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There are also benefits from a diversified business model that includes a significant portion of our revenues being derived from multiple sources. 31% of our revenues during the third quarter were generated from sources other than one-time capital equipment sales.Our distribution expansion initiative is moving along according to plan. The office we opened in Japan last quarter is working out well. We have hired direct sales and marketing staff, as well as set up people with peer capability with service technicians. This office will not only service the Japanese market, but help support our efforts in other Asian countries. Our business outside North America continues to strength. It now accounts for 38% of our overall product and service revenue.
Palomar Medical Technologies CEO Discusses Q3 2010 Results – Earnings Call Transcript
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- Medium
- cotton, plastic, metal
- Measurements
- (a) 52.5 cm (centre back) 58.0 cm (sleeve length) (jacket)
(b) 55.0 cm (centre back) 32.5 cm (waist, flat) (skirt)
- Place/s of Execution
- Melbourne, Victoria
- Accession Number
- 2008.576.a-b
- Department
- Australian Fashion and Textiles
- Credit Line
- National Gallery of Victoria, Melbourne
Purchased 2008
- Gallery location
- Not on display
- Description
- Suit comprising navy blue cotton twill jacket and matching pencil skirt. a) Navy blue cotton twill high-necked jacket constructed from eight vertical panels. The jacket has broad shoulders, long sleeves and narrows at the waistline before flaring out over the hipline. The front finishes at mid-thigh in waistcoat-style sharp points while at rear the jacket finishes at the waist with a short peplum. The jacket fastens with seven purple plastic buttons down centre front. There are two angled false pockets at front hipline, a back waist tab with two buttons and two decorative buttons at each cuff. b) Navy blue cotton twill pencil skirt with straight front and narrow waistband. Fastens at rear with a short, exposed metal zip which ends above three tiers of short ruffles across centre back panel.


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Hotel de l'Arve
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Really nice hotel with very friendly staff. The decor is typical of the area and this helps to create a real holiday feeling. The hotel is right next to the Arve River and is a short walk from the centre of town. Most of the water in the Arve River comes from the many glaciers in the Chamonix Valley, so I imagine it must be pretty cold.
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
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Miller tied his career high with 10 strikeouts and the Double-A Lookouts did not allow a baserunner after the second frame as three Chattanooga pitchers combined to one-hit the Jacksonville Suns in a 1-0 shutout.
Selected by the Dodgers in the first round of the 2009 Draft, Miller surrendered one hit and three walks over five innings. Red Patterson fanned a pair of batters across three perfect frames, and Shawn Tolleson struck out the side in the ninth to seal the win.
Texas native Miller walked two batters in the first inning, but a double-play ball and an inning-ending strikeout helped the left-hander escape unscathed.
Kyle Skipworth then recorded the only hit off Miller with one out in the second -- a line drive to right field -- but he was stranded in scoring position as Miller went on to retire the final 11 batters he faced.
Chattanooga's relief corps was equally stingy, and between Miller and the 'pen, the Lookouts set down the final 23 batters in order.
Left fielder Kyle Russell's solo homer in the top of the fourth -- his first longball of the year -- proved to be the sole run of the contest.
Miller's outing stood in contrast to his first start of the season, during which he allowed one run on five hits and a walk through 2 2/3 innings but didn't factor into the decision against the Tennessee Smokies.
The 24-year-old's 10 strikeouts matched a personal best last achieved with Class A Advanced Inland Empire in a 4-3 loss to Rancho Cucamonga on April 9, 2010.
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Nov 30, 1999
|Filing date
|Apr 14, 1995
|Priority date
|Apr 7, 1993
|Fee status
|Also published as
|Publication number
|Mohammed Eljamal, John S. Patton, Linda Foster, Robert M. Platz
|Original Assignee
|Inhale Therapeutic Systems, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (2), Non-Patent Citations (14), Referenced by (59), Classifications (31), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation-in-part of application Ser. No. 08/417,507 (attorney docket no. 15225-000410), filed on Apr. 4, 1995, now abandoned which was a file wrapper continuation of application Ser. No. 08/044,358, now abandoned the full disclosures of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates generally to compositions and methods for delivering nucleic acids to the lungs of humans and other animal hosts. More particularly, the present invention relates to compositions which are formed by incorporating insoluble nucleic acid constructs within a hydrophilic excipient matrix which is stored and utilized in dry powder form.
A form of human gene therapy which is receiving increasing interest relies on the in vivo delivery of functional nucleic acids, usually structural genes, to certain target cells within a human or other host. The nucleic acids may be incorporated into carriers such as viruses, liposomes, or the like, and will be delivered under conditions which result in uptake of the genes into the target cells, with subsequent expression of the genes for an extended period of time.
Of particular interest to the present invention, it has been demonstrated that nucleic acid constructs can be delivered to the lungs of mice and rats by different routes, including intratracheal administration of a liquid suspension of the nucleic acids and inhalation of an aqueous aerosol mist produced by a liquid nebulizer. Although holding great promise, both methods for the delivery of nucleic acids to the lungs suffer from certain drawbacks. Intratracheal administration is not suitable for routine therapeutic use in humans and has a very low patient acceptability. Moreover, intratracheal instillation often results in very uneven distribution of a dispersion in the lungs, with some regions receiving very little or no material. The use of a liquid nebulizer enjoys higher patient acceptability and achieves better distribution, but requires time-consuming equipment set-up, can require prolonged periods of treatment to achieve an adequate dosage, can inactivate a viral carrier, and can result in undesirable aggregation or degradation of the nucleic acids within the aerosol mist. Aggregated nucleic acids will generally be less suitable for uptake into host target cells.
For these reasons, it would be desirable to provide improved compositions and methods for the aerosol delivery of nucleic acids. The compositions will preferably be in a dry powder form which can be readily dispersed in a flowing air stream to provide a dry aerosol for delivery to a patient. The dry powder formulations will permit delivery of required dosages of nucleic acids in a very rapid manner (typically in several or fewer breaths) and will be suitable for storage over extended periods. The dry powders are delivered to particular target regions within the host and are readily dispersed over the internal surfaces of lung, where the powder dissolves in the moist layer over the surfaces to thereby release nucleic acids to interact with the target cells.
2. Description of the Background Art
Stribling et al. (1992) J. BIOPHARM. SCI. 3:255-263, describes the aerosol delivery of plasmids carrying a chloramphenicol acetyltransferase (CAT) reporter gene to mice. The plasmids were incorporated in DOTMA or cholesterol liposomes, and aqueous suspensions of the liposomes were nebulized into a small animal aerosol delivery chamber. Mice breathing the aerosol were found to at least transiently express CAT activity in their lung cells. Rosenfeld et al. (1991) SCIENCE 252:431-434, describes the in vivo delivery of an α1-antitrypsin gene to rats, with secretion of the gene product being observable for at least one week. The gene was diluted in saline and instilled directly into the rat trachea. Underwood et al. (1991) J. PHARMACOL. METH. 26:203-210, describes the administration of dry powder bronchodilators in a lactose carrier to pig lungs. U.S. Pat. No. 5,049,388 describes the delivery of liquid aerosols containing liposomes to the lungs. Friedman (1989) SCIENCE 244:1275-1281 is a review article describing human gene therapy strategies. The presence of certain polyvalent ions can reduce transfection efficiency in vitro using liposomes. Feigner and Ringold (1989) NATURE 387-388. Multivalent anions such as citrate or phosphate can induce fusion of positive-charged liposomes used for transfection. Gershon et al. (1993) BIOCHEMISTRY 32:7143-7151.
According to the present invention, dry powder nucleic acid compositions comprise insoluble nucleic acid constructs (typically small particles) dispersed within a matrix of hydrophilic excipient material to form large aerosol particles. Usually, the nucleic acid particles will be present in excess powdered excipient material, usually being the same excipient which forms the matrix. The powdered aerosol particles will have an average particle size in the range from 0.5 μm to 200 μm, usually being in the range from 0.5 μm to 5 μm for lung delivery with larger sizes being useful for delivery to other moist target locations. The nucleic acid constructs may comprise bare nucleic acid molecules, viral vectors, associated viral particle vectors, nucleic acids present in a vesicle, or the like.
The dry powder nucleic acid compositions may be prepared by suspending the insoluble nucleic acid constructs in an aqueous solution of the hydrophilic excipient and drying the solution to produce a powder comprising particles of the nucleic acid construct dispersed within the dried excipient material, usually in the presence of excess powdered excipient. The weight ratio of nucleic acid construct to hydrophilic excipient in the initial solution is in range from 2:1 to 1:100, preferably from 1:1 to 1:10, and the solution may be dried by spraying droplets into a flowing gas stream (spray drying) or by vacuum drying to produce a crude powder followed by grinding to produce a final powder.
In the case of particles intended for lung delivery, having a particle size from 0.5 μm to 5 μm, each particle may contain from 10 to 107 nucleic acid constructs, usually from 102 to 105 nucleic acid constructs, and preferably from 103 to 104 nucleic acid constructs. The constructs may be uniformly or non-uniformly dispersed in each particle, and the particles in turn will often be present in excess powdered excipient, usually at a weight ratio (nucleic acid construct:excipient powder free from nucleic acids) in the range from 1:1, to 1:103 usually from 1:10 to 1:500.
In a preferred aspect of the present invention, aqueous solutions containing the liposome vesicles as nucleic acid constructs will be substantially free from buffering agents and salts. It has been found that drying, particularly spray drying, of such neutrally charged solutions results in powders having enhanced transfection activity compared to powders formed by drying the same liposome vesicles in buffered solutions. In contrast, aqueous solutions containing viral vectors as the nucleic acid constructs usually will be buffered to enhance stability of the viral vectors.
In a second preferred aspect of the present invention, the dry powder nucleic acid compositions will be prepared by spraying droplets of the liquid solution into a heated gas stream over a short time period, typically 50� C. to 150� C. over a period from 10 msec to 100 msec, in a spray dryer. The resulting powder comprising particles containing nucleic acid constructs (and usually containing powdered excipient free from nucleic acids) will then be collected in a partially cooled environment, typically maintained at 5� C. to 50� C., and thereafter stored at a temperature from 5� C. to 25� C. at a low humidity, typically below 5% RH. It has been found that such collection and storage conditions help to preserve and stabilize the compositions and to enhance transfection efficiency.
Methods for delivering nucleic acid constructs according to the present invention comprise directing the dry powder containing the nucleic acid constructs to a moist target location in a host, where the hydrophilic excipient matrix material of the particles will dissolve when exposed to the moist target location, leaving the much smaller nucleic acid construct particles to freely interact with cells. In a preferred aspect of the present invention, the target location is the lung and the particles are directed to the lung by inhalation.
Compositions of the present invention are particularly advantageous since the hydrophilic excipient will stabilize the nucleic acid constructs for storage. Excess powdered hydrophilic excipient can also enhance dispersion of the dry powders into aerosols and, because of its high water solubility, facilitate dissolution of the composition to deposit the nucleic acid constructs into intimate contact with the target membranes, such as the lung surface membrane of the host.
FIGS. 1 and 2 are graphs comparing transfection efficiencies among nucleic acid constructs present in powders, stored liquids, and fresh liquids, as described in detail in the Experimental section.
The nucleic acid constructs of the present invention will comprise nucleic acid molecules in a form suitable for uptake into target cells within a host tissue. The nucleic acids may be in the form of bare DNA or RNA molecules, where the molecules may comprise one or more structural genes, one or more regulatory genes, antisense strands, strands capable of triplex formation, or the like. Commonly, the nucleic acid construct will include at least one structural gene under the transcriptional and translational control of a suitable regulatory region. More usually, nucleic acid constructs of the present invention will comprise nucleic acids incorporated in a delivery vehicle to improve transfection efficiency, wherein the delivery vehicle will be dispersed within larger particles comprising a dried hydrophilic excipient material.
A first type of such delivery vehicles comprises viral vectors, such as retroviruses, adenoviruses, and adeno-associated viruses, which have been inactivated to prevent self-replication but which maintain the native viral ability to bind a target host cell, deliver genetic material into the cytoplasm of the target host cell, and promote expression of structural or other genes which have been incorporated in the particle. Suitable retrovirus vectors for mediated gene transfer are described in Kahn et al. (1992) CIRC. RES. 71:1508-1517, the disclosure of which is incorporated herein by reference. A suitable adenovirus gene delivery is described in Rosenfeld et al. (1991) SCIENCE 252:431-434, the disclosure of which is incorporated herein by reference. Both retroviral and adenovirus delivery systems are described in Friedman (1989) SCIENCE 244:1275-1281, the disclosure of which is also incorporated herein by reference.
A second type of nucleic acid delivery vehicle comprises liposomal transfection vesicles, including both anionic and cationic liposomal constructs. The use of anionic liposomes requires that the nucleic acids be entrapped within the liposome. Cationic liposomes do not require nucleic acid entrapment and instead may be formed by simple mixing of the nucleic acids and liposomes. The cationic liposomes avidly bind to the negatively charged nucleic acid molecules, including both DNA and RNA, to yield complexes which give reasonable transfection efficiency in many cell types. See, Farhood et al. (1992) BIOCHEM. BIOPHYS. ACTA. 1111:239-246, the disclosure of which is incorporated herein by reference. A particularly preferred material for forming liposomal vesicles is lipofectin which is composed of an equimolar mixture of dioleylphosphatidyl ethanolamine (DOPE) and dioleyloxypropyl-triethylammonium (DOTMA), as described in Felgner and Ringold (1989) NATURE 337:387-388, the disclosure of which is incorporated herein by reference.
It is also possible to combine these two types of delivery systems. For example, Kahn et al. (1992), supra., teaches that a retrovirus vector may be combined in a cationic DEAE-dextran vesicle to further enhance transformation efficiency. It is also possible to incorporate nuclear proteins into viral and/or liposomal delivery vesicles to even further improve transfection efficiencies. See, Kaneda et al. (1989) SCIENCE 243:375-378, the disclosure of which is incorporated herein by reference.
Hydrophilic excipient materials suitable for use in the compositions of the present invention will be able to form a dried matrix in which the nucleic acid constructs are dispersed in order to stabilize the nucleic acid molecules during storage, facilitate dispersion of the nucleic acids in dry powder aerosols, and enhance wetting and subsequent contact of then nucleic acids with the moist target locations within a patient or other treated host. A sufficient amount of hydrophilic excipient will be present to form a dry powder matrix in which the nucleic acids are dispersed, typically being present in the resulting particles at a weight ratio (nucleic acid construct:particle) in the range from 1:1 to 1:1000, usually from 1:10 to 1:500. Suitable hydrophilic excipient materials include those listed in Table 1.
______________________________________TYPE OF HYDROPHILIC MATRIX MATERIAL EXAMPLES______________________________________Protein and Peptides Human serum albumin; Collagens; Gelatins; Lung surfactant proteins, and fragments thereof. Hyaluronic acid Hyaluronic acid. Sugars Glucose; Lactose; Sucrose, Xylose; Ribose; and Trehalose. Sugar alcohols Mannitol. Oligosaccharides Raffinose and Stachyose. Other carbohydrates Dextrans; Maltodextrans; Dextrins; Cyclodextrins; Maltodextrins; Cellulose; and Methylcellulose. Amino acids Glycine; Alanine; and Glutamate. Organic acids and salts1 Ascorbic acid; Ascorbate salts; Citric acid; and Citrate salts. Inorganic salts1 NaCl; NaHCO3 ; NH4 HCO3 ; MgSO4 ; and Na2 SO4.______________________________________ 1 The use of organic acids and salts, and inorganic salts, as a matrix material is less preferred in the case of liposomal transfection vesicles, where the salts and acids can interfere with the stability of the vesicle.
The dry powder formulations of the present invention may conveniently be formulated by first suspending the nucleic acid constructs, which are generally insoluble in water, in aqueous solutions of the hydrophilic excipient. The relative amounts of nucleic acid construct and hydrophilic excipient material will depend on the desired final ratio of nucleic acid to excipient. Conveniently, the ratio of nucleic acid construct to excipient will be in the range from about 2:1 to 1:100 (nucleic acid:excipient), preferably from 1:1 to 1:10, with a total solids concentration in the aqueous suspension being usually less than 5% by weight, more usually being less than 3% by weight.
In the case of nucleic acid constructs comprising liposomal transfection vesicles, the aqueous solutions are preferably free from polyvalent buffering agents (particularly citrate and phosphate), salts, and other negatively charged species (other than the nucleic acids and in some cases the hydrophilic matrix material), which have been found in some cases to reduce transfection efficiency of the resulting dried powders. It is presently believed that such charged species will interact with the liposomal constructs in a deleterious manner as the compositions are dried.
In the case of nucleic acid constructs comprising viral vectors, it is usually desirable that the aqueous solution be buffered in order to enhance the activity of the viral vectors after drying.
The aqueous solution can then be spray dried under conditions which result in a powder containing particles within a desired size range, typically but not necessarily having a mean particle diameter in the range from about 0.5 μm to 50 μm, with the precise particle size depending on the eventual use. For lung delivery, the particle size will typically be in the range from 0.5 μm to 10 μm, usually being from 0.5 μm to 7 μm, and preferably from 1 μm to 4 μm. The mean particle diameter can be measured using conventional equipment such as a Cascade Impactor (Andersen, Ga.).
Higher total solids concentrations within the aqueous solution will generally result in larger particle sizes. Powders having an average particle size above 10 μm, usually in the range from about 20 μm to 50 μm, can be thus formed, and are particularly useful for nasal, dermal, surgical, and wound applications where it is desired that the powder rapidly settle on a target location.
Dry powders can also be formed by vacuum drying, either at room temperature or under freezing temperatures (lyophilization). Usually, it will be desirable to start with an aqueous solution having higher total solids content, typically above 0.1% by weight, more typically above 0.2% by weight. For smaller particles having a size from 0.5 μm to 10 μm, the liquids will usually have an initial solids content from 0.2% to 1% by weight. For larger particles of 10 μm and above, the solids content will usually be from 15% to 10% by weight. The vacuum drying results in a crude powder which can then be further ground, typically by jet milling, to produce a product having a uniform particle size and a desired particle size, typically within the 1 μm to 50 μm range set forth above.
Specific methods for preparing dry powders of a type which are useful in the present invention are described in copending application Ser. No. 08/423,515 (attorney docket no. 15225-001400), filed on the same day as the present application, entitled Devices, Compositions and Methods for the Pulmonary Delivery of Aerosolized Medicaments, the full disclosure of which is incorporated herein by reference.
The dry powder compositions of the present invention are suitable for delivery to a variety of target locations within a patient or other treated host, with moist membrane locations, such as the lungs, nasal membranes, mouth, throat, stomach, intestines, vagina, and the like being preferred. The compositions may also be used to deliver the nucleic acid constructs the subcutaneous or intramuscular compartment by dry powder injection, or to open wounds, including surgical wounds, in order to deliver genes to exposed tissue.
In the case of delivery to the lungs, the dry powders will have a mean particle diameter in the range from about 1 μm to 5 μm, and may be efficiently dispersed and delivered in a flowing gas stream for inhalation by the patient or host.
A particularly suitable device for dry powder delivery is described in copending application Ser. No. 07/910,048, assigned to the assignee of the present application, and filed on Jul. 8, 1992, the full disclosure of which is incorporated herein by reference.
The following examples are offered by way of illustration, not by way of limitation.
1. Viral Vector Coated with Mannitol Prepared by Spray Drying
A respirable powder incorporating the human cystic fibrosis transmembrane conductance (CFTR) gene and having a particle diameter from 1 μm to 5 μm is formed as follows. The CFTR gene is linked to the adenovirus (Ad) late promoter, the resulting expression cassette is incorporated into an adenovirus vector, as taught in Rosenfeld et al. (1991) SCIENCE 252:431-434. The adenovirus vector has a deletion in the E3 region, thus permitting encapsidation of the recombinant genomic DNA including the CFTR gene. The vector further has a deletion in the Elq region, preventing viral replication.
Sufficient adenovirus vector is added to a phosphate buffered saline solution (0.15 mM NaCl, 2.7 mM KCl, 8.1 mM Na2 PO4, 1.5 mM KH2 PO4, pH 7.2) containing 5 mg/ml mannitol at 4� C. to provide approximately 108 plaque forming units (pfu)/ml. The resulting solution is spray dried in a commercially available drier from suppliers such as Buchi and Niro.
After spray drying, the powder is collected and stored at less than 10% relative humidity. The powder may be incorporated into inhalation delivery devices as described in copending application Ser. No. 07/910,048.
2. Plasmid Vector in Liposome Coated with Maltodextrin Prepared by Spray Drying
A respirable powder incorporating the α1-antitrypsin (α1AT) gene and having a particle diameter in the range from 1 μm to 5 μm is formed as follows. A plasmid vector carrying the α1AT gene is prepared as described in Gormon et al. (1982) PNAS 79:6777-6781 and Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. The α1AT gene is fused to the human cytomegalovirus (CMV) immediate early promoter/enhancer element. The plasmid is then purified by alkaline lysis and ammonium acetate precipitation, and the nucleic acid concentration is measured by UV absorption.
Plasmid DNA (0.75 mg/ml) is dispersed in an aqueous solution of double distilled water containing 1.35 mg/ml of DOTMA/DOPE liposomes at a 1:1 molar ratio. The resulting mixture is sonicated for 20 minutes in a water bath. Maltodextrin is added to the mixture after sonication at a concentration of 5 mg/ml. The mixture is then spray dried as described in Example 1.
3. Plasmid Vector in Liposomes, Freeze Dried, and Jet Milled
Plasmid DNA (0.75 mg/ml), prepared as described in Example 2, is mixed with a multilamillar dispersion of cationic fusogenic liposomes (1.5 mg/ml) by gentle agitation at 23� C. for 24 hours in a solution containing 10 mg/ml human serum albumin (HSA). The solution is freeze dried in trays, and the resulting powder is jet milled with high purity nitrogen in a conventional jet mill until a mass median aerodynamic diameter of 1 μm to 4 μm is achieved. The resulting respirable powder is stored at less than 10% relative humidity until it is needed for dispersion in a dry powder device for inhalation.
4. Transfection of Cells with Lipid:DNA Complexes and Adenovirus Vectors
Respirable dry powder aerosols containing lipid:DNA complexes or adenovirus vectors for the delivery of active genes to mammalian cells were prepared and tested. Dispersible dry powders containing either vehicles were made with mannitol and/or glycine as bulking agents and HSA as a surface modifier to help disperse the powders. Transfection activities in CFT1 cells (cells from the airways of cystic fibrosis patients) and virus titers of the resulting powders were measured and compared to liquid controls. The dispersibilities and aerodynamic particle size distributions of select powders that retained their transfection activities were also measured. The transfection activities of the lipid:DNA powders, formulated without buffer, were better than both the liquids they were made of and the freshly prepared liquid formulations. Lipids and DNA were complexed with each other at least 15 minutes prior to cytofection. The titers of the virus in the best powder formulation and its liquid control were 76% and 16% of the expected values, respectively. The dispersibility and the respirable fractions of the selected powders ranged from 40 to 64% and 60 to 80%, respectively. These data demonstrate the ability to obtain respirable and stable dry powder formulations of both cationic lipids complexes and adenovirus delivery systems.
1. DMRIE:DOPE (50/50, mole ratio, Vical, San Diego, Calif.). The lipids (DMRIE:DOPE) were formulated to generate 1.56 mM solution by resuspending 5 mg vial in 2.4 ml deionized water and vortexing at full speed for 1 minute.
2. DOTMA/DOPE (50:50, mole ratio, Megabios, San Francisco, Calif.).
DNA Plasmid
1. pCMVβ (Genzyme, Framingham, Mass.). pCMV-β-gal: Cytomegalovirus promoter was linked to the Escherichia coli Lac-Z gene, which codes for the enzyme β-galactosidase. The activity of this enzyme was visualized with the reagent X-gal (b-D-galactoside). The DNA plasmid (pCMVβ, 4.26 mg/ml) was formulated to generate 960 μM by adding 0.145 ml of the DNA suspension to 1.9 ml 1 mM tris buffer, pH 8.
2. pCIS-CAT (Megabios, San Francisco, Calif.). pCIS-CAT: Chloramphenicol acetyltransferase (CAT) fused to the human cytomegalovirus (CMV) immediate early promoter/enhancer element.
Lipid:DNA Complex
The complex was formed by first adding DNA plasmids (pCMVβ) to a certain volume of bulking and excipient materials solution to attain the desired concentration then the preformed lipids (DMRIE:DOPE) were added to form the complex at least 10 minutes prior to processing into powder. The lipid:DNA ratio was molar.
Ad2-CMV-LacZ-2 (Genzyme, Framingham, Mass.). AD2-CMV-Lac-Z: Cytomegalovirus promoter was linked to the Escherichia coli Lac-Z gene and was incorporated into replication deficient recombinant virus. Takiff et al. (1984) J. VIROL. 51:131-136 and Gilardi et al. (1990) FEBS LETT. 267:60-62.
1 mM Tris buffer pH 8 (0.14 mg/ml solids)
(1) Dissolved 60.6 mg Tris base (J T Baker, lot #x171-07) in 500 ml deionized house water to make a 1 mM solution. (2) Dissolved 78.8 mg Tris HCl (J T Baker, lot #4103-1) in 500 ml deionized house water to make a 1 mM solution. To the magnetically stirred Tris base solution, Tris HCl was slowly added to obtain pH 8.
Tris/Mannitol/HSA (5.07 mg/ml solids)
Dissolved 1,363.0 mg mannitol (Mallinckrodt, lot #6208 KLRP) and 156.7 mg HSA (Miles, lot #204) in 300 ml of the 1 mM Tris buffer.
Glycine/HSA (I) (5.44 mg/ml solids)
Dissolved 60.6 mg HSA and 1,028.0 mg glycine (J T Baker, Lot #A28732) in 200 ml filtered and deionized house water, pH 6.4.
Glycine/Mannitol/HSA (5.57 mg/ml solids): Dissolved 50.6 mh HSA, 540.0 mg glycine and 524.0 mg mannitol in 200 ml of filtered and deionized house water, pH 6.4.
Phosphate buffer (PB) pH 7.4 (1.89 mg/ml solids)
Dissolved 200.1 mg KCl (J T Baker, Lot No. 3040-01), 1,451.4 mg Na2 HPO4.7H2 O (Mallinckrodt, Lot No. 7896 KJPE) and 242.1 mg KH2 PO4 (J T Baker, Lot No. 3246-01) in one liter of the house deionized water to make pH 7.4.
Phosphate/HSA (3.93) mg/ml solids)
Dissolved 203.8 mg HSA (Miles, Lot No. 204) in 100 ml of the phosphate buffer pH 7.4.
Mannitol/HSA in PB (60.05 mg/ml solids)
Dissolved 1,403.1 mg mannitol (Mallinckrodt, Lot No. 6208 KLRP) in 25 ml phosphate/HSA. Stored below 5� C.
Glycine/HSA (I) in PB (28.40 mg/ml solids)
Dissolve 611.8 mg glycine (J T Baker, Lot No. 0581-01) in 25 ml phosphate/HSA. Stored below 5� C.
Glycine/HSA (II) in PB (10.5 mg/ml solids)
Dissolved 613.8 mg glycine (J T Baker, Lot No. 0581-01) and 1 ml (250 mg) HSA (Alpha Therapeutic, lot #NB2049A) in 100 ml phosphate/HSA. Stored below 5� C.
Glycine/HSA (II) in water (8.6 mg/ml solids)
Dissolved 612.4 mg glycine (J T Baker, Lot No. 0581-01) and 1 ml (250 mg) HSA (Alpha Therapeutic, lot #NB2049A) in 100 ml de-ionized water. Stored below 5� C.
Mannitol/Glycerine/HSA in PB (45.09 mg/ml solids)
Dissolved 700.2 mg mannitol (Mallinckrodt, Lot No. 6208 KLRP) and 328.8 mg glycine (J T Baker, Lot No. 0581-01) in 25 ml of phosphate/HSA. Stored below 5� C.
Adenovirus (40.20 mg/ml)
Dissolved 305.3 mg sucrose (Sigma, Lot No. 69F0026), 77.9 mg NaCl (VWR SCI., Lot No. 34005404) and 0.1 ml of Ad2-CMV-LacZ virus (1011 iu/ml with particle concentration of .sup.˜ 5�1012 /ml in PBS+3% sucrose, Genzyme) in 10 ml phosphate buffer. This solution was prepared and used cold on the same day and was stored frozen at -70� C. Also, it was used again 10 weeks later, it underwent only one freeze/thaw cycle.
Powder processing
All the powders were processed in a Buchi-190 mini spray dryer. Briefly, the solution is atomized into liquid droplets and is dried to solid particulate with adjunct stream of air heated to a specified temperature (inlet temperature). The airborne particulate are fed into a cyclone (outlet temperature) where they are separated from the air into a collection cup.
Dispersibility of the dry powder was determined using a dry powder inhaler (generally as described in application Ser. No. 08/309,691, the full disclosure of which is incorporated herein by reference) or a test bed. Briefly, a blister pack filled with 5.0�0.5 mg powder was loaded and dispersed in the device. The resulting aerosol cloud in the device chamber was immediately drawn at a suction flowrate of 30 LPM for 2.5 seconds and was collected on a 47 mm, 0.65 μm pore size, polyvinylidene fluoride membrane filter (Millipore). Dispersibility is the fraction of powder mass collected on the filter relative to mass filled into the blister pack.
Particle size (Horiba)
The particle size distribution (PSD) of the powder samples was measured using the Horiba CAPA-700 centrifugal sedimentation particle size analyzer. Approximately five mg of powder was suspended in approximately 5 ml of Sedisperse A-11 (Micromeritics, Norcoss, Ga.) and briefly sonicated before analysis. The instrument was configured to measure a particle size range of 0.4 to 10 μm in diameter, and the centrifuge was operated at 2000 rpm. The particle size distribution was characterized by mass median diameter, and by the mass fraction less than 5.0 μm.
Particle size (cascade impactor)
The particle size distribution of aerosolized powders (aerosol from blister using prototype 1B device) was obtained using an IMPAQ 6-stage (16, 8, 4, 2, 1, 0.5 μm cut off diameters) cascade impactor (California Measurement, Sierra Madre, Calif.). A glass Throat, described in the European Pharmacopoeia, was fitted over the intake of the cascade impactor. The glass throat was designed to simulate particle deposition in the human throat when aerosol is sampled in the cascade impactor. The impactor airflow was set to 14.5 LPM, the calibrated operating flow of the instrument. To measure the particle size of the aerosol, a blister pack filled with approximately 5 mg of powder was loaded into the prototype inhaler, the device was actuated and the aerosol cloud drawn from the chamber into the glass throat/cascade impactor set up. The particle size was determined gravimetrically by weighing the powder on the glass throat, impactor plates and the backup filter and plotting the results on a log-probability graph. The mass median aerodynamic diameter (MMAD) and the mass fraction less than 5 μm were determined from the graph.
Cationic Liposomes Dry Powder
The following formulations were made to develop aerosol liposomes in dry powder format. Cationic lipid (34.5 mg (25 μMoles) DOTMA:DOPE, 1:1, Megabios) was dispersed in 100 ml of 6.75 mg/ml mannitol solution. This solution (7.1 mg/ml solids) was processed into powder according to the following spray drying parameters:
Solution feed rate: 5.8 ml/min
Inlet/Outlet Temperatures: 137/73� C.
Atomizer air flow rate: 800 LPH
The powder yield was about 6% and could not be filled into blister packs. The resulting powder was sticky, possibly due to liposomes presence on the surface of the powder. This possibly resulted from the cationic liposomes on the surface of the dry particles strongly interacting with each other. In order to solve this problem, Human serum albumin (HSA) in solution to increase the dispersibility of the powder by modifying its surface morphology.
Two liquid formulations containing HSA (Alpha Therapeutic, 12.5 g/50 ml solution), lipids (DOTMA:DOPE) and mannitol were dried in the Buchi-190 spray dryer. The liquid solution was fed at 3 ml/min and the inlet/outlet temperatures ranged between 95-105� C./55-70� C. We found that both the yield and the dispersibility of the dry powder was improved with the addition of HSA (see Table 1).
TABLE 1______________________________________Summary of Lipids/Mannitol aerosol formulations. Composition Formula HSA/Lipids/Mannitol Yield Dispersibility No. (mg/ml) Percent Percent______________________________________1 0.00/0.35/6.75 6 -- 2 0.40/0.35/6.40 55 36 � 6 3 0.91/0.35/6.40 54 59 � 4______________________________________
DNA Powder
To investigate whether this process would preserve the integrity of DNA molecules, pCMVβ in Tris/Mannito/HSA solution (7.5 mg/ml solids) was spray dried according to the following conditions:
Solution feed rate: 4.3 ml/min
Inlet/Outlet Temperatures: 120� C./70� C.
Atomizer air flowrate: 800 LPH
The resulting powder was reconstituted in de-ionized water and was run in gel electrophoresis (1.3% agrose in 0.5�TBE plus 0.5 μg/ml ethidium bromide, 100 volts for four hours). Unprocessed DNA molecules were also run in the same gel. The powder was tested for transfection activity in vitro as follows:
Cytofection Assay
Cell Preparation
Cells of choice (CFT1, airway cells from cystic fibrosis patients) were placed into 96-well plates at 20,000/well in growth medium the day before cytofection. Just prior to cytofection, the cells were observed, and approximate confluencey estimated.
Lipid:DNA Preparation
The lipid was formulated to 670 mM and the DNA to 960 mM. The complex was formed by adding the lipid to the DNA for 15 minutes, and then 100 μl of the complex was added to the cells (media previously aspirated). Cytofection occurred over 6 hours before the addition of 50 μl 30% FCS-OPTIMEM. The following day, 100 μl of 10% FCS-OPTIMEM was added to each well. The assay began 48 hours after start of cytofection.
1. Remove media and wash cells twice with 100 μl PBS
2. Add 25 μl lysis buffer (250 mM Tris-HCl, pH8.0, and 0.15% Triton X-100) and incubate at RT for 30 minutes.
3. Freeze plate at -70� C. for 20 minutes, thaw at RT for 15 minutes.
4. Break up cells by carefully vortexing plate for 15 seconds.
5. Freeze plate at -70� C. for 20 minutes, thaw at RT for 15 minutes.
6. Add 100 μl PBS followed by 150 μl of CPRG substrate (1 mg/ml chlorophenol red glactopyranoside, 60 mM disodium hydrogen phosphate pH8, 1 mM magnesium sulfate, 10 mM potassium chloride, and 50 mM β-mercaptoethanol)
7. Incubate at 37� C. for 2 hrs until red color develops and read at 580 nm in microplate reader.
Similar bands were observed for both processed and unprocessed DNA in the gel electrophoresis. As expected the reconstituted DNA (without any delivery vehicle, cationic lipid or adenovirus) powder did not show any transfection activity.
Lipid:DNA Powder
Three sets of cationic lipid:DNA formulations were prepared, processed into dry powder and characterized:
1. The lipid:DNA complex was formed in Tris/mannitol/HSA solution (5.07 mg/ml solids) with the following concentration ratios of lipid:DNA (μM:μM)-0:0, 0:6.9, 20.9:12.8, 10.4:12.8, 5.2:12.8, 10.4:6.9, 5.2:6.9, 2.6:6.9, 0.4:3.5, 5.2:3.5 and 2.6:3.5.
2. The lipid:DNA complex was formed in glycine/HSA (I) in water 5.44 mg/ml solids) with the following lipid:DNA concentration ratios (μM:μM)-20:20, 20:15, 10:10 and 10:5.
3. The lipid:DNA complex was formed in glycine/mannitol/HSA solution 5.57 mg/ml solids) with the following ratios (μM:μM)-20:20, 20:15, 10:15, 10:10 and 10:5. The solutions were processed into powder according to the following spray drying parameters:
Solution feed rate: 3.8 ml/min
Inlet/Outlet Temperatures: 115-125� C./70-85� C.
Atomizer air flowrate: 700-800 LPH
Aliquots of the liquid formulations and the resulting powders were kept refrigerated and duplicates were sent on ice pack to be assayed for transfection activity in vitro (as described above) and also to be compared with freshly prepared suspensions of Lipid:DNA with similar concentration ratios. Select powders from sets 2 and 3 were characterized using the Horiba, IMPAQ 6-stage cascade impactor and a dry powder inhaler.
A comparison of β-gal expression in vitro (CFT1 cell line) between the powder and the two liquid (stored control and freshly made control) formulations are shown in FIGS. 1 and 2. The powders were reconstituted in double distilled de-ionized water. The transfection activities of the liquid and powder formulations of set 1, which contained the Tris buffer, were considerably less than freshly made liquid formulations (FIG. 1). In the powders, which contained no buffer, there was a 75% increase in the transfection activity of the 20:20 and 30% increase in the 20:15 as compared with liquid formulations (see FIG. 2). The measured physical parameters of the selected powders that showed superior transfection are listed in Table 2. The glycine/HSA and glycine/mannitol/HSA powder formulations had similar transfection activities (FIG. 1) but the glycine/HSA powders dispersed better than the glycine/mannitol/HSA (Table 2).
TABLE 2______________________________________Lipid:DNA powder physical characteristics. Dipersi. Formula Bulking (% RSD) HORIBA Cascade Impactor ratio Material (n = 3) MMD* MMAD** % ≦5 μm______________________________________20:20 Glycine 61(20) 2.0 3.9 60 20:15 Glycine 64(1) 2.0 2.4 75 20:20 Gly/Man 47(12) 2.0 3.0 70 20:15 Gly/Man 51(12) 2.4 4.1 60______________________________________ *MMD: Mass Median Diameter **MMAD: Mass Median Aerodynamic Diameter
Dry Powder Aerosol Development
This developmental study included two sets of experiments. In the first set, the effects of bulking agents in phosphate buffer (PB), (i) mannitol/HSA, (ii) glycine/HSA and (iii) mannitol/glycine/HSA, on the infectivity of the adenovirus dry powders were investigated. In the second set, we investigated the effects of buffer removal and the process outlet temperature on the infectivity. All solutions were used and stored cold (about 5� C.).
1. Five mannitol/HSA in PB formulations were prepared. (i) To 4�3 ml mannitol/HSA in we added 0.1 ml of adenovirus solution to obtain 3.2�107 iu/ml and about 60 mg/ml solids, and the fifth was used as a control with no virus. Two of the virus formula were diluted with deionized water to about 9 mg/ml solids. (ii) Two formulations of 6.3 ml glycine/HSA (I) in PB plus 0.4 ml adenovirus solution were made (29 mg/ml solids, 6.3�107 iu/ml). One of them was diluted with de-ionized water to 9 mg/ml solids. (iii) Two formulations of 4.1 ml mannitol/glycine/HSA in PB plus 0.4 ml of virus solution were made (45.1 mg/ml solids, 8.89�107 iu/ml). One was diluted with deionized water to 9 mg/ml. The adenovirus solution was freshly made on the same day and was kept cold on ice.
2. Four formulations were prepared, two contained 25 ml of glycine/HSA (II) in PB plus 0.4 ml of adenovirus solution (10.5 mg/ml, 1.6�107 iu/ml) and the other two contained 25 ml of glycine/HSA (II) in water plus 0.4 ml of adenovirus solution (8.6 mg/ml, 1.6�107 iu/ml). The adenovirus solution underwent only one freeze/thaw cycle before usage in the above preparations. It was prepared around 10 weeks ago and was stored frozen at -70� C.
These formulations were processed into powders in the Buchi-190 spray dryer according to the following parameters:
Solution feed rate: 3.5-6.0 ml/min
Inlet/Outlet temperatures: 100-140/70-90� C.
Atomize flowrate: 700-800 LPH
The resulting powder was kept refrigerated and was sent for testing on dry ice. Prior to testing for β-gal expression or for virus titers, the powders were reconstituted with phosphate buffered saline (PBS).
None of the mannitol powder formulations showed any β-gal expression in the standard 6-well test and therefore they were not titered for virus infectivity. The glycine/HSA (I) and glycine/mannitol/HSA in PB from set one were equal in their β-gal expression and were tittered for virus infectivity. Their titers ranged from 7% to 15% of the expected values. The particle size distribution (HORIBA), dispersibility and the aerodynamic size distribution (IMPAQ 6-stage) are listed in Table 3 for the two glycine/HSA in PB powders.
Set two powders and 0.1 ml of the adenovirus solution (V) frozen to -70� C. were sent on dry ice for titer measurements (Table 4). Powders manufactured with and without the phosphate buffer retained 76-54% and 2-1.4% of their virus infectivities, respectively (Table 4). Lowering the outlet temperature by 5� C. increased the buffered formulation virus infectivity by 22% but it lowered the unbuffered one by 6%.
TABLE 3______________________________________Glycine/HSA adenovirus formulations. Formula Dipersi. HORIBA Cascade impactor % infectivity(mg/ml) (% RSD) MMD MMAD % <5 μm retained______________________________________29 40(25) 2.6 2.8 70 14 9 51(1) 2.3 1.8 80 7______________________________________
TABLE 4______________________________________Adenovirus powders in buffer and without buffer titer results. Outlet Temp. Expected Measured Formulation � C. iu/ml iu/ml______________________________________V N/A 1.0 � 109 1.6 � 108 Buffered 77 1.0 � 108 5.4 � 107 Buffered 72 1.0 � 108 7.6 � 107 Unbuffered 77 1.0 � 108 2.0 � 106 Unbuffered 72 1.0 � 108 1.4 � 106______________________________________
Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
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|U.S. Classification
|International Classification
|Cooperative Classification
|European Classification
|Jul 17, 1995
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Qatar recalls envoy to Iran after attacks on Saudi missions
PRECIOUS-Gold hits 7-week high as worries over China hurt stocks
MIDEAST STOCKS-Emaar drags down Dubai; Gulf markets weak
Saudi Q3 GDP growth edges down to 3.6 pct y/y
The tech giant reveals what people in the Emirates Googled the most this yearDecember 16, 2015 12:52
Beemers and Mustangs ruleJuly 13, 2015 9:00
Google releases the list of the top trending searches in 2014. As a UAE-based media company, Kipp looks at the top 10 most searched local brands in the UAE in 2014.December 17, 2014 4:28
Google released its annual Zeitgeist report today, a look at 2011 through the collective eyes of the world on the web.December 19, 2011 5:12
With the latest Facebook-Google (Foogle? Goobook?) shenanigan-slash-PR fiasco, Alex McNabb takes us through what exactly we're letting these companies get away with in terms of online privacy.May 17, 2011 1:06
The Haitian government has ended the search and rescue phase for the survivors of the earthquake.January 23, 2010 8:01
While the King of Pop stole the top spot, many international entries have found a place in Google's fastest rising searches in 2009 list.December 6, 2009 5:19
As the Dubai-based property developer shuts its doors in Ireland, Google technology reveals that the Irish have been recently shown keen interest in Damac Properties.August 24, 2008 12:51
At least that's what Google's new tracking application seems to say. And many people in Dubai are also googling the city's police.August 10, 2008 8:41


text:
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Rush Team Free FPS Multiplayers
Jan. 15, 2016
Rating: 0
Hey Rush Team Soldiers !!!!
- Operation Share for an EXP boost !
- Check out https://www.facebook.com/RushTeamFPS/posts/966340153440221 …
Rush Team http://www.asr-games.net/
Voir la traduction
Apr. 15, 2015
Rating: 1
Hello Soldiers,
Google Chrome have released the version 42 of their web browser and it don't support unity webplayer now. If you just see 'Download Unity' button, follow this step.
1. Enter chrome://flags/#enable-npapi in URL bar
2. Click Enable NPAPI (will be in yellow)
3. At the bottom of the page 'Relaunch Now' button will appear. Click on it.
Or the other solution, is to use Firefox : https://www.mozilla.org or Opera : http://www.opera.com/
Have Fun.
Roka [Rush Team Leader]
Sep. 03, 2012
Rating: 10
The Forum is now open http://www.asrprogs.net/Forum/index.php
Sep. 01, 2012
Rating: 6
Game Updated to 2.0.3 Version the 1 september 2012
- The bullet hole when you use the melee has been removed.
- We have fixed the display bug when sometime you see one players who use the P90 and have the ak47 in his hand.
- We have fixed the chat length message in game.
- We have fixed the camera height , so now the players behind some boxe for exemple can't see you when you can't see him.
- The Katana has been add.
- The M16 has been add.
Have Fun , Roka !
Aug. 31, 2012
You can create an account without email ....
Aug. 21, 2012
Rating: 9
The error as been fixed now , youcan play and have fun , sorry
Rating: 7
Hello , i have just see that the website is down , i try to Check why but i'm not at my home so it's hard to find the problem thank you
Aug. 17, 2012
Ok guy , just some news about what i do with the game.
I have started to implement the melee to the game with a lot of fixe (coding ,map and lightmap).
I work on some new stuff too but i don't talk about that for the moment.
Again big thank to all players who understand that i'm the only one to work on this project and who are patient and come play the game everyday.
For all players who think that using glitch in underground is cool , play it normaly , what's you have more after the match if your score is like 15/0 or 2/15 ? nothing more , just the sensation to have lose some time , so let's play for fun and try to get some skill because in the futur you will need it for get one positive score ;)
Have Fun Roka
Aug. 03, 2012
Rating: 44
I have found how to reduce 15%/20% of lag this evening , the game will be updated this week end
Jul. 29, 2012
Hello crazy players ,
I have make again one big work for reduce the game lag and size and the result seem to be very good.
I have fixed some bugs discovered with you and normaly i will update the game Tuesday evening or Wednesday.
The priority for me is to get one stable version for one better game play.
Thank again for testing it and for your feedback.
Have fun , roka
Jul. 26, 2012
Game Updated to 1.9 Version
- We have reduce the size of the game for a better and more fast loading.
- We have reduce the size of many textures and worked on the code for reduce the lagg of some players with low computer.
- We have change some light system for improve performance in game.
If you have get some lagg issue in the past , now it's fixed.
Jul. 25, 2012
Rating: 2
I have done maybe 75% of the my work for reduce de lagg and size of the game and i have get one nice result yesterday. If all work fine and if i finish quickly my first work tomorrow , i will update it tomorrow evening. Roka
Jul. 24, 2012
Rating: 3
Hello everybody , i work to reduce the game size and the size of some textures because some players with low computer get a lagg in game. This process can take a little bit of time but i try to do it as fast is possible. After , i will work to add more weapons and the melee in game for more fun . Roka
Jul. 19, 2012
I have fixed the bug "invalid password" this morning , when one player have one password with special character.
Jul. 18, 2012
Rating: 1548
Thank you everybody for your feedback , currently i work to add one new map and fixe some bug that i have discovered with you. I don't know the release date but be sure as soon is possible (only me work on this project).
Have a good day , Roka
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I am looking for a really good restaurant with mariachis in San Diego next Friday or Saturday, April 1st or 2nd.. We have big family celebration coming up.
Hi Guys,
As you have seen your Covers Contests Data is no longer available on your Space page.
Dont worry this is the first step for the launch of our new and improved Contests platform.
Coming in October we will be launching the new platform completely and all your information will once again be available but just in a different location.
We appreciate your patience and we know that you will be more than happy with the new platform!
You can continue to track your contest picks in Covers League Contests
You can continue to track your contest picks in Covers Survivor Contest
Copyright © 1995-


text:
NYPD Continues Hunt For Brooklyn Hit-And-Run Suspects That Left 51-Year-Old Woman DeadPolice continue to search for two men responsible for crashing a vehicle and then fatally injuring a female pedestrian while harming others along the way.
Police: Woman Killed During Brooklyn Hit-And-Run, Others InjuredPolice are searching for two men who allegedly crashed a stolen vehicle into another car and hit two pedestrians on Sunday afternoon.
Cops Seek Leads In Death Of Man Found Brutally Murdered In Canarsie's Bayview HousesA mother's grief was on display in Brooklyn as a family begged for help in finding her son's killer.
Brooklyn DA: Gun-Smuggling Ring Sold More Than 100 Weapons To Undercover CopEight people have been charged in a gun-running scheme to use cheap bus services to smuggle dozens of illegal guns across state lines into New York City.
Police: Man Wanted In Rape, Attempted Rape In Canarsie, BrooklynPolice have now linked a rape in Canarsie, Brooklyn to a subsequent attempted rape in the same neighborhood.
Police: Man Raped Woman Behind Building In Canarsie, BrooklynPolice continued to search late Tuesday for a suspect in the rape of a woman in Canarsie, Brooklyn this past weekend.
Police Release Surveillance Video, Sketch Of Man Wanted In Canarsie RapePolice are hoping the public can help identify a man wanted for raping a 19-year-old woman in Brooklyn over the weekend.
Man Gets 18 Years For Running Georgia-To-New York Gun RingThe leader of an illegal Georgia-to-New York gun ring was sentenced to 18 years in prison Thursday, after being convicted of selling 151 weapons to an undercover officer in Brooklyn in a period of seven months.
Mystery Lottery Winner Has Until Friday To Claim PrizeThe last day to claim the prize is Friday.
35-Year-Old Mother Of 3 Killed In Brooklyn Hit-And-Run CrashSidney Ramsarup, 35, was hit by an SUV just before 5:30 a.m. at East 108th Street and Glenwood Road in Canarsie, police said.
Police: 2 Suspects Wanted For Robberies, Assaults In Canarsie, BrooklynPolice are searching for a pair of robbers they say are targeting young victims in Canarsie, Brooklyn.
14 Injured In MTA Bus Accident In CanarsieA B6 bus was struck by a vehicle that ran a stop sign at Glenwood Road and East 102nd Street, according to the Metropolitan Transportation Authority.


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"Pawpaw – In Search of America's Forgotten Fruit" tells a story of a uniquely American treasure that was lost but is being found again. Like many, I had never heard of the pawpaw, North America's largest native fruit. I discovered it while learning about permaculture plantings, and I wanted to know more. "Pawpaw" takes you on a virtual tour of pawpaw territory, comparing the history of the pawpaw in a region with the state of the pawpaw in that area today. It's an eye-opening journey that speaks not just to the fate of the pawpaw, but to the fate of local food systems as a whole. It also offers hope for the revival of local food systems under the watchful eyes of a growing number of passionate foodies who want to recapture the way food used to taste. [Read more…]
Posts may contain affiliate links, which allow me to earn a commission to support the site at no extra cost to you. Thank you!


text:
SAN FRANCISCO Facebook Inc (FB.O) is harnessing satellite, drone and other technology as part of an ambitious and costly effort to beam Internet connectivity to people in underdeveloped parts of the world.
The world's No. 1 social network said on Thursday it has hired aerospace and communications experts from NASA's Jet Propulsion Lab and its Ames Research Center for the new "Connectivity Lab" project.
"Today, we're sharing some details of the work Facebook's Connectivity Lab is doing to build drones, satellites and lasers to deliver the internet to everyone," Facebook Chief Executive Mark Zuckerberg said in a post on Facebook.
He gave few specifics and did not specify a time frame.
The move extends the social networking company's Internet.org effort, aimed at connecting billions of people who do not currently have Internet access in places such as Africa and Asia. Facebook has been working with telecommunications carriers to make Internet access more available and affordable.
"We're going to continue building these partnerships, but connecting the whole world will require inventing new technology too," Zuckerberg said in his post.
Facebook envisions a fleet of solar-powered drones as well as low-earth orbit and geosynchronous satellites delivering Internet access to different regions of the world. Invisible, infrared laser beams could allow Facebook to dramatically boost the speed of the Internet connections provided by the various aircraft, Facebook said on a Web page that explaining the project.
Facebook's plans to take to the skies underscore the company's rising ambitions to exert its influence beyond the confines of its 1.2 billion-member social network and to set the pace for new technology that will shape society. On Tuesday, Facebook announced plans to acquire Oculus VR Inc, a maker of virtual reality goggles that Facebook hopes could become the computing platform of the future.
Facebook is following in the steps of Google Inc (GOOG.O), the world's largest Internet search engine, whose Google X division is working on a variety of so-called "moonshot" projects including self-driving cars and wearable computers.
Google announced plans last year to use solar-powered balloons to deliver Internet access to remote regions of the world.
Among the jobs openings posted on Facebook's website on Thursday were roles such as Antenna Systems Engineer and Electro-Optical Network Access Hardware Engineer.
Facebook also said it had hired a five-member team that worked at Ascenta, a British company whose founders created early versions of the Zephyr, which Facebook said held the record for the longest-flying solar-powered unmanned aircraft.
Shares of Facebook were up 28 cents, or less than 1 percent, at $61.25 in after-hours trading on Thursday.
(Editing by Matthew Lewis and Andrew Hay)
Trending On Reuters
Solar-powered plane lands in Arizona on round-the-world flight
A solar-powered airplane midway through a historic bid to circle the globe completed the tenth leg of its journey on Monday, landing in Arizona after a 16-hour flight from California, the project team said. Full Article | Video
Reuters Showcase
Reuters India App
Download the Reuters app from the Apple App Store, Google Play and Amazon. Select the India edition from settings. Full Coverage


text:
The Mediterranean Diet for Every Day: 4 Weeks of Recipes & Meal Plans to Lose WeightNEW YORK TIMES BESTSELLER Experience the amazing benefits of the Mediterranean Diet in just one month. The Mediterranean Diet is certified as one of the healthiest diets by trusted organizations from the American Heart Association to the Mayo Clinic. With a focus on flavorful produce, healthy fats, whole grains, and wholesome natural foods, the Mediterranean Diet is a lifestyle change that is
The Mediterranean Diet for Beginners: The Complete Guide - 40 Delicious Recipes, 7-Day Diet Meal Plan, and 10 Tips for SuccessNEW YORK TIMES BESTSELLER Embrace a healthy Mediterranean diet through whole, natural foods prepared with heart. Hailed by the Mayo Clinic as a "heart-healthy diet," the Mediterranean diet is praised as one of the healthiest ways to eat in the world. The Mediterranean Diet for Beginners is your complete guide to understanding this low-fat, nutritious diet for optimum health and w...
105 Recipes Already Hiding in Your KitchenUsing checklists and smart strategies, Jennifer Chandler helps readers unearth dozens of Southern meals waiting to be discovered in their well-stocked pantries. Nothing can discourage a home cook quite like being unprepared—running to the store for that one item, getting halfway through a recipe and realizing something is missing, or simply not knowing quite where to begin. Kitchen pro and
Recipes and Lessons from Paris and ProvenceA master class in fresh, delicious, French-inspired cooking Since 1995, students have waited months and sometimes years for the privilege of learning to cook with Patricia Wells at Chanteduc, her eighteenth-century Provençal farmhouse, and at her Parisian cooking studio. Now, the culinary legend invites home cooks into her life in France, making the recipes from her popular classes available to
$7.99Aunt Bee and her friends have stirred up a cookbook that brings home all the flavor of "The Andy Griffith Show's" Mayberry. You'll enjoy most of the 300 mouth-watering recipes (but not all?included is the recipe for Kerosene Cucumbers) for the foods served by Aunt Bee and others in Mayberry. From good old-fashioned, down-home cooking to some of Mayberry's more unusual meals, you'll discover
Home Cooking with Jean-GeorgesJoin one of the world's greatest chefs in his most personal book yet, as Jean-Georges Vongerichten shares his favorite casual recipes in Home Cooking with Jean-Georges. Though he helms a worldwide restaurant empire—with locations in New York, Las Vegas, London, Paris, and Shanghai—Jean-Georges counts his greatest joy in life as family first, then food. In Home Cooking with Jean-Georges, he brings
The Southern Slow Cooker Bible
365 Easy and Delicious Down-Home RecipesSome days just call for the comforting flavors of the South. And while those days are often the busiest, your slow cooker is ready to do nearly all the work for you! Tammy Algood's The Southern Slow Cooker Bible serves up 365 recipes, covering a year's worth of dishes that let you effortlessly pair the flavors of the South with the convenience of slow cooking. Algood covers all the favorites—from
Pressure Cooking Chinese Recipes: How to Cook Delicious Chinese Food FastThis recipe cook book gives you 30 Chinese food recipes that you can cook with the pressure cooker. Each recipe comes with photos of the cooking process so that you will have a good idea how to cook it. I have covered a range of recipes for meat, fish, vegetables, noodles, rice and soups. These recipes are my own tested recipes using authentic Chinese sauce, spices and ingredients. A list of
$0.99Jamie Geller, "The Jewish Rachael Ray" (New York Times) and founder of the Kosher Media Network, including Joy of Kosher with Jamie Geller magazine and JoyofKosher.com, shares more than 200 ideas for fast, fresh family-friendly recipes, each with tips on how to dress them up for entertaining or dress them down for everyday meals. Accompanied by gorgeous full-color photos, Joy of Kosher includes
$13.99Wholesome, delicious Japanese comfort food, hot pot cooking satisfies the universal desire for steaming, gratifying and hearty meals the whole family can enjoy. In Japanese Hot Pots, chef Tadashi Ono and food journalist Harris Salat demystify this communal eating tradition for American home cooks with belly-warming dishes from all corners of Japan. Using savory broths and healthy, easy-to-find
150 Recipes and Ramblings from America's Best Restaurants on WheelsIt's the best of street food: bold, delicious, surprising, over-the-top goodness to eat on the run. And the best part is now you can make it at home. Obsessively researched by food authority John T. Edge, The Truck Food Cookbook delivers 150 recipes from America's best restaurants on wheels, from L.A. and New York to the truck food scenes in Portland, Austin, Minneapolis, and more.John T. Edge
$13.99How many minutes should you cook green beans? Is it better to steam them or to boil them?What are the right proportions for a vinaigrette?How do you skim off fat?What is the perfect way to roast a chicken?Julia Child gave us extensive answers to all these questions–and so many more–in the masterly books she published over the course of her career. But which one do you turn to for which solutions?


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Grace Kelly is a former French Teacher & Events Host who swapped her hectic London life & career for a life of doing work she loves whilst living in sun drenched southern Italy.
In this seminar Grace will share how she managed to escape the Rat Race once and for all despite the recession and fears of failure and how she managed to turn her once London sideline into a gold mine whilst living abroad.
The objective of this session is to enable you to breakthrough the fears that cloud your vision and prevent you from doing work you love and living the life you dream of.
During the session Grace will teach you:
1. Why your life is so damn hard and how to simplify things today.
2. How to gain clarity on what you'd really love to do next so you can consider leaving the rat race once and for all.
3. The 3 biggest lies running your mind that keep you stuck in a life or career that don't inspire you.
4. How your environment can impact negatively on your wellbeing and therefore sabotage your success and ways to choose the right locations for you.
5. How to overcome the fear of failure so you can create a career and life you'll love from anywhere in the world.
6. Techniques to help you build trust in yourself, your brand and your future so you can take the leap and move abroad.
7. The NO.1 way to escape the City and live and earn from abroad today .
Grace Kelly is a Mentor, Teacher & Blogger. Her mission involves assisting burnt-out & bored professionals in achieving better health and greater wealth doing work they love.
As creator of the CityGirl Career Style™ System, Grace helps women in Cities identify and create careers and/or businesses more in line with their values, purpose and personal style. She speaks globally on the link between vocation and vitality having overcome burnout not through medical means but by finding a way to work she enjoys.
Grace offers transformative intensives and retreats in cities around the world and is currently based in Puglia Italy having escaped the Rat Race in London as little as 18 months ago.
Hi All,
Just wanted to say a big Thank You again for attending , I'm having a lovely time getting to know you even more in the free sample sessions, these are available for ALL attendees so if you didn't reserve yours feel free to be in touch and I'll schedule you in. Hugs and Gratitude, Grace x
1 · July 9, 2013
Enjoyed this talk or missed it? check out Grace Kelly's teaser at http://www.interestingtalks.co.uk/gracekelly/
0 · July 10, 2013
This was a really good talk - very meaningful and inspiring - thank you
1 · July 3, 2013
I really enjoyed this talk too and more importantly found it inspirational. The excercises were really good and useful, I learned a lot about myself and also seeing that following a dream can be done and that there are other people who feel the same is reassuring. Thanks to Matt for putting this on and to Grace for her talk.
1 · July 3, 2013
A massive thank you to Gracy Kelly for coming all the way from Italy to speak at Interesting Talks. This talk will be online next week. You can sign up to the membership site today for just 99p and we will let you know when it is online http://www.interestingtalks.co.uk/specialoffer/
1 · July 3, 2013
Grace I'm sorry not to be able to get there and see you tonight. Have had a day of accounts as I'm in the midst of changing accountants, deadlines are looming and I am between accountants and it's a mess. Have spent the whole day either on the phone or searching through files. It's still ongoing too.
Hope it all goes really well and catch you next time, I hope. Heather x
1 · July 2, 2013
So, looking forward to seeing you Grace and hearing you speak. You really inspired me at your last gathering in London :-) x
I believe I'm doing what I love, now, though it's taken some time to craft it all out and I'm sure there will be more changes, but how does one let go of trying to control the "outcomes" of what one desires and just trust?
3 · June 25, 2013
Hi, I wasn't able to attend a previous paid for event and asked that the fee be transferred to another event. Can this be the event please?
0 · June 3, 2013
Looking forward to hearing Grace speak again and being inspired by her.
1 · May 15, 2013
Refunds offered if:
Additional notes: Cancel with more than 24 hours and I will credit you onto a future talk of the same value. Cash refunds are not available.
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Friday, April 20, 2012
0 Comments | Copyright: IANS
National Award winning Bengali actor Prosenjit Chatterjee's character of the suave and dynamic politician in Dibakar Banerjee's "Shanghai" is inspired by and modelled upon acclaimed filmmaker Shekhar Kapur.
Banerjee's team was asked to dig up pictures of how Kapur looked 15 to 20 years back. These were used as direct reference points for Prosenjit's physical appearance in "Shanghai".
"The fact is, when I wrote the role of the fantastically sexy and suave politician, it was Shekhar Kapur I had in mind. But we needed someome in his 40s. So, while we wisely chose Prosenjit for the politician's role, it was Shekhar Kapoor we had in mind," said Banerjee.
The filmmaker admits that watching Prosenjit in "Shanghai" will remind viewers of the way Kapur walks, talks and dresses.
"Shekhar had this absolutely crazy dress sense which worked perfectly for him. He would mix and match the most unlikely clothes and colours and still look dynamic," he said.
"We took out Shekhar's sartorial adventures from internet and we used pictures to design Prosenjit's clothes," he added.
However, more than the clothes, it's Kapur's attitude that's been imbued in Prosenjit's character.
"When Shekhar talks, he doesn't have to raise his voice to be heard. There's an inherent magnetism and wisdom to his personality that we've used for Prosenjit's character."
"I think his character will give to the middle-aged women in the audience a new sex symbol. He is a younger Shekhar Kapur and Naseeruddin Shah," said Banerjee, known for films like "Khosla Ka Ghosla", "Oye Lucky! Lucky Oye!" and "Love Sex Aur Dhokha".
Prosenjit says Kapur was a "thinking sex symbol" of his times.
"That's how Dibakar has visualised my character. We sat down together and thought of a look for me. We thought of the way Shekharji used to look 15 to 20 years ago."
"Women of a certain age would swoon over his suave serene styling and demeanour. He was the thinking woman's sex symbol. I love the idea of being in the same space. But I don't mind being the non-thinking woman's sex symbol either," said the 49-year-old.
"Shanghai", a political thriller, also features Abhay Deol, Emraan Hashmi and Kalki Koechlin. It is set to release June 8.
Priyadarshan is very demanding: Zayed Khan
Boldness a state of mind: Bengali actress Paoli Dam (Interview)
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text:
The organization called Seycor j & m S.L., with business activities related to other building installation and construction, with three filed documents at the Companies Registry Office, registered in the Barcelona company register, incorporated in Spain, the province of Barcelona, with address in the Sol street of Barcelona, at the postcode 8012, is a corporation of type "sociedad limitada". We have information about 2 current appointments. The business company was visited recently seven times in the last two years from two different countries and appointed one Joint And Several Administrator (a man) after its incorporation .
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Jim Cramer explains what to watch ahead of the open, including the outlook for toy companies, and coal. » Read More
These stocks can be trusted, even in an environment where it's hard to trust at all.
What follows is a roundup of corporate earnings reports for Monday, April 19.
As the nation emerges from recession it appears one of the first things parents want to do is go toy shopping. What's the trade?
The Dow pulled off a gain Monday as Goldman Sachs shares finished higher following news that the SEC vote to sue was close — raising doubt that the charges will stick. Citigroup jumped 7 percent after blowing past earnings forecasts.
What follows is a look at stocks in the S&P 500 displaying unusual volume in today's trading session.
Stocks pushed higher Monday after a sharper-than-expected rise in leading indicators and an earnings beat from Citigroup.
U.S. stock index futures were lower ahead of the open Monday as investors were still reeling from Friday's news that the Securities and Exchange Commission charged Goldman Sachs with fraud, with banks leading European indexes lower.
Uncertainty surrounding Goldman Sachs will likely overshadow the positive news from dozens of major corporate earnings reports in the week ahead. Some analysts say the Goldman spacer fraud charges could be the event that will trigger a much anticipated stock market correction.
Cramer makes the call on viewers' favorite stocks.
Hasbro has pushed back near an all-time high, and one trader is positioning for a correction in the next two months.
Given Cepia's runaway success with its Zhu Zhu Pet hamsters last year, it should come as no surprise that there was no shortage of hamsters at this year's American International Toy Fair.
Judging by the toys featured at the American International Toy Fair, the industry isn't ready just yet to place huge bets on an economic recovery. Click to see some of the coolest products from the show.
The American International Toy Fair, the industry's main trade show, kicks off Sunday in New York. Here are some noteworthy toys to watch for.
The more people who go on vacation, the more money this company can return to shareholders.
Mattel recently asked its fans to vote on Barbie's next career, and after more than half a million votes were tallied, the winner was...news anchor.
So far, that is. Read on for one of Cramer's retail favorites right now.
International growth helped Discovery Communications earnings grow 46 percent, in line with expectations, while revenue grew seven percent.
A big dividend hike in January has the Mad Money host bullish on this company's prospects.
Plus, find out an even more powerful driver behind the markets these days.
Cramer answers questions viewers mail on Google, Apple and more


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My LO still takes a bottle when he naps and goes to bed at night. I'm in no rush to get rid of it. It's special time for us. And he still gets up for 1 during the night which I hate but when he is ready to stop he will.
if you have failed as a mother than so have I. While my son doesnt co sleep or get ricked to sleep, those were his choices. he let us know when he was ready to sleep alone. I do not let him self soothe. I will alsways soothe him when he cries and he loves his bottle and his pacifier.
Tatiana (20) Married my best friend Elijah (22)BeautifuL son EJ (born 7-1-2011)
Angel Baby M/C November 30, 2012
Mommy to a beautiful little girl (7/11) :)
Praying for God to send us our rainbow!
Always remembering & missing my 4 angels
-- Alicia
Momma to: My lil' cowboy Chase David 7/19/02 & Brock Austin 6/29/11
Everyone has different parenting skills..if it works for you guys that's what matters most. I'm doing lot parenting skills some people don't like but oh well my babies, my life =) and my babies are happy lol
Army Wife 5/24/08
DavidJ 8/7/09
Felicia 7/15/11
T ♥ M Tied the knot for Time & Eternity in 2007
Mommy to S (5), I (2) & Baby L (0)
Learn more
Look how far your child has come.
Review your child's growth milestones.
Read about 4 years 8 months : Week 3.
Your growing child
Your 4 3/4-year-old: Discouraging tattling
Your 4 3/4-year-old: Bedtime habits
Your 4 3/4-year-old: Prepping for shots
Your 4 3/4-year-old: Listening skills
Your 4 3/4-year-old: Saying "I'm sorry"
Learn more
What's next for your growing child?
Get a glimpse of what's to come.
Read about 4 years 10 months : Week 1.


text: John Reed: The Making of a Revolutionary By Granville Hicks, John Stuart No cover image John Reed: The Making of a Revolutionary By Granville Hicks, John Stuart Read preview Related books and articles Books Journals Magazines Newspapers Encyclopedia A Yankee Saint: John Humphrey Noyes and the Oneida Community By Robert Allerton Parker G.P. Putnams Sons, 1935 Read preview Overview African-American Traditions in Song, Sermon, Tale, and Dance, 1600s-1920: An Annotated Bibliography of Literature, Collections, and Artworks By Eileen Southern; Josephine Wright Greenwood Press, 1990 Read preview Overview Reforming Women's Fashion, 1850-1920: Politics, Health, and Art By Patricia A. Cunningham Kent State University Press, 2003 Read preview Overview Pleadings & Filings By THE JOURNAL RECORD, August 8, 1989 Read preview Overview FREE! Reed, John The Columbia Encyclopedia, 6th ed., 2016 Read VIEW ALL RELATED BOOKS AND ARTICLES


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I want to create a View (or modify an existing one) so that the menu item type is MENU_LINKS_TO_PARENT, so that when I have sub menu's displayed for a page, the page has a link in the sub menu. This is exactly the same has having a default task, I just want it as a default menu item.
Is this possible, and how?


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Bernie Buescher, the incumbent Colorado secretary of state defeated by Republican Scott Gessler in November, has found a new job: overseeing the state lawyers who represent Gessler.
Buescher on Tuesday was hired as the deputy attorney general overseeing the Department of Law's state-services section, the division in the attorney general's office that provides legal advice and representation to other state agencies and elected officials. Among those elected officials who are frequently represented is the secretary of state.
The irony of Buescher's appointment is underscored by the fact that it was made by Attorney General John Suthers, the most senior Republican elected to statewide office in Colorado.
In a statement, Suthers said it came down to qualifications.
"I am pleased to welcome Bernie to my senior staff," Suthers said. "Bernie is a proven leader and manager. His breadth of experience as an attorney, an elected official and a statewide executive will make him an excellent addition to the Office of the Attorney General's management team. I look forward to working with him in this new capacity."
Gessler, though, seemed bewildered by the appointment.
"It's very unusual, that I can say," Gessler said. "I'm not going to be in the business of telling John Suthers how to run his shop. All I can expect of Bernie Buescher and all members of the attorney general's staff is that they're going to represent this office vigorously."
That's just what Buescher said he would do.
"My job is to make sure that the statutes and the constitution are interpreted and implemented properly," Buescher said. "In all these situations, the client is the one who ultimately calls the shots."
Buescher, a Democrat who served in the state House before being defeated for re-election in 2008 and then appointed by Gov. Bill Ritter to fill the vacant secretary of state job, also served as president of West Star Aviation Inc. and executive director of the Colorado Department of Health Care Policy and Financing under Gov. Roy Romer. He worked as a law clerk for Colorado Supreme Court Justice Paul Hodges from 1974 to 1975 and then practiced law in Grand Junction for a number of years.
He is a 1974 graduate of the University of Colorado Law School and a 1971 graduate of the University of Notre Dame.
"I'm assuming that Bernie Buescher is going to be a professional attorney about this," Gessler said, "but yeah, it's awkward."
Buescher agreed but said, "As professionals, you just work through these things."
Tim Hoover: 303-954-1626 or firstname.lastname@example.com