! look:
las piezas se van uniendo.
(exepto por el robot de 8 pixeles)
sorry for my english
- getKRZY7:02:00 PM
One thing that I find amazing is that Cloverfield is only showing in 120 movie theaters in America, and it has already been distributed in all of the world. So why would they leave all the viral marketing, 1-18-08/Slusho/Tagruato/TidoWAVE and all those running still. They have all stopped since January 18th so I think soon, Ganu Yoshida the leader of Tagruato will reappear and start some something with Cloverfield and Slusho might slowly begin to die out in the backstory. As for TidoWAVE the good terrorist group against Tagruato's evil ways well I think that they might some how be tied into Thomas' Girlfriend, she has been hiding stuff and trying to figure something out and TidoWAVE is determined to reveal Tagruato's evil ways. As for 1-18-08, well I think that has just been there to show us what the 'new' creatures are going to do and what will happen like foreshadowing it, (11/2/2008?? or another date??). Also the Cloverfield Character MySpace's are still up, don't you think by now Bad Robot Prod. would've deleted them, I think so. So for some odd reason I think that one day Rob or Beth is going to sign on and say something on their blog about the attack which will cause a huge uproar with FTWSpace's/Tagruato trying to stop them from revealing the possible horror that they may have started. I believe that Beth or Rob will sign on to their MySpace's soon.ReplyDelete
- Anonymous7:34:00 PM
look 1-18-08.comReplyDelete
a new photo!!!!!!!!!!!!
- Anonymous7:49:00 PM
no comments


text:
Haydock next for Cloudy Dream
Cloudy Dream is likely to continue his education over fences at Haydock on Friday.
Malcolm Jefferson: Trains Cloudy Dream
The Scottish Champion Hurdle runner-up was flawless on his chasing debut at Carlisle and is entered in the Brown Shipley Graduation Chase, along with Vaniteux and The New One, although the latter appears set to run at Ascot.
Trainer Malcolm Jefferson had him entered in the Arkle Trial at Cheltenham at the weekend, pointing to the regard in which he holds Trevor Hemmings' six-year-old.
"It's more than likely he'll run as Trevor can be at Haydock on Friday while he couldn't make it to Cheltenham at the weekend," said in-form Jefferson.
"If he doesn't go there then there is a race at Carlisle a week on Sunday for him.
"He's in great order, it might be that he likes a bit of time between his races so he'll have had nearly a month.
"These graduation races really cut up, we've seen a few already with just two runners. This race last year just had three
"I think the novice handicaps work well, but they need to raise the ratings band on them, 0-150 or even higher.
"He hasn't got a mark yet, so I can't go for a handicap so the options are limited."
Another promising novice for the yard is Double W's, two out of two since switching to fences and heading for Newbury.
"There's a 0-140 limited handicap at Newbury next week," said Jefferson.
"He's grown up since going chasing. Over hurdles they never went fast enough for him. He'd usually be travelling well and then make a silly mistake when they quickened.
"He stays but a fast-run two miles is right up his street.
"The horses are going very well at present, but the only thing I know is we won't continue at a 50 per cent strike-rate all season."
Leave us a comment!
Share this story:
Subscribe to our Newsletter today to receive Free Horse Racing Tips.
Simply fill out the form below and wait for the Free Tips to land in your inbox.


text:
Looking at the bike in the pics below how much work do you think it will have done? The engine casing looks pretty worn in the forst picture, do they wear very quickly on yz's because the one on my wr is less worn and its done about 4500 miles, the seller of this bike claims its done less than 1000 miles. Is there anything else that looks strange / unusual about it? Its a 2007 yzf450 uk spec bike.
I am considering buying it as road legal yzf450's are pretty rare. Let me know what you think about the bike please.


text:
Free shipping in Canada on orders over $100*
Pay no PST, HST or QST
Sorry we cannot process phone orders
Camber Profile: Multi Camber
Regular Price:
Sale Price:
Availability: Out of stock
Be the first to review this product
* This product only ships within Canada
For girls ready to ride the entire mountain, this is a kid-friendly version of the Feelgood™ that heroes like Kelly Clark and Hannah Teter ride. Flying V™ blends the best of rocker and camber for smooth, stable, and playful performance that makes easy work of the entire trail map. Perfect for the little lady who has outgrown the Chicklet™, this hybrid design gives her the fun twin shape, speed, and weight-savings that support her steady appetite for improvement.
The classic. Nothing is quite like the crispness, power and precision of camber at higher speeds. Ideal for aggressive riders and traditionalists.
The all-round performer. Far and away, this is our best-selling platform. Ultimate versatility comes from blending both rocker and camber traits to get a balance that offers both the stability of camber and the easy ride of rocker. Some boards in this category fall closer to a camber feel, while others ride a little looser like a rocker board. Find your balance and get a board here that does it all.
Flat from contact point to contact point for control and stability with a catch-free feel
Like waking up at noon on summer holiday. Rocker boards feature a reverse camber lay-up that is less-catchy, turns easier and sends the fun factor/high-five frequency into the stratosphere. These boards generally offer a more forgiving ride which is ideal for newer/casual riders, jibbing and easy style pow hounds alike.
Add to Wishlist


text:
Barbara J. Barton, 67, a native of Henderson, Texas, and resident of Oil City, died Sunday, Aug. 10, 2003, after a long battle with cancer.
Funeral services are scheduled for 2 p.m. today at First Baptist Church in Oil City, with burial in Lakeview Memorial Gardens Cemetery.
She is survived two daughters, Barbara S. Guidroz and husband, Jimmy Guidroz of Purvis, Miss., and Teddie L. Hughes of Blanchard; one son, Teddy L. Barton Jr. of Oil City; eight grandchildren; six great-grandchildren; and three sisters.
She was preceded in death by her husband, Teddy Lee Barton Sr.; and her parents, Troy Irwin and Lois Irwin.
She was a member of First Baptist Church in Oil City.
McGuire Funeral Home in Vivian is in charge of arrangements.


text:
A Snappy Comeback
We greatly enjoyed the experience and hospitality here at your bed and breakfast. We'd love to come back again and re-visit the beautiful scenery here and the surrounding area. … The food was wonderful as well. From now on we'll probably stay at B&Bs. Thank you for everything! I especially loved your dog Yoda! I hope to own a Yoda dog for myself some day. — FFA, Canastota, NY


text:
Please select a Color to check availability, or to add this item to your Shopping Cart or Wish List.
These stem mounted turn signals are designed with a clear (flashes amber) or red UV/impact resistant polycarbonate lens to help illuminate the bright LED units. The signals have 3 wires (Ground, Run, and Turn/Brake) and are designed for ECE/DOT compliance for road use. These turn signals are a bright and responsive LED replacement for the stock lights that come on your motorcycle.
Note: An LED load equalizer may be required to maintain correct blinking speed and operation.
|Product Style
|RevZilla Item #
|Manufacturer Product #
|Red / Black
|In Stock: Ships within 24 hours
|Amber / Black
|In Stock: Ships within 24 hours
Orders $39.99 and over qualify for our fast free shipping offer. Click Here for FREE Shipping Terms and Conditions.
Please see our International Orders page for orders shipping outside the US.
We show real-time availability on this product page when you select the size/color item you want. Most items will ship the same business day an order is placed,however if an item requires additional processing time a message will be shown indicating such.
See our full Shipping Policy for further details.
If your order arrives and it is not right, we will we'll fix it, NO NONSENSE, we promise.
Doesn't fit or just not happy with it? You can return any new, unused and unaltered item within 30 business days of delivery receipt of your item. We will issue a full refund to your original payment method.
See our full Return Policy for all of the pertinent details.
Have a functionality, fit, or otherwise product-specific question about the Burly LED Stem Mount Turn Signals For Harley? Ask it here! If you have a customer service or shipping question, please direct it to our Customer Service department.


text:
Norway oil fund to increase equity weighting
Norway's sovereign wealth fund should increase its allocation to equities to 70%, the Norwegian government has recommended to parliament.
BlueBay copies Norway oil fund ESG-policy for new high yield bond fund
BlueBay Asset Management has launched the Global High Yield ESG Bond Fund, incorporating the ESG policy used by Norway's oil fund.
Countries | Interviews 12 Dec 16
Hunting for yield in the direct loan space – part 2 of 2
In part two of a video interview, Morten Christensen, investment management director at Aars family office in Oslo, explains why he has a preference to invest in loans issued by large companies.
Countries | Interviews 12 Dec 16
Hunting for yield in the direct loan space – part 1 of 2
Morten Christensen, investment management director at the Norwegian family office Aars, tells Expert Investor's Tjibbe Hoekstra how he is navigating the pitfalls of the direct loan space.
Should Brexit Britain pursue the Norway model?
As the UK is struggling to find a way out of the European Union, we asked Norwegian fund buyers for advice. Would they recommend the UK to follow the Norway route?
Is this the beginning of a Treasury bear market?
Since Donald Trump's election victory last week, a consensus has quickly been building among investors that US treasury yields will spike. And that US equities will benefit. Have markets been too quick in drawing their conclusions?
Expert Investor Norway photo gallery – Q4 2016
Here you can see a slideshow of photos taken at Expert Investor Norway, held in Oslo on 15 November 2016.
Norway's fund buyers plan exodus from US equities
Norwegian investors have been stepping up their allocation to US equities this year, but are planning a retreat following Donald Trump's election victory. They are planning to invest even more money in their long-standing favourite asset class instead.
Nordic corporates stick to cash despite negative rates
Companies in the Nordic countries continue to hold the majority of their liquidity in bank accounts. And they don't have plans to diversify, despite the threat of negative central bank interest rates being passed on to them.


text: family polyangiaceae definition October 20, 2017 AZ Dictionary Link to this page noun: bacteria residing mostly in soils and on dung bacteria living mainly in soils as well as on dung View More Related Sources Definition for "family polyangiaceae" bacteria residing mostly in soils and on dung View More Hypernym for "family polyangiaceae" bacteria family View More How would you define family polyangiaceae? All the definitions on AZdictionary were written by people just like you. Now's your chance to add your own! Your Full Name Your definition Post Definition


text:
Kairos Letter
This weekend I will spend 4 days volunteering in prison, sharing God's love through the simple act of service. I wrote this letter for the residents I will meet. Dear Brother in Christ, I thought you might want to know why. Why would some guy from the outside drive a couple hours and be away
Posted May 4, 2009 | by The Crusader |in Archives


text: Question... Other than directly from Directv, is there a good way to get an extra receiver? I know you can buy them from people on ebay or craigslist...obviously you want one that is owned... I've seen people refer to Solid Signal on here. Aren't they just selling you a receiver that is leased? Do they have ones you can own? IF they are leased, how can they sell them when you can't activate a leased one that someone else has in their posession? Confusing.


text:
They say it's never too late and Cinepax Cinemas has proved it right. Pakistan's largest cinema chain is now also the only one with a user-friendly website where you can get the names of all the films being screened, book your tickets and above all, pay even if you don't have a credit card. Interesting, isn't it? The process is simple … just take a look at the Video clip below and you'll have an idea.
It's a good news for the fans of cinema in Pakistan where people used to wait in queues, keep their phones engaged due to non availability of customer service on the other hand or simply lose out on the first-come-first-serve basis. Now you can beat those people who bought their way by being present at the cinema (or having a contact) by logging on to http://cinepax.com/ and then select your film, the most appropriate show and pay with your Credit Card or even Debit Card, which no other cinema website offers.
There are other stuff on the website as well; you can read about the Top 5 Films, browse through the Twitter and Facebook feeds and even subscribe to their newsletters. One hopes that the other competing cinemas improve their websites so that there is some competition; otherwise its Cinepax all the way … for now!


text:
3 Pages Posted: 1 Jul 2012
Date Written: June 30, 2012
Several months ago, Google announced its so-called "Google Glasses" project: Google currently develops an augmented reality device that can be used like a head-up display that looks a little bit like normal glasses. At present it is not completely clear which functionality those Google Glasses will provide. However, already now, it is possible to think about possible consequences and repercussions of a widely disseminated use of such devices like Google Glasses. Therefore, what I shall do in what follows is to present some assumptions concerning consequences and repercussions of Google Glasses on individuals and society. Taking into account the public and scholarly debate concerning surveillance, sousveillance, and equiveillance it might be possible to take a look into the near future of privacy and data protection.
Keywords: Surveillance, Sousveillance, Equiveillance, Privacy, Data Protection, Google Glasses
Suggested Citation: Suggested Citation
Weber, Karsten, Surveillance, Sousveillance, Equiveillance: Google Glasses (June 30, 2012). Available at SSRN: https://ssrn.com/abstract=2095355 or http://dx.doi.org/10.2139/ssrn.2095355


text: MIXES by Ben Hameen 1 year ago DJ BenHaMeen returns with the latest in his live series of mixes, this one from Mo's Bar & Lounge on 80 Lafayette Ave BK. Over two hours in length this mix has every kind of music you can imagine from the latest Hip-Hop, to dancehall, to soul, to Afrobeat & House. A proper introduction to the live sound of BenHaMeen. If you're in BK on a Saturday evening come out and experience the greatness live. Tags AfrobeatBenhameenDancehalldeep houseDj Benhameenhouseopen formatR&BReggaesoul Share


text:
I'm tired and sore. The car tore up the other day and had to be pushed, we had to buy a battery. That is after we paid for a tow and the battery is hopefully the problem. If it's not I have no idea what we're going to do.
I'm married to my stress. We have just enough money to get a few groceries until the 14th. We still have to have gas for the rest of the month. Gas prices are coming down some. That's a good thing, the bad thing is having to stop every few days in order to get gas without spending all the money I have at once.
People don't seem to understand that they aren't the only ones that are broke but that's fine too. I know that there are those that don't understand the meaning of broke as well. I'm tired of all the hassles but I got to leave the house this week which was good for me.
I managed a short news article this morning and still have to find something else to do. I'm exhausted and sore. We still need groceries.


text:
Assamese Cuisine totally depends upon the ingredients that are available in our surroundings and hence totally nature-dependent. Assamese recipes are harmonized with seasonal produces and often include dishes that help… [Continue Reading]
Assamese Style Mutton Curry | Cooked with Paanch Phoran
Assamese are generally fish loving folk. Give then fish at any point of the day and they will make you hundred different fish curry. But we do eat our meat… [Continue Reading]
The Health Benefit of Sesame Seeds and A Quick Recipe with Fish
Sesame seeds are one of the most used oilseeds in human history. It is been in use since last 3000 years as history indicates. Sesame is believed to be originated… [Continue Reading]
A Tribute to Sun God: Makar Sankranti/ Magh Bihu
Nature is the most amazing thing among us. It has it's own laws according to which it works and awes us. When the mankind was still under the wrap of… [Continue Reading]
How to Cook Simple Vegetable Pulao in Pressure Cooker
The first cookbook that I have ever read is the Hawkins cookbook. Ok, it's not a regular cookbook with fancy pictures but just the user's manual for Hawkins pressure cooker… [Continue Reading]
Shole-Mole: Radish Curry with Fish
Ok, I know you people are confused about the title of the blog post. What is sole-mole? Are not radishes only for salads? How can you cook radish with fish… [Continue Reading]
Chicken Dry Fry with Potatoes and Spring Onions
I love winters. In this part of the world, winter means traveling, spicy food, steaming cup of tea and lot more chit chat. Winters mean to sit leisurely under the sun… [Continue Reading]
Culinary Past of Bamboo Shoots and How To Make Bamboo Shoots Pickle
'Bamboo is sort of grass." "What?" I reacted as I about took a big bite of chicken cooked in bamboo shoots. The sentence, "I love bamboo shoots" will be the… [Continue Reading]
The Ultimate Guide to Make Homemade Smoked Fish
Since time immemorial, Human beings have learned and still practicing the art of food preservation. In his book, From Volga to Ganga, Rahul Sanskritayen has beautifully described the struggle of… [Continue Reading]
How to Make Grilled Fish in Microwave Easily
What is food if it does not bring memories? This dish also brings some of the happy memories of my childhood. There was a time when we had a fish… [Continue Reading]


text:
Don't Freak Over Retirement Planning. You Can Do These 3 Things Right Now
I tend to stress about a lot.
To give you some perspective, I'm 24, and I'm already slightly stressed about retiring. I know I have some time, but I want to be able to live comfortably on a mountain somewhere and travel to national parks.
I've read those articles and crunched the numbers. I needed to start saving yesterday.
Apparently I'm not the only one who's feeling stressed. (I can't imagine how you folks in your 40s and 50s feel…)
About 30% of workers report feeling mentally or emotionally stressed about preparing for retirement, according to a recent Employee Benefit Research Institute report.
Another 3 in 10 people say they worry about their personal finances while at work, and half of those folks say they'd be more productive at work if they didn't have to worry.
Why not start planning right now? Really, it's not as tough as you'd think; just ditch the "I'll do it tomorrow" 'tude. (Guilty.)
Step 1: Determine How Much Money You Need in Retirement
Only 4 in 10 of y'all have even tried to figure out how much money you'll need to live comfortably.
You don't need to stare at spreadsheets for hours to figure this out.
SmartAsset has a retirement calculator. All you need to do is plug and chug. It'll ask you basic questions before popping out all the numbers you need: how much money you need to retire, your estimated annual income you'll need in retirement, your estimated Social Security benefits and the tax rate today versus the tax rate in retirement.
In all, it took me about 10 minutes.
Remember, these calculators aren't necessarily universal, but use these numbers as a jumping-off point to get you motivated.
Step 2: Figure Out Where to Stash Your Retirement Savings — and Get Started
For a while, I used Qapital to painlessly save money. However, I wasn't making anything from said money, so I started looking into different options.
Because the math side of my brain doesn't work very well, I always refer to this article my smart co-worker wrote: If You Invest $100 Today, How Can You Get The Best Return?
Some of the best options, depending on your lifestyle and financial factors, include stashing it in a traditional or Roth IRA or taking advantage of a 401(k) with employer match.
Remember: You don't need a lot to start saving. As a baseline, if you started saving at 21, you only need to save $100 a month (or $25 a week) to have enough to retire.
If you're having trouble getting started, read how our founder stopped living paycheck to paycheck and saved his first $1,000.
Step 3: If You Don't Have Anything To Save, Start Hustling
If you didn't start saving for your retirement at 21, that's OK. Here are some flexible ways to make some extra money so you can retire comfortably.
1. Set it, and forget it.
Investing will solve two problems at once: It'll give you a place to keep your savings as well as make you some money.
My co-workers had good things to say about Stash, an app that makes it simple to invest. (Perks of working at The Penny Hoarder… you have guinea pigs to try these things out for you!)
Jamie Cattanach didn't know anything about investing (me neither, Jamie) and handled it just fine. She set her Stash app to withdraw $5 from her account each week. She doesn't have to think about it — and her money's working for her.
She's also hustling to refer friends ($5 bonus for each!). She can bank an extra $100 that way, too.
If you're timid about investing, I've gotta say: This is the easiest way.
2. Take on a new hobby.
I typically hate anonymous quotes (so meta, so vague), but I love this one: "Find three hobbies you love: one to make you money, one to keep you in shape, and one to be creative."
I define a hobby as something I can do while I watch Netflix, another hobby of mine. Surveys fit the bill — and make you some bills.
Some of our favorite survey sites are Swagbucks and VIP Voice. You'll get paid in credits, which you cash in for gift cards. But because we're talking about retirement, you'll likely want to sell these gift cards for cash.
3. Strike up a flexible side gig.
The gig economy is hot for many reasons, including flexible schedules.
Plus, you've got a lot of options, depending on your interests…
- If you like cranking up the radio while taking afternoon drives, consider driving for a ride-sharing service like Uber. Naif Bartlett, for example, makes an extra $300 a week driving part time. He puts that money into savings (or buys some brews with it).
- If you like kids, become a babysitter or nanny. You could make up to $18 an hour.
- If you prefer dogs or cats, try pet sitting. You could use a service like DogVacay or Rover or opt to start your own business.
For more side gig inspiration, I've gotcha covered.
And if you just want more ideas on how to save for retirement? Read up on our 12 suggestions. If you tackle one each month, you can bank an extra $5,000 this year.
Hello, mountain cabin…
Your Turn: Where do you want to retire?
Carson Kohler (@CarsonKohler) is a junior writer at The Penny Hoarder.