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US 5696090 A Abstract Polypeptide pharmaceutical compositions having improved stability and resistance to aggregation, particle formation and precipitation comprising a polypeptide pharmaceutical and poloxamer surfactants alone, or in combination with polysorbate surfactants. Preferred polypeptides stabilized are bactericidal/permeability increasing (BPI) protein, biologically active fragments of BPI, biologically active analogs of BPI, and biologically active variants of BPI.
Claims(4) 1. A method of solubilizing or stabilizing a polypeptide that is bactericidal/permeability-increasing (BPI) protein or a BPI fragment thereof, or a BPI analog of said bactericidal/permeability-increasing protein or BPI fragment thereof in aqueous solution, comprising the step of contacting said polypeptide with polyoxypropylene-polyoxyethylene block copolymer (poloxamer) surfactant in a solubilizing/stabilizing concentration.
2. The method of claim 1 wherein the polyoxypropylene-polyoxyethylene block copolymer (poloxamer) surfactant is poloxamer 188.
3. A method of solubilizing or stabilizing a polypeptide that is bactericidal/permeability-increasing (BPI) protein or a BPI fragment thereof, or a BPI analog of said bactericidal/permeability-increasing protein or BPI fragment thereof in aqueous solution, comprising the step of contacting said polypeptide with concentrations of polyoxypropylene-polyoxyethylene block copolymer (poloxamer) surfactant and polyoxyethylene sorbitan fatty acid ester (polysorbate) surfactant in a solubilizing/stabilizing concentration.
4. The method of claim 3 wherein the polyoxypropylene-polyoxyethylene block copolymer (poloxamer) surfactant is poloxamer 188 and the polyoxyethylene sorbitan fatty acid ester (polysorbate) surfactant is polysorbate 80.
Description This is a divisional application of U.S. application Ser. No. 08/190,869, filed Feb. 2, 1994, now U.S. Pat. No. 5,488,034, which is a continuation-in-part of U.S. patent application Ser. No. 08/012,360 filed Feb. 2, 1993, now abandoned. The present invention relates generally to pharmaceutical compositions and more specifically to improved protein and polypeptide pharmaceuticals for use as parenteral drugs. Recent advances in the development of genetic engineering technology have made a wide variety of biologically active polypeptides available in sufficiently large quantifies for use as drugs. Polypeptides, however, can be subject to particulate formation and loss of biological activity by a variety of chemical and physical means including denaturation due to heating or freezing and by exposure to extreme pH or other chemical degradation. Particulate formation and loss of biological activity can also occur as a result of physical agitation and interactions of polypeptide molecules in solution and at the liquid-air interfaces within storage vials. It is believed that the polypeptide molecules adsorb to an air-liquid interface, unfolding to present hydrophobic groups to air with the hydrophilic groups immersed in the aqueous phase. Once so positioned at the surface, the polypeptide molecules are susceptible to aggregation, particle formation and precipitation. It is also believed that further conformational changes can occur in polypeptides adsorbed to air-liquid and solid-liquid interfaces during compression-extension of the interfaces such as occurs from agitation during transportation or otherwise. Such agitation can cause the protein to entangle, aggregate, form particles and ultimately precipitate with other adsorbed proteins. Particle formation due to surface denaturation can be somewhat controlled by appropriate selection of the dimensions of storage vials and by minimizing the air volume (headspace) in those vials. In this regard, partially filled containers represent the worst case for vibration induced precipitation. Particle formation can also be controlled by incorporation of surfactants into the protein containing composition in order to lower the surface tension at the solution-air interface. Classic stabilization of pharmaceuticals by surfactants or emulsifiers has focused on the amphipathic nature of molecular groups containing both hydrophilic and hydrophobic properties within the surfactant molecule. Thus, the an teaches that one can make a stable solution of immiscible molecules such as oil-in-water or water-in-oil by selecting an appropriate surfactant as a compatibilizer. One example is the stable emulsification of soybean oil using poloxamer 188 (PLURONIC F-68, BASF Wyandotte Corp., Parsippany, N.J.). Another example is the use of polysorbate 80 (TWEEN 80, ICI Americas, Inc., Wilmington, Del.) to emulsify oil-soluble vitamins A, E and K in aqueous solution for administration via oral and vascular routes. Work by Krantz, et al., "Sugar Alcohols--XXVIII. Toxicologic, Pharmacodynamic and Clinical Observations on TWEEN 80," Bull. of the School of Med., U. of MD., 36, 48 (1951) laid the groundwork leading to the listing of polysorbate 80 as a drug ingredient for which USP/NF requirements have been established in U.S. Pharmacopeia XXII. Of interest to the present invention is the work related to use of polysorbate 80 for stabilization of antibody-based product formulations as described in Levine, et al., J. Parenteral Sci. Technol., 45, 3, 160-165 (1991). This work disclosed that the amount of surfactant required for stabilization was in excess of the theoretical minimum required to reduce surface tension. The work further showed that the need for excess surfactant beyond the theoretical minimum could be attributed to (1) the concentration required to maintain an intact protective layer on a turbulent interface during random shaking; and (2) to surfactant loosely associated with protein and bound to container walls. Regulatory requirements limit the types and specific identities of surfactants that can be incorporated into parenteral compositions for injection into the human body. Generally accepted surfactants having a history of use and listed in the U.S. Pharmacopoeia XXII include poloxamer and polysorbate polymers. However, either of these alone may provide less than complete stabilization for the pharmaceutical compositions when used at concentrations of 0.1% or lower. Elevated concentrations of surfactant may pose increased risk of toxic effects, earlier onset of hemolysis, and observed changes in neutrophils and platelets, both of which are involved in blood complement activation. The highest safe concentration for poloxamer 188 in approved parenteral solutions is 2.7% when it is used in limited doses as a blood substitute and is diluted as much as 10 fold in the bloodstream. Similarly, polysorbate 80, approved in parenteral solutions for over 20 years, is rarely used in concentrations greater than 0.1% in solution volumes of 100 mL or more. Krantz et al., supra, identifies the onset of hemolysis in the dog for a polysorbate concentration of 0.1% at 90 minutes. Neonatal deaths have been associated with the use of polysorbate 80 at concentrations of greater than 1%. Accordingly, there exists a need in the art for pharmaceutical compositions providing improved protein stability which comprise only those components which are regarded as safe and are included in parenterals approved by regulatory authorities for commercial use. The present invention relates to pharmaceutical compositions of polypeptides and is directed to the discovery that poloxamer surfactants and combinations of poloxamer surfactants with polysorbate surfactants enhance the solubility/stability of bactericidal/permeability increasing (BPI) protein, biologically active fragments of BPI, biologically active analogs of BPI, and biologically active variants of BPI (produced by either recombinant or nonrecombinant means) in aqueous solution. The invention particularly provides for solubilization/stabilization of bactericidal/permeability increasing proteins which are biologically active amino-terminal fragments of BPI or analogs and variants thereof. Amino-terminal fragments of BPI, such as those designated rBPI23 or any amino-terminal fragment comprising from about the first 193 to about the first 199 amino-terminal amino acid residues of BPI, are believed to be particularly susceptible to loss of stability in aqueous solution. The present invention is directed in particular to the discovery that a combination of two specific types of surfactants provides a surprising improvement in protein stability to pharmaceutical compositions compared to either surfactant alone. Specifically, it has been found that a pharmaceutical composition comprising the combination of a poloxamer (polyoxypropylene-polyoxyethylene block copolymer) surfactant and polysorbate (polyoxyethylene sorbitan fatty acid ester) surfactant provides improved stability and resistance to aggregation, particle formation and precipitation of protein pharmaceutical agents. The combination of these two types of surfactants provides improved stability and resistance to surface denaturation, aggregation, particle formation and precipitation compared with either surfactant alone. The poloxamer surfactant component is preferably present in a concentration of from about 0.01% to about 1% by weight with a concentration of 0.1% to 0.2% by weight being preferred to stabilize protein solutions comprising less than or equal to 2 mg/mL. The polysorbate surfactant component is preferably present in a concentration of from about 0.0005% to about 1% by weight with a concentration of 0.002% by weight being preferred. Most preferred is the combination comprising 0.1% to 0.2% by weight of poloxamer 188 and 0.002% by weight polysorbate 80. This combination is particularly useful for preventing particle formation of extremely degradation sensitive proteins such as bactericidal/permeability increasing protein (BPI) but is also useful for promoting the stability of other polypeptide pharmaceuticals. It is contemplated that the combination of poloxamer and polysorbate surfactants may be used alone or in combination with additional surfactants. Moreover, the invention is not limited to a single poloxamer surfactant in combination with a single polysorbate surfactant and can include one or more poloxamer surfactants in combination with one or more polysorbate surfactants. A further aspect of the invention relates to the discovery that a poloxamer surfactant is particularly useful for the solubilization/stabilization of compositions comprising an aqueous solution of BPI protein or biologically active fragments, analogs, or variants of BPI protein (produced by recombinant or nonrecombinant means). The invention provides a method of solubilizing/stabilizing such polypeptides by contacting the polypeptide with a poloxamer surfactant. Without being bound by a theory of the invention, it is believed that poloxamer surfactants stabilize BPI protein products not by a mechanism involving lowering the surface tension of the aqueous solution, but, at elevated temperatures, by stabilizing unfolded and partially unfolded BPI protein molecules and preventing precipitation of those molecules. Preferred poloxamer surfactants are characterized by a HLB value greater than about 14 and a surface tension between 10 and 70 mN/m as measured in aqueous solution at room temperature and at a concentration of 0.1%. More preferred is a poloxamer surfactant which has an HLB value between about 25 and 35 and has a surface tension between 30 and 52 mN/m as measured in aqueous solution at room temperature and at a concentration of 0.1%. Most preferred is poloxamer 188 available commercially as PLURONIC F-68 (BASF Wyandotte, Parsippany, N.J.) which is characterized by a surface tension of 50 mN/m and by an HLB value of 29. A preferred polysorbate surfactant preferably has a surface tension between 10 and 70 mN/m as measured in aqueous solution at room temperature and at a concentration of 0.1%. More preferably, the polysorbate surfactant is characterized by a hydrophilic/lipophilic balance (HLB) value of about 15 and by a surface tension between 40 and 50 mN/m as measured in aqueous solution at room temperature and at a concentration of 0.1%. Most preferred is polysorbate 80 (sorbitan mono-9-octadeconoate) which is available commercially as TWEEN 80 (ICI Americas Inc., Wilmington, Del.). FIG. 1 is a graph depicting survival results over time of an actinomycin-D sensitized mouse model; FIG. 2 is a graph depicting survival results according to BPI dose in an actinomycin-D mouse model; and FIG. 3 is a graph depicting turbidity measurements of various BPI proteins with and without the preferred surfactants of the invention. FIG. 4 is a graph depicting surface tension measurements of rBPI21 Δcys solutions with varying surfactant concentrations of polysorbate 80 (PS80) and poloxamer 188 (F68). FIG. 5 is a series of graphs of differential scanning calorimetry results of rBPI21 Δcys with various concentrations of the surfactant poloxamer 188 (F68). FIG. 6 is another series of graphs of differential scanning calorimetry results of rBPI21 Δcys with various concentrations of poloxamer 188 (F68) FIG. 7 is a plot of the denaturation and precipitation temperatures of rBPI21 Δcys over varying concentrations of the surfactant poloxamer 188 (F68). FIG. 8 is a series of graphs of differential scanning calorimetry results of rBPI21 Δcys with various concentrations of polysorbate 80 (PS80) alone or in combination with 0.1% poloxamer 188 (F68) by weight. FIG. 9 is a set of graphs of differential scanning calorimetry results of rBPI21 Δcys with the surfactant polysorbate 80 (PS80) at two different concentrations. FIG. 10 is a set of graphs of differential scanning calorimetry results after a solution of rBPI21 Δcys and poloxamer 188 (F68) was heated to a temperature higher than the denaturation/unfolding temperature but lower than the precipitation temperature, and then was cooled down for repeat scanning. The present invention provides improved methods and materials for maintaining the stability of polypeptide pharmaceuticals and preventing surface denaturation of such biologically active polypeptides. Specifically, the invention relates to the discovery that a combination of two specific types of surfactant molecules provides synergistic improvements in stabilization from surface denaturation of polypeptide pharmaceuticals. The invention also relates to the discovery that poloxamer surfactants have unique properties in the solubilization/stabilization of BPI-related proteins. While specific embodiments of the invention are directed to stabilization of bactericidal/permeability increasing protein (BPI) and biologically active fragments and/or analogs or variants thereof which are particularly susceptible to denaturation and particle formation, the utility of the invention extends generally to all protein and polypeptide pharmaceuticals. BPI and active fragments and analogs thereof useful with the present invention include recombinant produced proteins such as described in U.S. Pat. No. 5,198,541. Co-owned, copending patent application Theofan et al., U.S. Ser. No. 08/064,693 filed May 19, 1993, which is a continuation-in-part application of U.S. Ser. No. 07/885,911 filed May 19, 1992, addresses BPI-Immunoglobulin fusion proteins which are variants of BPI protein comprising at the amino terminal a BPI protein or a biologically active fragment thereof, and retaining the same biological activity of BPI protein. Particularly preferred BPI materials include recombinant produced polypeptides produced according to the method of co-owned and copending Theofan et at. U.S. application Ser. No. 08/013,801 filed Feb. 2, 1993 and entitled "Stable Bactericidal/Permeability-Increasing Protein Products and Pharmaceutical Compositions Containing the Same," the disclosure of which is herein incorporated by reference. A preferred BPI fragment is characterized by about 1 to 199 or about 1 to 193 of the amino-terminal amino acid residues of the mature human BPI molecule as set out in Gray et at., J. Biol. Chem., 264, 9505-9509 (1989) except that residue 185 is glutamic acid rather than lysine as specified in Gray. The recombinant expression product of DNA encoding BPI amino acids 1 to 199 has been designated rBPI23. The recombinant expression product of DNA encoding BPI amino acids 1 to 193 has been designated rBPI(1-193). A preferred BPI fragment analog comprises the first 193 amino acid residues as set out in Gray except that residue 185 is glutamic acid rather than lysine and the cysteine at position 132 is replaced with a non-cysteine residue such as alanine. Such a protein is designated rBPI21 Δcys or rBPI(1-193)ala132. In this example, tests of various surfactant systems were conducted to determine their utility for surface stabilization of a polypeptide pharmaceutical (rBPI23). The rBPI23 was provided at a concentration of 1 mg/mL in citrate buffered saline (0.02M citrate, 0.15M NaCl, pH 5.0). Various surfactants were then added to this preparation in order to determine their utility as stabilizers. According to this test, rBPI23 BR-1! characterized by about 1 to about 199 of the first 199 amino acids of the mature human BPI molecule and produced according to the methods of Theofan et al., U.S. Pat. application Ser. No. 08/013,801 filed Feb. 2, 1993 was filled by hand to 5 mL in sealed sterile 6 mL molded glass vials (total capacity 8.4 mL, Wheaton) in the desired formulation buffer. The vials to be tested were set horizontally on a flat bed shaker (S/P rotor V) and fixed to the shaker by tape. Vials were then shaken at 150 rpm at room temperature. At 0 hours, 2-4 hours, and 18 hours, 150 μl samples were withdrawn in a biosafety cabinet using a 1 mL syringe fitted with a 21 gauge needle. The starting, in process, and ending soluble rBPI23 concentrations were determined by an ion exchange HPLC assay and visual observation of cloudiness of the solution was also recorded. The results are shown below in Table 1 in which acceptable stability was determined by visual inspection after the shake test. Testing of protein preparations comprising single surfactants showed good results for use of octoxynol-9 (TRITON X-100, Rohm & Haas), laureth-4, (BRIJ 30, ICI Americas), poloxamer 403 (PLURONIC P123, BASF Wyandotte) and telomere B monoether with polyethylene glycol (ZONYL FSO-100, E. I. DuPont de Nemours). While these surfactants are capable of reducing surface tensions to low levels, they are not included in approved parenteral pharmaceuticals due to suspected toxic effects or unknown biocompatibility. Testing of other surfactants as shown in Table 1 shows that surfactants producing a surface tension lower than 35 mN/m are capable of stabilizing rBPI at surfactant concentrations of 0.1%. This example further shows that both polysorbate 80 (TWEEN 80) and poloxamer 188 (PLURONIC F-68) were incapable of stabilizing the protein preparation alone under the shake test conditions employed. The incorporation of polysorbate 80 did, however, have the effect of clarifying a cloudy solution of BRIJ 30 which is not readily water soluble without the help of an additional solubilizer. TABLE 1__________________________________________________________________________ Surface Tension mN/m at 0.1% Conc. at Room Temp. in Surfactant Visual rBPI23 Conc. by HPLC Stability asExp Surfactant Water (w), Concentration Observation (mg/mL) Determined byNo. Used Buffer (b)1 in Form Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Visual Inspection__________________________________________________________________________1 ZONYL 17.sup.(w) 0.100% -- Clear 0.96 -- 1.00 Stable FSO-1002 PS-80 41.sup.(b) 0.100% -- Cloudy 1.11 -- 0.02 Unstable3 BRIJ 30 27.5.sup.(b) 0.500% Cloudy Cloudy 1.08 -- 1.14 BRIJ 30 alone is cloudy.4 TRITON 32.sup.(b) 0.100% Clear Clear 1.00 1.01 0.98 Stable X-1005 PLUR 34.3.sup.(w) 0.100% Clear Clear 1.08 1.08 1.08 Stable P1236 BRIJ 30/ -- 0.1%/0.125% Clear Clear 1.19 1.21 1.17 Stable PS-807 PLUR 46.sup.(b) 0.100% Clear Haze 1.23 1.22 0.95 Marginal F-68 stability. Slight haze, specks.8 PLUR 44.sup.(b) 0.200% Clear Haze -- -- 1.04 Marginal F-68 Stability. Slight haze with a few specks.9 PLUR 47.sup.(b) 0.1%/0.001% Clear Clear 1.14 1.09 Stable. Crystal F-68/ clear with a few PS-80 specks.__________________________________________________________________________ Surface tensions with superscript w are obtained from the surfactant manufacturer. Surface tensions with superscript b are obtained experimentally using Wilhelny plate method. In this example, additional comparisons were carried out according to the methods of Example 1 using various surfactants alone and in combination to stabilize a rBPI23 preparation. The results are shown below in Table 2 in which acceptable stability was determined by visual inspection after the shake test. The results, particularly those of experiments 52-58 show the unexpected utility of the combination of poloxamer 188 and polysorbate 80 for stabilizing the rBPI23 composition at concentrations where either surfactant alone is incapable of equivalently stabilizing the material under the conditions of the test. The experiments show that various combinations of concentrations of the two surfactants exhibit synergistic effects but that the preferred combination specific to rBPI23 at 1 mg/mL concentration is that having 0.1% by weight poloxamer 188 and 0.001% by weight polysorbate 80 in citrate buffered saline (0.02M citrate, 0.15 NaCl, pH 5.0). The results with polysorbate 80 at concentrations lower than 0.001% produced prompt cloudiness after 18 hours of shaking, but with only a small loss of protein as determined by ion-exchange HPLC MA7C column (Bio-Rad, Hercules, Calif.). Nevertheless, the cloudiness is unacceptable for appearance and suggests lowered stability. Testing with polysorbate 80 at concentrations of 0.005% and above all give good stability at up to 18 hours of shaking with little sign of protein loss by HPLC. Nevertheless, these higher concentrations of polysorbate 80 may provide less stability during long-term storage at 4� C. and at stress temperatures of ambient room temperature or above. TABLE 2__________________________________________________________________________ Surfactant Conc. by HPLCExp Conc. in Visual Observation (mg/mL) Stability as DeterminedNo. Surfactant Used Form. Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr by Visual Inspection__________________________________________________________________________ 1 ZONYL FSO-100 0.100% -- Clear 0.96 -- 1.00 Stable 2 PS-80 0.100% -- Cloudy 1.11 -- 0.02 Unstable 3 Dextran Sulfate 1 mg/mL -- Cloudy -- -- 0.00 Unstable 4 Glycerol 10.0% -- Cloudy 0.86 -- 0.02 Unstable5 HSA 5.0% -- Cloudy 0.92 -- 0.00 Unstable 6 Control-5 mL Fill -- -- Cloudy 1.13 -- 0.03 Unstable Volume 7 Control 8.4 mL -- -- Clear 1.13 -- 1.04 Stable. One speck of precipitate. 8 Control-5 mL -- Cloudy Cloudy 1.16 0.21 0.00 Unstable (partial) Fill Volume 9 TRITON X-100 0.500% Clear Clear 1.04 0.99 1.11 Stable10 PS-80 0.500% Clear Cloudy 1.12 0.95 0.59 Unstable11 PLURONIC P123 0.500% Clear Clear 1.15 -- 1.13 Stable12 BRIJ 30 0.500% Cloudy Cloudy 1.08 -- 1.14 BRIJ 30 alone is cloudy.13 TRITON X-100 0.100% Clear Clear 1.00 1.01 0.98 Stable14 TRITON X-100 0.010% Slt. Haze Cloudy 0.96 0.84 0.04 Unstable15 PLURONIC P123 0.100% Clear Clear 1.08 1.08 1.08 Stable16 PLURONIC P123 0.100% Clear Clear 1.23 1.26 0.94 Stable17 PLURONIC P123 0.050% Clear Slt. Haze 1.21 1.18 1.11 Unstable18 PLURONIC P123 0.010% Cloudy Cloudy 1.14 0.00 0.00 Unstable19 BRIJ 30/PS-80 0.1%/0.125% Clear Clear 1.19 1.21 1.17 Stable20 BRIJ 30/PS-80 0.075%/0.094% Clear Clear 1.22 1.20 1.18 Stable21 BRIJ 30/PS-80 0.03%/0.038% Slt. Haze Cloudy 1.20 1.05 0.41 Unstable22 BRIJ 30/PS-80 0.01%/0.013% Cloudy Cloudy 1.14 0.48 0.00 Unstable23 PLURONIC F68 0.100% Clear Slt. Haze 1.23 1.22 0.95 Marginal Stability24 PLURONIC F68 0.100% Clear Slt. Haze -- -- 1.00 Marginal Stability25 PLURONIC F68 0.150% Clear Slt. Haze -- -- i .06 Marginal Stability26 PLURONIC F68 0.200% Clear Slt. Haze -- -- 1 .04 Marginal Stability27 PLURONIC F68 0.300% Clear Slt. Haze -- -- 1.10 Marginal Stability28 PLURONIC F68 0.500% Clear Slt. Haze -- -- 1.08 Marginal Stability29 PLURONIC P123 0.070% Clear Clear 1.06 1.08 0.97 Marginal Stability30 BRIJ 30/PS-80 0.05%/0.063% Clear Clear 1.04 1.01 1.01 Stable31 PLUR F68/PS-80 0.1%/0.1% Clear Clear 1.05 1.06 1.10 Stable32 PLUR F68/BRIJ 30 0.1%/0.03% Clear Clear 1.05 1.05 1.03 Stable33 PLUR F68/BRIJ 30 0.1%/0.01% Clear Clear 1.06 1.04 1.05 Stable34 PLURONIC F88 0.100% Cloudy Cloudy 1.07 0.87 0.56 Unstable35 PLURONIC F98 0.100% Cloudy Cloudy 1.04 0.77 0.39 Unstable36 PLURONIC F108 0.100% Clear Cloudy 1.04 0.87 0.55 Unstable37 PLURONIC F127 0.100% Clear Clear 1.06 1.04 0.98 Marginal Stability38 PLUR F68/BRIJ 30 0.075%/0.01% Clear Clear 1.12 -- 1.11 Stable39 PLUR F68/BRIJ 30 0.05%/0.01% Clear Clear 1.12 -- 1.09 Stable40 PLUR F68/BRIJ 30 0.025%/0.01% Clear Clear 1.10 -- 1.04 Stable41 PLUR F68/BRIJ 30 0.01%/0.01% Cloudy Cloudy 1.07 -- 0.64 Unstable42 PLURONIC F127 0.100% Clear Clear 1.12 -- 0.93 Marginal Stability43 PLURONIC F127 0.075% Clear Clear 1.10 -- 0.61 Unstable44 PLURONIC F127 0.050% Clear Slt. Haze 1.09 -- 0.20 Unstable45 PLURONIC F127 0.025% Slt. Haze Cloudy 1.07 -- 0.00 Unstable46 PLURONIC F127 0.010% Cloudy Cloudy 1.06 -- 0.00 Unstable47 PLUR F68/BRIJ 30 0.05%/0.01% Clear Clear 1.04 -- 1.01 Stable48 PLUR F68/BRIJ 30 0.05%/0.008% Clear Clear 1.01 -- 1.01 Stable49 PLUR F68/BRIJ 30 0.05%/0.005% Clear Clear 1.00 -- 1.03 Stable50 PLUR F68/BRIJ 30 0.03%/0.008% Clear Clear 1.06 -- 0.99 Marginal Stability51 PLUR F68/BRIJ 30 0.03%/0.005% Clear Cloudy 1.01 -- 0.79 Unstable52 PLUR F68/PS-80 0.1%/0.05% Clear Clear 1.14 -- 1.11 Stable. A few specks.53 PLUR F68/PS-80 0.1%/0.01% Clear Clear 1.14 -- 1.11 Stable. A few specks.54 PLUR F68/PS-80 0.1%/0.005% Clear Clear 1.15 -- 1.10 Stable. A few specks.55 PLUR F68/PS-80 0.1%/0.001% Clear Clear 1.14 -- 1.09 Stable. A few specks.56 PLUR F68/PS-80 0.1%/0.005% Clear Cloudy 1.12 1.09 1.02 Unstable57 PLUR F68/PS-80 0.1%/0.0001% Slt. Haze Cloudy 1.09 1.09 1.02 Unstable58 PLUR F68/PS-80 0.05%/0.001% Clear Cloudy 1.08 1.00 0.72 Unstable__________________________________________________________________________ In this example, a study was conducted to compare the efficacy of rBPI23 formulated with and without the preferred formulation of the invention in an actinomycin-D sensitized mouse model according to Pieroni et al., Proc. Soc. Exp. Biol. & Med.; 133, 790 (1970). According to this example, ICR mice were administered an intravenous injection of actinomycin-D (800 μg/kg). Immediately thereafter, groups of 15 mice each received an injection of one of several doses of rBPI23 BR-1! characterized by about 1 to about 199 of the first 199 amino acids of the mature human BPI molecule and produced according to the methods of Theofan et al., U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993 at 1 mg/mL in citrate buffered saline (0.2M citrate, 0.15M NaCl, pH 5.0). The mouse injections were at dosages of 0.03, 0.1, 1.0 and 3.0 mg/kg. As a control, some animals received the formulation buffer with or without the poloxamer and polysorbate surfactants. Deaths were recorded over seven days. The results are shown in FIGS. 1 and 2. FIG. 1 shows the number of mice surviving on each study day in the buffer and 3.0 mg/kg rBPI23 treatment groups. For both buffer groups (with or without poloxamer and polysorbate surfactants), mortality was 80% overall. In contrast, rBPI23 in the presence of excipients was even more potent than either buffer or rBPI23 without excipients. FIG. 2 summarizes the data for the different dose groups at day 7 (final survivors). Beginning at the 0.1 mg/kg dose level, rBPI23 formulated with the preferred surfactant formulations provided significantly greater protection to the lethal effects of LPS (P<0.05 or better) than did rBPI23 in the absence of added excipients. In this example, experiments were conducted to determine the turbidity of various rBPI-containing pharmaceutical compositions with and without the preferred surfactant formulation of the invention. In this context, turbidity refers to the tendency of pharmaceutical compositions to engage in unfolding (i.e., loss of tertiary protein structure) and/or particle formation (interactions between individual proteins to form larger (>10 μm) particles). The pharmaceutical compositions tested contained either rBPI(1-199)ala132, rBPI(1-193)ala132 or various samples of rBPI23 produced according to co-owned and co-pending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993 in either a citrate buffer (20 mM sodium citrate/150 mM sodium chloride, pH 5.0) or a citrate buffer containing 0.1% poloxamer 188 and 0.002% polysorbate 80. Samples were analyzed to determine their resistance to turbidity over time at increasing temperature and at pH 7.0. Prior to analysis, all samples were diluted to a concentration of 0.1 mg/mL in 50 mM potassium phosphate at pH 7.0. Turbidity measurements were obtained by placing samples in quartz cuvettes for use in a Shimadzu UV-160 UV-Vis spectrophotometer equipped with a temperature-controlled cuvette holder attached to a recirculating water bath. Upon equilibrating the cuvette holder at 57� C., absorbance at 280 nm was measured to confirm that samples had been diluted to the proper concentration. Following this, the absorbance of samples at 350 nm was measured every 2 minutes for 1 hour to determine the change in absorbance over time. Results are presented in FIG. 3 showing a lower rate of change in turbidity (i.e., a lower rate of increase in absorbance over time), indicating increased stability against the formation of particles. As shown in FIG. 3, the addition of the preferred combination of surfactants resulted in increased stability (resistance to particle formation) of all compositions tested. Moreover, the rBPI(1-199)ala123 and rBPI(1-193)ala132 exhibited greatly improved resistance to particle formation relative to wild-type compositions rBPI23 !. In this example surface tension measurements were made of polysorbate and poloxamer surfactants or combinations of the two in solutions of the BPI protein product rBPI21 Δcys according to the procedure set out in the Kruss Digital Tensiometer K10ST Users Manual, Chapter 4: Measuring with the Plate. A decrease in surface tension indicates an increase in the surface activity of the surfactant, which has conventionally been thought to be the mechanism by which surfactants stabilize proteins. These procedures established that poloxamer surfactants provide advantageous results by a different and unexpected mechanism. Specifically, a 2 mg/mL solution of unformulated rBPI21 Δcys (lot 30216) was diluted with 20 mM sodium citrate, 150 mM sodium chloride, pH 5.0 rendering a 1 mg/mL solution. 15 mL of this solution was placed into a 50 mL glass beaker containing a mini stir bar. Surfactants poloxamer 188, polysorbate 80, or combinations of both were added incrementally up to 0.10%. Before each surface tension measurement, the platinum plate was heated above the reducing zone (blue flame) of a gas burner until the plate just began to glow red. The platinum plate was heated for about 10 to 15 seconds while turning the plate from side to side and then suspended back into the instrument. Each addition of surfactant was gently mixed using a magnetic stirrer and the solution was allowed to stand for 2 minutes on the thermostat vessel equilibrated at 4.6� C. The value for the surface tension was read after five minutes. The first part of this experiment evaluated the surface activity of the surfactants alone in buffer. Using the citrate saline buffer (20 mM sodium citrate, 150 mM sodium chloride, pH 5.0) as the baseline, surfactants were added incrementally. FIG. 4 is a plot of surface tension dependence on surfactant concentrations; the corresponding data is presented in Table 3. The open squares represent the citrate saline buffer in varying concentrations of poloxamer 188 while the closed circles represent the same buffer in varying concentrations of polysorbate 80. The citrate-saline buffer solution alone had a surface tension of about 75 mN/m at 4.6� C., similar to H2 O. With increasing concentrations of surfactants, the buffer solution showed decreasing surface tension. With 0.10% poloxamer 188, the surface tension of the solution was 55 mN/m. On the other hand, with 0.10% polysorbate 80, the surface tension of the solution was 45 mN/m. The decrease in surface tension indicates an increase in the surface activity of the surfactant, i.e., the lower the surface tension, the higher the surface activity. The results indicate that polysorbate 80 is more surface active than poloxamer 188. In the second part of the experiment, the surface activity of rBPI21 Δcys in the presence of surfactants was evaluated. The results show that rBPI21 Δcys at 1 mg/mL in citrate saline buffer, pH 5.0, is surface active with a surface tension of about 54 mN/m at 4.6� C. The addition of polysorbate 80 (PS80) alone up to 0.0005% did not change the surface tension of rBPI21 Δcys solution either (FIG. 4, closed triangles). At concentrations of polysorbate 80 exceeding 0.0005%, the surface tension of rBPI21 Δcys follows that of buffer with PS80 alone (no BPI), in which the surface tension of the solution decreases as the concentration of polysorbate 80 is gradually increased. For buffer with PS80 alone, the surface tension of 54 mN/m was reached when the PS80 concentration was increased from 0.0005%. These results indicate that when PS80 concentration is less than 0.0005%, the surface activity of the solution is dominated by rBPI21 Δcys. On the other hand, at PS80 concentration above 0.0005%, the surface activity of the solution is modulated by polysorbate 80 the addition of poloxamer 188 (F68) alone to rBPI21 Δcys up to 0.10% did not change the surface activity of rBPI21 Δcys solution significantly (FIG. 4, open triangles). TABLE 3__________________________________________________________________________ 8 2 4 6 ΔCys + 10 Buffer + Buffer + ΔCys + 0.1% F68 + ΔCys +1 F68 3 PS80 5 F68 7 PS80 9 PS80% F68 (mN/m) % PS80 (mN/m) % F68 (mN/m) % PS80 (mN/m) % PS80 (mN/m)__________________________________________________________________________1 0.00000 75.4 0.00000 75.1 0.00000 54.2 0.00000 53.7 0.00000 54.92 0.00001 74.9 0.00001 66.8 0.00001 54.7 0.00001 53.4 0.00001 55.03 0.00003 74.3 0.00002 60.0 0.00002 54.2 0.00002 53.3 0.00002 53.24 0.00005 68.2 0.00003 60.0 0.00003 54.9 0.00003 53.9 0.00003 53.35 0.00007 65.9 0.00005 60.0 0.00004 54.8 0.00004 53.9 0.00004 52.86 0.00010 64.0 0.00007 57.4 0.00005 55.0 0.00005 53.5 0.00005 52.47 0.00013 65.8 0.00010 56.6 0.00006 55.2 0.00006 53.5 0.00006 53.38 0.00015 65.4 0.00015 57.2 0.00007 55.4 0.00007 53.4 0.00007 53.69 0.00017 66.5 0.00020 56.7 0.00008 54.8 0.00008 53.8 0.00008 53.810 0.00020 65.7 0.00050 55.6 0.00009 55.0 0.00010 53.4 0.00009 53.211 0.00023 66.0 0.00070 55.3 0.00010 54.9 0.00020 53.5 0.00010 53.512 0.00027 64.4 0.00100 54.2 0.00030 55.3 0.00030 53.2 0.00020 53.213 0.00030 63.8 0.00300 52.7 0.00050 54.5 0.00050 52.3 0.00030 53.014 0.00033 64.1 0.00700 49.2 0.00070 55.5 0.00070 51.5 0.00050 52.015 0.00037 63.1 0.01000 48.3 0.00100 54.9 0.00100 51.0 0.00070 51.216 0.00040 64.2 0.03000 46.5 0.00500 54.9 0.00200 50.6 0.00100 50.517 0.00043 61.8 0.07000 45.3 0.01000 55.4 0.00500 50.1 0.00130 50.418 0.00047 62.4 0.10000 45.4 0.05000 53.6 0.01000 48.6 0.00170 49.819 0.00050 63. 0.10000 53.7 0.05000 45.6 0.00200 48.820 0.00060 61.6 0.10000 45.0 0.00500 47.721 0.00070 62.5 0.01000 46.722 0.00080 62.0 0.05000 45.423 0.00100 61.7 0.10000 45.024 0.00300 61.225 0.00500 59.326 0.00700 58.927 0.01000 58.428 0.03000 56.629 0.07000 56.130 0.10000 55.1__________________________________________________________________________ Protein samples were analyzed by Differential Scanning Calorimetry (DSC) to study the unfolding (or denaturation) of the protein. The starting materials for DSC analysis were identical to those used in the surface tension measurement. A series of rBPI21 Δcys solutions was prepared with varying concentrations of surfactants, poloxamer 188, polysorbate 80 or combinations of both, and diluted with buffer (20 mM sodium citrate, 150 mM sodium chloride, pH5.0) to give a final rBPI21 Δcys concentration of 1 mg/mL. A series of buffer solutions was also prepared with surfactants at the same concentrations as in the rBPI21 Δcys solutions to serve as blanks for DSC. Each solution was filtered and placed into a 2 mL sterile plastic vial. The samples were packed into a 4� C. cold box until subjected to DSC Analysis. The behavior of rBPI21 Δcys was evaluated as the temperature of the solution was gradually increased from ambient temperature to about 90� C., at a rate of 1 � C. per minute. As the temperature is increased two events occur. The first event is an unfolding reaction, which is endothermic, and is illustrated by an upward peak in the scans. The second event is precipitation, which is exothermic, and is depicted by a downward peak in the scans. In the scans depicted in FIGS. 5, 6 and 8-10, each scan is offset to facilitate analysis of data. In the rBPI21 Δcys solution not containing surfactants (FIG. 5, Scan 1) the unfolding of the protein at 65� C. was followed immediately by the second event, precipitation of the protein at 66� to 67� C. With low poloxamer 188 (PLURONIC� F68) concentrations ranging between 0.001% to 0.01%, the unfolding and precipitation events are similar to the rBPI21 Δcys solution without surfactants (FIG. 5, Scans 2 to 5), i.e. as rBPI21 Δcys unfolds, precipitation takes place immediately. With poloxamer 188 concentrations exceeding 0.05%, the unfolding of rBPI21 Δcys still occurs at 65� C., but precipitation does not occur until the temperature reaches 85� C. (FIG. 5, Scan 6). FIG. 6 shows that at poloxamer 188 concentrations between 0.01% and 0.05%, there is a gradual transition of delayed precipitation of unfolded BPI. These results suggest that at poloxamer 188 concentrations higher than 0.01%, unfolded rBPI21 Δcys can be stabilized and the occurrence of precipitation is delayed. A plot of denaturation and precipitation temperature dependence over the surfactant (poloxamer 188) concentration is shown in FIG. 7. The effects of poloxamer 188 appear to delay the precipitation of rBPI21 Δcys to a higher temperature but not to stabilize its native structure as the Tm (denaturation temperature) and ΔH (energy of denaturation) did not change. rBPI21 Δcys formulated with polysorbate 80 at concentrations up to 1% was likewise analyzed. The isotherms were similar to rBPI21 Δcys solution without surfactants (FIG. 8: Scans 1 and 8-13, FIG. 9: Scans 11, 12). Polysorbate 80 did not maintain the rBPI21 Δcys in solution at higher temperatures. The stabilization of unfolded rBPI21 Δcys is thus unique to poloxamer 188. The two formulations using combined poloxamer 188 and polysorbate 80, namely 0.1%F68/0.001%PS80 and 0.1%F68/0.002%PS80, showed the same scan profile as rBPI21 Δcys containing 0.05% and 0.1% PLURONIC F68, with unfolding at 65� C. and precipitation at 85� C. (FIG. 8: Scans 14, 15). In addition to determining the melting behavior of rBPI21 Δcys, rescanning was done with rBPI21 Δcys formulations containing 0.05% and 0.10% poloxamer 188 to determine if unfolding is a reversible process. The temperature of the rBPI21 Δcys solution was first increased to 75� C. (temperature after denaturation/unfolding but before precipitation), then was cooled down for repeat scanning. FIG. 10 shows that the addition of poloxamer 188 to rBPI21 Δcys does not make unfolding reversible. Profiles A5,1 and A6,1 show the scanning to 75� C., while profile A5,2 and A6,2 are repeat scanning after cooling the system from 75� C. If unfolding were a reversible process, 6 and 7 scan profiles would have been obtained. The experimental results described above demonstrate that poloxamer surfactant alone is capable of stabilizing BPI-related polypeptides in solution and delaying the occurrence of precipitation by a mechanism that does not appear to involve modulation of the surface tension of the aqueous solution. This property is unique to poloxamer because other surfactants such as polysorbate 80 do not affect the precipitation phenomenon and do involve modulation of the surface tension of the aqueous solution. The rate of rBPI21 Δcys precipitation during shipping was simulated in the laboratory by adjustment of the speed of the horizontal shaker. During five cycles of surface shipping, about 70% of the unformulated (surfactant free) rBPI21 Δcys precipitated. By varying the speed (rpm) of the flat-bed shaker, shake tests were then constituted such that 70 to 90% of unformulated rBPI21 Δcys subjected to the shake test precipitated. No rBPI21 Δcys was precipitated when the unformulated product was shaken on a flat bed shaker at 110 rpm or less for 18 hours at 4� C. Shaking at 140 rpm (rather than at 150 rpm as in Examples 1 and 2) most closely simulated the agitation occurring during five cycles of surface transport. Changes in the flow dynamics of the liquid in the vial are substantially different at 140 rpm versus 150 rpm. Compositions including various concentrations of surfactant combinations were screened using the 140 rpm shake condition and the results obtained are set out in Table 4. It was determined that the optimal surfactant concentrations for protection from precipitation were 0.2% poloxamer 188 with 0.002% polysorbate 80 and 0.15% poloxamer 188 with 0.005% polysorbate 80. TABLE 4______________________________________Summary of Shake Test at 140 rpm for rBPI21 Δcys at 4�C.Poloxamer PS80 Visual Concentration by MA7C HPLC (mg/ml)188(%) (%) (see note) Before After Loss (%)______________________________________ 0.005 5 2.14 1.54 280.075 0.010 4 2.10 1.56 26 0.020 1 2.14 1.68 21 0.002 5 2.24 1.85 17 0.005 4 2.14 1.85 140.100 0.010 1 2.10 1.87 11 0.020 1 2.13 1.94 9 0.002 3 2.19 1.92 12 0.005 2 2.08 1.95 60.150 0.010 1 2.19 1.94 11 0.020 1 2.06 1.96 5 0.002 2 2.19 1.98 100.200 0.005 1 2.19 1.95 11 0.005 5 2.14 1.54 28 0.010 1 2.22 1.95 12______________________________________ Note: The scoring for visual observation is as follows: 1. Clear 2. Clear with few particulates 3. Slightly hazy 4. Hazy 5. Cloudy Based on the above data, a preferred formulation for 2 mg/mL rBPI21 Δcys to be stored at 4� C. would contain 5 mM citrate, 150 mM NaCl, pH 5.0, 0.2% poloxamer 188 and 0.002% polysorbate 80. An alternative formulation for 2 mg/ml rBPI21 Δcys to be stored at 4� C. would contain 5 mM citrate, 150 mM NaCl, pH 5.0, 0.15% poloxamer 188 and 0.005% polysorbate 80. In summary, aggregation/precipitation is one of the major causes of protein instability and can occur when proteins at the air-liquid interface unfold and expose hydrophobic domains. If left unprotected, proteins self-associate through the interaction of the exposed hydrophobic domains, resulting in aggregation and/or precipitation. With the use of the surfactants and surfactant combinations of the invention, protein can be stabilized in two ways. First, exposed hydrophobic regions at the air-liquid interface are shielded by poloxamer surfactants. Second, additional stabilization can be provided by polysorbate surfactants through conventional modulation of the surface activity of the solution. Numerous modifications and variations of the above-described invention are expected to occur to those of skill in the art. Accordingly, only such limitations as appear in the appended claims should be placed thereon. Patent Citations
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Apple Is Going To Build A Mac In The USA
Apple CEO Tim Cook says an "existing" line of Macs is going to be assembled in the U.S. starting next year.
Apple CEO Tim Cook is on the interview circuit, with "exclusive" interviews given to both Bloomberg Businessweek and Brian Williams on NBC's Rock Center. There is one very important piece of news contained within them: Next year, Apple is going to have one of its "existing" lines of Macs made here in the United States. Which, for some reason, feels a little bit like that moment in Return of the Jedi when Mon Mothma informs the rebels, "But most important of all, we've learned that the Emperor himself is personally overseeing the final stages of the construction of this Death Star."
We've been working on this for a long time, and we were getting closer to it. It will happen in 2013. We're really proud of it. We could have quickly maybe done just assembly, but it's broader because we wanted to do something more substantial. So we'll literally invest over $100 million. This doesn't mean that Apple will do it ourselves, but we'll be working with people, and we'll be investing our money.
The line of Macs that Cook is referring to could be the iMac, given that some of the new models have already shipped with "Assembled in USA" etched onto their aluminum skin. This will no doubt satiate some critics who unfailingly press for more manufacturing jobs in the United States, but Macs are in fact a tiny business for Apple compared with the iPhone and iPad — Apple sold nearly 27 million iPhones and 14 million iPads last quarter, compared to just under 5 million Macs — and one that has a comparatively limited life as computers become increasingly mobile. Cook is, of course, quick to point out that the iPhone and iPad's processors are also made in the U.S. (albeit, by Samsung), but iPhones and iPads are mostly assembled in China. Which should reinforce the point that assembling Macs here does nothing to change the fact that most of its parts are still going to be produced abroad by foreign companies, even if they're going to be put into place and fastened together on a line somewhere here in the U.S. by whoever Apple partners with to make them.
Cook himself explains to Brian Williams why making consumer electronics entirely in the USA on the scale that Apple does is a staggeringly difficult proposition: It's "not so much about price; it's about the skills… Over time, there are skills that are associated with manufacturing that have left the U.S." Part of the reason Foxconn workers went on strike, in fact, is because of the iPhone 5's insane difficulty to manufacture. And The New York Times' report on the "iEconomy" makes what Cook is referring to exceedingly clear, with giant numbers:
Apple's executives had estimated that about 8,700 industrial engineers were needed to oversee and guide the 200,000 assembly-line workers eventually involved in manufacturing iPhones. The company's analysts had forecast it would take as long as nine months to find that many qualified engineers in the United States.
In China, it took 15 days.
Putting the iMac together here isn't going to change that anytime soon. But as Cook says, it is, if nothing else, "another step."

subset: commoncrawl


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Monticchiello is a pretty medieval village in the heart of the Val d'Orcia, which origins lose themselves in the time.The first official document dates back to the 973, when the marquis Lamberto Aldobrandesco surrendered the castle to the Badia Amiatina for a conspicuous sum of money. In the following years Monticchiello will belong to the Roman church and particularly to the Teutonici riders (until 1230), that being an order accustomed to fight, resulted proper to administer a territory of frontier. The first document relating the free Commune of Monticchiello brings the date of the 1243. From this moment, for beyond three centuries, the castle remained faithful to Siena until his capitulation in 1559 with the Frank-Spanish war, becoming, like the whole state of Siena, part of the Granducato of Tuscany. After five centuries the Commune of Monticchiello came under the jurisdiction of the Commune of Pienza in 1777.
Teatro Povero di Monticchiello The poor Theater of Monticchiello, born in 1967, realizes a life and culture experience particularly interesting. The inhabitants of the place, in fact, from the end of July for almost all month long of August, put in scene a drama, in the main square of the village, with daily replicas, that is completely written, directed and staged by themselves; the same staff creates also the music and the scenery. In the last years the performances, "recited" in the typical Val d'Orcia dialect; have been divided in two parts, the first concerning experiences of the past country life during the first half of the 20th century, the second dramatising themes of current events. The theatrical experience of the people of Monticchiello from "popular experiment" has become a meaningful example of making theatre in Italy, with flattering judgments of public and review.


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Hefei Castle
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Battle of Hefei Castle | |||||||
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Forces | |||||||
Wu | Wei | ||||||
Commanders | |||||||
Sun Quan (234) Zhuge Ke (253) | Cao Rui (234) Man Chong (234) Sima Shi (253) | ||||||
The Battle at Hefei Castle (合肥新城の戦い, rōmaji: Gappi Shinjō no Tatakai) or literally "Battle of Hefei New Castle" in the Asian ports, is one of the conflicts during the Battles of Hefei featured in the Dynasty Warriors series. Historically, Sun Quan led an assault on the castle that Wei built within He Fei and it was his last known military conflict with Wei. In the games, the conflict takes place during the final year of the castle's sieges and it is the decisive battle between Wu and Wei.
[show]Role in GamesEdit
Dynasty WarriorsEdit
In Dynasty Warriors 3, Wei's centered in and around the northern part of the map in a castle. The Wu army leads resistance in the center, and to the eastern and western parts of the stage with Zhen Ji and Sun Shang Xiang respectively. At the northeastern most corner of the stage lies bridge layer ruins with a drop on each side and stairs to get back up with, at the end of which archers and a deadly set of crossbow units are placed and a special item is often at the end of the second bridge layer ruins (which has a broken end unlike the other and features little-to-no enemies around the initial non-isolated side and past the first layer ruins).
As gates open, the various set traps in the western part of the stage can be seen (hidden arrows, and gusts of wind). Wu is found along the shoreline of the southern part of the map. In Wu's scenario, players have the option of going either left, right or through the center of enemy resistance. The left path offers hidden arrows and wind gusts, of which cannot be ceased, the right path features high enemy resistance, and the center path triggers a fire attack. Walking on the central northern lake with the bridge formation (just outside and in front of the castle's most northern and final palace where Sima Yi/Cao Cao resides) will trigger an archer ambush cutscene, and will occur on both Wei and Wu scenarios with the archers being of the opposing side regardless.
In both scenarios, Wei reinforcements will appear in the southeast near Wu's main camp after a set amount of time. Near the Wu forces, the central south area of the castle is an ambush that will prevent the player's forces from entering through there, and they must open the gates on the other side from the west and eastern point entries. This also applies to the Wei forces as well, where Lu Xun will declare a fire attack on the Wei forces who try to exit out the castle in the central entrance via a planned trap (due to their greater control over the castle grounds in Wei's scenario).
Despite not being an actual stage by itself in Dynasty Warriors 4, a portion of the incomplete (and currently-under-construction) castle is used as part of the default He Fei map and its variations.
In Dynasty Warriors 5, Wei is within He Fei Castle's inner, and outside areas. Wu's front is to the east and west of the outer region of the castle. Oscillating statues that shoot fireballs can be seen on the east and west sides of the stage, but only damage Wu officers. Rams will try breaking the central outer gates of He Fei Castle. There are critical ambush points in each region that one must be wary of. The inner pathways leading to the courtyard are also filled with fire breathing lion statues. Like in the fourth installment, incomplete traces of the future castle can be seen on the west side of the previous He Fei stage as a foreshadowing to the New Castle battle.
In Dynasty Warriors 6, the Wu forces besiege the castle from three sides. The castle itself contains three traps ― One central conveyor belt and two fire pits in the western and eastern sectors of the castle. The conveyor belt leads to the inner main gate and activates every ten seconds or so, moving every soldier or item on it into a pool of water below. To deactivate the belt, the player must destroy the inner main gate. The fire pits are courtyards accompanied by traveling machines that are able to breathe fire. In Wu's scenario, the pits can be stopped by destroying all of the courtyard's machines. By contrast, in Wei's scenario, the pits will be stopped if the defending officer is defeated.
The castle siege in Dynasty Warriors 7 takes place after Sun Quan's death. Zhuge Ke leads Wu troops after gaining momentum from Dongxing. To counter their push towards Wei, Sima Shi reinforces Sima Zhao and Zhuge Dan's whittled armies.
Warriors OrochiEdit
During the first game of the series, it serves as the stage for the last Wei gaiden. Cao Cao leads a siege against Orochi at the castle, telling his army to invade from three different directions. Da Ji acts as the enemy main strategist and triggers the castle's traps as the Wei army moves in. To aid their advance, Cao Cao sends engineers to break the gates blocking the mountain path. Keiji appears to guard it. If the plan succeeds, the ally army will have a morale boost.
As the battle progresses, Sun Ce, wanting to repay his debt to Cao Pi, appears to reinforce Cao Cao from the eastern docks. Da Ji also has engineers ready to burn down Cao Cao's main camp while he leaves it undermanned. Lu Bu also tires of Da Ji's orders and charges the invading army at whim. If Cao Cao's earlier plan succeeded, he will be impressed with the invading army after his defeat.
In Warriors Orochi 2, the stage is used as a dream stage for three master strategists: Zhuge Liang, Zhou Yu, and Shingen. They work together to defend the castle from Sima Yi's invading army.
Historical InformationEdit
After Sun Quan suppressed the Shan Yue tribes in Jingzhou and the minority in Wiling, he desired to recapture Wu's Hefei Castle and push back Wei. The previous defender of the area, Cao Xiu, died shortly after his defeat at the Battle of Shiting so his position was replaced by Man Chong. In Sun Quan's first offensive in 230, he marched his army near a location that was close to a dam. Since his advisers feared a water attack, he abandoned the expedition. A year later, he ordered Sun Bu to feign surrender to the Governor of Yangzhou, Wang Ling, and sow discord between him and his subordinate, Man Chong. The Wei retainers saw through the plot and ended it before it began.
In 233, Man Chong abandoned Wu's Hefei Castle and decided to build a new castle in Hefei, literally translated as "Hefei New Castle" for emphasis, and which became the subject of a long string of defensive and offensive battles. It is generally debated that Man Chong did this on purpose to throw off the Wu army. Researchers note that the older castle would have been difficult to protect in long drawn out sieges and, though its design indicates that it was well defended, it was impractical for the Wei army to keep for long due to Wu's familiarity with the area. The new castle's water ways prevented heavy naval assaults and allowed the Wei army to strategically station ambushes along Wu's retreats. The Wu army continued to attack the fortification but avoided long-term sieges. During the same year, Sun Quan led an army to take the western area around the new Hefei Castle, but was driven back by the military forces that were there.
In 234, Sun Quan gathered an army of 100,000 to take the new castle. This attack happened at the same time as Shu's Fifth Northern Campaign. While Sun Quan stationed himself in Jingzhou with Lu Xun and Zhuge Jin, he ordered Sun Shao and Zhang Cheng to attack from their respective locations. Marching towards the fortification from three fronts, Sun Quan ordered a full-scale charge. Man Chong countered with a massive fire attack, which crippled Wu's siege. After losing one of his nephews, Sun Tai, and additionally seeing his troops plagued by illness, Sun Quan abandoned the idea of taking the castle and ordered a retreat. It is said that the battles made him realize his foes' ferocity and was the reason why he did not commit to waging war with Wei for the rest of his days.
As a side note, Wei had predicted Sun Quan's assault but Sima Yi was busy dealing with Zhuge Liang's offensive. Cao Rui led troops as Hefei's reinforcements yet Sun Quan's army had already retreated by the time he arrived.
Romance of the Three KingdomsEdit
Romance of the Three Kingdoms wrote about the last encounter in chapters 102 and 103. In accordance with their alliance, Zhuge Liang requested for Sun Quan to aid the Shu army by raising arms. The Emperor of Wu agreed to do so and coordinated three hundred thousand men to march immediately toward Xincheng, Xiangyang and Huaiyang. Sun Quan would ride to Xincheng, Lu Xun and Zhuge Jin were to take Xiangyang, and Sun Shao and Zhang Cheng were ordered to take the last location.
As Sima Yi was fighting Zhuge Liang, Cao Rui led a three way defensive force to counter Wu's army. Cao Rui went with Man Chong into Hefei to counter Sun Quan. Liu Shao was sent to Jiangxia and Tian Du led the army at Xiangyang. Spotting a huge fleet from Wu, Man Chong devised a night raid and a fire attack on Zhuge Jin's men. The plan succeeded and Zhuge Jin scarcely fought and fled. When Lu Xun heard of the news, he gathered his officers into a meeting and all agreed to have Sun Quan cancel his siege of Xincheng. He planned to trap the pursuing Wei army in a pincer attack, with himself leading the frontal assault. Lu Xun sent a messenger but his envoy was captured by Wei's army.
With Zhuge Jin's troops suffering from illness and his plot discovered by the enemy, Lu Xun advised the army had best withdraw. Sending a memorial to Sun Quan for a retreat, Lu Xun and Zhuge Jin delayed their departure and feigned a strong front so that the three armies could safely escape.
This battle article is a stub. You can help the wiki by expanding it. |


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» GE Profile does not clean properly |
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GE Profile does not clean properly
Model Number: PDW7880J00SS Brand: GE Age: 5 - 10 years
Leaves residue on dishes. 8 years old. Does not rinse properly. Only silverware cones out clean. We use good quality dish washing detergent. Any suggestions? Related Models |
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Here are your parts
Replacement parts for General Electric PDW7880J00SS Dishwasher | AppliancePartsPros.com Often if you click on a part's picture a new page will open with more info about that part. Check that all the holes in the spray arms are clean. Check that the unit is filling properly. Most units fill to just under the heating element. One way to ensure this is to add a couple quarts of hot water after each fill during a cycle. Just be careful to not overfill the unit. If it then washes OK the problem is probably a dirty screen filter on the input side of the fill valve. Remove and check/clean the fine filter (Item 451 in Section 4). Check in the sump (Item 505 in Section 4) for debris or something wrapped around the chopper. Be careful as there is often broken glass in the sump.
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Won't clean properly- water luke warm | ERob | Dishwasher Repair | 0 | 11-30-2011 07:50 AM |
GE profile doesn't drain properly. | flunkymonkey | Dishwasher Repair | 0 | 04-18-2011 05:41 PM |
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How to Properly Clean your Coffee Maker | Dennis Andrews | General Talk | 3 | 12-16-2008 03:11 AM |


text:
(1735 quotes found)
"The best way to persuade people is with your ears - by listening to them."
Dean Rusk
"If you spend more time asking appropriate questions rather than giving answers or opinions, your listening skills will increase"
Brian Koslow
"What Women Want: To be loved, to be listened to, to be desired, to be respected, to be needed, to be trusted, and sometimes, just to be held. What Men Want: Tickets for the world series."
Dave Barry
"Do not follow the ideas of others, but learn to listen to the voice within yourself. Your body and mind will become clear and you will realize the unity of all things."
"Set up the listening. Prepare who you are talking to for what you want them to hear. Get people to listen as a possibility rather than a problem."
Mal Pancoast
"" Evervybody hears, but few listen"
Bobby Knight
"If you make listening and observation your occupation you will gain much more than you can by talk."
Sir Robert Baden-Powell
"If the person you are talking to doesn't appear to be listening, be patient. It may simply be that he has a small piece of fluff in his ear."
Winnie the Pooh
"When anyone tells me I can't do anything... I'm just not listening any more."
Florence Griffith Joyner
"Make sure you have finished speaking before your audience has finished listening"
Dorothy Sarnoff


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Max Occupancy: 4 guests (up to 3 children)Standard Room, Multiple Beds, Non Smoking, Refrigerator & Microwave
Max Occupancy: 6 guests (up to 5 children)Standard Room, Non Smoking, Refrigerator & Microwave
Max Occupancy: 8 guests (up to 7 children)Standard Room, 1 King Bed, Accessible, Non Smoking
Max Occupancy: 2 guests (up to 1 child)Standard Room, 1 King Bed, Accessible, Bathtub
Max Occupancy: 2 guests (up to 1 child)Suburban hotel with free breakfast and indoor pool
Situated in the suburbs, this hotel is within 3 mi (5 km) of The Edison Birthplace, Milan Historical Museum, and Norwalk Reservoirs. Eagle Creek Golf Club and Summit Motorsports Park are also within 6 mi (10 km).
Hotel Features
An indoor pool, a fitness center, and a 24-hour business center are available at this smoke-free hotel. Free buffet breakfast, free WiFi in public areas, and free self parking are also provided. Other amenities include coffee/tea in the lobby, laundry facilities, and a 24-hour front desk.
Room Amenities
All 58 rooms feature comforts like pillowtop beds and premium bedding, while conveniences include refrigerators and microwaves. Free WiFi and coffee makers are standard, as are flat-screen TVs with cable channels.
Information missing or incorrect? Tell us!Hotel Amenities
Hotel Amenities
Best Western Norwalk features an indoor pool and a fitness center. A computer station is located on site and high-speed wireless Internet access is complimentary. This hotel offers access to a 24-hour business center. Guests can enjoy a complimentary breakfast each morning. This Norwalk hotel also offers coffee/tea in the lobby, complimentary newspapers in the lobby, and laundry facilities. Onsite self parking is complimentary. A total renovation of this property was completed in May 2014.
Best Western Norwalk is a smoke-free property.
- 24-hour business center
- RV, bus, truck parking
- Number of buildings/towers - 1
- Fitness facilities
- Number of floors - 2
- Coffee/tea in lobby
- Free newspapers in lobby
- Express check-in
- Express check-out
- Free WiFi
- Computer station
- 24-hour front desk
- Total number of rooms - 58
- Laundry facilities
- Free self parking
- Smoke-free property
- Indoor pool
- Television in lobby
- Free breakfast
Family Friendly Amenities
- Indoor pool
- Free Wi-Fi
- Laundry facilities
- Refrigerator
- Microwave
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking, RV, bus, truck parking
Room Amenities
- Connecting/adjoining rooms available
- Microwave
- Desk
- Refrigerator
- Premium TV channels
- Coffee/tea maker
- Premium bedding
- Hair dryer
- Air conditioning
- Free weekday newspaper
- Phone
- Daily housekeeping
- Private bathroom
- Free toiletries
- Shower/tub combination
- Cable TV service
- Free local calls
- Iron/ironing board
- Flat-panel TV
- In-room safe
- Pillowtop mattress
- Free WiFi
Where to Eat
A complimentary hot/cold buffet breakfast is served each morning between 6 AM and 9:30 AM.
Nearby Things to Do
Recreational amenities at the hotel include an indoor pool and a fitness center.
Children under 16 years old are not allowed in the swimming pool or fitness facility without adult supervision.
The recreational activities listed below are available either on site or nearby; fees may apply.
- Golfing nearby
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
- Accessibility equipment for the deaf
- Accessible bathroom
- Accessible path of travel
- Braille or raised signage
- Accessible parking
- In-room accessibility
- Roll-in shower
Hotel Policies
Minimum check-in age is 21
Check-in time starts at 3 PM
Check-out time is 11 AM
Payment types
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
Only service dogs are allowed
No rollaway/extra beds available
No cribs (infant beds) available
- A 4.5 percent city/local tax will be charged
- 7.25 percent destination fee will be charged
We should mention
Only registered guests are allowed in the guestrooms. The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.


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Lehman College | |
Course Prefix: | ENG Course Number: 304 |
Course Title: | The Structure of Modern English |
Subject: | English |
Minimum Credits: | 3.0 Maximum Credits: 3.0 Hours per week: 3.0 |
Grammatical theory and linguistic descriptions of modern English (such as traditional, descriptive, and transformational grammars), with emphasis on the formal properties of grammar and the formal characterization of language. Samples of modern English to be studied will be drawn from literary works from the early modern English period to the present. |
Prerequisite: | Satisfactory completion of 60 college credits or of two 200-level ENG courses, or Departmental permission. |
Start Date: 08/31/2001 End Date: |


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As you are preparing for Thanksgiving and worrying you will accidentally burn the 15 pounds of turkey you have spent hours dressing, just remember, it could be worse. You could burn four million pounds of turkey. That's what happened to a turkey producer in a 1988 opinion of the Western District of Virginia in which the court found that under the terms of an excess fire policy the "actual cash value" of an insured turkey producer's damaged stock (four million pounds worth) was not the mean price for which the stock would have sold on the date of loss, but rather, was the price for which the stock would have been sold had no loss occurred.
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- Membership
- Publications
- Meetings
- Competitions
- Community
- Programs
- Students
- High School Teachers
- Faculty and Departments
- Underrepresented Groups
- MAA Awards
- MAA Grants
- News
- About MAA
As with Phase I, students engage in two different kinds of activity during Phase II. In the first, students write their own simple webpages. They learn to handle text, links and images. Usually there are a few HTML aficionados in the class who can serve as consultants to the other students. This activity usually takes up very little class time, since the students already know how to edit and post webpages. There are about half a dozen HTML tags they need, and then they are directed to the many sources of HTML information on the Web. The usual assignment for this part of the course is for the students to make a moderately interesting web page. The page should say something about them, and it should also contain a link to some interesting mathematical item on the Web. (We always get links to the Math Forum and to some part of the MAA web pages). A second pass through the webpage creation phase gives students the opportunity to add background and foreground images to their pages.
The other Phase II activity involves studying the source code for some of the games that we played in Phase I and at the code for the several calculators that we used for our mathematical work in the course. We often spend half an hour in each of several classes discussing in general terms how a few of these pages work, so that students get an idea of how programs are conceptualized and then actualized. The game programs were mentioned in the Section 2. Some of the calculators are
Dennis DeTurck, "Mathematical Thinking through Web Programming - Making HTML pages," Loci (October 2004)
Journal of Online Mathematics and its Applications


text:
The CBitmapHandling class was designed to calculate the contour points in a bitmap. It looks at the bitmap and finds pixel points different from white, it then places these start and stop pixel points in a Vertex of CPoint class. When it has found the outline of the bitmap, it then generates a Windows region. If you use this region in your OnInitDialog and then set the window region using SetWindowRgn(m_WinRgn, TRUE ), your window will then be shaped like the outline.
SetWindowRgn(m_WinRgn, TRUE )
The code consists of a header file and a CPP file.
// #include "BitMapHandling.h" using namespace HandlingBitmaps; BOOL CMyShapedWindowDlg::OnInitDialog() { CDialog::OnInitDialog(); VERIFY( SetWindowPos( NULL, 0, 0, m_nW, m_nH, SWP_NOMOVE | SWP_NOOWNERZORDER ) ); // size and pos. of window // cut of areas of region, which you do not really need. // fx. maybe you have bitmap buttons drawn under your bitmap window. CBitmapHandling bh; bh.BitMapContourToWinRgn(&m_WinRgn, IDB_BITMAP); VERIFY( SetWindowRgn(m_WinRgn , TRUE ) ); return TRUE; // return TRUE unless you set the focus to a control } //
This article, along with any associated source code and files, is licensed under The Code Project Open License (CPOL)
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#51535377 -
Farbige Pillenbox
© von Lieres - See portfolioPricing
Author | von LieresSee portfolio |
Image reference | 51535377 |
Country | Germany |
Descriptive Category | Objects Health/Hygiene Pharmaceuticals |
Keywords |
blister capsule colours drug medicine pharmacy pill pillbox sorted
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text:
Dear All 1990 ex-Andersonians,
Please note that the event of the day- 20 YEARS LATER-
batch's reunion event will be held as follows:
Date: 5th June 2010
Location: Anderson School Hall
Time: 8.00pm - 11.00pm
It's official!! its a time for reconciling, restoring old friendship and most important re-establishing and forging greater ties amongst the elite batch of 1990!!
No matter where you are, it is hoped that today's function will rekindle the flame and spirit of Class of 1990 and bring back its elegence and thunder!!
Most important tonights event will lead us to forge the strength that we had before. so please attend tonights event and leave with the zest and redefined spirit of class of 1990!ensure the networking is restored for the future of ourselves and our children.
To strive, seek , and not to yield! ONCE AN ANDERSONIAN, ALWAYS AN ANDERSONIAN.
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text:
"Batman v Superman: Dawn of Justice" Trailer Officially Released
Could Monkey D. Luffy and the Straw Hat Pirates finally be getting their due in North America?
They're the stars of Eiichiro Oda's long-running comedy-adventure One Piece, Japan's best-selling manga that's sold more than 176 million volumes since its debut in 1997. (Publisher Shueisha printed more than 3 million copies of the series' 57th volume alone.) On this side of the Pacific, however, the series hasn't been nearly as popular, overshadowed by the likes of Naruto, Bleach and Fullmetal Alchemist.
But this week, undoubtedly aided by Viz Media's accelerated release schedule, One Piece lands five volumes on The New York Times' Graphic Books bestseller list. (It's probably worth noting that nine of the 10 spots in the manga category are filled by Viz Media releases.)
Yes, the 47th volume of Masashi Kishimoto's Naruto still places higher on the chart than One Piece. And, yes, The New York Times' bestseller lists employ an arcane formula that no one seems to understand (a complex combination of numerology, calculus and chanting within a magic circle, most likely).
Still, it just may be a sign that the tide is turning for the crew of the Going Merry — or is that Merry Go? — on the coasts of North America.
I really hope so. It's a great series.
Finally! One piece will get popular in North America since 4kids mess it up.


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Ash's Frogadier is a water-type Pokémon and is the first Pokémon obtained by Ash in the Kalos region.
Frogadier first appeared as a Froakie when he observed Pikachu shocking Bonnie, Clemont and Ash outside the Lumiose Gym. It followed the group to a nearby park, where it watched the trio talking from a tree. When the group were attacked by Team Rocket, Froakie protected Pikachu from its own Electro Ball, which had been rebounded by Wobbuffet's Mirror Coat. He then used the sticky bubbles on his back, called Frubbles, to hit Wobbufet, and helped Ash defeat Team Rocket. After Team Rocket were sent blasting off, Froakie fainted and Ash took him to Professor Sycamore's lab.
Eventually, Froakie was healed at Sycamore's lab. Froakie was present when Team Rocket tried to take control of Professor Sycamore's Garchomp and assisted Ash in his attempt to help. When Garchomp was about to fall off the Prism Tower, Froakie used his Frubbles to stick Garchomp's feet to the floor and prevent it from falling. This then allowed Pikachu to destroy the machine controlling Garchomp with Iron Tail. At the end of the episode, Froakie appeared in front of Sycamore's lab with a Poké Ball and asked Ash to catch it, which Ash did so gladly.
In A Battle of Aerial Mobility!, Froakie quickly attacks a Fletchling that made Bonnie cry and a Dedenne leave, despite Ash telling him to stand down and let Pikachu attack. Fletchling easily evades every attack and almost knocks Froakie out with a devastating Razor Wind, but Ash pushes him away to save him. Although the group is intially mad, they learn of Froakie's motives, and make a plan for him to take down Fletchling.
They lure Fletchling to an area with many rocks at many levels, which allows to use Froakie to move freely and be on even grounds with the flying type. However, Fletchling's aeiral speed allows him to evade all of Froakie's attacks, although Froakie evades his just as well. Fletchling then gets a solid hit on a hidden Froakie, that was actually a fake made of frubbles. Froakie then finally lands a hit with his newly learned Bubble, and Ash attempts to capture it, but fails. Froakie then evades the powerful Razor Wind and finishes Fletchling off with Water Pulse, allowing Ash to capture it.
Froakie is used again in Battling on Thin Ice!, using his frubbles for Pikachu and Froakie to dodge, in preperation for Ash's rematch against Viola. In Clemont's Got a Secret!, Froakie is sent out to battle Clemont's Magnemite and Magneton, and despite the type disadvantage, Froakie single handedley incapacitates them with his Frubbles. In An Appetite for Battle!, Froakie is used to battle Clemont's Chespin for practice since Chespin was getting lazy and obese, but Froakie overcame another type disadvantage and easily beat it with his new Pound attack.
Later, Froakie was used to battle Sanpei and his Frogadier. But because of Frogadier's intense speed and power, it lost. Froakie then tried to become super fast and strong by trying to learn Quick Attack. However, when Team Rocket attacked, it instead learned Double Team.
Froakie was then used to battle Grant's Onix in Ash's second Kalos gym battle, which Froakie successfully did when a newly formed strategy called the Rock Tomb Climb. Froakie then battled Grant's Tyrunt but was easily defeat with a powerful Draco Meteor.
Froakie was later used to battle a Hawlucha Ash made friends with after settling a rivalry with a Machamp. However, the battle ended when Hawlucha's Flying Press collided with Froakie's Pound attack, knocking both Pokémon out in a draw.
Sometime later, Froakie was doing some training sessions with Ash and his Pikachu. But when a log came hurdling down from a waterfall, Froakie defended Pikachu's life and learned Cut to stop the log from falling on Pikachu. Froakie later got into a squabble with Hawlucha and began to fight each other. Their fighting didn't last long until a Trevenant kidnapped Ash and dragged him into the forest. When Froakie and Hawlucha attempted to save their trainer, they learned that the reason why Trevenant kidnapped Ash is because it needed his help in saving its companions, a Bonsly and a Sudowoodo, who were caught in an electric net set up by Team Rocket. Hawlucha and Froakie both worked together to save the two Pokémon, send Team Rocket flying, and settle their differences, becoming best friends in the process.
Froakie was later used in a first-touch battle against Ash's new rival, Tierno and his Squirtle. But was quickly overwhelmed by Squirtle's dance movements.
Sooner or later, Froakie tag-teamed with Sanpei's newly evolved Greninja to battle against a ninja named Saizo and his Pokémon, Barbaracle. During the battle, it evolved into a Frogadier and learned Aerial Ace in the process.
Frogadier was later used in Ash's gym battle against Ramos and his Weepinbell. Although Weepinbell managed to defeat both Fletchinder and Hawlucha, Frogadier defeated the Flycatcher Pokémon with a single hit from Aerial Ace. He later fought against Ramos' Gogoat. During the battle, he was weakened by Gogoat's strong Grass-type moves. But, after recalling the lesson Ramos taught Ash before the battle, Ash and Frogadier managed to come up with a strategy and defeat Gogoat with a blast of Water Pulse, thus earning Ash the Plant Badge.
Froakie seems to be a tough Pokémon in reference to his personality. He left many of his previous trainers as his natures didn't meet with them. He finally chose Ash after seeing his bravery. Froakie even has the tendency to blush when it is thanked and praised by other Pokémon and people. For example, when it used its frubbles to tidy up Fennekin's tail, it thanked Froakie for its thoughtfulness.
Known movesEdit
Move | Episode |
Water Pulse + | Kalos, Where Dreams and Adventures Begin! |
Bubble | A Battle of Aerial Mobility! |
Pound | An Appetite for Battle! |
Double Team (move) + | A Rush of Ninja Wisdom! |
Cut + | Forging Forest Friendships! |
Aerial Ace + | A Stealthy Challenge! |
+ indicates this Pokémon used this move recently.* - indicates this Pokémon normally can't use this move. |
Improvised movesEdit
Voice actorsEdit
- Yūji Ueda (Japanese)
- Billy Bob Thompson (English)
- Frogadier's capture marks the first time since catching Heracross that Ash's first capture in a region has not been of the Flying-type.
- Kalos is the second region where Ash catches only one starter Pokémon. The first was Hoenn where Ash caught Treecko.
- Frogadier is the first of Ash's Pokémon to be owned by more than one trainer before its capture by Ash.
- Frogadier is Ash's first Water-type starter Pokémon to evolve.
- Frogadier is also the second Water-type Pokémon of Ash's to evolve. The first being Kingler.
- ^ XY002: Lumiose City Pursuit!, Ash mentions Froakie being a guy


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Check out some available loans that are similar to this one!
In 2004, Diosdada joined GDMPC to gain access to financial services to help improve her living conditions and enable her to engage in business activities. She has successfully repaid a previous loan of 10,000 PHP from GDMPC. Diosdada is requesting a new loan of 25,000 PHP which will be used to repair her vehicle for better service. This will be her 14th loan from GDMPC. She plans to use the additional revenue generated from the business to improve the general living conditions of her family.
About GDMPC:
GDMPC is one of the most progressive cooperatives in Mindanao, Philippines. It has four branches and three unit offices covering the provinces of Zamboanga del Sur, Lanao del Norte and Misamis Occidental. Its membership is composed mostly of farmers, fisherfolks and small entrepreneurs.
Note: To maximize efficiency and reduce costs, this loan was posted using a template to automate the translation by Kiva. Given that manual translation of entrepreneur profiles is costly, Kiva created this template for our Field Partner to allow them to continue to post loans in English while providing quality and accurate information to Kiva lenders.
Additional Information
About GDMPC
Gata Daku Multi-Purpose Cooperative (GDMPC) was founded in 1992 by 32 farmer-members to empower people by providing livelihood opportunities and other support services. Today, it has over 15,000 members located in upland, lowland and coastal regions of the Philippines, and offers credit, savings, education, and various social services.About Philippines
252View loans »
Success!! The loan was 100% repaid
A loan of $575 helped Diosdada to repair her vehicle for better service.
- Repayment Term
- 3 months (Additional Information)
- Repayment Schedule
- At end of term
- Pre-Disbursed:
- Dec 22, 2010
- Listed
- Feb 1, 2011
- Currency Exchange Loss:
- Possible
- Ended:
- Mar 15, 2011


text:
Public-Private Partnership Helps Customers, Environment, Bottom Line
Jan. 11, 2013 /3BL Media/ - In a video address to a global audience, U.S. Postal Service Postmaster General and Chief Executive Officer Patrick Donahoe, and UPS Chairman and CEO Scott Davis announced a unique partnership designed to offer better service to customers, reduce greenhouse gas emissions and optimize operational efficiencies.
"We've named our partnership 'Blue and Brown Make Green,'" said Donahoe. "By working together, the Postal Service and UPS are able to reduce costs, serve our customers better and achieve sustainability goals, such as reducing our carbon footprints."
"It certainly describes what the Postal Service and UPS have been working on together," said Davis. "The world is changing dramatically as we become a more global economy. And, it takes big ideas and bold moves to keep up. This collaboration to reduce the carbon footprint of our respective supply chains is the sustainability wave of the future," Davis added.
Read the rest of the release on the UPS Pressroom.
Tweet me: Brown and Blue Make Green: @UPS and @USPS team up to serve customers better and reduce their impact on the environment http://bit.ly/13ls3KQ
Contact Info:
Kara Ross
+1 (202) 675-1725
Darlene Casey
+1 (202) 268-3440
KEYWORDS: UPS, USPS, logistics, sustainability, Green, customers, csr


text:
Most Promising Wood Products Story of 2012 Update
By Chuck Ray | Posted: 07/14/2013 10:02AM
You may remember what I called "the Most Promising Wood Industry Story of 2012," the public/private partnership between the U.S. Forest Service, community groups, and the Southwestern timber industry.
Patrick Rappold, the Wood Utilization and Marketing Specialist for the State of Arizona, recently shared some nice follow-up comments and photos on the project.
"Just wanted to pass along some interesting news from Northern Arizona.
The Vaagen Brothers hosted an open house on Wednesday May 29, for the opening of the Four Corners Forest Products sawmill in Eagar, AZ. A mobile HewSaw is being used to process small diameter trees that are being removed as part of the White Mountain Stewardship Contract on the Apache-Sitgreaves National Forest. The open house for the mill coincided with the two-year anniversary of the Wallow Fire. What eventually became Arizona's largest wildfire; the Wallow Fire devastated 538,049 acres of forests. While most of the acres burned did occur on the Apache-Sitgreaves National Forest, the fire did cross over into New Mexico and also burned some forests managed by the White Mountain Apache. Salvage of the timber is still ongoing.
The HewSaw [operation] is not associated with Pioneer Forest Products and the mill is exclusively utilizing material from the Apache-Sitgreaves National Forest. The Forest Supervisor for the Apache-Sitgreaves National Forest (Jim Zornes) was instrumental in bringing the operation to Eagar, AZ. Current timber management plans for the Forest allow for existing industry to coexist with the HewSaw. Several large diameter sawmills still operate in the region and are taking most of the larger logs from the Wallow Fire Salvage operation.
The Vaagen Brothers are operating the mill using a batch system. An onsite log merchandising system sorts the logs based upon diameter, sweep, and length. At the open house on May 29, the HewSaw was primarily producing pallet cants. The yield looked pretty good. The clean chips produced from the HewSaw are being marketed to different businesses. There is wood heating pellet plant nearby that is currently turning the chips into pellets. When the 24MW biomass fueled electrical plant (Snowflake Power) comes back online, chips will likely be marketed to that operation also.
Below are some production numbers for the mill:
This Hew Saw has the capabilities of producing over 100,000 board feet of lumber per shift. That amount of lumber is roughly 20 log loads per shift, producing lumber and chips to go into the local markets. This Sawmill will employ 15 to 30 people directly; the number of logging jobs in the forest will be approximately 25 to 50."
About the Author


text:
Scarlet tiger moth
Scarlet Tiger Moth | |
Scientific classification | |
Kingdom: | Animalia |
Phylum: | Arthropoda |
Class: | Insecta |
Order: | Lepidoptera |
Family: | Arctiidae |
Genus: | Callimorpha |
Binomial name | |
Callimorpha dominula |
Synonyms | |
The Scarlet Tiger Moth (Callimorpha dominula, formerly Panaxia dominula) is a colourful dayflying moth of Europe and the near East. It belongs to the tiger moth family, Arctiidae.
The caterpillars feed mostly on comfrey (Symphytum officinale). The moth imago flies by day, and has both cryptic colouring on the front wings, and warning colour on the hind wings. If disturbed it jinks the front wings forward to show the red warning flash on the hind wings.
It can occur in rare colour morphs, one with yellow hindwings and body and one with extended black on hindwings. Yellow, black and red are common warning colours in insects and other animals.
The three morphs occurring in the population at the Cothill reserve in Oxfordshire have been studied for many years, including research by E.B. Ford and R.A. Fisher.[1][2]
McNamara (1998) describes how amateurs can rear this species.[3]
References[change | change source]
- ↑ Fisher, R.A. and Ford E.B. 1947. The spread of a gene in natural conditions in a colony of the moth Panaxia dominula L. Heredity 1:143–174 PDF 1.8 MB
- ↑ Ford E.B. and Sheppard P.M. 1969. The medionigra polymorphism of Panaxia dominula. Heredity 24:112–134.
- ↑ McNamara, Don (1998). "Notes on rearing Scarlet tiger moth Callimorpha dominula (L.)". Amateur Entomologists' Society. http://www.amentsoc.org/mcnamara.htm. Retrieved 2006-08-15.


text:
Find commute times to your work, school, gym and more.
Alpine Commons
Contact this property
Floor Plans
Move In Special
Refer a Friend Receive $300! When you refer a friend. Call for details.
Prices, specials, features and availability subject to change.
Contact for details (413) 377-2789
Extra Storage, On Site Maintenance, On Site Management
Grill(s) Available, Off Street Parking, Onsite Parking, Planned Community Activities, Recycling Available
Property Details
High-Speed Comcast Internet and Basic Expanded Comcast Cable Inlcuded! (SAVE OVER $1500)!! Washer/Dryer Appliances in all 4 bedroom Apts!Minutes From UMASS! On Bus Route! Plenty of Parking!!
Our spacious four bedroom apartment homes have two full baths, a kitchen with a breakfast bar and modern appliances, beautiful oak cabinets, plush wall-to-wall carpeting, washer/dryer and private patio or balcony. 24-hour emergency maintenance and storage units are available for your convenience. Enjoy the experience of living in a charming New England town with the perfect location: just minutes from Amherst College, UMASS, the center of town and I-91. A friendly and attentive on-site management and maintenance staff are waiting to serve you. Visit us today! Ask about our one easy payment plan and our roommate finding service... call today to find out more about college rentals in Alpine Commons!
Leasing Terms
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Pet Policy
Cats Allowed. Dogs Allowed. Call for service animal policy.
Community Information
52 units
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Product Announcement from Tubacero S de RL de CV
Double Submerged Arc Welding is a process which applies an inner and outer seam to the pipe followed by a cold expansion process to enhance its dimensional characteristics. The pipe is then submitted to a hydrostatic test as well as visual and dimensional inspections according with the required specifications. DSAW is applied to pipes with an outside diameter of 18" or greater.
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text:
Latest step by Step Installation
guide for dummies: Nutch 0.9 By Peter P.
Wang, Zillionics LLC Try the search engine I developed for The Christian Life: Malachi Search Please support my effort by using the best free/low price web hosting: 1&1 Inc To add your comments, please go to: http://nutchtube.blogspot.com/2008/02/latest-step-by-step-installation-guide.html
Run it by clicking the Configure Tomcat icon below. Click the Start button below to start Apache Tomcat Service. Then you will be able to see the following screen in the
browser if you go to http://localhost:8080
<?xml version="1.0"?> <?xml-stylesheet type="text/xsl" href="configuration.xsl"?>
<!-- Put site-specific property overrides in this file. -->
<property> <name>http.agent.name</name> <value>Peter Wang</value> <description>Peter Pu Wang </description> </property>
<property> <name>http.agent.description</name> <value>Nutch spiderman</value> <description> Nutch spiderman </description> </property>
<property> <name>http.agent.url</name> <value>http://peterpuwang.googlepages.com </value> <description>http://peterpuwang.googlepages.com </description> </property>
<property> <name>http.agent.email</name> <value>MyEmail</value> <description>email@example.com </description> </property>
Once things are configured,
running the crawl is easy. Just use the crawl command. Its options include:
For example, a typical call
might be: bin/nutch crawl urls -dir crawl -depth 3 -topN 50 Typically one starts
testing one's configuration by crawling at shallow depths, sharply limiting the
number of pages fetched at each level (-topN), and watching the output to check that desired
pages are fetched and undesirable pages are not. Once one is confident of the
configuration, then an appropriate depth for a full crawl is around 10. The
number of pages per level (-topN)
for a full crawl can be from tens of thousands to millions, depending on your
Set Your Searcher Directory
Next, navigate to your nutch webapp folder then WEB-INF/classes.
Edit the nutch-site.xml
file and add the following to it (make sure you don't have two sets of
<configuration></configuration> tags!): <configuration> <property> <name>searcher.dir</name> <value>your_crawl_folder_here</value> </property> </configuration>
For example, if your nutch directory resides at C:\nutch-0.9.0
and you specified crawl as the directory after the -dir
command, then enter C:\nutch-0.9.0\crawl\ instead of your_crawl_folder_here. e.
Reload the Application. Use
the Tomcat Manager and simply click the "Reload" command for nutch, or restart Tomcat using the windows services tool. Open up a browser and enter
the url http://localhost:8080.
The nutch search page should appear. As long as
you've defined the correct location of your nutch
index directory (as shown above), clicking search should yield results. Congratulations!
It rocks! Peter P.
Wang firstname.lastname@example.com |


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Puppy - up to 1
Your New Puppy
Making the most of your small or toy breed dog's vet visit
Are you ready for your vet visit? Preparing is a good way to maximize the experience. Here are some handy strategies for your next exam.Picking a breed
Selecting the breed of puppy that will best suit your family and lifestyle is the first step in finding the perfect new addition to your home.
Where to get a puppy
There are several places to get a puppy, learn more about finding a puppy at a shelter, breeder, rescue organizations or local newspaper.
Bringing your puppy home
Bringing a new puppy into your home is an exciting time, a few important tips will help this process go smoothly.
Collar, leash and identification
Selecting the right leash, collar and identification for your new puppy is a big decision and should not be taken lightly, here are some tips.
Safe toys and games
Make sure the toys your puppy is playing with and the games you play with your pup are appropriate and safe.
Puppy Proofing Your Home
Playful and intelligent puppies investigate objects by touching, chewing and tasting them.
House Training Your Puppy
The principles underlying housetraining are very simple.
Getting Started
You can turn your cute, rambunctious puppy into super puppy by taking a little extra time to shape the behaviors and personality you want him to have as an adult.