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Tag "samsung moment"
New Update to Hit Samsung Moment on November 3rd
Owners of a Samsung Moment, rejoice! Sprint announced an update for your beloved phones. Well, update? This so called update, named DJ07, fixes only two minor bugs of the phone:
911 call restrictions after modem crash related to airplane mode and data lockup
Proximity sensor and screen lock enhancements
Sure, it's fun to have those things fixed, but didn't you expect more? The update will be available OTA soon, direct downloads will be available on November 3rd.
Sprint Expects to Launch Android 2.2 "In the Near Future"
In a recent announcement on Twitter, Sprint has said that it expects to launch Android 2.2 Sprint expects to launch Android 2.2 "in the near future." On the Sprint's message boards, a Sprint employee said
"As work on finalizing the software is under way, Sprint expects to launch Android 2.2 in the near future. It also will be available as an upgrade on the recently launched HTC EVO 4G."
Ok, For Real Guys… Android 2.1 Available for Samsung Moment
Stop us if you've heard this one. Android 2.1 is available for one of Sprint's handsets… Word on the streets is that the Samsung Moment is finally rolling out Android
Sprint Loves Your Mom, Do You?
Wireless provider Sprint is doing what they can to have you looking like a champ this coming Mother's Day. Hesse and Co. have put together a list of Android apps
First Pic of Alleged Samsung Moment 2 Arrives
It doesn't seem to be taking Samsung and Sprint very long to follow up last fall's Moment handset. What you are looking at above is rumored to be the first
Sprint's Android 2.1 Updates Due in "Coming Weeks"
Samsung Moment and HTC Hero owners will be excited to learn that Sprint plans to roll Android 2.1 out to their customers in the "coming weeks". As the carrier gears
Sprint Readying Android 2.1 for Early Q2
Many of the new handsets being unveiled this week are being announced with Android 2.1 installed as the OS. Invariably, this is the time where current Android owners start wondering
Android 2.1 Plans "Going Very Well" for Sprint
A member of the Sprint team took to their official message board to bring everyone up to speed in terms of their Android efforts. As you may know, the carrier
Maintenance Update for Sprint's Samsung Moment
Sprint just announced through their twitter account, and their support forum, that they have released a Maintenance Update for their Samsung Moment handset. Pleased to announce Maintenance Release CL14 is
Official: Sprint's Hero and Moment Getting Android 2.0
Those of you who are Sprint Android owners will be happy to learn that your handset will be getting an upgrade to Android 2.0 in the first half of 2010.Â
Best Buy Mobile Offering 5 Android Under $100
It's a great time to be shopping for Android handsets. Best Buy has announced that they will be carrying five Android powered smart phones at $99 for the holidays. They're
Sprint Working on "Maintenance Release" for Hero
Those of you who own the Sprint Hero will be happy to know that a maintenance release for Android is in the works. While it's not an upgrade to 1.6
Samsung Moment and T-Mobile Cliq Available Now
For those of you looking to get your hands on either the Samsung Moment or the Motorola Cliq, wait no more. You can now head down to your local carrier
Sprint Offers Up Android Anecdotes
sYesterday, David Owens, Sprint's VP of consumer marketing, took part in an hour-long online chat with customers where he dropped a few Android bits that are worth sharing.
The Great Debate
Sprint has finally unveiled details about its upcoming "Instinct Q," which we now know to be the "Moment." Now, I pose a question. So do you go ahead and get
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Chainsaws - Sheep T-Shirts.
Design ID: 11540573
Bernie Sanders Silhouette Women's T-Shirts Designed by 5507177
Tammy Comedy Movie Mahalo Bear Shirt Designed by Mackentee
Nah Rosa Parks Quote T-Shirts Designed by The Shirt Yurt
Beast Jacket Shirt Designed by Winterwolfjon
Hoodini Vanoss Gaming T-Shirts Designed by 3982143
Skeleton X-ray Maternity Shirt Designed by gladditudes
The Devil Made Me Do It Women's T-Shirts Designed by fluffshop
This premium t-shirt is as close to perfect as can be. It's optimized for all types of print and will quickly become your favorite t-shirt. Soft, comfortable and durable, this is a definite must-own and a Spreadshirt recommended product.
still to fill

subset: commoncrawl


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I am creating an simple email client and I want the inbox to display the date that the email was received in the format:
Today at 13:17
Yesterday at 20:38
13 January at 17:15
21 December 2012 @ 18:12
I am retrieving the data from a database, outputting it to xml (so everything can be done through AJAX) and printing the results to a
<ul><li> format.
The Date and Time are stored separately in the format:
I see that something like that is possible with php. Here - PHP: date "Yesterday", "Today"
Is this possible using javascript?


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This profile was last updated on 7/31/15 and contains information from public web pages and contributions from the ZoomInfo community.
Ms. Carol ZernialWrong Carol Zernial?
Executive Director
Phone: (210) ***-**** ext. ****Email: c***@***.net
WellMed Medical Management Inc
8637 Fredericksburg Rd. Suite 360
San Antonio , Texas 78240
United States
Company Description: The WellMed Charitable Foundation was established in 2006 by Dr. George Rapier III, a noted physician who is also the Chairman of the Board. The Foundation is a... more
Pro2015_Iteration9_NoTests.148[05] SEIDXWK-41


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Continuing Studies in Education offers several credit courses in partnership with the Faculty of Education. These courses are offered online. Online learning allows working professionals and others to complete courses without attending face to face classroom sessions.
The following courses are undergraduate credit courses offered at The University of Victoria. To register for these course, students must either be admitted to The University of Victoria or register through Thompson Rivers University. See our Registration page for how to register.
If you're looking for professional development opportunities, see our workshops page.
Assessment Tools - Telling the story of the learner!
10/23 ELL Workshop - Fasten your seatbelts!
10/23 Water You Waiting For?
Search for More


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There are numerous calorie calculators online that go by age and weight to figure out how many calories you burn during the day (aside from riding).
Anyone found one to be more accurate than the others as far as figuring out how many calories are burned during the day?
I have also heard the general estimate of 15 cal/lb thrown out for basic calorie intake for someone with a moderate activity level.
Thoughts? Trying to make sure I'm eating enough during the day.
Results 1 to 5 of 5
Thread: Estimating calorie output?
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Brandon Patrumwrote a review of Costa Man O War Polarized Sunglasses - Costa 580 Polycarbonate Lens on June 27, 2011
My Favorite Costas
I've owned three different pairs of Costas and these are by far my favorite. They were comfortable and kept the sun at bay. The sidearms are not so big that you get a ridiculous tan line from being on the water all day. The lens are by a far the best set of lens that I have ever looked through. I would recommend these to anyone.


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Snow Leopard to warn Apple users of malware - reportsOnly days before the launch of Apple's upcoming operating system upgrade, Snow Leopard, numerous online reports have alleged that the final build includes an anti-malware feature.
Apple's advertisements often boast that their systems are virus-free. While Mac OS X has had a number of widely reported vulnerabilities, despite being well-publicized, they never amounted to much.
Still, in this latest move, if true, it would appear that Apple is not taking its chances, and will provide users with a system that scans files for potential malware. In a screenshot that has been widely circulated, originating from the intego Mac Security Blog, Snow Leopard is shown to recommend that a .DMG file be moved to the trash. The OS claims that the file includes "OSX.RSPlug.A" malware.
That Trojan horse, which was discovered in 2007, runs on OS X and changes the system's DNS settings. It is easily removed and does little damage.
Writing for ZDNet's Zero Day security blog, Ryan Naraine states that Apple is not using the open source ClamAV engine. He believes Apple may have contracted with a third-party antivirus company for the reported new Snow Leopard feature.
If true, the anti-malware inclusion in Snow Leopard would mean that both Apple and Microsoft will soon officially offer some sort of free system-wide protection for their respective operating systems. Microsoft's "Morro" is expected sometime this year.
Late last year, some noticed that Apple began encouraging the use of antivirus for Mac OS X, though the company later removed that support note.
On Topic: Mac OS X
- iMovie update adds support for 4K video, 1080p 60fps, importing projects from iOS
- New Office for Mac 2011 update fixes Outlook sync bug with OS X El Capitan
- Third beta of Apple's OS X 10.11.1 El Capitan released to developers, public testers
- Adobe reverses course, says Metal support in After Effects 'one possibility' [u]
- Apple issues second public beta of OS X El Capitan 10.11.1


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Median Listing Price
Median Listing Price Per Sq. Ft.
# of Sales (30 days)
% Turnover (12 months)
What's the most popular storage unit size in Safford?
Those who rented self-storage in Safford were more likely to book a 5x10 storage unit than any other size.
Do I need a climate controlled storage unit in Safford?
The forecast high for Safford is 85 degrees today. The record high for today in Safford is 96 degrees. High temperatures and humidity can cause mold and mildew to thrive. If you're considering storing items that are susceptible to mold and mildew during this period, consider renting a climate controlled storage unit.
How does seasonality affect Safford storage prices?
Storage prices in Safford tend to be somewhat volatile when compared to other cities. Prices can vary by about $30.00 monthly depending on the time of year. Safford storage prices are highest in February, and lowest in October.


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News & Events
25th Annual General Meeting (AGM) – 14th September 2015 ...-Tue,15 Sep 2015
25th Annual General Meeting (AGM) – 14th September 2015 ...-Tue,15 Sep 2015
Notice regarding AGM : ...-Wed,09 Sep 2015
NOTICE ! NOTICE !! ...-Sun,06 Sep 2015
Funeral Procession of summiteers was organized on 21 April 2014, which started from Sherpa Sewa Kendra, Boudha.
The route of the procession was as follows:
Sherpa Sewa Kendra- Chuchhepati- Chabahil- Mitrapark- Siphal- Naxal Bhagwabati- Durbarmarg- Kantipath- Employees Provident Fund, Thamel- Lainchaur
Speaker of Constituent Assembly Subash Nembang, Deputy Prime Minister Prakash Man Singh, high government officials, political leaders, representatives of different related tourism organizations paid last tribute to the summiteers at Lainchaur.


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There is 1 private schools located in District of Columbia that offer Rock Climbing as an interscholastic sport.
Rock Climbing Private Schools
- School Location Grades Students
Grades: K-8 | 226 students
4400 36th Street NW
Washington, DC 20008
Recent Articles
September 20, 2015
It is easy to fall into the trap of not keeping your skill-set current. There's no time and no need anyway. We look at why you need to keep things current and offer some suggestions as to how to proceed.
September 07, 2015
Choosing the right school for your child is a major project. We look a five reasons why you might be looking at the wrong schools.
August 30, 2015
Scandals in private school are bound to raise questions for those of us thinking about sending our children to private school. Regardless of how sensational these scandals may appear, they are actually few and far between.


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Gearbelt Idlers
2 Results Found that Include 2 Products
- Gearbelt Pulley Idler, 1/2 PitchGearbelt Pulley Idler,1/2 Pitch
- Item # 6AGN9
- Mfr. Model # N16H100
Gearbelt Pulley Idler, Pitch 1/2 In, Gearbelt Type H, Number of Grooves 16, Bore Dia 1 In, Outside Dia 2.50...More
Price $120.40 / each
Catalog Page # 171
- Gearbelt Pulley Idler, 3/8 PitchGearbelt Pulley Idler,3/8 Pitch
- Item # 6AGP1
- Mfr. Model # N22L100
Gearbelt Pulley Idler, Pitch 3/8 In, Gearbelt Type L, Number of Grooves 22, Bore Dia 1 In, Outside Dia 2.60...More
Price $107.40 / each
Catalog Page # 171
Position belts away from obstacles and prevent belt slippage with gearbelt idlers from Grainger. Gearbelt pulley idlers are for use with drive tighteners and tightener shafts. They take up extra belt length and provide adjustment for the tensioning belt drive. You can apply them to either the inside or the backside of belts. Order your gearbelt idlers online at Grainger!


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Indoor Arcade Basketball Games and Options
Here is some help for you when researching a purchase for
an arcade basketball game for your home use. First, we will look at
manufacturers of these types of games. Lifetime Products, Spalding,
and Escalade Sports are the largest manufacturers of these types of
systems – there are many other smaller makers as well. Of these 3
main players in this market, Spalding and Escalade Sports offer a
more than 1 system. Each of these manufacturers offer very similar
products in quality and play. However, certain minor differences
between their product offerings may sway you one way or the other.
We will look at each unique portion of their systems next.
Using the Lifetime Double Shot Arcade Basketball Game Review
After about 6 months of ownership, I figured I should give an update of this system and how it has worked out for my family. First off, my new high score on this system 170. From speaking with a couple of people who work at Lifetime Products, they know of one of their employees that claims to have scored higher than 200. I am skeptical of that; though, I must say that the time I scored 170 I still had room for improvement. Back to the topic at hand, I wanted to share some observations of this system. I figured I would talk about the basketballs, the frame of the Double Shot, and the playability.


text: February 13, 2014 Mickey With Olympic Hockey Coverage Podcast FeedMore From Drew & Marc Red Wings Color Analyst Mickey Redmond dialed in today and discussed the Red Wings currently in Sochi at the Olympics. Listen right here for the coverage and schedules. 16:08Mickey_Redmond18Download Podcast http://detroitsports1051.com/wp-content/uploads/sites/23/2014/02/Mickey_Redmond18.mp3 By: Bruce Bennett Getty Images Sport Related Intern Joanna Rose Bowl Coverage From Pasadena Jim Leyland: HOF Voting, Fielder, Life After Coaching Bestselling Author John U Bacon Talking With Tirico


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Free Agency: The Hits and Misses
Matt Murnane 4:20 PM With the AFL's free agency period set to start on Friday, we look at some of the notable signings, both good and bad, since free agency started at the end of 2012.
Top Stories
Could this Cat become a Lion?
4:20 PM AFL Geelong premiership star Steve Johnson could extend his AFL career next season at the Brisbane Lions.
Ponting to testify in Cairns perjury case
Jon Pierik 3:25 PM CRICKET Former Australian captain Ricky Ponting among those who have been called to give evidence at the trial of Chris Cairns, who is facing charges of lying in court about his involvement in match-fixing.
Austin Waugh is now the one to watch
Jamie Marcuson 2:46 PM CRICKET What do you do when your father is a former Australian cricket captain and your uncle is considered one of the most elegant and gifted strokemakers ever?
Franklin to miss Swans best and fairest
Jon Pierik 12:11 PM AFL Lance Franklin will not attend the Sydney Swans' best-and-fairest count as he continues to recover from his mental health issues.
Victoria crush CA XI in Matador Cup
3:40 PM CRICKET The Cricket Australia XI have been on the end of another massive domestic one-day cap defeat, going down by nine wickets to Victoria at Hurstville Oval in Sydney on Wednesday.
Heartbroken Courtney ruled out of Bathurst
3:53 PM MOTOR SPORT Injured V8 Supercars star James Courtney is confident of returning to racing in a fortnight despite receiving the heartbreaking news he must pull out of this weekend's Bathurst 1000.
Melbourne Victory crack 25,000 members
2:50 PM SOCCER Melbourne Victory have become the first A-League club to surpass 25,000 members, beating out all but two NRL clubs in the membership stakes, as well.
Nikolic's racetrack ban lifted
Mark Russell 11:14 AM HORSE RACING Racing Victoria decided not to re-licence former jockey Danny Nikolic, saying one of the reasons for the decision was his racetrack ban. Nikolic may now appeal.
NBL 2015/16: Title prospects and key players
Roy Ward 9:21 PM BASKETBALL A look at how all the clubs look ahead of the new NBL season.
Preferment to bypass Caulfield Cup
Chris Roots 7:52 AM Racing Trainer Chris Waller has confirmed jockeys for four runners in next week's Caulfield Cup, but doesn't intend on running Preferment, which will be saved for the Cox Plate.
Nadal struggles in win over former ball boy
11:54 AM Rafael Nadal struggled to a win on Tuesday at the China Open against a man who used to toss him balls and towels.
Count me out for award, Goodes says
Larissa Nicholson AFL The award recognises retiring players who have contributed the most to the game and the broader community. Adam Goodes asked not to be considered.
Tigers 'tired' of missing grand finals
Daniel Cherny 8:18 PM Richmond president Peggy O'Neal says the Tigers are "tired of watching someone else play in the grand final," and labelled the conclusion to the club's season a "stinging disappointment."
Garland re-signs with Melbourne
Larissa Nicholson 1:03 PM Colin Garland has re-signed at Melbourne and will remain a Demon until the end of 2018.
Competition the key to City's success
Michael Lynch 11:17 AM For Melbourne City skipper Patrick Kisnorbo, it's the C-words that will determine whether his team can build on its fourth-placed finish from last season.
Siddle may be squeezed out of Vics opener
Daniel Cherny 8:25 PM Peter Siddle was supposed to be preparing for a Test match scheduled to begin on Friday. Now he might be squeezed from Victoria's side for the Matador BBQs One-day Cup clash on Wednesday.
Rugby Union
Wallabies face biggest challenge
Chris Dutton 6:38 AM A "gutted" Michael Hooper has backed Sean McMahon and Ben McCalman to stand tall in his absence, adamant the Wallabies have the back-row depth to continue their World Cup charge.
Horse Racing
Press Statement's barrier draw 'no problem'
Patrick Bartley 9:25 PM Australia's most successful trainer, Chris Waller, was unperturbed after his three-year-old Press Statement drew barrier 14 in Saturday's $1 million Caulfield Guineas.
NBL, Nine team up for weekly free-to-air game
Roy Ward 9:14 PM The NBL will get a weekly free-to-air game this season after signing a deal with Channel Nine.
Rugby League
Cowboys, Broncos and Roosters to represent NRL in World Club Series
Adrian Proszenko 5:39 PM North Queensland, Brisbane and the Roosters have officially accepted invitations to represent the NRL in next year's World Club Series.
Motor Sport
F1 could be sold this year: Ecclestone
9:35 AM Formula One could have new owners by the end of the year with three parties currently interested, the sport's commercial supremo Bernie Ecclestone said on Tuesday.
Kyrgios wins, three other Aussies out
8:46 AM Nick Kyrgios has spared Australia's blushes in a day of injury and implosion for his countrymen at the Japan Open.
Aussie Murdaca turns professional
10:56 AM Talented young Australian golfer Antonio Murdaca is turning professional.
Armstrong settles with promoter over bonuses
Tom Korosec Lance Armstrong settled a cycling promotion court fight on confidential terms but still has a lawsuit over a US Postal Service sponsorship to contend with.
More Sport
Bolt swaps lycra for lederhosen
David Sygall ATHLETICS Looking as resplendent in lederhosen as he does in lycra, the world's fastest human, Usain Bolt, has been getting into the spirit of Oktoberfest.
Baseball team takes stadium food to next level
David Sygall BASEBALL American sports stadium caterers seem intent on making their patrons ill. Starting with a cotton candy hot dog.
Hayne explains fumbled punt return
Jarryd Hayne has refused to point fingers at any of his San Francisco 49ers teammates, but says everyone needs to come together to break their losing streak.
Tony Modra winds back the clock
Got a favourite Tony Modra memory? Well, now you can add another to the list.
NRL wins grand final weekend
"Could you write a better script?!" cried commentator Phil Gould as the Cowboys drew level.
Bennett's golden point gripe is disrespectful
The Broncos coach couldn't help himself after his side lost the NRL grand final.
Eagles CEO wants more games at the G
Eagles CEO Trevor Nisbett wants more games at the MCG next season
Jose Mourinho: I will not resign
Chelsea keeep losing games, but Jose Mourinho says he will never resign from the club.
Hawks celebrate third Premiership win
Hawthorn's victorious premiership team take to the stage at Melbourne's Glenferrie Oval.
Hawthorn win 2015 AFL premiership
Hawthorn easily account for West Coast by 46 points to win their third straight AFL Grand Final.
Greatest Grand Final: The famous 1989 game
Brereton's KO, Ablett's nine goals, the Hawks hang on desperately - was this the greatest of all time?
It may have been so different for Lyon
In a recent interview on the ABC, Malcolm Turnbull alluded to the role of luck in his life.
Hodges speaks of 'relief' after hearing
Justin Hodges can play "one more game" after being cleared by the NRL judiciary to play in the Grand Final on Sunday.
Chelsea loses to Porto
Uncomfortable return for Jose Mourinho to Porto, the team he previously coached to Champions League success.
Warner gets life ban
Disgraced former FIFA vice-president, Jack Warner, has been banned from football for life.
AFL weekend wrap
Penultimate weekend of footy in 2015 sees the Hawks and Eagles secure their grand final spots.
Greatest grand finals: No.5, 1970
Jezza's mark, Pies' 44-point lead, Blues storm home - was this the greatest grand final of all time?


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Latest NewsGo to News
Core World Changes
10.05.2015 Drawing inspiration from the success of last year's 12x XP boost and the more recent Epic Story XP Boost, in Fallen Empire we've altered the core leveling experience from 1-60 for all players, making it quicker and more story-centric.
Combat Changes in Fallen Empire
10.02.2015 With the launch of Knights of Fallen Empire, we are planning quite a few general changes to combat.
Cartel Market Changes Coming in Fallen Empire!
10.01.2015 I wanted to take a moment and let you all know of some exciting changes coming to the Cartel Market with the release of Knights of the Fallen Empire
New Media ItemsGo to Media
Latest Videos
09.15.2015 Get a behind-the-scenes look at the story and writing behind Knights of the Fallen Empire, the upcoming expansion for Star Wars™: The Old Republic™!
08.05.2015 Take a first look at new gameplay coming from BioWare's Knights of the Fallen Empire, the upcoming expansion for Star Wars™: The Old Republic™!


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Went to this zoo because it was recommended in my traveling guide.
I can tell you I was far from disappointed, it's a great place to spend a few hours. Luckily we chose a day were there weren't a lot of people. Walked through the whole facility which took about 4 hours total. If you don't have that much time there's a train running through the park, a few meters above the ground, which drives every 30 minutes, also you could rent what I would call "bikes", but it's more like combined bikes kind of like a golf cart. They have a huge variaty of animals and pretty much all of them are in quite spacious, open areas without cages, bars or anything like that. Also the staff which drives around in golf carts always greets you, which is very nice. Also very impressive: A huge area with hundreds of different birds.
Only thing that keeps me from rating it as excellent is, that I expected the areas in which the animals are held to be a bit bigger (especially the snakes and frogs), about four type of animals were not in there areas ("off exhibit"), also as it wasn't a busy day only half of the food stands were open.
Own or manage this property? Claim your listing for free to respond to reviews, update your profile and much more.


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NL Central Smackdown : Right Fielders
Center Fielders | Poll
I'll save the left fielders for Monday since it's a pretty and talented group. Your right--field warriors after the break...
|3-Year Warp-3 Average
|3 Year Warp-3 Projection
|Rob's Ranking
|Kosuke Fukudome
|Corey Hart
|4.5 (2 years)
|Skip Schumaker
|1.5 (1 year)
|Ryan Ludwick
|2.7 (1 year)
|Hunter Pence
|6.4 (1 year)
|Ken Griffey Jr.
|Xavier Nady
The Justification: I think I sold Hunter Pence short on this one before I looked at the numbers. I just thought he was more useful as a center fielder and a little hacktastic for my tastes. Griffey Jr. probably deserves the top spot as a lifetime achievement award and if you think he can stay healthy, he probably still deserves it. Honestly, any of the top four are probably worth the top spot and when Rick Ankiel gets shifted back here, he'll make it even a little more interesting. But I took Hart for now, a rather successful 2007 campaign and youth on his side. I just didn't know what to expect out of Fukudome, and while the early returns are fantastic, he won't be able to keep his offensive numbers up averaging a homer a month. Nady's had a nice start to the season, but we all know he'll fade and the Cards platoon is just a placeholder until Colby Rasmus is ready and Ankiel gets shifted back.
There's another poll for you guys to fill out below this and I'll leave the old polls open until we finish this.
Please note, you do have to register to vote. This isn't some scheme to solicit registrations, it's just an anomaly of the site and the poll software that's built in. Trust me, I'd love to fix it, but I haven't found another piece of integrated software that does these ranking polls which I really like. Plus if you register, you get a better user experience as I tend to test everything as a registered user and you get an uncached version of the site. You can also view the results of the poll after you vote, instead of having to wait until I close it. It takes all of two minutes to register and unlikes some sites, I'll be more than happy to cancel your account if you wish. You'll never receive any spam from us either...promise.
Re: NL Central Smackdown : Right Fielders
This is very tough; really a close grouping and alot of moving parts.
I ranked as the following:
Fukudome (homer vote, but he is a 4-tool player)
Hart (slow start though)
Pence (would be higher, except for slow start; based on last year, he could be #1)
Griffey (Fading fast)
Cards (They are hitting great, but I hate platoons)
Re: NL Central Smackdown : Right Fielders
Tough one for me. I went Fukudome, partly out of homerism, then Pence, Hart, Nady, Cards, and Griffey.
Re: NL Central Smackdown : Right Fielders
I take Hart over the lot at this point, then Fuku, Pence, Griffey, Nady, Cards. Hart is young and is already a pretty complete hitter, plus I think he handles CF at least as well as Pence and Fuku. Pence has the best chance of being a real superstar, but he also seems most likely to flop due to his hacktastic approach. Fuku gets second based on defense and plate discipline. Griffey would be tops if he weren't such an injury risk and if he weren't so old and more expensive than the rest.
Nady doesn't seem all that valuable any more, but maybe that's just me.
Speaking of Fuku
is his lack of power concerning to anyone else? The bill of goods we got when he was signed was that he'd be good for at least 15 and probably 20 homers a year. He's got good gap power but the weight shift in his swing, in my mind, keeps him from putting more power in his swing. Anyway, I hope he starts hitting for a bit more power soon, as we need it out of our #5 hitter.
Re: Speaking of Fuku
I'd be more concerned if he wasn't on pace to hit 50+ doubles. I'll take 50 doubles and a .400+ OBP out of my 5th hitter any day, especially when he has DeRosa and Soto hitting behind him.
Re: Speaking of Fuku
Yep. Not concerned. There are other ways to win besides Chadball.
Re: Speaking of Fuku
Hey! 50 doubles is Chadball through and through!
Re: Speaking of Fuku
50 doubles isn't Chadball!
No steroids, no homeruns, no watching the ball exit the park followed by cocky strutting around the bases. No boombox in the locker room annoying everyone else.
I think Fuku is a few hat size short of Chadball.
Re: Speaking of Fuku
Sorry pal, but I'm the authority on Chadball. And 50 doubles is Chadball material. Chad likey the slugging percentage. last year when DLee wasn't hitting the ball over the fence but bouncing them off of it, I was totally ok with that. Walks and singles as your offensive set is not Chadball but Mark Grace was.
Re: Speaking of Fuku
personally, i didnt expect more than 15-20 homers at best.
however, i do/did expect him to get 40-50 doubles.
i expected him to K 100-120 times and though i knew he'd walk im personally impressed by how many so early.
to make a sloppy comparison...i pretty much expect mark grace in his prime at the plate (with more Ks and a touch more speed).
Re: NL Central Smackdown : Right Fielders
Vorare: Exactly! Soto hitting behind Fuku is brilliant, and I hope it continues.
Fuku is still adjusting to a lot of pitching he has never seen, and his contact style is unique. He pulls off the ball much more with two strikes on him. He gets into the two-strike situation because he is taking pitches in an attempt to see more (whether that is patience or just feeling out the pitcher, I don't know). The great thing is that he isn't afraid to hit with two strikes, and he's a damn good hitter in that situation. He is Ichiro with more power, and a slightly lower average due to his slower top speed. I see Ichiro leg out a lot of those infield singles which drives his average from the .320-.330 range to the rarefied air he has been privy to the last few seasons.
Re: NL Central Smackdown : Right Fielders
Identical to Rob's. A year or two ago Jr might have been top of the list, but not now.
Re: NL Central Smackdown : Right Fielders
I'm reading over other people's votes and I feel I should say it really pained me to vote Corey Hart first. He's a douche.
Re: NL Central Smackdown : Right Fielders
Me too, though I have nothing against Hart personally, I just can't stand the fact he plays for the Brewers. Is there some more specific reason you call him a "douche"?
Re: murt...
a trade?
Re: murt...
He's in there tonite, batting third.
2 for 2 as the DH.
Re: murt...
weird...they had Kroeger DH as the DH, now its murton on milb.com
Re: NL Central Smackdown : Right Fielders
Gagne blows a two-run lead in the ninth in Houston to send it into extras. Go 'Stros.
Gagne blows another save
this time a 6-4 lead in HOU. I guess the Brewers closer will be David Riske pretty soon.
Re: NL Central Smackdown : Right Fielders
A. Soriano lf
R. Theriot ss
D. Lee 1b
K. Fukudome rf
M. DeRosa 3b
G. Soto c
M. Fontenot 2b
R. Johnson cf
J. Marquis p
I wish Lou would expirement with hitting the pitcher 8th
because with this line-up it makes alot of sense, with Reed's good OBP and Marquis being a solid hitter.
Re: I wish Lou would expirement with hitting the pitcher 8th
the lineup's full of ob% guys...why let the pitcher move up a slot to replace a far better hitter?
and though i can understand not using cedeno today the guy should get some starting time one day...maybe.
...AND END THIS SORIANO BATTING 1ST CRAP. and stuff...
we got a RBI guy missing (aram) and we got a powerhitter looking to cash in the 7/8/9 hitters.
When did Augie Ojeda get good?
I think he has more hits today aganist Santana and the NLDS last year then he did his entire Cubs career.
cuz some of you likes the foots balls.
AUSTIN, Texas (AP) -- Chicago Bears running back Cedric Benson failed a sobriety test while operating a 30-foot boat, then resisted arrest before being hit with pepper spray and dragged ashore by officers.
Re: cuz some of you likes the foots balls.
Well I thought the Bears had a foolproof protocol for this kind of situation...
Why didn't he just dive into the water, dial 911 and tell the police that his boat was stolen?
Bill Hall just had a mild collision
with Prince trying to get a foul pop-up.
Pence hits a walkoff
to complete the sweep of the Brew Crew.
Recent comments
CUBSTER: I would think a cab or a limo would get Motte to the airport faster than a golf cart. The maximum speed of a golf cart is (what?) maybe 15 MPH, which could actually be dangerous on an expressway.
from the Sunday Trib...
Reliever Jason Motte waved as he left the visitor's clubhouse at Miller Park on a golf cart en route to the airport to catch a flight to Arizona to start a rehab assignment.
Motte has been on the 15-day disabled list since Aug. 24 because of a right shoulder strain, but there's a possibility he could rejoin the Cubs if they advance deep in the playoffs.
Motte is scheduled to pitch in simulated games and in Instructional League games in Arizona.
8 in a row to end the year...let's do this playoffs thing.
shelby miller (ATL) finishes the season with a win, snapping his 24-game winless streak...because ATL bats are non-existent.
3.02 era, 1.25 whip, 205.1ip, 171K
6-17 w/l...ugg.
Totally independent of the Cubs winning or losing today, Dan Haren came into today's game having earned $2M of his potential $3M in performance bonuses for 2015.
By making the start (his 32nd start of the season) he picked up $500K, and he'll get the remaining $500K if he can throw at least 8.2 IP in the game (he was at 181.1 IP coming into the game, and he needs to reach 190 IP to get the remaining $500K),
It's a bit like the old "Bowling for Dollars" TV show, but of course Haren is actually "Pitching for Dollars."
holy crap, MIL is just...awful.
i think he's currently playing for the Santo Domingo Meths. he's an Adderall-Star player there.
I'm longing for a Neifi sighting.
if you have stable/fast internet you can stream with and you don't mind giving up $20 a month check out "sling tv".
TBS, TNT, ESPN 1+2, etc...100% legal.
68 pitches (50 strikes)
love seeing that...especially with the batters doing very little with it.
Yeah, keep him off the playoff roster.
Or, he's looked exactly like Ted Williams.
Lately he hasn't looked like Ted Williams...
Reading between the lines on some of his comments he seems to know his longish swing doesn't play well with a man on third and two outs, and two strikes on him. I doubt a playoff adjustment is happening, just something he'll need to figure out next year. I have this odd feeling he will.
Mark Gonzales @MDGonzales 4m4 minutes ago
Bryant leads NL rookies in HRs (26), RBIs (99), 2B (31) and runs (86). Last rookie to reach 26 HR, 99 RBI, 31 2bs, 86 runs and 74 BBs?
Mark Gonzales @MDGonzales 4m4 minutes ago
Boston's Ted Williams in 1939


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Yarl's Wood Befrienders
We aim to affirm human dignity and restore self-esteem by listening and offering befriending support.
What Yarl's Wood is
Yarl's Wood Immigration Removal Centre has the capacity to hold around 400 detainees. It has two units for women, an admission unit and a smaller unit for couples and families with adult dependents. It is the main facility for the detention of women in the UK. In December 2010, it was finally decided that there would be no more children held at Yarl's Wood. From February 2012 it will also incorporate a short-term holding centre for males who have been detained upon arrival in the country, whilst they undergo an initial screening process.
What we do
Detainees can be vulnerable, isolated and frightened. Many are young, speak no English, and may not know anyone in this country. Whether a detainee has recently arrived in the UK or has been here for many years, the experience of detention can be traumatic. They are held for indefinite periods of time, and do not always understand the system that will decide their future.
Our volunteers undertake to visit a detainee regularly, listening and caring about what happens to them. In this way, although we cannot affect the outcome of their case, we can help to reduce their isolation, acting as a contact with the outside world. The visitor notices when anxiety and depression, or other medical problems, are becoming serious.
We put great emphasis on training and supporting our volunteers, who meet regularly for mutual support. The organisation is run by a Management Committee. The group is linked to organisations which visit other detention centres through AVID (the Association of Visitors to Immigration Detainees).
We can assist with small practical needs, such as toiletries, second-hand clothing, international phone cards, and small amounts of money to destitute people being deported.
We are non-party-political and are funded entirely by non-governmental grants and personal donations, enabling us to remain completely independent. Although we do not campaign, we use our insight into the experiences of detainees to inform policy, work towards the improved treatment of detainees and to challenge negative attitudes towards immigrants, particularly asylum seekers.
If you would like to become involved in our organisation, or contact us for any other reason, please follow this link
We are very grateful to all our funders and donors, who make the continuing work of Yarl's Wood Befrienders possible. The following organisations have supported us in 20014/15:
- The Eranda Foundation
- The Harpur Trust
- The Leigh Trust
- Cathedral & Abbey Church of St Alban
- The Bromley Trust
- Steel Charitable Trust
- St Andrew's Church Bedford
- Wixamtree Trust
- House of Industry
- Noel Buxton Trust
- Swan Mountain Trust
- St Peter de Merton Church, Bedford
- St Cuthbert's Church, Bedford
We have also been greatly assisted by support and advice from
AVID – Association of Visitors to Immigration Detainees


text:
The Buffalo Bisons organization was saddened to learn about the passing of former Bisons pitcher Rudy Minarcin.
Minarcin pitched five seasons in the International League (1951, 1954, 1956, 1958-59) with his best season coming in Buffalo. For the 1951 Bisons, the righty was 16-12 with a 3.20 ERA in 33 games. That season, he made 31 starts and tossed an amazing 17 complete games.
Minarcin also appeared in three seasons in the Major Leagues, 1955-1957, winning five games for the Cincinnati Redlegs in 1955. That year, he was a teammate of future Bisons manager, third baseman Rocky Bridges.
Minarcin passed away on October 15 in Cabot, PA at the age of 83.
Today on MiLB.com
Top Stories on MiLB.com
- Brainstorming at the MiLB Promo Seminar
- Listen: Rays' Blake Snell joins MiLB podcast
- Batting Around: PawSox looking for new home
- Rawlings, Minors announce Gold Glovers
- Contreras, Williams nab Cubs' Minors awards
- D-backs' Byler, Padres' Dimock suspended
- Toolshed: Five things we learned this season
- Harvey, Bundy heading off to Orioles' instructs


text:
johnm's blog
The "Community" is Always Right?
In Sue Spielman's Practical JSTL article, she writes:
Java's Inferiority Complex?
The TechnoTourette blogger
writes I don't want to hear about it Mr. dotnet and that got me thinking about how Sun and so many Java folks react to the dreded "Evil Empire" that is MS.
Java Servlet v2.4 and JavaServer Pages (JSP) v2.0 "Final" Specifications released
The JavaServer Pages v2.0 specification was released.
Be sure to also check out Greg Wilkins' critque of the Java Servlet v2.4 specification.
Architecture: Abstract vs. Manifest
Some seemingly random thoughts struck me as I read
Satya Komatineni's blog Architecture is language agnostic...:
China options push Sun to consider open-source J2EE app server?
Hmm... Opening up the Chinese market (that won't pay the ridiculous licensing fees anyway) through the hearts and minds of the developers and hoping that will convince them to buy (expensive) Sun hardware? I'm not so sure about that. Will they buy Sun's services? Perhaps but I'm not going to hold my breath.
Lame "survey" on Java reliability
First off, let me question the fact that nowhere is it listed in the article that we can't actually get a copy of the survey and survey results without signing up for one of Wiley Technologies seminars (i.e., sales pitches). Tsk, tsk, tsk.
Apache Software Foundation working on new/updated software licenses
There are a number of proposed changes to the various Apache softwware license agreements.
The Java Servlet Specification v2.4: The Good, the Bad, and the Ugly
Greg Wilkins, creator of Jetty and co-founder of the Core Developers Network wrote this in-depth critique of the Java Servlet Specification v2.4.
Enforcing Model-View Separation in Template Engines
Terence Parr, creator of ANTLR, writes on why
Enforcing Model-View Separation in Template Engines is a Good Thing(tm).
Why Humans Should NOT Have to Grok XML
Terence Parr, creator of ANTLR, wrote an excellent essay on why humans should not have to grok XML.


text:
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Klear Vu Corona DelightFill 2-Pack Chair Cushions
web ID: 7377104
Refresh your look with Gripper® chair cushions that are as functional as they are fashionable.
- slip-resistant latex backing eliminates the need for string ties
- knife edge border adds dimension
- color-coordinated shapes work well with almost any décor
Corona DelightFill® chair cushion is polyester/rayon/cotton with recycled polyester filling and a latex foam/polyester back. Package of 2, each measuring 16Wx15Dx2"H. Spot clean. USA.
Rated 4 out of 5 by HC7456 Stays put These chair cushions actually stay put on the chair and do not slide off when one is getting up and down. The pattern has enough colors to be useful in any color scheme. July 1, 2015
Rated 5 out of 5 by Scooter2008 very nice looking We really like these, love the colors and really enjoy having not to tie them, great concept ! March 4, 2015
Rated 4 out of 5 by piatrouble pretty not at all practical Perfect colors to tie together for my decor, price was right...BUT its only been a couple of weeks & the 2 most used are flat. Hopefully will last long enough to find good replacements. Save your money unless you're planning on only having them for show. December 17, 2014
Rated 5 out of 5 by vic333 chair cushion I love this chair cushion it does have the grip on the bottom so it doesn't slide or need to be tied. Great quality and colors. January 1, 2015
Rated 5 out of 5 by happiestcustomer Very satisfied These went wonderfully with my dining chairs and warmed up the extra folding chairs I needed for Thanksgiving dinner! Check out was easy, shipping was fast and arived within two days of ordering! Way to go JCP ! December 3, 2014
Rated 5 out of 5 by Batkiss Just Perfect No tie cushions work really well., no sliding at all and I didn't want the country look u get with ties. And, the colirs were true and as shown on my computer and match perfectly with my colors. Comfortable and good quality as well August 27, 2014
Rated 4 out of 5 by rosiefam Design and color make the difference on these cushions! Pleasantly surprised with these chair cushions, while they are not thickly padded, like my previous ones, they are well made. Have only had them a few weeks, family has "noticed" pattern & color. The gripping on cushion really works, even when active grandkids are visiting-they stay put! August 2, 2014
Rated 5 out of 5 by nunziata7 better than expected These cushions are much better than I expected. A great value. They fit snugly on the chairs.and look fantastic. September 24, 2014
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text:
So, yeah, I got the game on launch day, and I enjoy it very much
For those of who who have the game, I will share the ID of my levels soon.
Harem Escape Ch.1The IntroductionHarem Escape Ch.1 by Krocken
Schala groaned as she began to regain her senses. She had no idea what happened earlier. She had just started walking home after some quality time with Krocken,, when she felt some overwhelming force hit her, then…nothing.
Schala sighed , noticed she was sitting, then decided to stand up to get her bearings…and found herself rooted in place. She blinked in confusion before her senses sharpened.
Her eyes bulged as she saw her situation.
First, her purple dress was gone, replaced by a rather…scandalous outfit: A harem dancer outfit. Her top was a glorified bra, purple in color with golden tassels, and it hugged her breasts tightly, showing a bit of her cleavage, Her midriff was exposed, with a purple gem placed firmly in her navel. Her legs were covered by purple puffy harem pants with golden waistband, and her feet had Persian slippers covering them. But that was not the most shocking thing about her situation.
She was severely tied up. Her arms
Pirate Quest Ch. 23Pirate Quest Ch. 23 by Krocken
Saying Goodbye
"The old bastard didn't stand a chance one you decided to just ice him." Korra said with smirk to her captain.
"Yeah, but doing that always tires the hell out of me." Krocken sighed as she dragged the block of ice to the group.
Garna looked into the ice, and made out what appeared to be a very large heart, with surface that seemed to be a mix of molten rock and iron. "Say," he reached for it. "Just what is this any-"
He was halted when Krocken grabbed his arm in a firm grip. "Don't touch, and you may regret what you see." He said simply.
"Er…all right." Garna withdrew his hand.
Krocken sighed. "Now the, let's get this thing aboard and-"
A cock of a gun caught his attention, and he turned to see Noel pointing her weapons directly at his head.
"N-N-Noel?! What are you doing?!" Momo stammered din shock.
"I'd like to know as well."
Pirate Quest Ch. 22Pirate Quest Ch. 22 by Krocken
Nicol Bolas
The Opal Serpent soon reached the mouth of the cave, and the Krew began to prepare them to go in.
"All right, everyone." Krocken said as chest and weapons were gathered. "Arm yourselves well. We have no idea what else is in that cave. We need to prepare accordingly." He fingered Tasogare as his face hardened.
"Wow," Lawrence said to Ibuki as he heaved up an empty chest. "He always makes sure to be prepared…then again, that's quite reasonable, especially after all we dealt with today."
"Yep. It's how one survives in the wilderness." Ibuki replied. "Drop your guard, and you can wind up dead." Lawrence shuddered slightly but nodded.
As the small lifeboats were prepared, a groan could be heard form someone coming up. All eyes turned to see Korra, slightly dazed but otherwise looking quite all right.
"Oh, my head…" she groaned. "What did I miss?"


text:
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text:
Workshops and Events
- Cooperative Education Information Session
- Balance your studies with related experience! If you are considering a Co-op, this 45 minute presentation will inform you on how to get the process started. Select a date below to register!
- Mallory Hall, Suite 116 Conference Room
- Thursday, October 1st
- Thursday, October 29th
- Tuesday, November 3rd
- Thursday, November 5th
- Wednesday, November 11th
- Monday, December 7th
- Resume Writing Workshop
- Increase your understanding of how your resume is used in the job search and make sure it targets your career goal. Learn how to compose a resume that reflects your greatest strengths and talents. Explore formats that say, "Interview me for the position!" Don't forget to bring your resume.
- Sokol Room
- Wednesday, September 30th
- Wednesday, October 14th
- Tuesday, November 10th
- Thursday, December 3rd
- Monday, March 14
- 4pm-5pm
- Internship and Job Search
- This workshop will answer your questions about how to start the process of looking and finding part-time or full-time jobs and internships. It will provide specific steps on how to decide what to look for, where and when to search, and who to talk with. You will leave with resources to begin your research and the job search process.
- Sokol Room
- Thursday, October 8th from 5pm-6pm
- Lab Support
- Back by popular demand! Meet representatives from Lab Support and learn about what Human Resource Managers look for in the science resume. You will also learn about what makes candidates stand out in the interview process. This presentation will include an overview of Lab Support. Refreshments will be served. Reservations are required.
- Sokol Room
- Thursday, October 15th from 3pm-4pm
- Registration Required
- Writing Cover Letters
- Sokol Room
- Tuesday, October 27th from 12pm-1pm
- Are you LinkedIn or Left Out
- You will leave this presentation knowing the tools and tricks to create an online presence for employers, professional networks and more!
- Sokol Room
- Wednesday, November 4th from 3pm-4pm
- Thinking about a career as a college professor?
- If you have ever considered a career as a college professor, then this workshop is for you. Eric Cameron, Assistant Professor at Passaic Community College and author for Pearson Education, will discuss career opportunities as a faculty member while discussing the differences between employment at 4-year and 2-year colleges and universities.
- Sokol Room
- Monday, November 9th from 3pm-4pm
- Registration Required
- Internship and Job Search
- Sokol Room
- Thursday, November 19th from 5pm-6pm
- Making the Most of a Career Fair
- A workshop to help know what to expect at career fairs. Topics include: how to plan a strategy for most effective and efficient use of time at a fair; the introduction - making the first impression a good one; statistics on number of interviews and jobs offered at career fairs; follow-up after the fair.
- Wednesday March 2, 2016 (3pm-4pm)
- Monday March 14, 2016 (12pm-1pm)
- Career & Internship Fair
- March 16, 2016


text:
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SIRT1 is a NAD+-dependent histone H4K16 deacetylase that controls several different normal physiologic and disease processes. Like most histone deacetylases, SIRT1 also deacetylates nonhistone proteins. Here, we show that two members of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, hMOF and TIP60, are SIRT1 substrates. SIRT1 deacetylation of the enzymatic domains of hMOF and TIP60 inhibits their acetyltransferase activity and promotes ubiquitination-dependent degradation of these proteins. Importantly, immediately following DNA damage, the binding of SIRT1 to hMOF and TIP60 is transiently interrupted, with corresponding hMOF/TIP60 hyperacetylation. Lysine-to-arginine mutations in SIRT1-targeted lysines on hMOF and TIP60 repress DNA double-strand break repair and inhibit the ability of hMOF/TIP60 to induce apoptosis in response to DNA double-strand break. Together, these findings uncover novel pathways in which SIRT1 dynamically interacts with and regulates hMOF and TIP60 through deacetylation and provide additional mechanistic insights by which SIRT1 regulates DNA damage response.
Silent information regulator 2 (Sir2) was originally identified in a genetic screen for genes involved in controlling expression of silent mating type loci in yeast (58). Initially, the interest in Sir2 was limited to the studies of mating type interconversion or gene silencing in yeast. This situation, however, took a remarkable sharp turn with the intriguing finding that Sir2 promotes longevity in Saccharomyces cerevisiae (34). Subsequent studies by Imai et al. unexpectedly revealed that Sir2 is an NAD+-dependent histone deacetylase (HDAC), opening a new area of Sir2 research (30).
Of the seven human Sir2-like proteins (sirtuins), SIRT1 is most similar to the yeast Sir2 protein, which is the prototypic class III HDAC (3, 23, 25, 46). Although several studies suggest that sirtuins might not directly regulate aging via originally proposed mechanisms (6, 33), it is clear that sirtuins, particularly SIRT1, regulate metabolism, physiological homeostasis, and stress response and suppresses age-associated diseases (26, 27, 60, 72, 86). Most Sirt1-deficient mice die perinatally, and outbred background Sirt1-null animals are sterile with retinal, bone, and cardiac defects, reinforcing the notion of a pivotal function of SIRT1 in physiologic and developmental processes (12, 45). Despite its biological importance and the surge of interest in SIRT1 this past decade, there remain multiple gaps in our knowledge and many questions not yet answered regarding the functions and regulations of this deacetylase.
Consistent with its histone deacetylation function, biochemical studies revealed that SIRT1 deacetylates histone H4K16 and H3K9, interacts with and deacetylates histone H1K26, and mediates heterochromatin formation (75). Also, SIRT1 associates with LSD1, and together they play concerted roles in histones H4K16 deacetylation and H3K4 demethylation to repress gene expression (49). In addition to histones, over 30 nonhistone proteins have been reported to serve as substrates for SIRT1 (60, 72), and many of the effects of SIRT1 are attributed to its role in nonhistone deacetylation. For example, SIRT1 regulates energy metabolism by inducing gluconeogenic while repressing glycolytic gene expression through deacetylation of PGC-1α (59). SIRT1 deacetylates the tumor suppressor protein p53, Hif-1α, Hif-2α, HSF1, FOXO1, FOXO3, and FOXO4 to regulate cell growth, apoptosis, and the stress response (5, 19, 42, 44, 48, 77, 79). Deacetylation of NF-κB, AP1, and Foxp3 by SIRT1 modulates the inflammation and immune system (74, 84, 85, 89). Deacetylation of DNA repair proteins Ku70, NBS1, Werner syndrome protein (WRN), and xeroderma pigmentosum group A protein (XPA) by SIRT1 regulates genomic stability (14, 22, 31, 40, 88). In the current study, we describe the identification of two SIRT1 substrates, hMOF (human ortholog of the Drosophila males-absent-on-the-first) and TIP60 (HIV-1 TAT-interacting protein of 60 kDa), that belong to the MYST family (MOZ, Ybf2/Sas3, Sas2, and TIP60) of histone acetyltransferases (HATs).
MOF, a histone H4K16-specific HAT, was initially found to be a key component of the male-specific lethal (MSL) complex required for dosage compensation in Drosophila (1, 28). hMOF (KAT8 or MYST1) is a 458-amino-acid protein and, like the Drosophila homolog, possesses histone H4K16-specific HAT activity and contains a conserved MYST catalytic domain, a chromodomain, and a C2HC-type zinc finger (50). Through acetylation of H4K16, hMOF regulates chromatin organization and gene transcription. In addition, hMOF plays an important role in DNA damage response (DDR), DNA repair, cell cycle progression, and cell growth (24, 57). Biochemical purification revealed that hMOF exists in at least two multisubunit protein complexes, MSL and MOF-MSL1v1 (7, 32, 41, 63, 80). Both of the hMOF complexes acetylate histone H4K16, but the MOF-MSL1v1 complex acetylates additional nonhistone proteins, including K120 of p53 (p53-K120). hMOF-mediated acetylation of p53-K120 after DNA damage influences the cell's decision to undergo apoptosis instead of a cell cycle arrest (67). hMOF is bound to the oncogenic MLL protein and also partners with MRG15 to activate expression of the B-Myb gene (20, 54). Therefore, like other members of the MYST family, hMOF may have a role in the cause and development of cancer (2, 83).
TIP60 (KAT5 or HTATIP) is an acetyltransferase that is the closest relative of hMOF within the human MYST family (about 50% similarity over 513 amino acids). It is the catalytic subunit of the NuA4 HAT complex, which is involved in the activation of genes by acetylation of histones H4 and H2A (8, 21, 70). Like hMOF, TIP60 has been implicated in cell growth and arrest, apoptosis, and DNA repair (29). Also like hMOF, TIP60 acetylates p53-K120 to modulate the decision between cell cycle arrest and apoptosis (69). At DNA double-strand breaks (DSBs), the MRE11-RAD50-NBS1 (MRN) complex targets TIP60 to histone 3 trimethylated at K9 (H3K9me3). The MRN-TIP60 interaction activates TIP60, which leads to the acetylation and activation of the ATM kinase and initiation of DSB repair (65, 66). The Tip60 of Drosophila (dTip60) catalyzes exchange of phospho-H2Av with unmodified H2Av, the homolog of human histone variant H2A.X, at DNA DSBs (35).
In this work, we found that SIRT1 binds to and deacetylates hMOF and TIP60 in vitro and in vivo. SIRT1-hMOF/TIP60 interaction is dynamically regulated in response to DNA damage. Deacetylation of hMOF/TIP60 has multiple effects. First, deacetylation of hMOF/TIP60's enzymatic domains inhibits their autoacetylation and HAT activity. Second, deacetylation unexpectedly cross talks with ubiquitination and promotes proteosome-mediated degradation of hMOF and TIP60. Third, deacetylation of hMOF/TIP60 inactivates their DDR functions. Together, these results expand the diverse tasks of SIRT1, uncover a novel function of SIRT1 as a negative regulator of HATs in DDR, and furnish a new model of a complex reciprocal network of regulations for HATs and HDACs.
Myc-SIRT1, glutathione S-transferase (GST)–SIRT1, and Myc-tagged SIRT1 with an H363Y mutation [Myc-SIRT1(H363Y)] have previously been described (36). Expression plasmids for Flag-hMOF and Flag-TIP60 were derived from full-length hMOF (NP_115564) and TIP60 (NP_874369) cDNAs, respectively. Flag-hMOF and Flag-TIP60 deletion mutants were generated by subcloning PCR products containing hMOF/TIP60 fragments into the pcDNA3.1-Flag vector. Lysine-to-arginine mutants and the dominant negative (DN) mutants of Flag-hMOF/TIP60 were generated using a QuikChange Multi Site-Directed Mutagenesis Kit from Stratagene. The Flag-p53 expression plasmid was constructed by inserting a full-length human p53 cDNA into the p3XFlag-CMV-7.1 vector.
Mouse anti-Flag, rabbit antihemagglutinin (anti-HA), and mouse anti-β-actin antibodies were purchased from Sigma-Aldrich. Rabbit antiacetyllysine antibodies were purchased from Cell Signaling Technology and from Immunechem. Rabbit polyclonal anti-AcH4K16 (H4 acetylated at K16) antibody and rabbit polyclonal anti-TIP60 for immunoblotting of endogenous TIP60 were purchased from Millipore. Rabbit anti-p53 (FL-393), mouse anti-p53 (DO-1), and rabbit anti-c-Myc antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-acetyl-K120-p53 and mouse monoclonal anti-hMOF for detection of endogenous hMOF were purchased from Abcam. The anti-PUMA (p53 up-regulated modulator of apoptosis) antibody was purchased from Cell Signaling Technology.
Experiments using stable-isotope labeling by amino acids (SILAC) were performed according to protocols previously developed by Mann and colleagues, with minor modifications (52, 53). Reagents including lysine- and arginine-deficient SILAC medium, dialyzed FBS, and heavy and light amino acids were purchased from Thermo Scientific. Briefly, Sirt1+/+ and Sirt1−/− murine embryonic fibroblasts (MEFs) were grown in light (normal l-arginine and l-lysine) and heavy (l-[13C6 15N4]arginine-HCl and l-[13C6]lysine-2HCl) media, respectively, for at least six doubling passages. Cells were harvested, and 1 mg of nuclear extracts was prepared from each cell population, combined, and subjected to immunoprecipitation with 50 μg of a mixture of two different antiacetyllysine antibodies, which were cross-linked onto protein A-agarose by dimethyl adipimidate. After incubation overnight at 4°C, antiacetyllysine agarose was washed five times with NETN buffer (150 mM NaCl, 20 mM Tris-Cl [pH 8.0], 0.5 mM EDTA, 0.5% Nonidet P-40). Immunoprecipitates were resolved on a 4 to 12% Novex gradient gel, followed by Coomassie brilliant blue staining. To reduce complexity, samples were excised from gels and divided into 12 slices before being subjected to trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Peptide identification and protein quantification were done using MaxQuant software (16, 17). Pathway analyses were performed with MetaCore software from GeneGo, and functional classifications were done using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources.
MEF, HEK293T, U2OS, and H1299 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. Stable HeLa cell lines (HeLa-C and HeLa-S5) were cultured under the same conditions with the addition of 0.25 μg/ml of puromycin. Transfections were performed with Lipofectamine 2000 (Invitrogen) or Fugene HD (Roche), according to the manufacturer's instructions. All transfections were normalized for total DNA using vector plasmids.
Immunopurified Flag-hMOF and Flag-TIP60, expressed in 293T cells, were subjected to LC-MS/MS analysis as previously described (88, 90).
For immunoprecipitation, cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail. Lysates were incubated with primary antibodies overnight at 4°C. Immunoprecipitates were washed three times with the same buffer and resolved by SDS-PAGE. For Western blotting, proteins were transferred onto nitrocellulose membranes, which were then incubated with the antibodies indicated in the figures. Proteins of interest were visualized using a chemiluminescent detection kit (Pierce).
For in vitro deacetylation assays, immunopurified Flag-hMOF/TIP60 was incubated with 1 μg of either GST-SIRT1 or GST in 200 μl of HDAC buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10% glycerol) in the presence or absence of 50 mM NAD+ for 2 h at 30°C.
HAT assays were performed using a colorimetric HAT assay kit from Millipore according to the manufacturer's instruction. Briefly, 10 μg of nuclear extracts was incubated with biotin-conjugated H4 (residues 1 to 21) peptides and acetyl coenzyme A (acetyl-CoA) in a streptavidin-coated microwell plate for 1 h at 30°C. After samples were washed with Tris-buffered saline (TBS), reaction mixtures were incubated with antiacetyllysine antibodies for 1 h at room temperature, followed by washing with TBS-Tween 20 and then horseradish peroxidase (HRP)-conjugated secondary antibodies for 30 min at room temperature. Acetylated H4 peptides were visualized by tetramethylbenzidine substrate and measured by spectrometry at a wavelength of the optical density at 450 nm (OD450).
After transfection, cells were grown in medium containing 600 μg/ml of G418 for 2 weeks. Stable pools of G418-resistant cells were seeded onto chamber slides (Lab-Tek) and either treated with 50 μM etoposide or 10 Gy of ionizing radiation (IR) or left untreated. Twenty-four hours later, cells were fixed with prechilled methanol and stained with 2 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) for 20 min. For each condition, at least 500 cells were examined for nuclear morphology on a microscope, and the percentage of the apoptotic cells was calculated.
Neutral comet assays were performed according to the manufacturer's suggestions (Trevigen). Cells were lysed, mixed with low-melting-point agarose gel, spread on slides, and subjected to electrophoresis in neutral Tris-borate-EDTA (TBE) buffer at 21 V for 30 min (Fisher midi-horizontal system FB-SB-1316; 27-cm-wide tank). The nuclei on slides were stained with SYBR green and visualized by microscopy. Images were captured and measured by an automated comet assay analysis system (HCSA 2.3.3; Loats Associates). The extent of DSB damage was represented by the parameter of tail moment, a combination of the amount of DNA and the length in the tail of comet. At least 100 cells were measured for each sample, and each experiment was repeated twice.
Twenty-four hours after transfections, cells were sparsely seeded onto 10-cm dishes in triplicates. After being cultured in selection medium containing 600 μg/ml of G418 for 2 weeks, cell colonies were stained with Giemsa for 30 min and counted. All experiments were repeated at least three times.
A long-term goal of our research is to comprehensively identify proteins whose acetylations are regulated by HDACs. Toward this goal, we began a study to test the feasibility of identifying cellular proteins that become hyperacetylated in the absence of Sirt1. Stable-isotope labeling with amino acids in cell culture (SILAC) was used to identify, characterize, and quantify acetylated proteins in Sirt1+/+ and Sirt1−/− fibroblasts (see Fig. S1A in the supplemental material). In total, 213 proteins were unambiguously identified to be hyperacetylated in the absence of Sirt1 (see Table S1 in the supplemental material). Each potential Sirt1 substrate was assigned to a functional group based on gene ontology molecular functions or biochemical process groups or on previously published literature (see Fig. S1B). Pathway identification and rank analysis, performed using the MetaCore software from GeneGo (Thomson Reuters), suggest that DNA damage response and repair pathways are key Sirt1 targets (see Fig. S1C). Consistent with our previous observations that SIRT1 binds to and deacetylates NBS1 and DNMT1 (56, 88), MRN components and the DNMT1 protein were identified as Sirt1 substrates in this global screen. Other previously reported SIRT1 substrates that were also identified in the present study include p53, p300, and p300/CBP-associated factor (PCAF) (4, 44, 55, 77). However, the majority of proteins identified in this screen were targets that have not yet been reported to be influenced by SIRT1.
Of the proteins whose acetylation levels were increased in the absence of Sirt1 in three independent experiments, histone modification enzymes such as HATs and their interacting proteins are highly represented (see Fig. S1B in the supplemental material). A large number of proteins that are involved in DDR or DNA repair also appear to be potential SIRT1 substrates. Two of these proteins are members of the MYST acetyltransferase family, hMOF and TIP60. Given the important role of hMOF, TIP60, and SIRT1 in DDR and chromatin modification, we focused our analysis on the regulation of hMOF/TIP60 by SIRT1.
To confirm that hMOF and TIP60 are SIRT1 substrates, we expressed Flag-hMOF and Flag-TIP60 in 293T cells, immunoprecipitated the two proteins, and analyzed the acetylation and protein abundance by Western blotting. As shown in Fig. 1A, overexpression of SIRT1 resulted in a small reduction of Flag-hMOF (about 3-fold decrease) and Flag-TIP60 (less than 2-fold decrease) proteins. Acetylation of Flag-hMOF and Flag-TIP60, however, was significantly decreased (more than 10-fold on average). Treatment of cells with a class III HDAC inhibitor, nicotinamide, resulted in hyperacetylation of hMOF and TIP60, further supporting the notion that SIRT1 is responsible for deacetylation of hMOF and TIP60 (Fig. 1B). In vitro, GST-SIRT1, but not GST alone, efficiently deacetylated hMOF and TIP60 in the presence of NAD+ and in the absence of nicotinamide (Fig. 1C). Additionally, endogenous hMOF became hyperacetylated in a HeLa cell line depleted of SIRT1 (56) compared to control cells (Fig. 1D). Taken together, these results confirm that hMOF and TIP60 are SIRT1 substrates.
Like many nonhistone HDAC substrates that interact with HDACs and consistent with the observation that hMOF and TIP60 are SIRT1 substrates, Myc-SIRT1 coimmunoprecipitated with Flag-hMOF and Flag-TIP60 (Fig. 1E). Endogenous SIRT1 also coimmunoprecipitated with endogenous hMOF and TIP60 (Fig. 1E). Further, TIP60 coprecipitated with endogenous SIRT1 as determined by mass spectrometry analysis (L. Peng, B. Fang, and J. Koomen, unpublished data).
To identify the acetylated lysines in hMOF and TIP60, Flag-tagged hMOF and TIP60 were expressed in 293T cells and treated with HDAC inhibitors. Immunopurified Flag-hMOF and Flag-TIP60 were analyzed by mass spectrometry. Our results indicate that both hMOF and TIP60 are abundantly acetylated, with 18 sites on hMOF and 15 sites on TIP60 distributed throughout the two proteins (Fig. 1F).
To elucidate the region of hMOF/TIP60 that interacts with SIRT1 and to narrow down the area of deacetylation, truncation mutants were generated, and the acetylation levels of these mutants and their interactions with SIRT1 were examined. As shown in Fig. 1G, SIRT1 interacts with the middle (residues 121 to 232 of hMOF; residues 75 to 285 of TIP60) and the C-terminal (residues 232 to 458 of hMOF; residues 285 to 513 of TIP60) regions of both proteins. Both the middle and C-terminal regions contain parts of the enzymatic MYST domain. SIRT1 did not interact with the N-terminal region of either protein containing the chromodomain (residues 1 to 121 of hMOF; residues 1 to 75 of TIP60). Although mass spectrometry analysis of intact hMOF/TIP60 revealed that acetylated lysines exist outside the C-terminal region, acetylation in the N-terminal and middle regions was undetectable in the absence of the C-terminal regions, most likely because of the requirement of the C terminus (containing hMOF-K274 and TIP60-K327) for autoacetylation (87). Also, acetylation of the C-terminal regions was completely erased by SIRT1. Our results, therefore, indicate that SIRT1 preferentially interacts with and deacetylates the enzymatic MYST domains of hMOF and TIP60 although at this time we cannot rule out the possibility that SIRT1 might also act on the chromodomains of these two proteins. Our finding is also consistent with the idea that autoacetylation of hMOF/TIP60 requires the MYST domains.
To assess the consequences of hMOF/TIP60 deacetylation, a series of lysine-to-arginine mutations was generated at the sites of acetylation (see Table S2 in the supplemental material). Mutations of acetylated lysines in the chromodomains of hMOF or TIP60 (C mutants) did not result in a decrease of acetylation in either protein (Fig. 2A). In contrast, acetylation of hMOF and TIP60 was severely compromised with mutations of acetylated lysines on the MYST enzymatic domains of hMOF or TIP60 (E mutants [hMOF-E-mut or TIP60-E-mut, respectively]). These results, together with the observation that autoacetylation of hMOF/TIP60 requires the MYST domains (Fig. 1G), suggest that deacetylation of the enzymatic domain of hMOF/TIP60 inhibits their autoacetylation and HAT activity. As a further confirmation, we found that E mutants of hMOF and TIP60 completely lost their abilities to acetylate histone H4 in vitro (Fig. 2B). Also, compared to wild-type hMOF/TIP60, E mutants had significantly less H4K16 acetyltransferase activity in vivo (Fig. 2C). Consistent with an earlier report that SIRT1 binds strongly to TIP60 only when TIP60 is acetylated (78), the hMOF E mutant does not interact with SIRT1 (Fig. 2D).
Additional lysine-to-arginine mutants were generated to determine precisely which lysine(s) within the MYST domains is critical for hMOF/TIP60 autoacetylation and HAT activities. Consistent with recent reports that K274 of hMOF is a major autoacetylation site and functionally important (32, 43, 64, 87), mutation of K274 to arginine alone (K274R) was sufficient to abrogate hMOF autoacetylation and enzymatic activity (Fig. 2E and andG).G). Similarly, a deacetylation mimic of K327 in TIP60, corresponding to hMOF-K274 (see Fig. S2 in the supplemental material), inhibited TIP60 acetylation and enzymatic activity (Fig. 2F and andG).G). Surprisingly, lysine-to-arginine mutation at residue 357 of TIP60 behaved identically to the K327R mutation, with a complete loss of TIP60 acetylation and HAT activity. Although at this time we do not know if the corresponding lysine hMOF-K304 undergoes acetylation, a K304R mutant also abrogates hMOF acetylation (Fig. 2E). Deacetylation of other acetylated lysines on MYST domains, including hMOF-K351, TIP60-K296, and TIP60-K404, had no effect on hMOF/TIP60 acetylation and HAT activity (Fig. 2F and andG).G). Therefore, in addition to previous reports that hMOF activity is regulated by autoacetylation of K274 (43, 64, 87), our data indicate that hMOF-K304, TIP60-K327, and TIP60-K357 on the MYST domains also control the acetylation of hMOF/TIP60 and HAT activity.
TIP60 protein is regulated by ubiquitination and proteasome-dependent degradation via interaction with MDM2 (15, 38). Whether the stability of hMOF is regulated in a similar fashion is unknown at this time. During the course of our study, we noticed that mutations of acetylated lysines mimicking a deacetylated state in the enzymatic domains of hMOF and TIP60 (E mutants) resulted in reduced protein levels compared to wild-type levels or mutations of acetylated lysines in the chromodomains (C mutants) (see Fig. S3A and B in the supplemental material). The amount of protein decrease was more apparent with an increase in the number of lysines mutated in the MYST domain of hMOF and TIP60. Further, DN mutants of hMOF and TIP60 (hMOF-DN-mut and TIP60-DN-mut, respectively), which do not possess autoacetylation activity and are not acetylated, behaved similarly to hMOF-E-mut and TIP60-E-mut, further suggesting that deacetylation of hMOF/TIP60 is important in the regulation of hMOF/TIP60 protein abundance.
To extend and confirm that deacetylation in the enzymatic domain of hMOF/TIP60 affects ubiquitination-mediated degradation, protein levels were determined for wild-type and hMOF/TIP60 mutants in the presence of the protein synthesis inhibitor cycloheximide. In the absence of protein synthesis, E mutants and DN mutants of hMOF and TIP60 turned over much more rapidly than the wild type and C mutants, and the increased protein degradation was reversed with the proteasome inhibitor MG132 (Fig. 3A and andB).B). In the presence of cycloheximide, the amount of protein degradation was directly proportional to the number of lysines mutated in the MYST domain of hMOF. For example, a mutant with seven lysines replaced by arginines (7KR) was degraded slightly faster than a 3KR and K274R mutant but much more slowly than the E mutant (Fig. 3C).
Overexpression of SIRT1 reduces hMOF and TIP60 protein levels (Fig. 1A and and3D;3D; see also Fig. S3C in the supplemental material). The diminution of protein as a result of SIRT1 is more pronounced in hMOF than in TIP60. Protein levels of hMOF-E-mut were not affected by SIRT1 (Fig. 3D), suggesting that deacetylation of the MYST domain is a prerequisite for hMOF/TIP60 degradation. Also, overexpression of SIRT1 had no effect on the expression of Flag-hMOF-E-mut or a green fluorescent protein (GFP) under the control of the same cytomegalovirus (CMV) promoter used to express hMOF/TIP60 (Fig. 3D) (L. Peng, unpublished data), ruling out the possibility that SIRT1 represses transcription from promoters used to drive expression of hMOF/TIP60.
To provide further support that deacetylation of hMOF/TIP60 promotes protein degradation and that the effect is mediated by SIRT1, we assessed endogenous hMOF protein levels in HeLa-S5 cells (56). Compared to control cells (HeLa-C), endogenous hMOF is much more abundant in the HeLa cell line depleted of SIRT1 (HeLa-S5) (Fig. 3E). Also, we repeated the SIRT1 overexpression experiments in the presence of cycloheximide and nicotinamide. As shown in Fig. 3F and andG,G, the effects of SIRT1-mediated decrease in hMOF/TIP60 protein levels were more pronounced in the presence of cycloheximide, and the effects could be reversed with the SIRT1 inhibitor nicotinamide. These results suggest that SIRT1-mediated deacetylation reduces hMOF and TIP60 protein levels in the absence of new protein synthesis and causes a degradation of these two proteins.
Because the stability of many proteins is determined by whether they become ubiquitinated, we sought to determine if lysine deacetylation in the enzymatic domain of hMOF/TIP60 promotes ubiquitination and subsequent proteasome-dependent degradation. As shown in Fig. 4A and andB,B, wild-type hMOF and TIP60 were readily ubiquitinated, and the ubiquitination was enhanced in the presence of MG132. Consistent with the observation that E mutants (MYST domain deacetylation mimics) of hMOF and TIP60 are less stable than wild-type hMOF/TIP60, the ubiquitination of hMOF and TIP60 E-mutants is much greater than that of wild-type hMOF/TIP60. Also consistent with the premise that the amount of hMOF/TIP60 degradation is increased corresponding to an increase of lysines deacetylated in the MYST domains, ubiquitination of the hMOF-E mutant is much greater than that of hMOF-K274R, which in turn is greater than that of wild-type hMOF (Fig. 4C).
To confirm that SIRT1 deacetylase enzymatic activity is necessary and sufficient to promote ubiquitination of hMOF and TIP60, ubiquitination of hMOF and TIP60 was assessed in the presence of either overexpressed SIRT1 or a SIRT1 with a single point mutation (H363Y) that renders the enzyme inactive. As shown in Fig. 4D, wild-type SIRT1 promotes hMOF/TIP60 ubiquitination much more efficiently than catalytic-deficient SIRT1. Further, endogenous hMOF is less ubiquitinated in Sirt1 knockout MEFs than in wild-type MEFs (Fig. 4E).
Our results that lysine-to-arginine substitutions in the MYST domains of hMOF/TIP60 increased ubiquitination indicate that different lysines are targets for deacetylation and ubiquitination. Mass spectrometry-based proteomics identified two sites of ubiquitination, K432 and K444, on hMOF, and one site, K282, on TIP60 (see Fig. S4A, B, and C in the supplemental material). Treatment of cells with the sirtuin inhibitor nicotinamide, but not treatment with the class I and II HDAC inhibitor trichostatin A (TSA), resulted in a decrease of ubiquitination at all three sites (see Fig. S4D), confirming again that deacetylation by class III HDAC(s) promotes ubiquitination of hMOF and TIP60.
Next, to further confirm hMOF/TIP60 ubiquitination target sites, hMOF-E-mut and TIP60-E-mut were modified to contain additional mutations at the identified ubiquitination sites (see Table S2 in the supplemental material). As shown in Fig. 5A and andB,B, hMOF-E-mut-K432R and hMOF-E-mut-K444R (hMOF-E-mut with additional lysine-to-arginine substitutions at K432 and at K444, respectively) are less ubiquitinated and more stable than hMOF-E-mut. Similarly, TIP60-E-mut-K282R is less ubiquitinated and more stable than TIP60-E-mut (Fig. 5C and andD).D). Collectively, these data provide strong evidence that SIRT1 deacetylation of enzymatic domains of hMOF/TIP60 accelerates the degradation of hMOF and TIP60 by upregulating the ubiquitination of these proteins.
Because both hMOF and TIP60 participate in the DDR and have critical roles in DNA DSB repair, we determined the interaction between SIRT1 and hMOF/TIP60 during the DDR. Interestingly, DNA DSB damage induced by gamma irradiation or etoposide dissociates the SIRT1-hMOF/TIP60 complexes transiently. Reassociation of SIRT1 with hMOF/TIP60 begins between 2 and 4 h after initiation of DNA damage, and interactions are completely restored by 6 h after initiation of DNA damage (Fig. 6A). Corresponding to the initial decrease in SIRT1-hMOF/TIP60 interaction, hMOF/TIP60 became transiently hyperacetylated upon exposure to etoposide (Fig. 6B). These data argue that SIRT1-hMOF/TIP60 interaction and the acetylation status of hMOF/TIP60 are tightly controlled by DNA damage signals.
Both hMOF and TIP60 acetylate K120 of p53 to influence the cell's decision to undergo apoptosis instead of cell cycle arrest (10, 67, 69). A p53-K120R mutation selectively blocks the transcription of proapoptotic target genes such as PUMA, and depletion of hMOF and/or TIP60 inhibits the ability of p53 to activate PUMA transcription (67). To examine the effects of deacetylation of hMOF/TIP60 on p53 signaling, hMOF/TIP60 E mutants were compared to wild-type hMOF/TIP60 for their ability to acetylate p53-K120 and to regulate PUMA expression. Under ionizing radiation, p53-K120 acetylation and PUMA expression were lowered in hMOF-E-mut and TIP60-E-mut compared to wild-type hMOF and TIP60, respectively, in U2OS (Fig. 7A) and H1299 (see Fig. S5 in the supplemental material) cells. Consistent with this finding, deacetylation mimics of hMOF/TIP60 (hMOF-E-mut/TIP60-E-mut) no longer promote apoptosis as effectively as wild-type hMOF/TIP60 after gamma irradiation or treatment with etoposide (Fig. 7B).
Knockdown of hMOF/TIP60 by siRNA or overexpression of hMOF/TIP60 dominant negative mutants (DN mutants) impairs DNA DSB repair and inhibits cell proliferation (24, 63). Comet assays were performed to assess the ramifications of hMOF/TIP60 deacetylation on DNA damage and repair. As shown in Fig. 7C, following ionizing radiation, U2OS cells overexpressing E mutants of hMOF and TIP60 had a slower DNA repair response than cells overexpressing wild-type hMOF/TIP60. Additionally, without DNA damage or exogenous stress, overexpression of hMOF/TIP60 E-mutants resulted in significantly less colony formation (Fig. 7D) than in cells expressing wild-type hMOF/TIP60, consistent with the premise that deacetylation of the MYST domains of hMOF/TIP60 negatively affects cell growth.
Recent studies suggest that hMOF is autoacetylated at K274, and this autoacetylation is required for HAT activity and protein substrate binding (43, 64, 87). Lu et al. propose that SIRT1 modulates hMOF autoacetylation and regulates hMOF recruitment to chromatin (43). Likewise, Wang and Chen showed that SIRT1 regulates autoacetylation and HAT activity of TIP60 though the site(s) of acetylation was not identified (78). In addition, Yamagata and Kitabayashi have shown that SIRT1 interacts with TIP60 (81). In this study, we confirm that hMOF and TIP60 are SIRT1 substrates. We discovered multiple novel acetylated lysines on hMOF and TIP60, and mutations of hMOF-K274 or hMOF-K304 and TIP60-K327 or TIP60-K357 affect acetylation and HAT activities of these proteins. SIRT1 binds to and deacetylates the MYST domains of hMOF and TIP60. Most important, we found that SIRT1 negatively regulates the activities, functions, and stabilities of hMOF and TIP60.
A previous study suggests that HATs and HDACs interact to mutually balance their activities toward histones in vivo (82). It was proposed that by physically forming complexes, HATs and HDACs execute rapid cycles of coordinated histone acetylation and deacetylation in the same regions of chromatin. Our findings that hMOF and TIP60 plus five other HATs (p300, CBP, PCAF, GCN5, and TAF1) are SIRT1 substrates, together with a previous report that SIRT1 deacetylates p300 and PCAF (4, 55), further support the idea that HATs and HDACs do not act independently but, rather, that their activities and functions are interdependent on each other. Unlike the previous study, however, we propose here an alternative, although nonmutually exclusive, model in which HDACs, and SIRT1 in particular, modify the activities and functions of HATs by directly deacetylating HATs. This model is reminiscent of many kinase proteins, whose activities are activated or repressed by phosphorylation/dephosphorylation in signaling cascades.
Unlike other HATs, which usually target multiple lysines on different histones, a remarkable feature of hMOF is its specificity for histone H4K16 (1, 63, 71). In parallel, although there are reports that SIRT1 can deacetylate all four core histones in vitro, SIRT1's chief target site on histones is H4K16 (75, 76). Therefore, SIRT1 does not only deacetylate acetylated H4K16 but also prevents hMOF from acetylating nonacetylated H4K16. Given the importance of H4K16 deacetylation in higher-order chromatin organization, transcription repression, cellular life span, and X inactivation in flies and mammals (18, 61, 62, 76), it is reasonable to predict that SIRT1 might control these processes both directly by deacetylation of H4K16 and indirectly by inhibiting hMOF.
The ε-amino group of lysine is accessible to many different modifications including acetylation, methylation, ubiquitination, sumoylation, neddylation, biotinylation, propionylation, butyrylation, and crotonylation (11, 68). One type of modification could exclude another modification on the same lysine residue within a protein. Lysine acetylation often inhibits ubiquitination-dependent, proteasome-mediated protein degradation by this principle (9). The stability of a number of proteins has been shown to increase after acetylation as a result of lysine site competition preventing ubiquitination. In these situations, by converting acetylated lysines to nonacetylated lysines, HDACs accelerate protein degradations by exposing the same lysines for ubiquitination. One of the most intriguing results from our study is that SIRT1 deacetylation of hMOF and TIP60 regulates their degradation in a proteosome-dependent manner. Ubiquitination and degradation of hMOF/TIP60 were dependent on both deacetylation of particular lysine(s) in the MYST domains and the total number of lysines that are deacetylated. However, unlike proteins whose stability is regulated by competition between acetylation and ubiquitination of the same lysine residues, acetyl-acceptor sites on hMOF and TIP60 are distinct from the sites of ubiquitination. Mutations of acetylated lysines to arginines in the MYST domains or deacetylation of hMOF/TIP60 by SIRT1 does not simply provide access of previously acetylated lysines for ubiquitination. Rather, deacetylation of hMOF/TIP60 more likely induces protein conformational change, alters protein subcellular localization, or changes interaction of hMOF/TIP60 with other cellular proteins, which ultimately favors ubiquitination on nonacetylation targets.
Our findings that deacetylation regulates hMOF and TIP60 add to the growing list of posttranslational modifications that control the activities and functions of hMOF and TIP60. In addition to acetylation and ubiquitination, TIP60 undergoes sumoylation at K430 and K451, and sumoylation promotes TIP60 HAT activity in response to UV irradiation (13). Also, phosphorylation of S86 and S90 of TIP60 has been shown to facilitate TIP60 activation (10, 39). To the best of our knowledge, for hMOF, phosphorylation or sumoylation modification has not yet been reported, and our work here is the first demonstration of ubiquitination. Using mass spectrometry analysis, we discovered that S86, S90, S155, S199, Y158, and Y401 of TIP60 and Y45 and Y418 of hMOF are phosphorylated, suggesting additional modifications that can regulate hMOF/TIP60 (Peng et al., unpublished). As increasingly sophisticated and sensitive techniques to identify posttranslational modifications become available, we predict there will probably be even more modifications that will be discovered for hMOF and TIP60. The next step in furthering our understanding of the mechanisms and cellular functions of hMOF/TIP60 is to determine which modifications are physiologically relevant and how different modifications work together with acetylation/deacetylation to extend the functions of hMOF and TIP60.
A proper DNA damage response (DDR) is critical for genome stability. Sir2 enhances DNA repair through several different mechanisms in yeast (37, 73). Similarly, SIRT1 plays a positive role in promoting DNA repair (22, 31, 40, 51, 88). hMOF/TIP60 also have previously been implicated in DDR, and, in this study, we found that they operate in a common pathway with SIRT1. Without exogenous stress, SIRT1 binds to and deacetylates hMOF/TIP60, maintaining hMOF/TIP60 in a low physiological concentration and in an inactive state. Upon DNA damage, hMOF and TIP60 are transiently released from SIRT1 and then become acetylated and activated. After a burst of hMOF/TIP60 activity, SIRT1 reassociates with hMOF/TIP60, inhibits them from activating p53 and expressing proapoptotic genes, and prevents excessive apoptosis. Thus, SIRT1 not only directly enhances DNA repair but also functions indirectly in DNA repair by maintaining a critical level of active hMOF and TIP60 during DDR.
hMOF and TIP60 are closely related in both sequence homology and in some biological functions, and results in this study suggest that SIRT1 regulates both proteins via similar mechanisms. However, there are also clear differences between hMOF and TIP60. For example, while hMOF selectively acetylates H4K16 within histones, the histone substrate specificity for TIP60 is less restrictive. hMOF and TIP60 exist as distinct multiprotein complexes in vivo. Interestingly, in this study, not all of the acetylated lysines in hMOF and TIP60 identified by mass spectrometry are conserved between hMOF and TIP60. For example, while four acetylated TIP60 residues, K296, K327, K357, and K404, are conserved in hMOF (corresponding to K243, K274, K395, and K341, respectively), we did not find acetylation of K243 or K395 in hMOF. Reciprocally, several lysines that are acetylated in hMOF are not conserved in TIP60. This, then, suggests that the regulation of hMOF and TIP60 by HATs and HDACs could be much more complicated than we anticipate. Further experiments are necessary to understand how SIRT1 deacetylation might regulate both similar and different biological functions of these two proteins.
By deacetylation of histones, changing chromatin conformation, or altering histone-cellular protein interactions, HDACs are commonly believed to play a key role in the repression of gene transcription. This is indeed the case for Sir2 in yeast, especially at telomeric sequences, the MAT loci, and the ribosomal DNA locus (47). Unexpectedly, a study found no evidence of failure of gene silencing in Sirt1 null animals, suggesting either that SIRT1 has a different role in mammals than it does in Saccharomyces cerevisiae or that its role in gene silencing is confined to a limited subset of mammalian genes (45). In our study here, a casual inspection revealed no significant difference in protein expression from cells derived from Sirt1 knockout animals compared to the wild type (L. Peng, and J. Koomen, unpublished data), confirming that SIRT1 probably does not induce generalized global transcription repression. Rather, SIRT1 most likely carries out many of its functions by targeting nonhistone substrates such as hMOF and TIP60. Current work in our laboratory is focused on understanding the biological and physiological relevance of other potential SIRT1 substrates identified in this study.
We thank Tony Kouzarides for plasmids and John Neveu, Bogdan Budnik, Renee Robinson, Vicki Izumi, Umut Oguz, Xiaotao Qu, and the Moffitt Cancer Center Core Facility for their technical support. We are grateful to Xiang-Jiao Yang (McGill University), Yali Dou (University of Michigan), Jacques Cote (Centre hospitalier universitaire de Québec), and John Lucchesi (Emory University) for advice.
This work was supported by grants to E.S. from the NIH (R01GM081650) and the Kaul Foundation and by a fellowship to L.P. from the American Heart Association.
Published ahead of print 14 May 2012
Supplemental material for this article may be found at http://mcb.asm.org.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information 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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Sand Creek Middle School Science Teacher James Brown recently found out he is one of about 100 teachers who was named by President Barack Obama as a recipient of a Presidential Award for Excellence.
Brown will receive his award, which includes $10,000 from the National Science Foundation to be used at his discretion, on an unknown date in the Fall at the White House. The teachers will also receive an expense-paid trip to Washington, D.C. for the awards ceremony, as well as several days of educational events with members of Congress and science agency leaders.
In order to qualify for the awards, the teachers needed to be nominated. Brown said his nomination was made by Sand Creek Principal David Perry. The winners are then chosen by a panel of scientists, mathematicians and educators on the federal level, after they have been selected by a similar panel on a state level.
There is no higher calling than furthering the educational advancement of our nation's young people and encouraging and inspiring our next generation of leaders, Obama said in a written statement. "These awards represent a heartfelt salute of appreciation to a remarkable group of individuals who have devoted their lives and careers to helping others and in doing so, have helped us at all."
Brown said he was not aware that he had won the award until this week. He also said he is extremely excited.
For more on this story, check back at
www.spotlightnews.com, or read the Wednesday, July 15 print edition of the Colonie Spotlight.
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