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BRUSSELS, Belgium – Free access to Belgium will continue as normal this summer. Recent speculation that controls on air arrivals will be extended to land borders is erroneous. In particular coach groups can expect continued "borderless" access when arriving from any of the neighbouring states.
Peter de Wilde, Chief Executive of Visit Flanders says: "We know there has been some confusion in the marketplace, and so I am delighted to confirm that there are no plans to impose systematic checks on vehicles at the Belgian border. We know that Flanders features in many pan-European itineraries, and it is important that incoming visitors – especially those from long haul origin markets – know that they are welcome. Part of that welcome is the continuation of the unfettered access allowed to those coming in from our neighbouring countries."
"Belgium" said Tom Jenkins, CEO of ETOA "lies at the heart of Europe. Brussels is the centre of the EU. With its incomparable architectural heritage, its culture and cuisine, Brussels embodies much of what makes Europe attractive and unique. It is vital for visitors to Europe that they can have unimpeded access to such an important destination."


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Three things:
I want Pippa's dress.
I want the name Pippa!
I want to drive a wine-powered Aston Martin.
After our all-nighter, Basil and I took a (pre-nap!) walk around the gardens today.
Cool and dewy…
the sun did peek out while we were out there.
new trees.
soft leaves.
when you live by the golf club…
ivy by the river.
better view of the river:
Basil Dachshund…
Happy Friday!

subset: commoncrawl


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Latest blow to Microsoft´s modeling strategy: Doug Purdy moving to Facebook
The announcement is quickly spreading: Doug Purdy is leaving Microsoft and moving to Facebook to "improve Facebooks relationship with the community, including addressing bugs, improving responsiveness and other points of developer support"
This is just the latest step in the (downhill) Microsoft MDD strategy. First, Oslo (in short, Microsoft main initiative for model-driven development consisting in the M modeling language + the Quadrant modeling environment + a model repository) development team was merged with another Microsoft team .
Then, Oslo itself was renamed SQL Server Modeling , something that the communty interpreted as Oslo being demoted to become an environment for designing and managing SQL Server databases.
Later, Microsoft dropped Quadrant and now, finally, Doug Purdy (one of the key people behind Microsoft MDD strategy) leaves Microsoft.
While this may not be Microsoft´s fault (the reasons behind this departure have not been yet explained), this may be the last (and final, at least for a while) blow to Microsoft´s efforts on MDD.


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Politics World
FLASHBACK: Panama Papers Reveal Clinton's Kremlin Connection
[ditty_news_ticker id="25027″]
Almost lost among the many revelations is the fact that Russia's biggest bank uses The Podesta Group as its lobbyist in Washington, D.C. Though hardly a household name, this firm is well known inside the Beltway, not least because its CEO is Tony Podesta, one of the best-connected Democratic machers in the country. He founded the firm in 1998 with his brother John, formerly chief of staff to President Bill Clinton, then counselor to President Barack Obama, Mr. Podesta is the very definition of a Democratic insider. Outsiders engage the Podestas and their well-connected lobbying firm to improve their image and get access to Democratic bigwigs. – READ MORE


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Look Ma, No Lens!
Pinhole Cameras You Can Build Or Buy Page 2
Why is a larger pinhole image better? As with any unsharp lens, the larger the negative, the better the image appears because of better tonal gradation. For this reason most companies making "serious" pinhole cameras or kits use 120 roll film or 4x5 sheet film. You can buy medium format kits that can be assembled without tools, ready-made cameras, or get creative with my suggested modifications.
The 120 rollfilm pinhole kits make it easy to start with medium format images. Beseler, of enlarger fame, has a fold 'n tape cardboard camera that produces a generous 60x90mm negative. Due to some ingenious folding, the camera is both light-tight and does not scratch the film. The camera was developed by Peter Olpe, professor at the Basel School of Design, Switzerland.
The Beseler pinhole lens is laser drilled into a thin sheet of metal and the shutter is a simple cardboard slide mechanism. As with most pinhole cameras, framing is somewhat of a guess. But there are printed lines on the top of the camera to suggest the approximate viewing angle of the lens. According to Beseler, the average exposure with ISO 400 film in bright light is 2 seconds.
The Beseler camera has no tripod socket, which is a shame as it cannot be handheld due to the long exposures. A couple of heavy-duty elastic bands can lash the camera to a tripod, or you can hold the lightweight body steady on a pile of books. The kit is available wherever Beseler products are offered for under $40. A web search for "Beseler Pinhole" easily locates many sellers, including AAA Camera.
Upsized And Upscale
For those wanting a truly breathtaking and highly functional pinhole camera, the multi-format rollfilm model from Zero Image in Hong Kong is worth investigating. These cameras are designed by pinhole enthusiast Zerniki Au and are handmade using exotic teak and solid brass fittings. The camera joints are specially designed with reinforced bondings, allowing the camera to shrink and swell with weather changes without affecting the performance (read: no light leaks). The Zero Image offers multiple formats from 6x4.5 to 6x9 on 120 film by adjusting panels inside the camera. It is fitted with a brass tripod socket, exposure calculator dial, and ingenious wooden sliding "shutter." Build quality is so high that there is no need for foam light baffles or seals!
While it is certainly possible to make similar images with lesser cameras, many will find using a Zero Image model a superior pinhole experience due to the general handling, properly sized pinhole, and high-grade construction. The difference between using a Zero Image camera and the cardboard kits is like shooting with a Leica vs. a disposable camera.
Naturally, this kind of experience has its price. At $149.95 plus shipping, many casual experimenters will want to try other methods. But it is one of the most wonderful bits of construction I have ever seen, and pretty enough to leave out for display when not in use. The only negative is that the fine finish is delicate. This may prevent photographers from "roughing" with it. If you get one, use it. Don't just stare at it.
Another Budget Approach
Again, there is a much less expensive way to produce medium format pinhole pictures. Many cameras from the 1940s, '50s, and '60s are simply light-tight boxes with easily removable lens/shutter assemblies at the front. They are cheap and ready for pinhole conversion.
Simply find an old 120 box camera, remove the lens and replace it with a pinhole. With some cameras you'll be able to keep using the "click-clack" shutter if it has a bulb or time setting. Depending on the camera, you'll have anything from a 6x6 negative to a 6x9.
My homemade 120 Spartus "Full-Vue" pinhole conversion is a good example. I simply removed four large screws on the front, snapped off the plastic-mounted lens, replaced the cover, and taped on a pinhole lens. The shutter has a "B" setting where it remains open until released. I placed a rubber band around the bottom of the camera to hold the shutter lever open for longer exposures. As you can see, this is not rocket science.
One word of caution: Many simple box cameras use 620 size film that is no longer commonly available. There should be something written on the camera indicating the film used. If not, look for the thicker 120 spool vs. the much thinner 620 spool. You cannot put 120 film in these cameras without tedious respooling.
"That's The Ugliest Camera I Have Ever Seen!"
That was my wife's reaction to my other "Frankenstein" pinhole camera assembled from odd parts. For those who are adventurous, or who simply have a lot of camera parts in a box somewhere, the possibilities can be amusing. My Frankenstein camera is an ancient wooden box camera minus the lens, a Graflok back from a $10 oscilloscope camera, and a rollfilm back from a Mamiya RB or a Singer 70mm back. The shutter was black tape, although I experimented placing an old shutter from a folding camera in front of the pinhole for some shots.
The tools were Scotch double-faced mounting tape and a lot of black electrical and duct tape. Mounting tape is much thicker than simple double-sided tape and fastened the Graflok back to the camera body. It's better than glue, as the tape can be removed if you change your mind. It also conforms to variations in the wood body.
Black electrical and black duct tape seal any area that may leak light. Please note that not all black plastic tape is light proof. Most common brands have a thickness of only .008". When you hold a strip up to a light bulb, you see a dull glow through it. The solution: Add more tape! In my case, I used a couple of wraps of electrical tape topped by the wider black duct tape. The duct tape added a more finished look and prevented the tape underneath from coming loose.
I Want It Now
For those needing instant print gratification, nothing is faster than a pinhole Polaroid! These cameras range from a factory-made kit to a variety of home-brew models. Due to the faster speed of some Polaroid films, exposures can be greatly reduced. Indoor "available light" shots that take hours with film can be done in a minute or two. And, of course, you find out in a matter of seconds whether your efforts are successful.
The cheapest way to make a pinhole Polaroid is to locate an old Polaroid "pack" camera that uses the still-available Type 80 color or Type 667 black and white film. Then, using whatever tools work for you, remove the entire lens and bellows assembly and any supporting struts. This leaves the working film back and a large area to attach a box, can, or other material. Again, please wear eye protection, as bits of camera sometimes fly off unexpectedly.
- Here's Why One Photographer Says "Please Stop Asking About Camera Settings" (VIDEO)
- British Landscape Pro Adam Karnacz Reveals the Best Camera Settings for Great Nature Photos (VIDEO)
- 5 Lightroom Secrets That Will Change Your Landscape Photography Forever (VIDEO)
- Unlock the Hidden Power of Photoshop with These 5 Tips and Tricks (VIDEO)
- Photoshop Trick: Create Dramatic B&W Photographs by Isolating the Key Object in an Image (VIDEO)


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iHerb Customer Reviews
works great, fair price
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works great, fair price
I find this to be a good selenium supplement for thyroid support. It defiently does has been helping along with other suplements. These tablets disolve easily as they pass my 'tablet disolving test' with flying colors. What you do when you are unsure of the absorbality of a tablet you put it in water that is body temp not room temp a big difference and you time how long it takes them to disolve. If they disolve in like 5-10 minutes you have a very absorbalbe product (which these do) and 15-20 is still pretty good. I don't go over 30 minutes even for large tablets. At this point I replace them with another suplement and if I cannot find one I grind them up.
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Gives me diarrhea - metalloestrogen - caution for use with abnormal immune system
Posted by Health-Is-Wealth on Mar 21, 2014 | Verified Purchase
Have tested multiple times - side effect is unpleasant and no other benefits derived. I note with interest one major study that showed selenium supplementation on its own actually *increased* risk for breast cancer and how other studies failed to mention this outcome. High doses of selenium antagonise vit D, manganese, magnesium, zinc, chromium, sulfur - all essential for endocrine health and/or thyroid function and/or reducing disease/inflammation. Also interesting the number of people who gravitate to the selenium/sulfur combos vs sodium selenite or sodium selenate. I've tried four different kinds of selenium - Selenomethionine, Se-Methyl L-Selenocysteine, Sodium Selenite and Sodium Selenate. While this one (selenomethionine) at least offers a more realistic dose and is easier to cut tablets in half for a fifty microgram serving, I wonder re the number of people noting physiological benefits I'd be more inclined to attribute to the sulfur. If this review was helpful to you, please click YES below - thank you. For reviews of all the products I have purchased from iHerb, please click my username above.
works great, fair price
Posted by iHerb Customer on Oct 24, 2010 | Verified Purchase
I find this to be a good selenium supplement for thyroid support. It defiently does has been helping along with other suplements. These tablets disolve easily as they pass my 'tablet disolving test' with flying colors. What you do when you are unsure of the absorbality of a tablet you put it in water that is body temp not room temp a big difference and you time how long it takes them to disolve. If they disolve in like 5-10 minutes you have a very absorbalbe product (which these do) and 15-20 is still pretty good. I don't go over 30 minutes even for large tablets. At this point I replace them with another suplement and if I cannot find one I grind them up.
Great for thyroid function, reducing antibodies, and energy
Posted by iHerb Customer on Dec 02, 2009 | Verified Purchase
Great supplement for thyroid function. It has really helped both my husband and I with converting thyroid hormones T4 to T3 so we have a lot more energy. Selenium is also great for reducing antibodies. It has helped me reduce my antibodies from my Hashimotos Hypothyroidism. Great product!
Posted by iHerb Customer on Apr 27, 2009 | Verified Purchase
Affordable and seems to be helping in my overall regimen to combat low thyroid, adrenal fatigue and cloudy memory.
Creates a Relaxed State of Well-Being
Posted by rabbit on Aug 29, 2012 | Verified Purchase
I have symptoms which indicate that I may have problems with my thyroid, so based on my research, I take this selenium along with iodine. I started taking selenium soon after I discovered iodine. I must have been iodine deficient because the first time I took an iodine supplement, within minutes, I began to feel an overwhelming feeling of well-being, relaxation, and euphoria. I immediately felt better than I had since I was a kid. I could breathe so much easier and my mind felt clear and happy. I couldn't believe that something so simple could make me feel so much better so fast. It was such a relief! I am vegan, so I don't eat fish and I rarely eat seaweed products. I had no idea that iodine was so important. It has really lessened my symptoms of hypothyroidism. Do your research on Hashimoto's thyroiditis before using iodine as a treatment because it is not always recommended for this autoimmune disorder and has also been thought to trigger it. However, selenium has been said to counter this and protect the thyroid while taking iodine, so do your research and make up your own mind. You may only need to take selenium. I now use a stronger variety of iodine as both a supplement and as an antibiotic alternative. After I started taking the stronger variety, I had a cyst burst which was very painful. I think the cyst burst because iodine is known to shrink cysts and rid the body of them entirely in some cases. I am glad that the cyst burst because it was causing some pain, but with it came the risk of infection. I couldn't really afford to go to the doctor, so I increased my regular dose of iodine to protect me from getting an infection. I've felt fine since. Some people have even used iodine to cure their cancer! If you don't eat fish or ocean-based products, be sure you are getting enough of this crucial vitamin. After I started taking selenium with the iodine, I feel even better. I feel even more relaxed and at ease, especially right after taking the supplement.
Now Foods, Selenium, Yeast Free, 100 mcg, 250 tablets
Posted by iHerb Customer on Jul 16, 2008 | Verified Purchase
Selenium is important for thyroid function. I have used this product for years.
Great Value
Posted by iHerb Customer on Jul 19, 2008 | Verified Purchase
This is a great price and makes it possible to take it long term. My doctor wants me on Selenium as it is a great help for my thyroid.
Posted by iHerb Customer on Jun 25, 2012 | Verified Purchase
Dont skip this one, if you are serious about cancer prevention, and dementia prevention. 200mcg a day would be my recommendation, along with a good liquid mineral supplement.
I think it works
Posted by iHerb Customer on Nov 30, 2011 | Verified Purchase
In addition to Kelp and L-Tyrosine. Taking for goiter and low thyroid. I have astounding energy, the goiter has shrunken and my hair fall is almost non exsistant. Added this a month ago along with the L-Tyrosine but have been taking the Kelp for almost 3 months.1 pill a day was all it took. Thank the lord for these personal reviews. I have a Doc appt just to see where my levels are..
Good stuff, cheap
Posted by iHerb Customer on Aug 15, 2010 | Verified Purchase
Selenomethionine seems to be one of the superior forms of selenium. *interesting tidbit* -The real life Mark Whitacre (from the movie "The Informant") has done extensive research on selenium and has an interesting website with info on it.


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Sheepdog Is Afraid of Sheep [VIDEO]
I have three little dogs. Two of them have no idea they're small and have no fear, but one of them is afraid of everything, including leaves blowing in the breeze and other animals. But the dog in this video is a border collie. This breed is supposed to herd sheep. That's their job. It's in their genes. Except for Ci, who is afraid of sheep, and has been his whole life. His person, Jane Lippington, says he has a bit of the instinct, but not quite enough to be very effective at herding, but the sheep do seem to enjoy following him as he runs away!


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Antipater, (born c. 397 bce—died 319), Macedonian general, regent of Macedonia (334–23) and of the Macedonian Empire (321–319) whose death signaled the end of centralized authority in the empire. One of the leading men in Macedonia at the death of Philip II in 336, he helped to secure the succession to the Macedonian throne for Philip's son, Alexander the Great, who upon departure for the conquest of Asia (334) appointed Antipater regent in Macedonia with the title of general in Europe. Antipater's main task was to hold the northern frontiers against hostile tribes and to keep order among the Greek states. He ruled Greece by cooperating with the League of Corinth but was unpopular because he supported oligarchic governments. The settlement of the satrapies (provinces) of the Macedonian Empire by the new regent, Perdiccas, at Babylon in 323, immediately after Alexander's death, left Antipater in control of Macedonia and Greece, though as former regent his status in relation to Perdiccas was not clearly defined. Antipater then took the side of the Macedonian generals Antigonus, Seleucus, and Ptolemy, who were opposed to the claims of Perdiccas. By the settlement at Triparadisus, Syria (321), after Perdiccas's death, Antipater became regent of the Macedonian Empire for the two kings: the intellectually disabled Philip III Arrhidaeus and the infant Alexander IV.
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text: As Sheetz grows its fleet of stores, it looks to add new employees. Altoona, Pa.-based Sheetz plans to hire up to 250 new employees in greater Pittsburgh by the end of 2012 as it opens new stores and renovates others, Bizjournals.com reported. Within the next three months, the c-store chain is set to open two


text:
Web Content, Digital Assets, Form Builder, SEO Tools, Decision Support
Multisite Management, Multilingual Support, Site Synchronization, Deployment Options, Security, Scalability
Personalization, Email Marketing, Mobile, Social Media, Analytics
Integration Platform, Connector for Salesforce, Connector for Microsoft Dynamics CRM, Connector for Marketo, Connector for SharePoint
Product Catalogs, Multiple Storefronts, Targeted Offers, Payment Processing, Order Management
Custom Content Types, UI Framework, ASP.NET MVC Support, Visual Studio Support, Comprehensive API
Behaviour Tracking, Data Integration, 360° Customer Profile
Audience Segmentation, Predictive Analytics, Campaign Recommendations, Campaign Tracking
Marketing Attribution, Conversion Tracking, Persona Profiling, Lead Scoring
Predictive Personalization, Persona-Based Targeting
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The government shutdown is perilous in many ways—but the immediate impacts are concentrated on some people, in some places, rather than others—at least for the moment. Which Americans are really paying attention? Also, we'll discuss how President Obama and House Speaker John Boehner each plan on "winning" the government shutdown, and we'll hear why there's so much money in bull riding.
What Does the Shutdown Crisis in DC Mean to the Rest of America?
The government shutdown is perilous in many ways—but the immediate impacts are concentrated on some people, in some places, rather than others--at least for the moment.
President Obama has now cancelled his upcoming trip to Asia to deal with the government shutdown. But Republicans continue to attack him for refusing to negotiate. House Speaker John Boehner seized on a quote attributed to a "senior administration official" to say the President's not really serious. Manu Raju is a senior Congressional Reporter for Politico.
The government shutdown is having ripple effects beyond the furloughing of hundreds of thousands of federal workers. The CDC has delayed this year's flu program; food-safety operations have been curtailed; so have some Head Start programs. Direct benefits to veterans and the disabled could be disrupted. If the debt ceiling's not lifted, the world's most powerful nation won't pay its bills. But, despite the endless debate in Washington, that's not what most people are talking about in New York City. In New York City and rural Kentucky, it's hard to find people directly feeling the loss of government services. Elsewhere, it's a different story.
Katherine Rushton, The Telegraph (@kerushton)
Dee Davis, Center for Rural Strategies (@dailyyonder)
Dante Chinni, American Communities Project (@Dchinni)
Chris Littleton, Littleton and Associates (@clittleton)
Michael Kazin, Georgetown University
The Professional Bull Riders Association was formed in 1994, and 10 or 15 founding fathers each contributed one thousand dollars. When they sold out 4 years ago, each one made more than 4 million.
Luke Snyder is a professional bull rider. He holds the record for the longest streak of competing in consecutive events: from 2001 'til 2010.
Luke Snyder, Pro Bull Rider (@LukeSnyder82)
Vote! What question should we answer next? Vote now to help Curious Coast choose what to investigate next. Is there something you're curious about? Let us know! What do you want to know more about in or… Read More
Inside the charming animal metropolis of Kozyndan You want to be friends with artists Kozyndan. Their work is relatable, subversive, funny and quintessentially LA. Kozy and Dan Kitchens are a married Highland Park-based couple. They create worlds… Read More


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Having a touchscreen on my laptop, I really miss Modern IE. I'm told by someone on the inside that a touch browser is coming.
It's ridiculous that neither the Xbox Music nor Windows Phone app can rip a CD that I ALREADY OWN and sync it to my phone. For that I need to use Windows Media Player on the Desktop. That's fine...
SuperSite for Windows
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2013 Rhinos' Charge
Come and join the best family club in the Bulldogs' district! Registrations now being taken for all Under 6s, 7s, 8s, 9s, 10s, 11s, 12s and 13s - don't miss out!
Registration to be held at:
The Rhinos' Clubhouse, Neptune Park, Edinnburgh Drive, Revesby Heights
on Thursday, 21 February, 2012
5.30 pm - 7.00 pm
Come and join the Rhinos' herd - be part of the family!
Comment Guidelines: The SportsTG Network is made up of players, families and passionate sports followers like you who have a strong opinion about sport. That's great - we want you to have your say and share your thoughts with the world. However, we have a few rules that you must follow to keep it fun for all. Please don't be rude, abusive, swear or vilify others. Apart from some pretty serious sport sanctions, we also can ban you and report you if things get out of hand. So play fair and have fun, and thanks for your contribution.


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Arizona Diamondbacks: Who Gets Named the No. 5 Starter for 2013?November 14, 2012
The Arizona Diamondbacks have a deep rotation heading into the 2013 season. The top four spots, in no particular order, are comprised by Ian Kennedy, Trevor Cahill, Daniel Hudson and Wade Miley. After those four, there are three young guys who will be given the shot at the No. 5 spot.
Lefties Patrick Corbin and Tyler Skaggs, along with Trevor Bauer, will likely be given every opportunity in Spring Training to win the job. Prior to Spring Training, though, it's useful to look at who has the upper hand.
Corbin likely has the early lead in the competition. He had the most major league experience of the three last season, appearing in 22 games and starting 17 of them. He put together a 6-8 record with an ERA of 4.54 over 107 innings pitched. Corbin was very hittable, however. Opposing batters hit .280 against him with 14 home runs. He'll have to learn to miss bats as he progresses.
Skaggs likely has the best stuff of the trio, though that didn't lead to success in his only stint in the majors last season. In six appearances—all of which were starts—Skaggs posted a 1-3 record with an inflated ERA of 5.83. That came over 29.1 innings. Skaggs put a ton of runners on in his brief stint, producing a WHIP of 1.47. Much of that comes from inexperience and the fact that he was adjusting his stuff to major league talent.
Bauer, assuming he's even on the team come Spring Training, will also be given a shot. He wasn't very good in his four starts last season, pitching to a 6.06 ERA in 16.1 innings. His WHIP of 1.65 has much to do with him allowing 11 earned runs (13 total) during his starts. Bauer looks like the third option at this point. His standing with the organization is in question, and he was the least impressive of the trio last season.
In my opinion, Corbin should be the No. 5 starter. He has the most experience and was the most effective in 2012. It may make the rotation a little lefty-heavy with Miley also being a left-hander, but that will work to neutralize the left-handed batters in the National League West.
All three pitchers have bright futures ahead of them. Corbin just seems to be the furthest along in his development at this point.


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Pawlenty's slight of music library still rankles, but Chatfield band plays on
CHATFIELD, MINN. — It's been more than four years since then-Gov. Tim Pawlenty took a shot at the sheet music lending library in this pleasant community of 2,700 in southern Minnesota.
And folks here haven't forgotten.
Pawlenty took a shot at the little music library in 2008 when he vetoed a bonding bill item that would have given the city $400,000 to move and upgrade the unique facility.
The governor singled out the library, from among the many items he struck from the bill, and criticized DFL Rep. Ken Tschumper, who ended up serving only one term.
"We're going to have a sheet music museum, Rep. Tschumper? Is that the priorities for the state of Minnesota?" Pawlenty said on his radio show that spring. Republicans jumped on the bandwagon, so to speak, and attacked DFLers for the music library funding plan in the next cycle of elections.
The memory stings.
"We remember. You betcha," said Carol Lenz, who plays clarinet in the Chatfield Brass Band.
"That was so mean," said another resident.
Despite the slight, the music library keeps lending and the town's brass band associated with it keeps performing, as if it doesn't even matter what they say in St. Paul.
(Pawlenty made another Chatfield-related criticism in 2010, when he pooh-poohed plans to fund the city's arts center. The Legislature wouldn't expand a sex offender treatment program or homeland security facility, he said, "Yet they have money for an art or pottery facility in Chatfield." It was quite a gaffe from the governor. There is no pottery facility; it's the Potter Auditorium in the city's arts center, which looks marvelous after much renovation.)
A return visit
I'd visited Chatfield in the spring of 2008, to write a story after Pawlenty's veto, and found some bustling activity in the sheet music library's modest cement building on the edge of town, next to the sewer plant. Thousands of pieces of music for flute, tuba, trombone and trumpet were being lent out each year to community bands around the country, and in Japan and Australia.
While visiting the town again last week, I wanted to see what's changed at the Chatfield Brass Band Music Lending Library and while there, I lucked out and got a chance to listen in on a bit of the brass band's practice session at the new elementary school, up on the hill.
Chatting briefly with several band members, I found some still feel Pawlenty distorted the issue to fit his political agenda. And by doing so, they said, a fine institution, doing the Lord's work, or at least Sousa's work, was badly maligned.
They still seem really perplexed at the negative rap aimed at the library, and by extension, the town.
Strike up the band
The town's first band was organized in 1883 but had languished until it was revived in 1969 by Jim Perkins. Now it's well-known in the region for its summer concerts in the town park, as well as holiday shows and special events.
The music library began in 1971 as an off-shoot of the community band. It was hard to find sheet music for the various instruments, so Perkins put out a call to high schools to send any unneeded music. Tons came in. They stored it first in Perkins' garage, then at city hall. He did get a $50,000 grant in 1978 to build the current 3,000-square-foot facility on land Perkins donated.
By 2008, it was too small and outdated. But Pawlenty's veto meant it wouldn't move into the old school on Main Street.
Moving forward without public funds
Instead, they've worked hard at the old location to keep the operation running on a shoestring. The library is open only three days a week and the three employees each work 21 hours a week.
They need a new furnace and electrical work, but rather than relying on state aid, they're working on seeking foundation grants, said Teresa Cerling, the library manager.
The library gets no ongoing public funding; they rely on donations and charge a handling fee when they lend music to bands.
We're not talking big money: For a full set of music, there's a $15 handling fee for members; $18 for non-members. (Annual membership cost run $15 to $100.)
Last year, the library had 700 orders, with 34,808 individual pieces of music checked out.
The previous year it was about 31,000 pieces.
One big improvement in recent years: the library's collection is now listed in the SELCO (Southeastern Libraries Cooperating) system. That's increased its reach and made it easier for bands to order music and for the library to lend.
Still, there are boxes and boxes of unsorted music sitting atop the old filing cabinets inside the drafty building, said Theresa Hayden, president of the band/library board.
And they aren't looking for new music, at this time.
"We can't accept anymore music; there's the problem with space and we don't have the money to pay staff to sort and catalog and file," she said. "There are at least 1,000 boxes that we need to go through, so accepting more isn't the right thing.
"Since that brouhaha with the governor, we did receive two large grants administered by SELCO to go through lots of boxes, enough to tell that we've made a dent."
The library got some unexpected help last spring: a retired band conductor and his wife spent two weeks helping catalog music during the day and rehearsing with the band at night.
"They worked like crazy," Cerling said. "We thought they'd want to do some sightseeing, but they just wanted to help up out."
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Comments (5)
Please forgive Tim
It can be really hard to be as hungry as Tim is, and pretend you're just a guy with a normal apetite.You can't be as greedy for significance as Tim and not slip up once in a while. Know that Tim cares, probably not as much as Norm Coleman cares, but he does care. He wants a good paying job like Norm's, he wants the respect of someone who has not bent over for the party. So forgive Tim. His party took him for a ride, gave everything to Norm that was rightfully his, and his pettiness just slipped a bit. Certianly there is a bit of compassion for one so wronged.
An issue in the current election
The Chatfield Library was raised again as an issue by the Republicans in their losing bid to control the Legislature. In Dist. 11-B against eventual winner Tim Faust the R's not once but twice in mailers cited "frivolous things like a brass band sheet museum." (Yes, you read right-band members must be bringing in their bed sheets to be displayed according to the R's.) But don't worry you're in good company Chatfield-they also referred to ducks as "birds in the Caribbean" and funds for the MN Zoo as "Gorilla cages."
And if you're moved to support the library...
... here's the link: http://chatfieldband.lib.mn.us/join-2/
Toss a few bucks their way.
Thanks for the great story about this labor of love in Chatfield. Also, thanks for the reminder about Gov. Pawlenty's crassness (and lack of proper research: Potter Auditorium = a pottery?!)-- his snide remarks at the time seemed particularly mean, even for a guy who sneered reflexively at anything involving art or beauty.
A treasure
I'm pretty sure I've played from some of the Chatfield Brass Band's sheet music. Having this resource in a tiny town in Minnesota is a treasure, not something to be sneered at.
And in the end, Mr. Pawlenty left the state to do bigger, better things with money, while leaving the state in a hole. I'm not sure how much hope one can hold out for our economy if Tim Pawlenty is the voice Wall Street in Washington.
Library And Keeping Book
Book Keeping and library both are related to each other. In this type of concerns people might think to gather books of different types to improve their knowledge. Many people are there who like to write books about the people to whom they love very much. So many book stores are there to help the people to get so many books in one place and can develop their information power too. So many wedding books are also available to know about the different marriage types. I know it can be hard to see past the day itself and you're just doing whatever you can to craft a beautiful event, but there is often so much waste that goes into a wedding.
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D LA 4 years ago
Drew Doughty thinks the Kings can do a better job at getting to rebounds on St. Louis goaltender Brian Elliott.
"He's a good goalie. He makes the first save pretty much every time," Doughty said. "I think the one area struggles is he lets some rebounds, and I think we haven't been doing a good enough job of pouncing on those rebounds and putting in the second effort." There have been just seven goals scored in three games this series, so both teams are looking for more offense.


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A snare drum sample.
good sound! Thx for sharing =D
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It's like sparkling wine, but more conveninent. De Bortoli has launched what it claims is Australia's first sparkling wine with a screw-top closure.
The wine company has teamed up with Guala Closures to produce Viiva, a high-pressure screw-cap that is not only easy to open, but is resealable, retaining carbonation. This is particularly useful, according to the company, when it comes to by-the-glass sales of sparkling wine in the on-trade. The bottle's even been made taller so that it sits at the same height as other sparkling bottles.
De Bortoli is initially introducing the closure for its Willowglen Sparkling Brut NV, with stocks currently on the way to the UK.


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Stock Report: J & J Snack Foods Corp. (JJSF) Report Updated: Sep 16, 2017 | Print This Page
Report Features:
Stock Analysis
|Fundamental Grade:
|Sales Growth:
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|Earnings Surprises:
|Analyst Earnings Revisions:
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Rating: Monthly View
Rating: Weekly View
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J & J Snack Foods Corp.© quotemedia
Company Profile
J&J Snack Foods Corp. manufactures, markets, and distributes various nutritional snack foods and beverages to the food service and retail supermarket industries in the United States, Mexico, and Canada. The company operates through three segments: Food Service, Retail Supermarkets, and Frozen Beverages. It offers soft pretzels under the SUPERPRETZEL, PRETZEL FILLERS, PRETZELFILS, GOURMET TWISTS, MR. TWISTER, SOFT PRETZEL BITES, SOFTSTIX, SOFT PRETZEL BUNS, TEXAS TWIST, BAVARIAN BAKERY, SUPERPRETZEL BAVARIAN, NEW YORK PRETZEL, KIM & SCOTTS GOURMET PRETZELS, and SERIOUSLY TWISTED! brand names. The company also provides frozen juice treats and desserts primarily under the LUIGIS, WHOLE FRUIT, PHILLY SWIRL, ICEE, and MINUTE MAID brands; churros under the TIO PEPES, CALIFORNIA CHURROS, and OREO brand names; and dough enrobed handheld products under the PATIO, SUPREME STUFFERS, and SWEET STUFFERS brands. In addition, it offers bakery products, such as biscuits, fig and fruit bars, cookies, breads, rolls, crumbs, muffins, and donuts under the MRS. GOODCOOKIE, READI-BAKE, COUNTRY HOME, MARY BS, and DADDY RAYS brand names; and frozen beverages primarily under the ICEE, SLUSH PUPPIE, and PARROT ICE names. Further, the company provides soft drinks and funnel cakes under the FUNNEL CAKE FACTORY brand name. It serves snack bars and food stands in chains; department and mass merchandising stores; malls and shopping centers; fast food outlets; casual dining restaurants; stadiums and sports arenas; leisure and theme parks; convenience stores; movie theatres; warehouse club stores; schools, colleges, and other institutions; and independent retailers. The company sells its products through a network of food brokers and independent sales distributors; and direct sales force. J&J Snack Foods Corp. was founded in 1971 and is headquartered in Pennsauken, New Jersey.
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Barron's Buzz: The Top Hedge Funds Are ...5/16/2014 4:14PM
Jack Hough previews Barron's report on top hedge funds and explains why Larry Robbins' Glenview Offshore Opportunity landed at the top of the list. Photo: Roger Hagadone for Barron's. Photo: Roger Hagadone for Barron's.
This transcript has been automatically generated and may not be 100% accurate.
I ... I ... I ... the twenty eight at the new issue of Barron's magazine ... and Amy now as Barron's senior editor Jack how ... wonderful to see you thanks for being here ... what a cover story in the top one hundred hedge funds a highly anticipated list ... as we know hedge funds can be a little opaque so I did this come up with this little we have a variety of sources are really just a few months of reporting to track down the numbers on their five copies made for things or after fees and get a good ranking ... anyone can federal which neutral funds are doing well and as you said ... it's not as easy with hedge funds we have the top one hundred performers ... and what we find is that in recent years we've done this at near the top a list and it has front of him playing in ... mortgage backed securities in things like that now it's all about stocks and was near the top of a list have made ... serve clustered bets on stocks that no really well ... and in the top Alyssa's is Larry Robbins of Glenview Offshore opportunity Fund ... he's made a bet on Obamacare figured out which stocks and that benefit from ObamaCare and a construct that so ... that a cluster of stories read it to rankings ... the custom profiles of a fund managers and also ... some stocks that activist investors are targeting now have there been any upset so this last any major movement up to buy up or down well we have seen that those the those guys are making it ... the Tesla Mortgage Backed another asset backed securities that have dipped down and ... some awesome a great stock that's risen to the tops of their husbands to turn over what ... a lot of people a whale LSAP semi fell ... into a data so-called Warren ... Buffett that age that is selling off all of his Chinese property if the Fed announced ... that he act any housing bubble has finally officially bars well I think it's in the process that this is Lee cushioning the has sold more proper there with a cover story in Barron's that last year ... the warning about the conditions in the China property market we have seen prices fall this year in and a lot of markets there and China ... aam and so we take the opportunity to look a what's going on in the market now it's not so much liquidity event is just ... sort of the structural oversupply there and if you have investments in China not just wheels investors stocks were you thinking about maybe ... bargain hunting in China right now I did and this is the story for you to figure out where we are in the property cycle there and what is it seeing what happened in the long term will there be another rebound to the way our bubble here rebounded because I think you have to be really patient there some ... the the did the government are trying to get some ... for sample banks into it with you don't get more mortgages people banks are really keen you do that right now because the prophets are free to sell the products to be sewn make better money for them so ... it's kind of a ... It's tough and Chiron attitude to spur the set to revive its property markets in the dust ... in a bit more downside than you'd be careful here I finally say that this summer might be the right time to start alternating it's based on the chamber's ... really dear to my are forget about the ... hedge the fish Chinese property ... of beef prices are up nine percent this year don't work is up five percent ... on we hadn't arrived in grain prices and some farmers had to reduce aside the birds is also known as swine virus awesome supply issues and ... those could take a few years to play out sweet communities ... elevated prices that the parties are recommends that have a BBQ your house ... party invited to a neighbors are very a ... pic taken at the time to ... being really friendly with your neighbors Dhaka thinking so much on that


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Inbreeding depression and heterosis in sweet corn varieties Manis Madu and Bakti-1
Saleh, Ghizan and Yusop, Mohd Rafii and Yap, Thoo Chai (1993) Inbreeding depression and heterosis in sweet corn varieties Manis Madu and Bakti-1. Pertanika Journal of Tropical Agricultural Science, 16 (3). pp. 209-214. ISSN 0126-6128
Official URL: http://www.pertanika.upm.edu.my/Pertanika%20PAPERS...
S1 and full-sib progeny families developed from selfing and crossing between Manis Madu and Bakti-1 sweet corn varieties were evaluated to estimate inbreeding depression and heterosis in the populations. Selfing has caused a significant decrease in the measurements of all characters taken in both selfed populations, except for days to tasseling which has shown an increase. Midparent heterosis estimates for the characters evaluated ranged from -2.83% to 22.34% for the Manis Madu X Bakti-1 cross progeny population (MMB1), and from -2.65% to 16.57% in the Bakti-1 X Manis Madu cross progeny population (B1MM). The two varieties revealed good potential to be used as parents for crosses between improved populations or inbred lines developed from them.
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NRA members compared to those of TFL?
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April 21, 2013, 10:18 AM
Alabama Shooter
Senior Member
Join Date: December 20, 2012
Location: Sweet Home
Posts: 886
The point is, on average, TFL membership is far more educated on the issue than is the general public. That's why we poll differently. Plus, we are by definition, the "enthusiasts", which also lends to different polling.
That was partly my point. There is a lot of preaching to the choir here and shouting down those who hold a slightly different view point. It goes beyond education and more into a fervent belief. That is the same reason the anti's lose so often. The only chance they have is when they can generate enough of an emotional response to cause people to shut down their brains.
We don't need to become that.
Once people lose their objectivity they have lost their cause and don't even realize it. Facts, data, proper analysis of such is the proper way of doing things.
Tomorrow is the most important thing in life. Comes into us at midnight very clean. It's perfect when it arrives and it puts itself in our hands. It hopes we've learned something from yesterday.
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C For You Cologne
By Cindy C. for Men
C For You Cologne by Cindy C., Coat yourself with the rugged, masculine scent of c for you, a men's fragrance introduced in 2003 by the design house of cindy c . Its citrusy mandarin orange and petitgrain top notes awaken your senses for an invigorating blast of nutmeg, cedar, and sandalwood notes. With long-lasting earthy base notes, this fragrance stays with you throughout the day. Bold and charismatic, this magnetic scent captures the essence and the beauty of the great outdoors. All products are original, authentic name brands. We do not sell knockoffs or imitations. ... Read More
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Athenian Academy looks to move to Clearwater
Athenian Academy, a long-time charter school in Pinellas County, plans to move to a new facility in Clearwater for the 2015/16 school year.
The charter, which focuses on language immersion in Greek and Spanish, would move from its current location in Dunedin in two years, if the change is approved Tuesday by the Pinellas School Board. Charter officials also have requested a slight boost in enrollment for the 2015/16 school year, from 320 students to 400 students.
The charter school would like to move from 2817 St. Marks Drive in Dunedin to 2289 N. Hercules Ave. in Clearwater. The new location hasn't been completed yet so approval of the move would be contingent upon a satisfactory inspection at a later date.
Athenian Academy, which opened in 2000, serves students in kindergarten to eighth grade. It boasts being the first Greek immersion charter in the country. Students learn both Spanish and Greek daily, according to the school's website.
Charters are publicly funded but privately run.


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On-pack label manufacturer Yerecic Label Co. is one of only 15 employers nationwide to receive the U.S. Department of Defense's highest honor for demonstrating exceptional support for National Guard and Reserve employees in 2010.
The Secretary of Defense Employer Support of Freedom Award went to the New Kensington, Pa., company in late 2011, according to a news release from the Defense Department. Vice President Joe Biden and Defense Secretary Robert Gates personally met with Yerecic's president, Art Yerecic, to thank him for setting a good example for other employers.
The Yerecic firm was one of almost 2,500 employers nominated by Guard and Reserve personnel and their families to receive the honor.
At the time of the award, Yerecic had three employees actively serving in the U.S. military. Support efforts by the company included sending care packages and providing support to the employees' families.
The annual employer award was instituted in 1996 at the suggestion of the Employer Support of the Guard and Reserve organization within the Defense Department to recognize exceptional support from the nation's employers.
Yerecic Label Co. has been a maker of on-pack labels for fresh produce products and a variety of other goods for more than 40 years.


text:
Stock Report: () Report Updated: Sep 16, 2017 | Print This Page
Report Features:
Stock Analysis
|Fundamental Grade:
|Sales Growth:
|Operating Margin Growth:
|Earnings Growth:
|Earnings Momentum:
|Earnings Surprises:
|Analyst Earnings Revisions:
|Cash Flow:
|Return on Equity:
|Quantitative Grade:
|Total Grade:
Rating: Monthly View
Rating: Weekly View
|This Week:
|no change
|Last Week:
|no change
|Two Weeks Ago:
|no change
© quotemedia
Company Profile
Apollo Education Group, Inc. provides private education services in the United States and internationally. The company offers online and on-campus undergraduate, graduate, certificate, and non-degree educational programs and services to working learners. It operates through University of Phoenix, Apollo Global, and Other segments. The University of Phoenix segment provides undergraduate and graduate degrees in the areas of business, education, and nursing; and a range of non-degree education programs for lifelong learners and students. The Apollo Global segment offers accounting and law programs; recognized and government approved non-degree certificates, diplomas, and other qualifications in a range of disciplines and industries, including health and wellness, business management and services, and community services; and graduate and undergraduate degrees, high school, and executive education in various program areas and disciplines, including medical, dental, and communications. It also provides postsecondary education services through distance education and at campuses; undergraduate and graduate degrees primarily in the areas of business, education, and technology; and degree and non-degree programs focused on arts and technology. The Other segment offers non-degree information technology bootcamp programs, as well as skills training services; and online financial services education programs, including graduate degrees in certificate programs, financial industry regulatory authority securities license training, and continuing education courses. It also provides undergraduate and graduate degrees in business, information technology, and behavioral science areas; and various programs for employers. The company was formerly known as Apollo Group, Inc. and changed its name to Apollo Education Group, Inc. in November 2013. Apollo Education Group, Inc. was founded in 1973 and is headquartered in Phoenix, Arizona.
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text:
Today: Rowdies vs. Miami
When: 4 p.m.
Where: Steinbrenner Field, Tampa
Broadcasts: Live video feed on fctampabay.com; radio on 1010-AM
Records: Tampa Bay 5-7-5, 20 points, fifth in USL Conference; Miami 2-6-9, 15 points, sixth in NASL Conference
Tickets: Game day prices range from $14 to $32 and can be bought at fctampabay.com or by calling toll-free 1-888-757-6934.
Promotion: Fans can buy family four-packs, which start at $52, that include four tickets, four hot dogs and four small sodas.
Past meetings: This is the third of four regular-season meetings between the in-state rivals, who play for the Coastal Cup. The teams drew two previous league matches. On July 4, Tampa Bay couldn't hold on to the lead after Jeremy Christie scored in the 82nd minute and tied Miami 1-1 at Steinbrenner Field. On May 1, the teams tied 1-1 in Fort Lauderdale. In a non-league second-round U.S. Open Cup match June 22, Miami beat Tampa Bay 2-1 on Abe Thompson's penalty kick in the 98th minute.
Offensive struggles: The Rowdies haven't scored since Christie's goal against Miami, a span of more than three games and 278 game minutes. Overall, Tampa Bay has scored just two goals in its past eight league games.
At home: This is the third of a seasonlong four-game homestand for Tampa Bay. The Rowdies are 2-2-3 at home.
The slump: The Rowdies are 0-6-2 in their past eight league games, their last win coming May 29, 2-1 over Puerto Rico. To say the Rowdies have been streaky would be an understatement. At the time they were unbeaten in seven games and were 5-1-3 and in second place in the USL Conference with 18 points. Now, they are tied with Puerto Rico for the eighth and final playoff spot in the league.
New additions: Midfielder Jonny Steele played 90 minutes in Tampa Bay's 1-0 loss to Rochester. Steele and midfielder Ricardo Sanchez, who could be available today, were acquired last week from Vancouver.
Weird fact of the day: All three goals against Miami this season have come in the 82nd minute.
Injury report: Forward Aaron Wheeler (shin stress fracture), midfielder Erik Ustruck (broken leg), Scott Buete (hernia) are out. Defender Gordon Kljestan (knee) did not practice Saturday and is doubtful. Yendry Diaz (ankle) practiced and could return today after missing Thursday's game.
Eduardo A. Encina, Times staff writer


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Not the typical roadside memorial in National City
"Yeah, eight years. We come out every year on the night it happened."
Edward Magdaleno noon, Sept. 21
File under: sympathy for the cameraman. Filming butterflies is hard, dude. Even slowed down, the action can be a little hard to follow, so here are a few stills with accompanying explanations.
Our hero, barely visible as he clings to an orange flower, is content in his bachelorhood, happily minding his own business.
But of course, his tranquility is shattered when a fetching female flits by.
Before he can think what he is doing, he has left his happy bachelor's perch behind and is off in mad pursuit.
But love can be cruel. In vain, he searches for the vision he once glimpsed, wandering alone through a barren land festooned with barricades that block his progress.
Then, just as he is about to lose hope - she appears!
But what's this? Our hero is not alone in his pursuit! A rival enters the scene!
What happens next? I don't know! They went into this crazy love spiral and then disappeared! W.C. Fields was right - never work with children or animals!


text:
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Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell–derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell–derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.
Parkinson disease (PD) is a neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons, with subsequent reductions in striatal dopamine levels. While initial pharmacological treatment with L-dihydroxyphenylalanin (L-DOPA) can attenuate symptoms, the efficacy of this treatment gradually decreases over time. The development of motor complications then requires additional treatments, including deep brain stimulation and fetal DA neuron transplantation (1–3). Both studies of animal models and clinical investigations have shown that transplantation of fetal DA neurons can produce symptomatic relief (4–8). The technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissue, however, have limited the application of this therapy.
ES cells are self-renewing, pluripotent cells derived from the inner cell mass of the preimplantation blastocyst. These cells have many of the characteristics required of a cell source for cell-replacement therapy, including proliferation and differentiation capacities (9). We previously discovered that a strong neuralizing activity, which we called stromal cell–derived inducing activity (SDIA), is present on the surface of stromal cells. In the absence of exogenous bone morphogenic protein–4, mouse ES cells differentiate efficiently into neural precursors and neurons when cultured for 1 week on SDIA-expressing mouse stromal cells (PA6 cells) (10). Recently, SDIA induction has also been applied to primate ES cells, which generated large numbers of neural precursors and postmitotic neurons when cultured on PA6 cells for two weeks (11). The SDIA method is both technically simple and efficient, producing significant numbers of midbrain DA neurons (10, 11).
Self-renewing, multipotent neural progenitors can be cultured as neurospheres (12). In this study, we generated neural progenitors from monkey ES cells, then expanded them as neurospheres, which contained progenitors of DA neurons. In addition, we analyzed the effect of FGF20, a novel member of the FGF family of growth factors that is expressed exclusively in the substantia nigra of the brain and is reported to have a protective effect on DA neurons (13). We observed increased DA neuron induction following treatment with FGF20. Furthermore, we transplanted neurons generated by this method into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated (MPTP-treated) cynomolgus monkeys, a primate model for PD. We found that transplanted cells were able to function as DA neurons and could diminish Parkinsonian symptoms. This is the first report, to our knowledge, demonstrating the efficacy of transplantation therapy using ES cell–derived DA neurons in an experimental primate model of PD.
To enrich graftable neural progenitors, we first cultured monkey ES cells on PA6 stromal feeder cells, then detached the cells from the feeders for expansion as neurospheres. ES cells began to differentiate on PA6 cells, with cells immunoreactive for neural progenitor markers, such as N cell adhesion molecule (NCAM) and Musashi-1 (11, 14), emerging within three days. ES cells proliferated and differentiated on the feeder layer by forming colonies, and cells positive for neural markers increased in number until 2 weeks into the culture period. The percentages of the colonies including at least 1 NCAM- or Musashi-1–positive cell reached approximately 100% at 2 weeks of culture (NCAM = 90.4% ± 7.5%, Musashi-1 = 97.5% ± 4.6%, n = 100 from 3 independent cultures; Figure Figure1A).1A). At this time point, 78.3% ± 7.5% of the total cells were immunoreactive for NCAM, 75.0% ± 15.4% for Musashi-1, and 72.3% ± 9.1% for another neural progenitor marker, Nestin (14). These results indicate that the majority of the ES cells were committed to the neural lineage by day 14 of culture on feeder layers.
To further enrich neural progenitors, we detached the ES cells from the feeder layer on day 14, then continued to culture the cells on noncoated dishes in serum-free medium containing FGF2, EGF, and leukemia inhibitory factor (LIF). During the next few days, the cells formed spheres morphologically resembling those formed by neural progenitor cells (Figure (Figure1B).1B). These spheres were positively stained with antibodies specific for neural progenitor cell markers NCAM, Musashi-1, and Nestin (Figure (Figure1,1, C–E). To determine the potential of these cells to differentiate, we expanded the cells as spheres for 7 days, then induced differentiation by culturing the cells on poly-l-ornithine and laminin–coated (OL-coated) slides for 7 days. We removed the mitogens FGF2, EGF, and LIF from the medium, instead adding neurotrophic factors such as brain-derived neurotrophic factor and neurotrophin-3. Immunofluorescence analysis revealed that the cells differentiated into mature neural cells expressing the neuronal markers TuJ1 (52.8% ± 16.0% of DAPI) and Map2ab (38.3% ± 7.5% of DAPI), the astroglial marker glial fibrillary acidic protein (GFAP) (28.6% ± 17.6% of DAPI), and the oligodendroglial marker galactocerebroside C (GalC) (0.6% ± 0.4% of DAPI) (Figure (Figure2,2, A–D and I). Further analyses demonstrated that these neurons derived from ES cells were immunoreactive for γ-amino butyric acid (GABA) (28.6% ± 10.7% of TuJ1), glutamate (14.3% ± 5.3% of TuJ1), choline acetyltransferase (ChAT) (0.7% ± 0.3% of TuJ1), and serotonin (3.3% ± 1.7% of TuJ1) (Figure (Figure2,2, E–H and J). These results suggest that ES cell–derived spheres contain neural progenitor cells.
Effective treatment of PD requires substantial quantities of DA neurons. The percentage of tyrosine hydroxylase–positive (TH-positive) cells derived from neurospheres was only 5.4% ± 1.8% of the TuJ1-positive cells (Figure (Figure2J).2J). To increase the percentage of these cells, we examined the effects of various combinations of growth factors on neurosphere culture. The percentage of TH-positive neurons out of total Tuj1-positive cells increased to 24% in the presence of FGF2 and FGF20 (Figure (Figure3,3, A–E). Neither FGF2 nor FGF20 alone caused an increase in the number of TH-positive neurons. In the presence of FGF2, however, FGF20 increased the rate of TH differentiation in a dose-dependent manner (1 pg/ml = 3.8% ± 1.8% of TuJ1, 10 pg/ml = 8.8% ± 4.8% of TuJ1, 1 ng/ml = 24.3% ± 9.8% of TuJ1).
RT-PCR analyses showed that the cells derived from SDIA-induced neurospheres expressed mesencephalic DA neuron markers such as Pax2, Ptx3, Nurr1, Lmx1b, and TH (15, 16). The expression of these markers was more abundant in FGF2- and FGF20-treated cells than in FGF2- and EGF-treated cells (Figure (Figure3F).3F). Furthermore, FGF2- and FGF20-treated cells released 55.6 ± 19.9 pmol per 106 cells of dopamine in response to high K+ depolarizing stimuli as assayed by HPLC (n = 5; Figure Figure3G).3G). These results indicate that monkey ES spheres treated both with SDIA and with FGF2 and FGF20 generate a significant number of functional DA neurons in vitro.
To determine if the isolated TH-positive neurons function as DA neurons in vivo, ES cell–derived neurospheres were grafted into the putamen of the monkeys with MPTP-induced PD. In this primate model of PD, we administered MPTP intravenously to Macaca fascicularis (cynomolgus monkeys), hereafter M. fascicularis, and evaluated their behavior by scoring for neurological symptoms, such as motility and tremor (Table (Table1).1). Only the animals that exhibited stable deterioration for periods longer than 12 weeks were used for transplantation. ES cell–derived neurospheres used for grafting were prepared by treating with SDIA for 14 days and, subsequently, with FGF2 and FGF20 for 7 days, as described above. Using MRI images obtained for each monkey, we determined the necessary coordinates to stereotactically transplant the cells (300,000–600,000 cells per side) into the bilateral putamen. After injection, we continuously analyzed the behavior of postoperative monkeys by assessing neurological scores. We observed a slight recovery in the behavioral symptoms even in the sham-operated animals. At 10 weeks after transplantation, however, the mean scores of ES cell–transplanted monkeys significantly improved over the levels observed in sham-operated ones (Figure (Figure4A,4A, n = 6 and 4, respectively). In our evaluation of symptoms after transplantation, posture recovery was the most prominent improvement seen in ES cell–transplanted monkeys, with significant improvements in motility also observed. There were, however, no significant changes in head-checking movement. Consciousness was not disturbed in any preoperative animals, and no deterioration was observed postoperatively. In addition, none of the treated animals developed dyskinesia. Positron emission tomography (PET) at 14 weeks after transplantation revealed increases in 18F-fluorodopa uptake at the putamen of the ES cell–transplanted animals (Figure (Figure4,4, B and C).
After the PET study, animals were sacrificed and subjected to immunohistochemical analysis. Grafted cells, which were labeled by BrdU treatment during sphere culture, were detected in the putamen of ES cell–transplanted monkeys (7,996 ± 3,300 cells per side; Figure Figure5,5, A–C). DA neurons were detected by TH (Figure (Figure5,5, B, C, E, and F; ref. 11) or dopamine transporter (DAT) (Figure (Figure5,5, H and I; ref. 17) staining. An average of 2130 ± 645 TH-positive cells per side survived. Double labeling immunofluorescence microscopy revealed that 65.5% ± 4.3% and 50.3% ± 6.1% of the TH- and DAT-positive cells were immunoreactive for BrdU, respectively. Thus, the estimated number of TH/BrdU–double-positive cells was 1395 per side, 17.4% of the total of BrdU-positive cells. In contrast, in sham-operated monkeys, only a few TH-positive cells and scattered fibers were detectable in the putamen. No tumor formation or Ki-67–inducing reactivity was observed. As for other neuronal phenotypes, GABA-positive cells were detected in the graft at a slightly higher frequency than TH-positive cells, while few serotonin-positive cells were present (data not shown).
Following initial work by Thomson et al. reporting a method for establishing primate ES cells (18), Suemori et al. (19) recently devised a similar scheme for generating M. fascicularis ES cells. Lee et al. recently reported a 5-step method to induce DA neurons from ES cells through the induction of neural progenitor cells from embryoid bodies (20). Transplantation of induced DA neurons derived from mouse ES cells improves the neurological symptoms of rats with a Parkinson-like syndrome induced by treatment with 6-hydroxydopamine (6-OHDA) (21). We have also reported a method of inducing DA neurons based on SDIA resulting from coculture of ES cells on a PA6 stromal feeder layer. By the use of this method, mouse ES cells are diverted to a neuronal fate with TH-positive DA neurons composing 30% of total TuJ1-positive neurons (10). Furthermore, this method produced similar results with M. fascicularis ES cells (11). In this study, we produced a highly enriched population of proliferating neural progenitors derived from SDIA-treated monkey ES cells. Furthermore, treatment of these cells with a combination of FGF2 and FGF20 induced the generation of a large population of DA neurons from ES cell–derived neural progenitors. Using MPTP-treated monkeys as a primate model for PD, we analyzed the effect of administration of DA neurons generated from monkey ES cells in vivo. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons, attenuating the MPTP-induced symptoms.
In our preparation of graftable cells, we made two major modifications to our previously published protocol (10, 22). First, we induced the formation of neurospheres, expecting an enrichment of neuronal progenitor cells. We detached the ES cells from the feeder layer on day 14 and cultured them on noncoated dishes. Under these conditions, the cells formed floating spheres composed of neural precursor cells. Since the serum-free culture medium was suitable for neural cell growth, any contaminating nonneural and PA6 cells were likely eliminated as a result of a low proliferation rate and/or adherence to the bottom of the dish. Our previous report (22) demonstrated that, when grafted into the brain, fully maturated TH-positive neurons survived less efficiently than DA neuron progenitors induced by SDIA, probably due to their susceptibility to mechanical stress. This result is consistent with the fact that transplantation of mesencephalon tissues from early gestation stage embryos undergoing neurogenesis of DA neurons resulted in good survival of TH-positive cells (1300–18,000) and increased dopamine concentrations in the caudate nucleus of MPTP-treated monkeys, whereas these effects are not observed when mesencephalon tissues from later stages are transplanted (23). Thus, ES cell–derived neuronal progenitors competent to generate DA neurons appear to be more suitable for transplantation than DA neurons matured in vitro.
Another important modification to our previous protocol is the use of FGF2 and FGF20 treatment to enhance the generation of DA neurons. The percentage of TH-positive neurons generated from neurospheres was 5.4 ± 1.8% of TuJ1, much lower than that generated by culturing on PA6 cells (35%; ref. 10). This discrepancy most likely results from mechanical damage caused by detaching the cells or inappropriate culture conditions for spheres. To increase the number of DA neurons differentiated from neurospheres, we examined the effects of various additional growth factors. Ascorbic acid and Sonic hedgehog, which were used in the 5-step method (20), did not increase the proportion of TH-positive cells (data not shown). In contrast, FGF20 treatment in combination with FGF2 was able to efficiently increase the proportion of TH-positive cells. FGF20 is a secreted protein that is preferentially expressed in the substantia nigra pars compacta of the rat brain (13). The expression profile of FGF20 is quite different from that of other FGF family members, which suggests that FGF20 plays a unique role in the brain. Furthermore, recombinant FGF20 enhances the survival of primary DA neurons (13). FGF receptor–1c, the receptor through which FGF20 activates the mitogen-activated protein kinase pathway, is also preferentially expressed in the substantia nigra pars compacta (24). Our results raise the possibility that FGF20 in combination with FGF2 may support the survival or promote the proliferation of progenitors of DA neurons, resulting in the enrichment of DA progenitor cells in spheres. The mechanism by which this combined stimulation of FGF2 and FGF20 facilitates the production of DA neurons remains to be clarified.
FGF2 and EGF are reported to play different roles in the differentiation of neural precursors. Although FGF2 and EGF promote proliferation of neural precursor cells, the former promotes neuronal differentiation, while the latter induces glial differentiation (12, 25, 26). They also have different effects on the differentiation of embryoid bodies derived from human ES cells (27). In the present study using neural precursors derived from primate ES cells, FGF2 increased differentiation of ES cells into DA neurons, while EGF suppressed this process even in the presence of FGF2 and FGF20. It is possible that EGF interferes with the differentiation of SDIA-treated spheres into DA neurons directly or indirectly by promoting astroglial induction. Alternatively, EGF may stimulate proliferation or differentiation of a different cell population than that stimulated by FGF2. The differential effects of growth factors present an intriguing topic for future investigation.
In primate studies, functional neuroimaging is a useful tool for in vivo assessment of differentiation, survival, and functional integration of grafted cells. PET imaging of presynaptic targeting reagents such as fluoro-dopa, fluoro-metatyrosine, or 2β-carbomethoxy-3β-4-fluorophenyltropane (CFT) determines whether cells implanted in vivo have the molecular machinery necessary for dopamine synthesis and/or storage (28–30). In this study, we examined the uptake of fluoro-dopa at 14 weeks after transplantation. The significant increase in the mean Ki value in the putamen of ES cell–transplanted animals indicated that the grafted cells functioned as DA neurons. A postmortem examination of the ES cell–transplanted monkey in Figure Figure4C4C revealed that more TH-positive cells survived within the right putamen. This finding reflects the correlation of the PET results with the survival of DA neurons. For future studies, detailed analyses using additional tracers, including postsynaptic markers such as fluoro-raclopride, should allow for further understanding of the functional aspects of grafted cells. In this study, we detected significant differences in the mean Ki values from the entire putamen between the ES cell–transplanted monkeys and the sham-treated control monkeys 3 months after surgery. Widner et al. (8) reported that striatal uptake of fluoro-dopa was unchanged 5 to 6 months postoperatively, but increased markedly at 12 to 13 and 22 to 24 months in patients who received fetal mesencephalic grafts. Freed et al. (4) reported an improved Ki value from the entire putamen 6 months after transplantation. Given the lengthier monitoring periods in these reports, our evaluation of mean Ki values at 3 months may still be premature; further PET studies at later time points may result in even greater changes.
MPTP is a neurotoxin that causes selective destruction of DA neurons in the substantia nigra pars compacta, inducing PD-like symptoms in primates (31, 32). Following repetitive intravenous injections of MPTP (approximately 17 mg in total per animal), monkeys stably exhibited PD-like symptoms more than 12 weeks before transplantation surgery. With a blind evaluation based on neurological scores, we detected significant behavioral improvements in the ES cell–transplanted monkeys 10 weeks after transplantation. Recently, 2 double-blind placebo-controlled clinical trials of fetal nigral transplantation (4, 7) demonstrated that younger patients and patients with mild symptoms improved after treatment, with behavioral recovery first observed in the 3 to 4 month period following surgery. In this study, posture and motility were the symptoms showing the most marked improvement. These results are comparable with clinical reports (4, 33) demonstrating improvements in rigidity and hypokinesia.
PET and immunofluorescence studies demonstrated that a substantial number of the grafted cells survived in the putamen to function as DA neurons. We transplanted 300,000–600,000 cells into each side of the brain in each monkey. The number of surviving cells detected by BrdU staining was approximately 8,000 per side. Thus, the survival rate of the grafted cells was 1.3% to 2.7%, although the actual value could be higher (discussed below). Through TH staining, the number of surviving DA neurons was shown to be approximately 4,300 per brain. In normal brains, there are no DA neuron cell bodies in the striatum, only fibers. Thus, these TH-positive cells were considered to be derived from the grafted ES cells. While the grafted cells were labeled by BrdU prior to transplantation, only 65% of these TH-positive cells were immunoreactive for BrdU. This may have resulted from incomplete labeling of the input cells; as the cells were treated with BrdU while being cultured as spheres, the labeling rate was not 100%, but 68.8%. In addition, grafted cells might proliferate in vivo, reducing the concentration of BrdU in the cells. Furthermore, intrinsic striatal TH-positive neurons may be recruited, as reported previously (34), which may explain the observation of a few TH-positive neurons even in control monkeys.
According to an earlier clinical report, the number of TH-positive cells in the postmortem brain of a PD patient was approximately 200,000 (35). In 2 recent double-blind trials, however, the number of surviving TH-positive cells was determined to be 50,000–240,000 (4, 7). Given that the volume of the monkey putamen is 10% of that of the human putamen (36, 37), it is likely that the required number of TH-positive cells in the monkey (M. fascicularis) brain is 5,000–24,000. The results of this study remain in keeping with observations made in human patients, suggesting that ES cells are a promising candidate for a donor source for cell transplantation treatment of PD. It should be noted, however, that the MPTP-treated monkey is a model of acute selective nigral destruction whereas human PD patients also experience progressive deterioration and pathological changes of other neural systems (15, 38, 39).
Although the results presented here encourage the development of strategies involving ES cell–derived neurons for treatment of neurological diseases, further studies will be needed to address the long-term efficacy and safety of using these cells. For instance, the low survival rate of the grafted cells or neurons is comparable to that noted in previous reports (40). To increase the number of viable DA neurons produced by grafts in vivo, we used DA neuron progenitors in the present study. Multiple-target grafting (41) is also a strategy that should be considered. Notably, we observed a number of GABA-positive cells in the graft, suggesting that other types of neurons and/or glial cells in the graft may contribute to both the differentiation and function of transplanted DA neurons (42). However, the optimal cellular composition of the graft remains to be determined. In addition, while previous studies with rodents have demonstrated that tumor formation can be associated with ES cell grafts (43–45), we did not observe tumor formation or Ki67-positive cells within the first 3 months after transplantation. In the future, however, it will be important to examine late tumor formation as well as the possible long-term effects of ES cell grafts on motor behavior. It will also be necessary to use non–TH-positive cells in control grafts to exclude the possibility that the effects of this treatment are mediated by non-DA cells.
Finally, we would like to emphasize that our system (MRI, surgery, PET, etc.) is applicable to humans. Previous work has shown that monkey ES cells have characteristics similar to those of human ES cells (18, 19, 46). In addition, it was recently demonstrated that neural precursors induced from human ES cells were able to survive in rodent brains (47, 48). The SDIA method is applicable to human ES cells, allowing for enrichment of DA progenitors (unpublished data). These results suggest that transplantation using ES cells as a clinical therapy for PD is approaching the point of technical feasibility. Two recent double-blind, sham surgery–controlled trials of embryonic mesencephalic transplants for the treatment of PD, however, showed only modest improvement (4, 7), suggesting the potential limits of cell transplantation. Many basic issues, especially regarding stem cell therapy, remain to be resolved (38). Before the clinical application of human ES cell transplantation can be attempted, extensive studies assessing the safety and efficacy of ES cell transplantation in monkey models will be necessary.
Cynomolgus monkey ES cell lines were established and their pluripotency confirmed by teratoma formation in mice with severe combined immunodeficiency, as previously described (19). Undifferentiated ES cells were maintained on a feeder layer (i.e., STO) of embryonic fibroblasts treated with mitomycin C (Wako Pure Chemical Industries Ltd.) in DMEM (Sigma-Aldrich)/F-12 (Invitrogen Corp.) supplemented with 0.1 mM 2-mercaptoethanol (2-ME) (Sigma-Aldrich), 1,000 units/ml LIF (Chemicon International), 20% knockout serum replacement (KSR; Invitrogen Corp.), and 4 ng/ml FGF2 (Upstate Biotechnology Inc.). ES cells were subcultured using 0.25% trypsin (Invitrogen Corp.) in PBS with 20% KSR and 1 mM CaCl2 (Wako Pure Chemical Industries Ltd.) as described (11, 19).
PA6 cells plated on type I collagen–coated chamber slides (BD) or gelatin-coated dishes (gelatin from Sigma-Aldrich; dishes from BD) were used as a feeder cell layer. To avoid contamination by incidentally differentiating cells, we manually selected undifferentiated ES cell colonies with stem cell–like morphology (tightly packed cells exhibiting a high nucleus to cytoplasm ratio). Undifferentiated ES cell colonies were washed twice with Glasgow minimum essential medium (GMEM) (Sigma-Aldrich) supplemented with 10% KSR, 1 mM pyruvate (Sigma-Aldrich), 0.1 μM nonessential amino acids, and 0.1 mM 2-ME. After trypsinization, partially dissociated ES cell clumps (10–50 cells per clump) were plated on PA6 cells at 1000 clumps per 10-cm dish. Cells were then cultured in GMEM supplemented with 5% KSR, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, and 0.1 mM 2-ME for 2 weeks. Differentiated colonies were detached from feeder cells using a papain dissociation system (Worthington Biochemical Corp.). Isolated colonies were cultured in neurobasal medium (Invitrogen Corp.) supplemented with B27 supplement (Invitrogen Corp.), 20 ng/ml FGF2, and 20 ng/ml EGF (R&D Systems) for 1 week. In the experiments examining the effecs of growth factors on neurosphere culture, the indicated concentrations of FGF20 (1pg/ml, 10 pg/ml and 1 ng/ml) were added to medium with or without FGF2 and EGF. In order to evaluate the expression of neural progenitor cell markers by each cell, spheres were incubated with papain at 37°C for 10 minutes and then mechanically dissociated into single cells. After incubation with a papain inhibitor, the dissociated cells were plated on OL-coated slides (OL from Sigma-Aldrich) at a density of 105 cells/cm2. After 16 hours, they were fixed in 4% paraformaldehyde (Sigma-Aldrich) and evaluated by immunofluorescence.
Spheres were isolated manually and plated on OL-coated slides in neurobasal medium to which had been added B27 supplement, 20 ng/ml brain-derived neurotrophic factor (Sigma-Aldrich), and 20 ng/ml neurotrophin-3 (Sigma-Aldrich). After 1 week of culture, spheres were fixed or used for additional experiments.
Total RNA was isolated from differentiated cells using a TRIzol kit (Invitrogen Corp.) according to the manufacturer's instructions. cDNA synthesis was carried out using the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen Corp.). PCR was performed using KOD-plus polymerase (Toyobo Co.), with the following cycling conditions: 30 seconds at 94°C, 30 seconds at 60.5°C, 60 seconds at 68°C × 30 cycles for Pax2, Ptx3, Nurr1, and GAPDH; and 30 seconds at 94°C, 30 seconds at 66°C, 60 seconds at 68°C × 30 cycles for Lmx1b and TH in a thermal cycler (Astec Co.). The experiments were repeated 4 to 7 times for confirmation, and PCR products were sequenced to rule out false positives. The primers used are as follows: Pax2, TGTGTCAGCAAAATCCTGGGCAGGT and TGCTGAACTTTGGTCCGGATGAT; Ptx3, TTCGCCTTCAACTCGGTCAACGT and CCCAGGGTCTGAAAGGGGTG; Nurr1, CTCCCAGAGGGAACTGCACTTCG and CTCTGGAGTTAAGAAATCGGAGCTG; Lmx1b, GCAGCGGCTGCATGGAGAAGATCGC and GGTTCTGAAACCAGACCTGGACCAC; TH, GACTGCTGCCACGAGCTGCTGGG and TCTTGGTAGGGCTGCACGG; and GAPDH, GTGAAGGTCGGAGTCAACG and GGTGAAGACGCCAGTGGACTC.
Spheres treated with FGF2 and FGF20 were plated onto 35mm OL-coated dishes at a density of 50 spheres per dish and made to differentiate for 1 week. Then, after being rinsed in a low-KCl (4.7 mM) solution, the cells were incubated in 1 ml of a high-KCl solution (20 mM HEPES-NaOH, pH 7.4, 85 mM NaCl, 60 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 11 mM glucose) for 15 minutes. Concentrations of dopamine were determined by HPLC using a reverse-phase column and an electrochemical detector system (HTEC 500; Eicom Corp.) as previously described (49).
Adult male cynomolgus monkeys (M. fascicularis) weighing 2.5–3.5 kg were given intravenous injections of MPTP HCl (0.4 mg/kg as free base, Sigma-Aldrich) twice a week until persistent Parkinsonian behavioral disturbances, such as tremor, bradykinesia, and impaired balance, became evident. Animals were given an average of 14.7 MPTP shots and exhibited stable Parkinsonism for approximately 30 days. To prevent any possibility of spontaneous recovery, only those monkeys that presented stable deterioration for a period greater than 12 weeks were used for transplantation experiments. All animals were fed with commercial pellets and fresh fruits and had free access to clean water. Monkeys were cared for and handled according to Guidelines for Animal Experiments of Kyoto University and the National Cardiovascular Center (Osaka, Japan) and the Guide for the Care and Use of Laboratory Animals from the Institute of Laboratory Animal Resources (ILAR; Washington, DC, USA).
For accurate orientation of the putamen, animals (n = 10; 6 for ES transplanted and 4 for sham-operated group) were subjected to MRI examination using a 3.0 Tesla Signa system (General Electric). Following anesthesia by intramuscular injection with ketamine hydrochloride (15 mg/kg; Sankyo Co.) and xylazine (1.5 mg/kg; Boehringer Ingelheim Vetmedica Inc.), animals were positioned into the magnet using an MR-compatible headholder. T1-weighted images were used for further examination.
Following anesthesia with pentobarbital (7.5 mg/kg, intramuscularly [i.m.]; Dainippon Pharmaceutical Co.) and ketamine (10 mg/kg, i.m.), monkeys were fixed in a surgical frame (Narishige SN-1N; Narishige Co.). Neural progenitors from monkey ES cell cultures in two 6-cm dishes (300,000–600,000 cells per dish) were collected for each animal. While the ES cells were expanding as spheres, BrdU (5 μg/ml; Sigma-Aldrich) was added to the culture medium for 7 days to label the ES cells. Using an electric injector (Muromachi Kikai Co.), we transplanted donor cells into the putamen bilaterally, using the MRI findings for each monkey and the M. fascicularis brain atlas (36, 50). To cover the mid to posterior putamen, 3 targets that were 2 mm apart in anterior-posterior position and 1–2 mm in medial-lateral position were set, and 4 injections (1 μl/60 seconds for each injection) were made along each tract. After surgery, all animals were given antibiotics for 1 week and a daily immunosuppressant (cyclosporin A, 10 mg/kg, i.m.; Carbiochem) until sacrifice. Monthly blood analyses performed over the course of the experiment confirmed the levels of circulating cyclosporin A, which averaged 393 ng/ml. In control monkeys undergoing sham operations, the equivalent volume of culture medium was administered without cells. Afterward, antibiotics and immunosuppressants were injected as for the cell-grafted monkeys.
For in vitro studies, cells were fixed in 4% paraformaldehyde for 10 minutes for microscopic observation. For in vivo studies, after administration of deep anesthesia with pentobarbital and ketamine, monkeys (n = 10; 6 for ES cell–transplanted and 4 for sham-operated group) were transcardially perfused with 4% paraformaldehyde. Excised brains were frozen and cut with a microtome at a 50-μm thickness. Sections were then stained by the free-floating method. Slides were first incubated in 0.3% Triton X with 5% skim milk in PBS for 30 minutes. Samples were then incubated with one of the following primary antibodies: rabbit anti-NCAM (1:300; Chemicon Inc.), rat anti–Musashi-1 (1:200; gift from H. Okano, Keio University, Japan), mouse anti-Nestin (1:300; Chemicon Inc.), mouse anti-TuJ1 (1:300; BabCO), rabbit anti-GFAP (1:1000; Chemicon Inc.), mouse anti-Map2ab (1:250; Sigma-Aldrich), rabbit anti-GalC (1:100; Chemicon Inc.), rabbit anti-glutamate (1:200; Chemicon Inc.), goat anti-ChAT (1:1000; Chemicon Inc.), mouse anti-GABA (1:1000; Sigma-Aldrich), mouse anti-BrdU (1:200; BD), rabbit anti-TH (1:60; Chemicon Inc.), rabbit anti-serotonin (1:2000; DiaSorin Inc.), or rat anti-DAT (1:100; Chemicon Inc.) in 2% skim milk in PBS overnight at 4°C. After 3 washes in PBS, samples were incubated with the appropriate secondary antibodies for 1 hour at room temperature: FITC-labeled anti-mouse Ig (1:200; Jackson Immunoresearch Laboratories Inc.) and Cy3-labeled anti-rabbit Ig (1:200; Jackson Immunoresearch Laboratories Inc.). For nuclear staining, 200 ng/ml of 4′,6′-diamidino-2-phenylindole (DAPI) was added in the final wash. After being washed in PBS, samples were mounted and analyzed using a Fluoview FV300 laser confocal microscope (Olympus Optical Co.). For in vitro studies, cells in 5 randomly selected fields, each of which included 100–500 cells, were counted for 3 to 5 independent cultures. For in vivo studies, the number of immunoreactive cells was quantified in every sixth section throughout the graft and surrounding tissue. Statistical analysis was performed by one-way ANOVA and post hoc multiple comparison by the Dunn test. A P value of less than 0.05 was considered significant.
Parkinsonian behavior was evaluated using a rating scale previously proposed by Akai et al. (51), with modifications (Table (Table1).1). To prevent subjective biases, the evaluation was performed by a trained examiner who was not involved in the culturing and transplantation of the cells and not informed of the specific procedure to which each monkey was subjected. Student's t test was used to compare the 2 groups, and a P value of less than 0.05 was considered significant.
To evaluate the in vivo DA function of the grafted cells, PET scans using 18F-fluorodopa were performed on each animal prior to perfusion. Under generalized anesthesia with a continuous infusion of propofol (4 mg/kg/h; Zeneca Pharmaceuticals) and vecuronium-bromide (0.25 mg/kg/h; Boehringer Ingelheim Vetmedica Inc.), analyses were performed using an ECAT EXACT HR+ PET scanner (Siemens/CTI). After intravenous injection of 185MBq of 18F-fluorodopa, brain radioactivity was assessed for 90 minutes in animals that had received carbidopa (10 mg/kg) 30 minutes prior to the PET scan. Parametric images of the dopamine-irreversible metabolic rate of Ki (min–1), considered to be a measure of presynaptic DA function, were generated using the time course of radioactivity in each voxel by multiple-time graphical analysis (52) using the bilateral occipital lobes as a reference region. The 18F-fluorodopa Ki image was coregistered to the corresponding T1-weighted MRI image, which was obtained by an inversion-recovery prepared fast spoiled gradient recalled acquisition in the steady state (IR-FSPGR) sequence (Tr = 9.4, TI = 600, TE = 2.1 in msec) using a 3.0 Tesla Signa System (General Electric) and realigned to a standard space of M. fascicularis (36, 50). DA function was evaluated by visual inspection of Ki images and by quantitative Ki values in the bilateral striatum identified by the corresponding MRI image. This evaluation was performed in a blind manner to ensure objectivity. Student's t test was used to compare the results.
The authors thank H. Sameshima and T. Yamamoto (Shin Nippon Biomedical Laboratories Ltd.) for technical help with monkeys, H. Okano (Keio University) for kindly providing the Musashi-1 antibody, and Hiroyuki Nawa (Niigata University) for helpful advice regarding the dopamine release assay. We also thank Y. Yanagi and T. Gomibuchi for their secretarial help. This study was supported by the following: Grants-in-Aid for Scientific Research, Grants in Kobe Knowledge Cluster, and Establishment of International Center of Excellence for Integration of Transplantation Therapy and Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology; Health Sciences Research Grants in Research on Human Genome, Tissue Engineering and Food Biotechnology from the Ministry of Health, Labour and Welfare; and Grants in Organization for Pharmaceutical Safety and Research.
See the related Commentary beginning on page 23.
Nonstandard abbreviations used: ChAT, choline acetyltransferase; DA, dopa-miner-gic; DAT, dopamine transporter; GABA, γ-amino butyric acid; GalC, galactocere-bro-side C; GFAP, glial fibrillary acidic protein; GMEM, Glasgow minimum essential medium; i.m., intramuscular(ly); LIF, leukemia inhibitory factor; ME, mercaptoethanol; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NCAM, N cell adhesion molecule; OL, poly-l-ornithine and laminin; PD, Parkinson disease; PET, positron emission tomography; SDIA, stromal cell–derived inducing activity; TH, tyrosine hydroxylase.Conflict of interest: The authors have declared that no conflict of interest exists.
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On Mon, Jun 22, 2009 at 2:46 PM, Thomas DuBuisson <email@example.com> wrote:

 All,
 I've started to add to the network trac [1] - its just framework for
 now. ĀPlease do add proposals, organized comments, and feel free to
 alter the framework. ĀI'm not sure how formal we'd like to make this
 so I haven't even tried to make guidelines for proposals. ĀI'll add
 proposals and perhaps try my hand at guidelines throughout this week.

Lets just get some concrete proposals up there and see if we need some kind of guidelines and what those guidelines should be. FWIW I like the PEP style.

-- Johan