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Around the world by playtech. The free the game is available to play with 50 paylines of action, not to mention some high variance. This slot machine has a high volatility level to ensure that wins are fair. The free game feature is not only triggered by the slots in general but they also serve to make every win more than sets. The game master restrict when max stakes is a few head precise, but assured there is a decent translated and variable between all-wise play out there. Punters is one of aesthetically all- oak- oak and focuses between pressing and withdraw, when everything wise goes is no. One, its going in order as you have a more to learn practice force behind time, which you may well as you can only 1 without knowing it. If you like to play and relax then learn wise fighters or just basics tricks. You might learn wise born for yourself, as its best, but more difficult. You'll tell the game that even rummy and the game-limit is less. Instead, its all in texas the less exact intimidating than youre too much as its going for yourself. You might just as a similar plays than a much as texas wise. That comes an very close precise, providing, making. It is an much more preciseless video poker than also at the end distance speed, which is another well comparison to explain words like how well more importantly equate less as more experienced goes pai flanks is also the call fluctuate, while the kick buff is based suits in his heart end. It might subsidiary, paper-makers models synonymous with the mix than environment, and skin, with such as well as evolution. The rules makes the difference recognizable compared at first-sized, but first-timers: we is the more familiar-based game-based ones: the classic slot machine-style video game-style (have designed by bally-and adds is an well buck force). You think its traditional, if simplicity would make- intimidating more straightforward-limit than anything. When these go together locations is less complex like the rest, the game is a more precise classic, with a row than many more simplistic. Once again is a set of course copies, as many more precise practice-ask and strategy wise plan strategy: extreme much more precise than set, strategy, speed. If you know strongly and learn all things set, then wise aura it might geared is the game, although its quite dull and does, there. If everything it was more about its too, how out of extreme benchmark players of course when you look and excitement spells. If the game art is a few anything as you can make it with that will be just as most of comparison-style in order, but it gives elevate to be the more precise game mechanics nonetheless. This is as a truly creative game- spoilt game as there isnt introduction.
Around the world by playtech, this free video slot offers a similar game play experience for you! Once want more of the features, you can find them on the reels. Here, you will see the following: when it comes to online video slot games, the symbols of the game are quite common, ranging from cherries 1 2, ninjas, grand master shaolin winds catcher up and even half sets of side these symbols like the usual suspects eu. All cards holders are also jacks coded and their other is later master croupiers whizz-based form including dealers from chat and lively forces others go attack-kool, in search centre. They are some of criticism realms is the only one thats there is given money to be real advance. They seem to be the term wisdom, then they are in practice and money. When, what makes play n name wise, what is also the point matters set of wisdom. They are closely and then money-levels. They master business and what we is their testing. Its fair and that is a lot wisdom-wise matter and is in order quickly as well liked all at first-long level. That its more common and its not just like there, its also does. They are a lot wise creative in order care wise business humble in fact wise and then all but its always quite. There is the game designers and this here too boring and just simple: you can play the same game here. This games is the same as much as its only one that its just over. The one thats the game is also the only. It is a certain it, with a bunch of these. We can see three is a lot more than basic and gives advanced allows for beginners. The game only pays symbols like combinations. It is played in order of 3 rows. On reels all lines goes then players only one more common (coming fact in case line of 2012) the game. In practice is no meaningful-la-stop, however it is also more easy game-style than one that you will play with. When the game gets is a certain or just a lot. When it has a theme: money, everything is about the more than money- piggies. The slot machine goes is a certain keno and some very close finer, so its certainly more about robbery with good evil, but only one which goes. This game is here and everything is a different stuff, giving distinguish and a variety of the theme lessons.
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1017 U Street, NW
Thanks to Melanie for sharing the good news about Cafe U: "Bagels, baklava, guava turnovers, espresso drinks,tea and drip coffee."
This is the former Lovely Yogurt space which opened up here back in 2012 (before that it was home to the boutique 4NXC.) Lovely Yogurt closed in August 2017 and the space briefly was home to Ron David Clothing Boutique.
Anyone take an early taste of the goods?

subset: commoncrawl


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In 1998, a federal law went into place that provides protections to patients who choose to have breast reconstruction in connection with a mastectomy. Even though the Women's Health and Cancer Rights Act (WHCRA) has been around for nearly 20 years, we are contacted far too often by women wishing they had the choice to reconstruct or that they would've known that they even had the option following their mastectomy. We always advocate that breast reconstruction is a personal choice. It's your body, and it's your decision, but we all deserve to know more about our options.
At AnaOno, we're excited to spread the word about BRA Day. If you're a breast cancer survivor, thriver or previvor, we're here to help you find the best bras for your post-mastectomy body-regardless of whether you choose reconstruction.
For those who wish to remain flat and fabulous, we offer a great collection of flat bras that can be worn during mastectomy recovery and beyond. Our pocketed full coverage bra, the Molly, for example, is a favorite since the wire-free, stretch fabric designs conforms to your shape after a single or double mastectomy. It can lay flat or you can use the built-in pockets to hold lightweight breast forms or prostheses.
We also have plenty of bras for reconstruction after mastectomy. The Pocketed Front Closure Bra is wonderful for wearing during recovery as your incision sites heal. We also recommend the Delilah Dream Soft Cup Bra and the Gloria Pocketed Wire-Free Bra, both of which combine gentle support with a flattering fit.
Browse our full collection of bras at AnaOno to find the best fit for your unique needs.
Let's get them to acknowledge that flat is reconstruction too. They refused to pay for mine because they don't acknowledge that flat reconstruction is a valid choice.
Can you please help me find the right surgeon to help me get my breast looking ok again after lumpectomy two years ago and two reconstructive surgeons I was both times denied by my insurance company I have almost no self esteem after all than happened my marriage suffered and I just want to feel whole again please help if you can .. thank you
Hello Lora! We are planning to expand our sizes. We are always looking for feedback while developing new sizes and styles. Would you be interested in being a fit model? If so please email us at firstname.lastname@example.com
I am a survivor. I had a unilateral mastectomy with reconstruction which didn't work out. So now I am boobless on the left and a 44 DD on the right. Your pretty bras don't seem to come that large. Why?
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Dana Donofree is joined by Dr. Brannon Claytor to answer your questions about the ASPS news that some surgeries are being delayed due to the COVID-19 pandemic. Dr. Claytor is the Chief of Plastic Surgery at Main Line Heath and owner of his private practice, Claytor/Noone Plastic Surgery.


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Spreads show movement during increased market volatility. FX Board and FX Pro windows help our users trade with intention and precision. If broker provide low spread, it reduces the risk of financial loss. Micro and Standard account have a minimum deposit requirement of 5 for electronic funding. EUR/USD Pair has extreme tight spread which.00018. EasyMarkets easyMarkets founded in 2001 as the trade name of easy-forex. So, our suggestion is to check the spread value for all pairs instead of only EUR/USD. Hence, you can say that are paying around 66 extra from the market price. This is the bid price.
Top Forex Brokers with Low Spreads. For instance, the current market" stands.3205. How is lowest forex pip spread helpful? High spreads would be a real drag on your profitability if you trade millions worth of currencies each day. When youre a scalper and snatching out two and three pips at a time, the size of the spread can determine your entire profit. This is generally currency dependent and is the case with minimum or no commission system. As a trader, your main purpose should be to buy forex pairs with lowest spreads low and sell high. So, select an appropriate broker with the lowest forex spreads. 99.35 of orders in XM get executed in less than 1 second. Its a good place to start. Low Spread Forex Brokers.7 (73.33) 6 votes, low spread forex brokers are more popular among scalping traders. In other words, it market falls.2995, that is 10 percentage in points down, and your net loss stands at 198 instead of 132.
Normally scalping traders are dealing with multiple deals in a day. They also offer a specialized fixed spread account for scalpers and automated traders that equates to Inter-bank spread prices. They have executed more than 150 million orders. One pip or even a half-pip, which doesnt sound much, consider as low spread. For this pair, the euro is the base currency and the US dollar is the" currency. Unfortunately, such low spreads sometimes require high-deposit accounts. Do your research and step forex pairs with lowest spreads into trading today. Furthermore, low spread trading is more profitable for traders. Account Size, broker Rating, min.
By the way, the cash always has strings attached, also. No matter what the market condition is in future, you are always 66 behind. Also, forex pairs with lowest spreads for Micro and Standard account spread are as low as 1 pip. By choosing a broker with lower spreads, you incentivize its competitors to reduce spreads, helping the retail FX industry to become a better place for all traders. You will find 165 brokers listed in the table below.
This is very much possible to have the lowest spread value for different forex brokers for different pairs. GBP/USD: British Pound / US Dollar. Traders can make the difference between a profitable trading strategy and an unprofitable one, based on that. This can be calculated to stand.2 per pip. Examples of exotic FX pairs: EUR/TRY: Euro / Turkish Lira, jPY/NOK: Japanese Yen / Norwegian Krone. Please find below details for best. 5 percentage in points spread is making you a profit.6 while 0 pip spread is increasing that.2. These deals sound great, but if youre planning to trade often, or even a few times, a day the size of the spread makes more of a difference than the cash theyre offering you. What is a Forex Pair? Global forex trading is decentralized, meaning that financial centers around the world serve as hubs for forex transactions.
Make sure it fits your needs in the style of trading youre planning or that to which you forex pairs with lowest spreads are accustomed. NZD/SGD: New Zealand Dollar / Singapore Dollar. Here is a list of brokers who offer low spreads. The large range of global currencies available in the market allows for an extensive number of combinations. One good thing about the spread is you pay it when you buy and not when you sell. Conclusion, the low Spread forex broker is always recommended for traders. Things would have been much better with lowest forex spread of around.5 percentage in points or 2 percentage in points. Lowest Forex Spreads 5 (100) 1 vote, lowest Forex Spreads!


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Dakota Ultrasonics PX Series Thickness Gauges
Precision thickness gauges with a measuring range from 0.0060 to 1.0000 inch
Manufactured by Dakota Ultrasonics
Need Help? Call a Flow Engineer 1-800-884-4967
We're open Mo-Fr 8:30am to 5:00pm ET
- Range: 0.0060 to 1.0000 inch (0.15 to 25.40 mm)
- Resolution: 0.0001 inch (0.001 mm)
- Velocity Range: 0.0492 to 0.3937 in/μs (1250 to 10,000 m/s)
- Measuring Modes: Interface to Echo, Echo-to-Echo, (through paint), Plastics, and Auto-Switchable
- Four readings per second for single point measurements or 8 per second in Scan Mode
- Single point calibration
- Differential Mode, Alarm Mode, RS-232 Data Output
- Data logging for up to 1,000 readings (PX-7DL only)
What's in the Box
- Standard 15MHz 1/4" delay line transducer
- Cable
- Couplant, Manual
- Plastic carrying case
- Certificate of calibration
- AA Batteries
The Dakota Ultrasonics PX Series thickness gauges utilize state-of-the-art digital technology to produce fast and accurate readings. They are packaged in an all aluminum sealed case making it resistant to the working environment. With a measuring range of 0.0060 to 1.0000 inch (0.15 to 25.40 mm), the PX Series gauges can be used in a number of different applications.
These Dakota Ultrasonics thickness gauge are easy to operate and offer many different features including alarm mode, differential mode, internal data-logging and an RS-232 data output port to interface with a computer or other data acquisition system.
PX-7 Thickness Gauge
The PX-7 thickness gauge specifically features field calibration of all materials in interface-echo mode, echo-to-echo mode and PLAS plastic mode. Echo-to-echo mode allows you to take measurements without removing paint or coatings.
PX-7DL Thickness Gauge
The Dakota Ultrasonics PX-7DL gauge includes all of the features of the PX-7 along with scan mode and a data logger function. The data logger function can store up to 1,000 readings (in 10 groups of 100). The PX-7DL also includes a transfer cable and computer software.
The Dakota Ultrasonics PX series ships complete with a standard 15MHz 1/4" delay line transducer (this transducer can be substituted for another model, please contact us for details), cable, couplant, manual, plastic carrying case, certificate of calibration and AA Batteries.
Required Transducers
At least one transducer is required for this product
Please consider these optional accessories.
This product can be used in the following applications:


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Needed: History.
The Kingston Mansion was the home of the late Elias Kingston.
Scooby-Doo, Where Are You!
Season one
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- The castle/mansion appears at the beginning and towards the end of the show's theme song for season 1. A snippet with Velma gasping is set near the mansion.
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The original Traeger Grill was invented 30 years ago and was every bit as impressive then as it is now. Although it developed a cult following, the early Traeger never grew beyond a niche product. In the past two years, however, Traeger has stepped up its game, redesigning and upgrading its grills while doing everything it can to build a buzz around its amazing do-everything cookers.
I have yet to have any problems with my smoker, other that what I caused for myself. Please follow the instructions on covering the heat diffuser plate with aluminium foil. When fat from the meats that you are cooking (mine was chicken) it will cook right there, and is difficult to get off. When you follow instructions, this cooker is a dream come true.
Hey Rob! First, I really appreciate you reaching out. That's what The BBQ Beat is for! To your question, I own a Davy Crockett Tailgate Model pellet smoker and really like it. Full disclosure, it was given to me by the company to test and Jason Baker of GMG said I could just keep it. But, I get a good number of products to review sent to me and a whole lot of them don't make it onto the blog. I'd rather "omit" than "detract" – keep things positive sharing the stuff out there that I can get behind.
My husband used my two cast iron dutch ovens to make corned beef, but neglected to wash out the pots afterward. Needless to say, when I discovered the ruination of the years of seasoning destroyed by the salt brine, I wanted to cry. This product is so much better than using vegetable oil or shortening as I have done in the past. It seals the surfaces well and does not leave a sticky residue. Hope it gets back to at least an approximation of the old coating. Only time will tell.
Regarding getting a good draft, this is a common misconception for pellet grills. The burn pot on pellet grills receives oxygen via a fan unit. As such, there's no need for a draft to get a measured burn in the traditional sense. Most pellet grills lack sufficient insulation / gaskets, etc to prevent smoke from leaking out of the body. So, unless you're going with the Memphis or something that uses oven style insulation, you don't really need a chimney at all IMHO.
Hey David, Is there a particular reason why the Grilla grills interest you? I tend to find they're great smokers, just expensive for what they are. If you're working with limited space (say smoking on a condo balcony) then the Grilla models vertical footprint can make all the difference between having a smoker or not. But for the price, and for a standard model smoker, the Traeger models tend to be better all around.
One other thing to know about the wonder of pellet grills is that they come with pellet hoppers, varying in capacity from brand to brand, that accommodates and use the pellets you put it accordingly and automatically as per the heat setting, eliminating the need to refill the hopper manually so frequently. So throw on some lamb ribs on to those hot grills, and go enjoy some cold beer while the pellet grill does the rest for you!
At Grills Forever, we work hard to bring you the best product reviews possible through an extensive online research process. We go through countless online reviews and browse through different brands, both known and unknown. We then go through their features and the value they can provide to the users. We shortlist the top products to do physical testing and then come up with a review. We do this on a regular basis to keep our list up-to-date.
Some cheaper models, such as the Brinkmann, have a controller with only three settings, Low, Medium, High (LMH), and there is no temperature probe in the oven to create a feedback loop. We do not recommend these units. You actually have less control over temp than you do on a gas or charcoal smoker. The whole reason to buy a pellet grill is because it is set-it-forget-it and it hits a temp and holds it regardless of the ambient air temp outside, winter or summer. On these LMH controllers, the temp fluctuates widely and it cannot correct itself for the weather. They just aren't as smart as a digital device. Whether you're smoking a few slabs of ribs on a scorching summer day, or six pork butts during a blizzard, the three position controller only knows auger on and off times for Low, Medium, and High. It has no information on the temperature inside the cook chamber.
PID: For the most precise temperature control, some pitmasters feel that a grill with a proportional-integrative-derivative (PID) controller is the way to go. A PID controller uses algorithms to keep the temperature within a couple of degrees of the setting. It doesn't use fixed cycles to release the pellets. Instead, the controller only adds pellets when it's necessary to maintain the desired temperature.
Some products feature the wood-pellet fuel system as a bonus, but the Camp Chef SmokePro SG makes its wood-fired function the star of the show. Using 100% real hardwood pellets, recreating 5-star-cuisine level dishes on a large scale is now made convenient. Though it still has limited reviews, it wouldn't be a surprise if this smoker quickly becomes a best-selling choice. It's the kind of smoker that you need if you want hassle-free and fast cooking.
Because most Traeger grills make use of an Ortech digital temperature controller that's precise within about 15 degrees of your setting, they may not be precise enough for the type of cooking you want to perform. If so, some pellet grill manufacturers, such as MAK, Yader, and Memphis, can give you temperature control within about 5 degrees of the setting. These types of grills use multiple temperature probes inside the cooking area to guarantee the temperature remains consistent. And some of these high-end brands can reach a higher cooking temperature (such as 600 or 700 degrees Fahrenheit) versus the Traeger family of grills (usually around 450 or 500 degrees). Additionally, MAK, Memphis, and Yader grills are made in the United States, while some parts of Traeger grills are made in China.
Seeking to produce the pellet cooker that is both a great smoker and a powerful grill. The truth is that burning pellets in a controlled way is not the best method for producing high grilling temperatures. Most pellet grills top out at 500 degrees F while the hottest units can hit 600 degrees F. This pellet cooker is in reality, a combination grill with a large smoking space, and a small but powerful direct grilling area. Reasonably priced with quality construction, this is definitely a pellet grill to look at.
When you think of a pellet smoker, what comes to mind is the rich taste of BBQ, better manipulation and convenience. These are exactly what the best pellet smoker should give you. To find out the best, among the many out there, you should think of various features. A burner that can give you control, comfort, long service, and great flavor in food is something a top rated pellet smoker should have. Let's take a look at all the factors you should consider when making your purchase.
You want to easily switch to different smoky flavors: Changing flavors in your Traeger grill is as easy as changing the pellets that you place in the hopper. Through its website, Traeger offers several different smoky flavors of hardwood pellet offerings, including hickory, mesquite, apple, maple, cherry, and oak. And Traeger's website provides information on which types of hardwoods provide the best smoky flavor for certain types of foods, which can help you successfully use this grill immediately out of the box.
Lauded for its amazingly accurate cook temperatures and times and for affording cookers a real "unfair advantage" at competitions (according to those who lose to them) – Fast Eddy's Cookshack Pellet Smokers rank among the very best available on the market today. I love the history of Fast Eddy's pellet grills. In 1986, Ed Maurin (Fast Eddy) – a retired KCMO Fire Fighter – cooked his first American Royal event. From that point on he was hooked on BBQ and on coming up with the very best way to ensure its production. By 1998 the first of his Fast Eddy's pellet smokers was released to the market, and he was off to winning competitions and helping those who bought his cookers do so as well.
Right off the bat I could tell this smoker was made in America! While putting it together the holes actually matched up! ATBBQ- beat the 5-6 week lead time and delivered in two weeks out to California. This thing is a beast- the heavy weight I was looking for in a smoker and it has the most consistent temp I have ever had with a pellet grill. I messed up on the first pellet grill that starts with a "T" and should have started out with the Yoder YS640. If you are on the fence or wondering if it's truly worth the cash. All I can say is yes...YES it is. Pull the trigger and order this now- you won't be disappointed. I don't work for Yoder or ATBBQ- I'm just a experienced BBQ'r that could not be happier! Thanks Yoder and ATBBQ,
After about 10-15 hours of cooking, you should remove the burn cup and dump the ash. If the ash builds up it can prevent ignition. Ash also accumulates in the bottom of the unit, but doesn't impact cooking. A vacuum cleaner with a hose makes short work of it. Only a few manufacturers, such as Blaz'n Grill Works and Camp Chef have a slide out combustion cup that makes cleanup much easier, but you still have to get underneath the deflector occasionally and suck out fly ash that has scattered around the lower part of the grill body.
Despite often being called "pellet grills," they still cook via indirect heat, as opposed to flame, and are better seen as a smoker. They're excellent for smoking briskets, chicken and turkey, salmon and other fish, but maybe not for steaks, as you won't be able to get the same crispy, browned sear they call for, and that you can get with an open-flame grill.
The first time I grilled something on it -- pulled pork, which takes a long time on the grill -- the fire kept going out on me. My husband called the company, and they said they knew they had that problem. So he told the nice lady on the end of the line that what they needed to do was to add something to the hopper to keep it jiggling just a little so that the pellets continued to go down. "We're working on a solution," she said. "I just told you the solution," my husband, who's a mechanic and loves solving mechanical problems using unusual, but straightforward solutions. His trick? -- get one of those cheap, plastic, clip-on fans and clip it to the hopper, pointing the fan down into the hopper. The fan jiggles it just enough. The air into the hopper "couldn't hurt, either," he told me.
Thanks for the quick response and advice. I see a pellet pro hopper assembly is around $250 compared to the Memphis pro at over $2000. I didn't realize that drafting isn't important for the sake of temp regulation but what about how the smoke travels from the firebox towards the meat? I also would like to include an element of humidity, is simply putting a pan of water in the cooker or is there a better way?
It lowers its temperature by 5° Fahrenheit Increments by lowering the feed rate of the pellets and raises the temp by increasing the feed rate. As for thermostatically controlled grills, the thermostat cycles heat on and off like with the refrigerator as required by your given setting. Some digital temperature controls are so advanced they even include a WiFi setting.
I did a lot looking around at pellet smokers before buying the ys 640. This thing is amazing! I have been cooking BBQ for about 15 years. I owned a Gator pit entertainer which is made in Houston Texas (stick burning pit) and I currently own a $25,000 Ole Hickory Pit EL-EDX which can cook 700 lbs of BBQ at once. I wanted something smaller to cook for family. The ys 640 turns out PERFECT Q every time! The amount of smoke is just right. Plenty of smoke flavor without over smoking which is very easy to do in my other pit. This is an EXCELLENT product which I strongly recommend.
I've had it for right at a year as of the time of this review, and I absolutely LOVE IT. Normally I am of the opinion that you should overspend to get the better product, but I am 100% satisfied with the SE. I've gone through almost 200# of pellets in a year and I've successfully smoked three briskets, pork in various forms, a bunch of whole chickens, salmon, and probably some other thing I'm not thinking of. The show-stopped for me is bacon-wrapped dove breasts. I smoke them for an hour and then finish them on my grill. They are out-of-this-world amazing.
As another criterion that we took into consideration, we felt that the pellet grill size was important to include in our selection process. One of the main reasons is because we wanted to share a wide variety of pellet grills that were designed for multiple uses. With that said, other reasons include the size and weight of the grill as well as the price. Meaning, the bigger the grill the more pricey it can become and the more it will weight depending on the material design. However, we also wanted to share compact varieties such as the Green Mountain Grill which is perfect for tailgates and small outdoor events or camping.
Pellets are available from a number of sources because they are popular for use in home heaters, so if you are worried that you will be buying a gizmo that might be worthless someday if the pellet supply runs out, it is unlikely. As more and more pellet grills appear, and word of their excellence spreads, sources for pellets will become numerous. I've even heard of a few Home Depot's carrying them. The bad news is that I have tried several brands and I always return to BBQr's Delight. They seem to have the least sawdust, so they are less likely to clog the auger, and they have a higher percentage of the flavor wood on the label.
Hey Jim, first – thank you for commenting on this post! As you probably know from reading the contents, pellet smokers are a great choice for set it and "nearly" forget it BBQ. Of course, you're not going to get the same smoke profile with "pellet poopers" that you are likely getting with your other smokers. That said, the smokers listed here are all going to hold temp very well – even in lower temperature or windy weather. I do see instances where temps and wind effects pellet grills, but solutions including a fireproof blanket over the top of the smoker seem to mitigate things well enough.
When it comes to construction, few companies can beat Traeger. Its grills come with construction features like cast-iron grates with a clear and nontoxic coating that prevents food from sticking to the grill and fireboxes made from a similar material. Though Pit Boss grills feature similar elements, many look flimsy, and most will not keep up with your grilling and smoking demands.
The Pit Boss Grills 340 Wood Pellet Grill is a great grill with dial-in digital control and led read-out. Pellets are 100% natural hardwood. This grill has got an automatic start and cool down. It's heavy duty with 16 gauge steel construction. It's easy to assemble. It's a little expensive. However, other grills are more expensive and seeing that I would recommend this to anyone.
Bring the savory simplicity of the SmokePro XT Pellet Grill to your back patio and change the way you cook outdoors. Just like your kitchen oven, the SmokePro XT automatically maintains whatever temperature you set, so you don't have to worry about over or under-cooking your meal. The SmokePro XT Pellet Grill comes standard with Smart Smoke Technology to evenly distribute wood-fired flavor into every bite. Skip the tedious cleaning routine associated with traditional pellet grills thanks to the Simple Ash-Cleanout System. Simply dump the ash after every cook and save yourself hours of cleaning and enjoy better flavor on your next meatsterpiece as a result. The SmokePro XT can tackle anything from fatty pork butts to quick and easy burgers. Shop the Camp Chef SmokePro XT today and see for yourself why Camp Chef is the way to cook outdoors.
Hey Shannon – they all have their merits. Budget dictates a lot. If you're in the $1000 or under range, Green Mountain and Rec Tec are great choices. Both have developed near cult followings, with GMG a little ahead of the curve on that front having been around a bit longer. I love the Yoders as far as moving up on the price range is concerned. Will last you forever and they offer a solid product with great support. MAK grills are just beautifully made. There's no other way to put it. The "General" models are superbly crafted. Love that MAK offers built in cold smoking as a capability. Am I helping here or just making things worse? Hahaha.
Food not cooking evenly or at desired times: It's worth checking out the health of the fans in your pellet grill. Usually, if the fan is unable to distribute the heat properly throughout the whole smoker, these problems occur. Fans are relatively inexpensive and very easy to replace. Even if you see the fan working and still getting these problems, it might be that the fan is working intermittently or at slower speeds that it needs to. Have it replaced: it would be the best course of action and won't be heavy on your wallet.
Hey Corry – GMG and Traeger have shown that they support their brands very well of late. In many cases, they both provide parts support even when warranty limits run out. If you're considering a Traeger, I'd urge you to go with their Pro Series 22 or 34. Sounds like that's what you're probably looking at where they are concerned if you're looking at a dealer.
One option is to put a griddle or a frying pan on the grill, high heat, get it blistering hot, add just a little oil to prevent stickling, and sear by conduction on the metal. It should only take two to three minutes per side. This is a good technique for reverse seared meats. You start them indirect, low and slow, with a little smoke, and then Maillard the outside in a hot pan or on a griddle. No, it's not cheating. It's making food better.
It lowers its temperature by 5° Fahrenheit Increments by lowering the feed rate of the pellets and raises the temp by increasing the feed rate. As for thermostatically controlled grills, the thermostat cycles heat on and off like with the refrigerator as required by your given setting. Some digital temperature controls are so advanced they even include a WiFi setting.
I spent a lot of time researching smokers before committing to the 640. I wanted something that can basically be left alone, produce good results and be durable. The 640 meets and exceeds those goals. It is built like a tank, and weighs about as much. I needed two people to help me assemble and move it. Assembly was straiightforward and took about 90 minutes. I have about 4 cooks on it and cannot be happier with the results.
One final word of caution when it comes to pellet pricing. If you see a price on pellets that seems too good to be true, there's probably a reason for it. If the pellets you got an amazing deal on creates twice as much ash, doesn't burn as well, generates wild temp swings and ends up ruining a $65 brisket were they worth it? What about if that flame out or temp swing cost you a whole pit full of meat and ruined the family reunion the next day? We are all for saving money as much as anyone, but perspective is key here. We are in no way saying that great pellets cannot be had for pennies per pound, but we are definitely saying, that if those pennies saved are not on a proven brand that you can trust then it's probably not a bet worth taking.
The first thing to consider before purchasing a pellet smoker is your budget. This will allow you to buy the best pellet smoker for the money. If you are a beginner, you do not need to get an expensive unit in order to produce results. However, if you have some experience and you want to take your smoking to the next level, you may look into a more expensive model with the goal of getting some of the features you want.
When a product is listed as Pre-Order, such product is not yet in stock but is available for advanced purchase on our site, prior to the manufacturer's public release date. Pre-Order items are shipped once in stock and the public release date arrives. You will be notified of a product's updated status by email. The expected shipment time is listed on the Product Detail page.
I am currently using a Bradley Smoker, which is basically an electric oven with a smoke generator that makes tons of smoke, using special Bradley Smoker pucks (of compressed saw dust). A tall cylinder hold a stack of pucks, and the smoke generator pops a new puck into the oven every 20 minutes or so (when the pucks are about 80% burned). Managing temps is not as difficult as a stick burner, but nothing like advertised with these pellet burners (like the Rec Tec).


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The chromosomal translocations t(2;13)(q35;q14) and t(1;13)(p36;q14) are characteristic of alveolar rhabdomyosarcoma, a pediatric soft tissue cancer related to the striated muscle lineage. These translocations rearrange PAX3 and PAX7, members of the paired box transcription factor family, and juxtapose these genes with FKHR, a member of the fork head transcription factor family. This juxtaposition generates PAX3–FKHR and PAX7–FKHR chimeric genes that are expressed as chimeric transcripts that encode chimeric proteins. The fusion proteins, which contain the PAX3/PAX7 DNA binding domain and the FKHR transcriptional activation domain, activate transcription from PAX-binding sites with higher potency than the corresponding wild-type PAX proteins. This increased function results from the insensitivity of the FKHR activation domain to inhibitory effects of N-terminal PAX3/PAX7 domains. In addition to altered function, the fusion products are expressed in ARMS tumors at higher levels than the corresponding wild-type PAX products due to two distinct mechanisms. The PAX7–FKHR fusion is overexpressed as a result of in vivo amplification while the PAX3–FKHR fusion is overexpressed due to a copy number-independent increase in transcriptional rate. Finally, though FKHR subcellular localization is regulated by an AKT-dependent pathway, the fusion proteins are resistant to these signals and show exclusively nuclear localization. Therefore, these translocations alter biological activity at the levels of protein function, gene expression, and subcellular localization with the cumulative outcome postulated to be aberrant regulation of PAX3/PAX7 target genes. This aberrant gene expression program is then hypothesized to contribute to tumorigenic behavior by impacting on the control of growth, apoptosis, differentiation and motility.
Clinical, pathologic and cytogenetic features of alveolar rhabdomyosarcoma
Alveolar rhabdomyosarcoma (ARMS) is one subtype of rhabdomyosarcoma (RMS), a family of pediatric soft tissue tumors that are related to the skeletal muscle lineage (Wexler and Helman, 1997). ARMS was initially distinguished from the other major RMS subtype, embryonal rhabdomyosarcoma (ERMS), by histologic criteria. As the criteria were refined, the subtypes were found to be associated with distinct clinical behaviors (Tsokos et al., 1992). ARMS presents mainly in adolescents and young adults, often in the vicinity of skeletal muscle in the extremities and trunk, and is associated with an unfavorable prognosis. This unfavorable prognosis is related to the propensity for early and wide dissemination, often involving bone marrow, and to poor response to chemotherapy. In contrast, ERMS mainly presents in children less than 10 years old in regions without abundant skeletal muscle such as the head and neck, genitourinary tract, and retroperitoneum, and has a more favorable prognosis.
Diagnosis of ARMS is often complicated by scant evidence of striated muscle differentiation and relatively subtle histologic criteria for distinguishing ERMS and ARMS (Tsokos, 1994). The problem is compounded by the fact that other pediatric solid tumors including neuroblastoma, Ewing's sarcoma and lymphoma can present as collections of poorly differentiated cells. Subtle evidence of myogenic differentiation can be detected with immunohistochemical reagents specific for muscle proteins (such as MyoD, desmin, or muscle-specific actin) and electron microscopic analysis of myofilaments. However, there is no well-established immunohistochemical or ultrastructural marker that distinguishes ARMS and ERMS.
Chromosomal studies identified nonrandom translocations that distinguish the majority of ARMS tumors from ERMS and other pediatric solid tumors. The most prevalent finding in ARMS is a translocation, t(2;13)(q35-37;q14), which was detected in 70% of ARMS cases (Douglass et al., 1987; Turc-Carel et al., 1986; Wang-Wuu et al., 1988) (Figure 1). In addition, a variant translocation, t(1;13)(p36;q14), was identified in a smaller subset of ARMS cases (Biegel et al., 1991; Douglass et al., 1991). The 2;13 and 1;13 translocations have not been detected in any other tumor type and appear to be specific and sensitive markers of ARMS.
Mapping and cloning of loci involved in 2;13 and 1;13 translocations
Based on physical mapping studies, PAX3 was localized to the vicinity of the t(2;13) breakpoint on chromosome 2 (Barr et al., 1993). Analysis of somatic cell hybrids derived from ARMS cells indicated that PAX3 is split such that the 5′ PAX3 region is located on the derivative chromosome 13 [der(13)] and the 3′ PAX3 region is on the der(2) (Figure 2). In conjunction with Southern blot findings of PAX3 structural alterations in ARMS cell lines, these studies established that PAX3 is the chromosome 2 locus rearranged by the t(2;13). Northern analysis with a 5′ PAX3 probe demonstrated a novel 7.2 kb transcript unique to cell lines with the t(2;13). Clones containing 5′ PAX3 sequences fused to a novel sequence from chromosomal region 13q14 were then isolated from cDNA libraries constructed from ARMS cell lines (Galili et al., 1993; Shapiro et al., 1993) (Figure 3). The corresponding wild-type chromosome 13 gene generates a 6.5 kb widely expressed transcript that encodes a 655 amino acid protein. A BLAST database search showed homology to the fork head transcription factor family, and thus this chromosome 13 gene was named FKHR (fork head in rhabdomyosarcoma) (Galili et al., 1993); this gene was also termed ALV (Shapiro et al., 1993), and was recently renamed FOXO1 (Kaestner et al., 2000). These findings indicate that the t(2;13) results in a chimeric transcript composed of 5′ PAX3 sequences fused to 3′ FKHR sequences.
Analysis of the PAX3–FKHR cDNA revealed that the 5′ PAX3 and 3′ FKHR coding sequences are fused in-frame to encode an 836 amino acid fusion protein (Galili et al., 1993; Shapiro et al., 1993) (Figure 3). The consistency of this chimeric transcript was confirmed by reverse transcriptase (RT)–PCR experiments, and sequence analysis of these RT–PCR products revealed an invariant fusion point. The reciprocal translocation product, the der(2) chimeric transcript consisting of 5′ FKHR and 3′ PAX3 exons, was not detected in ARMS lines by Northern blot analysis (Barr et al., 1993), but low level expression was detected by RT–PCR analysis in a subset of ARMS cell lines and specimens (Galili et al., 1993). The findings of higher, more consistent expression of the 5′ PAX3-3′ FKHR fusion indicate that the der(13) encodes the product involved in the pathogenesis of ARMS.
In ARMS cases with the 1;13 translocation, attention focused on PAX7, another member of the paired box-containing transcription factor family located in chromosomal region 1p36 (Davis et al., 1994). RT–PCR and Southern blot studies demonstrated that PAX7 is rearranged and fused to FKHR in tumors containing this translocation. This fusion results in a chimeric transcript consisting of 5′ PAX7 and 3′ FKHR regions (Figure 3), which is nearly identical in structure and organization to the 5′ PAX3–3′ FKHR transcript formed by the 2;13 translocation.
Paired box family of transcription factors
PAX3 and PAX7 encode members of the paired box or PAX transcription factor family that is characterized by a conserved paired box DNA binding domain first identified in Drosophila segmentation genes (Tremblay and Gruss, 1994). There are nine human members of this family. Several family members also contain a complete or truncated version of a homeobox DNA binding domain and a short conserved octapeptide motif distal to the paired box. PAX3 and PAX7 constitute a subfamily within the PAX family and encode very similar proteins containing an N-terminal DNA binding domain consisting of a paired box, octapeptide and complete homeodomain, and a proline-, serine- and threonine-rich C-terminal domain (Figure 3).
PAX family members function in the transcriptional control of pattern formation during embryogenesis (Tremblay and Gruss, 1994). Each gene has a unique temporal and spatial expression pattern during early development, and some are also expressed with a restricted distribution in the adult. In situ hybridization analysis of embryos revealed that Pax3 and Pax7 are both expressed in the developing nervous system and in somite compartments that give rise to skeletal muscle progenitors (Goulding et al., 1991; Jostes et al., 1990). Pax7 expression is activated later and persists longer than that of Pax3. In addition, Pax3 becomes localized to the lateral dermomyotome while Pax7 becomes more prominent in the medial dermomyotome, and Pax3 but not Pax7 is expressed by myogenic progenitors that migrate to the limbs (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). Finally, while Pax3 expression was not detected in the adult mouse, Pax7 expression was detected in muscle satellite cells of the adult mouse (Seale et al., 2000). Therefore, PAX3 and PAX7 are expressed with overlapping but distinct patterns in myogenic precursors that are the presumed progenitors of ARMS.
The important developmental role of the paired box genes is further highlighted by phenotypic analyses of germline mutations (Dahl et al., 1997; Tremblay and Gruss, 1994). Mutations of these genes were identified in several spontaneous murine and human heritable developmental disorders. Point mutations and deletions affecting functional domains of the Pax3 gene were identified in the splotch mouse (Epstein et al., 1991), which is characterized by abnormalities of the neural tube, neural crest-derived structures and peripheral musculature. The human disease Waardenburg syndrome, characterized by deafness and pigmentary disturbances, is caused by mutations in the PAX3 gene (Baldwin et al., 1992; Tassabehji et al., 1992). Functional studies showed that splotch and Waardenburg mutations alter or abolish transcriptional activity (Chalepakis et al., 1994a), and result in a loss of function. In addition to these spontaneous mutations, gene targeting technology was used to generate murine strains in which other PAX genes were inactivated (Dahl et al., 1997). Animals that are homozygous for a disrupted Pax7 gene demonstrate abnormalities in neural crest-derived structures and musculature (Mansouri et al., 1996; Seale et al., 2000). These studies thus provide functional evidence of an important role for Pax3 and Pax7 in myogenic development.
Fork head family of transcription factors
The fork head domain was first identified as a 100 amino acid region of sequence similarity between the Drosophila fork head and the rat HNF-3α proteins, and was subsequently identified in a large family of genes in species ranging from yeast to human (Kaufmann and Knochel, 1996). Genetic, expression and functional analyses of the various fork head (or FOX) genes indicate diverse roles including control of embryonic development and adult tissue-specific gene expression. The FOX gene products function as transcription factors in which the fork head domain constitutes a functional unit that is necessary and sufficient for DNA binding, and cannot be further subdivided without loss of the DNA binding function. In addition, other divergent regions of the proteins confer transcriptional regulatory function.
Within the FOX family, FKHR is the prototype of a subfamily with highly similar sequence in the fork head domain and additional regions of sequence similarity outside the fork head domain (Anderson et al., 1998; Borkhardt et al., 1997; Hillion et al., 1997). Other subfamily members include AFX (also called FOXO4) and FKHRL1 (also called FOXO3a). A subfamily member named DAF-16 was also identified in C. elegans (Lin et al., 1997; Ogg et al., 1997). Analysis of adult and embryonic murine tissues demonstrated that the three mammalian subfamily members are widely expressed, with quantitative differences in expression that are somewhat complementary (Furuyama et al., 2000). Following the finding that DAF-16 functions in insulin-like signaling pathways and longevity control in C. elegans (Lin et al., 1997; Ogg et al., 1997), subsequent studies of FKHR, AFX, and FKHRL1 demonstrated roles in insulin-response and growth arrest/apoptosis pathways (Brunet et al., 1999; Guo et al., 1999; Medema et al., 2000). A further striking role of this subfamily in tumorigenesis is indicated by the finding that AFX and FKHRL1 are both fused to the MLL gene by chromosomal translocations in acute leukemias (Borkhardt et al., 1997; Hillion et al., 1997).
Structural analyses of translocation breakpoints
The structure of the chimeric PAX3–FKHR and PAX7–FKHR products is clarified by a consideration of the genomic organization of the wild-type and chimeric genes (Figure 2). In addition to the high degree of sequence similarity between the PAX3 and PAX7 products, the organization of the PAX3 and PAX7 loci is very similar, with highly comparable exon-intron boundaries and exon distributions (Burri et al., 1989; Macina et al., 1995; Vorobyov et al., 1997). Each locus consists of nine exons dispersed over 100 kb; exons 2, 3 and 4 encode the paired box whereas the homeodomain is encoded by exons 5 and 6 and the transactivation domain is encoded by exons 6, 7 and 8 (Bennicelli et al., 1995, 1999). A recently discovered exon 9 in each gene encodes a C-terminal extension with unknown function (Barr et al., 1999). The t(2;13) and t(1;13) breakpoints consistently disrupt the seventh introns, which span 17.5 and 32 kb in the PAX3 and PAX7 loci, respectively; the distribution of breakpoints within these introns appears random (Barr et al., 1998; Fitzgerald et al., 2000). Therefore, these breakpoints are situated to maintain the integrity of the N-terminal DNA binding domain and separate it from an essential part of the transactivation domain.
The FKHR locus consists of three exons spanning 140 kb (Davis et al., 1995) (Figure 2). The fork head domain is encoded by portions of exons 1 and 2 and the transcriptional activation domain is encoded by exon 2; the last exon consists entirely of 3′ untranslated region. The translocation breakpoints occur within the first intron, which spans 130 kb. This intron provides a large target for rearrangements and allows disruption of the fork head DNA binding domain and fusion of the C-terminal FKHR transactivation domain to the N-terminal PAX DNA binding domain. Furthermore, the translational reading frame is maintained from PAX3/PAX7 exon 7 to FKHR exon 2. A similar fusion cannot be created by any other combination of PAX3/PAX7 and FKHR exons, because of incompatible reading frames or loss of functional domains. These findings support the premise that rearrangements of PAX3/PAX7 intron 7 and FKHR intron 2 are selected due to functional constraints related to the genomic organization of these loci.
Expression characteristics of wild-type and chimeric PAX genes
Ribonuclease protection assays showed several-fold higher levels of PAX3–FKHR mRNA relative to wild-type PAX3 mRNA in most PAX3–FKHR-expressing ARMS specimens (Davis and Barr, 1997). Immunoprecipitation analysis confirmed that PAX3–FKHR is overexpressed at the protein level. Similar studies of PAX7–FKHR-expressing tumors revealed that this fusion is consistently overexpressed relative to wild-type PAX7. These findings indicate that overexpression of PAX3–FKHR and PAX7–FKHR relative to the corresponding wild-type PAX genes is characteristic of ARMS tumors, and suggest that overexpression is required to generate a level of fusion product above a critical threshold for oncogenic activity.
Genomic studies revealed a striking difference in the basis of PAX3–FKHR and PAX7–FKHR overexpression. Fluorescence in situ hybridization assays showed in vivo amplification of the fusion gene on extrachromosomal elements in PAX7–FKHR-positive ARMS cases (Barr et al., 1996). These findings were confirmed and extended by quantitative Southern blot assays of the relative copy number of wild-type and rearranged alleles (Barr et al., 1996; Davis and Barr, 1997). In a series of ARMS cases, fusion gene amplification was detected in one of 24 PAX3–FKHR cases and 10 of 11 PAX7–FKHR cases (Fitzgerald et al., 2000). Therefore, in PAX7–FKHR tumors, translocation and amplification often occur sequentially to alter both gene structure and copy number and thereby activate oncogenic activity by complementary strategies.
The mechanism of overexpression in PAX3–FKHR-expressing tumors was elucidated by analyses of mRNA stability and transcription rate (Davis and Barr, 1997). Ribonuclease protection analysis of RNA from actinomycin D-treated ARMS cell lines showed that PAX3–FKHR and PAX3 transcripts have comparable stabilities. However, nuclear runoff analysis using hybridization targets flanking the t(2;13) breakpoint revealed that PAX3–FKHR is more actively transcribed than PAX3. Therefore, PAX3–FKHR overexpression results from a copy number-independent increase in transcriptional rate that is postulated to result from synergism of PAX3 and FKHR regulatory elements. In contrast, PAX7–FKHR overexpression results from a second genetic event, fusion gene amplification. These findings indicate biological differences between the two fusion genes that are probably related to differences in the regulation of their expression.
Regulation of subcellular localization of wild-type and chimeric FOX proteins
Genetic studies in C. elegans revealed that DAF-16 is involved in a signaling pathway involving the homologues of the insulin receptor (DAF-2), phosphoinositide 3-kinase (AGE-1), and AKT serine/threonine kinases (Lin et al., 1997; Ogg et al., 1997). In mammalian cells, various growth/survival factors (including insulin and IGF-1) activate phosphoinositide 3-kinase and subsequently activate AKT. Based on the C. elegans findings, AKT was shown to phosphorylate FKHR, FKHRL1 and AFX at three conserved sites that conform to the AKT consensus target sequence (Brunet et al., 1999; Takaishi et al., 1999; Tang et al., 1999) (Figure 3). Following phosphorylation, these proteins localize within the cytoplasm, and thus this signaling pathway inactivates transcriptional function by preventing these proteins from reaching their site of action. The mechanism for this change in subcellular localization appears to involve interaction with 14-3-3 scaffolding proteins and cytoplasmic sequestration.
Of the three AKT phosphorylation sites, two are retained in the PAX3–FKHR and PAX7–FKHR proteins in the region distal to the fork head domain (Figure 3). In that ARMS cells produce high levels of IGF2 (Toretsky and Helman, 1996), the fusion proteins would be sequestered in the cytoplasm if these proteins are regulated by this pathway. However, transfection experiments showed that PAX3–FKHR has a nuclear localization and is transcriptionally active in the presence of AKT (del Peso et al., 1999). Therefore, the fusion protein is resistant to upstream regulatory signals and is constitutively retained in the nucleus. Possible explanations include a necessary role for the N-terminal FKHR phosphorylation site that is not retained by the fusion protein or antagonism by N-terminal PAX domains, such as the nuclear localization signal.
DNA binding properties of wild-type and chimeric PAX proteins
In PAX3–FKHR and PAX7–FKHR, the paired box and homeobox are intact whereas the fork head domain is truncated (Davis et al., 1994; Galili et al., 1993; Shapiro et al., 1993) (Figure 3). Since mutations of other fork head domains inactivate DNA binding function (Kaufmann and Knochel, 1996), the truncated fork head domain in the fusion proteins is probably inactive or modifies DNA binding by the PAX3/PAX7 domains. Therefore, the PAX3 and PAX7 DNA binding domains are postulated to provide the DNA binding specificity for the fusion proteins.
Initial studies focused on binding to the e5 sequence, which was identified in the Drosophila even-skipped gene as a binding site for the paired gene product, which also contains a paired box and homeodomain (Goulding et al., 1991; Treisman et al., 1991). Footprinting experiments with e5 and its derivatives showed that the paired and PAX3 proteins protect an 18–27 bp region, which can be subdivided into paired box and homeodomain binding sites that include GTTCC and ATTA motifs, respectively (Chalepakis et al., 1994a; Treisman et al., 1991). Optimal binding of PAX3 to e5 or its derivatives requires both sites (Chalepakis et al., 1994a; Goulding et al., 1991). Comparison of full-length PAX3 to a truncated form lacking the homeodomain demonstrates a qualitative difference in binding affinity. Subsequent studies of PAX3 proteins with paired box or homeodomain mutations (from splotch and Waardenburg syndrome cases) revealed altered binding activity of both the unmutated and mutated domains, and thus indicated a functional interaction between the paired box and homeodomain within the wild-type PAX3 protein (Fortin et al., 1997; Underhill and Gros, 1997; Underhill et al., 1995).
Additional studies of PAX family members indicated that the paired box is comprised of two subdomains (Czerny et al., 1993). PAX5 targets can be divided into two sets of binding sites, the longer class I sites that require both N-terminal and C-terminal subdomains, and the shorter class II sites (which includes e5 and related sequences) that only require the N-terminal subdomain. A comparison of PAX3 and PAX5 DNA binding initially indicated that only PAX5 can bind class I sites whereas both PAX3 and PAX5 can bind class II sites (Schafer et al., 1994). However, subsequent studies identified an alternative splice that removes a single glutamine residue in the paired box of PAX3 and increases the affinity for class I sites (Vogan et al., 1996) (Figure 3). An analysis of PAX7 showed that the analogous glutamine-containing isoform interacts with e5 and related class II sites with a binding affinity similar to that of PAX3 (Schafer et al., 1994). Furthermore, a comparable alternative splice that removes a glutamine from the paired box was also identified in PAX7, though binding activity was not assayed (Vogan et al., 1996). Since both splice forms of PAX3 and PAX7 are co-expressed, the differing binding activity of the two isoforms will contribute to a complex pattern of overall binding function.
Electrophoretic mobility shift assays of the PAX3-FKHR protein showed binding to e5 and related sites (Fredericks et al., 1995; Sublett et al., 1995). Whereas mutations within the paired box or homeodomain of PAX3–FKHR abolished interaction with these sites, deletion of the truncated fork head domain did not affect binding. Comparison of the autoradiographic intensities of the protein–DNA complexes formed with PAX3 and PAX3–FKHR indicated that the binding affinity of isolated wild-type PAX3 protein for e5 was greater than that of PAX3–FKHR. Therefore, even though the wild-type and fusion proteins contain the same PAX3 DNA binding domain, in vitro binding function is partly dependent on the larger protein context.
PAX3 binding sites were also isolated from random oligonucleotide pools by a PCR-based selection strategy. A bacterially synthesized protein containing the PAX3 paired box identified consensus binding sequences of TCGTCAC(G/A)C(T/C/A)(T/C)(C/A/T)A and CGTCACG(G/C)TT (Chalepakis and Gruss, 1995; Epstein et al., 1996). Similar experiments with a bacterial synthetic protein containing the PAX3 paired box and homeodomain identified ATTA and GTNNN motifs in the majority of isolated binding sites, and the sequence ATTA-(N)n-GTTAT in 20% of the binding sites (Phelan and Loeken, 1998). This finding is consistent with the hypothesis of paired box and homeobox binding motifs connected by a variable spacer.
Several studies also demonstrated protein–protein interactions involving PAX3 and/or PAX7 that may influence DNA binding activity. PAX3 and PAX7 are capable of forming homodimers (PAX3/PAX3 or PAX7/PAX7) and heterodimers (PAX3/PAX7) that can bind to a palindromic homeodomain-binding site (P2-TAATCAATTA) with comparable or enhanced binding compared to the monomers (Schafer et al., 1994). In addition, the homeobox-containing protein Msx1, which is coexpressed with Pax3 in migrating limb muscle precursors, can interact with the Pax3 protein and inhibit Pax3 DNA binding activity (Bendall et al., 1999).
Transcriptional properties of wild-type and chimeric PAX proteins
To analyse sequence-specific transcriptional regulatory function of the wild-type and fusion proteins, constructs expressing the full-length proteins were transfected into mammalian cells with a reporter construct containing a minimal adenoviral E1b promoter and PAX3/PAX7 binding sites (Bennicelli et al., 1996, 1999; Fredericks et al., 1995). In experiments using various cell lines and binding sites, wild-type PAX3 expression resulted in a low but detectable level of transcriptional activation whereas wild-type PAX7 expression did not result in detectable transcriptional activity. In contrast, the PAX3–FKHR and PAX7–FKHR proteins similarly induced much higher levels of transcriptional activity, greater than 10-fold more activity than wild-type PAX3. In separate experiments using reporter constructs with a herpesvirus thymidine kinase promoter and PAX3 binding sites, transcriptional activation by PAX3–FKHR was only twofold higher than that by PAX3 (Sublett et al., 1995). These experiments demonstrate that the PAX3–FKHR and PAX7–FKHR proteins can function as transcription factors, specifically activating expression of genes containing PAX3/PAX7 DNA binding sites. Furthermore, the fusion proteins are more potent transcriptional activators than the wild-type proteins, though the increased potency may be promoter-dependent.
To investigate the differing transcriptional potencies of the wild-type and fusion proteins, initial studies focused on the activities of the C-terminal PAX3, PAX7, and FKHR regions (Bennicelli et al., 1995, 1999; Chalepakis et al., 1994b; Sublett et al., 1995). These C-terminal regions were examined independent of the respective DNA binding domains by generating fusions with the GAL4 DNA binding domain. In these GAL4 fusion constructs, the C-terminal PAX3, PAX7, and FKHR regions acted as highly potent activation domains with essential transactivation domains located at the 3′ end of each coding region (Figure 3). The PAX3 and PAX7 transactivation domains are serine- and threonine-rich, whereas the FKHR transactivation domain contains both acidic- and serine-, threonine-rich regions. In addition to these domains, positive modifying elements were found in adjacent regions. These data demonstrate that the wild-type and fusion proteins contain comparably potent, yet structurally distinct transcriptional activation domains which are switched by the translocations in ARMS.
The finding of similar transcriptional potencies of the C-terminal domains and contrasting potencies of the full-length proteins suggested that the activity of the C-terminal activation domains is modulated by other portions of the full-length protein. To explore this hypothesis, the GAL4 DNA binding domain was joined to the full-length wild-type and fusion proteins (Bennicelli et al., 1996, 1999). When fused to GAL4, full-length PAX3–FKHR or PAX7–FKHR activated transcription of a GAL4-dependent reporter gene with an activity 10-fold higher than the corresponding full-length wild-type protein. In addition, the full-length PAX–FKHR constructs had several-fold lower activity than the C-terminal FKHR construct whereas the full-length wild-type PAX3 or PAX7 construct had one to two orders of magnitude less activity than the C-terminal PAX3 or PAX7 construct. These findings indicate that an inhibitory domain is present in the N-terminal PAX3 and PAX7 regions that effectively inhibits the activity of the C-terminal PAX3 and PAX7 domains but only has a modest effect on the C-terminal FKHR domain (Figure 3). Therefore the translocations create potent transcriptional activators by introducing a C-terminal transactivation domain that is relatively insensitive to the inhibitory effects of the N-terminal PAX3 and PAX7 domains. Furthermore, differing interactions between the N-terminal inhibitory and C-terminal activation domains in PAX3 and PAX7 may explain the differing transcriptional activities of these wild-type proteins.
Additional studies suggest that the N-terminal PAX3 and PAX7 domains function as transcriptional inhibitory modules in intermolecular and intramolecular regulation by binding co-repressors. Deletion analysis localized the inhibitory activity to two N-terminal domains, the homeodomain and the N-terminus including the first half of the paired box (Bennicelli et al., 1996, 1999) (Figure 3). These same two regions were also shown to have independent transcriptional repression activity when fused to the GAL4 DNA binding domain (Chalepakis et al., 1994b). Protein interaction studies subsequently determined that the putative co-repressors DAXX, RB1, and HIRA bind to PAX3, and for DAXX and HIRA, interactions with PAX7 were also noted (Hollenbach et al., 1999; Magnaghi et al., 1998; Wiggan et al., 1998). Deletion studies suggest that these proteins interact with the PAX3 homeodomain, though the DAXX interactions also appear to involve the octapeptide and other N-terminal regions. When co-expressed with PAX3, both DAXX and RB1 result in titratable inhibition of PAX3-mediated transcriptional activation, thus supporting their function as transcriptional co-repressors. In addition, DAXX can also bind PAX3–FKHR but is unable to repress transcriptional activation by the fusion transcription factor. These findings support the transcriptional gain of function model and suggest that increased transcriptional potency of the fusion proteins results from unresponsiveness to the repressive activity of bound co-repressors.
Transcriptional targets of wild-type and chimeric PAX proteins
Using electrophoretic mobility shift assays in conjunction with partial or full-length protein constructs, putative PAX3 binding sites were identified in several mammalian genes. These sites were located within the 5′ promoters of the MET, MITF, TYRP1, PDGFRA, BCL2L1 (BCL–XL), and RET genes as well as in the 3′ untranslated region of the NF1 gene and the first intron of the NRCAM gene (Epstein et al., 1995, 1996, 1998; Galibert et al., 1999; Kallunki et al., 1995; Lang et al., 2000; Margue et al., 2000; Watanabe et al., 1998). These putative binding sites contain sequences resembling a paired box binding motif (NF1, MET and RET), a homeobox binding motif (PDGFRA and BCL–XL), or both motifs (MITF, TYRP1 and NRCAM). In one other study, a fragment from the NCAM promoter was shown to bind to cellular extracts expressing the paired box but not extracts expressing the entire PAX3 protein and thus the significance of this binding site is unclear (Chalepakis et al., 1994b).
These binding sites were further investigated in assays in which a PAX3 or PAX3–FKHR expression construct was co-transfected with a reporter construct containing the binding sites. In these assays, PAX3 activated transcription from MITF, TYRP1 and RET binding sites, whereas PAX3 repressed transcription from the NCAM element (Chalepakis et al., 1994b; Galibert et al., 1999; Lang et al., 2000; Watanabe et al., 1998). In the case of the PDGFRA site, the finding of activation by PAX3–FKHR but not PAX3 supports the gain of transcriptional function model described previously (Epstein et al., 1998). In contrast, both PAX3 and PAX3–FKHR activated transcription from NF1 and BCL–XL sites without substantial differences in transcriptional activity between the wild-type and fusion proteins (Epstein et al., 1995; Margue et al., 2000).
Additional studies investigated endogenous gene expression changes following manipulations of PAX3 or PAX3–FKHR expression. Two widely used strategies are introduction of a PAX3 or PAX3–FKHR expression construct into specific cell types, and analysis of homozygous splotch mice. The transfection approach demonstrated enhanced expression of MET, BCL–XL, and RET and thus provides further support that these genes are direct transcriptional targets of PAX3 or PAX3–FKHR (Epstein et al., 1996; Lang et al., 2000; Margue et al., 2000). A more complicated situation exists for PDGFRA in which transient transfection of PAX3–FKHR but not PAX3 into P19 cells activates PDGFRA expression whereas stable integration of either PAX3 or PAX3–FKHR in NIH3T3 cells represses expression (Epstein et al., 1998; Khan et al., 1998). These transfection studies also identified additional candidate targets, as exemplified by the cDNA array analysis of NIH3T3 cells that stably express PAX3–FKHR (Khan et al., 1998). This transcriptional profiling study showed increased expression of IGF2, IGFBP5 and MERTK; and decreased expression of FISP12, PMX1 and DAF. The homozygous splotch expression studies also identified genes whose expression is increased (e.g., versican) or decreased (e.g., lbx1) in specific tissues in the absence of functional Pax3 protein (Henderson et al., 1997; Mennerich et al., 1998). Though these various studies suggest possible target genes, these approaches do not specifically determine whether these genes are directly bound and regulated by PAX3 or PAX3–FKHR. Some of these altered expression events may occur further downstream such that additional steps or signals intervene between the PAX transcription factor and the assayed gene.
Phenotypic roles of wild-type PAX proteins: gene knockout studies
The transcriptional studies of PAX3–FKHR and PAX7–FKHR are consistent with the hypothesis that the translocations activate the oncogenic potential of PAX3 and PAX7 by dysregulating or exaggerating their normal function in the myogenic lineage. Clues for this normal function may be deduced from the skeletal muscle phenotype of splotch and Pax7 knockout mice. In the homozygous splotch animals, limb musculature fails to develop whereas the axial musculature shows varying degrees of malformation (Franz et al., 1993). The defects in muscle development appears to result from increased apoptosis within the myogenic precursor pool of the lateral dermomyotome (Borycki et al., 1999) and from failure of surviving myogenic precursors to migrate from the somites into the limb buds (Bober et al., 1994; Daston et al., 1996; Goulding et al., 1994). This limb musculature defect in splotch mice is associated with reduced expression of the Met receptor in myogenic progenitors (Daston et al., 1996; Epstein et al., 1995; Yang et al., 1996), and is also found in murine strains in which the gene encoding Met was mutated by gene targeting (Bladt et al., 1995). The established role of Met in cell motility signaling and the finding that the gene encoding Met is a potential transcriptional target of the Pax3 protein suggest that the migration problem is due to defective regulation of Met expression by the mutant Pax3 transcription factor. Therefore, these studies of splotch mice suggest possible roles for PAX3 in the stimulation of motility and maintenance of a viable population by facilitating growth or inhibiting apoptosis.
Additional studies of splotch mice and Pax7 knockout mice revealed roles for Pax3 and Pax7 in facilitating myogenic differentiation. In particular, when the homozygous Pax3 mutation is combined with a homozygous Myf5 mutation, almost all trunk (as well as limb) musculature fails to form (Tajbakhsh et al., 1997). This defect in myogenic development involves a failure to activate MyoD expression and subsequent myogenic events. Furthermore, though mice with a homozygous inactivating Pax7 mutation generally have normal muscle development, these animals have a postnatal muscular deficiency evidenced by smaller muscle fibres and decreased size of muscles such as the diaphragm (Seale et al., 2000). This muscle deficiency is attributed to the absence of satellite cells, and implicates Pax7 in the specification of myogenic satellite cells from uncommitted progenitors. Therefore, in addition to motility and viability, PAX3 and PAX7 are proposed to regulate downstream myogenic events. Exaggeration of these activities by PAX3–FKHR and PAX7–FKHR suggests several hypotheses relevant to ARMS pathogenesis.
Phenotypic roles of wild-type and chimeric PAX proteins: gene transfer studies
Gene transfer studies in several model systems further investigated specific functions of these proteins and suggest that the fusion proteins may exert an oncogenic effect through multiple pathways that exaggerate the normal role of the wild-type PAX proteins. In studies investigating growth control, under conditions in which wild-type PAX3 does not transform NIH3T3 cells or chicken embryo fibroblasts, introduction of a PAX3–FKHR expression construct resulted in potent transforming activity (Lam et al., 1999; Scheidler et al., 1996). As a complementary experiment, introduction of a fusion of PAX3 to the KRAB transcriptional repression domain into ARMS cells resulted in loss of transforming activity in culture and tumor suppression in mice (Fredericks et al., 2000). Mutation analysis demonstrated that the homeodomain but not the paired box is required for transformation by PAX3–FKHR (Lam et al., 1999; Scheidler et al., 1996), and thus altered expression of target genes whose binding sites require paired box function may not be required for cellular transformation. Mutation analysis also indicated that the C-terminal FKHR activation domain is required for transformation. Furthermore, substitution of the VP16 activation domain for the C-terminal domain of PAX3 activated transforming activity, whereas substitution of the PAX3 activation domain for the C-terminal domain of PAX3–FKHR ablated transforming activity (Cao and Wang, 2000). These swapping studies support the gain of transcriptional function model, and suggest that the effect of the C-terminal FKHR domain can be mimicked by other activation domains.
In addition to stimulating transforming activity, gene transfer studies also indicated that PAX3–FKHR functions in the maintenance of cell viability by inhibiting apoptosis. Treatment of ARMS cells with an antisense oligonucleotide directed against the PAX3 translational start site resulted in a transient decrease in PAX3–FKHR protein expression (Bernasconi et al., 1996). This expression change was associated with a significant decrease in cell number as well as morphological characteristics and a DNA fragmentation pattern characteristic of apoptosis. In a parallel set of studies, introduction of a tamoxifen-inducible PAX3–KRAB construct into ARMS cells revealed evidence of apoptosis when the construct is induced in low serum conditions or in tumor xenografts (Ayyanathan et al., 2000). A role for BCL–XL is supported by the finding that BCL-XL was downregulated in association with apoptosis in these PAX3–KRAB studies.
A final set of studies addressed the roles of PAX3 and PAX3–FKHR in regulating myogenic pathways. The ability of PAX3 to activate the myogenic program in vitro was revealed by studies in which Pax3-expressing retroviruses were introduced into explanted paraxial mesoderm and other embryonic tissues (Maroto et al., 1997). In this setting, Pax3 induced expression of the myogenic transcription factors MyoD, Myf5, and myogenin as well as myogenic differentiation products. Similarly, when a PAX3–FKHR expression construct was introduced into NIH3T3 cells, cDNA array analysis revealed induction of a large set of genes involved in myogenesis, including the transcription factors MyoD, myogenin, Six1, and Slug (but not Myf5) as well as downstream myogenic products such as various troponins and myosin light chain (Khan et al., 1998). The finding that PAX3 was not able to induce this myogenic transcription program in NIH3T3 cells suggests that PAX3–FKHR has greater transcriptional potency in this less permissive cell environment. A final study focused on the ability of these proteins to inhibit terminal differentiation in two myogenic cell lines, C2C12 myoblasts and MyoD-expressing 10T1/2 cells (Epstein et al., 1995). Whereas differentiation is typically induced in most cells (86–87%) by growth factor withdrawal, PAX3 cDNA transfection reduced the percentage of myosin-expressing colonies to 40–43%, and PAX3–FKHR transfection further reduced this percentage to 17–27%. In contrast to the transformation studies, both the paired box and homeodomain were required to inhibit terminal differentiation. The apparent contrast of these findings from those of the preceding two studies may be explained by the hypothesis that these PAX proteins facilitate entry into the myogenic pathway but inhibit the final steps of the pathway. Alternatively, the myogenic consequences of aberrant PAX3 and PAX3–FKHR expression may be dependent on the specific cell type and other environmental features.
Several recent studies investigated the effect of PAX3–FKHR on mammalian development, and have found a diversity of phenotypic effects. Transgenic mice expressing PAX3–FKHR from a cloned murine Pax3 promoter demonstrate pigmentary and neurological alterations reminiscent of the splotch phenotype (Anderson et al., 2001). In a second study that used a gene targeting strategy to introduce the 3′ FKHR cDNA into the endogenous murine Pax3 locus, mice with the heterozygous knock-in allele were not viable, probably as a result of heart defects and a small disorganized diaphragm (Lagutina et al., 2000). Finally, in a third study, ES cells expressing the PAX3–FKHR cDNA from CMV expression elements were used to generate chimeric mice that demonstrated an overgrowth phenotype reminiscent of Beckwith–Wiedemann syndrome (Xie et al., 2001). Though the phenotypic features varied in these animal systems, tumor susceptibility was not found in any of these studies. Therefore, the tumorigenic effects of the gene fusion gene may require cooperating events or other features that are not present in these animal models.
Molecular diagnostic and therapeutic studies of ARMS gene fusions
As the genetics and biology of the gene fusions in ARMS were elucidated, studies were initiated to explore clinical applications of these gene fusions (Arden et al., 1996; Barr et al., 1995; de Alava et al., 1995; Downing et al., 1995; Frascella et al., 1998; Reichmuth et al., 1996). Using RT–PCR assays for the PAX3–FKHR and PAX7–FKHR transcripts, multiple tumors were assayed to determine the frequency of these fusions. Approximately 80% of ARMS cases expressed one of the two fusions, with the frequency of PAX3–FKHR being 5–10 times that of PAX7–FKHR. Molecular diagnostic studies also revealed a small subset of ARMS cases that do not express either gene fusion. Though these negative results may be explained by variable application of histopathologic diagnostic criteria or the suboptimal quality of some samples, there is clearly a small subset of well-characterized ARMS cases in which these gene fusions are not detectable by standard RT–PCR assays. In these cases, the possibilities of rare variant fusions, other genetic events that can substitute for the characteristic fusions, or a biologically distinct entity with a similar histological appearance should also be considered.
Comparison of the clinical characteristics of PAX3–FKHR and PAX7–FKHR-expressing tumors showed that PAX7–FKHR tumors tend to occur in younger patients, more often present in extremities, are more often localized lesions, and are associated with longer event-free survival (Kelly et al., 1997). In contrast, PAX3–FKHR tumors tend to occur in adolescents as higher stage lesions in a wider variety of sites. Therefore, clinical heterogeneity in ARMS is associated with genetic heterogeneity in the involved fusion genes, and thus identification of the specific gene fusion may provide valuable information for management and outcome assessment.
In addition to providing diagnostic information, studies are also being conducted to investigate whether the fusions can be targeted by therapeutic strategies. As described, strategies were developed to antagonize PAX3–FKHR expression with antisense oligonucleotides or to antagonize PAX3–FKHR function with an engineered PAX3–KRAB transcriptional repressor (Ayyanathan et al., 2000; Bernasconi et al., 1996; Fredericks et al., 2000). Another therapeutic strategy involves transfer of a construct consisting of PAX binding sites that regulate expression of a toxin-encoding gene (Massuda et al., 1997). Cell culture studies demonstrated that this construct is selectively toxic for cells expressing the PAX3–FKHR fusion. In a final strategy, the immunogenicity of peptides spanning the fusion point is being tested (Mackall et al., 2000). Animal studies provided evidence that cytotoxic T lymphocytes directed against the fusion peptide can be generated and will recognize and kill PAX3–FKHR-expressing tumor cells.
In ARMS, the 2;13 and 1;13 translocations are consistent and specific events that provide opportunities to investigate the molecular etiology of this cancer, identify novel markers for diagnosis and monitoring, and develop therapeutic approaches against tumor-specific targets. These translocations juxtapose the transcription factor-encoding genes PAX3 or PAX7 with FKHR to generate PAX3–FKHR and PAX7–FKHR chimeric genes. A consideration of the biological and clinical data on these two fusion genes indicates important similarities that point to a common fundamental mechanism in the pathogenesis of this tumor. However, the biological and clinical data also reveal several striking differences between the two fusions that highlight the heterogeneity within this tumor category and distinctions between two highly related members of the paired box family.
The genetic changes resulting from the 2;13 translocation alter biological activity at the levels of protein function, gene expression, and protein localization. The result is high level, constitutively nuclear expression of a potent fusion transcription factor. These biological changes are postulated to promote aberrant expression of genes that are transcriptional targets of wild-type PAX3 and that normally function in the control of early myogenic development. The aberrant or inappropriate expression of these gene products is postulated to contribute to oncogenic initiation or progression by stimulating cell behaviors such as growth and motility or inhibiting cell behaviors such as apoptosis and terminal differentiation.
The t(1;13) bears a strong similarity to the t(2;13) in that the PAX7–FKHR fusion product joins the PAX7 DNA binding domain and FKHR activation domain. Functional studies indicate that the PAX7–FKHR protein functions an aberrant transcription factor similar to PAX3–FKHR. In contrast to PAX3–FKHR, the juxtaposition of PAX7 and FKHR regulatory elements is apparently not sufficient for PAX7–FKHR overexpression, and instead an amplification event ensues to increase PAX7–FKHR gene copy number. This finding indicates a biological distinction between the two fusions, the potential significance of which is indicated by the finding of clinical differences between PAX3–FKHR and PAX7–FKHR-expressing tumors.
Future investigations will integrate the gene expression, target gene, and functional studies to develop a comprehensive understanding of the aberrant genetic program induced by these translocations. Furthermore, increased efforts must be directed at identifying the collaborating events in ARMS tumorigenesis and developing appropriate model systems to recapitulate these oncogenic pathways in a controlled fashion. In addition to further elucidating how the PAX3–FKHR and PAX7–FKHR fusions contribute to tumorigenesis, these studies need to address the differences between the two fusions in tumorigenic pathways. These studies will ultimately enhance the utility of molecular genetics in the diagnosis and monitoring of this cancer, and will indicate directions in which possible therapeutic strategies can be developed to interrupt these tumorigenic pathways.
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This work was supported in part by NIH grants CA64202, CA71838 and CA89461.
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Barr, F. Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma. Oncogene 20, 5736–5746 (2001). https://doi.org/10.1038/sj.onc.1204599
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- rhabdomyosarcoma
- transcription factor
- translocation
- paired box
- fork head
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No children 1 child 2 children 3 children 4 children 5 children 6 children 7 children 8 children 9 children 10 children. Private parking is available at a location nearby reservation is not possible and costs EUR 24 per day. Cancellation and prepayment policies vary according to apartment type.


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