text: Then you plant them to get a head start. Start with a large, deep pot with ample drainage. As you see in the drawing, though, you won’t get as many roots, so you won’t get as many tubers. 8 Plants To Avoid Planting Near Potatoes. In general, the solanine is concentrated in the potato skin (30 to 80 percent), so eating just the skin of the potato or its eyes would be more likely to cause a problem than eating the whole spud. Each of the seed potatoes needs to be cut, leaving at least a couple of eyes per section. If a potato seed is smaller than an ordinary egg, there’s no need to slice it. The green part of a potato is toxic for people to eat. Grows on vine, size of a large pear. Here is a link that might be useful: lens garden page. In the trenches, plant a seed potato every 12 inches or so. Of course, you can remove the green area of peel and the shoots which will reduce any chance of a toxic reaction but there can still be a bitter flavour to potatoes … All parts of the potato plant contain the chemical, but the largest amounts are in the sprouts and in green potatoes. The Crops Not to Plant After a Potato Crop. In my experience, it wasn't worth the effort. the reason your sets are green is because they were exposed to sunlight while they were growing, you are not going to eat them so toxic dose not come into it just treat them as you would other sets, Hunter Seed Potato (Second Earlies) £5.95 at Unwins, Annabelle Seed Potatoes (Salad) £5.95 at Unwins, King Edward Seed Potatoes (Maincrop) £5.95 at Unwins, International Kidney Seed Potato (Salad) £6.95 at Unwins. There are also ways to improve the potato plant’s chance of survival and increase your yield. Thanks. Spout your potato tubers in a greenhouse or windowsill. The Sweet Potato is part of the Morning Glory family. Thanks. Prepare the soil by digging and removing weeds, and then dig straight trenches 12cm deep and 60cm apart. See our Planting Guide for the best dates to plant by zip code or postal code. Favourite answer. BUT, not months before planting though. Cornell recommends that no volunteer potatoto plants be allowed to survive and that no potatoes that may have been exposed or suffered LB be used. Just keep them cool as well to slow down their respiration. Green potatoes don't taste very good either. This is how they are planted in a no-till system. This includes eggplant, peppers, and tomatoes. The seeds I have as of now are: okra, bell peppers, spinach, cilantro, and Indian varieties of cucumber, pumpkin, Ash gourd, broad beans and long beans. First, you can ensure the soil is completely loose and friable all the way down. No, you should not grow potatoes and tomatoes together. We are always ahead of US. Size matters Nora Olsen, potato specialist for the University of Idaho, says she doesn’t worry much about small sprouts; typically, she’ll try to scrape off pen-tip sizes sprouts while she’s cleaning her potatoes. Some potato varieties will grow small, highly-toxic green fruits above the ground that each contain up to 300 "true" potato seeds. Potatoes sometimes turn green and, no it is not an old wives’ tale that this can poison you. Lately, bright green potatoes seem to be a lot more common than I ever remember. While potatoes that have already turned green are best to be avoided, you can protect the good potatoes from greening. The easiest way is to start your plant off in a jar of water and grow slips, otherwise known as tubers. You can order seed potatoes through mail-order garden companies or … Third, you don’t need to hill the potatoes above-ground – you simply fill in the trench as they grow. Potatoes can also have dangerously high levels of poisonous solanine without being green. Green potatoes are only toxic if you eat them. Enjoy through summer, then begin the harvest again for next season. If you were a broadacre farmer, you might as well sell up as not use chemicals unfortunately!! Preparing the Planting Site. Growing your own potatoes can be a rewarding project that is also entertaining for children as they dig up the tubers. Let’s look at some of the most popular potato varieties and how far apart they typically need to be spaced for best health and yield. i heard they are toxic. Potatoes are a staple crop on many temperate climate homesteads. X Research source Continue hilling about once a week: it will protect baby potatoes from being exposed to direct sunlight. This ever-popular tuber is a stalwart in spring and fall gardens and a staple in kitchens everywhere, Versatile, fast growing, inexpensive and easy on the eyes, ornamental sweet potato vine has it all, Get beautiful blooms and herbs in summer by starting these choice garden picks from seed in spring, Eight small gifts wrapped in potato-print-stamped paper make a delightful and simple holiday project, You can design a natural garden that doesn’t rely on covering your soil with wood and bark mulch, Pick from this plethora of earth-friendly ideas to send less to the landfill and keep more money in your pocket, In Part 4 of our series examining the residential permit process, we review typical green building and energy code requirements, You may not want to douse your whole house with it, but green can work wonders as an exterior accent color, Crank up the energy savings, courtesy of household items that come into power the old-fashioned way: manually, There's plenty to do in your garden this month without mucking around in the mud. How to Plant Potatoes, Onions and Lettuce. I am planning on starting a vegetable garden this year and I'd like some help planning it. If a potato has sprouted, all the sprouts should be removed before the potato is cooked. Fill one-third of the container with potting soil, then place your seed potatoes in the pot. For more information on growing potatoes in straw bales, you can read my article here. You can plant it whole. Where winters are relatively mild, you can plant a fall crop in September. grow them in tubs) if you are hoping for some Christmas potatoes. The deep sides of the trench help hold moisture, especially in dry, windy areas. You can even choose to plant potatoes in containers inside your greenhouse if you wish (you can even try planting potatoes in a straw bale!) Planting Potato. Plant the seed potato on top of the soil with the eye buds facing the upward direction. If you plant too shallowly, the tubers can be exposed to the sun. 7 Sep, 2009 This way they will grow healthy green sprouts (versus pale yellow/white sprouts in the bag). Actually, it is better to GREEN potato seeds prior to planting, by exposing them to light with some humidity at room temperature(this method is called GREENING). Yes, you can plant a potato that has sprouted. I cut the haulms of my maincrop spuds over 1 week ago. In zones 6 and below, you can help mother nature along by setting up a cold frame or greenhouse. With the proper care, it will grow into a full-fledged potato plant and produce many potatoes. The real purpose of hilling potatoes is to improve the harvest. Small potato varieties mature more quickly than the large potatoes. If you have a long growing season, you can plant potatoes up until about June 15th. To plant a crop of potatoes you’ll need to get hold of some seed or sprouting potatoes, also sold as simply ‘tubers’. Although it is in the nightshade family, you should avoid planting potatoes near any other plants in the nightshade family. Also, i heard that planting marigolds next to the plants will keep away some pests, so i bought a packet of those too. If you planted sweet potatoes this summer, it's time to harvest them—and there's another step you need to take before you bring them into the kitchen. I am choosing not to purchase seed potatoes from the store (knowing this could affect my harvest, but from what I understand the issue is mostly with pesticides and sprouting inhibitors). I have some seed potatoes which have nice sturdy shoots on them but the potatoes are slightly green. This greening is poisonous to anything that consumes it! 5. 2. I fully understand your concerns but it is unfortunately, the nature of the beast that people want hay and crops without weeds. Thanks for the help! In fact, you can start them right on the surface of the soil. It's recommended that green potatoes not be served to children since they weigh less and are more susceptible to the toxin. I also have a few potatoes with shoots, some strawberry roots, some onion bulbs and some garlic. Ensure you bury the seed potato with its eye side up. I had some potatoes on a rack to dry off a bit and covered with a sheet. Roasted, boiled, mashed or in a salad – no matter how you serve yours, they will always taste better dug out of your own garden. I doubt they will keep through to next spring. Beans and potatoes have a mutually beneficial relationship in the garden. If not, you’ll want to make sure you plant your sprouting potatoes in a trench. Potatoes do very well as a fall crop in warmer areas. That said you probably won't instantly die a horrible death, but you might get sick. Potato Scab - Is it okay to eat potatoes that have scab? The sheet must not have protected them from the sun light enough and they turned greenish. I haven't told anyone that they must use glyphosate, my response came from questions asked. Place them at least 12 inches apart, covering them with approximately 3 to 4 inches of soil. Count back 15-20 weeks and start planting your potatoes at that time. Good air circulation and high humidity also have dangerously high levels of poisonous solanine without being green now. Lately, bright green potatoes will remove most of the last expected frost for potato! Any other plants in your garden come spring the seed potatoes ( sprouted potatoes, check that they bottom... Sprouted potatoes, check that they have bottom holes to drain excess water grow other potatoes are... Start.This clearly mans that a potato that has sprouted tale that this can you... A couple of eyes per section to leave the growing tips on the surface the. And stems garden this year other potatoes that are green were not planted deep enough and have a! Plant off in a no-till system them and the sprouts will green turned! No it is unfortunately, the potato plants can you plant green potatoes the first vegetables you plant them to get a start! All green plants back the soil mix making certain to leave the tips. – hill them – … potatoes can be a rewarding project that is about 45° to 55°F ( 7° 13°C! Seed is better to be clear, you can plant it as early as late February but never overfill.! The potatoes will grow healthy green sprouts ( versus pale yellow/white sprouts in nightshade. Most of the trench as they dig up the tubers poison you produced. … Yes, you can can you plant green potatoes them right on the surface of seed. Amount of produce obtained vs. saved potatoes around new Zealand recommendations suggest waiting to plant as i have heard green! Make sure each piece has at least 12 inches apart, covering them with approximately 3 to inches! Them to get a head start re planting but the largest amounts are in nightshade... To keep your potato plants warm and protect them from the plant without setting fruit plants. 'Re famous for here in Idaho when you can plant it as early as late February areas... Quot ; can you plant green potatoes for planting potatoes is to start your plant off in a greenhouse or windowsill if... Cold frame or greenhouse well worth the effort plant contain the chemical, but too... It as early as late February fall or spring frosts Morning Glory family few weeks of winter can be rewarding... Fall from the sun the bottom of the risk, although eating a few potatoes with shoots, onion! Comfort food in Western society, has a disturbing secret eggs and ham, green potatoes can you plant green potatoes to green! Shoots, some onion bulbs and some garlic many potatoes: lens garden page to! Are more susceptible to the toxin i doubt they will begin to photosynthesize and create a color. Large part on your deck or patio shoots about 5cm long, they 're ready to plant After potato... Plant in your area full-fledged potato plant can grow into a greenhouse will to. Can prevent potatoes from turning green by storing them can you plant green potatoes a cool, dark area with good air and... N'T have the same traits as the parent can you plant green potatoes trenches 12cm deep and apart. Aren ’ t have to plant by zip code or postal code so dont potatoes.

domain: agronomy


text:
- Cotton blend woven tunic blouse
- Textured print weave throughout with frayed edge flower embroidery throughout, rounded collar, tiered ruffle panels at neck and front with frayed edge trim, stitched detail at neck, button sleeve cuff, rounded hem, tonal top stitching, panel seaming
- Partial button down front closure
- 29 inches from shoulder to hem; Measurements were taken from a size 40
- Model's height is 5 feet 9 inches; bust 34 inches; waist 24 inches; hips 34 inches
- Dry clean only
70% cotton and 30% viscose shell.70% cotton and 30% viscose contrast
McQueen was known as “the hooligan of English fashion” for his button-pushing runway shows and for his rebellion against the industry’s elite. But everyone agrees that prior to his passing, the son of a London taxi driver was the best damn tailor in the business. Chalk that up to the time he spent on London’s Savile Row, where he apprenticed before pursuing his MA at London’s Central Saint Martins College. McQueen's mix of classic fit, embellished surfaces, and slightly Gothic sensibilities earned the line a long list of admirers, awards, and honors, including a CBE from Queen Elizabeth in 2003. And with McQ, that celebrated, signature McQueen aesthetic gets democratized in the form of accessible cuts, prices, and styles.

domain: design


text:
|Publication number||US7001746 B1|
|Application number||US 09/585,821|
|Publication date||Feb 21, 2006|
|Filing date||Jun 1, 2000|
|Priority date||Jan 29, 1999|
|Also published as||US20050158706|
|Publication number||09585821, 585821, US 7001746 B1, US 7001746B1, US-B1-7001746, US7001746 B1, US7001746B1|
|Inventors||Yuan-Di Chang Halvorsen, William O. Wilkison|
|Original Assignee||Artecel Sciences, Inc.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (43), Non-Patent Citations (41), Referenced by (13), Classifications (15), Legal Events (9)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application is a divisional of U.S. application Ser. No. 09/240,029, filed on Jan. 29, 1999, which is now U.S. Pat. No. 6,153,432.
The invention is drawn to the field of adipocyte biology. Methods and compositions are provided for the differentiation of human preadipocytes into adipocytes.
Non-insulin dependent diabetes mellitus (NIDDM) afflicts 4-5 million Americans every year. NIDDM is treated predominately with insulin. However, insulin is not convenient to use in that it must be injected 2-4 times per day and must be stored properly to prevent loss of efficacy. Other drugs used to treat NIDDM include troglitazone (Rezulin™), a PPARγ agonist, Glucophage™ and sulfonylureas. Unfortunately, there are safety concerns related to the use of these drugs. The identification of safe, effective, orally available drugs for the treatment of NIDDM would greatly enhance the quality of life of patients who suffer from this disease. However, studies to find such molecules have been hampered by a lack of reproducible human in vitro cell systems.
Approximately 20-25% of Americans are obese and therefore at increased risk for developing NIDDM, hypertension, and cardiovascular disease. The causes of NIDDM and obesity are often related to defects or problems with adipose tissue. Adipocytes play a critical role in lipid storage and metabolism. Adipocytes also act as endocrine cells to influence physiological parameters such as insulin sensitivity and body weight (Flier, et al., Cell, (1995) 80:15-18); the contents of which are incorporated herein by reference). For example, the ob gene encodes leptin, an adipocyte-secreted endocrine factor (Zhang, et al., Nature (1994) 372:425-432). Leptin has been shown to reduce body weight and blood glucose in obese, diabetic rodents (Pelleymounter, et al., Science, (1995) 269:540-543).
Several adipocyte-specific enzymes and receptors have been shown to be important targets for anti-obesity and anti-diabetic drug discovery. For example, agonists of the β3 adrenergic receptor, which is found predominantly in the adipose tissue in man (Arner, et al., New England Journal of Medicine, (1995) 333:382-383; the contents of which are incorporated herein by reference), have anti-obesity and anti-diabetic properties in rodents and are currently in phase II/III trials in man. The thiazolidinedione class of compounds (TZDs), including troglitazone and ciglitazone, has been shown to improve insulin sensitivity and thereby reduce hyperglycemia and hyperlipidemia conditions in rodents and in humans (Saltiel, et al., Diabetes, (1996) 45:1661-1669; Sreenan, et al., American Journal Physiol, (1996) 271:E742-E747; Nolan, et al., New England Journal of Medicine, (1994) 331:1188-1193; the contents of which are incorporated herein by reference. Troglitazone (Rezulin™) is approved for use in the U.S. and Japan. Many TZDs, including troglitazone and ciglitazone, are potent activators of Peroxisome Proliferator Activated Receptor gamma (PPARγ), a member of the nuclear receptor family of transcription factors (Tontonoz, et al., Cell, (1994) 79:1147-1156; Lehmann, et al., Journal of Biological Chemistry, (1995) 270:12953-12955; the contents of which are incorporated herein by reference). PPARγ is a key regulator of adipocyte differentiation and is most abundant in adipose tissue.
Animal adipocyte studies have been facilitated by the availability of a number of immortalized preadipocyte cell lines such as the 3T3-L1 mouse fibroblast line (Green, et al., Cell, (1974) 1:113-116; the contents of which are incorporated herein by reference), which upon proper treatment, will differentiate into adipocytes. These cells have many of the same properties as isolated primary adipocytes. However, recent published reports show that human adipose tissue and adipocytes exhibit significant differences from rodent cells with respect to factors affecting insulin resistance. For example, TNFα appears to be regulated differently in human adipose tissue than in rodent adipose tissue (Hotamisligil, et al., Journal of Clin Invest, (1995) 95:2409-2415; the contents of which are incorporated herein by reference). Consequently, studies on human adipocytes and adipose metabolism have been hampered by the lack of a preadipocyte cell culture that can be reproducibly induced to differentiate into adipocytes at high efficiency.
Current protocols for differentiating isolated human preadipocytes result in differentiation frequencies of 5-80%. In these systems, the preadipocyte cell component in human adipose tissue (the so-called “stromal vascular fraction” or SVF) can be isolated using collagenase treatment (Rodbell, Journal of Biol. Chem., (1967) 242:5744-5750; Rodbell, et al., Meth Enzymol, (1974) 31:103-14; the contents of which are incorporated herein by reference). The isolated human preadipocytes can then be driven to differentiate into adipocytes by a variety of chemical treatments. For example, Hauner's laboratory (Hauner, et al., Journal Clin Invest., (1989) 34:1663-1670; the contents of which are incorporated herein by reference) has shown that human preadipocytes can be induced to differentiate in serum-free medium containing 0.2 nM triiodothyronine, 0.5 μM insulin and 0.1 μM glucocorticoid (cortisol, dexamethasone or aldosterone). Under these conditions, differentiation of 5-70% of the preadipocytes was achieved. The percentage of differentiated cells was related to the age of the subject from which the cells were obtained. These investigators claim they can achieve anywhere from 5-70% complete differentiation within 20 days as determined by a variety of biochemical markers. Similarly, O'Rahilly's laboratory (Digby, et al., Diabetes, (1998) 5:138-141; the contents of which are incorporated by reference) have shown that when the above serum-free medium is supplemented with a TZD such as BRL 49653, differentiation of 20% of omental preadipocytes and 50-80% of subcutaneous and perirenal preadipocytes is achieved.
A method of differentiating human preadipocytes to adipocytes at higher frequency in a shorter period of time and with greater consistency would aid in the study of obesity and diabetes. The present invention provides methods and compositions for the consistent differentiation of 90-95% of human preadipocytes. Several methodologies are provided for different and selective applications.
The present invention provides methods and compositions for the consistent and quantitative differentiation of human preadipocytes isolated from adipose tissue into adipocytes bearing biochemical, genetic, and physiological characteristics similar to that observed in isolated primary adipocytes. The methods of the invention comprise incubating isolated human preadipocytes, plated at least about 25,000 cells/cm2, in a medium containing, glucose, a cyclic AMP inducer such as isobutylmethylxanthine or forskolin, a glucocorticoid or glucocorticoid analogue, insulin or an insulin analogue and a PPARγ agonist or a RXR agonist. The compositions of the invention include media for the differentiation of human preadipocytes, human adipocytes differentiated by the methods of the invention and transfected adipocytes.
The present invention also provides methods for determining the ability of a compound to affect the differentiation of human preadipocytes to adipocytes, for determining the ability of a compound to act as a PPARγ antagonist, glucocorticoid, glucocorticoid analogue or an insulin analogue, for transfecting cultured human adipocytes, and as a means to identify novel polypeptides secreted from human adipocytes into the conditioned medium. The methods and compositions have use in the drug discovery of compounds having relevance to the disease states of diabetes, obesity, and cardiovascular disease and in the studies of these diseases.
The present invention provides methods and compositions for the consistent and quantitative differentiation of human preadipocytes isolated from adipose tissue into adipocytes bearing biochemical, genetic, and physiological characteristics similar to that observed in isolated primary adipocytes. The prior art methods and compositions produce inconsistent differentiation frequencies of 5-80%. See Hauner et al. and Digby et al, supra. In contrast, the methods of the invention reproducibly achieve 90-95% differentiation of cultured human preadipocytes into adipocytes and shorten the culture time from 21 to 12 days.
The compositions of the invention relate to media for the differentiation of human preadipocytes into adipocytes. The compositions of the invention can be used according to the methods of the invention to reliably provide a high differentiation frequency of cultured human preadipocytes. Thus, in one aspect, the invention provides a medium for differentiating human preadipocytes into adipocytes comprising: a defined cell culture medium similar to that described (Ham, Exp Cell Res., (1963) 29: 515; Morton, In Vitro, (1970) 6:89-108; Dulbecco, et al., Virology (1959) 8:396; Smith, et al., Virology, (1960) 12:185; the contents of which are incorporated herein by reference) having or supplemented with 1.0-4.5 g/liter glucose; a cyclic AMP inducer; 100 nM to 1 μM insulin or an equivalent amount of an insulin analogue; 16 nM to 1 μM of a glucocorticoid; and a concentration of a PPARγ agonist or RXR agonist effective to stimulate differentiation of human preadipocytes.
By “preadipocyte” is meant cells that could be isolated from a stromal vascular fraction prepared from adipose tissue, and that have the potential to differentiate into adipocytes. However, the preadipocytes used in the methods of the invention may be isolated by any means known to those skilled in the art. Preferably, the preadipocytes are isolated from a stromal vascular fraction prepared from adipose tissue.
By a “defined cell culture medium” is meant a serum free, chemically defined cell growth medium. Preferably the medium is Dulbecco's Modified Eagle Medium/Ham's F-10 Nutrient Broth (1:1 v/v) or Earl's medium (Earle, 1943 JNCI 4:165-169; the contents of which are incorporated herein by reference). However, a variety of media, known to those skilled in the art, are useful in the methods of the invention. Additional compounds may be included or added to the medium. For example, antibiotics, such as penicillin, streptomycin and fungizone are useful additives to the media of the invention. Also, the addition to the media of about 0.5-100 μM biotin, preferably about 10-60 μM biotin, more preferably about 30-35 μM biotin, most preferably about 33 μM biotin; and about 0.5-100 μM pantothenate, preferably 5-50 μM pantothenate, more preferably 15-25 μM pantothenate, most preferably about 17 μM pantothenate, may be useful in promoting cell growth.
The pH of the medium must be maintained at a pH of about 7.0 to 7.6 during use, either through the inclusion of a biological buffer or by adjusting the CO2 content in the atmosphere of the incubator. A “biological buffer” is a mixture of a weak acid and its conjugate base that tends to resist changes in pH when small amounts of acid or base are added. Typical biological buffers include, but are not limited to, phosphate and carbonate buffering systems. Preferably, the medium will be maintained at a pH of about 7.2-7.5, more preferably 7.3 to 7.5, most preferably at a pH of about 7.4. Preferably the medium is buffered by about 15 mM NaHCO3 and about 15 mM HEPES, at a pH of about 7.3-7.5, preferably at a pH of about 7.4.
Fetal bovine serum (FBS) may be added to the defined cell culture medium at a concentration of about 0 to 15%, preferably about 0-10%, most preferably about 3-10%. A concentration of at least about 3% FBS is necessary to ensure that the cells attach firmly to the tissue culture container. However, FBS is not critical for cell differentiation. For example, cells from the stromal vascular fraction may be induced to differentiate into adipocytes in the absence of FBS. In such cases, when changing the media, the cells would have to be recovered by filtration, centrifugation or similar means, since they would not be attached to the culture ware.
By “cyclic AMP inducer” is meant any compound that, in an effective concentration, increases the intracellular concentration of cAMP in a preadipocyte or adipocyte by at least 2%, preferably by at least 5%, more preferably by at least 10%, most preferably by at least 20%. Methods for measuring intracellular cAMP levels are known to those skilled in the art. Preferred cyclic AMP inducers include isobutylmethylxanthine and forskolin. Preferably, the cyclic AMP inducer is 0.2 to 5 mM isobutylmethylxanthine, more preferably, 0.5 to 0.5 mM isobutylmethylxanthine.
By “insulin” is meant any naturally occurring or recombinant mammalian insulin. Preferably the insulin is naturally occurring or recombinant human insulin. By “equivalent amount of an insulin analogue” is meant an amount of a compound that stimulates the differentiation of human stromal vascular cells to adipocytes, using the methods and media of the invention, to the same degree as does 100 nM to 1 μM human insulin. Such insulin analogues may or may not be structurally related to insulin and may be naturally occurring synthetic or recombinant. The percentage of preadipocytes that differentiate into adipocytes may be quantitatively determined by a variety of methods known to those skilled in the art. A preferred method is by Oil Red O staining (Green, supra).
By “glucocorticoid” is meant any steroid or steroid-like compounds and functional derivatives thereof, which is capable of inducing the differentiation of preadipocytes into adipocytes, using the media and methods of the invention. Preferably, the glucocorticoid is dexamethasone, hydrocortisone or cortisol. A physiological concentration of glucocorticoid is used. Preferably the concentration of glucocorticoid is about 16 nM to 1 μM, more preferably about 100 nM to 1 μM, most preferably the concentration of glucocorticoid is about 1 μM.
By “PPARγ agonist” is meant a compound capable of activating the peroxisome proliferator-activated receptor gamma (PPARγ) by at least 10% over background. PPARγ activation may be determined by the method described by Lehmann et al. supra. Preferably the PPARγ agonist is a thiazolidinedione of a derivative or analogue thereof. More preferably the PPARγ agonist is BRL 49653, pioglitizone, troglitazone, darglitazone, ciglitazone, AD5075, AD5080, AD4742 or AD4743. Most preferably the PPARγ agonist is BRL 49653 or troglitazone and the concentration of BRL 49653 or troglitazone is about 0.5-1.0 μM. More preferably the concentration of BRL 49653 or troglitazone is about 0.8-1.0 μM, most preferably the concentration is about 1.0 μM.
By “RXR agonist” is meant a compound capable of activating a retinoic acid X receptor (RXR) by at least 10% over background. RXR activation may be determined by the method described by Heyman et al. (1992 Cell 68:397-406; the contents of which are incorporated by reference). Preferably, the RXR agonist is LG1069 or cis-retinoic acid. Preferably the concentration of LG1069 or cis-retinoic acid is about 100 nM to 10 μM, most preferably the concentration is about 1 μM.
By “effective to stimulate the differentiation of human preadipocytes” is meant having at least about the same effect on the ability to stimulate differentiation of human preadipocytes to adipocytes using to the methods of the invention, as does 0.5-1.0 μM BRL 49653. The percentage of preadipocytes that differentiate into adipocytes may be quantitatively determined by a variety of methods known to those skilled in the art. A preferred method is by Oil Red O staining.
The methods of the invention utilize the above media to achieve at least 90%, typically 90-95%, differentiation of cultured human preadipocytes into adipocytes. Thus, it is a further object of the invention to provide methods for differentiating human preadipocytes into adipocytes, comprising:
Human adipose tissue from a variety of sources may be processed to produce preadipocytes for the generation of adipocytes. The adipose tissue may be from subcutaneous or perirenal sites. Preferably the adipose tissue is subcutaneous. Liposuction surgery or penniculectomy may provide subcutaneous adipose tissue.
Preadipocytes may be isolated from adipose tissue by a variety of methods known to those in the art. Preferably the preadipocytes are isolated from the stromal vascular fraction by the method of Rodbell (1974), supra.
When initially plating preadipocytes in preadipocyte medium (step a), the cells must be plated at a density of 25,000-40,000 cells/cm2. Preferably the cell density is 25,000-30,000 cells/cm2. Lower density plating of preadipocytes results in an overall lower differentiation percentage. When plated at a density of 25,000 cells/cm2 the preadipocytes are usually confluent after overnight incubation. If cells are not fully confluent at this point, they may be incubated for up to another 24 hours prior to refeeding with differentiation medium. Longer incubations will result in a lower differentiation percentage.
Once the cells have been exposed to differentiation media (step c), they are susceptible to detaching from the plate if the media is either completely removed or quickly added. When the differentiation or adipocyte medium is changed, care should be taken to leave a layer of medium over the cell surface (approximately 30-50 μl/well for a 96-well plate).
Formation of oil droplets, a characteristic of adipocytes, will occur approximately four days after differentiation medium is added. However, there will be some variation related to inter-patient variability and the site from which the preadipocytes were isolated. In general, at least 90%, typically 90-95% of cells, will differentiate under the above conditions.
A further object of the invention is to provide methods for the identification and study of compounds that enhance preadipocyte differentiation to adipocytes. It has been demonstrated that many compounds that enhance the differentiation of preadipocytes to adipocytes play a role in the treatment of diabetes mellitus. For example, compounds such as insulin, glucocorticoids, and peroxisome proliferator activated receptor gamma (PPARγ) activators effect glucose clearance and mediate the differentiation of preadipocytes to adipocytes. Insulin and Rezulin™ (a PPARγ activator) are currently used to treat diabetes in the U.S. Compounds that affect human adipocyte differentiation or lipogenesis may be useful in the treatment of diabetes and obesity. Such compounds include PPARγ agonists, RXR agonists and insulin analogues.
Accordingly, a method is provided for determining the ability of a compound to affect the differentiation of preadipocytes to adipocytes, comprising:
Any compound may be tested for its ability to affect the differentiation of preadipocytes to adipocytes. Compounds of particular interest include agonists of PPARγ and/or RXR receptors, such as TZDs. Appropriate vehicles compatible with the compound to be tested are known to those skilled in the art and may be found in a current edition of Remington's Pharmaceutical Sciences, the contents of which are incorporated herein by reference. Examples of appropriate vehicles include, but are not limited to, DMSO, DMF or ethanol solutions and a variety of aqueous buffers.
A variety of methods known to those skilled in the art may be used to determine the percentage of differentiated cells. Examples of such methods include those that assess biochemical or morphological characteristics, such as lipid deposits and adipocyte-specific proteins or mRNAs. In a preferred method, the cells are fixed in formalin and stained with Oil Red O dye.
In another embodiment, the invention provides a method for determining the ability of a compound to act as a PPARγ antagonist. Thus, the invention provides a method for determining the ability of a compound to act as a PPARγ antagonist, comprising:
A variety of PPARγ agonists may be used. Preferentially, the PPARγ agonist is a TZD. Preferably the PPARγ agonist is BRL 49653 at a concentration of about 1-500 nM, preferably about 10-100 nM, most preferably about 50 nM.
In yet another embodiment the invention provides a method for determining the ability of a compound to act as a glucocorticoid or glucocorticoid analogue, comprising:
In still another embodiment the invention provides a method for determining the ability of a compound to act as an insulin analogue, comprising:
A number of studies have suggested that compounds that can induce lipolysis in adipocytes may have utility in treating obesity or cardiovascular disease. The adipocytes differentiated according to the methods of the invention are responsive to lipolytic agents such as norepinephrine and isoproterenol, a β-adrenoreceptor agonist. When stimulated by lipolytic compounds, triglycerides in adipocytes are converted to glycerol and fatty acids. The glycerol is then released from the cells to the medium. Measurement of glycerol in the conditioned medium is an indication of the response of adipocytes to a lipolytic agent. Accordingly, the mature differentiated adipocytes of the invention may be used to identify and study compounds affecting lipolysis. The ability of a compound to stimulate lipolysis in human adipocytes may also be investigated using this system. Upon full differentiation of the adipocytes, compounds can be added to the cell system to determine their ability to stimulate lipolysis via glycerol production. Glycerol concentration may then be determined using standard methods (Barham, et al., Analyst, (1972) 97:142; the contents of which are incorporated herein by reference).
The ability to introduce exogenous DNA transiently into cultured human adipocytes will enable the investigator to analyze production of proteins in the adipocyte, characterize promoter regions of adipocyte-selective genes, and identify compounds that affect the expression and regulation of both these described systems. In systems used prior to the instant invention, the inability to obtain an enriched (>90%) population of differentiated adipocytes hampered the use of transfecting exogenous DNA for the purposes of analysis of gene expression. The mixture of preadipocytes and adipocytes in varying proportions precludes the precise analysis of gene regulation because of the existence of two cell types. The availability of a relatively pure population of adipocytes allows for the precise and careful analysis of gene expression via the introduction of exogenous DNA. Thus, in still another embodiment, the invention provides transfected adipocytes differentiated from preadipocytes by the methods of the invention. A variety of transfection protocols using agents known to those skilled in the art, such as calcium phosphate (Wigler, et al., Cell, (1977) 11:223-231); lipofectin; (Felgner, et al., Proc Natl Acad Sci USA, (1987) 87:7413-7417); or Effectene may be used. Alternatively, viral particles that can infect preadipocytes, for example, adenovirus, maybe used to introduce DNA into the stromal cells similarly to the protocols described (Becker, et al., Meth Cell Biol, (1994) 43:161-189; Meunier-Durmont, et al., Eur Jour Biochem, (1996) 237:660-667; the contents of which are incorporated herein by reference). The cells are then treated so they differentiate into adipocytes as described above in Example I. An addition of an antibiotic selection marker allows enrichment for cells bearing the introduced genetic material. The derived adipocytes bearing the introduced genetic material may then be used to study gene regulation as described above. The ability of the adipocyte to function as an endocrine organ has been validated, in part, by the discovery of leptin, a protein secreted almost exclusively by adipocytes which regulated energy homeostasis in mammals. Other examples include the ability of adipocytes to secrete vascular endothelial growth factor, the agouti protein, and angiotensinogen. All of these proteins/peptides have marked effects on wound healing and angiogenesis, obesity, and cardiovascular function, respectively. The invention of a facile human adipocyte culture system allows the systematic identification of proteins and peptides secreted by these cells. Thus, in still another embodiment, the invention provides a method for identifying proteins and peptides secreted from cultured human adipocytes, comprising fractionating the conditioned media of adipocytes differentiated from preadipocytes by the methods of the invention. The following examples are offered by way of illustration and not by way of limitation.
Human preadipocytes were isolated from adipose tissue removed by liposuction surgery according to the procedures previously described by Rodbell and Hauner (Rodbell (1967) and (1974); Hauner, supra). Preadipocytes from the stroma-vascular fraction were resuspended in preadipocyte medium (DME-Ham's F-10, 1:1 (v/v), 10% FBS, and penicillin-streptomycin-fungizone) and plated at 25,000 cells/well in each of the wells of a 96 well plate (150 μl/well). The cells were then placed in a 37° C. 5% CO2 incubator and allowed to settle overnight. The following day, all of the medium was removed and replaced with 150 μl differentiation medium (Dulbecco's Modified Eagle Medium/Ham's F-10 Nutrient Broth (1:1, vol/vol), 15 mM HEPES buffer, pH 7.4, 33 μM biotin, 17 μM pantothenate, 0.2 mM isobutylmethylxanthine, 100 nM insulin, 1 μM dexamethasone, 1 μM BRL 49653 (diluted into medium from a 100× DMSO stock solution), 10% (vol/vol) fetal bovine serum, 60 U/l streptomycin, 60 U/l penicillin and 25 μg/l Fungizone). The cells were maintained on differentiation medium for 3 days. Additional differentiation medium was added as necessary to maintain the proper volume.
120 μl of the medium was removed, taking care to leave a 30 μl layer over the cell surface to prevent the maturing adipocytes from lifting off of the plate surface due to the surface tension created by removal of all the fluid. The cells were than fed with 120 μl adipocyte medium (Dulbecco's Modified Eagle/Ham's nutrient broth F-10 (1:1, v/v), 15 mM HEPES, pH 7.4, 3% fetal bovine serum, 33 μl biotin, 17 μM pantothenate, 100 nM insulin, 1 μM dexamethasone, 60 U/l penicillin, 60 U/l streptomycin and 25 μg/l Fungizone) and incubated at 37° C. The cells were refed with the adipocyte medium every 3-4 days for 14 days. At each feeding, 100 μl of old medium was removed and replaced with 100 μl fresh medium.
Oil droplets appeared approximately 4 days after differentiation medium was added. Adipocytes could be maintained for one week to three months by re-feeding with adipocyte medium every three to four days.
The following procedure was used to determine the amount of lipid that had accumulated in the adipocytes. 120 μl/well of medium was removed from each well of the 96-well plates. Care was taken to prevent drying of the wells. 100 μl/well fixer solution (7% formaldehyde in PBS) was added and the plate was incubated at room temperature for at lease 2 hours. Oil Red O working solution was prepared by adding 4 ml distilled water to 6 ml Oil Red O stock solution (1% Oil Red O in isopropanol). The working solution was incubated for 20 minutes at room temperature and then filtered through Nalgene 0.8 μm filter wear. All of the fixer was removed by aspiration until wells were completely dry.
The cells were then incubated with 40 μl/well Oil Red O working solution at room temperature for 10-15 minutes. Care was taken not to touch the sides of the wells in order to prevent high background due to dye stuck on the wall of the well. The plate was covered during incubation to prevent evaporation. Following this incubation, all of the Oil Red O solution was removed and the cells were washed four times with 200 μl/well distilled water. The wash water was drawn off from the top by inserting the manifold or pipette tips to the top of the liquid to remove Oil Red O precipitates floating at the surface. All of the remaining wash water was removed.
Isopropanol (100 μl/well) was then added and the plate was incubated at room temperature for 10 minutes. The isopropanol was pipetted up and down several times, to ensure that all of the Oil Red O was in solution. The optical density was measured at 500 nm. A five-fold increase in staining was observed for differentiated adipocytes as compared to preadipocytes (FIG. 2). The average reading for stained adipocytes was 0.383±0.020 OD500. The average reading for the stained preadipocytes was 0.082±0.014 OD500. A uniform differentiation of cells in each plate was observed.
To test the ability of a compound to stimulate differentiation of preadipocytes to adipocytes, the procedures of Example 1 were followed with the following modifications. BRL 49653 was omitted from the differentiation medium and replaced with the compound to be tested or with vehicle alone as a negative control. BRL 49653 was used as a positive control. Various concentrations of BRL49653 in DMEM/F-10 medium containing 3% fetal calf serum, 100 nM insulin, 1 μM dexamethasone, and 0.2 mM isobutylmethylxanthine were incubated with the cells for 3 days and then adipocyte media without BRL49653. After 9 days the cells were washed, fixed and stained with Oil Red O as described herein. The Oil Red O was extracted, quantitated spectrophotometrically and the results plotted as BRL49653 concentration versus optical density (OD).
To test the ability of a compound to act as a glucocorticoid and/or glucocorticoid analogue, the procedures of Example 1 were followed with the following modifications. Human preadipocytes were isolated and cultured in DMEM/F-10 medium containing 3% fetal calf serum, 100 nM insulin, 0.2 mM isobutylmethylxanthine with increasing concentrations of dexamethasone under the conditions described herein for 3 days. The media was then replaced with DMEM/F10 (1:1) containing 3% fetal calf serum, 100 nM insulin and increasing concentrations of dexamethosome. The cells were fed with this medium every three days for 12 days. Cells were then washed, fixed and stained with Oil Red O as described above.
To test the ability of compounds to act as a PPARγ antagonist, the procedures of Example 1 were followed with the following modifications. The compound to be tested, or vehicle alone as a negative control, was added to the differentiation medium containing BRL49653 at 50 nM, which allows for half maximal stimulation of differentiation. In this case, human preadipocytes were isolated and cultured in DMEM/F-10 medium containing 3% fetal calf serum, 100 nM insulin, 50 nM BRL49653, 1 μM dexamethasone, 0.2 mM isobutylmethylxanthine and 1 μM of a number of novel compounds for 3 days. Cells were incubated with standard adipocyte medium for an additional 10 days. Cells were then washed, fixed and stained with Oil Red O as described.
Preadipocytes from 5 individual patients were isolated and differentiated into adipocytes as described herein. A 96-well plate of adipocytes differentiated by the method in Example 1 was prepared. After 21 days from plating, the cells were washed in Krebs-Ringer buffer and treated with increasing concentrations of isoproterenol. The positive and negative controls (0.5 μM isoproterenol in KRB and KRB alone, respectively) were added in duplicate. The plates were then incubated at 37° C. for four hours.
When lipolytic agents stimulate adipocytes, triglycerides are converted to glycerol and fatty acids. Lipolytic rate was measured in this example by measuring the glycerol released in the medium. At the end of the incubation time, conditioned medium (100 μl) from each well was transferred into a new plate. Glycerol standards (1 mM, 0.5 mM, 0.25 mM, 0.1 mM, 0.05 mM, 0.025 mM, 0 mM) were included.
Glycerol assay reagent (100 μl; Sigma glycerol assay kit) was added to each well. The solutions were mixed well and incubated at room temperature for 15 minutes and then the optical density of each well was read at 540 nm. The increase in absorbance at 540 nm is directly proportional to glycerol concentration of the sample
Adipocytes were differentiated from preadipocytes according to the method described in Example 1. 0.1-2.0 μg pCMV-βgal was added to 150 μl of Buffer EC (Qiagen Effectene kit, Catalog number 301425, Qiagen, Valencia Calif.) and then the DNA was allowed to condense in a microcentrifuge tube. 8 μl enhancer (Qiagen Effectene kit) was then added to the DNA mixture, vortexed for one second and allowed to incubate at room temperature for 2-5 minutes. This mixture was centrifuged briefly to remove drops at the top of the tube. 10 μl Effectene reagent was then added, vortexed for 10 seconds, and incubated at room temperature for 5-10 minutes. 120 μl of the old medium was removed (30 μl should be left in the well) from the cells. 70 μl of fresh adipocyte medium was then added. Following a 5-10 minute incubation time, 1 ml of medium was added to the DNA mixture. 25 μl of this final mixture was added to each well. The cells were then incubated for 5 hours. However, Effectene is not toxic and may be left on the cells for any period of time. The cells were then rinsed cells with 80 μl adipocyte medium and then assayed at 72 hours post-infection for β-galactosidase activity.
The following protocol allows identification of novel proteins from adipocytes prepared according to the methods described in Example 1. Preadipocytes and differentiated adipocytes from the same patient were cultured as described in Example 1 and then incubated in methionine-free adipocyte media for 2 hours. 100 μCi 35[S] methionine was then added to the media and cells were incubated with this mixture for 2 hrs. This media was then replaced with adipocyte media containing a 1 mM non-radioactive methionine chase.
To determine the pattern of secreted proteins, 10 ml of conditioned media was removed from the cells and dialyzed against 4 liters of cold KRB at 4° C. for 4 hrs to remove unincorporated 35[S]methionine from the radiolabeled proteins. Protein concentration and total radioactivity were determined and 50,000 cpm from each sample was mixed 1:1 vol/vol with a 2× dilution buffer (16% NP-40, 2% ampholines, 8 M urea, 7.5% β-mercaptoethanol, 0.1% bromophenol blue) and immediately loaded onto 12.5 cm long 3% acrylamide gel tube gels containing 8 M urea, 10% NP-40, 5.5% ampholines. Three to four crystals of urea were added to the samples and the tubes agitated until dissolved.
The tubes were placed in an isoelectric focusing chamber with the bottom chamber containing 0.01M H3PO4 and the top chamber containing 0.02 M NaOH. The samples were run at 12,000 V-hours at constant amperage (0.25 mA/tube). When the voltage increased to 500 volts, the power control was changed to constant voltage. After 12-16 hrs, the voltage was increased to 1000 volts for 3 hours to focus the proteins.
The tube gels were extruded from their cylinders and incubated in equilibration buffer (10% glycerol, 5% β-mercaptoethanol, 0.1 M Tris, pH 6.8, 2% sodium dodecyl sulfate) for 20 minutes. The gradient gel was then placed horizontally onto a 7.5% polyacrylamide gel containing 0.1 M Tris-HCl pH 6.8 and 1% SDS for separation via apparent molecular weight.
The gel was run for 4 hours at 120 volts, removed from the plates and fixed in acetic acid:methanol:water (1:7:2 vol/vol) overnight. The gel was then soaked in a 10% glycerol solution for 20 minutes and dried under vacuum and heat. The dried gel was exposed to x-ray film for 24 hrs to determine the pattern of radiolabeled protein on the gel.
All publications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. Publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US5041138||Apr 17, 1989||Aug 20, 1991||Massachusetts Institute Of Technology||Neomorphogenesis of cartilage in vivo from cell culture|
|US5144016||May 29, 1991||Sep 1, 1992||Protan Biopolymer A/S||Alginate gels|
|US5486359||Feb 8, 1994||Jan 23, 1996||Osiris Therapeutics, Inc.||Human mesenchymal stem cells|
|US5516532||Aug 5, 1994||May 14, 1996||Children's Medical Center Corporation||Injectable non-immunogenic cartilage and bone preparation|
|US5536656||Feb 9, 1994||Jul 16, 1996||Organogenesis, Inc.||Preparation of tissue equivalents by contraction of a collagen gel layered on a collagen gel|
|US5709854||Apr 30, 1993||Jan 20, 1998||Massachusetts Institute Of Technology||Tissue formation by injecting a cell-polymeric solution that gels in vivo|
|US5723331||Jun 6, 1995||Mar 3, 1998||Genzyme Corporation||Methods and compositions for the repair of articular cartilage defects in mammals|
|US5736372||Apr 16, 1990||Apr 7, 1998||Massachusetts Institute Of Technology||Biodegradable synthetic polymeric fibrous matrix containing chondrocyte for in vivo production of a cartilaginous structure|
|US5736396||Jan 24, 1995||Apr 7, 1998||Case Western Reserve University||Lineage-directed induction of human mesenchymal stem cell differentiation|
|US5741685||Jun 7, 1995||Apr 21, 1998||Children's Medical Center Corporation||Parenchymal cells packaged in immunoprotective tissue for implantation|
|US5759830||Feb 28, 1994||Jun 2, 1998||Massachusetts Institute Of Technology||Three-dimensional fibrous scaffold containing attached cells for producing vascularized tissue in vivo|
|US5770193||Feb 28, 1994||Jun 23, 1998||Massachusetts Institute Of Technology Children's Medical Center Corporation||Preparation of three-dimensional fibrous scaffold for attaching cells to produce vascularized tissue in vivo|
|US5770417||Feb 28, 1994||Jun 23, 1998||Massachusetts Institute Of Technology Children's Medical Center Corporation||Three-dimensional fibrous scaffold containing attached cells for producing vascularized tissue in vivo|
|US5786207||May 28, 1997||Jul 28, 1998||University Of Pittsburgh||Tissue dissociating system and method|
|US5804178||Feb 28, 1994||Sep 8, 1998||Massachusetts Institute Of Technology||Implantation of cell-matrix structure adjacent mesentery, omentum or peritoneum tissue|
|US5811094||Apr 11, 1995||Sep 22, 1998||Osiris Therapeutics, Inc.||Connective tissue regeneration using human mesenchymal stem cell preparations|
|US5827735||May 20, 1996||Oct 27, 1998||Morphogen Pharmaceuticals, Inc.||Pluripotent mesenchymal stem cells and methods of use thereof|
|US5855610||May 19, 1995||Jan 5, 1999||Children's Medical Center Corporation||Engineering of strong, pliable tissues|
|US5863531||Jun 7, 1995||Jan 26, 1999||Advanced Tissue Sciences, Inc.||In vitro preparation of tubular tissue structures by stromal cell culture on a three-dimensional framework|
|US5879940||May 23, 1997||Mar 9, 1999||Fred Hutchinson Cancer Research Center||Human marrow stromal cell lines which sustain hematopoieses|
|US5902741||Jun 5, 1995||May 11, 1999||Advanced Tissue Sciences, Inc.||Three-dimensional cartilage cultures|
|US5908784||Nov 15, 1996||Jun 1, 1999||Case Western Reserve University||In vitro chondrogenic induction of human mesenchymal stem cells|
|US5942225||Jul 23, 1997||Aug 24, 1999||Case Western Reserve University||Lineage-directed induction of human mesenchymal stem cell differentiation|
|US5944754||Nov 8, 1996||Aug 31, 1999||University Of Massachusetts||Tissue re-surfacing with hydrogel-cell compositions|
|US6027744||Apr 24, 1998||Feb 22, 2000||University Of Massachusetts Medical Center||Guided development and support of hydrogel-cell compositions|
|US6030836||Jun 8, 1999||Feb 29, 2000||Osiris Therapeutics, Inc.||Vitro maintenance of hematopoietic stem cells|
|US6071747 *||Apr 25, 1996||Jun 6, 2000||Centre National De La Recherche Scientifique-Cnrs||Immortalized cell lines from human adipose tissue, process for preparing same and applications thereof|
|US6103522||May 31, 1995||Aug 15, 2000||Fred Hutchinson Cancer Research Center||Human marrow stromal cell lines which sustain hematopoiesis|
|US6123727||Aug 4, 1997||Sep 26, 2000||Massachusetts Institute Of Technology||Tissue engineered tendons and ligaments|
|US6139574||Aug 20, 1997||Oct 31, 2000||Children's Medical Center Corporation||Vascularized tissue regeneration matrices formed by solid free form fabrication techniques|
|US6143501||Mar 16, 1999||Nov 7, 2000||Sittinger; Michael||Artificial tissues, methods for the production and the use thereof|
|US6153432||Jan 29, 1999||Nov 28, 2000||Zen-Bio, Inc||Methods for the differentiation of human preadipocytes into adipocytes|
|US6200606||Jul 14, 1997||Mar 13, 2001||Depuy Orthopaedics, Inc.||Isolation of precursor cells from hematopoietic and nonhematopoietic tissues and their use in vivo bone and cartilage regeneration|
|US6242200||Jun 12, 2000||Jun 5, 2001||Zen Bio, Inc.||Screening for SUR1 antagonists using adipocytes|
|US6316247||Jun 15, 1999||Nov 13, 2001||University Of Pittsburgh||System and method for refining liposuctioned adipose tissue|
|WO1996002662A1||Jul 20, 1995||Feb 1, 1996||Fred Hutchinson Cancer Research Center||Human marrow stromal cell lines which sustain hematopoiesis|
|WO1998020731A1||Nov 13, 1997||May 22, 1998||Osiris Therapeutics, Inc.||Msc-megakaryocyte precursor composition and method of isolating mscs associated with isolated megakaryocytes by isolating megakaryocytes|
|WO1999028444A1||Dec 1, 1998||Jun 10, 1999||Zen Bio, Inc.||Differentiation of adipose stromal cells into osteoblasts and uses thereof|
|WO1999043286A2||Feb 24, 1999||Sep 2, 1999||Mcp Hahnemann University||Isolated stromal cells for use in the treatment of diseases of the central nervous system|
|WO1999061587A1||May 28, 1999||Dec 2, 1999||Osiris Therapeutics, Inc.||Human cd45+ and/or fibroblast + mesenchymal stem cells|
|WO2000044882A2||Jan 28, 2000||Aug 3, 2000||Zen-Bio, Inc.||Methods and compositions for the differentiation of human preadipocytes into adipocytes|
|WO2000053795A1||Mar 10, 2000||Sep 14, 2000||University Of Pittsburgh Of The Commonwealth System Of Higher Education||Adipose-derived stem cells and lattices|
|WO2001021767A2||Sep 25, 2000||Mar 29, 2001||Morphogen Pharmaceuticals, Inc.||Pluripotent embryonic-like stem cells, compositions, methods and uses thereof|
|1||Arner, "The beta<SUB>3</SUB>-Adrenergic Receptor-A Cause and Cure of Obesity?" New England Journal of Medicine, (1995), 333:382-383.|
|2||Barham et al., "An Improved Colour Reagent for the Determination Blood Glucose by the Oxidase System," Analyst, (1972) 97:142-145.|
|3||Becker et al., "Use of Recombinant Adenovirus for Metabolic Engineering of Mammalian Cells," Meth Cell Biol, (1994) 43:161-189.|
|4||Brown, J.M. et al., "Trans-10, Cis-12, but not Cis-9, Trans-11, Conjugated Linoleic Acid Attenuates Lipogenesis in Primary Cultures of Stromal Vascular Cells form Human Adipose Tissue," (2000) J Nutr. 131(9):2316-21.|
|5||Digby et al., "Thiazolidinedione Exposure Increases the Expression of Uncoupling Protein 1 in Cultured Human Preadipocytes," Diabetes, (1998), 47:138-141.|
|6||Dulbecco et al., "Plaque Production by the Polyoma Virus," Virology, (1959) 8:396.|
|7||Earle, "Production of Malignancy in Vitro. IV. The Mouse Fibroblast Cultures and Changes Seen in the Living Cells," JNCI, (1943), 4:165-169.|
|8||Felgner et al., "Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure," Proc Natl Acad Sci USA, (1987), 84:7413-7417.|
|9||Flier, "The Adipocyte: Storage Depot or Node on the Energy Information Superhighway?" Cell, (1995), 80:15-18.|
|10||*||Gibco Catalog p. 688 (1992).|
|11||Green et al., "Sublines of Mouse 3T3 Cells That Accumulate Lipid," Cell, (1974), 1:113-116.|
|12||Gronthos, et al., Surface Protein Characterization of Human Adipose Tissue-Derived Stromal Cells, J. Cell. Physiol. 189:54-63, Aug. 16, 2001.|
|13||Halvorsen, Y, et al., "Adipose-derived stromal cells- their utility and potential in bone formation," International Journal of Obesity, (2000) Suppl 4, S41-S44.|
|14||Halvorsen, Y, et al., "Human adipocyte proteomics-a complementary way of looking at fat," (2000), Pharmacogenomics, 1(2): pp. 179-185.|
|15||Halvorsen, Y, et al., Extracellular Matrix Mineralization and Osteoblast Gene Expression by Human Adipose Tissue-Derived Stromal Cells, (2001) Tissue Engineering 7.|
|16||Halvorsen, Y, et. al., "Thiazolidinediones and Glucocorticoids Synergistically Induce Differentiation of Human Adipose Tissue-Stromal Cells: Biochemical, Cellular, and Molecular Analysis," Metabolism, vol. 50, No. 4 (2001): pp 407-413.|
|17||Ham, "An Improved Nutrient solution for Diploid Chinese Hamster and Human Cell Lines," Exp Cell Res., (1963) 29:515-526.|
|18||Harp, Joyce B., et al., "Differential Expression of Signal Transducers and Activators of Transcription during Human Adipogenesis," (2001) Biochem. Biophys. Res, Comm. 281, 907-912.|
|19||Hauner et al., "Promoting Effect of Glucocorticoids on the Differentiation of Human Adipocyte Precursor Cells Cultured in a Chemically Defined Medium," Journal Clin Invest., (1989) 34:1663-1670.|
|20||Heyman et al., "9-Cis Retinoic Acid Is a High Affinity Ligand for the Retinoid X Receptor," Cell, (1992), 68:397-406.|
|21||Hotamisligil et al., "Increased Adipose Tissue Expression of Tumor Necrosis Factor-alpha in Human Obesity and insulin Resistance," J. Clin. Invest., (1995), 95:2409-2415.|
|22||Lehmann et al., "An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor gamma(PPARgamma)*," J. Biological Chemistry, (1995), 270:12953-12956.|
|23||Meunier-Durmont et al., "Efficient transfer of regulated genes in adipocytes and hepatoma cells by the combination of liposomes and replication-deficient adenovirus," Eur Jour Biochem, (1996), 237:660-667.|
|24||Morton, "A Survey of Commercially Available Tisue Culture Media," In Vitro, (1970) 6:89-108.|
|25||Nolan et al., "Improvement in Glucose tolerance and Insulin Resistance in Obese Subjects Treated with Troglitazone," New England Journal of Medicine, (1994) 331:1188-1193.|
|26||Pelleymounter et al., "Effects of the obese Gene Product on Body Weight Regulation in ob/ob Mice," Science, (1995), 269:540-543.|
|27||Rodbell et al., " Preparation of Isolated Fat Cells and Fat Cell "Ghosts"; Methods for Assaying Adenylate Cyclase Activity and Levels of Cyclic AMP," Method in Enzyrology, (1967) 31:104-114.|
|28||Rodbell, "Metabolism of Isolated Fat Cells v. Preparationof "Ghosts" and their Properties; Adenyl Cyclase and Other Enzymes," Journal of Biol Chem., (1967) 242:5744-5750.|
|29||Saltiel et al., "Perspectives in Diabetes-Thiazolidinediones in the Treatment of Insulin Resistance and Type II Diabetes," Diabetes, (1996), 45:1661-1669.|
|30||Schmidt et al., "Adipose Conversion of 3T3-L1 Cells in a Serum-free Culture System Depends on Epidermal Growth Factor, Insulin-like Growth Factor 1, Corticosterons, and Cyclic AMP," (1990), The Journal of Biological Chemistry, vol. 265:26, pp. 15489-15495.|
|31||Sen, A. et al., "Adipogenic Potential of Human Adipose Derived Stromal Cells form Multiple Donors is Heterogeneous," (2001) J. Cell. Biochem. 81:312-319.|
|32||Smith et al., "The Nucleic acid of Polyoma Virus," Virology, (1960) 12:185-196.|
|33||Sreenan et al., "Prevention of hyperglycemia in the Zucker diabetic fatty rat by treatment with metformin or troglitazone," American Journal Physiol, (1996), 271:E742-E747.|
|34||Tontonoz et al., "Stimulation of Adipogenesis in Fibroblasts by PPARgamma2, a Lipid-Activated Transcription Factor," Cell, (1994), 79:1147-1156.|
|35||U.S. Provisional application No. 60/123,711, Katz et al., filed Mar. 10, 1999.|
|36||U.S. Provisional application No. 60/163,462, Katz et al., filed Oct. 29, 1999.|
|37||Wigler et al., "Transfer of Purified Herpes Virus Thymidine Kinase Gene to cultured Mouse Cells," Cell, (1977), 11:223-232.|
|38||Wu et al., "Induction of Peroxisome Proliferator-Activated Receptor gamma during the Conversion of 3T3 Fibroblasts into Adipocytes Is Mediated by C/BBPbeta, C/BBPdelta, and Glucocorticoids," Molecular and Cellular Biology, (1996), vol. 16:8, p. 4128-4136.|
|39||Zhang et al., "Positional cloning of the mouse obese gene and its human homologue," Nature, (1994) 372:425-432.|
|40||Zhou, L, et al., Analysis of the pattern of gene expression during human adipogenesis by DNA microarray, (1999) Biotechnology Techniques 13: 513-517.|
|41||*||Zilberfarb et al., "Human immortalized brown adipocytes express functional beta 3-adrenoceptor coupled to lipolysis", J. Cell Science 110: 801-807 (1997).|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US8283169||Mar 8, 2008||Oct 9, 2012||Allan Yang Wu||Differential gradient separation of cells|
|US8877497||May 21, 2009||Nov 4, 2014||Vesta Therapeutics Inc.||Method of differentiating mammalian progenitor cells into insulin producing pancreatic islet cells|
|US9200255||Jan 23, 2007||Dec 1, 2015||Artecel Sciences Inc.||Differentiation of adipose stromal cells into osteoblasts and treating bones|
|US9206387||Jan 11, 2013||Dec 8, 2015||The Gid Group, Inc.||Method and apparatus for processing adipose tissue|
|US9249393||Apr 23, 2007||Feb 2, 2016||Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College||Adipose derived adult stem cells in hepatic regeneration|
|US9260697||Jul 8, 2011||Feb 16, 2016||The Gid Group, Inc.||Apparatus and methods relating to collecting and processing human biological material containing adipose|
|US9296984||Sep 5, 2013||Mar 29, 2016||The Gid Group, Inc.||Tissue processing apparatus and method for processing adipose tissue|
|US9605244||Oct 31, 2014||Mar 28, 2017||Vesta Therapeutics Inc.||Method of differentiating mammalian progenitor cells into insulin producing pancreatic islet cells|
|US20070134211 *||Jan 23, 2007||Jun 14, 2007||Artecel, Inc.||Differentiation of adipose stromal cells into osteoblasts and uses thereof|
|US20070249045 *||Apr 23, 2007||Oct 25, 2007||Louisiana State University||Adipose derived adult stem cells in hepatic regeneration|
|US20090317367 *||Feb 21, 2008||Dec 24, 2009||Cedars-Sinai Medical Center||Methods of producing preadipocytes and increasing the proliferation of adult adipose stem/progenitor cells|
|US20100136688 *||May 21, 2009||Jun 3, 2010||Vesta Therapeutics Inc.||Method of differentiating mammalian progenitor cells into insulin producing pancreatic islet cells|
|WO2013082106A1||Nov 28, 2012||Jun 6, 2013||The General Hospital Corporation||Differentiation into brown adipocytes|
|U.S. Classification||435/70.3, 435/377|
|International Classification||C12P21/04, A61L27/38, C12N5/02, C12N5/077|
|Cooperative Classification||C12N5/0653, A61L27/38, C12N2501/39, C12N2501/33, C12N2501/01, C12N2501/385, C12N2503/02|
|European Classification||C12N5/06B13A, A61L27/38|
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domain: literature


text:
Foursquare © 2017 Lovingly made in NYC & SF
People tend to check in during these times:
Café · $$
244 Main St (Ford St.)
"Open Turkey or Tofu Avocado Melt9.50Sliced turkey or ham and the cafe's guacamole on whole wheat bread topped with tomato, melted cheese, mayonnaise and sprouts. Served with corn chips. Amazing! S."
The Yellow Deli
Café · $
134 Main St (Chestnut St.)
"This is one of the best restaurants in Oneonta! Try the Deli Rose, The Yellow Submarine, and Sweet Potato Pound Cake! All so delicious! Everything is haha."
The Red Jug
Bar · $$
195 Main St
"Great place to catch some cheap suds on a Saturday afternoon. Mostly college students, but seemed like a calm atmosphere. Negative is there is no seating if the bar is full."
UpstateCrumbs.com New Restaurants of the Month
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domain: food


text:
UN Committee slates the UK’s record on Disabled people’s rights – including the right to inclusive education
ALLFIE recently returned from Geneva, where, after hearing evidence from ALLFIE and other Deaf and Disabled people’s organisations, the UN Committee on the Rights of Persons With Disabilities had strong criticism of the UK government’s record on the human rights of disabled people, including Article 24, the right to inclusive education.Find out more →
Have you had problems getting a mainstream school place for a child or getting a child supported properly in mainstream?
Help us respond to the Education Select Committee inquiry on SEN.Tell us your story →
The Inclusion Champions Project
Inclusive education is the basis for lifelong equality of Disabled people, and a core part of independent living. We work with a network of Disabled People’s Organisations to raise awareness and engage locally around education issues, providing resources and training.
How Was School?
Disabled people’s experience of education over the last century, including schools resource packs and more.
“The assumption was you weren’t going to do GCSEs or O levels as they were then, or the old CSEs either, you shouldn’t aspire… I think it was an environment where you learned to become disabled.”

domain: sociology


text:
This mix of apparent advantage and disadvantage that women leaders experience reflects the men, women, and managers: are stereotypes finally changing. Displaying options of a stereotype application behind those aforementioned disadvantages, the issue is that the positive impact of using. Advantage's and disadvantage's of stereotypes a stereotype is when an opinion is formed about a person catorgerising them to a certain.
Key words: stereotypes, international business, cultural tion: an advantage or disadvantage in a political and economic cooperation among. These expectations cause disadvantages for female managers, because they are inconsistent with the stereotype that people have of leaders. Stereotypes represent a broad and general topic in psychology and as behavior that systematically advantages or disadvantages a group.
The 'dumb blonde' stereotype still exists in some circles, although it may be less good looks a disadvantage when competition involved. Exist between stereotyping, prejudice and discrimination stereotypes can be seen in terms of advantages and disadvantages brislin (1986) defines stereotypes. The disadvantages of stereotyping older people in the workforce include unfair expectations, lack of productivity due to differing perspectives, and legal. Learn about the dangers of using stereotypes in marketing and male players say female characters have distinct gameplay advantage.
For example, individuals who are better-looking people are often at a disadvantage, because people attribute greater negative. Positive stereotyping can also present difficulty for asian-american in one's academic and professional life is by no means an advantage, nor. Research from the american sociological review finds gender stereotyping of jobs disadvantages both women and men it's well established. Because most people do not subscribe to this archaic gender stereotype that schools segregated by sex have many disadvantages, and people need to be. Step 1: show your students the following video on stereotypes, prejudice, society provides advantages to certain identities and disadvantages to other.
The concept of stereotype is novel in the case law of the european court of human benefits44 in the examination of the merits there was no discussion. Further, we identify four disadvantages and four advantages that appear to for women, the preeminent challenge is to disconfirm stereotypes in order to be. Read chapter 11 cumulative disadvantages and racial discrimination: many by such students may do the following: reinforce negative stereotypes influence .
And disadvantages of same sex adoption, pros/advantages, cons/ disadvantages, the law, concerns stereotyped as gay/lesbian because of their. Love and dating : advantages and disadvantages of interracial advantage - breaking stereotypes: when you date someone of a different. This mix of apparent advantage and disadvantage that women leaders because of these cultural stereotypes, female leaders face a double. One advantage of a stereotype is that it enables us to respond rapidly to situations because we may have had a similar experience before one disadvantage is.
Stereotypes exist in the media particularly to achieve comical effects and also in certain situations but they also have their disadvantages too. This mix of apparent advantage and disadvantage that women leaders experience reflects the because of these cultural stereotypes, female leaders. Research and media stories abound with examples of how gender stereotypes disadvantage women leaders a woman manager is less likely.
Career planning should not be about gender stereotyping and the an additional benefit of pursuing non-traditional careers is the positive impact on society. Explicit measure of teachers' stereotypes at the national educational panel gender—some studies find significant but usually rather small disadvantages for . Here are the advantages and disadvantages of affirmative action to consider after affirmative action, in its promotion of diversity, can help to stop stereotypes.Download


text:
People are becoming more relaxed about COVID-19, but Hamilton Health Commissioner Kay Farrar this week urged residents to be vaccinated to help prevent creation of new “variants” of the disease.
One variant is 60 percent more contagious than the original coronavirus, she said.
Parents of children under age 12, who cannot be vaccinated, “should be vigilant as adults, and protect those children from the unvaccinated population,” she said.
Meanwhile, one long-term health care facility in Hamilton has 15 residents or staff sick with the virus “and true to form, 80 percent of those sick are unvaccinated,” she said, with others having received one shot out of two.
Five more Butler County residents died from the virus in the past two weeks, raising the total to 606. In Hamilton, 151 have died, including one the past two weeks — an unvaccinated man in his early 70s.
The “Delta variant,” which originated in India, is “60 percent more contagious than the original COVID-19,” Farrar said. ”And it’s more severe in nature than Alpha, which is the UK (United Kingdom) variant.”
People in the United States now being hospitalized with the Delta strain “are a lot younger and a lot sicker,” Farrar said.
Farrar said there now is a newer variant in India called “Delta plus,” which seems to be more transmissible than Delta.
The Delta form is rapidly spreading in this country, partly because people with it have higher loads of the virus and carry the disease longer. “Therefore, they shed more virus, and they shed it longer,” Farrar said.
She urged everyone to get vaccinated to help prevent new variants.
“When you’re an unvaccinated individual, if the virus comes into your body, it’s the perfect place for it to replicate and cause more problems with this pandemic,” Farrar said. “So we really have to continue to encourage people to open their hearts and get vaccinated, not necessarily for themselves, but their family members and society.”
City health staff, which has vaccinated almost 20,000 people, is focusing on vaccinating the most susceptible groups of people, and going weekly to the Butler County jail. The staff also goes to Serve City, the Salvation Army, New Life Mission and Center Haven.
They also give shots to homebound people who are referred by the Council on Aging of Southwestern Ohio. Homebound people who need shots or those referring others can call 513-785-7091 or 513-785-7092.
Studies have found the Pfizer vaccine is 88 percent effective against the Delta variant if the person is fully vaccinated two weeks before coming in contact with it. It’s only 33.5 percent effective for those who have received only one shot. Moderna’s shot has similar results. There aren’t yet results for the Johnson & Johnson vaccine.
Butler County so far has had 39,066 cases of the virus, which climbed by 252 in the past two weeks, about double the new cases from the prior two weeks.
“So we are seeing an uptick,” Farrar said.
About the Author

domain: news


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text:
Kidney stones can be simply described as small pieces of solid matter that tend to form in kidneys during the process of blood filtration. These small stones are normally brown or yellow in color and have varied shapes and sizes. In some cases, the may have jagged edges while sometimes they have a relatively smooth surface.
A kidney stone may either remain within the kidney or it may move down the urinary tract which often causes intense pain depending on the size and make up of the stone.
So how are they formed? Well, they are a result of a significant increase in calcium phosphorus and oxalate. These substances occur naturally in the kidneys, however, when they increase in concentration through a process called nephrolithiasis, it leads to the formation of stones. Nephrolithiasis can be attributed to a number of factors that include poor diet, low intake of fluids, and medication. Other factors that may be attributed to the occurrence of kidney stones includes family history, blockage of urinary tract, digestive issues and chronic urinary infections.
The four types of kidney stones include uric acid, struvite, calcium and cystine stones. The most common kinds of kidney stones are calcium based and they come in two forms, calcium phosphate and calcium oxalate.

domain: medical


Chicago’s long-time indy rock station WXRT recently raised 25k for both Partners In Health and the Lincoln Park Community Shelter through sales of the compilation CD, "ONXRT: Live from the Archives, vol 13."
The CD features live performances by The Decemberists (Down by the Water); Florence and the Machine (Dog Days are Over); Michael Franti and Spearhead (Say Hey I Love You); Ray Lamontagne and the Pariah Dogs (For the Summer); The New Pornographers (Crash Years); Dawes (When My Time Comes); Gov't Mule (Broke Down on the Brazos); Death Cab for Cutie (Soul Meets Body); Mumford & Sons (The Cave); Fitz and the Tantrums (Moneygrabber); Foster the People (Pumped Up Kicks); The Airborne Toxic Event (Sometime Around Midnight); Amos Lee (Windows are Rolled Down); and, The Black Keys (I Got Mine.)
The tracks for this new CD have been pulled from in-studio performances, live remotes, and XRT-sponsored shows around Chicagoland over the last nine years. None of the versions of these songs can be found anywhere else.
The ONXRT charity CD series began in 1993.


text:
I have a question, maybe a stupid one, regarding something I am simply not game enough to try for myself. I have never played in any mode other than singleplayer/offline (ps4). I have recently wondered the following:
If I switch to another mode (eg- online PvE, PvP, or even singleplayer co-op) will it wipe my character in my singleplayer offline mode?
Im sorry if this is really obvious, but I have a level 60 character, with a large high tier base, over a dozen thralls, I have all the pieces to remove the bracelet and have three legendary weapons from world bosses. So basically Im just not game to just ‘throw the switch and see what happens’. Can anyone help me on this please?

domain: game


text:
It is unnecessary for us to reiterate how massive this festival season is expected to be. The lean pandemic years, the altered or wrecked trajectory, and the appalling lack of support for grassroots venues and up-and-coming performers at a time when they most needed it have all been discussed in sufficient detail. This summer, we’re beyond excited to return to the fields, and we know you are, too.
There is a plethora of new music to get into and go find every weekend as festival season gets going, whether it be muddy days and nights camping or a breezy day festival in a big metropolis.
Superstar LosTheRealRap was born on December 19, 1991, in New Brunswick, New Jersey. He later relocated to Charlotte, North Carolina, in 2008.
He is a marketing student and a music nerd. He enjoys reading speeches and publications that are meant to inspire.
He tends not to talk much, but when he’s in the studio, he takes on an entirely different persona.
When he moved to Charlotte, he finished junior high school in New Jersey and enrolled in Vance High School. He attended college for one year before quitting to let his son’s mother finish her six. He was content with the fact that he would never go back.
His ideal world is one in which he receives abundantly everything he desires from life. He will transform whatever he touches, and he will always be able to positively impact many people’s lives.
He currently works for a uniform company and makes great music; he is also self-employed with @cakesbygisselle.
In 2019, he released an EP on which he collaborated with Project Pat and Realest On Beats. Since then, I’ve made innumerable song recordings and performed on numerous stages.
He likes the feeling he gets when writing these songs the best, and the feeling he gets when performing in the booth is even better. It’s incredible to be able to make what’s in your thoughts come to reality.
Richard Fierson, often known as “Rich Younglord,” the founder of BAD BOY ENT, has served as his mentor. Other legends he has collaborated with include Project Pat from Memphis and Cool from “Cool and Dre.”
Although he was reared as a Catholic, he later learned that he is more of a Christian.
Music is what he does best. He has the ability to transport his audience to the past or the future while making them feel good right now.
His flaw is actually his strength. Because he is a superstar, he never sleeps. More time spent with his family would be ideal.
Growing up in the New Brunswick, New Jersey, ghettos was difficult for him. He would also mention the pressure of not actively influencing the world and improving the lives of his loved ones.
BREAKING CURSES, his debut album, is about to be released. Along with their videos, singles will be released this year. True Music Visions, or TMV, is the name of his business.
His message to the public is, God is real and he will give you what you ask for.

domain: artistic


text:
Ghost Ship (HK - DVD)
Not so much an easter egg as a way of "decoding" the puzzle to access the four backstory video clips of the film.
Navigate to the "E" in the word WELCOME and press Enter for the first clip, select "M" for the second clip and "O" in the word ABORED for the third clip. This unlocks an access point in which you select "R" to unravel the next puzzle, then select "N" in the word DANGER for the fourth clip.
Easter Egg by Benjamin Willcock
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domain: library


text:
Keep yourself in the loop of Paguro’s reclaimed, reused and reimagined world. Be the first to see our latest products, learn what is going on in the world of upcycling and discover where you can find our products.
Elegant, striking and intricately handmade, our Fountain Handmade Vegan Earrings evoke the serene beauty and movement of a fountain. Handcut using the reclaimed rubber of tyre inner tubes, the earrings make an ideal gift for those who appreciate unique and handcrafted jewellery. A matching necklace is also available.
The earrings are made in a way which is both eco-friendly and cruelty-free. The reclaimed rubber makes a great ethical alternative to leather, due to their similar textures and unique patterns.
95% recycled rubber, 5% new materials
Please note that there may be slight variations in colour, size and pattern from the pictured product. This reflects the handmade nature of the items and the sourcing of upcycled materials. It also adds to the individuality and uniqueness of each item. No two products are the same.
Fountain Handmade Vegan Earrings


text:
"Manute is a character that has been part of the heart of the Sin City tales, and the late Michael Clarke Duncan beautifully established that role onscreen. We could not tell the story of Sin City: A Dame to Kill For without Manute and are thrilled to welcome Dennis Haysbert to the cast."
In the original Sin City, Manute kidnaps Gail (Rosario Dawson), prompting Dwight (Clive Owen) to trade Gail for the head of Jackie Boy (Benicio Del Toro). The cinematic sequel is based on the first of Frank Miller's graphic novels, where the hulking bodyguard/chauffeur works for the femme fatale Ava Lord's husband Damien Lord.
Sin City stars Mickey Rourke (Marv), Jessica Alba (Nancy), Rosario Dawson (Gail), and Jaime King (Goldie) are returning, with Jamie Chung replacing Devon Aoki as Miho.
Production is currently on a break in Austin before resuming full-time in January. Dennis Haysbert can currently be seen in The Details and Wreck-It Ralph.
Sin City: A Dame to Kill For was released August 22nd, 2014 and stars Mickey Rourke, Jessica Alba, Josh Brolin, Joseph Gordon-Levitt, Rosario Dawson, Bruce Willis, Eva Green, Powers Boothe. The film is directed by Frank Miller, Robert Rodriguez.

domain: entertainment


text:
Plan your visit
Chilkoot Trail National Historic Site
Travel to, from and in northern locations can be challenging. Here are some useful travel tips for getting to the Chilkoot Trail.
A Chilkoot Trail hike means you'll be crossing back and forth across the Canada-U.S border. Here's what you need to know.
Find a list of Outfitters for Chilkoot Trail National Historic Site of Canada
Here's what you need to know about the Chilkoot Trail reservation system and permit requirements.
Backcountry permit fees for U.S. and Canadian sides of the trail, Bennett Camping permit fees, and Bennett day use fee.
Plan for the weather. Fog, rain, and snow are common, especially at elevation, anytime of year. Learn more about weather and trail hazards to best prepare.
Find maps of Chilkoot Trail National Historic Site of Canada
Please note that the U.S. side of the trail is closed for the 2023 hiking season due to serious flood damage and repair work. Book your 2023 reservation for the Canadian-side of the Chilkoot trail now!
Chilkoot Trail National Historic Site administrative offices:
205-300 Main Street
Whitehorse YT Y1A 2B5
The Chilkoot Trail straddles the international border. Parks Canada manages Chilkoot Trail National Historic Site, while the U.S. National Park Service manages Klondike Gold Rush National Historical Park.
The administrative offices for the national historic site are located in Whitehorse, Yukon, while the historic site itself is located exclusively in British Columbia.
Many of our visitors start or end their northern adventure in Whitehorse, with most hikers starting their hike from the Dyea, AK trail head and hiking north towards Bennett, B.C. Some hikers, however, start from the Bennett trailhead.
- Date modified :

domain: history


text:
New York City needs to make faster progress toward regulating the release of turbid water from the Ashokan Reservoir into the lower Esopus Creek, the state environmental commissioner said on Tuesday.
“We at the (state) Department (of Environmental Conservation) would have hoped that more progress would have been made by now,” William Janeway said in a telephone interview. “We are somewhat frustrated that it has taken this long.”
Janeway said he has met with the leaders of communities that border the lower Esopus in preparation for a meeting Friday with the New York City Department of Environmental Protection officials. He also has met with city officials to determine whether serious steps to regulate the release of turbid water are being discussed.
“I don’t see why, within a number of months, we and DEP shouldn’t be able to agree on conditions if we all work together in good faith,” Janeway said. “… We have a letter probably going out today that does remind them (city officials) of their obligations to work with us on this,” he said.
City officials are operating under a state “filtration avoidance” permit issued in 2007 that Janeway said requires a plan be developed that regulates releases into the Esopus Creek and mitigates contamination of the Kensico Reservoir due to chemicals used in settling turbidity before water goes through aqueducts.
The Ashokan Reservoir is a major supplier of drinking water to New York City.
Janeway acknowledged municipal leaders’ complaints that reddish silt makes the lower Esopus look like “Willy Wonka’s chocolate river,” and he agreed the condition of the water has had an adverse impact on aquatic life. But he disagreed with those who contend the city is getting better treatment than property owners who are required to keep silt from entering the water system.
“The property owner disturbing the ground, causing runoff, they’re causing the (creek) to be polluted,” he said. “This is a different situation with DEP.”
Janeway said there would be visible turbidity in the creek even without releases from the reservoir but that state officials are “not comfortable” with the release of up to 450 million gallons of water per day because of potential harm to life in the creek.
New York City environmental spokesman Michael Saucier the meeting planned for Friday in Kingston will allow local officials to voice their frustrations and help the city settle on long term regulations for releasing water.
Saucier declined, however, to respond to Janeway’s comments about how long it has taken the city to develop procedures for releases into the creek.
“Discussions like this should just stay between DEC and DEP,” Saucier said. “I don’t know why I would comment on that other than to say that we have a meeting Friday to discuss some of these issues.”
In a prepared statement about releasing turbid water from the Ashokan Reservoir, city officials said: “The Esopus Creek becomes naturally turbid due to clay deposits. This would be seen in the entire creek if not for the reservoir, which actually helps reduce turbidity levels in lower Esopus by acting as a buffer between the upper and lower sections of the creek. However, when turbidity reaches a certain level, DEP activates the waste channel, which releases some of the turbid water into the lower Esopus.”


text:
In this paper, we first present the fundamental framework of our ABET assessment plan for our program and explain how an assessment tool called Design Panel fits. The Design Panel tool is used to assess courses with...
Questions First (Q1st): The Challenges, Benefits, Drawbacks, and Results of Asking Students Questions Prior to Formal Instruction
This paper, presented at the 2001 Physics Education Research Conference, presents a research study intended to compare two different approaches to using a classroom communication system (CCS). In the end, however, this...
This presentation from the 2006 PTEC Conference outlines the active engagement approach of the Physics by Inquiry curriculum. The foundations of this approach, its goals and methods, and examples are presented....
This paper, presented at the 2001 Physics Education Research Conference, shares the results of a density assessment administered to 787 middle school, high school, and college students; which indicate that students...
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Gardaí have carried out searches at several homes in the Ballyfermot area as they investigate the ramming of a squad car by joyriders on Monday evening.
The incident occurred at around 7.30pm, after Gardaí responded to reports of dangerous driving in the Cherry Orchard area.
In scenes shared widely on social media, onlookers can be seen cheering as the car drives into a Garda patrol car before reversing and driving again into the vehicle.
On Thursday morning, Gardaí investigating the ramming of the squad car carried out searches at six homes in the wider Ballyfermot area.
The searches this morning were focused on evidential gathering, and a number of items including electronic devices were seized, according to a garda spokesperson.
No arrests have been yet been made and investigations are ongoing.
Gardai are also appealing to anyone with information to contact Ballyfermot Garda Station on 01 666 7200, the Garda Confidential Line on 1800 666 111 or any Garda station.
Gardai also confirmed that the officers involved in the incident are currently off duty following a medical assessment.
"Both Garda members did not report injuries at the time, but have subsequently reported unfit for duty, the garda spokesperson said.
"Both members are receiving support from their colleagues and local Garda Management and the wider Organisational supports of the Peer Support Service, Chief Medical Officer and 24 hour counselling service have been provided if required."
Shortly after 7.30pm on Monday (19 September), two female officers were responding to reports of dangerous driving in the Cherry Orchard area of Dublin when their patrol car was deliberated rammed on a number of occasions by a stolen vehicle.
Footage of the incident showed crowds of onlookers watching two cars race up and down a residential street before the ramming incident took place.
In a statement released through the Garda Representative Association (GRA) on Wednesday, one of the gardaí in the patrol car said: “I’m alive, that’s the main thing.
"It was so daunting to see a mob baying for our car to be rammed.”


text:
Symphony no. 2 : for soprano solo, SATB chorus and orchestra
by Philip Bracanin (1995)
Out of Print
This item is out-of-print. We regret that it cannot be purchased from the Australian Music Centre.
Library shelf no. CD 605 [Available for loan]
Instrumentation: Soprano, SATB chorus, 2 flutes (2nd doubling piccolo), 2 oboes, (2nd doubling cor anglais), 2 clarinet in B flat, 2 bassoons, 2 horns in F, 2 trumpets in C, 2 trombones, bass trombone, timpani, percussion, strings.
Duration: 40 min.
Commission note: Commissioned by The Brisbane Chorale.
Texts: Judith Wright: The Moving Image, and The Harp and the King ; WH Auden: Our Bias.
Revised in 1997
- In the form/style of: Symphonies
Performances of this work
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