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By Douglas Robson, Special for USA TODAY
NEW YORK The Grand Slam season closed Sunday at the U.S. Open, so what have we learned? What can be expected going forward? Some familiar names dominated the majors in 2007 — Roger Federer, Rafal Nadal, Justine Henin and the Venus and Serena Williams. But there were also some exciting revelations, history-making accomplishments and disturbing trends. Here are a few themes that hint at what might be down the pike in tennis.
Clearly No. 1, part 1
Justine Henin's unprecedented defeat of Serena and Venus Williams in claiming a seventh Grand Slam title has unassailably planted her name atop the women's game. Pound for pound, the 5-53/4, 126-pound Belgian might be the best female player in history.
"The quality I played in the last few matches is amazing," said Henin, who crushed No. 4 seed Svetlana Kuznetsova of Russia 6-1, 6-3 in Saturday's prime-time final to win her second U.S. Open without dropping a set. "It's just a great feeling because I had a tough draw, and I had a lot of things to prove to myself not to anyone else, just to myself. And I did it."
With a flowing, versatile and attacking style that belies her size, the 25-year-old has established herself as a clear No. 1. She has reached the final in the last seven Grand Slams she's entered, winning three, all while reconciling with her long-estranged family and going through a painful divorce. With her personal and professional lives in sync, Henin is primed to challenge Serena Williams, who has eight majors, as the game's active leader.
"When I was a little girl, I was dreaming of winning just one Grand Slam in my career, and I've won seven," Henin said. "It's still hard to believe."
Clearly No. 1, part 2
No man in history has had a better four-year run, which Federer punctuated by beating Novak Djokovic 7-6, (7-4), 7-6 (7-2), 6-4 for his 12th major and fourth consecutive U.S. Open crown.
His 11 majors from 2004-2007 are more than any player in any era, including an unparalleled four consecutive back-to-back Wimbledon and U.S. Open wins.
Credit, too, to Federer, who decided to split with coach Tony Roche and recovered from a shaky start to the spring and reassert his place at the top.
"I don't need to sit down and talk about an opponent for an hour," the coach-less Federer said this weekend about his ability to size up the competition on his own terms. "Takes me 15 seconds. I know everything I need to know."
Scarier still, The Unflappable One is one of the most durable athletes on tour, having missed no Grand Slam tournament since the 1999 U.S. Open.
Not so, Nadal. While the second-ranked Spaniard Nadal has had another stellar year, including a third consecutive French Open title and second Wimbledon final, the 21-year-old leaves New York with a few more questions regarding his health, schedule-management and hard-court game following another sub-par performance.
In terms of history, Federer's win Sunday pushed him past Bjorn Borg and Rod Laver into a tie for second place with Roy Emerson in total majors, and just two shy of recordholder Pete Sampras' 14. Federer also only trails good buddy Tiger Woods by one Grand Slam, 13-12.
Williams resurgence
For the first time since 2001, Serena Williams competed in all four Grand Slams. Venus Williams played in three. The upshot: Each won a major, Serena at the Australian Open and Venus at Wimbledon. The game gets a boost when they are healthy, competing and engaged, and their presence in New York added extra spice to a tournament the sisters owned from 1999-2002. If they can avoid the injuries that have plagued them in recent years, Venus, 27, and Serena, 25, should be challenging for majors consistently.
"It's been surprise and excitement that Serena and Venus won the first and third major of the year, but disappointment they weren't able to play more and gotten their ranking up to play these matches later," says CBS analyst Mary Joe Fernandez, referring to the Serena-Henin quarterfinal and Venus-Henin semifinal. "To me, these are the three best players in the world."
WTA injuries
Part of what made the U.S. Open sizzle was the healthy representation of marquee names. Among the top-10 players, only No. 7 Amelie Mauresmo was MIA. But that has not been the norm on the women's tour, which again is struggling to keep its best players active. Despite efforts to reduce player injuries and withdrawals, the Sony Ericsson WTA Tour is on pace to equal or surpass elite player pullouts (top-10 players) at tournaments this year. So far, 33 have withdrawn from events, which is equal to the 33 pullouts the tour cited in mid-October of last year when it announced bigger fines and fewer required events to mitigate the ballooning problem. The total in 2007 was 39.
On a positive note, the tour, which promises to deliver six top-10 players to its Tier I tournaments, has met that commitment in five of seven events after going 0-10 last year. "It's nowhere near satisfactory," WTA CEO Larry Scott says.
U.S. men
Americans have reason to be encouraged by strong performances from youngsters coming up the ranks. Eighteen-year-old Donald Young and towering John Isner, 22, both reached the third round in New York. The nearly 6-10 Isner, who in May helped lead Georgia to the NCAA team title, beat No. 26 seed Jarkko Nieminen and put a scare into Federer, winning the first set in a four-set loss. Young, a versatile lefty and former world junior No. 1, pushed Feliciano Lopez to four sets and is starting to show the promise many have expected after starting his ATP Tour career with 11 consecutive losses. There's also 19-year-old Sam Querrey. The 6-6 Californian didn't fare well at the U.S. Open but has already cracked the top 50.
"We have something positive to look out for from the summer," U.S. Davis Cup captain Patrick McEnroe says. "We have three young guys to watch."
Djokovic on rise
Novak Djokovic's best impression is of himself trying to crash the stranglehold Roger Federer and Rafael Nadal have held at the top of the men's game. With a stellar Grand Slam season — including semifinal appearances at Roland Garros and Wimbledon to go with his first final at the U.S. Open — this gifted 20-year-old from Serbia looks ready to make the Federer-Nadal duopoly a three-man show.
"Djokovic has played his way into being a major contender," McEnroe says of the world No. 3.
As he showed New Yorkers and millions of television viewers with his dead-on impersonations of Maria Sharapova and Rafael Nadal, he also has a compelling personality.
"I need to say in last two days the people were more congratulating me for the impressions than for my tennis," court jester Djokovic said with a smile following his semifinal win against David Ferrer. "I was wondering, 'Guys, am I here for the impersonation, entertaining, or to play tennis.' "
His rise in 1007 suggests fans will be treated to a little more of both in the months to come.
Conversation guidelines: USA TODAY welcomes your thoughts, stories and information related to this article. Please stay on topic and be respectful of others. Keep the conversation appropriate for interested readers across the map.


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Scots Crumpets (Scottish Pancakes)
READY IN: 18mins
Recipe by mollypaul
These are soft pancake-like or crepe-like fare but made larger and more thinly than pancakes. They can be spread with butter and/or jam and they are traditionally rolled up before eating. The quantities below will make about 16 crumpets. Posted from an online source in response to a recipe request. Cooking time is approximate.
Top Review by DancerADHD
This would be lovely with tea.
See Discussion:1 Review
Ingredients Nutrition
- 2 cups all-purpose flour
- 2 tablespoons granulated sugar
- 1 pinch salt
- 2 large eggs, separated into whites and yolks
- 2 tablespoons melted butter
- 15 ounces milk
Directions See How It's Made
- Beat the egg yolks and blend in the sifted flour, sugar, salt.
- Then add in the melted butter and milk to make a thin batter about the consistency of thin cream.
- Beat the egg whites to the soft peak stage and quickly add to the batter, folding in with a knife or metal spoon.
- Heat a lightly greased griddle or a frying pan; pour in large spoonfuls of the batter when the pan is hot.
- Each crumpet should spread thinly to about 4" to 5" in diameter (you may have to tilt the pan to achieve this).
- When the batter is brown underneath and slightly bubbly on top, turn and cook on the other side.
- Serve warm.


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There's no way around it: The U.S. first-quarter gross domestic product numbers were deplorable. But they're already ancient history.
The U.S. Commerce Department's final estimate of first-quarter GDP, released Wednesday morning, reported that the economy shrank at a 2.9-per-cent annualized rate in the quarter. Not only was that much worse than economists had anticipated (and a massive revision, even by Commerce's notoriously revision-happy standards, from its previous estimate of a 1-per-cent decline), it was also the worst economic performance since the 2009 first quarter – when the Great Recession still had the global economy by the throat. That's not a comparison anyone wants to see – least of all Canadians, whose own economic recovery is deeply dependent on an acceleration in U.S. demand for its exports.Report Typo/Error
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Ford is recalling 8,266 of its redesigned 2013 Escape compact crossovers -- that just went on sale -- for a problem that could interfere with braking, according to the National Highway Traffic Safety Administration.
The recall covers vehicles built March 8 through June 7 (build date is on a sticker on the driver-side door jamb).
The new Escapes have a piece of carpet padding in the center console trim panel that might be incorrectly installed. If so, it could cause the driver's foot to miss the brake pedal initially when moving from the gas pedal. This could increase stopping distance and could lead to an accident.
According to a statement from the automaker, "Ford has not received any field reports of increased stopping distance for this issue."
Ford will notify owners starting July 23, and dealers will replace the affected parts. Owners can call Ford at 866-436-7332 or NHTSA's vehicle safety hotline at 888-327-4236 for more info.
-- Jennifer Geiger, Cars.com's Kicking Tires
Chris Woodyard is an auto writer for USA TODAY who covers all aspects of motoring. He revels in the exhaust note of a Maserati and the sharp creases of a Cadillac CTS. Chris strives to live a Porsche life on a Scion budget. More about Chris


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Data from: Persistent natural acidification drives major distribution shifts in marine benthic ecosystems
Date Published: October 1, 2015
DOI: http://dx.doi.org/10.5061/dryad.0f7s7
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text: I copied this layout from a layout I found on here. "A Child's Love" all supplies are just basic except the Artist Paper the background for the photo. The black photo mat is just an old cardboard picture holder that I tore apart. I used ribbons button and musical notes, I haven't added the tags in the pocket yet (I don't have them) and the journaling.


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Definition Edit
DS-1 (also known as T-1) is
|a T-carrier signaling scheme devised by Bell Labs. DS-1 is a widely used standard in telecommunications in North America and Japan to transmit voice and data between devices. DS-1 is the logical bit pattern used over a physical T1 line; however, the terms DS-1 and T1 are often used interchangeably. [A T1 line] [c]arries approximately 1.544 megabits per second of data.[1]


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CEP report finds no clear benefit from school vouchers
According to the independent non-profit Center on Education Policy (CEP), a review of the past decade of research on school vouchers has found "no clear advantage in academic achievement," and mixed outcomes overall, for students who attend private schools using vouchers.
The report, "Keeping Informed about School Vouchers: A Review of Major Developments and Research," [PDF file] reviews and summarizes major studies on school vouchers from the past decade, and also summarizes current publicly funded school founder programs.
According to the report, the scope of voucher programs and proposals has broadened over the past decade beyond serving just low-income students in low-performing schools to serving middle-income or suburban families. "In expanding voucher programs to middle- and higher-income families and suburban families, policymakers risk alienating those who support vouchers as a means to improve education for low-income urban students," the authors write.
In general, the report finds, several prominent voucher studies released since 2000, examining voucher programs in Cleveland, Milwaukee and Washington, D.C., have concluded that achievement gains for students receiving publicly funded vouchers are similar to those for comparable public school students.
"We have a great body of research about the effects of vouchers that policymakers should draw on to inform current debates," study co-author Alexandra Usher said in a statement. "Before state legislators and Congress move quickly to enact new voucher programs, they should consider the evidence from programs already in place for several years to ensure they understand the impacts their policies would have."
The report notes that many recent voucher studies have been carried out or sponsored by pro-voucher organizations, and recommends steps to ensure that studies are designed, conducted and reported in an objective manner.
"We were surprised to find so many studies done by pro-voucher groups," study co-author Nancy Kober said in a statement. "While this doesn't mean researchers with definite positions on vouchers can't be objective, it speaks to the need for outside scrutiny of study methods and guidance from objective expert panels."


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Drunk In Love by Beyonce ft. Jay Z (Violin Cover) - Emmanuel Houndo MP3
Feedback is very much appreciated! Thanks : ) Took some time to put together but please enjoy. Drunk In Love by Beyonce ft. Jay Z (Violin Cover) - Emmanuel ...
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If you only recognize the space-agey steel and concrete structure inhabiting Queens' Flushing Meadows–Corona Park from its cameos in Men in Black and Iron Man 2, then it may be time to brush up on your outer-borough history: Built for the 1964 World's Fair, the New York State Pavilion has experienced several incarnations, including a roller-skating rink and a venue for concerts by the likes of Led Zeppelin, Fleetwood Mac and the Grateful Dead. Learn more about the iconic edifice on this rare tour, which kicks off a six-month celebration of the grand exhibition's 50th anniversary.
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text: Young Walt is using his ability to transcend time and space ( by using his "Mind's Eye" ) to appear as himself ( at 13 yrs old )and as future Walt( aka Matthew Abaddon ). When Abaddon visits Hurley at Santa Rosa, there is a hand-drawn picture of an Island scene behind Abaddon. It looks scary and as if a an adult drew it. When Walt visits Hurley, there is a hand-drawn picture of an Island scene behind Walt that is very colorful and looks like a child drew it. This picture doesn't appear behind any other characters who visited Hurley and represents Walt appearing as himself and future Walt( aka-Abaddon ).I believe one of the reasons Walt appears as himself and Abaddon to Hurley, is to find out what happened to his Father, Micheal. Because he is young he might not have the power to see the future.( Like Locke) As Abaddon he asks Hurley, "are they still alive? As Walt, he ask Hurley why they are lying. Hurley said to protect the people still on the Island. Walt then ask, "like my Dad?" When arranging the team with Naomi, she ask Abaddon "What if we run into survivors of Flight 815?" Abaddon answers abruptly and sternly "There are NO survivors from Flight 815. Walt knows at this point in time His Father, Micheal (aka Kevin Johnson) will be on the same exact boat that she will be on and says that in order to protect Micheal from whatever it is that's causing Hurley and the others to lie. When returning from the island, Walt and Micheal get into a fight and Walt goes and lives with his Grandmother. Walt refuses to talk to or see Micheal. When Micheal runs into Tom ( aka Mr.Friendly - an "other") off the Island, Tom asks where Walt was. Micheal gave him a look and Tom knew right away that Micheal told Walt that he killed Ana Lucia and Libby. Why would Walt be so upset that they were killed? WALT NEVER MET ANA LUCIA OR LIBBY !!!! When Micheal meets the Tallies, Brings them back to camp and eventually kills Ana Lucia and Libby - Walt has been held captive the whole time by the others.(23 Days) Juliet was branded and banished because she killed one of her people.(Picket-another "other") John Locke couldn't kill " Jones "( Aka young Widmore ) as he tried to escape, because he was one of his "people". Walt is very upset that Micheal killed Ana Lucia and Libby, because they were perspective or already "others".(Goodwin was fighting on behalf of Anna to Ben and she would have joined them as an "other") and Libby (Who never had her own flashback, is very mysterious and sports the whole "bug eye-blank stare" that the others seem to share) Walt (Abaddon) is the future Leader of the "others" and by the "Other's Law" has to banish and not speak to anyone who kills one of them.( That's how Tom knew Micheal told Walt he killed them - Walt wasn't with him ) When Micheal goes to see Walt at his Mother's, Walt is looking at Micheal through a window as he leaves. Poor young Walt still loves his father. That's why he asks about his dad when the 6 return and tries to protect him from Naomi and the others, But as the future Leader of the Others-He has to follow the rules. Locke(aka Jeremy Bentham )returned from the Island and was the only one to visit Walt. I believe Locke told Walt, because of the Island skips, the original outcomes of time have changed. He tells Walt that he has to intervene by going forward/backwards in time to convince the others to return to the Island. This includes convincing John himself, who will have no memory of the Island when Matthew Abaddon visits him in the past. First words out of Abaddon's mouth before we even see him are " Don't give up Mr Locke." John replies " Excuse me." Abaddon then replies " I'm just saying, Don't give up." Abaddon strikes me as a very powerful and confident man, but when Locke rebuttals slightly - he backs away and said something that sounds like it came straight out of the mouth of a ten year old boy " I'm just saying...." Abaddon tells John he needs to go on a Walk About, because he (Abaddon) went on a Walk about ( Walt's time on the Island or as the case may be "EYELAND")and He found out what He was made of - Who He was. Locke says " And here you are an orderly." Abaddon replies " Oh I'm a lot more than an orderly John." Abaddon asks John "Do you believe in miracles Mr Locke?" The first time Locke and Walt speak on the Island, Locke asks Walt if he wants to know a secret. Later when Walt is talking to Micheal, he tells him that Mr Locke has a secret and He (Locke) said "a miracle happened to him." When speaking to Locke at the Hospital, He refers to him as both "Mr.Locke" (as an apprentice)and "John" (authority to Locke) John Locke's motto is, "Don't tell me what I can't do" and he doesn't take orders from no one.(ask His high school teacher, the people at the Walk-About, His Dad, Helen, Jack, Ben, Eko and countless others) But when the fate of his people and the Island (FACING ULTIMATE DOOM ) rests in the hands of their leader John Locke....Locke receives and executes orders from a 10 year old boy, that he knew for 2 months, that appeared taller? Locke takes the orders and executes them, because Walt is ( will become his authority-Abaddon ) Walt further displays his authority by calling him "John". When Walt appears to Locke at the Dharma grave site, only a month has gone by (In island time)that Walt left the Island. It is highly unlikely that Walt grew so much in a month, that Locke would notice and tells the others. Walt is taller because it's actually 3 years later, after Locke and the others came home. Walt travels back in time to tell John to get up, he has work to do, because Locke himself told him to do it. During one of Island's flashes in time, Locke sees the light coming out of the Hatch, He tells Sawyer the story and SAWYER SAYS WHY DON'T YOU GO TALK TO YOURSELF SO YOU CAN DO THINGS DIFFERENT THE NEXT TIME. As the current leader of the Others, John has a lot of work to do. He enlists the only person naturally qualified for the job - his old pal Walt. While on the Island , Locke tells Micheal " you need to let Walt realize his full potential." John knows Walt will be his leader one day. Walt was chosen at a young age to become the future leader. A Dharmacakra (A wheel with eight spokes-like the underground wheel on the Island )appeared on the ceiling of Susan Lloyd and Brian Porter's house in "Special". Walt was tested by Locke and "the others" when he was on the island ,but like Locke (when tested by Richard at an young age) wasn't ready. That's why when he appears to Shannon a few times, he spoke backwards - He couldn't control his powers very well. 3 years have passed and with the help from Locke - he's a Pro. ( In fact Abaddon sports a bald head like Locke and is his "Carbon Copy"..."two players one dark - one light"...Hmmmm ) Ben, Locke, and Walt's Fathers were all sacrificed to the Island in one way or another. The Island demands a sacrifice before you become a leader. That's why Micheal couldn't kill himself or be killed. It wasn't his time. But when the time came, Christian Shephard appeared to tell him he could "go now." ( You have to be pretty big or be related to someone big to get a visit from Christian Shephard) Buddhist believe the "wheel" (aka Dharmacakra) has to be turned 3 times when the Dharma is lost or forgotten. Ben turned it, Locke turned it..now it's Walt's (Abaddon) turn. Let me Know what you think.


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MALVERN, Pa. (AP) IKON Office Solutions (IKN), which sells and leases copiers, printers and other office machines, has agreed to be bought by one of its suppliers, Ricoh (RICOY) of Japan, for $1.62 billion.
The cash offer of $17.25 per share represents a 33% premium to IKON's average stock price the last two months, the company said. IKON shares closed Tuesday at $15.56.
The deal will combine IKON's sales and service capabilities with Ricoh's engineering and manufacturing expertise, IKON said. It follows an "extensive review of our strategic opportunities," said IKON Chairman and Chief Executive Matthew Espe.
IKON, based in Malvern, Pa., also sells and leases equipment from other companies, including Canon and Hewlett-Packard. It was not immediately clear if IKON will maintain its relationship with those companies.
The deal, which has been approved by the boards of both companies, is expected to close in the fourth quarter. As a subsidiary of Ricoh, IKON will keep its headquarters in Malvern, it said.
Separately, Ricoh said it will finance the deal with a mix of internal and external funding.
Copyright 2008 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
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This nålbinding round-start tutorial illustrates one method for casting stitches onto a loop. Want to nålbind top-down hats, and toe-up socks? Make a flat medallion for (more…)
nålbinding archaeology project
An anthropology student at Camosun College commissioned two 12cm x 12cm nålbinding samples for an archaeology project she was doing. Nadine wanted to compare (more…)
nålbinding starting loops tutorial – method 1
Nålbinding begins with starting loops, and this easy-to-follow tutorial illustrates one method for creating starting loops for a chain of nålbound stitches. This method (more…)
wild strawberry stolon basket
Wild strawberry plants abound in our garden, and as I was teasing them out of my thyme, it occurred to me that I might be able to weave the stolons into a little basket. (more…)
mending logan's slippers
Logan loves wearing his slippers so much that he wore holes in them, in spite of the double layered soles I knit before fulling them. I should have (more…)
logan's nålbound hat
My son wanted a nålbound hat for Christmas, and I knew that this vintage yarn from my stash would suit him perfectly. When I test fit the hat on his sister, (more…)
upcycled leg warmers
When you want some knit legwarmers, but don't want to spend hours and hours knitting, consider upcycling a sweater from a thrift store. A fair isle man's sweater was (more…)
nålbound wool hat for largesse
I was asked to make a hat for Kingdom largesse, as we have a Norse King and Queen being coronated this weekend at An Tir 12th Night, and they would like to have some (more…)
elf shoe ornament
Because . . . well . . . just because! I just couldn't resist making a miniature version of Kimberley's elf slippers. I cast on half the number of stitches on a much finer wool, with (more…)
holding the knitting yarn
A few years ago I took some photos of how I hold the yarn for knitting, to remind my friends what I had taught them during a lesson. This is only one way to do it, but it (more…)


text:
Learn more about PERCY HARRISON (OPTICIANS) LIMITED. Check the company's details for free and view the Companies House information, company documents and list of directors.
Know about every new company document that is added!
Take advantage of our watch this company feature free of charge and have notifications sent directly to your inbox. Whenever a new document appears on this website, or if their particulars change, you will be immediately informed about such changes. To monitor these changes, all you need to do is click on watch this company.
|Fixed Assets
|£ 98,942
|Tangible Fixed Assets
|£ 26,942
|Current Assets
|£ 33,888
|Tangible Fixed Assets Depreciation
|£ 46,912
|Provisions For Liabilities Charges
|£ 2,431
|£ 4,433
|Shareholder Funds
|£ 28,216
|Profit Loss Account Reserve
|£ 28,214
|Called Up Share Capital
|£ 2
|Net Assets Liabilities Including Pension Asset Liability
|£ 28,216
|Total Assets Less Current Liabilities
|£ 55,233
|Net Current Assets Liabilities
|£ 43,709
|Creditors Due Within One Year
|£ 77,597
|Cash Bank In Hand
|£ 9,105
|Stocks Inventory
|£ 20,350
|Share Capital Allotted Called Up Paid
|£ 2
|Number Shares Allotted
|£ 51
|Tangible Fixed Assets Cost Or Valuation
|£ 60,666
|Tangible Fixed Assets Depreciation Charged In Period
|£ 13,188
|Intangible Fixed Assets
|£ 72,000
|Amount Due From To Related Party
|£ 53,538
|Creditors Due After One Year
|£ 27,017
Information about the Private Limited Company PERCY HARRISON (OPTICIANS) LIMITED has been prepared for information purposes only. It is not intended to be nor does it constitute legal advice. This is public information provided by the official company register.


text:
Defensive back Brad Mueller has added another trip to his official visit schedule.
"I'm going to Duke on December 6, and my other visit is to Wisconsin on December 13," Mueller said.
Mueller has already made an official visit to Boston College.
Does one school stand out to Mueller?
"I would say that Boston College is recruiting me harder than the rest of the schools," Mueller said.
"All the schools are pretty even right now. But I look for things to change in a month."
The three-star prospect plays on both sides of the ball. On offense Mueller had 230 carries and 1940 yards during his senior season. He also had 5 interceptions on defense.
The 6-foot-0 and 170 pounder from Mars, Pa. has scholarship offers from Purdue, Wisconsin, Boston College, Duke and Maryland.


text:
|2008-01-03, 22:35
|Link #681
Join Date: Dec 2007
In no specific order..
School Rumble: This is my all time fav. comedic anime despite the fact that the plot rarely advanced. It was quite entertaining. Hopes for 3rd season!
Suzumiya Harushi: I thought this was really intresting, the way the show could be watched. I thought the story was really intresting and im hoping for a second season!
ef - a tale of memories : I must say, this has got to be one of the best animes of 2007. The animation was top-notched and the story was fantastic. I loved it. I found the biggest turning point of the show was episode 7..
Code Geass: Lelouch of the Rebellion: What more could I ask for? Death Note and Gundam Seed combined? (Well not really but its somewhere along the lines) It was an amazing season and I can wait for the 2nd season!
Nodame Cantabile: Its was a fun series. I liked the music and the characters, and it was overall really good. Im hoping for a second season!
|2008-01-10, 21:14
|Link #692
Senior Member
Join Date: Sep 2007
Age: 26
1. Code Geass : Lelouch of The Rebellion
2. ef ~ a tale of memories~
3. Full Metal Panic series/ Death Note
4. Tengen Toppa Gurren Lagann/ The Melancholy of Haruhi Suzumiya
5. Ghost Hunt / Elfen Lied / NANA
Last edited by Razziell22; 2008-05-31 at 19:22.
|lists, top five
|Thread Tools


text:
We've caught the Blackberry Gemini, or Curve 8520, in the wild before, but now a picture has surfaced showing the phone with a T-Mobile logo, which means it just might be hitting shelves with the operator soon.
According to Softpedia, the handset is a upgrade of the Curve 8320, with one noticeable omission - no trackball for the first time on a BlackBerry, replaced by an optical trackpad. It's a quadband phone with Wi-Fi support but not 3G; slower GPRS/EDGE data speeds for this one. The full QWERTY is standard BlackBerry fare, and the phone also comes with a two-meg camera and 3.5mm audio jack.


text:
SportsDirect Inc.
Preview: Knicks at Spurs
The New York Knicks take the only undefeated record in the NBA to San Antonio, where they've lost nine straight meetings with the Spurs, in a matchup of two of the league's hottest teams on Thursday. New York is off to a 5-0 start for the first time since 1993-94, when it won its first seven games. The Knicks, who opened a three-game road trip with a 99-89 win at Orlando on Tuesday, are just two wins shy of matching the best start in franchise history.


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Florida State Road 298
|Route information
|Maintained by FDOT
|6.837 mi[1] (11.003 km)
|Major junctions
|West end:
|US 98 / CR 297 in Millview
|SR 727 in Myrtle Grove
|East end:
|SR 295 in West Pensacola
|Highway system
State Road 298 (SR 298), known locally as Lillian Highway, is a mostly east–west thoroughfare in Pensacola, Florida. It runs from its intersection with U.S. Highway 98 and Dog Track Road, skirting Perdido Bay on its way east to its terminus at U.S. Highway 90 (Cervantes Street). For a short stretch near its terminus, SR 298 runs along New Warrington Road. It serves as a major route through the communities of Myrtle Grove and West Pensacola.
In spite of the fact that it intersects US 98, the designation of SR 298 is not meant to imply that it is an auxiliary route of US 98.
Major intersections[edit]
The entire route is in Escambia County.
|US 98 (Lillian Highway / SR 30) / CR 297 south (Dog Track Road)
|Sand Cut
|SR 173 – Naval Aviation Museum, NAS Pensacola
|Myrtle Grove
|SR 727 (Fairfield Drive)
|West Pensacola
|SR 295 (North New Warrington Road)
|1.000 mi = 1.609 km; 1.000 km = 0.621 mi
Related route[edit]
|This section needs to be updated. (December 2013)
|Escambia County
State Road 298 Alternate (SR 298A, also known as West Jackson Street) is a former residential state highway in Escambia County, within the U.S. state of Florida. It was an alternate route to SR 298 in case there was a traffic accident on US 90/US 98 or any one of the roads intersecting SR 298. It was approximately 6 miles (9.7 km) long, passing the western suburbs, Myrtle Grove, West Pensacola, and Brownsville before ending at US 29 in Downtown Pensacola. Oddly, it was less than 1 mile (1.6 km) south of a major road. One of them is US 90. The entire road was only 2 lanes wide. The western terminus was at an intersection with SR 727, 2 blocks south of SR 298. Its eastern terminus was at an intersection with US 29 in Pensacola.
- ^ a b FDOT straight line diagrams Archived March 6, 2014, at the Wayback Machine., accessed March 2014
External links[edit]
|This Florida road-related article is a stub. You can help Wikipedia by expanding it.


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Vacations are ending, the weather is changing, and the kids are starting to freak out about entering a new grade. Ahhh end of summer! Although Fall is a favorite season for many, the transition from summer can sometimes be quite stressful. We've put together a few tips for staying healthy and stress-free as you get ready for Autumn!
Take a walk
It sounds simple enough, but taking a walk around your neighborhood or local park will not only help you clear your mind, but you'll be taking advantage of the last nice days of summer. If you've just spent four hours dragging your kids around the store picking up school supplies, they'll also appreciate a little quiet, and low-impact exercise is always a good thing!
Small Calendar Cushions
This tip for healthy destressing is probably the hardest to follow for most procrastinators, but stick with us! Creating small cushions throughout your daily schedule will work wonders for your state-of-mind! For example, if you usually get up at 8:00am to start your morning routine, set your alarm for 7:45am instead. Adding this tiny little fifteen minutes will make getting ready so much less rushed! Another example would be taking 20 minutes when you first get home to chat with your kids about the day. Don't just jump straight into chores and homework and your evening will be more stress-free!
STOP with the multi-tasking!
ESPECIALLY when it comes to meal-time. Put the gadgets away, turn the tv off, and sit at the actual dinner table (that's what it's there for!) In today's multi-connected world, it's a fallacy to think that multi-tasking is "easier." Sure the process may be more efficient, but it also means it's easy to ALWAYS have two or three things vying for your attention at all times, and that's no bueno. Once you have dinner on the table, it's a time for communication and togetherness.
Healthy Eating
You worked all spring to get your beach-bod ready to go, then spent all summer maintaining it. It's tempting to let the Fall pull you out of your routine (especially with PUMPKIN-EVERYTHING,) so don't let wearing a light jacket mean that you can eat whatever you want! Order that latte with skim milk and less sugar, keep fruits and veggies as a main staple of the diet, and stay away from those gingerbread muffins! Better yet, keep a bag of our ENLIGHTENED Crisps on hand for a delicious and healthy snack!
What's your favorite healthy recipe? Let us know and be sure to follow us here and on Instagram as we share tips for healthy living and healthy recipes all year long.
Follow @eatenlightened


text:
Brisbane Welcomes the World Program
Welcome to Ipswich Program
Welcome to Redlands Program
Scenic Rim Welcomes the World Program
Welcome to North Stradbroke Island Program
Course Registration
Frequently Asked Questions
News and Events
Brisbane – Australia's New World City and its surrounding stunning region is one of the most welcoming and friendliest destinations in the world. Brisbane Region Welcomes the World online training programs are all about our industry and communities continuing to enhance this fine reputation.
It's our shared aim to provide visitors with a very personal connection to our destination, our culture and our events that will be cherished for years to come and leave visitors wanting to be part of it again.
To enable us to consistently make a world of difference to our visitors, our local communities and tourism industry have enthusiastically developed a number of "Welcome to …" online training programs so that we continue to nurture well-informed, passionate ambassadors.
Being part of a team of great welcoming hosts across our great destination involves...
– Understanding our role as hosts and the roles that others play in welcoming visitors
– Understanding how we can all work together consistently and cohesively to enhance the visitor experience across our destination
– Understanding and learning more about our destination and how it is managed to address the "whole experience" … visitor needs and wants from destination journey start to end
Anyone within our tourism industry and communities who plays a role in welcoming and hosting visitors – in Brisbane and across the region – can participate. Participants may include those working in hotels, venues, transport, events, attractions, retail, restaurants, cafes and bars or volunteers with a passion and drive to make a difference.
Each individual participant will receive an official Certificate of Achievement for each online training course fully completed.
Participants may register for one or all courses.
Note: No one ever fails these courses (if you do get some answers incorrect, the system will always share the correct answer with you). There is no scoring process or level to achieve. It's an information share and more about awareness raising and participation than anything else.
The Brisbane Region online welcome programs training is FREE.
The focus of each Brisbane Region Welcome Program is on treating visitors as guests; highly engaging visitors through story telling; readily assisting visitors with wayfinding around the destination.
Click here to view Industry Feedback
Each online Welcome Program takes no longer than two hours to complete. It's also a flexible training process – participants are able to undertake the program in as many sittings as they wish – just saving the completed module components as they go.
Click here to view current and available online welcome program options. Participants may register for one or all courses.
Click here to complete the Course Registration Form - you are able to subscribe to more than one.
If you have any further questions, please do not hesitate to contact our Manager- Destination Programs on firstname.lastname@example.com or 07 3006 6216.


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Here's an easy dinner recipe that rates high in all categories that matter to me: It's quick and easy, healthful and ethnic.
It's not going to rate among the finest Asian chicken dishes you've ever had, necessarily, but for a busy weeknight meal that feeds a family you can't beat it. The recipe suggests using Jasmine rice, which always complements Asian food so nicely.
I'm a nut about using brown rice, however, because it's a high-fiber complex carb and I substitute it for white rice in all my recipes. In this one, it works just fine. I also upped the spices a bit. The first time I tried it I found it a little bland, so I added Chinese Five Spice powder, minced garlic and 1 teaspoon of salt.
If you're really pressed for time and don't mind spending a little extra money, use chicken tenders instead of chicken breasts. You can stir-fry the tenders whole, saving yourself the time and trouble of having to slice the breasts and sanitize the cutting board and knife.


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Miley Cyrus, 18, is going to host Saturday Night Live on March 5, Lifeline Live has confirmed. It will be the first time the star has done the honors.
We'll be watching to see if Miley be a guest on Vanessa Bayer's The Miley Cyrus Show, featuring the SNL cast member as the teen star, with Jason Sudeikis playing dad Billy Ray. In December, Vanessa spoofed Miley's recent bong incident. "The moral of the story: Don't let your friend videotape you while you're doin' stuff."
No matter what, we're betting it'll be "pretty cool!"
Sign up for Lifeline Live e-mail alerts


text:
IBPS Mains Clerk 6 Expected Cut Off 2016-17 Result Joining Dates
(IBPS) Institute of Banking Personnel Selection has recently released a notification regarding the IBPS Mains Sectional & overall Expected Cutoff for the academic year 2016. The examination was conducted at various pre-location centers. The IBPS is an organization which is responsible for every year recruiting applicants with their skills and performance in written examination and interview rounds. The IBPS Mains Exam was conducted in the month of December and January 2016-17.
Key Points on IBPS Mains Exams
As the applicants were first invited so as to fill the application form and thereafter they were able to appear for the exams. As soon as the exam was over all of the applicants started looking for the expected cutoff with according to their category. Here is good news that the IBPS organization is now going to upload the entire expected cutoff with category wise. In this article, we have given complete information on IBPS Sectional & Overall Expected Cutoff for the academic year 2016.
Key Points on IBPS Mains General OBS SC ST Expected Cutoff 2016
The IBPS Examination is one of the toughest exams in the bank field. As every year many applicants appear for the exam and many of them get qualified as per their performance and interview selection. So the here competitive numbers of applicants are every year increasing. Applicants are working hard and get appear for the exam. Recently it had conducted IBPS Mains Exam for the academic year 2016 so applicants, after exams are now looking for the exams cutoff and applicants, can download it from the official website of IBPS.
Methods to Download IBPS Mains Exam Cutoff for the academic year 2016
- Step 1: Applicants need to go to official website to download the Mains Exam Cutoff.
- Step 2: Now see for the expected cutoff link.
- Step 3: Click on the link and download it.
- Step 4: After that check your cutoff.
According to the previous cut off the expected cut off for Gen/OBC/SC/ST could be : 14+/135+/130+/120+
Click me to reach the IBPS Portal directly.


text:
You purchased your D200 because its reliable transport and versatility agreed with your nature; so why would you be okay with second best when it comes to the highest quality auto parts? Dodge car, truck, or SUV drivers have come to expect a certain level of dependability when driving their D200 around town. Your Dodge dealership isn't the only place to locate trusted parts to repair your D200. It's not all the time that you realize that you have to buy a replacement Brake Line & Hose for your automobile.
A reliable and intact brake system gives you and your passengers a sense of safety when you want to slow down. That means your Dodge D200 brake lines or hoses need to be free of leaks at all times. Hydraulic brake lines connect the brake master cylinder to the calipers and/or wheel cylinders on your car or truck. Every time you apply the brakes on your Dodge D200, the brake fluid goes from the master cylinder to each wheel and works its magic to slow down your car. If there's a leak in a brake hose, then you will likely see a large puddle of fluid under your car when you press the brake pedal.
Don't let a Brake Line & Hose repair spell the end of your car, truck, or SUV, and don't risk lowering its resale value by installing anything but the best repair parts. Buy all the car parts you will ever need from carpartsdiscount.com and realize incredible savings on all your upcoming repairs. Given all the hassles associated with installing a part to fix your car or truck, let www.carpartsdiscount.com be the number one store you go to make purchasing it easy. If your car or truck was built in 1980, 1979, 1978, 1977, 1976, 1975, then you will find the correct Dodge D200 Brake Line & Hose parts right here.


text:
October 24, 2012
How many libertarians are there? And how do they vote? Those are just two of the questions examined in The Libertarian Vote: Swing Voters, Tea Parties, and the Fiscally Conservative, Socially Liberal Center, the new ebook from David Boaz, David Kirby, and Emily Ekins. David Boaz will discuss those issues and what libertarians can hope for from the upcoming election.


text:
Sign up for our Free Newslettermore >>
- Get Our Emails
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Free Samples, Contests, Coupons and other Great Free Stuff for Canadian Women.
Sign up for our Free Newslettermore >>
May 17, 2013 | Coupons & Savings, Household
Have a Brita pitcher in your fridge but can't remember the last time you changed the filter? It might be a good idea to do so now, especially when you can save $2 with this coupon!
Sign up with Brita to receive your coupon, and email reminders to tell you when it's time for a new filter!
You work too hard for your money to throw it all away on full price when you don't have to!
© adjump 2011 All Rights Reserved No part of this website or any of its contents may be reproduced, copied, modified or adapted without adjump's prior written consent.


text:
If an item you ordered from TextbookRush does not meet your expectations due to an error on our part, simply fill out a return request and then return it by mail within 30 days of ordering it for a full refund of item cost.
Learn more about our returns policy
Description: An engaging overview of today's most effective technical analysis tools and techniques Technical analysis helps market players predict the price behavior of a stock, commodity, or other financial instrument, giving clues about the course your trade may take in the coming weeks and months. This approach to the markets has proven itself time and again, and now, with Bloomberg on Technical Analysis, you'll discover how to put it to use in your everyday trading endeavors. Filled with in-depth insights and expert advice, Bloomberg on Technical Analysis pulls together the expertise of market professionals, analytics from Bloomberg, and the knowledge of the author himself, an experienced technical analyst who deals with traders and investors every day, to present the most up-to-date information in this field. Reviews technical indicators that traders have found to be extremely useful Taps into one of the author's great strengths: his extensive day-to-day interaction with financial professionals Contains a detailed analysis and explanation of two informative technical analysis studies performed by Bloomberg technical analysts Written with the serious trader in mind, Bloomberg on Technical Analysis offers you the tools you need to improve your performance and make the most out of your time in today's markets.
Limited time offer:
Get the first one free! (?)
All the information you need in one place! Each Study Brief is a summary of one specific subject; facts, figures, and explanations to help you learn faster.
List price: $90.00Copyright year: 2011Publisher: John Wiley & Sons, IncorporatedPublication date: 9/27/2011Binding: HardcoverPages: 336Size: 6.50" wide x 9.50" long x 1.50" tall Weight: 1.342Language: English
*A minimum purchase of $35 is required. Shipping is provided via FedEx SmartPost®
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text:
This is a partial transcript of "The Big Story With John Gibson," March 14, 2006, that has been edited for clarity.
JOHN GIBSON, HOST: Well, he's just doing his job as spokesman for the tobacco industry. Sometimes it pays to put an interesting spin on things. Jane Skinner joins us now with a sneak peek at a new film, "Thank You for Smoking."
JANE SKINNER, CORRESPONDENT: John, what is it like to be the guy responsible for defending Big Tobacco. Who does that job? How does he explain it to his kids? Well, this movie takes a satirical look at lobbyists, and is, as The Washington Post calls it, a wicked little comedy, and it comes to us courtesy of director Jason Reitman, who must have a wicked little sense of humor.
I saw the movie last week. It's very funny. There was probably a danger that it may seem kind of dated. We're talking about tobacco, settlements and all of that several years ago. But it's not. It's very relevant because of the lobbying scandals on Capitol Hill. I was wondering if you wrote Jack Abramoff a thank you note.
JASON REITMAN, DIRECTOR, "THANK YOU FOR SMOKING": Well, the hard part was pushing those indictments that happened right around the release of the film. FOX is very powerful.
SKINNER: Tell me, this just doesn't rip on lobbyists, It rips on journalists, Hollywood types, and you got incredible performances out of these people like Rob Lowe, William H. Macy. And the star as the lobbyist that we're talking about, he's great. But also, the side performances from really well known people make it all work.
REITMAN: We were very fortunate. It was an embarrassment of riches. Everyone kept on saying yes. And we wound up with a wonderful cast.
SKINNER: Robert Duvall as well?
REITMAN: No kidding.
SKINNER: This is your first feature film. How did you go about getting these people on board?
REITMAN: They really responded to the words. It's based on a book called "Thank You for Smoking" by Christopher Buckley. I often ask them because I have no idea why they said yes to doing the film. They wanted to say smart and funny things and the book and screenplay provided that.
SKINNER: We should point out that your last name may be familiar. Your father, Ivan Reitman, is a really well known director, "Animal House," "Ghostbusters" and a whole string of big hits. How much pressure is there, being the son of a famous director, when you talk about your first feature film — you are 28 years old?
REITMAN: Well, the pressure didn't come from that. There's enough pressure just making the movie. My father is my hero, my role model. He told me to be myself and follow my heart.
SKINNER: John was just asking you in the break if you went to film school. Sounds like with your life, you didn't need to go to film school. Your life was film sets.
REITMAN: Right, I grew up on film sets and movie theaters. By the time I got to college, I thought an English degree would suit me better to become a good storyteller.
SKINNER: What has been the reaction so far. You skewer a lot of people in the movie. What's the reaction so far?
REITMAN: We go after everybody and everybody seems to enjoy it. One of the book's great compliments was that Democrats thought it was their book and Republicans thought it was theirs. Hopefully we're getting that. Lobbyists like it so far in particular.
We were in D.C. at a screening and a couple liquor lobbyists pulled me over and said, "All my friends in guns and cigarettes can't wait to see this."
SKINNER: It's not just cigarette lobbyists who are featured. It's also gun lobbyists, liquor licenses. Tell me about that little group, that cadre there.
REITMAN: This is a group called The MOD Squad, Merchants of Death, comprised of the head lobbyist for Big Tobacco, big guns, big liquor played by Aaron Eckhart, Maria Bello and David Koechner. They meet everyday and discuss the going ons of their life and often compete for the highest death toll.
SKINNER: That's one of the funniest parts when they are exposed in The Washington Post by this reporter, played by a sassy Katie Holmes, and in case anybody doesn't know, soon to be Mrs. Tom Cruise. And this cause added little bit of controversy when it was screened at the Sundance Film Festival.
You got a lot of P.R. out of it. There was a sex scene missing from the screening and people wondered if Tom Cruise came down and said I don't want my future wife to be shown in a compromising position. What happened?
REITMAN: Well, first of all, there are no compromising positions in the film. It's a very tame scene. I don't want anybody to be disappointed when they see it. It's nothing but humorous humping really. And the last time I talked to Tom Cruise, I was 14 years old and he made no mention of the scene then.
SKINNER: I did see it and I wasn't actually sure if it was a scene or not a scene.
REITMAN: That's the funny thing. People talked about it seeing it the way they wanted, talking about her being nude and the scene being sultry. So, people are looking for a scene that's not there.
SKINNER: A little hype doesn't hurt. Jason Reitman, good luck to you. "Thank You for Smoking" is the film.
Content and Programming Copyright 2006 FOX News Network, LLC. ALL RIGHTS RESERVED. Transcription Copyright 2006 Voxant, Inc. (www.voxant.com), which takes sole responsibility for the accuracy of the transcription. ALL RIGHTS RESERVED. No license is granted to the user of this material except for the user's personal or internal use and, in such case, only one copy may be printed, nor shall user use any material for commercial purposes or in any fashion that may infringe upon FOX News Network, LLC'S and Voxant, Inc.'s copyrights or other proprietary rights or interests in the material. This is not a legal transcript for purposes of litigation.


text: Die Young Printings/Rarity: Cost: CMC: 2 Card Type: Sorcery Oracle Text: Choose target creature. You get (two energy counters), then you may pay any amount of . The creature gets -1/-1 until end of turn for each paid this way. Flavor Text: "When the time comes, let go. Nothing lasts forever." Retrieved from "http://mtg.wikia.com/wiki/Die_Young?oldid=70333" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Kaladesh Common Energy Cards CMC: 2 Black Sorcery


text:
There is nothing that could please me more than when Wikia actually makes a feature or updates a feature that actually could be useful. Here comes the forum feature!
It has been no secret that I really wanted a forum feature for Ghostbusters wiki (and Thundercats wiki too) for ages. I was hoping for a feature that allowed topics and input without wiki know-how. I wanted it easy for non-wiki editors to be able to talk with use as well as us wiki editors.
The blog about the forum feature is here.
The test forum is here.


text:
- This event has passed.
Sons of the Pioneers in concert
August 21, 2014 @ 7:00 pm - 9:00 pm$1 - $23
Event Navigation
For over 75 years the Sons of the Pioneers have been proud to perform the music of the American West. They embrace Western heritage in their music and strive to honor it. Their songs are unabashed love songs to the West.


text:
Chandrasekhar Gurramkonda
With three-years of research experience as Research Assistant Professor at the Technology Research Centre, Department of Biochemical Engineering, University of Maryland Baltimore County, where I was involved in research activities and managed the laboratory work. I was more familiar with research projects which involved the development of cell-free expression and its application in recombinant protein production. Also worked on screening the dengue vaccine candidates (Two patent applications). Earlier, I was trained at Helmholtz Centre for Infection research, Braunschweig, Germany and at the International Centre for Genetic Engineering and Biotechnology, New Delhi, India. Being a driving force worked on applied research involving design and development of recombinant vaccines for HBsAg, putative dengue vaccine candidate, Human Insulin development, HCV diagnostic kit development. In the past, also worked at Shantha Biotech (A part of Sanofi, India) for the development of erythropoietin.
Additionally, serving as reviewer for prestigious journals like Biotechnology advances, Protein expression and purification, PLoS ONE, Microbial cell factories, Preparative Biochemistry and Biotechnology, Korean Journal of applied biological society and Biochemical engineering Journal and etc.,
Biochemistry Biotechnology Infectious Diseases Microbiology Molecular Biology Virology


text:
Consdesocial is committed to protecting your privacy and implementing technology that gives you a rewarding and safe online experience. We want you to feel secure when visiting our site and are committed to maintaining your privacy when doing so.
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Generation of a reference transcriptome for evaluating rainbow trout responses to various stressors
Fish under intensive culture conditions are exposed to a variety of acute and chronic stressors, including high rearing densities, sub-optimal water quality, and severe thermal fluctuations. Such stressors are inherent in aquaculture production and can induce physiological responses with adverse effects on traits important to producers and consumers, including those associated with growth, nutrition, reproduction, immune response, and fillet quality. Understanding and monitoring the biological mechanisms underlying stress responses will facilitate alleviating their negative effects through selective breeding and changes in management practices, resulting in improved animal welfare and production efficiency.
Physiological responses to five treatments associated with stress were characterized by measuring plasma lysozyme activity, glucose, lactate, chloride, and cortisol concentrations, in addition to stress-associated transcripts by quantitative PCR. Results indicate that the fish had significant stressor-specific changes in their physiological conditions. Sequencing of a pooled normalized transcriptome library created from gill, brain, liver, spleen, kidney and muscle RNA of control and stressed fish produced 3,160,306 expressed sequence tags which were assembled and annotated. SNP discovery resulted in identification of ~58,000 putative single nucleotide polymorphisms including 24,479 which were predicted to fall within exons. Of these, 4907 were predicted to occupy the first position of a codon and 4110 the second, increasing the probability to impact amino acid sequence variation and potentially gene function.
We have generated and characterized a reference transcriptome for rainbow trout that represents multiple tissues responding to multiple stressors common to aquaculture production environments. This resource compliments existing public transcriptome data and will facilitate approaches aiming to evaluate gene expression associated with stress in this species.
Fish under intensive culture conditions are exposed to a variety of acute and chronic stressors, including elevated rearing densities, sub-optimal water quality including decreased dissolved oxygen (DO) and high carbon dioxide (CO2), and thermal fluctuations
Studies which aim to characterize global gene expression in response to stress often use hybridization-based approaches (i.e. microarrays) to identify differences between challenged and control fish
analyzed; and it is usually not possible to distinguish different gene isoforms
In the absence of a genome reference sequence for rainbow trout, we used Roche 454 pyrosequencing technology to develop a reference transcriptome sequence to be used specifically in gene expression analyses associated with stress, including RNASeq based approaches. The library construction included a normalization process
Previous studies have evaluated the combined effects of temperature and salinity on physiological condition of rainbow trout. Niu
We demonstrate that each stressor produced a physiological response by reporting changes in plasma variables indicative of various phases of a stress response (cortisol, glucose, lactate, and chloride concentrations and lysozyme activity
Results and Discussion
Water Quality Parameters
Water quality parameters including temperature, DO, pH, CO2, and ammonia were measured at 0, 3, and 6 hours during the challenges and as summarized in Additional File
Additional File 1
Table S1 Water Quality Parameters, Table S2 Gene Ontology Annotation. Table S1 contains temperature, dissolved oxygen, carbon dioxide, pH, ammonia, and nitrite data for each of the time periods and treatments. Table S2 contains transcriptome annotation assignments including BLASTx hits and Gene Ontology terms. Additional file 1: Additional File 1 Water Quality and GO.xlsx, 11694K
Click here for file
Physiological responses
We measured plasma cortisol as an indicator of primary response and glucose, lactate, chloride and lysozyme as indicators of secondary stress responses to detect if stress responses occurred and to characterize differences in responses that are unique to each stressor or shared among stressors. Figures
Figure 1
Physiological Responses to Stress Treatments
Physiological Responses to Stress Treatments. The response of plasma glucose and lactate across all treatments and controls. Fish treatments included Control (Con), High Temperature (HT), Low Temperature (LT), Re-use Water (RW), Salinity (Sal) and Handling/Confinement (Han). Values from all control tanks were pooled and presented as a single mean, which was compared to the mean for each stress treatment.
Figure 2
Physiological Responses to Stress Treatments
Physiological Responses to Stress Treatments. The response of plasma chloride and cortisol concentrations compared across all treatments and controls. Fish treatments included Control (Con), High Temperature (HT), Low Temperature (LT), Re-use Water (RW), Salinity (Sal) and Handling/Confinement (Han). Values from all control tanks were pooled and presented as a single mean, which was compared to the mean for each stress treatment.
Figure 3
Physiological Responses to Stress Treatments
Physiological Responses to Stress Treatments. The response of lysozyme activity compared across all treatments and controls. Fish treatments included Control (Con), High Temperature (HT), Low Temperature (LT), Re-use Water (RW), Salinity (Sal) and Handling/Confinement (Han). Values from all control tanks were pooled and presented as a single mean, which was compared to the mean for each stress treatment.
Analysis of gene expression
Stress and its resulting increase in cortisol levels have been reported to affect the onset of the apoptosis mechanism
The caspases are proteolytic enzymes which are the core components of the intracellular apoptosis mechanism. Caspases often function in cascades, where an upstream caspase is activated by its interaction with a caspase adaptor
Figure 4
Differential Expression in Response to Stress
Differential Expression in Response to Stress. Figures 4a-c show the fold change in expression for genes involved in the stress response in gill tissues of fish within each treatment. Values represent fold change in expression compared to the control treatment, which was set at 1.0. Means without a common letter represent significant differences, P ≤ 0.05.
QPCR results for tumor suppressor gene p53 (
To compensate for osmotic gain of fluid and diffusive loss of ions, freshwater teleosts developed an active ion transport mechanism. Ions like Na+, Cl-, Ca2+ are actively transported across the gill epithelium to maintain high levels of ions in the blood. Euryhaline fishes that move from freshwater to salt water transform their gills from ion absorbing mechanisms to an ion-secreting epithelium, resulting in an increase of Na+/K+-ATPase (
Transcriptome sequencing and assembly
Newbler and MIRA3 were used to assemble 3,160,306 high quality reads generated by 454 pyrosequencing of the rainbow trout RNA samples; this information is available through the Short Read Archive in GenBank as Accession
Table 1
Detailed Information on the Assembly Results.
Newbler Reads
Total number of reads
Number of aligned reads
Number of aligned bases
Number of wholly assembled reads
Number of partially assembled reads
Number of singletons
Number of repetitive reads
Number of outlier reads
Number of too-short reads
Newbler Contigs
Total number of contigs
Total number of contig bases
Average coverage
Average contig size
502 bp
Contig N50
829 bp
Number of contigs with size > 100 bp
Number of contigs with size > 500 bp
Largest contig Size
16,649 bp
Newbler Isotigs
Total number of isotigs
Average contig count
Largest contig count
Number of isotigs with one contig
Average isotig size
1,337 bp
Isotig N50
1,639 bp
Largest isotig Size
24,959 bp
Newbler Isogroups
Total number of isogroups
Average isotig count
Average contig count
Largest isotig count
Largest contig count
Number of isogroups with one isotig
Number of isogroups with one contig
MIRA3 assembly of Newbler singletons
Reads assembled
Number of contigs
Contig N50
Largest contig size
Transcriptome characterization
For the Newbler assembly, only isotigs that are longer than 100 bp were selected for annotation. To avoid redundancy, all isotigs with 90% or higher identities were grouped in one cluster and only a representative from each group was selected for annotation. This step reduced the number of sequences from Newbler assembly from 83,935 to 62,071. Moreover, the 143,560 reads listed on MIRA3 results as debris (singletons), were masked with RepeatMasker using the INRA (National Institute for Agricultural Research, France) Rainbow Trout repeat and cGRASP Salmon masker databases, and 39,210 masked sequences that have more than 300 base pairs of continuous non repetitive segments were also included in the annotation process. After adding the 37,143 MIRA3 contigs; 138,424 sequences were included used for annotations.
Overall, 56,991 sequences were detected as BLASTx hits. The top 10 distributions of BLASTx species are shown in Figure
Figure 5
Top Blast Hits by Species
Top Blast Hits by Species. Figure 5 shows the species represented in the top BLASTx hit for each isotig.
The GO term distribution (see Additional File
Additional File 2
Gene Ontology Figures, Figures S1 and S2 in a PDF. Figures show the distribution of Gene Ontology hits by GO level as broken down by Biological Brocess (P), Molecular Function (F) and Cellular Component (C) (Figure S1). A second figure shows assignment of Gene Ontology categories broken down by Biological Processes (a), Molecular Functions (b) and Cellular Components (c) (Figure S2). Additional file 2: Additional File 2 Gene Ontology Results.pdf, 934K
Click here for file
Table 2
Keyword Search of Functional Annotation.
Search Term
BLASTx Hit Description
Gene Ontology Term
carbon dioxide
heat shock
Searching BLASTx Hit Descriptions and Gene Ontology Terms associated with isotigs from the reference transcriptome reveals significant representation of transcripts expected to be involved in stress responses.
Current rainbow trout transcriptome sequence datasets available in NCBI include 287,967 EST sequences in dbEST and the 454-pyrosequencing data submitted to SRA, which include 1,298,911 sequences of a Swanson double-haploid fish and 1,507,274 other rainbow trout cDNA sequences submitted by INRA. In addition, there are 116,463 sequence clusters generated from 391,356 rainbow trout mRNA and EST sequences in NCBI UniGene database and 161,818 rainbow trout transcriptome reference sequences generated by Salem
SNP Discovery
Four full sib families were used in this experiment, therefore assembly of the transcriptome data was expected to result in the identification of putative SNPs. A total of 57,207 SNPs were identified from the Newbler assembly which represents 30,293 contigs assigned to 21,335 isogroups. The average number of putative SNPs in a contig was 1.9 and in an isogroup 2.7. The maximum number of putative SNPs in a contig was 20, and 15,667 contigs had only one putative SNP. On average, one putative SNP was found every 483 bp. The maximum number of putative SNPs in an isogroup was 392, and 13,254 isogroups had at least two SNPs. Similarly, an additional 808 putative SNPs were identified from the MIRA3 assembly. Overall, the putative SNPs would include 37,358 transitions and 20,657 transversions, similar to the 2:1 ratio observed in some but not all species
It is likely that some of the putative SNPs are actually paralogous sequence variants (PSVs), and therefore are the result of gene duplication events and not sequence variation
Additional File 3
Table S3 Newbler SNP Discovery, Table S4 MIRA3 SNP Discovery. Table S3 contains information for 57,207 putative SNPs identified from Newbler reference transcriptome contigs. Table S4 contains information for 808 putative SNPs identified from MIRA3 reference transcriptome contigs. Additional File 3: Additional File 3 SNPs.xlsx, 11083K
Click here for file
We have generated and characterized a reference transcriptome for rainbow trout that represents multiple tissues responding to multiple stressors, and the unique expression portfolios for each. This resource complements existing public transcriptome data and will serve approaches aiming to evaluate gene expression associated with stress in this species.
All experiments were conducted under approval of the USDA/ARS National Center for Cool and Cold Water Aquaculture Institutional Animal Care and Use Committee, protocol #50.
Four unrelated families from the NCCCWA even year broodstock population under selective breeding for growth
Stress challenges
After acclimation, water conditions in each tank were brought to the experimental parameters over the course of three hours (time 3) and then maintained at stress conditions for three hours. The following stress conditions were used: 1) high temperature 25°C; 2) low temperature 2°C; 3) salinity 32‰ (addition of salt, NaCl); 4) Re-use water, presenting lower DO and higher CO2; and finally 5) handling/crowding stressors, following procedures previously described by Weber and colleagues
Plasma metabolite measurements
To identify the physiological responses to stress, plasma metabolite measurements including concentrations of plasma cortisol, glucose, lactate, and chloride in addition to lysozyme activity were measured. Whereas cortisol is a measure of the primary stress response, the others are measures of secondary stress responses. Glucose and lactate serve as measures of metabolic response, chloride as a measure of the osmoregulatory response, and lysozyme activity as a more direct effect of the stressor on the immune system.
Plasma cortisol was measured by radioimmunoassay following procedures described by Redding
Quantitative PCR
Differential expression among the stressors was determined by sampling gills from all the stress and control samples (160 samples) for quantitative PCR (qPCR). RNA was isolated from tissue using standard TRIzol (Invitrogen, Carlsbad, CA) extraction methodology according to the manufacturer's suggested protocol. RNA quality was visualized using agarose gel electrophoresis. RNA samples were from those extracted for creation of the pooled library, see below. Reactions optimized for the following pathways and genes: 1) apoptosis pathway (
Table 3
Quantitative PCR Gene and Primer Information.
Gene Symbol
Primer Sequences
Amplicon Size
Caspase 3
Caspase 8
Nuclear protein 1
Heat shock
protein 70 b
Elongation factor
Housekeeping genes were used to normalize the transcript expression values [Elongation factor (
Significant effects of stress on gene expression were detected using the general linear models procedure using PC-SAS (Version 9.1). In the event of a significant p-value (P ≤ 0.05), differences between treatments were detected using the Fischer's least significant difference (LSD) procedure. Data on metabolites were analyzed using SigmaStat (Version 3.1; Jandel Scientific, San Rafael, CA). Significant effects of stress on metabolite expression were detected by comparing among control and all treatment groups by Kruskal-Wallis One Way Analysis of Variance on Ranks, followed by Dunn's Method for all pairwise multiple comparisons of means. Treatment means were considered significantly different when P ≤ 0.05. All data are presented as means ± SEM (standard error of the mean).
Transcriptome sequencing
To identify the collection of genes expressed during response to stressors, RNA was extracted from gill, brain, liver, spleen, kidney and muscle from one random sample from each treatment tank plus three control samples (total of 108 RNA samples). All RNA samples were diluted in water and pooled together in a 500 ng/μl concentration. Library preparation and sequencing were performed at the Indiana University's Center for Genomics and Bioinformatics. Methodology was based on Meyer
Transcriptome sequence assembly
The generated rainbow trout cDNA sequence reads were quality trimmed and the adaptor's sequences were removed. Subsequently, GS
SNP discovery
For SNP identification, high quality sequence reads were first screened for repetitive elements with RepeatMasker
Sequence Annotation
Functional annotations of Newbler isotigs/contigs, MIRA3
Sequence datasets comparison
A total of 3,091,890 sequences that had been cleaned of vector and
Authors' contributions
CER, JY and CCS were responsible for overall study design and execution, GMW was responsible for physiological data, BMC for quantitative PCR, GG performed sequence assembly and annotation. All authors have read and approved the final manuscript.
The authors would like to thank Roseanna Long and Jill Birkett for their technical assistance with real-time PCR and performing the metabolite assays, and Dr. Keithanne Mockaitis for library preparation and sequencing. We acknowledge Mark Hostuttler, Clayton Birkett and Kevin Melody for setting up the established experimental conditions; Paul Peach and Kevin Melody for performing water quality measurements; Roseanna Long, Kristy Shewbridge, Brian Smith and Meghan Manor for their assistance with tissue sampling. We also thank Kevin Melody, Josh Kretzer, Kyle Jenkins and Jenea McGowan for their efforts in animal care and sampling. The authors acknowledge Dr. Ben Koop and Ben Sutherland for critical review of the manuscript. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.
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Heat shock proteins are not sensitive indicators of hatchery stress in salmon
Methods for reducing stressors and maintaining water quality associated with live fish transport in tanks: a review of the basics
FIsh transport in the aquaculture sector: an overview of the road transport of Atlantic Salmon in Tasmania
The influence of ploidy on saltwater adaptation, acute stress response and immune function following seawater transfer in non-smolting rainbow trout
Rearing oxygen level and slaughter stress effects on rainbow trout flesh quality
The stress Response in Fish
Fish welfare and quality as affected by pre-slaughter and slaughter management
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Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish
Sequencing and characterization of the guppy (Poecilia reticulata) transcriptome
Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches
Mining SNPs from EST databases
Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\ normalization and subtraction of cDNA libraries
Gene expression in the brain and kidney of rainbow trout in response to handling stress
Gene expression in the liver of rainbow trout, Oncorhynchus mykiss, during the stress response
Gene expression pattern in the liver during recovery from an accute stressor in rainbow trou
Heat shock proteins (Hsp70) induced by a mild heat shock slightly moderates osmolarity increases upon salinity transfer in rainbow trout (
Effect of Low temperature on sea water tolerance in Rainbow Trout,
Mortality of salmonids cultured at low temperature in sea water
Rearing oxygen levels and slaughter stress effects on rainbow trout flesh quality
Effects of Carbon dioxide exposure on intensively cultured Rainbow trout Oncorhynchus mykiss: Physiological responses and Fillet Attributes
A partial-reuse system for coldwater aquaculture
Evaluation of a stress response for use in a selective breeding program for improved growth and disease resistance in rainbow trout (Oncorhynchus mykiss)
Modification of the plasma cortisol response to stress in rainbow trout by selective breeding
Associations between plasma growth hormone, insulinlike growth factor-1 and cortisol, with stress responsiveness and growth performance in a selective breeding program for rainbow trout (oncorhychus mykiss)
Cortisol Response to a Crowding Stress: Heritability and Association with Disease Resistance to Yersinia ruckeri in Rainbow Trout
Stress-associated impacts of short term holding in fishes
The stress response in fish
Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids
Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchus mykiss) monocyte/macrophage cells in primary culture treated with cortisol
Cortisol inhibits apoptosis in carp neutrophilic granulocytes
Conservation of apoptosis as an immune regulatory mechanism: effects of cortisol and cortisone on carp lymphocytes
Gene ontology: tool for the unification of biology. The Gene Ontology Consortium
Environmental Requirements and tolerance of Rainbow trout (
The effects of carbon dioxide on performance and histopathology of rainbow trout
Cell suicide and caspases
Proteases for cell suicide: functions and regulation of caspases
Rainbow trout p53: cDNA cloning and biochemical characterization
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Nupr1: the Swiss-knife of cancer
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Heat shock protein genes and their functional significance in fish
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SSAHA: a fast search method for large DNA databases
Mira 3
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