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the very first elevate fellows have been announced!
The ELEVATE StartWell™ Challenge invited applicants to present innovative new concepts to improve the element of play in early childhood (0-8 years), reimagine urban spaces and playgrounds, and the way young children interact with space.
Entries were received from 12 countries, across disciplines that included design, architecture, early childhood, performing arts, technology and production. Ideas included pop-up spaces representing the local country, iPhone apps to encourage children to play outside within nature, spaces and concepts to enable full enjoyment for children with access requirements, and large-scale playgrounds inspired by the 'A Different Class' typologies presented by Lekker Architects with the Lien Foundation earlier this year.
The panel of judges – consisting of senior representatives from the Lien Foundation, Lekker Architects (Singapore), British Council, Yamaguchi Center for Arts and Media [YCAM] (Japan), St James' Church Kindergarten (Singapore) and the ArtScience Museum (Singapore), Demos (UK) and international playground manufacturer Lappset – was hugely impressed by the creativity of applicants in approaching the ELEVATE Challenge.
The following 12 applicants of the ELEVATE StartWell™ Challenge, representing a diverse range of nationalities, were judged the best by the panel, and have gone on to become ELEVATE Fellows for 2015:
- Dang Thi Minh Thu, Vietnam
- Grant Menzie, UK
- Chloe Meineck, UK
- Ben Barker, UK
- Paul Drury, UK
- James Sales, UK
- Sumitra Pasupathy, Singapore
- Francis Sollano, Philippines
- Ngai Yuen Low, Malaysia
- Fakhrizal Nashr, Indonesia
- Bronwyn Cumbo, Australia
- Neil Hobbs, Australia
In February, the 12 Fellows assembled in Tokyo and Yamaguchi, Japan, for a one week Innovation Camp, organised by the British Council, Yamaguchi City Foundation for Cultural Promotion and with the support of Yamaguchi Center for Arts and Media [YCAM]. The camp was also facilitated by UK experts Shelagh Wright and Ella Britton.
There, they worked with local and international experts to further develop and refine their ideas, experience new and innovative models of design and ideation, and collaborate across countries. At the end of the Camp, the Fellows resubmited their ideas for the opportunity to secure a portion of funding available to further develop their concepts.
In May 2015, The Lien Foundation and the British Council announced the recipients of $50,000 to create a more playful future for children across the world.
The recipients selected were:
- POP UP PARKS, by James Sale, UK ($20,000)
- GREAT URBAN CHALLENGE, by Francis Sollano, Philippines ($10,000)
- PLAYEUM, by Sumitra Pasupathy, Singapore ($10,000)
- PLAYFINDERS, by Paul Drury, UK ($10,000)
Developed and presented by the British Council, ELEVATE aims to build a global community of creative problem-solvers, who will meet practical challenges with innovative solutions.
ELEVATE provides a platform for innovative thinkers to come together, to discuss issues, to learn and to develop projects using culture and creativity in Health & Wellbeing; Education & Play; Creative Cities; Environment; Conflict & Safety.
ELEVATE Challenge: An open call in which applicants from Australia, East Asia and the UK are invited to put forward their best ideas in response to set challenges.
ELEVATE BT Connect: Applicants and local experts will share ideas and hear from leading international figures in the world of innovation and design, via teleconferencing technology from ELEVATE global partner, BT Global Services.
ELEVATE Innovation Camp: ELEVATE Fellows from around the world will come together for six days of workshops, training sessions, collaboration and ideas-sharing.
ELEVATE Digital Network: Launching April 2015, the ELEVATE Digital Network will be a web-based platform for creative innovators, the world over.


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Home > Gaming > PC, Mac Gaming > PC, Mac Games
THQ THQ Warhammer 40k: Dawn of War II Retribution (PC)
Ten years after the events of Chaos Rising and the fall of Blood Raven's Chapter Master to Chaos, Sub-Sector Aurelia remains in a state of constant warfare. The pirate Freebooter Orks have arrived to take advantage of this and loot the sector, the vile forces of Chaos continue to spread their blasphemy, the Eldar of Craftworld Alaitoc work to recover key artifacts from their past, and the feral Tyranids remaining from the failed Hive fleet invasion have suddenly reestablished contact with the Hive mind. Choose your race and determine the final fate of the entire sector, in Dawn of War II: Retribution the next installment in the critically acclaimed Dawn of War real-time strategy franchise.
Important Add-Ons
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- Main Website: www.thq.com/us
- Support Website: support.thq.com/thq/consumer/search.asp
Micro Center Return Policy:
We guarantee your satisfaction on every product we sell with a full refund — and you won't even need a receipt.* We want you to be satisfied with your Micro Center purchase. However, if you need help or need to return an item, we're here for you!
If an item you have purchased from us is not working as expected, please visit one of our in-store Knowledge Experts for free help, where they can solve your problem or even exchange the item for a product that better suits your needs.
If you need to return an item, simply bring it back to any Micro Center store for a full refund or exchange.
*If you are a Micro Center Insider or if you have provided us with validated contact information (name, address, email address), you won't even need your receipt.
General Return Policy
Desktop / notebook computers, tablets, processors, motherboards, digital cameras, camcorders and projectors, 3D printers, 3D scanners, and CD/DVD duplicators may be returned within 15 days of purchase. All other products may be returned within 30 days of purchase. Merchandise must be in new condition, with original carton / UPC, and all packaging / accessories / materials. If you need to return software, a game or a movie, remember that you may not retain any copies — it's not legal, and it's not nice.
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Using the software may require the use of a computer or other device that must meet minimum system requirements. It is recommended that you familiarize yourself with the system requirements before making your purchase. Software system requirements are typically found on the Product information specification page.
Aerial Drones
Micro Center is happy to honor its customary 30-day return policy for Aerial Drone returns due to product defect or customer dissatisfaction. Unfortunately, we cannot accept returns with damage due to pilot error. So let's be careful out there!
As of December 21, 2015 all drones, regardless of purchase date, between 0.55 lb (8.82 oz, 250 g, 0.25 kg) and 55 lb take-off weight are required by the FAA to be registered for outdoor use. Details regarding registration can be found at: http://www.faa.gov/uas/registration/
Wireless Phones & Devices
Wireless phones and devices may be returned within 14 days of purchase. When returning a wireless phone or device with a plan, you are responsible for ensuring that your service is cancelled with the carrier. If you don't cancel your service, you may incur additional charges from your carrier. All carrier charges are your responsibility.
Refunds will be credited to the credit card or debit card account used for the original purchase.
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If you need help with hardware or software products purchased from Micro Center, please contact one of our knowledgeable tech support reps by live chat or email.
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We accept the following credit cards: Micro Center VISA® credit card with WorldPoints® Rewards; VISA; MasterCard; American Express; Novus/Discover.
|Learn more about our exclusive Priority Care Program. You'll have access to our in-house Technical Support team of certified technology professionals trained to assist you on a variety of PC and Mac related support issues.

subset: commoncrawl


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We are proud to present the Ed, Edd n Eddy Seasons 1-6 (Plus Extras) DVD Box Set.
This DVD Set contains every episode of this hilarious Cartoon Network animated series, exclusively available at DVDRare.com
Ed, Edd N Eddy is one of Cartoon Network's longest running and most successful franchises and the longest-running of the channel's original Cartoon Cartoons. The series revolves around three adolescent boys collectively known as "the Eds," who hang around in a suburban neighborhood.
Unofficially led by Eddy, the Eds constantly scheme to make money off their peers, in order to purchase their favorite confectionery: jawbreakers. However, their plans usually fail, leaving them in various predicaments.
The Ed Edd n Eddy DVD collection is a box set as shown above, all episodes are organized from pilot to finale.
The DVDs include custom artwork and episode guides so you can find your favorite episode at anytime
The DVD collection is formatted region free so it will play on any DVD player, DVD-ROM X-Box or PS2 Worldwide
This ultra-rare DVD set contains every episode of the show, unedited and exactly the same as shown on Cartoon Network back in the day!
At DVDRare we pride ourselves on the fact that most of our customers come back and order more, because they trust that we will provide great, high quality, unofficial DVDs.
So as a token of our thanks to the more serious customers, we'd like to offer you free delivery on orders of 2 or more sets.
Special Offer
Order 2 or more DVD sets, and we will give you FREE DELIVERY.
Product 13/33
This product was added to our catalog on Sunday 17 July, 2011.


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Play Video
Lighting a fart on fire
Posted by Garrety on Jun. 03, 2009
Elizabeth Rage takes you through the steps to create the Enchantress look f...
On this episode, Master Chalk Artist Chris Carlson takes on an epic Star Tr...
Get you geek on and come check out these hilarious clips!
Posted by Garrety on Jun. 03, 2009


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That government of the people, by the people, for the people, shall not perish!
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The Declaration of Independence expresses the fundamental values of all people, and the United States Constitution formed a Republic to protect those fundamental values against unrestrained government intervention.
Promote the values expressed in the Declaration of Independence and preserve our republic as established by the United States Constitution.
To hold government - at all levels - accountable to the people!
Quotable Quote
"Property must be secured or liberty cannot exist." - John Adams
The Ninth Amendment to the Constitution states that, "The enumeration in the Constitution, of certain rights, shall not be construed to deny or disparage others retained by the people."
Recently I spent some time with a person I respect highly, who is very intelligent, and who has thought about and reached conclusions concerning America's Constitution. This person, who is…Continue
Tags: Scalia, Antonin, view, progressive, agenda
Started by Dr. Robert Owens in Constitutional Discussions Jul 16.
Endless violence. It becomes part of your psyché. But you never get used to it. You despise the pain and suffering. The endless terrorist attacks by the radical Islamic terrorists.We must never think…Continue
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Started by David Wallace in Federal- Defense Jul 3.
Abortion mills are not required to meet health standards as high as…Continue
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Started by Dr. Robert Owens in General Discussion Jul 1.
How can we possibly know the motive of the perpetrator of the Orlando Massacre? It is a mystery to the President, the Attorney General, his whole administration, and their cheerleaders in the…Continue
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Started by Dr. Robert Owens in Restore America's Mission Now! Jun 23.
Posted by David Wallace on October 27, 2015 at 10:01pm
Posted by David Wallace on September 30, 2015 at 8:44am
Published: 12 hours ago
Posted by Don on September 17, 2015 at 9:42am
Posted by Don on September 15, 2015 at 1:32pm
© 2016 Created by David Wallace. Powered by


text:
Traders Buying EIB CO2 May Have Privileged Data, IETA Says
Companies buying 2013 European Union carbon permits directly from the European Investment Bank may have "privileged" information that other traders don't have, according to an emissions trading lobby group.
"It's not very easy to assess the impact" of the sales, because they are confidential, said Simone Ruiz, Brussels-based European policy director at the International Emissions Trading Association. "There are many rumors and that's not helpful."
The lack of public information about the commencement of the sales means that "if you are a recipient of a large volume you are in a privileged position," Ruiz said today by phone. "The market wants more transparency."
The EIB may have last week begun selling privately the first tranche of 200 million EU permits for the third phase of the EU's emissions trading system, which begins in 2013, to fund renewable energy and carbon capture projects as part of the EU's drive toward a low-carbon economy. The permits are worth as much as 1.5 billion euros ($2 billion).
Nick Antonovics, a spokesman for the Luxembourg-based EIB, declined to comment on criticism of the sales process when reached today by phone and e-mail.
"The EIB has stated repeatedly, including on our website, that we would start sales soon after delivery but would not be making any other statements regarding timing," Antonovics said. "Results for December will be published by the middle of January."
IETA is continuing internal discussions on precisely what information and changes it will seek from the commission and the EIB, Ruiz said.
"There have been some accusations of market manipulation," Ruiz said, referring to price volatility in carbon contracts since the start of December. "I can't judge on that," she said.
On Dec. 2, the European Commission in Brussels delivered 300 million phase three allowances to the EIB, 200 million of which need to be sold by Oct. 2, 2012.
EU carbon for 2013 dropped to 7.78 euros ($10.13) a metric ton on Dec. 6, a record low at the time. It rebounded to as high as 9.18 euros a ton on Dec. 12, the day after nations at climate-protection talks in Durban, South Africa, decided to seek a global agreement by 2015. That's an increase of 18 percent in six days.
The contract fell 1.2 percent today to close at 7.69 euros on the ICE Futures Europe exchange in London. It dropped as much as 3.3 percent in intraday trading. The record low of 7.26 euros was reached on Dec. 14.
The bank has a mandate to impact the secondary market as little as possible while selling, Ruiz said. "We would have preferred them to start on exchanges" and it's unclear when they will start to do that, she said.
The market would probably like more information on the price structure used and number of parties the EIB is dealing with as it carries out the sales, Ruiz said.


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I have created a test project to understand reflection. I'm loading an assembly from .NET 4.0 GAC. (As I understand, .NET 4.0 maintains it's GAC in C:\WINDOWS\Microsoft.NET\assembly)
I wrote the code like this:
Assembly testAssembly = Assembly.ReflectionOnlyLoadFrom(@"C:\WINDOWS\Microsoft.NET\assembly\GAC_32\TestReflection\v4.0_22.214.171.124__7ff2353191526e8c\TestReflection.dll");
When I run this code,
GlobalAssemblyCache property always return FALSE although I'm loading assembly from GAC.
Can someone tell me the reason? Or Am I missing something?


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Poor Man's Guide to WSPR Spots
Okay, so I've been playing around with generating various kinds of visual signals on the MEPT subband on 30m. That's the 100hz wide band just above 10.140Mhz, and it's fun, because there are a number of "grabbers" which have the spectrum displays from their radio receivers available on the web. But there is a minor problem: there aren't that many of them here in the U.S., and propagation often makes it difficult to find them.
But there is an alternative: WSPR. WSPR is a beaconing mode designed by K1JT, Joe Taylor, a Nobel Prize winning Princeton physicist. He is also the author of the program WSJT, which is used by many EME and weak signal operators to allow them to exchange messages at very low signal levels. WSPR encodes a callsign, grid and power level as a 162 symbol long sequence of FSK tones 1.46 hz apart, lasting for about 110 seconds. Joe's code is open source, which is awfully cool, but a lot of it is written in FORTRAN and I've had difficulty getting it to run on my Macintosh. It's probably that I've got a slightly odd development environment, but it's been frustrating.
And here's the other thing: I really don't need all the heavy lifting to process receive level stuff. Ultimately, I'd like to be able to send this stuff with just a little microcontroller, with a fixed message. That isn't hard, but it's hard to get the exact details of the protocol description without digging into the source code.
But I did finally figure out how to generate a compatible message. It turns out that you can get a Windows command-line program from Joe's website, called WSPR.EXE. If you read really carefully, you will realize that it can be tricked into telling you which tone it sends in each of the 162 slots for a messag that you specify. I specified a message containing my call (KF6KYI) my grid (CM87) and my power (27dBm or 0.5 watt), and it dumped the tones. From then on, all the work I've been doing with writing CW and Feld Hell encoders helped, and I generated a little WAV file that had the right tones in it. I imported that into iTunes, and then played it into my FT-817 at the beginning of an even numbered minute. I then went to the automated WSPR logging site, and...
I think another hour's worth of work, and I could have the entire thing automated, and it would totally work. Very neat.
Thunderbirds are go!
Yesterday, I took the wife and the future daughter-in-law to Travis AFB for their airshow. It was a blast. Lots of cool planes, culminating in a nice show by the Air Force Thunderbirds. I snapped a lot of photos, this one probably being the best.


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International Edition
July, 28, 2016
About Us
Contact Us
Police authorities in Northern Taiwan recently busted a narcotic ring that planned to sell illicit drugs in a New Taipei rock festival to be held later this month, the Criminal Investigation Bureau (CIB, 刑事警察局) has announced.
Taipei 101 was awarded the top honor Thursday of the Leadership in Energy and Environmental Design (LEED) building rating system, achieving the highest level of Platinum in the category of Operation and Maintenance of an Existing Building.
Inspectors in full protective gear examine a geese farm in Hualien in this photo provided by the eastern county's animal and plant quarantine department on Friday, July 8. The inspection was conducted in the wake of reports about geese deaths at the farm. Test results showed that the dead geese there were infected by H5 bird flu viruses, the department said.
Consumers gather outside the entrance of the Fengyuan branch of Pacific Department Store in central Taichung City well before doors opened at 2 p.m., Friday, July 8. Many shoppers were eager to make use of the extra typhoon holiday. The city reported little wind or rain despite Typhoon Nepartak ravaging parts of Eastern and Southern Taiwan.
The China International Tourism Association (CITA) will hold an exhibition to mark its 20th anniversary, as well as the 60th birthday of its director-general in Taipei, July 9.
Keelung Mayor Lin Yu-chang Wednesday met two members of Assemble, a British group of young architects and designers who won the prestigious Turner Prize for art.
Gamblers and a dealer are seen at an illegal Texas Hold 'Em casino in Taipei on Thursday, July 7. Police said that the operator of the casino hired an attractive woman as a dealer and used messaging apps such as Line and WeChat to draw young gamblers.
Military and government authorities said Thursday that they are on standby for disaster response as Super Typhoon Nepartak, which is expected to bring heavy rain and strong wind to Taiwan, continued its approach toward the country.
None of the 60 venues designated for the 2017 Taipei Summer Universiade are currently ready for operation, a Taipei City councilor said on Wednesday.
With a decision looming over Farglory's (遠雄) contract for the Taipei Dome (台北大巨蛋) project, city officials estimated Thursday that the contractor was seeking NT$90 billion (approx. US$2.9 billion) from the city in compensation should its contract be terminated.


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Swarm/agent Technology For Small Unit Scalable Effects
Award Information
Small Business Information
Procerus Technologies LC
452 South 950 East, Orem, UT, 84097
Principal Investigator:
Blake Barber
Chief Engineer
(801) 224-5713
Chief Engineer
(801) 224-5713
Business Contact:
Todd Titensor
(801) 224-5713
(801) 224-5713
Research Institution:
AbstractProcerus Technologies, along with its partner Brigham Young University, propose to develop cooperative control technologies to enable multiple small, unmanned air vehicles (SUAVs) to carry out a mission involving search, tracking, imaging, and localization (STIL). The goal is to carry out these activities as a team of SUAVs, enhancing the performance and robustness of the mission execution. During this Phase I SBIR task, the Procerus/BYU team will carry out the following technical tasks: 1) Create a multiple SUAV simulation testbed for algorithm testing and development. 2) Develop probabilistic target motion models that capture target behavior based on situational awareness. 3) Formulate and test a multi-model tracker to estimate target vehicle location. 4) Design and implement a cooperative control architecture that enables multi-SUAV STIL. 5) Develop a search strategy that maximizes the probability of target detection based on a location probability prior. 6) Design and implement a cooperative tracking algorithm that maximizes tracking robustness. 7) Demonstrate the multi-SUAV STIL algorithms in a realistic mission simulation. The Procerus/BYU team will leverage their technical expertise in vision-based control of SUAVs and cooperative control of SUAVs to successfully complete the technical objectives of this proposal.
* information listed above is at the time of submission.
An Official Website of the United States Government


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text: 000 FXUS65 KPIH 210924 AFDPIH Area Forecast Discussion National Weather Service Pocatello ID 324 AM MDT THU JUL 21 2016 .SHORT TERM...Today and Friday. Hot...dry and breezy conditions will persist today as we remain in Southwest flow ahead of a Upper Level Low and associated dry cold front off the Pacific coast. May see isolated showers and thunderstorms across Eastern Lemhi County and Western Wyoming this afternoon into early evening. Winds will decrease today 5 to 10 mph over yesterday but remain breezy particularly in the mountains. Friday look for moderate to strong winds particularly over our Northern and Central areas as a dry cold front passes through the area. The Nam12 model is showing isolated afternoon thunderstorms along the Wyoming border Friday. Will likely see wind gusts near 30 mph in the Central Mountains and near 40 mph for the Upper Snake river plain Friday. Expect temperatures 5 to 10 degrees above normal today. Today will likely be the warmest day of the year so far with temperatures in the mid to upper 90s across the Snake river plain and 80s in the mountains. Temperatures will then drop to near normal on Friday. Wyatt .LONG TERM...Saturday through next Thursday. Weak high pressure ridging builds in Saturday before a very weak trough moves through Sunday with little impact...perhaps just a slight increase in winds. Monday into Wednesday expect dry Westerly flow before another system bringing moderate winds moves through the area on Wednesday night into Thursday. Temperatures will be near normal on Saturday before gradually warming back up through the period becoming 4 to 8 degrees above normal by Tuesday and Wednesday. Wyatt && .AVIATION...A dry SW flow aloft will continue across the region with VFR conditions today. Huston && .FIRE WEATHER...A dry SW flow aloft will continue across the region today driving Haines indices up into the moderate to high category. The NAM model was hinting at the potential for isolated thunderstorms popping up over ERN Lemhi County and WRN Wyoming late this afternoon and evening. The lack of moisture will likely play a limiting factor in any convective development today. A low pressure trough positioned off the NW coast will sweep inland tonight and then east along the Canadian border Friday with an associated dry surface cold front shifting through the region Friday afternoon. Strong gusty winds and low humidity will accompany the front across the region Friday afternoon and continue into the evening hours prompting the issuance of a Fire Weather Watch for the CNTRL mountains and Snake River Plain. A weak secondary trough spills into Washington Saturday and then works east Sunday as it continues to fill. For now, there appears to be little impact from wind or moisture associated with this system. A dry and modest west flow generally dominates through Wednesday as the next weak Pacific trough works east into the region Wednesday afternoon potentially providing the next opportunity for a wind event. Sub-tropical moisture begins to work north into Nevada by weeks end for a possible moisture source for isolated thunderstorm activity next weekend. Until then, we will continue to bake the fuels as daytime temps run above normal. Huston && .PIH Watches/Warnings/Advisories... Fire Weather Watch from Friday afternoon through Friday evening for IDZ410-422-425-475-476. && $$


text:
Acarbose doses should be adjusted based upon blood glucose levels taken one hour after a meal and blood HbA1c levels taken about three months after starting or changing the dose. (HbA1c is a chemical in the blood that is a good indicator of blood glucose control.)
DRUG INTERACTIONS: Acarbose may interfere with digoxin (Lanoxin) absorption thereby decreasing digoxin blood levels and its effect. Therefore, the digoxin dose may need to be increased if acarbose is begun. Since adding insulin or a sulfonylurea to acarbose therapy may lower blood glucose more than acarbose alone, the risk for developing hypoglycemia is greater when these drugs are combined. Caution should be used when combining these drugs. If mild to moderate hypoglycemia occurs while taking acarbose in combination with another anti-diabetic drug, the hypoglycemia should be treated with oral glucose (dextrose) instead of sucrose (table sugar) because acarbose blocks the digestion of sucrose to glucose, and hypoglycemia will not be corrected rapidly with sucrose. Acarbose alone does not produce hypoglycemia. Charcoal may absorb acarbose and digestive enzyme preparations such as amylase or pancreatin may breakdown acarbose and should not be adminstered with acarbose.
PREGNANCY: There are no studies of acarbose treatment during pregnancy in humans. Insulin therapy is recommended in pregnancy.
NURSING MOTHERS: Acarbose is excreted in the milk of lactating animals, but no human studies have been conducted. Acarbose use is not recommended for women who are breastfeeding.
Medically reviewed by Eni Williams, PharmD
REFERENCE: FDA Prescribing Information
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The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ~90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the inter-subunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic.
Diminished activity of human phenylalanine hydroxylase (PAH), which catalyzes the iron, dioxygen, and BH4 dependent oxidation of phenylalanine (Phe) to tyrosine, is the most common inborn error of metabolism; the most severe cases result in phenylketonuria (PKU) [1]. Despite the success of dietary therapy in preventing severe PKU-associated mental deficits, patients continue to struggle with lifelong control of blood Phe levels. Poor control can compromise behavior, learning, and executive function [2–3]. Thus, alternative therapies are sought, including small molecule therapeutics [1, 4]. Pharmacological chaperones that stabilize PAH structure are gaining support as a long term therapeutic approach [5–12]. The cofactor BH4, which is an active site directed pharmacological chaperone, is in clinical use with some success [13]. Although biological and chemical chaperones are generally viewed as assisting in all aspects of protein folding, a focus on quaternary structure dynamics provides a different perspective that is applicable to PAH. Pharmacological chaperones can function allosterically (e.g. not at the enzyme active site) to assist a protein in achieving and/or maintaining its most active structure. PAH is known to be an allosteric enzyme where allostery involves major conformational changes and an equilibrium of multimers including dimers and tetramers. Although the structural basis for allosteric activation by Phe is poorly understood, PAH was recently identified as a putative morpheein, and the current work builds on this hypothesis [14–15]. Unlike previous views of PAH multimerization equilibria, the morpheein model contains two different PAH tetramers separated by a large kinetic barrier that requires multimer dissociation, conformational change in the dissociated state, and reassociation to an alternate tetramer. Herein we 1) detail the rationale behind developing a morpheein model for PAH allostery, 2) present protein structure models for components of a PAH quaternary structure equilibrium, and 3) provide experimental evidence on purified full length rat and human PAH consistent with the proposed equilibrium. The current work sets the stage for our long-term goal, which is to develop allosteric pharmacological chaperones to expand the PKU patient population responsive to small molecule therapy.
A contemporary explanation for PAH dysfunction causing PKU identifies the disorder as one of PAH protein stability and/or folding [10, 12, 16–18]. For multimeric proteins, where the final step in the folding process is multimer assembly, perturbation of an equilibrium of functionally distinct alternative multimers provides both a mechanism for allostery as well as a mechanism for protein dysfunction [19–20]. Extensive literature describes mammalian PAH as an allosteric enzyme [21–22]. At low Phe levels the protein has basal activity which allows retention of sufficient Phe to support protein biosynthesis. At high Phe levels protein activation is accompanied by a large conformational change which dramatically changes various characteristics of the protein, including the fluorescence spectrum and the susceptibility to proteolytic digestion [23–25]. PAH allostery is associated with a quaternary structure equilibrium consisting of dimers and tetramers with some propensity to form larger aggregates [21, 23, 26–29]. Phe serves both as substrate and as allosteric activator; allosteric activation by Phe has been shown to draw an equilibrium of tetramers and dimers toward the tetramers [28, 30]. A structural basis for allosteric Phe binding causing stabilization of a specific, fully active conformation of the tetramer is proposed herein.
The new model for allosteric regulation of PAH, its relationship to PKU, and the promise of developing pharmacological chaperones that function allosterically follows our recently reviewed work with the prototype morpheein porphobilinogen synthase (PBGS) whose dysfunction causes the inborn error of metabolism known as ALAD porphyria [31–32]. Four important corollaries exist between the behavior of PBGS and the model proposed for PAH.
The goals of this study are to prepare and support a model for the quaternary structure equilibrium that governs PAH activity and to develop methods that can evaluate the position of the PAH quaternary structure equilibrium in a variety of physiologically relevant environments. The developed methods will allow future investigation of the effect of PKU-associated variants on the position of the PAH quaternary structure equilibrium. The developed protein structure models will assist in pursuit of allosteric pharmacologic chaperones that promote PAH to achieve and maintain its active multimeric assembly.
Both human PAH tetramer models are based on the human PAH structure 2PAH, which is a tetramer of a truncated form of PAH that contains only the catalytic and multimerization domains. A human regulatory domain homology model (residues 19–117) was prepared from the rat PAH crystal structure 1PHZ, which is of a truncated rat PAH containing only the regulatory and catalytic domains. Modeling was performed using our program MolIDE, which includes side chain rotamer optimization [49–50]. The low activity human PAH tetramer model was prepared using the program CHIMERA [51–52] to align the catalytic domains of the rat structure 1PHZ with the human structure 2PAH and the human regulatory domain model. Loop modeling (amino acids 113–121) was used to attach a regulatory domain to each of the human PAH subunits to yield our structure of 4mer*, using the program YASARA [53]. Following loop modeling, loop optimization included energy minimization by steepest descent methods. A final optimization of side chain rotamers was done in the context of the full multimer using SCWRL4 [54]. Preparation of the high activity human 4mer model started with the preparation of a dimer of the human PAH regulatory domain homology model, prepared by aligning two regulatory domains with the ACT domain dimer seen in crystal structure 1PSD. Following energy minimization and side chain optimization, the human PAH regulatory domain dimers were manually positioned to optimize the connection to the catalytic domains in the region of amino acid 118 of two catalytic domains with amino acid 117 of two halves of the regulatory domain homology model dimer. Loop modeling followed by side chain optimization and energy minimization completed the preparation of the human PAH 4mer model. Similar methods were used to prepare models of rat PAH 4mer* and 4mer.
The expression construct for a the MBP fusion protein of human PAH was a kind gift of Dr. A. Martinez and protein purification followed standard procedures on an amylose column. Expression constructs for full length rat and human PAH were a kind gift of Prof. P. Fitzpatrick. All gifted plamids were amplified and the sequence was confirmed to be as published. Expression procedures included small variations on the procedures used for expression of mammalian PBGS [55]. Expression typically was carried out at reduced temperature (15 – 25 °C), Fe(II) was added at the same time as IPTG (at various concentrations), and in some cases glycerol was included as a chemical chaperone. None of these variations had a significant impact on protein expression levels. Purification of rat PAH followed the procedure of Shiman wherein crude cell extract was passed over a 75 ml Phenyl Sepharose-CL4B column in the presence of phenylalanine, followed by extensive washes with a variety of buffered solvents, and elution of the protein following removal of Phe from the elution buffer, which contains 15% glycerol, all carried out at 4 °C [56]. This affinity purification is based on a major conformational change that has long been known to accompany the process wherein Phe activates mammalian PAH [21]. Although this purification works well for rat PAH, human PAH, which expressed poorly, emerged from the column requiring significant further purification, which was carried out by ion exchange chromatography (IEC) on a 1 ml HiTrap Q column in 30 mM Tris-HCl, pH 7.4 using a KCl gradient (0–0.4 M) at room temperature.
Analytical SEC (100 μl sample size) was carried out on a 24 ml Superdex 200 column at 0.5 ml/min using 30 mM Tris pH 7.4, 150 mM KCl buffer with or without various concentrations of Phe. Analytical IEC was carried out on a 1 ml HiTrap Q column at a flow rate of 0.5 ml/min using 30 M Tris pH 7.4 with a KCl gradient from 20 mM to 0.4M with a variety of additives (see results).
Electrophoretic methods used the GE PhastSystem using 12.5% acrylamide gels and either native or SDS buffer strips. Western blotting on both native and denaturing gels used the WesternBreeze Chromogenic Western Blot Immunodetection Kit, WB7103 (Invitrogen). The primary antibody was MAB5278 (Millipore,10 ug/blot), which recognizes an epitope in the catalytic domain that is common to human tryptophan hydroxylase, tyrosine hydroxylase, and PAH.
Rat PAH was subjected to limited proteolysis by trypsin (Roche sequencing grade). Trypsin stock solutions were prepared in 1 mM HCl, and the total amount of HCl added to each reaction was normalized. Digestion reactions were carried out at room temperature in phosphate buffered saline pH 7.4 with PAH held constant at 0.2 mg/mL. Ratios of trypsin:PAH were varied from 1:5 to 1:500, in the presence or absence of 1 mM Phe. At various times, aliquots of the digestion were removed and the digestion was terminated with 10 mM PMSF prior to resolution of the digestion products by SDS-PAGE.
PAH activity determination used a continuous fluorescence assay monitoring the production of tyrosine [57] adapted to single microcuvette format for use with a Photon Technologies International fluorescence spectrophotometer equipped with a xenon flash lamp. Fluorescence was monitored at λEx = 275 nm and λEm = 305 nm, with excitation and emission slit widths set to 2 nm. The standard assay (200 μL final volume) contained 20 mM Na-Hepes pH 7.3, 10 μM ferrous ammonium sulfate, 2 mM dithiothreitol, 1 mg/mL catalase (to yield 2000–5000 U/mL), 1 mM Phe, 75 μM BH4, and 1–5 μg PAH at 30 °C. For experiments monitoring "order-of-addition" effects, the assay components were allowed to equilibrate at 30 °C for 5 minutes (rat PAH) or 15 minutes (human PAH) prior to starting the reaction by addition of the final assay reaction component. Quantification of tyrosine was performed by constructing standard curves of tyrosine under assay solution conditions in the presence of the relevant concentrations of BH4, which can quench tyrosine fluorescence [57].
Human and rat PAH were diluted to 0.2 mg/mL in 20 mM Na-Hepes pH 7.3, 2 mM DTT, 10 μM ferrous ammonium sulfate and 200 mM KCl in the presence or absence of 1 mM Phe. Emission spectra were recorded at 25 °C with λEx = 295 nm, and the excitation and emission slits set to 2 and 5 nm, respectively.
A protein structure model for the full length human PAH (Fig 1b, coordinates included in Supplementary Information) was prepared by combining 1) a homology model of the regulatory domain drawn from the crystal structure of a truncated rat PAH containing both the regulatory and catalytic domains (PDBid 1PHZ) with 2) the crystal structure of a truncated human PAH containing both the catalytic and multimerization domains (PDBid 2PAH). Similar straightforward combined models of full length tetrameric PAH have been presented previously [58]: they provide the standard representation of tetrameric PAH [22]. In these models the most N-terminal ordered region partially blocks access to the Fe-containing enzyme active site (Fig 1b). It is well documented that removal of the regulatory domain renders the protein constitutively active; this form is insensitive to Phe activation and has a specific activity comparable to the Phe-activated form of the full length protein [21]. Thus the tetramer shown in Fig 1b is assigned to the full length protein assembly with low or basal activity, which we designate 4mer*. The conformational change that differentiates the low activity from the high activity multimers is posited to disallow this inhibitory interaction between the regulatory and catalytic domains. A similar model for rat PAH 4mer*, where the multimerization domain is homology modeled, is included in Supplementary Information.
Formulating a structural basis for PAH allostery draws on the comprehensive review of Kaufman [21], which describes extensive biochemical, biophysical, and kinetic data on full-length human and rat PAH isolated from mammalian tissue. Although not previously interpreted in this framework, published data are largely consistent with a dissociative allosteric model as detailed for the morpheein model of allostery [14–15, 19, 59–60]. The proposed dissociative mechanism for allosteric regulation of mammalian PAH consists of an equilibrium of two structurally and functionally distinct PAH tetramers (4mer*, which is low activity, and 4mer, which is high activity) where the spatial relationship between the regulatory and catalytic domains changes dramatically. The required conformational change is proposed to be sterically hindered in the tetrameric states, and to occur at the level of a dimer, hypothetically through the pathway illustrated in Fig 1b & c. The structure of the activated tetramer is conceived to include a protein-protein interaction, which is not present in the low activity tetramer, and which provides a hypothetical binding site for an allosteric Phe. Allosteric Phe binding is supported by published Phe binding stoichiometry studies [61–62]. Based on the behavior of ACT domains, described in more detail below, allosteric Phe binding is proposed to occur at an interaction interface between the regulatory domains of two subunits (Fig 1c, coordinates included in Supplementary Information). This proposition is consistent with a recent biophysical characterization of the isolated rat PAH regulatory domain, which was unexpectedly discovered to be both dimeric and capable of binding Phe [63]. Consistent with our model, early work on the kinetic characteristics of dimeric and tetrameric mammalian PAH yielded the hypothesis that an allosteric Phe binding site is present in a tetramer and absent in the dimer [64]. According to the proposed equilibrium, 4mer* 2mer* 2mer 4mer, allosteric Phe binding would stabilize the 4mer-specific interaction and draw the equilibrium toward 4mer. The active sites of 4mer, as modeled, have unfettered access to substrates (Fig 1c). Although the data presented herein do not address whether dissociation to the dimer is an essential component of the interconversion between the low activity tetramer (4mer*) and the high activity tetramer (4mer), we provide evidence for inclusion of a PAH dimer in the equilibrium as well as evidence for additional species in the equilibration of 4mer* with 4mer. A significant unknown in modeling the pathway between the tetramers is an uncertainty as to the conformation of the most N-terminal portion of the regulatory domain in the active 4mer (see below). Hydrogen/deuterium exchange studies show this region to be flexible both before and after Phe activation [37]. Indirectly supporting a role for dimers in this pathway, the same studies show significant flexibility in the C-terminal helical portion of the multimerization domain both in the presence and absence of the allosteric Phe.
A large portion of the PAH regulatory domain contains a sequence motif known as an ACT domain, named for three allosterically regulated enzymes containing this sequence (aspartate kinase, chorismate mutase, and TyrA (a.k.a. prephenate dehydrogenase)) [33, 35]. Modeling the structure of 4mer draws on the structure(s) of ACT domains in the PDB [34] as analyzed by ProtCID and PDBfam [65–66] as well as a recent review on the role of ACT domains in the regulation of amino acid and nucleoside metabolism [36]. Analysis of ACT domains in the PDB reveals that ACT domains commonly form dimers, as illustrated in Figure 2, where the ACT:ACT interface is often seen bound to a regulatory amino acid ligand. Publications on ACT domains often cite PAH as an outlier that does not include this interface [35–36]. However, applying this multimerization motif to the commonly accepted PAH tetramer structure, 4mer*, would require a ~90° rotation of the regulatory domain relative to the catalytic domain, not unlike the ~114° rotation seen in an ACT-like regulatory domain of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) (see discussion) [67]. In building the high activity or allosterically activated PAH tetramer model, the initial step was to dimerize the human PAH regulatory domain homology model by forming an interface between the ACT domain segments. The ACT domain dimer seen in the crystal structure of phosphoglycerate dehydrogenase (PDBid 1PSD, Fig 2b) was used as a template for orienting the PAH ACT domains. The resultant regulatory domain dimer(s) were attached to the human PAH tetramer (PDB code 2PAH) at the hinge region that connects the regulatory and catalytic domains to yield the 4mer model illustrated in Figure 1c. One aspect of the 4mer model which has low confidence is the conformation of the N-terminal 33 amino acids and its orientation relative to the ACT domain; the model simply retains these structural elements from the rat PAH crystal structure. The coordinates for the human PAH 4mer model and a similarly prepared rat PAH 4mer model are both included in Supplementary Information.
The models of 4mer* and 4mer are consistent with the well documented behaviors of PAH. For example, fluorescence ascribed to Trp120 and tryptic susceptibility in a highly basic region at the C-terminal region of the regulatory domain are two characteristics well documented to differentiate low activity PAH from Phe-activated PAH [23, 68–69]. Figure 3a illustrates that these regions of PAH are in different environments in 4mer relative to 4mer* consistent with published data. Using purified rat and human PAH, described below, we confirmed the 10 nm red shift in the PAH fluorescence spectra upon addition of sufficient Phe known to provide maximum activity stimulation (~1 mM Phe) [70]. We also show that the susceptibility of purified rat PAH to limited tryptic digestion is a function of whether or not Phe is present (Fig 3b). Under the conditions shown, 1 mM Phe provides nearly total protection from limited tryptic digestion, which has previously been shown to cleave within the basic region at the C-terminal end of the regulatory domain [69]. For rat PAH (Fig 3a) the surface accessibility of basic residues in this region decrease significantly when the conformation changes from the low activity to the high activity tetramers (e.g. for Arg111, Lys113, and Arg123, modeled surface accessibilities decrease to 69, 25, and 79 percent respectively). Hydrogen/deuterium exchange studies indicate that the entire interface between the regulatory and catalytic domains is more solvent exposed in the allosterically activated form, consistent with the 4mer (more exposed) and 4mer* (less exposed) models [37]. The proposed models also provide a structural explanation for some published data on disease-associated PAH variants. For example, ex post facto inspection of 4mer reveals that Gly46, Ala47, Thr63, His64, Ile65, and Arg68 correspond to the amino acid ligand binding sites seen in ACT domain dimers (compare Figs 2b, c, d, and e), and would thus be predicted to be in the vicinity of the allosteric Phe binding site on 4mer. Notably, and in strong support of our model for ACT domain dimerization forming the allosteric Phe binding site, in vitro characterization of PAH fusion proteins for the PKU-associated variants G46S, A47T, T63P/H64N, I65T, and R68S showed that these mutations eliminate Phe binding [71].
Although the proposed allosteric and structural models are consistent with published data on mammalian PAH, a unique aspect of the morpheein model is an equilibrium of two tetrameric assemblies separated by an unusual kinetic barrier which is proposed to include multimer dissociation, conformational change in the dissociated state, and assembly to an alternate multimer structure. In particular, the novel 4mer model suggests that the alternate tetramers can be separated using ion exchange chromatography (IEC); Fig 1d shows the enhanced anionic character of the 4mer surface relative to the 4mer* surface. IEC has been useful in distinguishing between the PBGS hexamer and octamer and in evaluating how disease associated variants shift the PBGS quaternary structure equilibrium [32]. We foresee IEC as a major tool in the future evaluation of disease-associated PAH variants. Using such classic biochemical techniques to establish an equilibrium of slowly exchanging protein quaternary structure isoforms can be straightforward if one presupposes the existence of such an equilibrium in the interpretation of experimental data. In addition to IEC, particularly useful and generally accessible experimental methods are native polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography (SEC). For an equilibrium of homo-multimers, native PAGE separates on the basis of multimerization stoichiometry because all multimers of a given protein have the same charge/mass ratio. SEC provides an independent estimate of multimerization stoichiometry (size). Both native PAGE and SEC can deviate from ideal behavior due to differences in shape, and such evidence is presented for PAH to support tetramers of significantly different shape (see below). Although these techniques are relatively low resolution, as described below, together they present compelling data in favor of a PAH quaternary structure equilibrium that consists of dimers and at least two architecturally different tetramers.
Experimental data has been obtained on mammalian PAH, heterologously expressed in Escherichia coli, from three different expression vectors. Because the human protein is reported to be unstable, most of the published work utilizes either a fusion protein or fragments lacking the regulatory domain. Below we describe studies of human PAH purified with a cleavable N-terminal maltose binding protein (MBP) as well as studies with full length human PAH expressed and purified without a tag. Fortunately, full length rat PAH is reported to be more tractable and expression, purification, and characterization of the tag-less rat protein informed our approach to working with tag-less full length human PAH. Our long-range goal of understanding how PKU-associated variants perturb a human PAH quaternary structure equilibrium demands establishing a baseline for the full length wild type human protein.
Initially human PAH was expressed as the MBP-PAH fusion and was found to largely follow published behavior [28], which is consistent with the proposed allosteric model. However, in the modeled fragile protein quaternary structure equilibrium, where the long-range plan is to evaluate disease-associated variants for their effect on said equilibrium, it is not reasonable to expect the MBP-PAH fusion to reflect a physiologically relevant equilibrium representative of full length PAH. For example, an N-terminal His-tag on DAH7PS has been shown to lock the position of its ACT-like regulatory domain and prevent its rotation [67]. Various conditions used to cleave MBP from the fusion protein resulted in either incomplete digestion or undesired nonspecific proteolysis yielding PAH dimers and tetramers that were heteromeric in size, surface charge, and charge/mass ratio, and thus unacceptable for the proposed methodology.
Full length (tag-less) rat and human PAH were expressed from published pET constructs with superior expression observed for the rat protein [72]. Following the Phenyl Sepharose affinity method of Shiman [56] the yield for rat PAH was ~12 mg at >90% purity (SDS PAGE in Fig 4a) from 9.1 g of E. coli cell paste with a specific activity of 7900 nmol min−1 mg−1 when addition of PAH is used to initiate activity. Expression of full length human PAH in E. coli used a variety of protocols, all of which provided relatively poor yield. Consequently 47.7 g of E. coli cell paste was used in the classic Shiman single column purification protocol. Unlike the behavior of rat PAH, human PAH activity eluted very slowly from the Phenyl Sepharose CL-4B (PS) column and the resulting PS pool measured 670 ml. This pool was concentrated to ~15 ml at ~ 1 mg/ml where SDS PAGE showed it to be of poor purity (Fig 4b) with a specific activity of only 1200 nmol min−1 mg−1. Nevertheless, an SDS Western blot yielded a single band for PAH (Fig 4b). A portion of the PS purified human PAH was further purified by IEC, described in more detail below, which yielded ~ 1mg of pure protein (Fig. 4b).
The PS purified rat PAH was used to illustrate dramatic differences in PAH catalyzed reaction rates during assays that vary only in the order-of-addition of reaction components; this kinetic behavior is one characteristic of morpheeins [60]. As illustrated in Figure 5, when the PAH assay is initiated by the addition of BH4, the initial rate is rapid and decays as O2 becomes limiting. When the assay is initiated by the addition of Phe, the initial rate is quite slow. An intermediate rate is seen when both Phe and BH4 are used to initiate the assay. One interpretation of these anomalies is that Phe stabilizes 4mer, yielding high initial activity, while BH4 (in the absence of Phe) stabilizes a form with low or basal activity, perhaps 4mer*. IEC purified human PAH showed nearly identical order of addition effects (not shown).
Considerable information on the behavior of PAH was derived from chromatographic and electrophoretic data. Native PAGE (Fig 6a) and SEC (Fig 6b) are consistent with the freshly purified rat PAH protein (at neutral pH with 15% glycerol) being predominantly tetrameric with a small amount of dimer; the dimeric component is more apparent in the Western blot relative to the Coomassie stained gel. Close inspection of the native PAGE results suggests two different rat PAH tetramers of different architecture. Close inspection of the SEC results suggests that rat PAH defrosted from −80 °C at 37 °C may contain more than one tetrameric component. This is not seen when the protein is defrosted at 25 °C, which is the temperature later used to defrost all other PAH samples. For comparison, a Coomassie stained SDS gel of freshly purified rat PAH, shown in Fig 4a, indicates a single band at the expected 53 kDa. To address whether or not IEC could resolve components of the quaternary structure equilibrium, purified rat PAH (~100 μg) was subjected to analytical IEC in a neutral pH buffer with a KCl gradient (Fig 6c) in the presence and absence of Phe. In the absence of Phe, the chromatogram showed predominantly one peak centered at 256 mM KCl. These same buffer conditions used for SEC analysis showed predominantly tetramer with a small amount of dimer (Fig 6b). Inclusion of 1 mM Phe in the IEC elution buffer, which is sufficient to fully activate rat PAH, dramatically altered the elution profile such that the main peak is centered at 294 mM KCl. The observed increased retention of rat PAH upon exposure to 1 mM Phe is consistent with the surface charge predicted for 4mer relative to 4mer* in rat PAH tetramer models (see Fig 1d). SEC (Figure 6b) indicates that 1 mM Phe does not dramatically alter the size of tetrameric PAH, although the precise elution of the PAH tetramer is slightly earlier in the presence of 1 mM Phe, consistent with the notion that 4mer* and 4mer have somewhat different shapes. The key demonstration that rat PAH exists in an equilibrium of multiple quaternary structure isoforms is its behavior on the analytical IEC column when 50 μM Phe is included in the elution buffers, but not the PAH sample; 50 μM Phe is sub-saturating for the allosteric activation of PAH. Under these conditions, rat PAH separates into both different tetramers, as well as additional forms (Fig 6c). At 50 μM Phe, rat PAH runs as an equilibrium of the form eluting at ~260 mM KCl (presumably 4mer*), the form eluting at ~300 mM KCl (presumably 4mer), as well as additional components that elute at higher KCl (~380 mM). The appearance of additional distinct chromatographically separable components is consistent with the participation of dimeric forms in the equilibration between alternate PAH tetramer structures.
Using knowledge gained from the IEC behavior of rat PAH, the low-purity PS-purified human PAH was further purified by IEC, yielding two pools of activity (Fig 7a). The first peak, well resolved from the others, appeared pure by Coomassie stained SDS PAGE (Fig 4b) and of high specific activity. The fractions were brought to 15% glycerol and stored as the central fraction (Pool A, 6800 nmoles tyrosine min−1 mg−1), and the edge fractions (Pool A1, 6100 nmoles tyrosine min−1 mg−1). The region of disperse low activity (Pool B) coeluted with other proteins and was of lower specific activity (700 nmoles tyrosine min−1 mg−1). Coomassie stained native PAGE and native Western blots of these Pools are illustrated in Fig 7b, which shows that although Pool A is pure PAH (see Fig 4b), it contains tetramer, dimer, and some higher order PAH multimers that barely enter the gel (see Discussion).
In order to investigate whether the IEC-resolved peaks of human PAH are in equilibrium, both Pools A1 and B were dialyzed against the PS elution buffer, and individually re-chromatographed by IEC. In case of Pool A1, rechromatography resulted in several peaks, notably one at 85 minutes. In the case of Pool B, rechromatography resulted in the reemergence of the original Pool B peaks plus a small peak at 74 min. The combination of these analyses supports the conclusion that the form of human PAH that elutes at 74 min is in equilibrium with a form of human PAH that elutes at 85 min. SDS PAGE (not shown) indicates that the protein subunits do not change molecular mass during this equilibration.
IEC purified human PAH (Pool A) was used to evaluate the effect of Phe on IEC mobility (Fig 7c). In this experiment 100 μl aliquots of protein were diluted to 1 ml in the presence of Phe at 0, 50 μM, and 1 mM, followed by a 1 hour incubation prior to IEC; the results showed the main peak of human PAH migrating at 214 mM KCl in the absence of Phe and at 247 mM KCl in the presence of 1 mM Phe. At 50 μM Phe a broad peak of intermediate mobility is seen. Without the incubation, the chromatographic changes were attenuated. Although interpretation of human PAH IEC chromatograms is not as clear-cut as a similar study done with rat PAH (Fig 6c), the data indicate that the Phe induced conformational change causes the protein to be further retained on an anion exchange resin.
To demonstrate that Phe stabilizes a PAH tetramer relative to the dimer(s), the predominantly tetrameric rat PAH (~ 1 mg/ml, Fig 6a), was incubated at ~ 0.2 mg/ml in the presence and absence of ligands. Under these conditions, time points (not shown) indicate that the protein slowly equilibrates between tetramer and dimer, yielding about a 50/50 mixture after 24 h at 25 °C (Fig 8). Phe at 1 mM provided complete stabilization of a tetramer, presumably 4mer; Phe at 50 μM did not appreciably stabilize the tetramer; BH4 at 75 μM appears to promote formation of a dimer; and this effect is modulated in the presence of both 75 μM BH4 and 50 μM Phe.
We demonstrate for the first time that mammalian PAH exists in an equilibrium consisting of tetramers of two distinct architectures and that one or more dimeric forms of PAH are part of the equilibrium. The published behavior of mammalian PAH is consistent with a dissociative allosteric mechanism [21–22], which is approximated by a morpheein model of PAH allostery consisting of dimers and tetramers in the equilibrium 4mer* 2mer* 2mer 4mer; 4mer is the most active assembly and has a distinct allosteric binding site for Phe that is not present in the other components of the equilibrium. 4mer stabilization is proposed as the mechanism wherein Phe acts as an allosteric activator of mammalian PAH. In 4mer the N-terminal portion of the regulatory domain cannot sample the active-site-blocking conformation illustrated for 4mer* and seen in the rat PAH two-domain crystal structure. Although the current work does not disallow a direct equilibration between the tetramers, e.g. 4mer* 4mer, which would complete a thermodynamic box, we propose this interconversion to be sterically forbidden. We are developing the necessary tools to address this question directly as we have done for PBGS [59, 73]. A key to this demonstration is the isolation of a heterologously expressed human PAH variant whose quaternary structure equilibrium is dramatically shifted, as we found in the F12L variant of PBGS. Such variants are expected to emerge from the planned characterization of select disease associated PAH proteins.
In formulating further hypotheses about the PAH quaternary structure equilibrium, we look to the behavior of human PBGS, where a morpheein model for allostery is 6mer* 2mer* 2mer 8mer, and the high activity 8mer is a fragile assembly [31]. The human PBGS protein assembly defaults to the low activity 6mer* in response to a variety of specific point mutations, to basic pH, and to the absence of active site ligands [31]. In fact the basic arm of the bell-shaped pH activity profile is attributed to a shift in the equilibrium from predominantly high activity 8mer to predominantly low activity 6mer* [74]. Specifically, all of the point mutations that encode variants associated with the inborn error of metabolism ALAD porphyria result in protein with an increased propensity to sample the hexameric assembly [32]. A variety of small molecule ligands have been described as acting at the active site as well as allosterically to modulate the human PBGS quaternary structure equilibrium and thus modulate PBGS activity [43–44, 47–48]. A plethora of literature on mammalian PAH suggests comparable behavior wherein a variety of factors such as pH and ligand binding are reported to affect enzyme activity and protein stability. Here we consider that the reported inability of some disease associated PAH variants to achieve an active conformation may reflect an altered quaternary structure equilibrium rather than a failure to properly fold the individual subunits. Accumulation of the low activity components of the 4mer* 2mer* 2mer 4mer equilibrium is not an accumulation of improperly folded PAH; it is an accumulation of protein in a physiologically meaningful state with basal activity. Although we cannot predict the percentage of PKU-associated mutations that shift the PAH quaternary structure equilibrium, based on our experience with small molecule modulation of the PBGS quaternary structure equilibrium, we are confident that small molecules will be found that effectively shift the equilibrium toward the active 4mer.
It is well documented that PAH has a tendency to "aggregate", or to form higher order multimers, and we see evidence for this in our native Western blots (e.g. Fig 7b). The formation of 4mer from two 2mers, stabilized by ACT domain dimerization, forms a closed system without the ability to further multimerize. This is illustrated schematically in Figure 9a. However, one can model the observed higher order multimerization through the interaction between ACT domains of the 4mer* and 2mer* components of the PAH quaternary structure equilibrium. The association of ACT domains between 4mer* and/or 2mer* components always leaves an exposed ACT domain interaction surface that can further multimerize (Fig 9b). It is unknown if these higher order multimers exist in vivo or if they have any meaningful place in PAH allostery. Other multimeric allosteric metabolic enzymes such as inosine monophosphate dehydrogenase and CTP synthase have been shown to form higher order multimers that appear to function meaningfully in aspects of cell cycle control [75].
DAH7PS (E.C. 184.108.40.206) catalyzes the first step in de novo aromatic amino acid biosynthesis via the shikimate pathway. DAH7PS, like PAH and PBGS, has evolved a phylogenetically diverse repertoire of mechanisms for allosteric regulation; for DAH7PS the regulation appears to be a classic feedback inhibition by tyrosine or phenylalanine. T. maritima DAH7PS is a homotetramer in which the structural basis for feedback inhibition was recently revealed to involve a large rotation (~114°) of an ACT-like regulatory domain relative to a catalytic domain [67]. Similar to the proposed allosteric regulation of PAH, the DAH7PS domain reorientation serves to gate access to the enzyme active site; this is not unlike the PBGS case wherein allosteric regulation modulates the opening/closing of an active site lid. In all three cases, the fundamental difference between the low activity state and the high activity state lies in the modulation of active site access, not in any repositioning of active site residues. Strikingly similar to the proposed PAH regulatory model, allosteric regulation of T. maritima DAH7PS involves dimerization of the ACT-like domain and tyrosine is seen bound at the domain interface (Fig S1). Strikingly dissimilar is the dimerization mode (Fig S1). However, unlike PBGS and PAH, there is no evidence to date that allosteric regulation T. maritima DAH7PS involves tetramer dissociation.
The authors gratefully acknowledge support from the Developmental Therapeutics program at the Fox Chase Cancer Center, from the National Institutes of Health, National Cancer Institute Comprehensive Cancer Center grant P30CA006927, and the National Institute of General Medical Sciences R01GM84453 (RLD). We acknowledge contributions from the FCCC Molecular Modeling Facility, the Glasswashing Facility, and the DNA Sequencing Facility.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Outdoor Hour Challenge
Bat Nature Study 2016
Outdoor Hour Challenge – Summer Bat Study:
Use the ideas found in this archived challenge to learn more about bats and their sense of hearing. There are also links to additional resources and a video that will make this study come alive even if you don't have access to a live bat to observe.
Need help getting started with your nature study using the Handbook of Nature Study? Check out the fresh "Getting Started" page here on the website!
Don't forget that Ultimate Naturalist and Journey level members have access to the brand new Learning About Birds ebook!


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The Malik Report
Practice post: Jurco iffy for Tuesday tilt, Howard traveling w/ team; Panthers need 4-point game
by George Malik on 01/26/15 at 05:35 PM ET
Comments (1)
Updated 2x at 7:47 PM: The Red WIngs took part in a less-than-revelatory afternoon practice in Taylor prior to flying to Miami to take on the Panthers (Tuesday @ 7:30 PM EST on FSD Plus) and Lightning (Thursday @ 7:30 PM EST on FSD).
Ahead of a trio of "trap games" (Saturday's home tilt against the Islanders included) and a very early 4 PM-scheduled flight for the Wings, the Macomb Daily's Chuck Pleiness and the Free Press's George Sipple added some intriguing twists to the mix.
Sipple revealed that Jimmy Howard's going to travel with the Wings...
Jimmy Howard said he skated for 15 minutes today and will go on trip.
Howard said he hasn't watched replay of injury.
Sounds like Jimmy Howard is still a couple weeks away from playing.
And that Tomas Jurco may or may not supplant Daniel Cleary from the Pulkkinen-and-Franzen-less lineup:
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Practice Tweets: Wings get back to work minus Howard
by George Malik on 01/26/15 at 03:00 PM ET
Comments (7)
Updated 2x at 2:38 PM: The Detroit Red Wings got back on the ice a little late, but they did return to work on Monday afternoon--ahead of a week of "trap games," starting with tomorrow night's tilt against the Kryptonite, I mean Florida Panthers (7:30 PM on FSD Plus)--and I'd gather that they're practicing in Taylor because the Joe was used for the auto show.
The Macomb Daily's Chuck Pleiness reports that the Wings' goaltending situation remains the same for the present moment...
No sign of Wings goalie Jimmy Howard on the ice at Taylor SportsPlex. It's Mrazek, McCollum and Gustavsson.
Xavier Ouellet on the ice. That most likely means Kindl still isn't ready to play and Ouellet will be in the lineup Tuesday in Florida.
(I'm just glad that Ouellet's sore wrist has healed faster than Kindl's elbow)
Tomas Jurco on ice. In White.
White jersey = bottom-couple lines jerseys, red = top lines jerseys, black = defensemen, goalies, gray = no-contact.
And this, we've talked about:
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A rare speculatory post: Are concussions catching up to Johan Franzen?
by George Malik on 01/26/15 at 02:19 PM ET
Comments (27)
So Wings fans are having a discussion on Twitter, and I suppose it's worth discussing here as well.
You know I don't like speculation--I'd rather discuss facts and educated guesses, and issue an honest, "I don't know" when we delve into the land of speculation, but now is as good a time as any to have this conversation.
Mike Richards is on waivers with a nearly immovable contract, and in a month, we might be calling what's happening with Richards (Kings tried to trade him, could find no takers) the "treatment" given to a waived, AHL-bound Jakub Kindl...
The 29-year-old Richards has "comparables" in terms of duration of contract and inconsistencies in one Johan Franzen, who's signed through the 2019-20 season (per SporTrac), and at 35, has increasingly missed time due to concussion-related issues over the past 3 years.
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The Red Wings' 'Western Canadian' road trip video chronicle concludes with 'Calgary'
by George Malik on 01/26/15 at 01:46 PM ET
Comments (0)
The Red Wings have posted the fourth and final installment of Dan Mannes and Kyle Gajewski's videos chronicling the team's Western Canadian road trip, and as so many of you have stated, while these videos are of brief duration, they're sure as hell better than anything from the 24/7 experiment. Far, far, far superior quality and quantity here:
Off to Vancouver!
Filed in: | The Malik Report | Permalink
The District Detroit's about more than a rink
by George Malik on 01/26/15 at 01:06 PM ET
Comments (0)
The Milwaukee Journal-Sentinel of all publications examined the Wings' rink-and-entertainment district "from afar," and the Journal-Sentinel's Don Walker penned the article to serve as a compare-and-contrast situation to the Milwaukee Bucks' push for a new, partially publicly-subsidized arena.
This part, we haven't talked about, and yes, it's worth posting on a hockey blog:
Once the housing and other development is built, will people come?
Robert Gibbs, president of Gibbs Planning Group, an urban retail planning consulting firm in suburban Birmingham, Mich., says there is a strong market in the downtown area for entertainment, restaurants and what he calls community service retail outlets like Target.
"Within five miles of the project, there are 300,000 people," he said. "A lot of people who live in that area have to drive to the suburbs for shopping," Gibbs said.
"There is no doubt Detroit is on a roll," added [Olympia Development's Steve] Marquardt. "The city and district are well positioned for growth in the years to come. People will be drawn to the district because they have high interest in the area."
"In general, it's a desert there for certain services," said Castaneda-Lopez.
Walker continues, and while the rink is most likely to be the one part of the project built by the summer of 2017--mostly because the surrounding economic development will probably be delayed due to City, County and State finaggling--but it's that surrounding development and the realization of the "neighborhoods" promised when Comerica Park was built that will tell us whether the $650 million investment is really worth it.
Filed in: | The Malik Report | Permalink
Hello out there, we're back on the air
by George Malik on 01/26/15 at 12:38 PM ET
Comments (24)
Today is both a practice day for the Wings and the first day "back at work" for me in three-and-a-half weeks. I know that some of you aren't thrilled when I talk about my health, but I weathered one of the worst depressive episodes I've had in a couple of years (and don't worry, you can scroll on down to actual hockey talk).
Filed in: | The Malik Report | Permalink
This Week
by Paul on 01/26/15 at 09:20 AM ET
Comments (0)
from Ansar Khan of Mlive,
Here are five things to watch this week
1. Florida sliding out of playoff contention
The New York Rangers, Washington and Boston have distanced themselves from Florida in recent weeks. The Panthers (20-14-10) are winless in four (0-3-1) and are seven points behind the Capitals and Bruins for the final wild card playoff spot in the Eastern Conference.
The Panthers have won both meetings this season from the Red Wings (4-3 and 3-2 in a shootout, both at Joe Louis Arena). Florida is 5-2-0 vs. Detroit since the start of last season, winning three times in a shootout.
Florida has difficulty scoring, with only two players in double figures in goals - Nick Bjugstad (15) and Jimmy Hayes (13).
2. Division lead on the line in Tampa Bay?
Thursday's game at Tampa Bay could be for the Atlantic Division lead, at least temporarily.
Tampa Bay (30-14-4) has won three in a row and is 10-3-0 in its past 13, but leads the Red Wings by only one point.
Filed in: | The Malik Report | Permalink
Gave: Wings don't expect Howard or Gustavsson to return until early February
by George Malik on 01/25/15 at 06:18 PM ET
Comments (4)
The Red Wings' situation in goal isn't particularly complicated over the short haul. According to Fox Sports Detroit's Keith Gave, Jimmy Howard (groin) and Jonas Gustavsson (shoulder) are at least a week or two out, so Petr Mrazek and Tom McCollum will continue to take the reins for the next week or two...
"With Gustavsson, the plan right now is for him to practice Monday, Tuesday, Wednesday and Thursday, and then we'll sit down on Thursday and have a conversation about what to do," [Red Wings GM Ken] Holland said, acknowledging that if Gustavsson is cleared to play there's a good chance he'll get a game or two with Grand Rapids before returning to the Detroit roster.
"With Howard, we're hoping he's to a point where he could do some off-ice work with our trainers," Holland said. "He just had five days off for the All-Star break. If he didn't go to Florida, he'd be looking at 9-10 days by himself. I would think he'd come with us so we can slowly start ramping him up."
And as such, Jared Coreau and Pat Nagle will tend the net in Grand Rapids. That works for now, as Gave notes...
Filed in: | The Malik Report | Permalink
Red Wings TV posts parts 2 and 3 of Western Canadian chronicle
by George Malik on 01/25/15 at 01:01 PM ET
Comments (0)
The Red Wings posted an "opener" of a behind-the-scenes set of videos that will chronicle their Western Canadian road trip on Friday...
Yesterday, they posted a behind-the-scenes clip of the team's visit to Vancouver...
Filed in: | The Malik Report | Permalink
AHL All-Star festivities cap off a busy week for Teemu Pulkkinen
by George Malik on 01/25/15 at 12:07 PM ET
Comments (0)
The AHL is holding its All-Star skills competition tonight at 8 PM EST and its All-Star Game tomorrow at 7 PM. The AHL will stream both events online, and Fox Sports Detroit (click for all the AHL ASG's TV options) will air the ASG live and tape delay the skills competition to 10 PM on Monday night.
Teemu Pulkkinen's the Grand Rapids Griffins' lone representative due to Xavier Ouellet nursing a sore wrist, but Pulkkinen will start for the Western Conference on Monday, and even after spending a week and a half in Detroit, he sits 2nd in AHL scoring with 21 goals, 22 assists and 43 points registered over the course of only 36 games played.
His sojourn with the Wings ended with Mike Babcock promising to send video to Jeff Blashill to ensure that Pulkkinen graduates to the NHL on a full-time basis having remedied his few deficiencies, and Pulkkinen telling Fox Sports Detroit's Keith Gave that he knows he has room for improvement, and tonight and tomorrow's activities represent the end of a particularly grueling schedule for Pulkkinen, as the Grand Rapids Press's Peter J. Wallner noted:
Filed in: | The Malik Report | Permalink
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About The Malik Report
The Malik Report is a destination for all things Red Wings-related. I offer biased, perhaps unprofessional-at-times and verbose coverage of my favorite team, their prospects and developmental affiliates. I've joined the Kukla's Korner family with five years of blogging under my belt, and I hope you'll find almost everything you need to follow your Red Wings at a place where all opinions are created equal and we're all friends, talking about hockey and the team we love to follow.
Most Recent Blog Posts
Red Wings to hold Hockeytown 'BrewHaHa' on Thursday, July 28th
Big Gap Exists With Petr Mrazek's Arbitration Numbers
The Latest On Danny DeKeyser And Petr Mrazek Before Their Arbitration Hearings
Looking back at Riley Sheahan's 15-16 campaign
Discussing Luke Glendening's pluses and minuses
'Eastside Elite Hockey League' All-Star Game goes well
Frans Nielsen's Role With The Wings
Examining the Wings' OHL-playing prospects
Introducing Hockey To The Detroit Youth


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Have you ever wondered if a lava lamp would work on Jupiter?
Yeah, me neither, but Neil Fraser did. To find out he built himself a centrifuge and put a lava lamp on for a spin. The resulting rotations cranked up the G-force on the lamp to around 2.3, or approximately the same that you'd find on Jupiter
Neil Fraser's Lava Lamp Centrifuge via We Interrupt
Are you on Twitter? Follow me (@sheagunther) there, I give good tweets.
And if you really like my writing, you can join my Facebook page.


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Read the July issue of Petfood Industry!
July 2016
The July 2016 issue of Petfood Industry covers a Canadian specialty pet food company with a "quality first, pets first" philosophy behind all of its products.
PTI-Europe, of Plastic Technologies Inc., offers Deep Grip packaging, which uses injection stretch blow molding to create container handles. The technology used enables grip depth of more than 1 inch on either side, with less than .01 inch grip "web" thickness. Applications include liquid and dry contents.


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NET Sports sat down with Team USA Volleyball player, Jordan Larson to talk about her time at the 2012 Olympics in London. In this question, Jordan explains the difference between playing college volleyball vs. professional international volleyball.
live streaming schedule & on demand videos
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Mile Hi-Fidelity playlist - 03.11.09
Ah, good to be back. After a week off, Andy and I were back behind the mike once again with two more hours of handpicked local goodness. Unfortunately, the stream was whacked again last night, so we weren't able to record the show, but as always, you'll find the complete playlist after the jump. It's too bad, really, that we don't have a recast today, because we were joined last night by Kurt Ottaway, a true icon of the Denver scene who's virtually done it all, from releasing a number of great records on his Denver Coffee Achievers imprint, to recording countless bands, to running the Tarshack, one of Denver's fabled underground spaces, to mixing countless other acts at the Climax, to fronting a trio of stellar outfits, Twice Wilted, the Tarmints and the Overcasters. Kurt, whose band is heading to SXSW next week, was the first of what will hopefully be many guests on a segment we've dubbed the Takeover, in which an active member of the scene -- be it a musician, fan or otherwise -- takes the show over for a set of songs. In addition to spinning an Overcasters track, he played and talked about tracks by the Fluid, Gangcharger, Blue Million Miles and Sonnenblume. Thanks, Kurt!
Mile Hi-Fidelity - 03.01.09
Alan Alda - Red Sky Morning
Magic Mice - Dreams of Reason
Black Market Empire - Drop Dead Disco
Petals of Spain - Starting Anew
Roger Roll - It's All the Things
Goodbye Champion - Outside Looking In
Overcasters - Way of the World
The Fluid - Candy
Gangcharger - Kathy In the Quarry
Blue Million Miles - Over the Fall
Sonnenblume - Hold On
L'elan Vital - Endlessly Indepted
Fresh Breath Committee - Why They Do What They Do
Spoke-In-Wordz - Bringing It Back
Air Dubai - Baby I'm a Mess
Bionik Brown - The Thug Inside
Life In Electric - Dead and Buried
Statewide Emergency - Midnight Creeper
Black Lamb - Widowmaker
CacheFlowe - Casio vs. Heavy Metal
Lib Exploration - Greg Harris Vibe Quintet
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Derek Steven West
Position: T
6-8, 309lb (203cm, 140kg)
Born: March 28, 1972 in Denver, CO
College: Colorado
Weighted Career AV (100-95-...): 0 (15060th overall since 1960)
Draft: Indianapolis Colts in the 5th round (149th overall) of the 1995 NFL Draft.
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Woven Wednesday: Free Shipping on 1+ Yards of any woven fabrics through 11:59 p.m. EDT July 27th.
I am a fashion designer and illustrator, with spoonflower I intend to broaden my horizons a bit. I never find the right fabrics for my designs. From now on I know this will change!
© 2008-2015 Spoonflower Inc


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James N. Miller, editor and proprietor of the Sac Sun, is a native of Pa., and was for several years editor of the Republican, published at Waynesburg. In 1870 he came to Iowa and located at Eldora, Hardin County, remained only a short time, then came to Sac City, and established the Sac Sun, which has grown to importance and is the official paper of the county. In 1878 He built the new office, which he now occupies. The paper is an eight-column folio, and has a circulation of eight hundred. Mr. Miller is prepared to do first-class job work of all kinds.
Biographical Sketch of James N. Miller
MLA Source Citation:Western Publishing Company History of Western Iowa, Its Settlement and Growth: A Comprehensive Compilation of Progressive Events Concerning the Counties, Cities, Towns and Villages--biographical Sketches of the Pioneers and Business Men, with an Authentic History of the State of Iowa. Sioux City, Iowa: Western Publishing Company. 1882. AccessGenealogy.com. Web. 24 July 2016.
https://www.accessgenealogy.com/iowa/biographical-sketch-of-james-n-miller.htm- Last updated on Aug 16th, 2012
This page is part of a larger collection. Access the full collection at Biographical Sketches of Sac County Iowa.


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- SA - Adelaide
Editor's Pick
National Galleries and Museums
A State by State Guide to Australia's Greatest Galleries and Museums
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