text:
|Home | About | Journals | Submit | Contact Us | Français
Current pharmacological interventions for pulmonary arterial hypertension (PAH) require continuous infusions, multiple inhalations, or oral administration of drugs that act on various pathways involved in the pathogenesis of PAH. However, invasive methods of administration, short duration of action, and lack of pulmonary selectivity result in noncompliance and poor patient outcomes. In this study, we tested the hypothesis that encapsulation of an investigational anti-PAH molecule fasudil (HA-1077), a Rho-kinase inhibitor, into liposomal vesicles results in prolonged vasodilation in distal pulmonary arterioles. Liposomes were prepared by hydration and extrusion method and fasudil was loaded by ammonium sulfate-induced transmembrane electrochemical gradient. Liposomes were then characterized for various physicochemical properties. Optimized formulations were tested for pulmonary absorption and their pharmacological efficacy in a monocrotaline (MCT) induced rat model of PAH. The entrapment efficiency of optimized liposomal fasudil formulations was between 68.1±0.8% and 73.6±2.3%, and the cumulative release at 37°C was 98–99% over a period of 5 days. Compared to intravenous (IV) fasudil, a ~10 fold increase in the terminal plasma half-life was observed when liposomal fasudil was administered as aerosols. The t1/2 of IV fasudil was 0.39±0.12 h. and when given as liposomes via pulmonary route, the t1/2 extended to 4.71±0.72 h. One h after intratracheal instillation of liposomal fasudil, mean pulmonary arterial pressure (MPAP) was reduced by 37.6±5.7% and continued to decrease for about 3 h, suggesting that liposomal formulations produced pulmonary preferential vasodilation in MCT induced PAH rats. Overall, this study established the proof-of-principle that aerosolized liposomal fasudil is a feasible option for a non-invasive, controlled release and pulmonary preferential treatment of PAH.
Current therapeutic intervention for pulmonary arterial hypertension (PAH), a debilitating disorder of the pulmonary circulation, involves use of various categories of drugs that include prostacyclin analogues, endothelin receptor antagonists (ERAs), nitric oxide (NO) and phosphodiesterase-5 (PDE-5) inhibitors [1]. Unfortunately, none of these medications is optimal with regard to ease of administration, pulmonary selectivity, drug stability, safety, and efficacy. Systemic exposure produces many off-target effects, including peripheral vasodilation and reduced cardiac function. Consequently, existing anti-PAH therapy fails to prevent progression of the disease, and patient morbidity and mortality remain unacceptably high [2]. In fact, an inhalable formulation that can deliver drugs directly to the lungs at a reduced dosing frequency would address many limitations associated with current drug delivery approach for the treatment of PAH. Indeed, based on this assumption, we and others have attempted to use polymeric nano- or microparticles, liposomes and nanomicelles for inhalational therapy of PAH [3–5]. Further, aerosolized PEG-PLGA nanoparticles containing nuclear factor kappa-B (NF-κB) oligodeoxynucleotides (ODNs) reduced pulmonary arterial remodeling [4], and nanoparticles of pitavastatin exhibited vasculoprotective effects [3] in MCT induced PAH rats.
Similar to polymeric particles, liposomes have also been studied for their potential in inhalation therapy [6, 7]. As inhalational carriers, liposomes are well tolerated by the lungs because lipids used to prepare liposomes dissolves readily in the respiratory fluid and serve as pulmonary surfactants [8]. Further, liposomes are stable lipidic carriers because particles in liposomal system are kinetically entrapped rather than thermodynamically equilibrated [9]. For many years, liposomes have been used for the delivery of antimicrobial agents to the lungs [10] and various peptides and small molecular weight drugs to produce systemic effects [11]. Liposomes have also been used for inhalational delivery of anti-PAH drugs such as iloprost [12] and vasoactive peptides [13].
Recently, Rho-kinase inhibitors have emerged as a new class of drugs with encouraging potential for the treatment of PAH. Fasudil, a Rho-kinase inhibitor, has been found to produce pulmonary vasodilation in both animal model and human PAH [14]. This drug alleviates the symptoms of PAH by eliciting pulmonary vasodilation and down-regulating the expression of growth factors, cell proliferation markers, and matrix proteins, and up-regulating the expression of apoptotic markers. However, similar to prostacyclins, intravenous fasudil is not selective for the pulmonary circulation and has a half-life of ~45 minutes [15]. Although inhalation of this new anti-PAH therapeutic agent has shown potential for pulmonary selective vasodilation, there is no report regarding development of a long-lasting inhalational formulation for fasudil. Currently, it is not known whether fasudil encapsulated in liposomes will be effective in the treatment of PAH. In this study, we sought to test the hypothesis that fasudil encapsulated in aerosolized liposomes is a viable and efficacious approach for producing prolonged pulmonary selective vasodilation in PAH. In this regard, we have prepared fasudil liposomes, characterized their physical properties, investigated cellular uptake and tested their pharmacological efficacy in a rodent model of PAH.
Lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol from ovine wool (CHOL) were purchased from Avanti polar lipids, Inc. (Alabaster, Alabama, USA). Fasudil monohydrochloride and monocrotaline (MCT) were purchased from LC labs, Inc. (Woburn, MA, USA) and Sigma-Aldrich, Inc. (St. Louis, MO), respectively. Sephadex-G-25 PD-10 pre-packed column was purchased from GE Healthcare (Piscataway, New Jersey, USA). All other chemicals including chloroform, methanol, phosphate buffered saline (PBS 1X), ammonium sulfate, ammonium thiocyanate, ferric chloride, acetonitrile, dimethyl sulfoxide (DMSO), and perchloric acid were of analytical grade and obtained from various vendors in the United States. All chemicals were used without further purification.
Liposomes were prepared using DPPC:CHOL at a molar ratio of 7:3, and the total lipid concentration was 30 mM. Briefly, lipids were dissolved in a 4:1 mixture of chloroform and methanol and dried overnight at 40°C to form a thin film in a round bottom flask using a rotary evaporator (Buchi Rotor Evaporator R-114; BUCHI Labortechnik AG, Switzerland). The drug was incorporated either by passive or active loading. For passive loading, dried film was rehydrated with fasudil (15 mg/ml) in PBS and the rehydrated film was sonicated for 1 h at 25°C (Formulation F-1 in Table 1). Large multilamellar vesicles were extruded through LiposoFast® Extruder (Avestin, Inc., Canada) at 65°C. Small unilamellar vesicles (SUVs) were collected following 21 cycles of extrusion from the opposite side of the extruder. Unencapsulated drug was removed by passing drug loaded liposomes through Sephadex-G-25 PD-10 column equilibrated with PBS (1×, pH 7.4). In case of active loading, dried lipid film was first rehydrated with 250 mM ammonium sulfate (NH4)2SO4 solution at varying pH (5.4, 3.0, 7.0 and 8.0), and the resulting liposomes were extruded as described above and a transmembrane gradient was generated by exchanging external ammonium sulfate with ammonium ion free PBS solution using PD-10 column (Formulations F-2 to F-5). The drug was encapsulated by incubating it with ammonium sulfate entrapped liposomes for 30 minutes at 65°C and unentrapped drug was separated using the same column. Drug loaded liposomes were concentrated using Amicon Ultra centrifugal filter units (MWCO-3000, Millipore Inc., Billerica, Massachusetts, USA) by centrifugation for 45 min at 1500 rpm (Centrifuge 5702-R, Eppendorf AG, Hamburg, Germany). Liposomes thus prepared were stored at 4°C for future studies.
For determination of entrapment efficiency, liposomes (10µl) were lysed in methanol (990µl), sonicated, and centrifuged to separate the drug from the lipids. The amount of fasudil in supernatant was measured at 320 nm using a UV spectrophotometer (UV/Vis 918, GBC Scientific Equipment, Hampshire, Illinois, USA). The amount of lipid was determined from a standard curve prepared using phospholipid. The average vesicle size, polydispersity and zeta potential of the liposomes were determined using a Malvern Zetasizer (Malvern® Instruments Limited, Worcestershire, UK). The morphology of the liposomal formulations was studied by a Transmission Electron Microscope (TEM) (Hitachi H-7650, Hitachi High Technologies America, Inc., Pleasanton, CA). The in-vitro drug release studies were performed in dialysis cassettes (Slide-A-Lyzer, 3500 MWCO, 0.1–0.5 ml, Thermo-Scientific, Waltham, MA) as reported previously [16]. Briefly, the dialysis cassettes were first hydrated with PBS (pH 7.4) and 500 µl of liposomes were loaded with a syringe. Plain fasudil was used as a control to evaluate whether dialysis cassettes themselves have any influence on drug release. Cassettes were immersed in 100 ml PBS in a beaker and incubated at 37°C with moderate stirring. Samples were drawn at predetermined time intervals and the media was immediately replenished with fresh PBS. The amount of drug released was estimated spectrophotometrically as described above. The stability of fasudil-loaded liposomes was evaluated for 4 weeks. For this study, liposomes (500 µl) were stored at 4°C and 25°C and samples were withdrawn on day 0, 7, 14, 21 and 28 and analyzed for particle size and drug content as described above. Further, liposomal stability after aerosolization was evaluated with a PennCentury Microsprayer® (Model IA–1B, PennCentury, PA). Briefly, an aliquot of liposomal sample in the form of suspension was aerosolized five times using the microsprayer device and fine droplets were collected in an Eppendorf® tube which were evaluated for vesicle size, polydispersity index and entrapment efficiency as described above.
Uptake of liposomes by macrophages was evaluated by incubating the liposomes containing FITC-Dextran with rat alveolar macrophages collected from the lungs of anesthetized male Sprague–Dawley rats (200–250 g). Briefly, the lungs were surgically removed and bronchoalveolar lavage (BAL) was performed by repeated washing of the lungs with Ca2+ and Mg2+ free Dulbecco's PBS containing 0.5 mM disodium EDTA as described in our previously published article [17]. The resulting BAL fluid was centrifuged to obtain pellets of macrophages, which was then suspended in Hanks Balanced Salt Solution (HBSS). The cells at a density of 4×105 cells/ml were then seeded onto coverslips placed in 12-well plates and incubated in a humidified chamber at 37 °C for an h. Following incubation, an aliquot of liposomes containing FITC-Dextran suspended in HBSS was added to the cells and incubated again for an h at 37 °C. The cells were then fixed with acetone:methanol (1:1) at room temperature and incubated with a blocking solution containing goat serum and Tween 20 in PBS. After this, cells were incubated with monoclonal anti-β-actin primary antibodies (Sigma-Aldrich, St. Louis, MO) and Alexa Fluor® 594 goat anti-mouse IgG (Invitrogen, Grand Island, NY) sequentially. Glass coverslips were placed onto glass slides and uptake of the liposomes was observed under a fluorescence microscope (IX-81, Olympus). For uptake of liposomes by rat pulmonary arterial smooth muscle cells (PASMCs, P-7), cells were seeded at a density of 5×103 cells/ml and left for overnight attachment. Next day, liposomes containing FITC-Dextran were incubated with cells for an h at 37°C in a humidified chamber. Following incubation, cells were processed as described above and observed under fluorescence microscope.
The pulmonary absorption of plain fasudil and fasudil-loaded liposomes were studied in adult male Sprague-Dawley rats (Charles River Laboratories, Charlotte, NC, USA) weighing between 250–300 g. Four groups of rats (n = 6–8) were first anesthetized by an intramuscular (IM) injection of ketamine and xylazine cocktail (90 mg/kg + 10 mg/kg) to receive the following treatments: plain fasudil via (i) intravenous and (ii) intratracheal routes at a dose of 10 mg/kg and, two liposomal formulations equivalent to 10 mg/kg fasudil, (iii) F-3 and (iv) F-4 administered intratracheally. Intravenous administration was performed via the penile vein and intratracheal administration was performed using a PennCentury Microsprayer® for rats [18]. Liposomal formulations were administered as dispersions in normal sterile saline. Following pulmonary or intravenous administration, blood samples were collected in citrated microcentrifuge tubes at predetermined time intervals for 12 to 24 h. The plasma was separated by centrifugation at 5,000 rpm for 5 minutes and stored in separate microcentrifuge tubes at −20°C until further analysis. The plasma levels of fasudil were determined according to a published HPLC method [19] with little modifications using a Varian HPLC equipped with an autosampler (Varian Prostar 320, Walnut Creek, CA). The plasma sample was first deproteinized with perchloric acid and an aliquot of the supernatant (30 µl) was injected into a C18 column (Inertsil 4 µ ODS-3, 4.6 × 250 mm, GL Sciences, Inc., CA, USA). The mobile phase was an isocratic solvent system comprised of 30% acetonitrile and 70% phosphate buffer (0.02M, pH 7.4) and was run at a flow rate of 1 ml/min. Fasudil eluted at 5.3 min and was detected by an UV detector at 320 nm. The drug was quantified from a previously prepared standard curve using plain fasudil.
The safety of the optimized liposomal formulations (F-3 and F-4) was evaluated using an MTT assay in two different cell lines: human airway epithelial cells (Calu-3) and rat PASMCs [5]. Cells were seeded into 96-well plates at a density of 5 × 104 cells per well and incubated overnight for cells to adhere to the wells. The cells were then incubated with liposomal formulation (F-3), upon dispersion in fresh media, for 24 h (100 µM; 100 µl per well). Saline and 0.1% SDS were used as negative and positive controls, respectively. After the incubation, cells were washed with saline and then incubated for an additional 4 h with 100 µl MTT (0.5 mg/ml in sterile PBS). Subsequently, the medium was pipetted off and the formazan crystals were dissolved in 100 µl DMSO by shaking for 1 h on a plate shaker. The absorbance was read on a SynergyMX microplate reader (Biotek, Winnoski, VT) at 570 nm.
BAL studies were performed to evaluate the safety of the optimized formulations after in vivo instillation as per our previously published procedure [20]. Male SD rats were anesthetized with ketamine/xylazine cocktail and divided into three groups to receive following treatments intratracheally: (i) saline (negative control), (ii) fasudil liposomes (F-3), (iii) sodium dodecyl sulfate (SDS, 0.1 %, positive control). Twelve h after administration of the formulations, animals were weighed; lungs were surgically removed and weighed. The wet lung weights were reported as g/100 g body weight. The lungs were then lavaged by instilling 5-ml of normal saline into the trachea and collecting the fluid after 30 s. The BAL fluid was centrifuged at 500 g for 10 min and then the supernatant was stored at −20 °C. Protein levels in BAL fluid were assessed by Bradford assay. The enzymatic activities of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in BAL fluid were determined by using commercial kits for assay of LDH and ALP (Pointe Scientific, Canton, MI).
The pharmacological efficacy of the formulations was studied in a rodent model of PAH. The model was developed by a single subcutaneous injection of MCT (50 mg/kg body weight) into adult male Sprague-Dawley rats weighing between 250 and 300 g (Charles River Laboratories, Wilmington, MA). MCT solution was prepared by dissolving an aliquot of MCT in 0.1 N HCl and then the pH was adjusted to 7.4 with 1 N NaOH. All animals were housed at TTUHSC Amarillo animal facility for 28 days with free access to food and water for developing PAH.
Four weeks later when the hypertension was established, PAH rats were anesthetized by an intramuscular injection of a cocktail of ketamine (90 mg/kg) and xylazine (10 mg/kg) and were divided into three groups (n=8) to receive various treatments. Before administration of the formulations, rats were catheterized for hemodynamic measurements. For catheterization, the ventral neck area and the dorsal area between the scapulae were shaved and cleaned by scrubbing with Betadine® and ethyl alcohol. The internal jugular vein was exposed by a 2–3 cm incision over the right ventral neck area and kept separated using a suture. The right carotid artery was exposed and a PE-50 catheter (BD Intramedic™, Sparks, MD) was inserted 3–4 cm into the artery and secured with a suture for measurement of systemic arterial pressure. For measurement of pulmonary arterial pressure (PAP), a polyvinyl (PV-1, Tygon®, Lima, OH) catheter was used with the top end curved at 60–65° angle, threaded into the pulmonary artery via the right internal jugular vein and secured with a suture. Following catheterization, mean pulmonary arterial pressure (MPAP) and mean systemic arterial pressure (MSAP) were measured using Memscap SP844 physiological pressure transducers (Memscap AS, Scoppum, Norway) and bridge amplifiers connected to PowerLab 16/30 system with LabChart Pro 7.0 software (AD Instruments, Inc., Colorado Springs, CO). Correct positioning of the PV-1 catheter was confirmed from characteristic shapes of the pulmonary arterial pressure tracings captured by PowerLab system as reported previously [21]. Following measurement of hemodynamic parameters at zero time point, rats received one of the three treatments of fasudil (3 mg/kg): (i) plain intravenous fasudil, (ii) plain intratracheal fasudil, and (iii) liposomal formulation of fasudil (Formulation F-3) administered intratracheally. Intravenous administration was performed via the penile vein and intratracheal administration was performed using the PennCentury Microsprayer® as described above. The anesthesia was maintained throughout the procedure, and blood pressures were monitored until the pulmonary arterial pressures returned to the baseline value observed at time zero. After completion of hemodynamic measurements, the animals were sacrificed by exsanguination. All animal studies were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals under a protocol approved by Texas Tech University Health Sciences Center (TTUHSC) Animal Care and Use Committee (AM-10012).
All in vitro and in vivo data are presented as mean ± SD and were analyzed by one-way ANOVA followed by Bonferroni post-hoc analysis using Graphpad Prism 5.0 software. Significance was defined as p value < 0.05. For pharmacokinetic analysis, standard non-compartmental analysis (Kinetica®, Version 5.0, Innaphase Corp., Philadelphia, PA) was performed to calculate the area of the plasma concentration-time curve from zero to 24 h. (AUC0→24), maximum plasma concentration (Cmax), and elimination half-life (t1/2).
Since fasudil is a weakly basic hydrophilic molecule (pKa = 9.727), it exhibits very low entrapment efficiency in lipid-based formulations. For this reason, we first optimized the entrapment efficiency of fasudil in liposomes using passive and active loading methods and by varying drug-to-lipid ratio. Liposomes prepared by the passive method at a drug-to-lipid molar ratio of 1.5:1 showed an entrapment efficiency of 27.4±1.2% (Fig. 1A). To enhance the entrapment, we switched to active loading using (NH4)2SO4 based transmembrane gradient method by varying pH of the rehydrating medium. Using this method, we prepared four sets of formulations at different pH values. Compared to passive loading, drug entrapment increased by 2.5 folds with (NH4)2SO4 based transmembrane gradient method at the same drug-to-lipid molar ratio at pH 5.4 (native) and changing the pH of the medium resulted in further increase in entrapment efficiency. To some extent, the data suggests that the drug entrapment by active loading method was influenced by the pH of the rehydrating medium and the highest increase in entrapment was observed at pH 3.0 and 7.0. However, when the pH was increased to 8.0, no further increase in entrapment was observed. We further tested the influence of drug-to-lipid molar ratio on active loading of fasudil (Fig. 1B). The entrapment efficiency of fasudil was 20–25% when drug-to-lipid ratios were 0.5:1 and 1:1. When the ratio was increased to 1.5:1, a two-fold increase in loading was observed (Fig. 1B), but no appreciable increase in entrapment efficiency was observed with the increase in molar ratio from 1.5:1 to 2:1. Based on these data, we considered that active loading at drug-to-lipid ratio of 1.5:1 was optimum with substantial drug entrapment.
These data agree with previous studies wherein (NH4)2SO4 based remote loading was used to increase entrapment efficiency of hydrophilic drugs. In fact, low encapsulation of hydrophilic drugs has been a limitation when small unilamellar vesicles (SUV) are to be used as drug delivery carriers [22]. Similar to fasudil, weak bases including doxorubicin and hydromorphone have been successfully encapsulated in preformed liposomes utilizing transmembrane gradient [23–26]. For example, Caelyx®, a PEGylated liposomal formulation of doxorubicin, was prepared using ammonium sulfate gradient for generating indirect pH-gradient. In this method, the presence of ammonium sulfate in the core of liposomes produces an excess availability of protons inside the vesicle because of removal of ammonium sulfate from the extraliposomal compartment resulting in a proton gradient across the liposomal membrane [27]. Unprotonated weak bases outside the vesicles can easily permeate through liposomal membranes. Once inside the vesicles, they are protonated in H+ rich environment and thereby trapped in the aqueous core of the vesicles [27]. Enhanced entrapment may also result from incubation of the drug with preformed liposomes at 65°C, which is consistent with previous assumption that unprotonated amphipathic bases are trapped within the liposomes when incubated at temperatures higher than phase transition temperatures of lipids for 30 minutes [28]. In fact, a 30-minute incubation time is reported to be optimal for maximal drug entrapment. Incubation beyond 30 minutes apparently reduces entrapment efficiency because of formation of neutral complexes between protonated fasudil and sulfate (SO4−2) ions, which tend to effuse though liposomal membrane [19]. No additional entrapment was observed with the increase in pH from 3.0 to 7.0 or 8.0 perhaps because of reduction of proton gradient that may result from reduced protonation of (NH4)2SO4 at pH as high as 7.0 or 8.0. The entrapment efficiency data suggest that ammonium sulfate based active loading is one of the most efficient methods of encapsulation of fasudil in liposomes.
However, this data raise questions regarding the influence of pH gradient on the entrapment efficiency. In fact, similar anomaly in entrapment efficiency was observed when Ishida et al. [19] varied pH and concentration of ammonium sulfate to study the influence of proton and ammonium sulfate gradient on the entrapment of fasudil into liposomes. Similar to the data presented in this manuscript, no appreciable increase in drug entrapment was observed when pH was varied. But the entrapment efficiency was increased when concentration of ammonium sulfate was increased. The authors theorized that pH gradient has perhaps a limited role in controlling entrapment of fasudil, a weakly basic drug (pKa of 9.727) [19]. Based on this published study and our data, we assume that ammonium sulfate gradient is perhaps the major player in enhancing drug loading.
Upon efficient encapsulation of liposomes, particle size, polydispersity index, and morphology of various liposomal preparations were evaluated. The gradient produced by the presence of (NH4)2SO4 in the vesicle core significantly influenced the size of liposomes. Compared to liposomes prepared without pH gradient, an overall reduction was observed in the size of liposomes prepared with (NH4)2SO4 in combination with pH gradient (Fig. 1C). In the absence of transmembrane gradient, the size of liposomes was 288.7±9.9 nm, which was decreased to 140.6±0.7 nm for the formulation with active loading at the pH of 5.4 (Fig. 1C). Such reduction in particle size can be explained by the changes in pH around the lipids during liposome preparation as observed by others [29, 30]. It has also been suggested that reduction in particle size results from an effect of osmotic stress produced by concentration and pH gradients. However, the size of liposomes prepared by (NH4)2SO4 gradient was in the range of 100 and 250 nm, which puts them under the category of SUVs as reported by others [31]. The SUV like size distribution of the liposomes suggest that hydration followed by sonication can be used for preparation of SUVs. Due to their small size, these liposomal formulations are expected to be optimal for in-vivo efficacy and avoid clearance by alveolar macrophages. Further, all liposomal formulations prepared by (NH4)2SO4 and pH gradient method had a polydispersity index (PDI) in the range of 0.15 and 0.2 (Table 1), suggesting that the formulations had a homogenous size distribution. Transmission electron microscopic (TEM) image presented in Fig. 1D exhibit round or oval liposomes with a smooth surface. The formulations showed little or no aggregations indicating that the vesicles were uniformly dispersed in extraliposomal fluid. TEM has previously been used for examining the lamellar morphology, bilayer organization, and identification of possible flaws in bilayers [32]. However, the TEM photographs showed no morphological or structural changes due to incorporation of ammonium sulfate in the liposome core. Rather, we observed a uniform bed of discrete spherical vesicles that agree with vesicle size data obtained by the dynamic light scattering technique.
The in-vitro drug release studies were performed to test the hypothesis that encapsulation of fasudil in lipidic SUVs produce sustained drug release in physiological fluid. The in-vitro release profile of the liposomal formulations obtained in PBS buffer at 37°C showed a continuous release of the drug for about 40 h (Fig. 2A). But 100% of plain fasudil was released in the media (data not shown) suggesting that dialysis cassettes were not the rate controlling membrane for drug release. The time required to release 50% of the drug was in the range of 8 to 12 h for F-1, F-2, and F-5 formulations and that for F-3, and F-4 was ~24 h. During the first 24 h, F-3 and F-4 produced 51.2±0.5% and 46.2±0.7% drug release, respectively (Fig. 2A). Since the amount of drug release was relatively small, one may question about the stability of the drug in release media. In fact, a published report suggests that fasudil is stable over an extended period of time (~24 weeks) at various storage conditions [19]. Further, we have used an HPLC method to quantitate fasudil in vivo which is sensitive to only intact fasudil rather than degradation products of fasudil. Thus it is unlikely that drug underwent degradation during the study period. Further, F-5 exhibited no burst release as evidenced from the negligible amount of drug released during first 30 minutes, but F-3 and F-4 formulations showed 9.1±1.4% and 11.8±2.7% drug release during the burst release phase (Fig. 2B). A slower and consistent release of fasudil from liposomal formulations suggests that drug release was due to diffusion of the drug though lipid membranes rather than disruption of the SUVs. These data agree with previous studies with liposomal fasudil [19], where carboxyfluorescien, an aqueous phase marker, was encapsulated in liposomes, and only 15% of the dye was released during a 14-day experiment [19]. In fact, the similarity among release profiles from various formulations stem from the fact that there were no differences in the type and amount of lipid used to prepare the formulations. The diffusion of the drug from the vesicles can be explained by interaction of protonated fasudil molecule with the sulfate ions in vesicle core. As discussed above, upon entering the hydrophilic core, unprotonated fasudil becomes protonated in H+ rich environment in the core and forms electrochemically neutral complexes with anionic sulfates (SO4−−) [33]. The resulting uncharged complex then diffuses out from the core via the lipid bilayer. Further, as the phase transition temperature for DPPC is 41°C, which is close to the physiological temperature, 37°C, it is reasonable to assume that the principal mechanism for drug release from liposomal formulations is diffusion of the electrochemically neutral fasudil complex through liquefied lipid membranes. This assumption is consistent with a previous study that evaluated temperature dependent phase transition of DPPC liposomal systems and showed that DPPC vesicles of >100 nm undergo phase transition at around 37–39°C [34]. Further, encapsulation of small molecules such as fasudil is unlikely to alter phase transition properties of lipid vesicles at high drug to lipid ratios. Overall, the in-vitro release data suggest that F-3 and F-4 formulations would be efficacious in delivering fasudil in the distal pulmonary arterioles for a prolonged period of time and would likely provide sustained vasodilation.
Reduction in drug entrapment due to drug leakage and aggregation of vesicles are major concerns with the stability of liposomal formulations. To examine the stability of liposomal formulations, we assessed the changes in particle size and drug entrapment of F-3 and F-4 formulations which showed maximal loading. The formulations were stored at 4°C and 25°C, and the particle size and drug entrapments were monitored for 4 weeks. No changes in vesicle size was observed in formulations stored at 4°C and 25°C, suggesting that the vesicles remained well separated from each other and formed no aggregates during the storage period (Fig. 3A). Therefore, no change in particle size can be attributed to the stability provided by the optimum zeta potential which prevents the particles from coalescing and aggregating. When stored at 4°C, no change in drug entrapment was observed for 21 days, but a 10–15% reduction in drug entrapment was observed upon storage for 28 days (Fig. 3B). However, storage at room temperature (25°C) resulted in about 30–35% drug loss over 28 days, which is 2–3 times higher than drug loss that occurred upon storage at 4°C. Moreover the lipids, cholesterol and phospholipid, used to prepare liposomes are unlikely to undergo degradation during storage at 4°C [27] and it has also been shown that liposomal formulations can release drugs from 2 weeks to as long as several months [35], indicating that the integrity of liposomes is unlikely to be adversely affected in the physiological fluid.
In addition to studying the stability at various storage temperatures, we also studied stability upon aerosolization. In fact, aerosolization breaks larger droplets into smaller ones and delivers the formulations in the form of fine mists. But the force applied for nebulization may rupture the liposomes and release the drug prematurely. This may also result in leakage of drugs from some liposomes. To test this, we nebulized the formulation 5 times to mimic intratracheal administration into rats and evaluated various physicochemical properties of liposomes. The data presented in Table 2 show that there were no significant differences in vesicle size, polydispersity index and entrapment efficiency before and after nebulization of the liposomes, suggesting that liposomal integrity was not affected due to the force applied to administer the formulations using PennCentury Microsprayers®.
Alveolar macrophages, present on lung epithelial surfaces and responsible for engulfing and clearing of inhaled particles, may influence the clearance of liposomal formulations. Further, previously published reports suggest that particles less than 250 nm can escape clearance by macrophages [36]. Microscopic images suggest little or no uptake of liposomes by macrophages (Fig. 4A), indicating that liposomes were not cleared via macrophageal uptake mechanism. Particles are intended to produce their therapeutic response in smooth muscle cells by inhibiting Rho-kinase and hence reduce mean pulmonary arterial pressure. To demonstrate this, we performed another set of experiment using rat PASMCs and observed that there was significant uptake of liposomes by these cells (Fig. 4B). Superimposed images of FITC (green) and TRITC (red) channels show the localization, as visualized by yellow fluorescence, indicating that liposomal formulations can act on vascular smooth muscles and produce therapeutic effect of the drug. Further transport studies using cells of airway epithelium are required to track the movement of fasudil or liposomes across the pulmonary vasculature.
In pulmonary absorption studies, the pharmacokinetics of two formulations with maximal drug loading (F-3 and F-4) were compared with that of plain fasudil administered via intravenous or pulmonary routes (Fig. 5). Intravenous administration of fasudil resulted in a quick rise in drug plasma concentrations with a Cmax of 298±10 ng/ml followed by a rapid decline (Fig. 5A), whereas pulmonary administration of plain fasudil resulted in an appreciable increase in plasma concentrations (Cmax = 66.2±13.6 ng/ml, Fig. 5A). Unlike intravenous route, the elimination of fasudil administered by pulmonary route was slow (Fig. 5A and Table 3), which is evident from the differences in elimination half-lives of fasudil administered by the two routes. The elimination half-life (t1/2) of fasudil administered by pulmonary route (1.17±0.21 h.) was significantly higher than that of fasudil given intravenously (t1/2 = 0.39±0.12 h.) (p<0.05) (Fig. 5A, Table 3). The relative bioavailability of fasudil after pulmonary administration was ≈40% (Table 3). However, the pharmacokinetic profiles observed after intratracheal administration of two liposomal formulations (F-3 and F-4) were completely different from those observed with plain intratracheal and intravenous fasudil (Fig. 5B). F-3 formulation showed a Cmax of 89.4±29.7 ng/ml at 8 h. followed by a sharp decline in plasma fasudil levels with no detectable concentrations at 18 h. Similarly, F-4 formulation produced a Cmax of 86.9±11.9 ng/ml at 2 h. Pharmacokinetic analysis of the in-vivo data revealed that fasudil liposomes resulted in a remarkable extension of elimination t1/2 (4.71±0.72 h. for F-3 and 3.44±0.49 h. for F-4). In fact, t1/2 of both formulations was ~3–4 and 10 times, respectively longer than plain fasudil after pulmonary and intravenous administration. The IC50 of fasudil against Rho-kinase is reported to be between 1.9–3.7 µM [37, 38]. Based on the published data we assume that the plasma concentrations of fasudil were above therapeutic levels for at least 15 h with liposomal formulations, which can be translated into a once-a-day or twice-a-day dosing regimen. Similar to in vitro data, in-vivo absorption profiles also show a trend of extended release although the two profiles were not similar. Differences between in vitro release and in vivo absorption profiles can be attributed to a number of factors including mechanisms of absorption, distribution and elimination of liposomes administered via the pulmonary, physicochemical characteristics of the aerosolized liposomes, macrophage dependent clearance, lipid degrading enzymes, and transport of intact liposomes from lungs to the systemic circulation, metabolic degradation and protein binding. Further, sustained release behavior of liposomes may also result from multiple factors. First, upon inhalation, liposomes may act as reservoirs that remain submerged in the respiratory fluid and release the drug continuously. In fact, particulate carriers with particle size below 1 µm can avoid phagocytosis by alveolar macrophages [39, 40] and thereby perhaps form aggregates in the respiratory mucosa and act like reservoirs or depot. Based on this assumption, the drug is expected to be released from the liposomes by diffusion though lipid bilayer. Release of the drug due to rupture of liposomal bilayer is unlikely since lipids used are resistant to degradation by enzymes present in respiratory mucosa [41, 42]. However, dissolution of liposomes in the surfactant rich respiratory fluid cannot be ruled out. A fraction of the drug is likely to be released due to dissolution of liposomes in the respiratory mucosa. In either case, drug released from liposomes will traverse the blood airway barrier and enter arterioles via the adventitial side to produce its vasodilatory effects. A second mechanism for sustained release of the drug may involve transport of intact liposomes or released drug to the systemic circulation via the air-blood barrier. Upon entering the systemic circulation fasudil may diffuse out of the liposomes to the systemic circulation. Fasudil in the circulating blood enters the smooth muscles of the pulmonary arterioles from the endothelial side to produce its therapeutic effect. Further studies using ex-vivo models such as isolated perfused lungs are required to assess the relative contribution of above described absorptive mechanisms upon intratracheal administration of liposomes. However, one of the important limitations of this in vivo study is that it was performed in healthy animals. Since PAH animals undergo several pathological changes, pharmacokinetics of the drug in PAH rats could be slightly different from those observed in healthy animals. Future studies should delineate the differences between the pharmacokinetics of the drug in healthy and diseased animals.
In-vitro cytotoxicity studies were performed to determine the effects of liposomal formulations on two different cell lines: Calu-3 and rat PASMCs. Cells were treated with saline (negative control) and 0.1% w/v sodium dodecyl sulfate (SDS) (positive control) showed cell viability of 99% and 15%, respectively (Fig. 6A). When Calu-3 cells were treated with 100 µM F-3 formulations for 24 h, the cell viability was 86.19±13.66% (Fig. 6A). One primary cell line (rat PASMCs) treated with 100 µM of liposomes for 24 h showed similar cell viability. In none of the cases was cell viability below 70%, suggesting that formulations were toxic neither to immortalized Calu-3 cells nor to primary PASMCs. These observations were expected since lipids used to prepare liposomes have long been known to produce little or no cytotoxicity.
To further evaluate the safety, BAL was performed after taking weights of wet lungs as discussed above in the method section. The weight of wet lungs, expressed in lung weight per 100 g of body weight (L/B), of saline treated animals was 0.397±0.0047 and that for SDS treated animals was 0.614±0.0368 (Fig. 6B), suggesting formation of edema due to accumulation of extracellular fluid into the epithelial cells of the respiratory wall in SDS treated lungs. For liposomal formulation containing fasudil (F-3), L/B ratio was 0.42±0.0116, indicating no substantial lung injury or edema formation. The levels of two injury markers, LDH and ALP, as well as total protein concentration in the BAL collected from animals treated with formulation F-3 were not significantly different from that observed in the lungs of animals that received saline (Fig. 6C and 6D) which were significantly higher in SDS treated animals. Overall, data presented in Fig. 6 establish the safety profile of liposome based formulations after single administration, but long-term safety profiles of fasudil formulations after multiple administrations should be evaluated.
The pharmacological efficacy of the formulations in reducing mean pulmonary arterial pressure (MPAP) was studied in an MCT induced rat model of PAH. The signs induced by a single subcutaneous injection of MCT (50–60 mg/kg) resemble those of clinical PAH that include increased MPAP, right ventricular hypertrophy, pulmonary vascular remodeling, and reduced luminal diameter of small pulmonary arterioles [43, 44]. In this study, the average MPAP was 38.0±9.4 mm Hg 4 weeks after MCT injection as compared to 15.6±4.4 mm Hg in sham animals. In agreement with earlier reports [14, 45, 46], intravenous administration of plain fasudil (3 mg/kg) produced a 45.6±17.0% decrease in MPAP, but its vasodilatory effect quickly subsided within 60 to 80 minutes (Fig. 7A). Intratracheal administration of plain fasudil also caused a 38.3±7.3% decrease in MPAP, although the duration of the vasodilatory effect was slightly longer than that produced by intravenous fasudil (Fig. 7A and 7C). However, when liposomal fasudil (Formulation F-3) was administered via the pulmonary route, the duration of vasodilatory effect was extended up to 200 minutes (Fig. 7A). The maximal reduction in MPAP produced by liposomal fasudil was 37.6±5.7% which was similar to that produced by plain fasudil administered via either route. However, unlike plain fasudil administered intravenously or intratracheally, liposomal fasudil continued to produce pulmonary vasodilation even 3.5 h post instillation, i.e., a 20.0±8.7% reduction in MPAP at 200 minutes (Fig. 7A). The data concerning the pharmacological efficacy presented in Fig. 7 reflects the absorption profile of the formulation presented in Fig. 5, wherein the t1/2 of liposomal fasudil was significantly longer than that of plain fasudil. To calculate the duration of pulmonary vasodilatory effects, we used 15% reduction in MPAP as baseline and plotted the values to compare the duration of three treatments. This baseline was chosen based on previous studies that show a 15 to 20% reduction in MPAP is therapeutically relevant and significant [47]. The liposomal fasudil maintained pulmonary vasodilatory effects for 3 h. (172±17 minutes) while intravenous and pulmonary plain fasudil caused vasodilation for 50.0±10.4 and 60.0±15.3 minutes, respectively (Fig. 7C). These data further confirm that liposomal fasudil produced a prolonged vasodilation that would offer therapeutic advantages over the plain drug. However, concerns may be raised regarding the candidacy of fasudil as a drug for development into controlled formulations. Since the effective dose in animal was 3 mg/kg, one might argue that a much larger dose (200–300 mg) would be required in humans and thus the proposed formulation would be not be viable for use in human patients. But calculation of human dose based on the allometric [48] and body surface area principles [49] suggest that 3 mg/kg dose in rats is equivalent to ~35–40 mg in 70-kg human subjects. In fact, fasudil used at a dose of 30 or 40 mg was found to effective in reducing MPAP in PAH patients [45, 50]. Thus this study provides convincing data in support of an inhaled controlled release formulation of fasudil for the treatment of PAH.
To evaluate the pulmonary selectivity of the formulation, we also measured the mean systemic arterial pressure (MSAP) and calculated the reduction in MSAP produced by the formulations (Fig. 7B). Intravenous and intratracheal fasudil produced a 45.6±8.4% and 38.1±10.8% reduction in MSAP, respectively (Fig. 7B). The extent of reduction of MSAP produced by plain fasudil was close to that observed in MPAP (Fig. 7A), suggesting that fasudil when given intravenously does not exhibit much selectivity toward the pulmonary circulation and thus produces peripheral vasodilation. However, intratracheal liposomal fasudil produced a 26.4±7.4% reduction in MSAP, which was significantly less (p<0.05) than that produced by intravenous and intratracheal plain fasudil. A point-by-point comparison of hemodynamic data presented in Fig. 7B also suggests that the reduction in systemic arterial pressure produced by aerosolized liposomal fasudil was consistently lower than that produced by plain fasudil administered either by intravenous or pulmonary route. In addition to pulmonary selectivity, fasudil encapsulated in liposomes is likely to reduce the dosing frequency and minimize the fluctuations in MSAP due to multiple injections or continuous infusion that leads to serious side effects including syncope and cardiovascular collapse [51]. Overall, the hemodynamic study suggests that intratracheal instillation of fasudil encapsulated in liposomes was therapeutically active and the drug was released from liposomes over time. There was a continuous influx of fasudil into the pulmonary arteries that provided sustained vasodilation with reduced effect on systemic arterial pressure than that of plain fasudil. These data are in agreement with in-vivo absorption profile wherein a continuous absorption of fasudil was observed. However, the in vivo efficacy study was limited to evaluation of only one of the pharmacological parameters, mean pulmonary arterial pressures. Evaluation of other parameters such as influence of the formulations in reducing vascular resistance and ameliorating other pathological changes in PAH would have provided more useful information regarding the therapeutic benefit of liposomal fasudil. Future studies will be directed toward assessing various cellular and vascular markers upon administration of the formulations.
In summary, this is the first study to investigate the feasibility of an aerosolized controlled release formulation of fasudil that produces a sustained pulmonary preferential vasodilation and ameliorates the severity of MCT-induced PAH. Liposomal formulations can be used as vehicles for pulmonary delivery of fasudil as demonstrated by favorable physicochemical properties. The drug exhibited an extended t1/2 following in-vivo administration in rats. Compared to intravenous fasudil, a single dose of the optimized liposomal formulations produced sustained pulmonary vasodilation for ~3 h and produced minimal impact on MSAP. Nanosized liposomes were safe after intratracheal delivery, escaped clearance by alveolar macrophages and were internalized by PASMCs. Thus, the data presented suggest that aerosolized liposomes of fasudil can be used for sustained and pulmonary preferential vasodilation at a reduced dosing frequency and can potentially be used as a viable anti-PAH therapy. However, further studies are required to establish the long-term efficacy of fasudil liposomes in providing protection against PAH related lesions.
The authors sincerely acknowledge Mr. Charles Linch at the Department of Medical Photography and Electron Microscopy of Texas Tech University Health Sciences Center, Lubbock, TX, for his help with the transmission electron-microscopy (TEM) experiments. This work was supported in part by an American Recovery and Reinvestment Act Fund, NIH 1R15HL103431 to Dr. Fakhrul Ahsan. Primary PASM cell lines were developed with a financial support from Cardiovascular and Pulmonary Research, University of Colorado, Denver and NIH-PPG # 2P01HL014985-36A1 (Adaptations to Hypoxia) to Drs. Eva Nozik-Grayck and Kurt Stenmark.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Company Overview of Germantown Trust & Savings Bank
Company Overview
Germantown Trust & Savings Bank offers personal and commercial banking services. The bank was founded in 1906 and is based in Breese, Illinois. Germantown Trust & Savings Bank operates as a subsidiary of Germantown Bancorp.
205 Germantown Road
Breese, IL 62230
United States
Founded in 1906
Key Executives for Germantown Trust & Savings Bank
Germantown Trust & Savings Bank does not have any Key Executives recorded.
Similar Private Companies By Industry
|Company Name
|DNB National Bank (Clear Lake, SD)
|United States
|Middleton Community Bank
|United States
|Farmers Bank & Trust
|United States
|Rocky Mountain Bank & Trust (Florence, CO)
|United States
|Union Bank & Trust Company
|United States
Recent Private Companies Transactions
|No transactions available in the past 12 months.
Most Searched Private Companies
|Company Name
|Geographic Region
|Lawyers Committee for Civil Rights Under Law
|United States
|NYC2012, Inc.
|United States
|Bertelsmann AG
|Rush University
|United States
|Greater Houston Partnership
|United States
Sponsored Financial Commentaries
Sponsored Links
To contact Germantown Trust & Savings Bank, please visit www.gtsb.com. Company data is provided by Capital IQ. Please use this form to report any data issues.
Our data partners will research the update request and update the information on this page if necessary. Research and follow-up could take several weeks. If you have questions, you can contact them at email@example.com.

subset: commoncrawl


text:
Leavitt Recreation & Hospitality Insurance Welcomes Patrick Powers
Leavitt Recreation & Hospitality Insurance (LRHI) is pleased to announce Patrick Powers has joined the agency as a claims coordinator.Read More →
Flanigan-Leavitt Insurance Agency Welcomes Nielsen and Shewmaker
Flanigan-Leavitt Insurance Agency is pleased to announce that Kyle Nielsen and Kelley Shewmaker have joined their agency in Reno.Read More →
Donovan Bell Joins Leavitt Group in Montana
Leavitt Group is pleased to announce that Donovan Bell has joined their Bozeman office focusing on insurance for the hospitality and tourism industry, such as dude ranches, resorts, restaurants, bars, and breweries.Read More →
Mike Vowles to Lead Independent Agent Association in Utah
Leavitt Group of Spanish Fork's managing principal, Mike Vowles, was recently sworn in as the new president of the Utah Association of Independent Insurance Agents (UAIIA) during their annual convention.Read More →
2015 Annual LGAA Conference Award Winners
The 37th Annual LGAA Managers Conference was held this year in Scottsdale, Arizona.Read More →
McDonald-Leavitt Insurance Agency Welcomes Jean Stevens
McDonald-Leavitt Insurance Agency is pleased to announce that Jean Stevens has joined their agency as an agent licensed in commercial insurance and employee benefits.Read More →
Leavitt Insurance Agency Welcomes New Benefits Agent
Leavitt Insurance Agency is pleased to announce that Preston Guthrie has joined their St. George office focusing on employee benefits and life insurance. Preston has a strong background in sales and life insurance.Read More →
Jason Ferree Joins Leavitt Group in Montana
Leavitt Group is pleased to announce that Jason Ferree has joined their Missoula agency. He earned his consulting license and specializes in designing custom benefit programs for employers.Read More →
Leavitt Group of Boise Welcomes Briana Giordano
Leavitt Group of Boise is pleased to announce Briana Giordano has joined their agency as a new operations specialist.Read More →
Archibald Insurance Center Helps Four Paws Pet Adoptions Win Nationwide Contest
Archibald Insurance Center, a Leavitt Group affiliate, recently helped Four Paws Pet Adoptions win second place in the Safeco Insurance "Make More Happen" spring contest.Read More →


text:
Alex Toys
My Workbench
- Color:
- multi
Qty: 1
- 1
Please select an available size.
Please select a size.
Description: Big stand-up workbench and tools for little builders!
- 37-piece set includes tools, robot, car, nuts and bolts.
- 17¼"W x 11"D x 26"H
- Suitable for age: 3 years and up
Material: Wood
Brand: Alex Toys
Origin: Imported


text:
The Plaza Hotel4
Belgard Road, Tallaght, Dublin 24, County Dublin
Price FromView Rooms
- Very Good
- 10 guest reviews
"Tired dated rooms, but for budget you expect, 3rd floor kept for proper fee paying guests not budget, noisy as due to nightclub and second s..."
"Nice and decent hotel. Incredible value for money. Room looked very nice, clean, high standard...."
"Friendly staff great location very clean hotel throughout would definately use this hotel again...."
"Overall this hotel was absolutely fine. The location isn't that great so it would be good if it made up for it with a new decor and just mod..."
"I have stayed here a few times the decor is a bit out dated but for the price was amazing. Rooms are spacious and have all that you would ex..."
About the hotel
The Plaza Hotel is a four star hotel ideally located on the Belgard Road, just off the M50 motorway. The property is situated 5 miles from the city centre and a 25 minute drive from Dublin Airport. Looking out of the Vista Cafe on the first floor you have a picturesque view of the Dublin Mountains.
Nestling at the foothills of the Dublin mountains, you will find the ingredients of good food, service and our commitment to make a recipe for success at The Plaza Hotel. Please note latest check-in time is 11pm. If arriving after this time please contact the hotel in advance to request late check-in.
Read MoreLess
Room Availability
Showing Saturday 10 October 2015 to Sunday 11 October 2015Change Search
Facilities Show/hide
- Main
- Hotel Non-Smoking Throughout , Disabled Facilities
- Hotel services
- 24 Hour Reception , Limited Hours Room Service , Wake Up Call Available , Currency Exchange
- Family friendly
- Cots available
- Other
- Wedding Licence , Doctor on call , Lift to all floors
- Parking
- Car parking available onsite
- Business
- Wi-Fi Available , Meeting Facilities , Wi-Fi Available In Public Areas , Wi-Fi Available In Rooms , Private dining services
- Dining
- Restaurant
- In room entertainment
- TV , Satellite TV
- In room facilities
- Tea/Coffee making facilities in each room , Hairdryer in each room , Iron And Board In Each Room , Telephone In Each Room
- Accessibility
- Disabled Access Public Areas , Disabled Access Bedrooms , Wheelchair Access , Wheelchair Access To Reception , Wheelchair Access To Restaurant , Wheelchair Access To Bar , Wheelchair Access To Conference Rooms , Wheelchair Access To Disabled Car Parking Spaces
Useful Info Show/hide
- From 14:00 hours
- Until 23:00 hours
- 08/11/2012 to 25/11/2016 - Latest check-in time is 11pm. If arriving after this time please contact the hotel in advance to request late check-in. Please be aware of limitations on food & beverages between 24th - 26th December. The price shown is based on the occupancy and dates you have searched for. Prices and availability are subject to change based on the arrival date, number of nights and occupancy searched for. All prices are inclusive of all local taxes.
- Pets are not allowed
Customer Reviews
Our reviews are from genuine LateRooms.com customers. We verify that the customer has stayed before leaving a review.
Very Good
70% would recommend it to a friend
Customer service
Food quality
Room quality
Fantastic from outside, dated and tired in room and so noisy
Tired dated rooms, but for budget you expect, 3rd floor kept for proper fee paying guests not budget, noisy as due to nightclub and second storey room and not a safety window appalling.
Nice and decent hotel.
Incredible value for money. Room looked very nice, clean, high standard.
Read More Less


text:
Fall is happening and it is time for our SUMMER CLEARANCE SPECIAL. Any item that spent the summer with us is ON SALE now. We have numerous styles of aluminum ATV trailers by Aluma at low prices. Do you enjoy rafting or fishing in our beautiful rivers? Why not consider a new trailer? We have galvanized Raft Trailers from SporTrail with wood or steel decks – prices are the lowest that they have been all year! Additionally, we are selling our dump and car hauler trailers from Midsota. These are high quality, nice looking trailers that will assist you in getting your jobs done.
The Kioti Mechron is a 22HP diesel utility vehicle with high clearance and superior tow / haul capacity. This machine gets your work done. We are taking $1000 off the list price PLUS throwing in a free sun canopy and glass windshield with the purchase. Considering a tractor? Hitchin' Post is offering lots of package deals on the Kioti compact tractors. There is a DK4510 with implements for $4000 off the list price. The CS2410 sub-compact tractor is also a favorite for home owners. We are interested in moving this inventory so come drive a tractor. It will be love at first drive……
Hitchin' Post Rentals continues to be the best spot to rent trailers. We have horse, stock, utility or an 18' flatbed trailer for rent. These trailers can be rented for hours to weeks thus assisting you with activities that require a trailer. The 27HP tractor with loader and implements continues to be a hot rental item. We are currently building up an inventory of rental tools so give us a call and see if we have an item that you could use.
Hitchin' Post has been repairing trailers and tractors in the Woodland Park, Divide, Buena Vista, Salida, and Fairplay area for over 15 years. Our mechanics and staff are highly qualified and committed to superior customer service. We value the community that we live in and appreciate your patronage. Stop in, send an email or call – we look forward to assisting you!
Sincerely - Deb Reuter (owner)
Product Manufacturers:
Kioti SPECIALS for October
Tractors: We have a large selection of compact tractors. Kioti is offering discounts of $1700 to $4500 depending on the horse power and size. We use that cash discount to offer you the lowest price possible for the tractor. We also have an excellent financing resource - 5.6% for up to 84 months - no down. This month we are adding trailers and implements into the LOW tractor prices.


text:
Top Definition
A state of extreme hunger.
Man, I'm really hungo bungo right now.
Anyone down for some hungo bungo's?
Anyone down for some hungo bungo's?
by the creator of the word August 28, 2010
Type your email address below to get our free Urban Word of the Day every morning!
Emails are sent from firstname.lastname@example.com. We'll never spam you.×


text:
Habitat for Humanity
At our core, we are a family helping families.
We strive to eliminate poverty housing and homelessness by constructing safe, decent, and affordable homes. We provide community service to our neighbors in the local community and beyond. Contact us at email@example.com.
- WAC_Habitat: RT @TaylorFrey1782: Love starting out week with convos on affordable housing in communities of high need. Excited to discuss further with NeighborWorks friends.
- WAC_Habitat: RT @TaylorFrey1782: Working with @washcoll @Habitat_org team to determine where to build houses and hope in the spring. #CollegiateChallenge @Habitat_at_WAC
- WAC_Habitat: RT @TaylorFrey1782: Great to catch up with @CumberlandHFH Exec. Director today. So excited to bring our @washcoll @Habitat_org @Habitat_at_WAC team to NJ soon.
- WAC_Habitat: RT @TaylorFrey1782: Missing the @Habitat_org worksite, and my @washcoll @Habitat_at_WAC family! #BuildingHousesBuildingHope http://t.co/Y8UNNMzQZA


text:
1863-S $10 XF40 NGC....Click the image to load the highest resolution version.
Luminous XF40 1863-S Eagle1863-S $10 XF40 NGC. The 1863-S was the highest-mintage eagle issue for the year, though with its only competition being the 1,218 business strikes produced at Philadelphia, even this issue with its minimal five-figure mintage scores an easy win. The present coin is luminous yellow-orange with small, scattered marks and a slightly soft strike. Census: 4 in 40, 27 finer (12/08).(Registry values: N2998) (NGC ID# 264C, PCGS# 8638)
Service and Handling Description: Coins & Currency (view shipping information)
Sales Tax information | NGC Coin Grading Guarantee | Terms and Conditions
Bidding Guidelines and Bid Increments
Glossary of Terms
Auction Info
Buyer's Premium per Lot:
15% of the successful bid (minimum $9) per lot.
Floor auctionsOpen for bidding: (View All)
Decorative Art
Ends on 10/10/2015
Illustration Art
Ends on 10/14/2015
Nature & Science
Ends on 10/15/2015
Musical Instruments
Ends on 10/24/2015
Arms & Armor
Ends on 10/25/2015
Old West
Ends on 10/25/2015
US Coins
Ends on 10/28/2015
Fine Art
Ends on 10/28/2015
US Coins
Ends on 10/29/2015
Jewelry & Timepieces
Ends on 10/29/2015
Rare Books
Ends on 11/04/2015
Sports Collectibles
Ends on 11/05/2015
Western & Texas Art
Ends on 11/07/2015
Opens about 10/16/2015
American Indian Art
Opens about 10/12/2015
Opens about 10/19/2015
Opens about 10/19/2015
Opens about 10/15/2015
Opens about 10/12/2015
Fine Art
Opens about 10/12/2015
Fine Art
Opens about 10/14/2015
Comics & Comic Art
Opens about 10/30/2015
Movie Posters
Opens about 10/30/2015
Decorative Art
Opens about 10/21/2015
US Coins
Opens about 11/13/2015
Fine & Rare Wine
Opens about 11/16/2015
Fine Art
Opens about 11/12/2015
Heritage membership
sold in the last year
- Past Auction Values (prices, photos, full descriptions, etc.)
- Bid online
- Free Collector newsletter
- Want List with instant e-mail notifications
- Reduced auction commissions when you resell your
Consignments wanted
Learn about consigning with us
With all these; friendly people, highly professional staff, superb web site makes you a winner.View More Testimonials
HA.com receives more traffic than any other auction house website. To compare for yourself, visit: compete.com
Recent auctions
You could win!
Take the Survey


text:
Add to Favorite List:
Create a new folder:
Next »
ShesNew Cam girl Russian Korean teen fucked facialized
Recognize a pornstar in this video?
Help make pornstars easier to find on Tube8 by telling us who is in this video.
Thanks! The pornstar names will be added to this video when enough users submit the same names.
You must enter a pornstar name that already exists in our database.
- Description: ShesNew Cam girl Russian Korean teen fucked facialized
- Tags: shesnew.com, young, camgirl, russian, korean, asian, european, pov, webcam, facial, cumshot, perkytits, smalltits, hd, barely-legal
- Pornstars: Add Pornstar
- Favorite by: 567
- Featured: 1 year ago
- Report This Video
Video Comments
Top Video Comments
All Video Comments (9)
1 year ago
1 year ago
Natural tits bouncing. Energetic fuck. Great facial. Nice 8 minutes.
- 0 people liked this -
1 year ago
1 year ago
She doesnt seem to be russian, not that it matters though cause her pussy is hot
- 1 people liked this -
1 year ago
1 year ago
yep- that accent gives it away, eh?
- 0 people liked this -
1 year ago
1 year ago
Formerly a cam girl on Myfreecams going by the name of Daisy Haze, she actually lost her virginity in a video she did last year.
- 0 people liked this -
Related Videos
AdBlock blows! Please disable the add-on to ensure you get the most out of Tube8's features and functionality. Read about why you shouldn't use AdBlock. Close


text:
Claude Dauphin: late leader in the commodities trading industry
He was jailed during a scandal about the dumping of toxic waste
Welcome to the new Independent website. We hope you enjoy it and we value your feedback. Please contact us here.
He was jailed during a scandal about the dumping of toxic waste
The likelihood of people developing diseases including type two diabetes and Crohn's could be affected by genes inherited from Neanderthals
Two Britons are among a group of 20 journalists working for Al-Jazeera facing criminal trial in Egypt on charges of aiding or joining a terrorist organisation – the latest press crackdown in a country which is facing growing instability.
For some language courses, a year in Paris is usual. But if you're reading Arabic, you have to travel a little further afield, as Rosie Collington found out
Emergency services were called to 25 Bank Street at around 8am this morning
Sarah Morrison finds out what traditional charities think of letting the poor spend it on what they like
Customs officials in Thailand and Singapore seized £3.5m worth of rhino horn en route to Vietnam
Government are now in discussions over how to remove the rabbit without damaging the statue of the anti-apartheid icon
Magistrate orders that the charges be dropped and that the former computer analyst be deported from the east African country on the basis that his visa has expired
At around 3pm on Monday afternoon the Delhi traffic police issued an unlikely-sounding message by means of social media.
Bailey was arguably the only failure in the Test side that claimed a 5-0 Ashes whitewash victory over England
Champagne looks to burst the bubble of the long-serving Fifa overlord in next year's presidential elections
A sports stadium in Mogadishu became a key base for al-Shabaab in Somalia as their influence expanded across eastern Africa. Photographer Johnnie Shand Kydd visited the arena after it was reclaimed by African Union forces
Homophobia is not the only motive behind these unsavoury measures
Report reveals there are just 250 adult lions after 'catastrophic collapse' in numbers in the region puts animals in critical danger
The Christian man who called himself Mad Dog allegedly dragged the Muslim stranger from the bus and ate part of his leg
First 800 will be airlifted from the makeshift Bangui camp to the Chadian capital N'Djamena later today


text:
- October 2: Nissan Versa Coil Spring Problems Cause Recall of 218,000 Cars
- September 4: Nissan Passenger Airbag Problems Lead to Investigation
- September 3: Nissan Recalls 300,000 Cars to Modify Console Plastic Trim
- September 3: Nissan Seeks Dismissal of Nissan Altima Floorboard Rust Lawsuit
- August 14: Nissan Recalls 2016 Maxima To Fix Fuel Sending Units
NHTSA — Seat Belts Problems
CarComplaints.com Notes: The 2013 Nissan Altima is showing multiple early problem trends, prompting us to give it our "beware of this clunker" badge.
Owners are complaining about a whining noise from the power steering pump and a concerning amount vibration related to the CVT (Continuously Variable Transmission). Not to mention constant windshield problems that appear to be the result of poor design.
10.0really awful
- Crashes / Fires:
- 2 / 0
- Injuries / Deaths:
- 2 / 0
- Average Mileage:
- 8,360 miles
About These NHTSA Complaints:
This data is from the NHTSA — the US gov't agency tasked with vehicle safety. Complaints are spread across multiple & redundant categories, & are not organized by problem.
So how do you find out what problems are occurring? For this NHTSA complaint data, the only way is to read through the comments below. Any duplicates or errors? It's not us.
seat belts / air bags problem


text:
- Thomas Keller born (1955)
- Thomas Keller born (1955)
Georgia Woman Discovers Giant 7-Foot KFC Bucket in Yard
Today on The Daily Meal
Recipe of the day
Something irrational inside us really wants a 7-foot Kentucky Fried Chicken bucket to showcase as a trophy on our front lawn (or like, surround with garden gnomes), but a Georgia resident is living the dream.
NBC affiliate WAGT reported on this mysterious KFC bucket that somehow popped up in front of Aleena Headrick's house. "I was driving by, I saw this giant Kentucky Fried Chicken bucket in my yard, and I thought for sure I was hallucinating," Headrick told WAGT, "so I called my teenagers who were at home and had them go outside." Sitting on her lawn: A giant, empty (unfortunately) bucket with Kentucky Fried Chicken's logo.
Turns out, Headrick's landlord actually collects old signs, so he decided to buy the bucket and place it in front of the property. The sign, which says KFC's full name, is about 40 years old, her landlord says, and he plans to display it permanently there. Luckily, Headrick doesn't mind.
"It's unusual, but it makes really good landmarks when people come to our house," Headrick said. "You can just say come down to the giant KFC bucket and turn right." Except now, we imagine, she'll crave fried chicken every time she leaves home. We know we would.
Be a Part of the Conversation
Join the Daily Meal's Community and Share your Thoughts
Have lots to say?
Contribute your own article.
Know a great recipe worth sharing?
We want it. Post it now!
Enjoyed (or not enjoyed) a restaurant experience?
Write a review.
Tasted a bottle of wine you\'d like to tell us about?
Write a review.


text:
Butterfly by Matthew Williamson Brown snakeskin print swimsuit
Item No.0640201033
Item No.0640201033
70% Off - Save a total of £29.40
From Butterfly by Matthew Williamson, this brown snakeskin print swimsuit has a self fastening tie halter neck with metallic butterfly details, a clip back fastening and lightly padded cups for a natural looking boost.
UK Home Delivery Free (UK orders over £30) or £3.49
Delivered within 5 working days
Next or Named Day Delivery
7 days a week between 8am - 6pm
Sunday to Friday - order by 9pm - £3.99
Saturday - order by 6pm - £3.99
Next or Named Evening Delivery
7 days a week between 6pm - 10pm
Monday to Sunday - order by Midnight - £6.99
Collect from store the next day - FREE
Order before 9pm for next day collection after 12pm midday
Show participating stores
Show full delivery details
Subject to exceptions, Debenhams is happy to exchange or refund your purchase within 28 days of delivery. Please check here for more details on our returns policy and exceptions.


text:
(757): View more from Virginia
Was it you I was with where I saw a guy open a beer with his butt?
Tap Here to view the Mobile Optimized TFLN
Was it you I was with where I saw a guy open a beer with his butt?
I dare you to find another dealer that delivers bud to your home along with deep fried vegan burritos
she used her teeth again, but this time it was out of love
the quiet that you are hearing is a silent suggestion that you should go fuck yourself
he sent me a green and gold dick pic and advised me I needed to come drive the snake from Ireland.
I'm still, like... really stoked about not having any STDs
Handcuffs. Recoverd. I'm a goddamn detective.
I think it's time to give up this life and become vikings. You in?
Your the only person I know that needed stiches after a Monday morning conference call. How are you in your 20s? How
So how do I get back in good graces for trying to trade you for superbowl tickets?
If my birthday doesn't end with my panties hanging from a ceiling fan, I'm holding you responsible.
He told me he wished he could shrink down to a small size so he could live inside my cleavage
I just have to point out that once I typed "fa" my phone filled in "fatass"
Is it possible to be sexually attracted to someone's hair?
We've been staking out a taco bell for 2 hours trying to find last night's one night stand


text:
Register or Log in Now→
"I had many inquiries from your site. I would use you again."
Tom M.Grass Valley, CA
Recently Listed
No Photo Available
2 Photos
1 Photo
Search Sold Mobile Homes in Harrogate TN
Advertise |
Terms of Use |
Privacy Policy |
About Us
Mobile Home |
Mobile Home Values |
Mobile Home Loans |
Mobile Home Insurance
Copyright (c) 2003 - 2015 MHVillage Inc.


text:
Register with us or sign in
Thanks for your favourable comments about the 'Afghan Girl'....a stunning shot & a masterpiece of simplicity.
Fritillary wrote (see)
Please feel free to tinker with the photo. Just puzzled how you can alter a photo on someone's post.
Please feel free to tinker with the photo. Just puzzled how you can alter a photo on someone's post.
I was hoping to lose the water butt, but wasn't able to without spoiling your pic.
Thanks, KEF....decided to give my new camera a twirtle in monochrome today ....hope I don't sound boring, but apart from aforementioned weather, the wind is bitterly cold.
Btw, I saw a black & white cow today, too.
PS. The one wiv the droopy horns is brown & white.
Crisp, clear, cold winter's day here today and I think this flooded river pic shows it.
Fritillary - like KEF, I'm pleased you have managed to post your pics....also, like KEF, I love those poppies. I was tempted to tinker-around and remove some of background.
Baz - I see you've had a few reliable tips elsewhere. However, from what you ask I would just add.
Sweet peas are best grown in the same pots from seed sowing to planting out, because they don't like root disturbance....hence no potting-on/off.
'Prick the leaves off'? I presume you mean pinching-out. Best to nip out the growing tips when they have two or three pairs of leaves (detailed pictures on the previous page of this thread).
They prefer a sunny, open situation and don't plant too thickly, otherwise they'll be troubled with mildew later in the season.
Finally, expect them to grow to at least 8 feet, so support accordingly.
Read this recently, which I think summed up the Toby Buckland era very well:
"Toby Buckland took over in 2009, and after a short-lived revamp of the show that used 60 minute-long episodes with added features which had been likened (amongst other things) to elements of programmes such as popular motoring show Top Gear (by featuring a 'cool wall') and CBeebies by virtue of featuring young children."
Rather a shame I think, as I saw him as a victim of someone else's stupidity.
Like bekkie, I'm not up to speed with happenings on this thread, having said that I send my best wishes to Matty and hope she makes a quick & complete recovery.
Googled pic:
Steve McCurry is a National Geographic photographer and some of you may have seen this portrait of the Afghan girl. This is inspiring to anyone who would aspire to take stunning portraits.
Anyone with nostalgic traits towards old TV progs, I would suggest getting a copy of The Victorian Kitchen Garden. Presented by Peter Thoday and ably assisted by the wonderful & talented head gardener, Harry Dodson.....the latter being on a par with the sainted Geoff Hamilton, IMHO.


text:
A new, leaked driver offers a preview of what mobile G-Sync will look like in the future. The software is still in early stages, but the technology is as impressive in laptops as it is in desktops.
AMD is claiming it can implement the advantages of G-Sync using freely available software and functionality baked into its existing GCN GPUs. Nvidia is claiming that it can't. Who's right?
Nvidia's G-Sync has now been demoed and tested independently, and the results, while early, are fairly good. Could this tech take over gaming?
Nvidia's new G-Sync technology is designed to finally fix the monitor tearing and synchronization issues that have plagued gaming for decades — and the early feedback from those who've seen it is that the technology is fabulous.
Subscribe Today to get the latest ExtremeTech news delivered right to your inbox.


text:
Need ideas arranging staff
0 I am a fairly new DON and I need guidance from all of you wise ones out there regarding staffing. How do you move nurses around, or change their schedule without dropping the morale and them end up quitting? I need to move an LPN from days to night shift very soon!! Plus I feel like we have too many CNAs at night. Ideas anyone?
About FieryGingerRN, BSN
FieryGingerRN has '6' year(s) of experience and specializes in 'Med-Surg, Ortho, CM, LTC, HH/Hospice'. From 'United States'; Joined Jul '08; Posts: 59; Likes: 67.1Jul 9, '13 by TBlaseI take this to mean you need one less day nurse, and one more night nurse? If I were DON, I would hold a staff meeting, to tell the staff that you are facing a situation, and why (be frank) , and then ask if any day nurses want to move to nights. Or any 3-11 want to move to nights, and then if any day shift nurse wants to move to 3-11. Or, you might even be suprised at solutions that might be offered up. If nobody jumps, you can then hire a night nurse, while informing your staff that in that event, a day nurse will be laid off- based on seniority, in my opinion. Fair, open, transparent, the way to minimize pain all around. I don't envy you. But I also wouldn't want to see you alienate staff, and cause more pain that must be caused? And even though nurses are a dime a dozen anymore, with no jobs to go around...being frank, honest, appreciative, rather than heavy handed, will do nothing but make your job easier?0Jul 10, '13 by wyogypsy@ TBlase - were you being facetious saying nurses were a dime a dozen with no jobs to go around? Wow, that would be the dream of so many places I know who are struggling to find enough nurses to cover shifts, there aren't enough nurses living in those areas to fill the needs.1Jul 10, '13 by SoldierNurse22, BSN, RN, EMT-BTBlase is correct... generally speaking, there aren't enough jobs to go around, and it's especially difficult to get hired as a new grad. There are (as always) exceptions to this rule in parts of the country, but the exceptions aren't the norm, that's for sure.
It isn't so much a lack of job availability, it's a lack of funds or lack of willingness ot adequately staff hospitals due to the cost of hiring an RN. It will take time to convince our nation's healthcare system that adequate staffing is important to outcomes and is less expensive in the long run, but it's a worthwhile fight.
OP, I agree with TBlase's idea. Suggest it to your own staff first and see if you get any bites. A lot of people would rather switch than lose their job in this economy.3Jul 10, '13 by TBlaseQuote from wyogypsyEveryone reading this forum is waiting for you to disclose these so many places that don't have enough nurses living in them, please:@ TBlase - were you being facetious saying nurses were a dime a dozen with no jobs to go around? Wow, that would be the dream of so many places I know who are struggling to find enough nurses to cover shifts, there aren't enough nurses living in those areas to fill the needs.1Jul 10, '13 by HeyHeyitsMaayThe issue is that most places don't have enough staffing to cover health care needs. We're faced with initiatives like INTERACT that require advanced critical thinking skills of new grads who aren't even taught to make a bed or why it's a critical first line intervention in reduction of facility acquired pressure ulcers. We have shrinking CMS funding and growing censuses with very complicated comorbidities. I've had more than a few nurses who I've hired who quit within weeks because they can't or don't want to handle the level of care and responsibility required of them. We do our absolute best to hold on to nurses and bleed care and concern for their needs as nurses and human beings. It's not enough when we're so heavily regulated that the level of complication of the requirements handed down from DADS and CMS is directly proportionate to the frustrations, lack of sound education inclusive of teaching the reality of how grueling nursing really is, and the millineal generation entering nursing with crazy expectations.0Jul 10, '13 by FieryGingerRN, BSNBasically I have a nurse in a position that I don't feel is a good fit for her and I want to hire someone to fill her spot and that would mean I would need to move her. It's a sticky situation but I need to make the change for the better of the facility.2Jul 11, '13 by TBlaseAh, the plot thickens. You don't have a staffing issue at all. You have a nurse you don't want or like or trust on day shift, but you need a night nurse, and are wondering if you can push here there, and keep her morale intact, and prevent her from quitting? You have to have a face to face with her, and be honest:"I am not sure you can handle the day shift. But I would like to keep you, because you're a good nurse. Unfortunately, the only other opening I have is on night shift- will you consider taking that?" She may, or she may quit. But since you've already made your mind up, you have to face that. But if the reality is that you want to get rid of her, and are trying to scheme ways to make her quit without you having to fire her, I say you owe it to her, on a professional level, to fire her straight off.0Jul 11, '13 by TBlaseQuote from wyogypsy@ TBlase - were you being facetious saying nurses were a dime a dozen with no jobs to go around? Wow, that would be the dream of so many places I know who are struggling to find enough nurses to cover shifts, there aren't enough nurses living in those areas to fill the needs.
Anybody out there, wyo? My bags are packed, I'm waiting for you to tell me where to go!
Must Read Topics


text:
Earth Island InstituteShared publicly -
We are excited to announce Earth Island Institute's newest Brower Youth Awards sponsor partner – Straus Home Ranch! Situated on 166 acres of certified #organic land in stunning west Marin County, California, their beautiful 150+ year old farmhouse was recently renovated and is now available as a vacation rental! Plan your escape from the city and relax in the country in this gorgeous eco-farmhouse that sleeps 8. Thank you for your partnership, Straus Home Ranch! http://www.StrausHomeRanch.com
Add a comment...


text:
Java Marvel Character Creator
editor's review
Given its long history on the market, Marvel's got quite the wide selection of superheroes, some of which are extremely famous, while others only got their fifteen minutes of fame. However, if not even that is enough for you, then you can also take matters into your own hand and try to create a superhero from scratch, using the powers you find the most useful, or just cool.
A simple tool to quickly generate superheroes
As Java Marvel Character Creator gives it away in the name, the application is all about allowing you to come up with the desired Marvel character, using a variety of stats, abilities, and talents. Although it might look a bit overwhelming at first, every field has its purpose and the interface is well-explained, so you won't stumble into things that lack description or function.
To begin with, you can easily give a name to your character and configure a few cosmetic attributes as well, including hair color, eyes, height, weight, and so on. However, the most important part is related to the powers, because that's what makes them great after all. As such, you can either generate a random configuration or choose them from a long list of options.
Export your creations and use them for various purposes
In fact, the stats are split into multiple categories, as follows: primary abilities, talents, contacts, weakness, and powers. Hence, the abilities are expressed in numbers and they include endurance, strength, and agility, while talents range from martial arts to psychiatry. The powers are also quite important, obviously, but they are much easier to choose, given the fact that it's the first thing you come up with.
After you are done customizing your character and adjusting his/her stats, you can easily export the result to a variety of formats, as well as in plain text. The sheets can be used for a variety of purposes, depending on what you need them for. In addition, entire databases can be created as well, which are designed to hold a large number of characters.
A small, yet powerful application aimed at enthusiasts
Although Java Marvel Character Creator is not for everyone, it does have a rather loyal audience, given the fact that there are not too many alternatives out there. Hence, if the subject interests you and you could benefit from a tool such as this, the fact that it runs on Java means that you can take advantage of it on basically any device out there.


text:
The Steampunk Empire
The Crossroads of the Aether
Added by Boilerplate
0 Comments
1 Like
Posted on July 31, 2010 at 9:38pm
1 Comment
Join The Steampunk Empire
Happy Birthday Boilerplate, I do hope it is a pleasant one, wherever you are.
View All Comments
Welcome toThe Steampunk Empire
Sign Upor Sign In
Or sign in with:
Retro Victorian Steampunk Goggles Glasses Welding Cyber Punk Gothic Cosplay
Vintage style Victorian Steampunk Goggles Glasses welding Cosplay Goth Punk
Steampunk Goggles Glasses Vintage Victorian welding Cosplay Goth Punk Costume
Gothic Boned Lace Up Steampunk Underbust Corset Bustier Waist Cincher Training
Vintage Victorian Steampunk Goggles Glasses Welding Cyber Punk Gothic Cosplay
© 2015 Created by Hephzibah Marsh.
Badges |
Report an Issue |
Terms of Service
Please check your browser settings or contact your system administrator.


text:
- 87Promote
Framed Art Print
Make it Spring...by Isabelle Lafrance Photography
Choose from a variety of frame styles, colors and sizes to compliment your favorite Society6 gallery, or fine art print - made ready to hang. Fine-crafted from solid woods, premium shatterproof acrylic protects the face of the art print, while an acid free dust cover on the back provides a custom finish. All framed art prints include wall hanging hardware.
beautiful Isabelle. love that vase
Thank you Beverly, it's a thrift store find :)
Very beautiful!
Thank you Bob!
so gentle
Thanks Sharon!
Pure delicacy, Isabelle!
Very pretty!
Beautiful :-)
Thanks Galaxy Eyes, Colleen, & Anna!
Thank you Syl!
This is beyond beautiful, Isabelle!
So soft and lovely!
Thank you very much Angela and TaLins!
Thank you Esther!
Soft, dreamy!
Thanks for your visit Catherine!
dreamy and pretty!
Thank you Sandra!
This is so pretty :)
so gorgeous isabelle
Very Pretty Isabelle
Thank you Kay, jamesy and Anthony :)
so soft & lovely!
Thank you Deepti!
So lovely and sweet!
Thanks Lisa!
Really lovely! :)
Thank you Christine!
This is so beautiful, Isabelle! I love the petals that have fallen... and what a sweet shade of pink.
A very gorgeous capture Isabelle.
Gorgeous shot Isabelle :)
So soft and lovely!
Thank you everyone!
beautiful shot!
such lovely, soft tones.
Thank you Vasare and Judi!
lovely !
Thank you Alexandros!
I really like this crop. Great picture!
. a soft beauty . lovely work :))
so soft and beautiful!
Thank you Leah, Sharon and Arevik :)
Oh so pretty!!
☆ ☆ ☆ ☆ ☆… Whether it be words or stars, I only comment on work I genuinely like, to provide you highlighted visibility now and the opportunity for additional exposure later! ;-) A great piece... this is five star work!
See More Comments


text:
Depending upon the setting, one candidate's cross to bear is another similarly afflicted candidate's dust on the shoulder.
On Tuesday, I attended the congressional debate staged by the Conservative Order of Good Government, a Rancho Bernardo-based political group that, in a bygone era, called itself the Conservative Order of Good Guys.
To give you a sense of the audience's political persuasion, Sheriff Joe Arpaio, the Arizona lawman famous for his self-reliant approach to enforcing immigration law, received a rock star's ovation when he walked into the same room nearly two years ago.
Before Tuesday's debate, termed-out San Diego Councilman Brian Maienschein performed a cameo to remind COGG members that they were the first group to support him when he was running for council in 2000. Maienschein is now running as a Republican in the 77th Assembly District, which includes his old council stomping grounds.
Though he has real opposition, the still-boyish Maienschein appears a front-runner in the safe GOP district. He's raised more money than his opponents, his website boasts. His endorsement list includes Pete Wilson and San Diego Mayor Jerry Sanders, moderate GOP icons.
As a councilman during two major wildfires, Maienschein was so popular in Rancho Bernardo and environs that older residents not only wanted to vote for him, they wanted to adopt him.
Scott Peters, one of two Democratic challengers to incumbent Rep. Brian Bilbray in the new 52nd Congressional District, represented District 1, San Diego's North County coastal district, for the same eight-year period.
As fate would have it, both Peters and Maienschein voted to underfund the municipal pension, a judgment widely considered to be somewhere between terrible and calamitous.
In Maienschein's Assembly race, the pension vote will no doubt figure in hit pieces, but it's an open question if there will be enough firepower behind the attack to hurt the popular figure in a fairly obscure race for a ticket to Sacramento.
Peters, on the other hand, can expect a barrage if he's polling near the top of what at this point is a high-profile five-person contest.
As a harbinger of what's to come, one of the COGG questions was tailored just for him: "In a fundraising letter, Congressman Bilbray called you 'The Father of San Diego's Pension Mess.' I'm sure you'd like to respond."
The audience laughed as if Peters had been, figuratively, tarred and feathered.
Peters was prepared for the paternity question. He did not try to absolve himself by blaming others or arguing the problem wasn't as bad as it was cracked up to be. He admitted poor judgment and then pivoted to measures that he'd supported to improve, if not completely fix, the hemorrhage in the city's budget.
"I'm the only one on this panel to do meaningful government reform," Peters said, doing his best to wring something drinkable out of Bilbray's lemon.
Peters ended up drawing some applause — not raucous Sheriff Joe level, but respectful — in a room that probably wouldn't vote for him if he enlisted in the border Minutemen.
Despite the old rugged cross he must bear, Peters, a lawyer who models himself as a moderate Democrat with a record of nonpartisan leadership, particularly on the environment, stands a fighting chance of finishing in the top two in the "open" primary. (The top two vote getters, regardless of party, run off in November.)
It won't be easy, though. Lori Saldaña, a termed-out Democratic assemblywoman, likely will appeal to a percentage of base Democratic voters.
For his part, Bilbray, a wily Republican pol with tremendous wingspan from far right to left, may have to fend off not one but two credible tea-party-style GOP candidates — John Stahl and Dr. Wayne Iverson. To make the June cut, Bilbray must mine the voter-rich sweet spot occupied by moderate Republicans and right-leaning independents who have not come down with a case of anti-incumbent fever.
Some liken this primary to "jungle ball," an anarchic form of hoops in which it's every player for himself.
The only unsafe congressional district in the county, the new 52nd is a near-perfectly balanced court upon which the two best political athletes will elbow themselves into position and score the most votes.
We're driving into the jungle. If you like politics, it should be a ball.


text:
All hotels in Eureka, California, United States of America
1 room, 2 adults
2223 4th St, Eureka, CA, 95501, United States of America, 800-491-6126
Clarion Hotel By Humboldt Bay, Eureka
Lowest price$81
Best Price Guarantee
No-frills Eureka motel with indoor pool
- Free continental breakfast and free parking
Main amenities
- 65 guestrooms
- Indoor pool
- Fitness center
- Business center
- 24-hour front desk
- Coffee/tea in the lobby
- Air conditioning
- Self-serve laundry
- Free newspapers in the lobby
What's around
- Blue Ox Millworks (0.2 mi / 0.4 km)
- Carson Mansion (0.7 mi / 1.1 km)
- Clarke Historical Museum (1.1 mi / 1.8 km)
- Morris Graves Museum of Art (1.2 mi / 1.9 km)
- North Coast Repertory Theatre (1.2 mi / 1.9 km)
Bright and clean. Very comfy beds. Excellent breakfast
Very comfortable beds. We lucked out that out room was next to guest laundry. Been…
TripAdvisor Traveller Rating: 3.5
From 133 reviews
Enter dates to check prices
Hotel Info: 866-539-8117from $81
- Suite, 1 King Bed, Non Smoking, Hot Tub
- Standard Room, 1 King Bed, Non Smoking
- Standard Room, 2 Queen Beds, Non Smoking
- Standard Double Room, 2 Double Beds, Non Smoking
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 65 rooms
- This hotel is arranged over 3 floors
- Check-in time starts at 3 PM
- Check-out time is noon
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets not allowed
- Free self parking
- Accessible parking
Nearby Two Star Eureka hotels
In the hotel
Food and drink
- Free continental breakfast
- Coffee/tea in the lobby
Things to do
- Indoor pool
- Fitness facilities
Working away
- Business center
- 24-hour front desk
- Laundry facilities
- Free newspapers in lobby
- Number of buildings/towers - 3
- Elevator/lift
- Disabled parking
- Accessible path of travel
- Braille or raised signage
- Accessibility equipment for the deaf
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
Home comforts
- Air conditioning
- Coffee/tea maker
- Iron/ironing board
Freshen up
- Hair dryer
Be entertained
- Cable TV channels
Stay connected
- Free local calls
Food and drink
- Refrigerator (on request)
- Microwave (on request)
Top Eureka hotels
Recently reviewed hotels in Eureka
- The Scotia InnFrom
"This is a charming historic property, built by the Pacific Lumber Company as a guest house and now run as a family business. The present owners have done a good job in restoring…"Aug 31, 2015
- Rodeway Inn Emerald Forest of TrinidadFrom
"We basically just slept here, but it was nice and clean and comfortable. Staff was friendly. It was a bit pricey, though, for a basic hotel (cabin)."Aug 23, 2015


text:
Free Website Template w/ jQuery Slideshow - Design
- Type: Website
- Item number: 51556
- Author: Big mama
- Share:
Please welcome the fresh free website template with jQuery slideshow and a jQuery slider in the homepage area. It was originally designed for an exterior design related project, but since the PSD sources are included you can customize the template for any other topic or purpose you may have. We encourage you to use this template for your own websites or for clients web design projects, however please do not resell it.
- Features
Animation: HTML plus Flash
- Sources Available
- Software Required
- Adobe Photoshop CS+
- For uncompressing a template ZIP package: WinZip 9+ (Windows); Stuffit Expander 10+ (Mac)