text:
If there is a Doomsday clock somewhere, one that tells us how many minutes we're away from 30 mushroom clouds over 30 NHL cities, you should know that it started ticking at 11 a.m. and there doesn't seem any way to stop it. Nobody knows exactly when midnight is, but the last faint hopes for a peaceful resolution before then will be exhausted today when Gary Bettman and his ownership committee are expected to reject the NHLPA's latest offer and counter with the one thing players vowed would kill the season on contact: A link between revenues and salaries.
With 87% of NHL players in a recent Hockey News poll saying they'd rather stay out than accept any form of cost certainty, and with Bettman needing only eight ownership votes to keep a salary cap on the negotiating table, it'll take a Christmas miracle to save the season.
"We're getting down to the last hours,'' said Oilers' player rep Ethan Moreau. "If the owners' next proposal is the same as their last one, with no concessions at all, just a salary cap, then we're in trouble. The season will be lost.''
"We're not going to accept a salary cap,'' added winger Brad Isbister. "If they reject it and come back with a cap, we're just that much farther apart.''
An NHL memo leaked yesterday confirms the league will indeed reject the PA's offer - something players knew last week when Bettman said a 24% salary rollback wouldn't fix serious systemic problems - but the memo doesn't spell out details of the counter-proposal. If there's a cap in it, all we can do is watch the clock.
"You'd have to be pretty naive to think they'd just agree to our terms ,'' said Moreau. "We knew they wouldn't accept it, but they have to work with us. If there's something in their proposal that gives us even a window to negotiate, that's all we want.
"If they come back with a hard cap, what do we do? Come back with another proposal? Give up our first-born, too?''
If that baby is wearing a salary cap, they'll talk. If not, get ready for a nuclear winter.
"I don't expect a quick resolution one way or the other,'' said Edmonton Investor's Group chairman Cal Nichols, who has shown steely resolve throughout the battle. "What we're going through is just a process; it's gone on longer than you wish it would have and I feel badly for those who miss the game as much as most of us do, yet there's no short-cutting this.''
That's the diplomatic way of saying "as long as it takes.''
Because of a gag order handed down by the NHL, Nichols isn't allowed to speak on whether or not the season can be saved, but he did say the PA's highly publicized rollback would not be enough to save the Oilers, who have "extended their thoughts and opinions to the negotiating committee.''
"My opinion, and that of the Edmonton Oilers, is that what's on the table is an enticement for teams to keep doing, essentially, what they've been doing,'' he said. "And the Edmonton Oilers aren't going to be here long term if that's what we're prepared to accept.''
Nichols says the one-time salary rollback is barely even a short-term fix.
"It was a recognition that we really do have significant financial problems. At least that much was accomplished by it, and appreciated,'' he said. "But the free spenders, what are they going to do the day (the deal) is in place?''
Spend. But if that's the case, Moreau says the owners' beef is with the owners, not the players.
"Why should we have to hold the hands of owners who are brilliant businessmen? Why do we have to baby sit them through the process? We're going to give them a new marketplace, then it's up them. It's frustrating that we have to protect them from each other.''
It's frustrating to everyone, but that's where we are. And until Colorado, St. Louis and Dallas are no longer using Edmonton and Calgary as farm teams, there's no hockey. And if 30 mushroom clouds go off, most of those 30 cities are going to be OK with that - the U.S. markets because they don't care, the Canadian ones because they do.
"Our fans have been outstanding,'' said Nichols. "They have a clear understanding of the philosophy of this team and what it's going to take to keep it here.
"As this goes on I just remain very much at peace and confident that it'll work out.''
Just not this year.


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31st January 2011
In July of 2010 British mountain biker Dan Atherton sustained a terrifying injury. A crash at his monster sized dirt jumps in North Wales resulted in a break of the C1 Disc in his neck. Just about the worst injury a rider could sustain and survive. Dan's constant commitment and dedication that he puts into his riding was then needed to get him through the recovery. With months in a brace and none of his life's usual manic activity, there were low moments along the way and plenty of time for contemplation. Dan's determination to recover was only enhanced by his desire to return to the highest level of the sport that he has achieved so much success in before.
On the 20th January, Dan received the results of a final CT scan from Dr Robert Bray, the spinal specialist who has been overseeing his treatment. Red Bull's Darren Roberts, the Atherton families personal trainer, accompanied Dan and had this to report, "The fractures have all healed and the tiny fragments of bone which are loose will be absorbed by the body over time, there was a slim possibility that an operation was required to remove these – but thankfully that op is not needed. More importantly Dr Bray said the words we've been waiting to hear since July 2010, 'you're good to go Dan, totally healed – get back on your bike and get riding hard. Try not to break it again'."
It is with great happiness that we can announce Dan is now back on a bike and in full training, this moment was never a certainty as the injury to the C1 Disc was one of the most catastrophic ever seen. With strength and conditioning training to get him back to full race fitness his aim is to return to World Cup competition at the second round of the series in Fort William, Scotland. Concentrating on the discipline of downhill he's hoping to take his new Commencal Supreme bike back to the podium glory he enjoyed before this incident.
The Atherton team and family would like to thank all the sponsors and friends that have stood by Dan through this difficult time and worked with him to get back to this point where he can continue to shine in a sport to which he has brought so much of his unique attitude, style and speed. Here's to the return of Dan Atherton to his rightful place, on a bike.

subset: commoncrawl


text:
Hey guys so let me tell u my situation:
1. Had a Soft Modded Wii 4.2 but i wanted to play Metroid other M so badly I formatted my Wii.
2. It still wouldn't let me update to 4.3 to play Metroid, nor did it let me install the update from the disc.
3. Re-Soft Modded my Wii to play off disc with NeoGamma and it worked fine up until a certain cutscene.
4. Installed to HDD and it stopped again at the exact same cut scene
5. Returned Metroid other M and got a new one
6. Played off NeoGamma and it stopped EXACTLY at the same part AGAIN
7. Re-Formatted my wii system memory
Please guys i just want to play this game! I REALLY NEED HELP!
1. First-time posters are asked to make a quick "hello" post in the Introductions section of the site;
2. We have a guide for this, in fact I blogged emuhack's guide on the main page;
3. Read it and follow it; if any questions, you are to post them within the guide --- not creating another thread.
4. Welcome to the site.


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Tag: Pope Benedict XVI
Catholic higher ed leader Father Stromberg dies
A priest who helped bring Pope Benedict XVI to Minnesota has died.
Pope Benedict has no regret, doubt about decision to retire, aide says
Retired Pope Benedict XVI has never doubted or regretted his decision to resign, knowing it was the right thing to do for the good of the church, said Archbishop Georg Ganswein, prefect of the papal household and personal secretary to the retired pope.
MN Catholics express sadness, thanks
Early Monday morning, Marcelle Diedrich was half asleep when she heard the news on her radio that Pope Benedict XVI will resign at the end of the month.
Pope's decision shows concern for good of the Church, say Archbishop Nienstedt, Bishop Piché
Archbishop John Nienstedt said he was surprised like everyone else around the world when he heard the news about Pope Benedict's resignation for health reasons.
My experiences of the Church in China
Last summer I decided to teach English in China with the Maryknoll Missionaries and six other seminarians as my summer apostolic work for the Pontifical North American College, where I currently reside for major seminary studies.
Quotes from this week's newsmakers – March 1, 2012
"The service of charity is a privileged form of evangelization in the light of the teaching of Jesus, who will count what we have done for our brothers and sisters — especially the smallest and most overlooked — as something done for him."
— Pope Benedict XVI, speaking Feb. 24 to members of a Rome-based charity
A 'faith app' would be helpful
One of the lesser effects of the adoption of the new translation of the Roman Missal is the ubiquitous presence of worship aids.
Quotes from this week's newsmakers — December 8, 2011
"If you've been away, come home to your parish, and visit CatholicsComeHome.org today."
— Part of the message of a new advertising campaign airing on major television networks Dec. 16-Jan. 8 sponsored by the Atlanta-based organization Catholics Come Home, which seeks to bring Catholics back to church


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This luxurious cream gently soothes, calms, hydrates and balances sensitive, irritated and reactive skin. Thoroughly moisturizes all skin types.
|• 100% Vegetarian
• Fragrance Free
• Paraben Free
• Organic Ingredients
• Natural Ingredients
• Cruelty Free
• Made in the USA
|1 oz jar:
[cc_product sku="roha-205″ display="inline" quantity="true" price="true"]
Combine a dime-sized amount of product into your palm with OHA serum and mist. Massage into skin with upward strokes to face and neck until absorbed. You may also apply to clean skin as needed.
distilled water, aloe barbadensis*, vegetable emulsifying wax, radish root ferment filtrate (leuconostoc), apricot kernel (prunus armeniacae) oil, coconut oil (cocos nucifera) oil*, cocoa (theobroma cacao) butter*, sunflower seed (helianthus annus) oil*, beeswax (cera alba), rice bran (oryza sativa) oil, shea (butyrospermum parkii) butter*, MSM(methylsulfonylmethane), green tea (cameilla sinesis) extract*, cranberry (vaccinium macrocarpon) seed oil, avocado (persea gratissima) oil, vegetable glycerin*, stearic acid, tocopherol vitamin E (non GMO), Ester-C (vitamin C), hydrolyzed oats, acacia senegal gum*, guar gum, xanthan gum
* Organic


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This is American Idol.
Other than "Georgia wins," that has to be my favorite phrase in the English language. Yes, folks, it's that time of the year again. The pop cultural phenomenon and TV juggernaut is returning for Season 8 on Tuesday (8 p.m., Fox). And this blog is going to be with you every step of the way until a new winner is crowned in May.
First things first, let me tell you a little about me. I'm a copy editor on the sports desk here at the Chronicle. But more importantly – as far as readers of this blog should be concerned – I'm an avid viewer of American Idol. I've been hooked ever since I stumbled upon the reality competition in the summer of 2002 and watched Kelly Clarkson, a virtually unknown singer-songwriter, become a pop superstar overnight. I host viewing parties for big episodes. I attend the Idols Live! concerts (I drove to Charlotte last year). I passionately debate friends – and sometimes myself – about the contestants' song choices and the judges' critiques. And I usually end up writing down those thoughts. So here we are.
You can expect recaps every night after episodes, which air Tuesday and Wednesdays along with Thursday on occasion. I'll also provide as much news and gossip as I can throughout the week. I'm going to want to hear what you have to say, too. Your thoughtful – and respectful – opinions are always welcome in the comments section.
I'll have more for you tomorrow. In the meantime, check out this USA Today article from over the weekend, which should serve as a good preview of the upcoming season. And then this Entertainment Weekly profile on Idol's new judge, Kara DioGuardi.


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Standard character mark
Electric apparatus for cooking foods, namely, electric grills, electric meat grills, electric barbecues grills
Business in Rumilly, FR


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Maurizio Bocchetta0000-0003-0447-3262
Maurizio Bocchetta graduated in biology in 1991 at the Sapienza University of Rome, Rome, Italy. After a fellowship at the Palo Alto Medical Foundation, Palo Alto, Calif, USA, he received his Ph.D. degree in human molecular biology at the Sapienza University of Rome in 1996. Dr. Bocchetta was a Postdoctoral Fellow at the Department of Medicinal Chemistry of the University of Illinois at Chicago (1996–1998) working under the mentorship of Dr. Alexander S. Mankin. During these years Dr. Bocchetta studied the structure and function of the peptidyl-transferase center of the E. coli large ribosomal subunit. In 1998, he joined the Loyola University Chicago Medical Center as a Research Assistant Professor. Dr. Bocchetta's primary research interest has been the pathogenic role of Simian virus 40 (SV40) in the development of malignant mesothelioma, the mechanisms of DNA viruses-mediated cell transformation, and interaction of the major SV40 oncogenes with primary mesothelial cells' proteins. More recently, Dr. Bocchetta's laboratory has shifted the focus of its research on hypoxic tumor microenvironment, the role that developmental pathways play for hypoxic tumor cell survival, quiescence, and regulation of metabolism. These investigations are aimed at finding novel therapeutic strategies for the treatment of hypoxic lung cancer and mesothelioma tumor microenvironment. Dr. Bocchetta's findings have been published in high-quality scientific journals, including Proceedings of the National Academy of Sciences, Cancer Research, and Oncogene.
Biography Updated on 14 August 2012


text:
Rudy: Not your grandparents' EC2 deployment tool.
WWW: http://solutious.com/projects/rudy/
make generate-plist
To install the port: cd /usr/ports/net/rubygem-rudy/ && make install cleanTo add the package: pkg install rubygem-rudy
cd /usr/ports/net/rubygem-rudy/ && make install clean
pkg install rubygem-rudy
PKGNAME: rubygem-rudy
SHA256 (rubygem/rudy-0.9.8.020.gem) = 3d511ec5d0b8117c90ec7a6becc9a9862a6db4cf4b76b02dfc95097719bc062c
SIZE (rubygem/rudy-0.9.8.020.gem) = 106496
NOTE: FreshPorts displays only information on required and default dependencies. Optional dependencies are not covered.
No options to configure
gem shebangfix
Number of commits found: 9
Convert USES=gem:autoplist to USES=gem since autoplist is default
create USES=gem and update rubygem- ports to use it
Note that for now ports still have to have USE_RUBY=yes to use USES=gem
Differential Revision: https://reviews.freebsd.org/D6070
Remove ${PORTSDIR}/ from dependencies, categories m, n, o, and p.
With hat: portmgr
Sponsored by: Absolight
- Add LICENSE_FILE
Approved by: portmgr (blanket)
- Fix shebangs
- Add NO_ARCH
Approved by: portmgr blanket
MFH: 2015Q3 (blanket)
- Add LICENSE
PR: ports/189034
Submitted by: sunpoet
- Stage Safe
Add NO_STAGE all over the place in preparation for the staging support (cat:
Rudy: Not your grandparents' EC2 deployment tool.
WWW: http://solutious.com/projects/rudy/
Feature safe: yes
Servers and bandwidth provided byNew York Internet, SuperNews, and RootBSD
No vulnerabilities have been reported in the past 14 days
* - modified, not new
All vulnerabilities


text:
The Top 8 Headlines You Might Have Missed / Haaretz Newsline, October 4
From the tension along the Turkey-Syria border to the first presidential debate; Haaretz.com brings you the top headlines you might have missed.
Turkey authorizes military operations outside Turkish borders if the government deemed them necessary, a day after artillery shelling from Syria killed five civilians in a Turkish town.
The European Union is poised to ban imports of Iranian gas into Europe as part of its efforts to ratchet up pressure on the Islamic Republic over its nuclear program.
The first presidential debate proved what the American people had plenty of opportunities to discover in the past few years: that both Barack Obama and Mitt Romney are not eager to risk being caught saying a provocative phrase.
Six months following the eviction of Jewish families, the Civil Administration rules that due to flaws in the agreement, the purchase cannot be approved.
Asylum seekers and Israeli human rights groups petitioned a Jerusalem court to prohibit Interior Minister Eli Yishai from 'hysterical and barbaric' decision to round up and jail Sudanese citizens in Israel.
Modi'in municipality's decision to bar non-residents from the main city park during the intermediate days of Sukkot led to some confrontations at the park's gate, when out-of-town visitors asked to enter the park.
A fervent congregation of about 35 people in San Nicandro has won the embrace of Italy's Orthodox Jewish mainstream community, part of a new effort to reach out to so-called Returning Jews in the south.
South African groups expressed support for Cyril Karabus, an elderly doctor arrested in Dubai in August on manslaughter charges stemming from a 10-year-old case.
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Rapture, the result of a failed utopian society lying rotting at the bottom of the Atlantic Ocean, our first encounter with this mysterious metropolis gave but a small glimpse into a world of 'moral freedom' and what man could really achieve when 'petty morality' is pushed to one side. With the success of the original title a sequel was sure to follow but can Bioshock 2 rekindle and even surpass its predecessor?
Bioshock 2 initially takes place in 1958, 2 years before Bioshock with an opening cinema as seen from the perspective of your character, it introduces both the villain and yourself before abruptly ending. Skip 10 years later to 1968, 8 years after Bioshock you find yourself, a big daddy, alone with no little sister to protect. Andrew Ryan has been dead for almost 10 years now and Rapture is ruled by the somewhat vindictive Psychiatrist Sofia Lamb. The introduction of Lamb into the Bioshock universe and the way she fits into the world is up for debate. As with the original Bioshock a lot of the story in the game is found on audio logs found around Rapture, these logs can give intimate details on a person's life or a broad overview of the state of Rapture during its fall from grace. In the audio recordings there are many in which Ryan is debating with Lamb over meaning of Rapture, this shows that Lamb was at that time a well known figure yet she wasn't mentioned in the first Bioshock and not only that the audio logs in Bioshock 2 go out of their way to put provenance on the fact that Lamb was there during the entire course of the first game, make of it what you will.
The story really has a timeless quality to it, where Bioshock chose the route of misdirection to prove a point and shock its players Bioshock 2 goes for the more direct approach where every twist is predictably clichéd and revelations can be guessed easily within the first few hours of the game. That's not to say the game is badly written, far from it. The character dialogue is on par with Bioshock if not even surpasses it in places with Ryan's famous "What is a man?" speech receiving a reworking to fit the mood of the sequel.
For the sequel there have been some pretty hefty changes to the combat with the most noticeable being the ability to dual wield plasmids with weapons at the same time, this gives the player the advantage to unleash a flurry of damage on enemies or continually refreezing a tougher enemy on the spot while you empty clip after clip of ammo into them.
You'd think with this new ability then the game would be a cake walk, far from it Bioshock 2 introduces two new forms of enemies, the first, the Big Sister is a female form Big Daddy who is more agile than yourself and can utilise plasmids much the same way you do, she only appears after you have either harvested or saved the all the little sisters in an area. The other addition is the Brute splicer, the Brute is similar to a Big Daddy in terms of damage caused to you and the amount of damage he can take, the brute is large swollen splicer who can lift and throw heavy objects to inflict damage. The Brute can really change the favour of a fight especially if you have to deal with several regular splicers all at once too.
All original Plasmids are present in all their destructive glory along with a few new additions such as the Scout plasmid which I personally didn't find to be very useful, but then again some of the original plasmids weren't all that useful either so no harm done. What is most impressive though is the level 3 plasmids, with the Electro-Bolt being turned into a handheld thunderstorm or the Fire plasmid becoming a handheld flame thrower, you can guarantee to really feel the power of this new level of plasmid with each and every enemy you take down.
Since you play the game as a Big Daddy you must be wondering "Do you get to use little sisters to collect ADAM like the little sisters from Bioshock did?" Well the simple answer is yes, as with the first game you will be pitted against Big Daddies though this time once you dispense with them you can either Harvest or choose to Adopt their little sister, if you Adopt a little sister then she will ride on your shoulder until you find a glowing corpse which she can draw ADAM from. And this is where the new weapon, the Rivet Gun comes into play. The Rivet gun is a must for Big Daddies as it has an ammo mode called 'Trap Rivets' trap rivets are rivets that when fired don't just disappear but instead stick to any surface they are fired at... including splicers, this means you can protect your little sister when she's harvesting ADAM from the waves of splicers that come your way.
You may think that all of this is a waste of time and yes you don't need to harvest ADAM to complete the game, either saving or harvesting the little sisters would be enough but if you do chose to push that little bit more you'll find you'll be able to afford all the health and EVE upgrades whenever you need them and you won't have to scavenge for ADAM as you'll always have an ample supply to buy new plasmids and stay one step ahead of the enemy.
The moral choice aspect from Bioshock returns in not just the form of whether to save or harvest the little sisters but during the course of the game you will meet characters each in different circumstances and you will be forced to either save them or kill them, these choices along with the little sisters can affect the ending on multiple levels which is a much more complex and personal way to determine the ending as not everyone will kill all the characters or only chose to kill one or two and leave one due to various reasons, all of these choices change the ending for better or worse.
Right from the beginning you'll be greeted with the regular Bioshock style when it comes to music, old 40's/50's tunes echo down corridors and give a sense of age and nostalgia to what can be described as walking around an old photograph. The voice acting as with the original Bioshock is fabulous, no cheesy, lifeless performances here instead you get to hear firsthand the bitterness of Lamb, dismay of Ryan or the loving compassion of Eleanor are all acted with sincerity and flesh the characters more and more with each passing line.
In 2007 I remember being in awe of how realistic the water effects were, the shimmering, the way it trickled down the screen and I'd wondered how they could possibly top it. Early in the game you enter a large dining hall with a wall made from glass, as you approach it a Big Sister cracks all the glass and runs off, the room immediately floods and you find yourself walking around underwater outside the dry interiors of Rapture. The visual effects during these sequences are simply eye candy, just like in Bioshock when Rapture is first revealed Bioshock 2 takes it that one more level by letting you walk around while the skyline of Rapture remains ever present in the background.
Although the underwater sequences are a sheer delight for the eyes there has been only minor fixes to the graphics engine for Bioshock 2. That being said the graphics still hold up extremely well compared to other big budget games being released today and surprisingly even when there is a lot of activity on screen there was rarely any slowdown.
When multiplayer was originally announced there was a flurry or angst from both sides, some called it the end of the franchise and that multiplayer would ruin what made Bioshock such a lonely and singular experience and others just wanted to set things on fire!
Unlike with most multiplayer games Bioshock 2's multiplayer begins with a prologue where you find your human character waking in an apartment after injecting yourself with what looks like ADAM, A TV in the corner flickers to life and a broadcast by Andrew Ryan welcomes you to Rapture, as with most interactive cut scenes in games I chose to explore the apartment rooms rather than listen to Ryan's ideological views on a utopian society. In the apartment there is a gene bank that can be used to change your arsenal of weapons and plasmids for use in multiplayer. When you first start out you have the basic three plasmids, Fire, Freeze and Electro-bolt with locked slots for status bonus plasmids, weapons wise to begin with you'll only be able to wield the Pistol and the Shotgun but you'll quickly gain access to other weapons and plasmids as you rank up.
As with most mainstream FPS's there is a rank system based on the amount of kills you rack up, these kills are translated into experience points, when you get enough points you level up and unlock new content, Bioshock 2 does nothing new here as the ranking system has you starting at level 1 and with each level you unlock a new ability, plasmid or weapon. It's not disappointing for 2K to implement their multiplayer in this way as it's a tried and tested method of ensuring players continue to play the game into the foreseeable future.
Once you've equipped your character it's time to setup or join a game, the game modes on offer are the standard FPS multiplayer fair but with a Bioshock twist such as 'capture the flag' is 'capture the little sister' and 'last man standing' is 'last splicer standing.' With so much potential given the nature of the series I would have expected 2K to have come up with new multiplayer modes that cleverly utilises the weapon/plasmid combo, as it stands the modes on offer do give you a wide range of play styles that should suit most players.
The actual experience of wielding the fire plasmid in multiplayer is something awesome indeed, it did take a short while to adjust to the plasmids being weaker than in single player but once you start you realise very quickly that the weapon/plasmid combination you choose and greatly affect your chances in a one on one confrontation with an enemy and if all else fails you can repeatedly hit him with your trusty wrench as he tries to reload. There have been a few glitches and random drop outs with the multiplayer but nothing more than has been experience with other games online.
Overall the multiplayer really does add another dimension to the game, the inclusion of the apartment as a means to customise your characters weaponry really helps give the illusion that it is in fact your apartment and you are a citizen of Rapture.
The main campaign in Bioshock 2 is short even exploring all areas and rescuing all the little sisters you're looking at completion time of around 6-7 hours compared to the original you'd be forgiven for thinking that Bioshock was longer which it is but in Bioshock there was a lot of backtracking to complete objectives, in Bioshock 2 however there is little backtracking as most of the game is pretty linear. Once you've completed the main game there is always the multiplayer but if you're not a person who plays online it might seem a little steep to spend full retail on a game you may have completed in a single sitting.
When everything is weighed up Bioshock 2 may not have the dynamic story from its predecessor but it excels with its tight game play and compelling voice acting, fans of multiplayer FPS's will be more than happy with what Bioshock 2 has to offer. The game is a complete package for anyone looking for something a little different to all the war based shooters out there, a must buy!


text:
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ZWINT encodes a protein that is clearly involved in kinetochore function although an exact role is not known. Additionally we are shipping ZWINT Antibodies (71) and ZWINT Proteins (10) and many more products for this protein.
Showing 4 out of 4 products:
Our results provide the first evidence that Zwint-1 is required to correct erroneous kinetochore-microtubule attachment and regulate spindle checkpoint function during meiosis.
the E3 ubiquitin ligase terf (show TRIM17 ELISA Kits) causes protein degradation of ZWINT and negatively regulates cell proliferation
These studies identify zwint-1 as a novel AurB (show AURKB ELISA Kits) substrate required for kinetochore assembly and for proper spindle assembly checkpoint silencing at metaphase
hZwint-1 bridges the inner and outer kinetochore: identification of the kinetochore localization domain and the hZw10 (show ZW10 ELISA Kits)-interaction domain
show that ZW10 interacting protein-1(Zwint-1) is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10 (show ZW10 ELISA Kits)) in HeLa cells
Stable hZW10 (show ZW10 ELISA Kits) kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint (show BUB3 ELISA Kits) arrest.
This gene encodes a protein that is clearly involved in kinetochore function although an exact role is not known. It interacts with ZW10, another kinetochore protein, possibly regulating the association between ZW10 and kinetochores. The encoded protein localizes to prophase kinetochores before ZW10 does and it remains detectable on the kinetochore until late anaphase. It has a uniform distribution in the cytoplasm of interphase cells. Alternatively spliced transcript variants encoding different isoforms have been found for this gene.
ZW10 interactor
, ZW10 interactor, kinetochore protein
, ZW10-interacting protein 1
, human ZW10 interacting protein-1
, zwint-1
, SNAP25 interacting protein 30
, SNAP25-interacting protein 30
, ZW10 interacting protein-1
, neuroprotective protein 8
, scoilin


text:
For some people, social media is baked into their lives – not a minute or a meal can pass without it being shared with the world. Those people won't want to use Relaxed, a tool that will respond to Facebook mentions and Tweets directed at you on your behalf. There's not the most advanced set of responses to choose from (although you can of course set your own) and it will always include the autorelaxed.com URL at the end, but if you're the kind of person that likes to take a step back from social media over the holidays, it's a simple tool for setting something akin to an out of office email responder. To use it, you just connect your chosen account(s) to the service, set a message and a date the auto-responder should start and stop. Once that's done, you're all set. It should be noted that while it will happily send your response to private (as well as public) messages on Facebook, it can't respond to Direct Messages on Twitter.
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"'Relaxed' Will Auto-Reply to Facebook and Twitter Messages for You" by @ShellyPalmer
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text:
Forget about buying guns, start storing food, NOW!
I like you can see what is coming for us. Just the hint of a gun grab sold out every gun and ammo store in america within days.
Americans bought over 12 billion rounds of ammo last year.
We already have over 300 million guns in our hands.
We are the largest armed population on the planet.
How many Americans have months of food stored?
Survival can be broken down into the rule of 3's
You can only survive….
3 SECONDS without HOPE,
3 MINUTES without AIR,
3 HOURS without SHELTER,
3 DAYS without WATER,
3 WEEKS without FOOD,
3 MONTHS without LOVE…
Anyone who studies military tactics knows the first thing you do to win a war is stop the distribution and flow of food to its population..
I am here to tell you it's not going to be lack of bullets that kill most of us, its going to be a lack of food.
For very little money you can store away months of essential food items that will be very valuable for you and your loved ones in the coming years. Properly stored items placed in Mylar bags with Oxygen absorbers can extend the shelf life of certain foods to well over 50 years, some say forever.
Start storing food and water now before its too late.
Here is a short list of links to get you started on how to store food for long term survival.
For the Price of one AR-15 rifle without any bullets, you can get a 6 month supply of food dropped at your door.
Here is what you get:
6-Month/1-Person Family Basics (Traditional) Package: (3463 Servings!)
Premium Whole Wheat Flour (96 Servings)
Fine Enriched and Unbleached White Flour (84 Servings)
Baker's Fine White Granulated Sugar (666 Servings)
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Liquid_Pestilence24 views


text:
House Party Leverages On and Offline Strategy to Compete in Viral Marketing
(Page 2 of 2)
conducts surveys before and after the parties for feedback on the attendees' purchase intent and receptivity toward their products. "There is an amazing amount of digital activity with impressions and engagement happening," Maher says. Users are encouraged to post on the House Party website photos, videos, and blog posts about their parties. "All that stuff gets loaded to our platform where the client [companies] can see all of that," he says.
House Party works with such brands as Unilever, Kraft, Ford, Budweiser, and cable channel AMC. The sponsors supply boxes of merchandise and promotional material that the party hosts receive for free to use and share at their private events. At most of the parties, sponsors put products directly in consumers' hands.
AMC is using House Party to promote the March season premiere of television series "Mad Men." Each party host will get a package that includes posters and "Mad Men" branded party supplies to share with their guests. While fans of the show at the parties get some swag, AMC locks in product placement in the home courtesy of House Party. "We're not just a public relations or sampling tool," Maher says. "We're a viable media channel for them."
These sponsored parties may be promotional, but the hosts do not have to buy anything nor do they sell anything to their guests. Would-be party hosts must apply online at House Party's website to receive sponsorship from the brands. The application process helps match products with the appropriate audience so a party primarily for kids does not get drink coasters and discounts on Courvoisier cognac. Maher says the individual parties typically range from 12 to 16 people though some may top 20 attendees.
Each brand sponsor typically works with House Party on campaigns to provide goodies for about 5,000 parties taking place across country, though some have topped 10,000 parties. Maher says House Party has 800,000 members (who are both guests and hosts) registered on its website. "Those are some of your biggest brand advocates, influencers, social viral people," he says. Based on the brands' objectives, House Party will inform this community about the types of parties being sponsored.
The parties include diverse groups, Maher says, from people interested in video games and toys to young adults who want nothing more than to go to a gathering sponsored by a beer company. That said, a significant portion of the hosts are mothers. "Our sweet spot is [consumer packaged goods] with products that are geared towards middle-age, middle-income moms," Maher says.
House Party generates revenue by collecting fees from the brands and marketers. "We're selling them an integrated marketing campaign," Maher says. At the high end —perhaps a campaign with 10,000 parties—brands pay as much as $1 million he says. "On average, $250,000 to $500,000 is what our clients spend," he says.
As House Party grows, Maher says the company is cautious about working with some types of sponsors such as political parties in order to avoid controversy. "Though we're not backing a candidate, we have to be sensitive with our community and how they respond to that," he says.
House Party was co-founded in 2005 by Gene DeRose, who was founder and CEO of Jupiter Communications, and Parker Reilly, whose media marketing career includes positions at Polygram Records, Time Inc., and Viacom. Maher says Reilly and DeRose launched House Party to put a new spin on word-of-mouth marketing. "Big brands have known for decades that the most powerful form of advertising," Maher says, "is getting a consumer to talk about a product to another consumer."
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text:
Hand of Faerun
Red Knight
A deity that Tetta serves, looking up to her as a role model.
The Red Knight is depicted as a dark haired woman in red plate armor who wields a longsword called Checkmate. The Red Knight's symbol is a red Staunton knight chess piece with stars for eyes. The Red Knight is described as looking to Tempus, the god who elevated her into godhood, as a father figure. She is the deity of strategy and battle tactics.


text:
Photographer's Note
This is a sunset shot from my recent visit to the beautiful hamlet, Lolegaon [@ 1850 m, 123 Km from Siliguri; overlooking Kalimpong] on the slopes of the pristine green hills of the Darjeeling district..It was taken from a sunset point atop a hill which requires a 500 m trek through unspoiled pine forest..We started the climb at around 4:30 pm and reached the top at around 5:15 pm..Its worth a million bucks as the view from the point is just breathtaking specially at sunset..These are hill dogs [not domesticated] with more fur than usual city dogs that we see in the plains..In the morning I had fed these dogs with biscuits and cake..Thanks a trillion to these beautiful animals that they actually followed us right up to the sunset point, giving me this photo opportunity..
Critiques | Translate
Hello Kaushik,
Thanks for sharing this beautiful photo with us. The colors and the silhouettes are very nice,
Best regards,
rajju (1345) 2007-12-28 20:48
silhouettes is really beautiful... very nice shot..
avid82 (26) 2008-05-09 21:30
i remember the story ...."call of the wild" sunset on a hill top..symmetrical wolfish dogs...gives a beatiful wild touch to the photo...very good capture kaushik
rhamza (118) 2008-11-18 22:13
Nice shot of the sunset ! I envy you!!
Photo Information
- Copyright: Kaushik Sengupta (kseng76) (144)
- Genre: Places
- Medium: Color
- Date Taken: 2007-12-10
- Categories: Nature
- Camera: KODAK EasyShare Z612
- Exposure: f/8, 1/80 seconds
- More Photo Info: view
- Map: view
- Photo Version: Original Version
- Date Submitted: 2007-12-20 2:23


text:
DENVER -- There wasn't time to look up any books on obstetrics before a woman gave birth in the Denver Public Library on Tuesday.
Library spokeswoman Celeste Jackson said the woman walked into the library, said she had been riding a city bus and was in labor.
"She was pretty far along. She delivered right there," just inside the entrance, Jackson said.
Staffers and security guards helped until paramedics arrived and took the mother and baby girl to Denver Health Medical Center.
A hospital spokeswoman said the mother was in good condition Tuesday night. But she said privacy rules prevented her from releasing any information on the baby.
Jackson said staffers didn't yet know their names but they want to send flowers.
"It's never happened before," she said.
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text:
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text:
Album Rating: 5.0nah Black Sabbath
Digging: Open Mike Eagle - Brick Body Kids Still Daydream
this sucks
josh sucks.
Album Rating: 4.5 | Sound OffSad Wings of Destiny is an amazing album
Digging: Sorcerer - The Crowning of the Fire King
Album Rating: 5.0damn no one likes josh aww
Album Rating: 5.0 | Sound OffStained Class rules hard.
Album Rating: 4.5Awesome album.
I had to wait to obtain this from the store cause i don't usually dig classic albums from the youtube. It spoils the magic.
Imo this isn't as good as Painkiller but it is definetely their best 70's album. The first track is pure British Metal.
Album Rating: 4.5I will definitely re listen to this, and i might 5'd it.
Album Rating: 5.05 for life
Album Rating: 4.0painkiller is better but this is m/ as well
Album Rating: 5.0necl
Album Rating: 4.0nah bro painkiller m/
This is better.
Album Rating: 4.5 | Sound Offhumm
haha I know you disagree Notrap.
Album Rating: 4.5 | Sound Offm/
Album Rating: 5.0its official dreamer deceiver is the best song ever made
Album Rating: 4.5Dad got me this on vinyl, so awesome.
Album Rating: 5.0sweet dad
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


text:
MILWAUKEE—No one can accuse the Brewers of being afraid to trade first-round draft picks.
In July 2008, the Brewers traded outfielder/first baseman Matt LaPorta, their first-rounder in '07, to the Indians in the deal that brought C.C. Sabathia to Milwaukee. The Brewers had no regrets about paying that price when Sabathia almost single-handedly led them to their first playoff berth in 26 years.
On the eve of baseball's Winter Meetings, the Brewers struck again, trading second-base prospect Brett Lawrie to the Blue Jays for righthander Shaun Marcum. Lawrie was the Brewers' first-round pick out of British Columbia in the 2008 draft.
"It was a steep price but it's something we felt we had to do," general manager Doug Melvin said. "We still feel we have a very talented team that can compete in our division and get to the postseason again."
Lawrie, 20, held his own as one of the youngest players in the Double-A Southern League in 2010. In 135 games for Huntsville, he batted .285/.346/.451 with 36 doubles, 16 triples, eight homers, 63 RBIs and 30 steals.
Drafted as a catcher but moved to second base at his request, Lawrie improved defensively but still has work to do after committing 25 errors at Huntsville.
Considered the Brewers' No. 1 prospect prior to the trade, Lawrie was slated to play at Triple-A Nashville in 2011 but was blocked at the big league level by Rickie Weeks. The Brewers are trying to work out a contract extension with Weeks, who can be a free agent after next season.
Melvin said Toronto GM Alex Anthopoulos had been persistent in trying to acquire Lawrie, a native Canadian. Different names were discussed but Melvin said only in recent days had the focus shifted to the 29-year-old Marcum.
Anthopoulos said he "swallowed hard" to trade Marcum, who went 13-8, 3.64 in 31 starts in 2010. But, in the end, he couldn't pass on Lawrie.
"Trading Shaun Marcum wasn't easy," Anthopoulos said. "I agonized over it. With the camaraderie and what he brought to the rotation, we didn't take that lightly. But we've been interested in acquiring Brett Lawrie for quite some time."
• The Brewers selected righthander Pat Egan, 26, from Baltimore in the major league phase of the Rule 5 draft, and will be given a chance to earn a spot in the bullpen in spring camp. He went 7-2, 3.44 in 49 relief outings at Double-A Bowie and Triple-A Norfolk.
• The Orioles selected righthander Adrian Rosario in the Rule 5 draft, a move that surprised the Brewers because Rosario, 21, pitched no higher than low Class A Wisconsin, where he went 4-0, 4.50 with 44 strikeouts in 32 innings.


text:
This just in from Gail Levy at TabletKiosk. TK is launching the Sahara Slate PC i400 series that feature both active digitizers and touch screens. There are three models of the new Sahara slates:
Sahara Slate PC i412T is the entry level touch screen model featuring a lower power, lower cost Intel® Celeron® M ULV 423 processor. The Sahara Slate PC i412T sports a wide-angle view passive resistive touch screen, perfect for accessing menu driven software such as Point of Sale applications and kiosk-based systems, and comes pre-loaded with Microsoft Windows XP Professional, Windows Vista Business or Linux.
For a more powerful touch screen solution, the Sahara Slate PC i440T features Intel® Centrino™ Duo Mobile Technology with an Intel® Core™ Duo LV L2500 processor, the same wide-angle view touch screen and also either Microsoft Windows XP Professional, Windows Vista Business or Linux. For maximum productivity, the Sahara Slate PC i440T is the most powerful slate style touch screen Tablet PC currently available.
For the ultimate Tablet PC experience, the Sahara Slate PC i440D, powered by Intel® Centrino™ Duo Mobile Technology with an Intel® Core™ Duo LV L2500 processor, features a revolutionary new input screen that converts from a pressure-sensitive active digitizer to a resistive touch screen at the push of a button. The Sahara Slate PC i440D will be available with the new Windows Vista Business OS which elevates the existing Sahara Slate PC brand into new performance territory.
The high-end i440D is a very sweet Tablet PC that Kevin and I got to see at the CES in January. Head over to the TabletKiosk site for full specs and pricing on all of the models. Nice job, TK!


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Gary Bettman says he won't be stepping down any time soon as commissioner of the NHL and the salary-cap loving owners aren't likely to give him the boot.
If only we could all be so lucky to hold our jobs for that long without accomplishing anything.
Bettman is the only commissioner the NHL has ever had and he came in 14 years ago with a mandate to sell the game in the United States.
Today, absolutely nobody is watching hockey on television in the United States. Less people than ever are watching it live in places like Boston, Chicago, St. Louis and Los Angeles, all hot hockey markets at one time.
There are teams all over the southern states, in places like Florida, Carolina, Atlanta, Nashville and Phoenix, where people would just as soon travel to the North Pole as enter a hockey rink.
Meanwhile, in Canada, two of our teams flew south on his watch and now the league has adopted a ridiculous schedule where the Canadian teams don't even play each other every year.
NBC big mouth Brett Hull had it right when he made this comment recently: "I don't think (Bettman) really understands the game. He doesn't understand the history, and at times I don't think he really gives a hoot. All he cares about is if he's making the owners happy."
It's time for Little Man Gary to either change his focus and start bringing hockey back to its roots or simply do the honourable thing and walk away.
FAREWELL NEIL: The Western Manitoba sporting community is mourning this week following the death of Neil Andrews, a man whose passion for the games we play was an inspiration to many.
Andrews, who died last Saturday of a rare brain disease, was a fine curler -- as his two senior provincial championships would attest -- but also threw his company name behind one of Brandon's biggest bonspiels and volunteered to head up the ice crew at every major event the Wheat City hosted over the last 20 years.
He was also a baseball lover who founded the Brandon Marlins senior team when the players were all juniors and eventually led them to numerous provincial titles. His efforts there were recognized when the City of Brandon renamed the baseball stadium in his honour.
There is no consolation to his grieving family, but perhaps some small comfort in the knowledge that his legacy will be long lasting and his memory held strong by those of us who knew him.
BACK TO LIGHTER FARE: Great news for NFL football fans -- Michael Irvin has been whacked as an analyst with ESPN. Now if you want to listen to utter nonsense on TV, you'll have to go back to the Teletubbies ... Now, back to important discussions, like whether or not Britney's hair is the father of Anna Nicole's baby ... There has been tons of talk about Sports Illustrated featuring singer Beyonce on the cover but not nearly enough chatter about the fact that many of the pictures in the mag are in 3D. Very cool ... Can't wait for the round-the-clock coverage of the NHL trade deadline on Canadian sports networks this week. You haven't lived until you've spent 14 hours listening to Pierre Maguire shouting about the importance of the third-round pick Dallas acquired for a Kazakhstani prospect ... Who would have thought that a week after the Peter Forsberg trade, Scottie Upshall would still be the most productive player in the deal? ... If the San Diego Chargers were going to dive into the recycling heap to get coach Norv Turner, why didn't they just dig a little deeper and snag Mike Ditka, Don Shula or Jimmy Johnson. Hey, if Marv Levy's still in the league, those guys can be too. And unlike Turner, at least those guys win occasionally ... Tennessee Titans running back Pacman Jones took more than $80,000 to a Vegas strip club recently to toss at the dancers. Forget the lap dances, for that kind of dough, one of those strippers better become Mrs. Pacman ...
QUICK HITS: Let's start off with T.J. Simers of the Los Angeles Times on the San Diego Chargers' decision to hire Norv Turner (58-82-1 career record) as head coach: "A wax likeness of Tom Landry would've been a more inspiring hire." ... Steve Rosenbloom of ChicagoSports.com on recent anti-gay remarks uttered by ex-NBA player Tim Hardaway: "Tim Hardaway, your plane is boarding -- and you have a window seat next to John Rocker." ... ABC funnyman Jimmy Kimmel on people criticizing Sports Illustrated for putting singer Beyonce on the cover when she obviously has nothing to do with sports: "What was Heidi Klum, power forward for the Atlanta Hawks?" ... Comedian Argus Hamilton, on golfer John Daly saying he has addictions to alcohol, tobacco, food and gambling: "The Nevada economy goes into recession every time John Daly gets a good night's sleep." ... When a season-low 2,640 fans showed up for a recent WHL game in his northern B.C. town, Prince George Citizen sports editor Jim Swanson saw a bright side: "The good news is none of the men in attendance is claiming to be the father of Anna Nicole Smith's daughter." ... Greg Cote of the Miami Herald, on Michael Waltrip getting busted after his intake manifold was found to contain a property associated with jet fuel at the Daytona 500: "Officials became suspicious when Waltrip's car made its qualifying run after launching from an aircraft carrier." Another from Cote: "Part of the legend of Red Sox pitching phenom Daisuke Matsuzaka is that he supposedly has a pitch called a 'gyroball.' Nobody knows exactly what it is, but it is very effective because batters cannot see the ball as it flies to the plate wrapped in Greek pita bread." ... From Dan Daly, of the Washington Times: "Finally found out what the hold-up was with Barry Bonds' contract. During his physical exam, it seems, team doctors found an unidentified additive in his air intake manifold." ... New York Mets outfielder Moises Alou on how he has managed to stay in great shape, even though he has hit 40: "I drink light beer." ... Jim Armstrong of the Denver Post on New England Patriots quarterback Tom Brady knocking up his ex-girlfriend Bridget Moynahan and currently dating Victoria's Secret model Gisele Bundchen: "And we thought Joe Namath got around." From Greg Cote, of the Miami Herald: "Barry Bonds signed a one-year, $15.8 million deal with the Giants. He said he planned to use the money to hire people to like him." ... Finally, this headline at fark.com summed the DUI arrest of Indianapolis Colts Super Bowl hero Dominic Rhodes: "Good news for the Bengals: He's a free agent."


text: I am looking to buy a set of new frames since my N six-one 95's are cracked. I feel comfortable using my Ncode 95 but would like to try new frames. How does the K factors compare to the old ncodes? I am looking at 90 and 95. How does the 90 play compared to the 95's. Also I am thinking of trying PDR since it will be way easier to play with than K factors but the control won't be there. Reason I am asking is I won't be able to demo until next week so would like some heads up.


text:
Barbarians plans thrown into chaos
November 27, 2007
The Barbarians' preparations for this weekend's eagerly-anticipated clash with world champions South Africa have been thrown into chaos with captain Mark Regan and fellow Premiership star Andrew Sheridan banned from taking part.
Premiership sides Bristol and Sale have both ruled their respective players out from Saturday's fixture that clashes with the final round of matches in this season's EDF Energy Anglo-Welsh Cup.
Bristol coach Richard Hill told The Guardian newspaper, "As far as we are concerned, Mark will not be involved in the South Africa match.
"He has received a letter from the club to that effect and we have taken a very strong line over this."
Bristol chief executive David White told BBC Sport: ``Premier Rugby have made their decision and we are supporting that.
``We have not released him, he can't play and we have the jurisdiction to fine him.
``If he does play he'll be breaking his contract and will have to face the consequences.''
Sale too moved quickly to rule Sheridan out of the game after he was announced in the Barbarians squad yesterday.
A statement released by the club read, "The news that Andrew Sheridan has been named in the Barbarians Squad to play against South Africa at the weekend is incorrect.
"Andrew is currently in training with his Sale Sharks team mates, preparing for Friday evening's EDF Energy Cup game at Leicester Tigers."
However, Sheridan's agent Mike Burton has insisted that the England international prop is free to play in the game as it was a clause in his recently signed contract.
Burton, who is also a member of the Barbarians' committee, commented, "The new contract he [Sheridan] signed with the club last week contained an unconditional clause that allows him to play for the Barbarians against South Africa on Saturday.
"Clearly Sale are in breach of that contract. I will be speaking to Andrew to get his views on the matter. Any decision will be his and his alone."
Premier Rugby have taken a firm stance on the release of players insisting, "the decision not to release players for the Barbarians was made at a meeting of all 12 Premiership clubs and it was a unanimous one."
The newspaper reports that the clubs would face a financial sanction if the players turn out for the Barbarians.
"We want to work with the Barbarians and will provide players for their end-of- season game against England and their summer tours," said the PRL chief executive, Mark McCafferty. "The autumn international window is another matter."
A similar row looms for those Magners Celtic League players slated to appear at Twickenham.
Cardiff Blues stars Martyn Williams and Tom Shanklin have also been named in the Barbarians squad even though both are expected to feature in their Anglo-Welsh clash with Bath on Friday night.
Similarly Ospreys scrum half Justin Marshall is also slated for the Twickenham fixture that clashes with the Welsh side's meeting with Harlequins on Sunday afternoon and Munster's Federico Pucciarello would be expected to feature against Leinster in the Magners League on Friday night.
Barbarians Squad:
Communication error please reload the page.


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text:
The hematopoietic hormone erythropoietin (Epo), regularly produced by the kidneys and the liver, is also expressed in neuronal tissue, where it has been found to mediate paracrine neuroprotective effects. In most studies exploring the rescue effects of Epo, apoptosis was exogenously induced by different cell death stimuli. Herein, we set out to study the expression and function of Epo in physiologically occurring apoptosis in a model of retinal development. We made use of an organotypic retinal wholemount culture system that resembles the physiological in vivo situation with cell connections still retained. Epo mRNA expression in the retina, liver, and kidney showed a significant increase during early development, coinciding with the anemia of the newborn. In the retina of Epo-green fluorescent protein transgenic mice, Epo-expressing cells were identified and found to be distributed in the retinal ganglion cell layer. Treatment of retinal wholemount cultures with recombinant Epo resulted in a significant decrease of apoptotic ganglion cells as well as photoreceptor cells throughout retinal development. Moreover, transforming growth factor-β-induced apoptosis was completely antagonized by Epo when both factors were simultaneously applied. Investigations on the signaling pathway revealed a decrease in Bax mRNA levels in Epo-treated retinal cells. We conclude that Epo exerts wide and prolonged neuroprotective activity in physiologically occurring apoptosis and thus contributes to proper retinal development.
- apoptosis
- erythropoietin receptor
- neuroprotection
- retinal ganglion cells
apoptosis is a widespread physiological process during development of the nervous system in general and in the retina in particular (4). During postnatal development of the retina, cells unable to form proper synapses and communication are eliminated by apoptosis (40). Therefore, apoptosis is essential for adequate murine retinal development and postnatally occurs in three waves or peaks at postnatal day (P) 2, 9, and 15 (31). At P2, cells of the inner neuroblastic layer are affected by apoptosis. This cell type later develops into ganglion cells and undergoes apoptosis again at P15, together with photoreceptor cells. At P9, most apoptotic cells are localized in the inner nuclear layer and are most likely amacrine cells (31).
Several factors have either stimulating effects, like transforming growth factor-β (TGF-β) (1, 14), or repressing effects on apoptosis, like insulin (39) and among others brain-derived neurotrophic factor (BDNF) (5). The interaction of these factors results in the control of retinal apoptosis (10).
The hormone erythropoietin (Epo) was first described as the main regulator of red blood cell production, inhibiting apoptosis of erythroid progenitor cells by binding to its specific membrane receptor EpoR (13, 23). Both Epo and Epo receptor (EpoR) null mutant mice exhibit reduced primitive erythropoiesis and die in utero because of severe anemia (42). However, EpoR was also found to be expressed by nonerythroid cells, e.g., neuronal cells (24, 34, 43). Experimental evidence suggested that Epo also exerts anti-apoptotic activity on developing neuronal tissue (7) and limits ischemic damage of the adult brain in stroke models (3). Moreover, Epo was found to rescue retinal photoreceptor cells from light-induced apoptosis (17, 18) as well as retinal ganglion cells from neurotrophic factor deprivation (41).
In most studies exploring the rescue effects of Epo on retinal cells, apoptosis was exogenously induced by different cell death stimuli in adult retinal cells (26). However, the expression and function of Epo during physiological development with naturally occurring apoptosis is still unknown.
Primary cell cultures provide an excellent opportunity to examine the effects of hormones and extracellular molecules in a controlled environment. Herein, we made use of an organotypic retinal wholemount culture system (9) to investigate anti-apoptotic effects of Epo during postnatal murine retinal development. In our experimental system, which closely resembles the physiological in vivo situation with cell connections still retained, we set out to identify the Epo-expressing cells and investigate which retinal cells are rescued from apoptosis by Epo. Furthermore, we studied whether Epo interacts with other factors like TGF-β and which pathways are activated by Epo. This study contributes to characterizing the importance of Epo for adequate retinal development.
Retinal cultures.
All experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the National Institutes of Health regarding the care and use of animals for experimental procedures. The study protocol was approved by the institutional animal ethics committee of the Universitätsklinikum Essen, Universität Duisburg-Essen. Eyes were dissected from specific pathogen-free C57BL/6J mice (Jackson Laboratory) on postnatal days (P) 2, 9, and 15. Lens, vitreous body, and pigment epithelium were removed, and the neural retinas were preincubated with 0.5 mg/ml hyaluronidase for 15 min at 37°C. Retinas were cultured as organotypic wholemounts in 2 ml chemically defined medium as described previously (9, 10, 20). In dose-response experiments, cultures were treated with 0.2, 0.5, 1, 2, 5, 10, and 20 U/ml recombinant human Epo (Boeringer Ingelheim, Ingelheim, Germany) or with 2, 5, 10, and 20 ng/ml recombinant human TGF-β1 (R&D Systems, Wiesbaden, Germany) to determine the optimal concentration of the factors to prevent or to induce apoptosis. Where indicated, in all further experiments, a concentration of 0.5 U/ml recombinant Epo or 2 ng/ml recombinant human TGF-β1 (R&D Systems, Wiesbaden, Germany) were added to the serum- and insulin-free basal medium (Dulbecco's modified eagle medium F-12; Sigma-Aldrich). Cultures were maintained for 24 h at 37°C in a 5% CO2 atmosphere.
Detection of apoptosis.
Apoptotic cell death was determined by counting 4′,6-diamidino-2-phenylindole (DAPI)-stained pycnotic nuclei of retinal single cell suspensions after dissociating cultured wholemount retinas. For cell scoring of DAPI-stained nuclei, retinas were dissociated into single cell suspension by incubation at 37°C with 50 U/ml collagenase (Sigma-Aldrich, Munich, Germany), 1.5 mg/ml trypsin (Sigma-Aldrich), 0.7 mg/ml hyaluronidase (Sigma-Aldrich), and 0.1 mg/ml DNase (Roche, Penzberg, Germany), followed by passes through a siliconized Pasteur pipette. Digestion of the tissue was stopped by addition of EDTA, and cells were fixed for 1 h in 8% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). An aliquot of the cell suspension was spotted on a slide using a cytospin at 700 rpm for 7 min. Cells were stained and mounted with DAPI (2 μg/ml; Sigma-Aldrich).
The effect of Epo and TGF-β treatment, respectively, was scored by counting at least 1,000 cells and at least 10 pycnotic nuclei as well as at least 10 fields of view using a NIKON ECLIPSE 1000 microscope.
To find out which cell types are rescued by recombinant Epo, we stained cytospins of retinal wholemount explants, cultured for 24 h in medium containing 0.5 U/ml recombinant Epo, with the ganglion cell marker Brna3a or the photoreceptor marker opsin (1).
The cytospins were preincubated with BGT1 (3 mg/ml BSA, 100 mM glycin, and 0.25% Triton) and with a Biotin-Avidin Endogenous Blocking Kit (DAKO) for 1 h. Before incubation with the polyclonal mouse Brn3a antibody (1:100; Chemicon, Schwalbach, Germany) or the polyclonal mouse opsin antibody (1:10,000; Sigma-Aldrich) at 4°C overnight, the cytospins were blocked with 15% normal goat serum in BGT1 for 30 min at room temperature. The reaction was visualized using goat biotinylated anti-mouse (1:400; Molecular Probes, Karlsruhe, Germany) IgG and CY3- or FITC-conjugated streptavidin (1:800; Molecular Probes). As controls in all cases, the primary antibody was substituted for PBS to test for nonspecific labeling. No specific cellular staining was observed when the primary antiserum was omitted.
RNA preparation and RT-PCR.
Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method (8). Total RNA (1 μg) was reverse-transcribed into cDNA with oligo(dT)15 as primer for avian-myeloblastosis-virus reverse transcriptase (Promega, Heidelberg, Germany). Real-time PCR was performed with SYBR green as fluorescent dye (Eurogentec, Köln, Germany) on the Gene Amp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). The cDNA standards for real-time PCR were prepared from the specific PCR products using a DNA purification kit (Roche). The amount of standard cDNA was determined photometrically. The standard concentrations ranged from 1,000 to 0.001 fg/μl. Quantification was done in a two-step real-time PCR with a denaturation step at 95°C for 10 min and then 45 cycles at 95°C for 15 s and at 60°C for 1 min (15). For primer sequences see Table 1.
Hematocrit and hemoglobin measurements.
For hematocrit measurements, blood was centrifuged in hematocrit capillaries for 10 min at 13,000 rpm, and hemoglobin levels were determined photometrically (546 nm) after 1:250 dilution in Drabkins' solution (0.5 g sodium bicarbonate, 100 mg potassium ferricyanide, and 25 mg potassium cyanide in 500 ml aqua dest).
Immunohistochemistry of cryostat sections of Epo-green fluorescent protein transgenic mice.
Eyes were dissected from 6- to 20-wk-old wild-type and Epo-green fluorescent protein (GFP) transgenic mice (17K-Epo-GFP and 22K-Epo-GFP lines) expressing GFP under the control of mouse Epo gene regulatory regions. The GFP cDNA is linked to 17-kb upstream sequences of the mouse Epo gene in the 17K-Epo-GFP transgene, whereas the 22K-Epo-GFP transgene contains 22-kb upstream and 162-kb downstream sequences from the Epo gene (37).
Eyes were fixed in 4% paraformaldehyde-PBS at 4°C overnight, washed two times with PBS, incubated in 20% sucrose at 4°C overnight, and then frozen in embedding medium (Serva, Amstetten, Austria) at −80°C. Frozen sections of these eyes were incubated for 1 h in 3% hydrogen peroxide-PBS, washed three times with PBS, and then incubated with blocking buffer (5% normal goat serum-1% BSA in 20% skim-powdered milk in PBS with 1% Triton; 1:5) for 1 h at room temperature. The slides were then incubated with rabbit anti-GFP polyclonal antibody (1:1,000; Molecular Probes) at 4°C overnight. After being washed with PBS three times, the slides were further incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:500; Biosource, Bethesda, MD) for 30 min at room temperature. Specific staining was visualized by incubation with 3′,3′-diaminobenzidine (DAB; Dako Cytomation). Hematoxylin (Shandon Therma) was used for counterstaining.
Statistical significance of differences was calculated by one-way ANOVA and Newman-Keuls posttest or Student's t-test.
Hypoxia-induced Epo mRNA expression during early postnatal retinal development coincides with the anemia of the newborn.
RT-PCR measurements revealed that Epo mRNA levels in the retina significantly increased during early postnatal development, peaked at P9, and later decreased, reaching levels similar to P0 (Fig. 1A). During this period, mice also experience the anemia of the newborn, a condition we confirmed by measuring hemoglobin and hematocrit levels. Hemoglobin and hematocrit levels significantly decreased during the first 3 wk after birth (Fig. 1, D and E), suggesting that the increase in Epo mRNA levels in the retina might be due to developing tissue hypoxia.
Kidney and liver of these mice also showed a significant increase in Epo mRNA levels and thus likewise reflect the hypoxic effects caused by the anemia of the newborn (Fig. 1, B and C).
Identification of Epo-expressing cells in the retina.
To identify Epo-expressing cells in the retina, Epo-GFP transgenic mice were used. These mice express transgenic constructs in which GFP was either linked to 17-kb upstream sequences (17K-Epo-GFP mice) or to 22-kb upstream and 162-kb downstream sequences (22K-Epo-GFP mice) (37) of the mouse Epo gene. Therefore, the detection of GFP indicates cells in which expression of Epo is turned on. GFP was detected by means of immunohistochemistry in cryostat sections of these mice. DAB-positive cells were localized in the ganglion cell layer of 17K-Epo-GFP and 22K-Epo-GFP transgenic mouse retina. Strikingly, those cells were without exception found in the outermost periphery of the retina of 6- to 20-wk-old transgenic mice, where the pars optica meets the pars ceca (Fig. 2, A–C). In contrast, retinas of P9 and P12 transgenic mice showed DAB-positive cells in the center of the retina, where the optic nerve leaves the retina (Fig. 2, D and E). No signal was detected in wild-type mice.
EpoR mRNA is continuously expressed in the retina during postnatal development.
We detected EpoR mRNA expression in the retina during all development stages and found no significant changes in expression levels, suggesting that EpoR could mediate Epo activity throughout postnatal development (Fig. 1F).
Epo mediates neuroprotective activity during postnatal development of murine retina.
In dose-effect experiments with organotypic murine retinal wholemount cultures, we first applied recombinant Epo in different concentrations between 0.2 and 20 U/ml to P15 murine retinas to test whether Epo is able to mediate neuroprotective activity and to determine the optimal concentration for rescue effects. The number of pycnotic nuclei significantly decreased after application of Epo compared with the controls. Moreover, 0.5 U/ml turned out to be sufficient to achieve the full anti-apoptotic effect (Fig. 3C). Application of 0.5 U/ml of recombinant Epo did not only rescue P15 retinal cells from apoptosis but also mediated neuroprotective activity during the first postnatal cell death period at P2 (Fig. 3A) and during the second at P9 (Fig. 3B).
Thus we demonstrate that Epo has an anti-apoptotic effect during all three postnatal periods of physiological apoptosis, which suggests an important role of Epo during murine postnatal development of the retina.
Epo rescues retinal ganglion and retinal photoreceptor cells from apoptosis.
Both ganglion and photoreceptor cells undergo apoptosis at P15 (31). To find out which cell type is rescued by Epo, we double-stained cytospins of retinal wholemounts, treated with 0.5 U/ml Epo, with DAPI and the ganglion cell marker Brn3a or the photoreceptor cell marker opsin. Analysis of the data revealed a significant decrease of ganglion (Fig. 4A) and photoreceptor (Fig. 4B) cell apoptosis after application of Epo. Thus the rescue effect of Epo is not limited to a single cell type in the retina but affects ganglion as well as photoreceptor cells, emphasizing the importance of Epo for proper retinal development.
Epo protects P15 retinal ganglion cells from TGF-β-mediated apoptosis.
TGF-β is one of the principal mediators of retinal cell death during postnatal development (1) and mainly affects retinal ganglion cells at P15. We therefore focused on investigating the interaction between Epo and TGF-β in P15 retinas. Previously, we have shown that application of TGF-β alone resulted in a significant increase of apoptotic cells compared with untreated controls with maximum effects at a concentration of 2 ng/ml (1). Simultaneous application of TGF-β and Epo did not only prevent the proapoptotic effect of TGF-β but even reduced the number of pycnotic nuclei compared with untreated controls (Fig. 5). Thus TGF-β-induced apoptosis was completely antagonized by Epo when both factors where applied simultaneously, suggesting that Epo can compensate for the well-described proapoptotic role of TGF-β during retinal development.
Epo prevents apoptosis by inhibiting Bax mRNA expression.
To investigate the signaling pathways underlying the neuroprotective activity of Epo, we concentrated on downstream signaling molecules involved in the mitochondrial apoptosis pathway. Our results revealed significantly lower Bax mRNA levels after application of Epo compared with the controls (Fig. 6A). Interestingly, Bcl-2 mRNA levels were not affected (Fig. 6B). These data suggest that Epo may shift the balance of pro- and anti-apoptotic factors toward the anti-apoptotic members of the Bcl-family by inhibiting the mRNA expression of the proapoptotic member Bax.
Epo is the primary regulator of red blood cell production. Besides its hematopoietic function, Epo is neuroprotective in the retina. However, this anti-apoptotic effect has usually been found in experiments where Epo prevented cell death induced by exogenous stimuli. Examples are light-induced photoreceptor degeneration (17, 18), axotomy (26, 41), glutamate-induced toxicity (28), or ocular hypertension (16). Epo was also found to support proper brain development by preventing apoptosis in the embryonic brain (43) and to promote neurite outgrowth of rat retinal ganglion cells after axotomy (2). Mice with a specific knock out for Epo or EpoR in the brain suffered from embryonic and adult neurogenesis defects (38), indicating an important role of Epo for proper brain development. However, the role of Epo in physiologically occurring retinal apoptosis, a process contributing to normal postnatal development, remained mostly unknown. Likewise, knowledge about Epo expression in the developing retina is missing. In this study, we investigated the physiological role of Epo during retinal development in an organotypic retinal wholemount culture system that maintains tissue organization and interaction between cells and may therefore provide a model that recapitulates the in vivo situation.
In mammalian species, hemoglobin levels decrease after birth because of the switch from fetal to adult hemoglobin causing the anemia of the newborn, which is accompanied by an exponential rise of serum Epo levels (22). Epo expression was first detected in the murine retina during the first 3 wk after birth and reached a significant increase in Epo mRNA levels with peak values at P9. Peak expression coincided with the reduction in hemoglobin. Epo mRNA levels also increased in liver and kidney of these mice, reflecting the body's response toward the anemia. Interestingly, the retina appeared to respond earlier when hemoglobin levels started to decline, whereas peak expression in kidneys and liver coincided with the nadir of hemoglobin concentration. Whether this is the result of different oxygen sensitivity in the respective tissues or can be attributed to tissue specific transcription factors remains to be studied (13). Because Epo expression in astrocytes (27) and neuronal cells (36) has been found to be hypoxia-inducible, we conclude that the developing anemia also causes hypoxic Epo expression in the retina during this particular early postnatal period of development.
Our data on the retinal Epo mRNA expression pattern during postnatal development are in accordance with the study of Chen and colleagues (6), who also found a decrease of Epo mRNA levels of P10 to P17 murine normoxic retinas. Elevated Epo expression caused by relative hypoxia caused pathological neovascularization in their model of retinopathy of prematurity (6). Thus changes in the physiological retinal Epo expression pattern contribute to developmental aberration.
In the retina, Epo was localized to the ganglion and inner nuclear cell layer by immunhistochemistry using an anti-Epo antibody (29). However, this method may identify both Epo-secreting cells and cells on which Epo is bound to the EpoR. In this study, we made use of adult transgenic mice expressing GFP under the control of Epo gene regulatory elements (30). Consequently, Epo-expressing cells in the retina were localized in the ganglion cell layer by means of immunohistochemical staining with an anti-GFP antibody. In retinas of 6- to 20-wk-old GFP transgenic mice, those cells were without exception found in the edge of the retina at the passage of the pars optica to the pars ceca where the Po2 is very low. Based on their morphology, these Epo-expressing cells appear to be neurons with the typical neuron specific shape and an axon leaving the soma. In contrast, at P9 and P12, the period of the anemia of the newborn and highest Epo mRNA expression, DAB-positive cells could also be localized in the center of the retina. These findings are supported by Chen et al. (6), who showed in their model of retinopathy of prematurity that hypoxia in the retina was predominantly located in the central part of the retina. In addition, the data from transgenic mouse analysis provided information on regulatory mechanisms of the Epo gene expression in retina, although further experiments are required. Because GFP expression is detectable in the 17K-Epo-GFP transgenic mouse retina, it is likely that retinal cis-regulatory domains are included in the 17-kb upstream sequences of the Epo gene. This means that the regulatory mechanism of Epo gene expression in the retina is different from that in hepatocytes, which is controlled by the proximal downstream region containing a hypoxia-inducible transcription factor binding site (33, 35).
The fact that we detected continuous EpoR mRNA expression in the retina throughout neonatal life suggests that Epo may exert its activity throughout postnatal development. These results correspond with other studies that provided evidence for EpoR expression in the retina (18) and in the embryonic brain (7). Application of recombinant Epo led to a significant decrease of apoptotic cells in murine organotypic retinal wholemounts during all three cell death periods at P2, P9, and P15. These data for the first time prove that neuroprotection by Epo is not limited to artificially induced retinal cell death but that it is also effective in a model of physiologically occurring apoptosis, indicating the importance of Epo throughout retinal development. Furthermore, we were able to demonstrate that retinal ganglion cells and photoreceptor cells can be rescued by Epo during development. Therefore, neuroprotection by Epo is not restricted to one single cell type but appears to have a wide influence on cell survival during retinal development. Because of the fact that the effect of Epo during retinal development is long lasting and affects a great number of retinal cells, we may conclude that Epo plays an important role for proper retinal development.
The highest Epo mRNA levels were detected during the cell death period at P9. Thus the high Epo levels immediately preceded the subsequent reduction in apoptosis potentially due to the anti-apoptotic action of Epo. This interpretation is supported by our former findings that proapoptotic factors exert their highest activity shortly before the onset of the cell death periods (1). Because apoptosis during retinal development is known to be induced by TGF-β (1, 11, 12), we performed cell culture experiments in which TGF-β and Epo were simultaneously applied to determine the efficacy of Epo-mediated neuroprotection. Our results show that Epo is not only able to completely compensate for TGF-β-mediated apoptosis but even lowers apoptotic rates below those of untreated controls. We may, thus, conclude that Epo expression coincides with periods of apoptosis in the developing retina of the mouse and can efficiently counteract proapoptotic stimuli.
Former studies addressing the signaling pathways activated by Epo in models of neuronal cell survival did not provide unequivocal data. In a model of axotomy-induced degeneration, extracellular signal-regulated kinase (ERK)-1/-2 but not protein kinase B (Akt) was found to be involved in retinal ganglion cell survival (26), whereas a dual activation of ERK-1/-2 and Akt was reported in Epo-mediated protection from focal cerebral ischemia (25). By contrast, other studies using a model of axotomy of rat retinal ganglion cells identified the phosphatidylinositol 3-kinase/Akt pathway as the predominant pathway activated by Epo, and the authors did not find any evidence for the involvement of ERK-1/-2 (41). More recent investigations upon Epo signaling in retinal cells supported the hypothesis that the individual expression pattern is mainly death stimulus dependent. This was concluded from the finding that the activation of Jak2 only led to a neuroprotective effect in induced but not in inherited retinal cell death (32).
In our study, we concentrated on signaling molecules that are known to be of great importance for the regulation of retinal apoptosis during development like the Bcl-2 family members Bax (21, 31) and Bcl-2 (10, 19). Because our study did not investigate induced but naturally occurring cell death, we were prepared to get results differing from those models using cell death stimuli. Whereas application of Epo in our model resulted in a significant decrease of mRNA for the proapoptotic Bax, mRNA levels of anti-apoptotic Bcl-2 remained unchanged. Consequently, our data suggest that Epo prevents apoptosis in the developing murine retina via the inhibition of the mitochondrial pathway by shifting the balance in favor of the anti-apoptotic members of the Bcl-2 family.
Perspectives and Significance
The data presented in this study clearly demonstrate that Epo exerts wide and prolonged neuroprotective activity during postnatal retinal development, rescuing retinal ganglion and photoreceptor cells from naturally occurring apoptosis.
This work was supported by the Yokochi Fund of the Kanehara Ichiro Foundation and the Interne Forschungsförderung Essen stipend program of the Medical Faculty of the University of Duisburg-Essen.
No conflicts of interest are declared by the authors.
We gratefully acknowledge the help of Dr. Naoshi Obara, Dr. Ken Matsumoto, and Dr. Xiaoqing Pan from the University of Tsukuba, Japan, and Dr. Stilla Frede, Dr. Christoph Wotzlaw, and Patricia Freitag from the University of Duisburg-Essen.
Present addresses: M. Yamamoto, Dept. of Medical Biochemistry, Tohoko University Graduate School of Medicine, Sendai, Japan; and N. Suzuki, Dept. of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.
- Copyright © 2010 the American Physiological Society


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French Grand Prix Technical Preview -- Q+A with Pascal Vasselon, Senior General Manager Chassis Q: Sixth place for Jarno and the best result of the season for the team at Indy. Satisfying? Pascal Vasselon: Jarno gave us an excellent...
French Grand Prix Technical Preview -- Q+A with Pascal Vasselon, Senior General Manager Chassis
Q: Sixth place for Jarno and the best result of the season for the team at Indy. Satisfying?
Pascal Vasselon: Jarno gave us an excellent performance. He was very honest about his concern after the incident with Robert Kubica in Montreal. I think Jarno was relieved at the eventual outcome and was able to come to Indianapolis with a clear head and focus fully on the American weekend. He even entered into the spirit of things by arriving every day on a Harley Davidson! His qualifying performance to start eighth was very satisfying, especially when you factor in the fuel load, and he was flat-out all race. He resisted some determined driving by Mark Webber and got us a great sixth place. With the two leading teams monopolising the top four positions again, it was a very strong performance from Jarno. Still, we cannot be totally happy with a sixth place.
Q: But Ralf's problems at Indianapolis struck again?
PV: There's no legislating for that. Ralf certainly doesn't seem to have a lot of luck at Indy. There was a lot of jockeying for position going into Turn 1, Ralf's tyres locked and he made contact with David Coulthard. That was the end of his afternoon, right there at Turn 1.
Q: Ralf set a very quick time in Q1 but then just missed out in Q2. What happened there?
PV: Ralf was actually doing a very good job. His lap time in Q1 was very strong, seventh quickest overall and almost as quick as Jarno's Q2 time. He had a very strong infield sector time but as we saw with a lot of drivers, it was very difficult to put the whole lap together. If Ralf had been able to do the same sector two time in Q2 he would have been in the top 10.
Q:How did Indianapolis go from the operational point of view?
PV: It was quite difficult, for reasons that applied to everyone. You have a combination of two things: first, low downforce because of the long front straight and, second, the asphalt on the infield is low grip, so that makes life difficult for the drivers. That's why we always see a lot of cars struggling for grip at Indy.
Q: We had the soft and medium Bridgestone Potenza tyres at Indy instead of the super soft and soft from Monaco/Montreal. How did the tyres perform?
PV: The tyres were working well without any of the extreme situations we saw to in Monaco and especially in Montreal with the super soft. Both tyres were easily raceable but still a difference between the compounds developed throughout the weekend. As usual the harder tyre started quite well. It is usually more consistent on a Friday but then when the grip develops the harder one starts to lose its potential. The soft was definitely the quicker race tyre.
Q: Some drivers said that the track did not seem to 'come to them' as much at Indy as the weekend developed. Was that down to the asphalt?
PV: Actually, for us the track developed as we would have expected. The evolution of the difference between the two tyres is usually a good indicator and we saw a fairly typical development of relative grip and consistency, which showed that the track was rubbering in.
Q: Looking ahead to Magny Cours, will you have any aerodynamic upgrades on the TF107?
PV: We have a few updates as we go back to a kind of baseline circuit in aerodynamic terms. Ever since Monaco we have been running with special aero packages, which are more race-specific than our baseline package. Monaco, of course, is high downforce, while Canada and Indy are two steps in the direction of a low downforce package. At Magny Cours we will be back to a similar baseline as Barcelona and even Bahrain and Sepang. We have been developing of course and will have an upgraded package.
Q: What do you think of Magny Cours?
PV: Well, I'm just curious to see how our British friends will celebrate what could be the last Magny Cours race because we know that they love the place so much!
Q: Doesn't the track itself have a rather unique surface?
PV: What's special at Magny Cours is that the asphalt is very dark. As soon as you have a bit of sunshine the surface temperature goes up very quickly so, even with reasonable ambient temperature between 25-30 degrees you can have 50 degree track temperature.
Q: What are the implications of that?
PV: It impacts significantly on tyre behaviour and balance.
Q: It has been suggested that reduced tyre grip and aero tweaks have made overtaking even tougher this year. What do you think?
PV: I'm not sure that the aero explanation is the main factor. The tyre factor is probably more dominant because, for sure, the tyres allow less sliding. It is much easier to make mistakes. The tyres have less grip and it's very easy to put them outside the right operating window. That makes it more difficult for the driver to attack a corner with the confidence that a bit of sliding won't put him in the gravel. It's actually quite hard to attack with these tyres. You have to be very clean and that explains the difficulties some drivers have, particularly those who are used to sliding the rear tyres. This season that approach doesn't work so well.
Q: Are you confident that Toyota can return to the head of the midfield group at Magny Cours?
PV: At Indianapolis we were comparatively not so strong in the low grip/low downforce configuration compared to where we were in Barcelona, although Jarno gave us a very good race. We are looking forward to being back to the baseline package which we have focused most of our effort on. We are hopeful of another strong points-scoring performance.
-credit: toyota