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Hello pals!
After spending this past semester abroad in Scotland, I am now interning in about the most opposite city I could possibly choose: Miami. I'm interning at FIU's Summer Treatment Program for children with behavioral disorders and more specifically, ADHD, Oppositional Defiant Disorder, and Conduct Disorder. This program, run by Dr. William Pelham, is the leading research center for ADHD and I am so blessed to be able to work for him and gain experience with these children since this is the area of research I am interested in continuing long term. As a counselor, we are basically administering behavioral modification treatment to the children and, as part of the study that is being conducted, their medication is altered daily which makes each day exciting to say the least. Day to day, we are with the children all day and take them through various activities which consists of 2 class periods, an art class, and 2 sports games. To prepare for the internship, we had to memorize a 400 page manual of all the rules the children have to follow and definitions of behaviors that we track for each child. To say the least, it has easily been the most challenging and exhausting job I have ever had. I absolutely love all of my children (and obvi have my favorites) but they are such a handful and I have gained an entire new outlook on ADHD as a disorder and the deficits that these children face.
One of my favorites parts about the internship though is that it is based right in Miami! Even though I'm from Orlando, Miami isn't a place that I visit often so living down here for 2 months has been such a fun time! There's so much to do every weekend; the beaches are beautiful, and The Keys are only an hour away! However, it is a lot different than I expected and is never a place I would want to live full time. But it is definitely a summer I will remember and I have made pals that I will for sure keep in touch with long term! Look below for some pics of me and the crew*~*~* MORE IMPORTANTLY: T-Minus 19 days until I'm back in Lex for preseason aka the happiest place and the happiest time of year!!
Peace n' luv n' GO GENNIES
Anna #14


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Not surprisingly, they have the highest burn rate in the very small industry of sled-delivered goods. ~NSHA
From the Submitter: I made this contraption to keep my laptop from overheating. It cooled from 69ºC to 39ºC within ten minutes. And yes, those are 2.1 kV transformers. And yes, that's a roll of duct tape.
Let me label that for you:
1: Laptop that he just can't let go of. 2: Books that he tried to read and failed. The Silmarillion (left) and Ulysses (right). 2 - again? come on dude, you only had to count to 5: Anyway, mentioned above. 3. CPU fan stolen from parent's desktop. 4: The crowning achievement of the piece, almighty duct tape.
Awww, all that effort and she didn't even take a swim!

subset: commoncrawl


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The Top 10 In Action Sports
01Why Do We Travel?
02Eccentric Adventures Episode 4: We Risked Life And Shin At The Cotswold Olimpicks
03Going Against the Brain: How Ski and Snowboard Head Injuries Re-Shaped Two Young Lives
04Easy Riders: Is This Vintage Bikes & Surfboards Festival the Coolest in Europe?
05Free Wheels East: Rejecting Society for Historic Adventure
06Eccentric Adventures Episode 3: We Took on Vikings at the Scottish Highland Games
07A Million Miles Away... In Your Own Backyard
08Brick By Brick: Camping Lego Style
0917 Cringe-Worthy Holiday Video Clichés That Every Skier Should Avoid
10Booze and Bikes: 14 Tours That Let Combine Two of the Best Things in the World
DIGBMX: Mike Miller Escaping Winter
Winters in England are normally pretty cold, but we never get snow like we did this year. It was savage and it really made me feel for the guys who get a ton more than us. Anyway, back to the point. Due to having soo much snow for ages me and Miller decided to head to the indoor parks to get in some much needed riding and along the way we shot this short edit. enjoy - sam
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Blogs Liberty and Power The First Thanksgiving and GunsNov 24, 2005 6:00 pm
The First Thanksgiving and Guns
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Kenneth R. Gregg - 11/25/2005
Sartell Prentice's article on "The First Thankgiving" ( http://www.fee.org/in_brief/default.asp?id=106&year=2005&month=11 ) is on FEE's website at this time. Sartell, an old friend of mine and author of a work on cathedrals ( http://www.forbesbookclub.com/BookPage.asp?prod_cd=IN6L0 ) was always quite proud of his essay. Sartell always argued with his brother, Perry Prentice ( http://www.cooperativeindividualism.org/prentice-perry_taxes-and-the-death-of-cities.html ), who was publisher and editor of the Architectural Forum and VP of Time, Inc. Sartell was a hard-core free market anarchist and his brother a Georgist. He died years ago, and quite old when I knew him in the 1970's,
Just as an aside, you might be interested in this: "Pilgrims Mistook Natives for Israelites" ( http://dsc.discovery.com/news/briefs/20050704/kennewick.html ) which is some recent research on Indian/Pilgrim relations.
Just a thought (and Happy Thanksgiving!).
Just Ken
- Kitty Genovese Killing Is Retold in the Film '37'
- Lithuania wants to erase its ugly history of Nazi collaboration
- Huckabee: Iran nuclear deal will march Israelis 'to the door of the oven'
- Connecticut Democrats drop Thomas Jefferson and Andrew Jackson names from annual fundraising dinner
- AP releases a million minutes of filmed history to YouTube
- Historian Howard Segal says the cost of paying for expensive commencement speeches is diverting funds from where they're most needed
- Historian Shelly Cline researches female Nazi guards
- Owen Chadwick, Eminent Historian of Christianity, Dies at 99
- Members of the University of South Florida's history department are finding new ways to get their jobs done after budget cuts
- Testing the U.S.-Israel Bond


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Insomniacs can't sleep without sheep, unless they get Serta.


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Bob Dylan Among Artists Linked to Sesac Bond Issue
Aug. 31 (Bloomberg) -- Sesac Inc., a performance-rights company, plans to sell $300 million of bonds linked to licensing agreements, copyrights and royalty payments for artists from Bob Dylan to Neil Diamond. Bloomberg's Tom Keene reports on Bloomberg Television's "Bloomberg Surveillance." (Source: Bloomberg)
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50:16 - Financial Insyghts President Peter Atwater examines social mood in economic confidence across the markets. He speaks with Bloomberg's Alix Steel and Joe Weisenthal on "What'd You Miss?" (Source: Bloomberg)


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Pak Army 'trust us' mantra not going to work, Osama spent years hidden
Islamabad: The long standing mantra of the Pakistani army 'trust us, guys' is not going to work further after the Abbottabad commission report revealed that Osama bin Laden spent years in Pakistan hidden, according to an editorial in a Pakistani daily.
A report in the Dawn News said that Osama being killed on Pakistani soil by US troops is surely one of the staggering national-security lapses in the country's history.
A large scale appeal made in this report has urged the Pakistani government to release the commission report on the slain al-Qaeda leader.
The report cited that the government of Pakistan did not release the Abbottabad Commission report for public, which was handed over to former prime minister Raja Pervez Ashraf in January.
According to the report, it is assumed that the responsibility for the secrecy lay with the military leadership.
But the country focuses more on the embarrassment that will be caused nationally and internationally by a comprehensive official account of such an episode that has led to undermine national security, report said.
To conclude, it will be inevitable for the Pakistani government to officially release the report because it will form basis for any prosecutions deemed necessary.
More from India
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- Punjab: Gurdaspur gunbattle ends, all terrorists killed
- Mumbai blasts case: Maharashtra govt opposes Yakub Memon's mercy plea in SC
- PM Modi condoles APJ Abdul Kalam's death
- Kalam's advisor recalls his last moments with the great scientist
- Politicos mourn death of former president APJ Abdul Kalam, hail him as 'Missile Man of India'
- When UN declared Dr APJ Abdul Kalam's 79th birthday as World Students' Day
- Gurdaspur terror attack: Blow-by-blow account
- SC hearing on 1993 Mumbai blasts convict Yakub Memon's curative plea


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A SIGNIFICANT drop in enrolment of Indian students at the University of Southern Queensland may be partly caused by fears of racist attacks in Australia.
USQ Vice Chancellor Professor Bill Lovegrove said negative media reports about racist attacks on Indian students in Sydney and Melbourne could be a factor in the decline.
He said this was despite positive reports from Indian students he had spoken to on campus in Toowoomba.
It is a bit early to state a definite outcome on international enrolments, but we are down by 10 to 15 per cent and any decline is a concern.
Violence towards Indian students in Australia may be playing a role, but there are other factors.
The Australian dollar has gone up and its also so much more difficult for students to get a visa.
Prof. Lovegrove said changes to student visa criteria meant international students needed to show they had $18,000 in a bank account, where in previous years they needed only $12,000.
The university has a dedicated arrivals team to assist international students to settle in as they enter the country.
USQ Indian students Praneeth Juwadi and Sudha Reddy Soma both said their experiences in Australia and Toowoomba had been positive.
Mr Juwadi said the decision to study at USQ was an easy one after he received positive feedback from friends who had studied at the Toowoomba campus.
I had a few friends who were studying here and all recommended the university to me and I am glad that I followed their advice, he said.
All of the academics are really friendly and helpful and so are the students.
It has made studying here very enjoyable.
Ms Reddy was thrilled to find Toowoomba and USQ to be culturally diverse and accepting of international students.
The university has excellent facilities and very supportive faculties, but it is the multicultural environment which has made me feel really comfortable.
Despite the drop in enrolments for Indian students, there has been a 27 per cent increase in domestic enrolments at USQ, well above the State average of 13 per cent.
Significant drop in Indian students in the University of Southern Queensland
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US trade Currency macroeconomic wars on the world and had ruined Rupee sending India Students packing
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4 Reviews
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Most Helpful First | Newest First
10 of 10 people found the following review helpful
5.0 out of 5 stars Just superb,
AquaViola (Nottingham) - See all my reviews
This review is from: Just A Classic Minute: Volume 5: v. 5 (BBC Radio Collection) (Audio CD)
If you are reading this, then you probably already have Just a Classic Minute 1 to 4. So what is different?
In the first place, these are 4 truly classic episodes - none of your curiousos such as when Kenneth Williams was chairman as in Just a Classic Minute 2. These episodes come from 1977 to 1988, and are just great.
Secondly, and very importantly, instead of an introduction to each episode by Nicholas Parsons, there are instead some long conversations between Nicholas and Paul Merton. Not only do they discuss the episode of JAM itself, but they also go much wider, and put different aspects, such as the role of women in the show, in greater context. This double CD set is worth getting, if only for these conversations.
Two excerpts from these episodes have appeared on the first compilation way back in 1992 called "Silver Minutes" (only available second hand on cassette, but very well worth listening to). However, if you have "Silver Minutes", then you obviously are the sort of collector who would want this because of the sheer quality of the episodes and the introduction.
Overall, get it! It's very well worth it, and long may there be additional releases of Just a Classic Minute.
1 of 1 people found the following review helpful
5.0 out of 5 stars Another great selection from a great show,
Mark Pack (London, UK) - See all my reviews
This review is from: Just a Classic Minute, Volume 5 (Audio Download)
As with previous editions in the series, this collection of Just a Minute episodes is a selection of four from across the decades of the long-running and successful Radio 4 comedy show, with some added commentary from Nicholas Parsons about each episode. This time Paul Merton joins Nicholas Parsons for those interludes, adding a welcome new angle as the Nicholas Parsons comments between episodes on previous volumes had started to get a little bit too similar to each other.
But of course the real fun comes from the episodes themselves, especially the one where Nicholas Parsons - briefly an impersonator early in his show business career - revives that skill to imitate Kenneth Williams, adding a moment of comic genius to that show.
We also get to enjoy an early example of the even more surreal than now Paul Merton along with a good spread of examples of how some new guests take to the show easily whilst others struggle. Of course even the experienced players find it tough at times and in one of the shows the others let Peter Jones struggle on with a subject deciding there were more jokes to be had in letting him repeat, deviate and hesitate as he dried up, rather than interrupting him. Even Nicholas Parsons joins in, only bringing the round to an end after 1 minute 15 seconds.
All in all, great fun.
5.0 out of 5 stars Classic fun,
M. Cusack (Ireland) - See all my reviews
This review is from: Just A Classic Minute: Volume 5: v. 5 (BBC Radio Collection) (Audio CD)
Excellent collection of Just a Minute classics with some humorous and informative introductions by Nicholas Parsons and Paul Merton. Well worth it - great to listen to in the car instead of all the current doom and gloom! Still funny after all this time - my kids enjoy them too.
5.0 out of 5 stars Five Stars,
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This review is from: Just A Classic Minute: Volume 5: v. 5 (BBC Radio Collection) (Audio CD)
great fun
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Just A Classic Minute: Volume 5: v. 5 (BBC Radio Collection) by Ian Messiter (Audio CD - 10 July 2008)
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Retailers stick with the standards for online marketing
The State of Retailing Online 2015 report finds search and email leading the pack with e-retailers.
Retailers stick with the standards for online marketing
The State of Retailing Online 2015 report finds search and email leading the pack with e-retailers.
Ellen DeGeneres' celebrity pals promote her e-commerce site
Justin Bieber, Madonna and Kim Kardashian-West tweeted about the launch of EDbyEllen.com.
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Total and comparable sales also grew, but not as much.
The Children's Place Retail Stores Inc. booked sales growth in the second quarter, led by the Internet.
For the quarter ended July 28, specialty apparel retailer The Children's Place, No. 111 in the Internet Retailer Top 500 reported:
"Despite the continued difficult economic environment, we believe lower apparel costs in the back half coupled with strengthening conversion, transactions and average transaction value, will result in positive comp sales and operating margin expansion in fiscal 2012," says Jane Elfers, president and CEO.
For the first six months:
The Children's Place plans to consolidate into a single U.S. distribution center in Fort Payne, AL, in order to more effectively manage its inventory and optimize capacity, the retailer says. The Fort Payne facility is the company's newest and largest. Its Northeast distribution center in Dayton, NJ, will close Dec. 31.
Closing that distribution center is expected to save more than $4 million annually, the company says. The distribution center changes are expected to result in charges of approximately $5 million in the third quarter and $11 million in the fourth quarter of 2012.
An IBM survey shows the most successful retailers set themselves apart by creating a centralized ...
Posted 08/03/15
With more B2B buyers demanding the option to buy from their suppliers online, companies need ...
Posted 07/31/15
If there's something you've bought offline that you weren't able to buy online, we're probably thinking about that."


text:
Do Uber and Lyft Redline Low-Income Communities?
Some critics allege that ultra-modern ride-sharing services resemble old-line taxi operations in declining service to poor communities. No way, say Uber and Lyft.
Some critics allege that ultra-modern ride-sharing services resemble old-line taxi operations in declining service to poor communities. No way, say Uber and Lyft.
So, you're a Doomsday prepper and you scan the skies for black helicopters like Henry Hill in Goodfellas. You need a car that's going to help you blend in with the crowd.
BestRide.com offers readers fun, informative and up-to-the minute automotive content alongside a nationwide inventory and a powerful search engine, plus integrated user tools like price alerts, inventory alerts and saved search.
Kyle Busch took the checkered flag at the 5-Hour Energy 301 at New Hampshire International Speedway on Saturday, and his trophy was a giant New Hampshire lobster. Apparently, his wife Samantha is more into tofu.
Mad Max: Fury Road is one of the summer's big hits. It's hard not to like a movie that takes place in a post-apocalyptic wasteland with crazily modified cars that have accessories like flamethrowers and machine guns. It couldn't possibly be better, unless you give Furiosa a lightsaber.
As though Tesla's Insane Mode wasn't ludicrous enough, the automaker announced recently that they will add "Ludicrous Mode" to their flagship sedan. The $10,000 add-on option will permit the all-wheel drive 762hp Model S P90D to achieve 0 to 60 mph in a mind numbing 2.8-seconds.
Ford says it's testing some new lighting technology that promises to make driving safer. Too bad American drivers probably won't see it for a long time — if ever.
It's a weird thing to review a car like the Ford Mustang. People love this car. They don't really need a reason because it's a 'Stang and who doesn't want one of those parked in the driveway? It's as if loving the Mustang is some rite of passage. You grow up. You get a license. You lust after a Mustang. Turns out, there are really good reasons why you should want this car in your driveway.
A fast-moving wildfire in San Bernardino County California about 45 miles from Los Angeles crossed the highway today igniting cars sitting in traffic on I-15. About 20 cars were destroyed. Fire crews were themselves stuck in traffic and could not get to the scene of the fires. Motorist fled the vehicles, and some took their keys, making it difficult for workers to remove the vehicles to clear the roadway. Fire retardant and water were dropped from helicopters to put out the blaze, but it didn't help very much. According to reports, the fires started off to the side of the I-15 freeway at about 2:30. About an hour later the fire had spread to 50 acres. Two hours later it covered 3,500 acres.
As the fire spread to the hills in the area, 40 mph winds fanned the flames. Five home had been destroyed at the time of this report and more threatened. Facts in this report courtesy of KTLA 5.
Senate panel killed a proposal to permit companies to continue renting vehicles that have been recalled. Thanks, guys. There go all of our Hertz homonym jokes.


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Biogenesis Scandal: MMA Can Survive Unless Sport's Top Fighters Implicated
Porter Fischer, the whistle-blower who exposed several major league baseball players for allegedly using performance enhancement drugs, now tells ESPN's Outside the Lines that athletes from other sports also cheated, including mixed martial arts.
Unless it's involving the sport's two best fighters, UFC welterweight champion Georges St-Pierre or UFC light heavyweight Jon Jones, the UFC can handle and survive a drug cheating scandal.
Fischer, a former employee of Miami clinic Biogenesis, turned over documents to the media last year that led to the suspension of Ryan Braun. Now he says the cheating includes athletes in MMA, NBA, boxing, tennis and other sports.
Cheating in sports is a betrayal of the public trust. We see our athletes as role models, heroes and people who are bigger and better than we are. But this feeling goes hand-in-hand with the nature of the sport and the history and tradition behind it. With baseball rooted in our culture, from Babe Ruth and Bobby Thomson to Mark McGwire and Barry Bonds, America, all of it, cares about what happens with America's pastime.
MMA is different. It's fighting. Even though it is a Pay-Per-View juggernaut, it's not rooted deep in our culture. It's not even as popular as it was three years ago. The athletes in mixed martial arts are among the best athletes in the world and certainly the best fighters on the planet. But their road to mainstream is still full of potholes. MMA isn't even allowed in the most important city and state in the world, New York.
Most people on an American street can't name an MMA fighter, and it's just harder to relate to the MMA experience for most observers and young children. When Lance Armstrong cheats, every kid on a bicycle feels betrayed. When Ryan Braun is accused of cheating, every kid who swings a bat feels the pain. It's different with MMA. The idea that an MMA fighter might be using performance enhancing drugs is a bummer, but not a betrayal.
The sport is not yet rooted deeply enough into our culture to understand how significant an MMA fighter cheating would be. Although it's by far not the same thing, there's already a significant number of fighters who take part in testosterone replacement therapy who fight and no one seems to care about that.
MMA is a sport on the rise and it will be hit by scandals of all types as it battles for an equal spot at the table with MLB, NBA, NFL and professional boxing. If MMA fighters are involved in the Biogenesis scandal, the sport will survive and the controversy will just be part of its growth and maturity.
Joshua Molina is a mixed martial arts and pro wrestling writer for rantsports.com. Follow him on Twitter @JECMolina and add him to your network on Google.
"Bigfoot" Can't Afford Another Stumble At UFC 190
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Michael Stipe Wants Fox News to Stop Using His Music
BY Jeremy Kinser
September 07 2012 11:33 AM ET
Musician Michael Stipe says his band, R.E.M., has demanded a cease and desist from Fox News after the group's 1991 hit "Losing My Religion"was used during the network's coverage of the Democratic National Convention last night as an easy comment on controversy surrounding the use of "God" at the DNC.
A message posted on the band's website reads:
R.E.M.'s "Losing My Religion" was used in the Fox News coverage of the Democratic National Convention last night. R.E.M. today, through its music publisher, Warner-Tamerlane Music, demanded that Fox News cease and desist from continuing its unlicensed and unauthorized use of the song. Michael Stipe said, "We have little or no respect for their puff adder brand of reportage. Our music does not belong there."
Earlier this week GOP vice presidential nominee Paul Ryan appeared on Fox & Friends to blast the change from the Democrats' 2008 platform. "I guess I would just put the onus and the burden on them to explain why they did all this, these purges of God," Ryan said.
However, on Wednesday President Obama ordered party leaders to restore a reference to God as well as a statement declaring Jerusalem as the capital of Israel to the party's 2012 platform document.
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text:
Files without tags
14355 files
Adorable Adoring Fan for Oblivion
updated 22:34, 30 Jul 2015 2 0 1kb SatansFetusLegs
BC-OCR-CTN-patch for Oblivion
Patch for Cyrodiil Transportation Networc- OC and Better Cities
updated 0:35, 31 Jul 2015 3 0 1kb fienyx
Alternative Death for Oblivion
You go into the chapel at weynon priory instead of dieng, Adds a "reserve" to make sure your charecter doesnt die naturally. can leave through the door after activating the alter or use the scroll of return that you have to buy after you activate the alter. youll also have to check your health through the menu
since the hud wont be a
updated 4:23, 30 Jul 2015 23 1 1,033kb thelawfull
Coral Island for Oblivion
An island with a coral reef
updated 3:25, 27 Jul 2015 65 4 5,098kb glowplug
Monkeys In Oblivion for Oblivion
Adds monkeys to random parts of oblivion
updated 7:42, 29 Jul 2015 36 1 1,060kb thelawfull
Order Of Silence WIP for Oblivion
The Order Of Silence, An old brotherhood comes back. Work in progress.
updated 7:43, 29 Jul 2015 2 0 213kb thelawfull
Thelawfulls Deer Overhaul for Oblivion
Makes deer slightly bigger, Have more ingrediants with high prices. And gives the charecter hunters sight toggle ring
updated 16:13, 28 Jul 2015 5 1 2kb thelawfull
Morrowind Racial Abilities for Oblivion
This mod overwrites (some of) Oblivions racial abilities and powers with the abilities and powers from Morrowind. Primarily for Altmers, Bosmers, Imperials and Nords.
updated 19:58, 25 Jul 2015 2 0 3kb InMeITrust
Thelawfulls Hero Party members for Oblivion
Adds a respawnable Apprentice, Rogue, Archer and paladin that follow you around to help you out with battles. Also adds some other things.
Open the console and click the ones you dont want and type disable and they wont show up, For charecters like thiefs who dont want to get caught very easy. Teammates die quickly so keep them safe!, The
updated 17:09, 29 Jul 2015 12 0 5kb thelawfull
Vilverin Start Stuff for Oblivion
Adds some op stuff for experienced/bored players
updated 3:01, 21 Jul 2015 19 0 2kb beka72988
No Loading Screens for Oblivion
Removes loading screens to be more immersive doesnt effect loading screens from mods.
Credit goes to glowplug for making the black .dds file. :)
updated 18:09, 25 Jul 2015 27 1 6kb thelawfull
Tepals Dress for Oblivion
updated 2:08, 29 Jul 2015 392 31 4,698kb Nailflan


text:
Jan von Holleben: Lily & Jonathan
There can be few more stressful things for a child – or their family – than a stay in hospital but photographer Jan von Holleben is doing his bit to help with his biggest ever project, having just installed massive new pieces at Oxford's John Radcliffe Children's Hospital. The magical tales of children's adventures – in space and underwater – have already been a big hit, both with the youngsters and those trying to help take their minds off their ordeals.
The panels follow the fun, fantastic tale of Jonathan and Lily and their journey from the depths of the oceans to the outer reaches of the universe, meeting and greeting some new friends along the way.
First conceived in 2011, Jan spent several weeks as an artist in residence at the hospital where he spoke to doctors, therapists, play specialists, art coordinators, nurses, anesthetists, porters, plus of course patients and their parents, to gather initial ideas, and he shot the pictures at his Berlin studio with the help of a gaggle of kids.
"There were nine screaming, happy, candy-eating kids on the floor, tons of toys, my assistant and my camera," Jan told It's Nice That.
Lily and Jonathan comprises 315 individual photographs and more than 40 composite panels but Jan's delight with the final pieces suggests the hard graft was more than worth it.
"This was by far the largest and most rewarding project to date for me. Working with an enormously varied group of people from doctors to patients and porters who push the beds down the corridors made this a complete commission.
"Being able to incorporate so many different ideas and fusing them with my personal storytelling is an incredible experience! I am utterly proud having created this work for such a tragic and emotionally-loaded environment. I hope that anyone – and particularly the kids – walking down this corridor will be able to dream a little bit.
"The project's story was designed so that it mirrors the child's own journey into the surgery room, therefore helping to construct individual stories for each child.
"In this way the children can drift away into the fantastic stories at just the right time. It is my biggest hope that the kids can relate to the story and be therefore distracted from their fears of the surgery."
And Erica Watson, senior play specialist at the hospital agreed the new works served as "a great distraction."
"We talk about coat hangers being used for birds and socks for fish and trying to see if there are pairs of socks. We also follow the children's journey either diving into the sea or going up into space and when we reach the end of the pictures I tell the children that they may find one in the anaesthetic room up on the ceiling and we need to look for it when we get there. The response has been very positive."
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text:
Town Square
Simitian pushes bill to keep parks open
Original post made on May 9, 2012
Read the full story here Web Link posted Wednesday, May 9, 2012, 10:41 AM
Comments (1)
a resident of another community
on May 9, 2012 at 12:23 pm
Local parks must be encouraged, not closed. This all-important healthful resource benefits everyone who cannot afford other entertainment. NEVER close a park.
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text:
Remaining British troops 'to leave Iraq in spring'
Wednesday 24 November 2010
Most of the 150 British troops still serving in Iraq are set to be brought home next spring, the military said today.
Iraq's council of ministers agreed on November 10 that a small Royal Navy team training the Iraqi navy and marines should be allowed to stay for another six months, until May 2011.
But the 100 or so UK sailors, based in the port of Umm Qasr in southern Iraq, are expected to be withdrawn after this.
Senior British military spokesman Major General John Lorimer said: "With the additional six months we probably can anticipate that the Royal Navy mission will complete then, having trained the Iraqi navy."
A small number of British servicemen and women will remain in Iraq after May as part of a separate Nato training mission to instruct Iraqi army officers.
Maj Gen Lorimer said it was his understanding that the decision to withdraw the Royal Navy team was not related to the defence cuts introduced after the UK's recent strategic defence and security review.
Umm Qasr, in the far south of Basra Province, is Iraq's only deep water port and receives 80% of the nation's grain supply.
It is also home to the Iraqi navy, which has a key role in protecting the country's two oil terminals out in the Persian Gulf.
The vast majority of British forces were pulled out of Iraq in May 2009.
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text:
The present invention relates to DNA encoding proteins contributing to the regulation of a plant's response to DNA damage. DNA according to the invention comprises an open reading frame encoding a protein characterized by a stretch of amino acids or component amino acid sequence having 40% or more identity with an aligned component sequence of SEQ ID NO: 3. Preferably the DNA encodes a MAP kinase phosphatase.
1. An isolated DNA comprising an open reading frame encoding a phosphatase protein contributing to repair of DNA damage in a plant cell, wherein said phosphatase protein is characterized by a component amino acid sequence having about 70% or more identity with an aligned component sequence of SEQ ID NO:3.
2. The DNA according to
3. The DNA according to
4. The DNA according to
5. The DNA according to
6. The DNA according to
7. The DNA according to
8. The DNA according to
9. An isolated DNA encoding a phosphatase protein, wherein said phosphatase protein comprises a component amino acid sequence having at least about 70% identity with an aligned component sequence of SEQ ID NO:3, and wherein said component amino acid sequence comprises at least one sequence motif chosen from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.
10. The DNA of
11. The DNA of
12. The DNA of
13. The DNA of
14. The DNA of
This is a continuation of International Application No. PCT/EP99/05413, filed on Jul. 28, 1999, and published in English under PCT Article 21(2), the contents of which are incorporated herein by reference.
The present invention relates to DNA encoding proteins contributing to the regulation of a plant's response to abiotic stress and in particular genotoxic stress.
Cells of all organisms have evolved a series of DNA repair pathways which counteract the deleterious effects of DNA damage and are triggered by intricate signal cascades. To be able to modify or improve DNA repair using gene technology it is necessary to identify key proteins involved in said pathways or cascades. Therefore it is the main object of the present invention to provide DNA comprising an open reading frame encoding such a key protein.
DNA according to the present invention comprises an open reading frame encoding a protein characterized by a stretch of amino acids or component amino acid sequence having 40% or more identity with an aligned component sequence of SEQ ID NO: 3. The protein characterized by SEQ ID NO: 3 is tracked down with the help of a T-DNA tagged Arabidopsis mutant showing hypersensitivity to methyl methanesulfonate (MMS). Said hypersensitivity as well as an observed hypersensitivity to other DNA damaging treatments such as UV light is indicative of the proteins' involvement in the repair of DNA damage, or in signaling pathways implicated in the response to similar genotoxic stress. The mutant is also sensitive to elevated temperature and anti-oxidant N-acetylcysteine. The mutant is not sensitive to osmotic shock, increased salinity, oxidative stress or elevated ehtylene levels. An important characteristic of the mutant is cell death in response to growth in small closed vessels. This phenotype can be complemented by addition of abscisic acid (ABA) to the growth media. Furthermore, the mutant is more sensitive to exogenously applied ABA compared with the wild type which supports the notion that the genes disclosed by the present invention (SEQ ID NO: 1) are involved in stress signaling mediated by ABA.
Sequence alignments of SEQ ID NO: 3 using commercially available computer programs such as BLASTP of the NCBI BLAST family of programs or TFASTA or BestFit of the Wisconsin Package Software, all based on well known algorithms for sequence identity or similarity searches, reveal that stretches of SEQ ID NO: 3 (component sequences) having more than 100 and preferably between 120 to 250 amino acids length can show between 20% and almost 40% sequence identity to aligned stretches of known phosphatases, particularly phosphotyrosine phosphatases, MAP kinase phosphatases or dual specificity phosphatases. Protein phosphatases are classified by their substrate specificities as either phosphoserine/threonine phosphatases (PSTPs) or phosphotyrosine phosphatases (PTPs). The dual specificity phosphatases (DSPs) dephosphorylate both phosphotyrosine and phosphoserine/threonine residues and represent a subfamily of PTPs. MAP kinase phosphatases (MKPs) belong to the family of DSPs. The sequence VHCCQGVSRS (SEQ ID NO: 4) found in SEQ ID NO: 3 can be interpreted as corresponding to the mammalian sequence motif IHCXAGXXRS (SEQ ID NO: 5) defining the family of PTPs, wherein the lie at the first position can be replaced by Val and the Ser at the last position can be replaced by Thr
The present invention defines a new protein family the members of which are characterized by component amino acid sequences of more than 100 amino acid length showing 40% or higher amino acid sequence identity to aligned component sequences of SEQ ID NO: 3. Preferably said component sequences are of more than 120, more than 160 or even more than 200 amino acids length. The amino acid sequence identity is preferably higher than 50% or even higher than 55%. Most preferred are identities higher than 70%.
An example of DNA according to the present invention is described in SEQ ID NO: 1. The amino acid sequence of the protein encoded is identical to SEQ ID NO: 3. After alignment a stretch of the protein having about 140 amino acids shows 36% sequence identity to the MKP-1 protein described by Sun et al (Cell 75: 487-493, 1993). The identity determined after alignment with MKP-2 and MKP-3 is determined as 34% and 26%, respectively. Thus, according to the present invention a protein family related to MAP kinase phosphatases can be defined the members of which after alignment of a stretch of more than 100 amino acids length show 40% or higher amino acid sequence identity to SEQ ID NO: 3. Preferably, the amino acid sequence identity is higher than 50% or even higher than 55%. When making multiple sequence alignments, certain algorithms can take into account sequence similarities, such as same net charge or comparable hydrophobicity/hydrophilicity of the individual amino acids, in addition to sequence identities. The resulting values of sequence similarities, as compared to sequence identities, can help to assign a protein to the correct protein family in border-line cases. Proteins of particular interest, within the scope of the present invention, are MAP kinase phosphatases the amino acid sequence of which comprises at least one of the following characteristic amino acid subsequences:
DNA encoding proteins belonging to the new protein family according to the present invention can be isolated from monocotyledonous and dicotyledonous plants. Preferred sources are corn, sugar beet, sunflower, winter oilseed rape, soybean, cotton, wheat, rice, potato, broccoli, cauliflower, cabbage, cucumber, sweet corn, daikon, garden beans, lettuce, melon, pepper, squash, tomato, or watermelon. However, they can also be isolated from mammalian sources such as mouse or human tissues. The following general method, can be used, which the person skilled in the art will normally adapt to his specific task. A single stranded fragment of SEQ ID NO: 1 or SEQ ID NO: 2 consisting of at least 15, preferably 20 to 30 or even more than 100 consecutive nucleotides is used as a probe to screen a DNA library for clones hybridizing to said fragment. The factors to be observed for hybridization are described in Sambrook et al, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, chapters 9.47-9.57 and 11.45-11.49, 1989. Hybridizing clones are sequenced and DNA of clones comprising a complete coding region encoding a protein with more than 40% sequence identity to SEQ ID NO: 3 is purified. Said DNA can then be further processed by a number of routine recombinant DNA techniques such as restriction enzyme digestion, ligation, or polymerase chain reaction analysis.
The disclosure of SEQ ID NO: 1 enables a person skilled in the art to design oligonucleotides for polymerase chain reactions which attempt to amplify DNA fragments from templates comprising a sequence of nucleotides characterized by any continuous sequence of 15 and preferably 20 to 30 or more basepairs in SEQ ID NO: 1. Said nucleotides comprise a sequence of nucleotides which represents 15 and preferably 20 to 30 or more basepairs of SEQ ID NO: 1. Polymerase chain reactions performed using at least one such oligonucleotide and their amplification products constitute another embodiment of the present invention.
Knowing the nucleotide sequence of the Arabidopsis MKP1 gene and the amino acid sequence of the encoded protein it is possible to identify proteins interacting with AtMKP1 and to clone their corresponding genes using well known techniques. For example radioactively labeled AtMKP1 protein can be used for interactive cloning on cDNA expression libraries. AtMKP1 protein or parts thereof can be used to generate polyclonal or monoclonal antibodies specific for AtMKP1. The AtMKP1 gene can be used to generate variants of AtMKP1 protein tagged with GST, MYK or His. Said antibodies and MKP1 variants allow to isolate native protein complexes by immunoprecipitation and to determine sequences of proteins present in these complexes by micro-sequencing. The resulting sequence information can in turn be used to clone corresponding genes. Alternatively, said antibodies or tagged MKP1 variants can be used to screen epitope libraries for epitopes which interact with AtMKP1 protein. The AtMKP1 protein and parts thereof, in particular the N-terminal 490 amino acid region and the C-terminal 492 amino acid region can also be used to search for interacting proteins with a Two-hybrid system (e.g. in yeast, in mammalian cells, or in bacteria). This allows to obtain sequence information about interacting proteins.
Based on the disclosed finding that AtMKP1 proteins are involved in a plant's abiotic environmental stress response, it becomes possible to engineer the corresponding signaling pathway, of which AtMKP1 is a part, to be chemically regulated due to chemical activation or repression of transgenes encoding AtMKP1 or proteins interacting therewith. Such plants can be obtained by transformation with the corresponding genes under control of chemically inducible promoters. Application of inducers is expected to modify the activity of the AtMKP1 signaling pathway and to result in altered adaptation to abiotic environmental stress. Alternatively, AtMKP1 protein or its interacting proteins can be used as targets for chemicals inhibiting or stimulating their activities which again is expected to modify abiotic stress responses.
Arabidopsis T-DNA insertion lines as produced by the INRA-Versailles and available from the Nottingham Arabidopsis Stock Center (NASC) are screened for sensitivity to methyl methanesulfonate (MMS) at a concentration of 100 ppm as described by Masson et al, Genetics 146: 401-407, 1997. Plants which die in the presence of 100 ppm MMS are found in the family AAN4. Thus, the corresponding T-DNA insertion mutation is assumed to give rise to this hypersensitive phenotype. This assumption is supported by genetic analysis showing co-segregation of the hypersensitive phenotype with the T-DNA insertion. Genomic DNA from the mutant plants is isolated as described by Dellaporta et al, Plant Mol Biol Reporter 1: 19-21, 1983. A fragment of genomic DNA flanking the right border of the inserted T-DNA is rescued essentially according to Bouchez et al, Plant Mol Biol Reporter 14: 115-123, 1996, with minor modifications. Genomic DNA is digested with Pstl, ethanol precipitated and resuspended in H2O. DNA of vector pResc38 (Bouchez et al supra) is digested with Pstl and dephosphorylated with shrimp alkaline phosphatase. The phosphatase is heat inactivated, the vector DNA is ethanol precipitated and resuspended in H2O. 2.5 μg of Pstl digested genomic DNA and 2.5 μg of Pstl digested and dephosphorylated vector are mixed and ligated overnight at room temperature in a total volume 100 μl in the presence of 10 units of T4 DNA ligase. The DNA of the ligation mixture is precipitated with ethanol, resuspended in 50 μl H2O, and digested with Xbal in a total volume of 100 μl. Xbal digested DNA is precipitated with ethanol and resuspended in H2O. A second overnight ligation reaction in the presence of 10 units T4 DNA ligase is performed in a total volume of 200 μl at room temperature to achieve circularization of DNA fragments. The DNA of the ligation mixture is again precipitated with ethanol, rinsed two times with 70% ethanol, dried and dissolved in 5 μl H2O. Two 2 μl aliquots are used for electroporation of electrocompetent E. coli XL1-Blue cells (Stratagene) according to the manufacturer's instructions. Transformants containing the inserted T-DNA and adjacent Arabidopsis genomic DNA sequences are selected on plates with 50 mg/l kanamycin. Single bacterial colonies are analyzed by isolation of plasmid DNA using QlAprep Spin Plasmid Kit (Qiagen) and restriction digestion with Pstl and Xbal. Plasmid pBN1 containing 3.7 kb of inserted T-DNA linked to 5 kb of Arabidopsis DNA is identified. Sequencing of the junction site is performed using a primer directed towards the flanking plant DNA and having the nucleotide sequence 5′-GGTTTCTACAGGACGTAACAT-3′ (SEQ ID NO: 14) complementary to T-DNA 41 nucleotides from the right border. Digestion of this clone with SstI allows isolation of a 960 bp fragment which when labelled with 32P can be conveniently used as a probe to screen wild type Arabidopsis genomic and cDNA libraries in order to identify the wild type gene affected in the mkp1 mutant line.
The 960 bp SstI fragment mentioned at the end of example 1 is labeled with 32P by random oligonucleotide-primed synthesis (Feinberg et al, Anal Biochem 132: 6-13, 1983) for use as a probe in the following hybridization experiments.
Southern blot analysis of Arabidopsis wild type and mkp1 DNA digested with EcoRV confirms that in the mkp1 genomic DNA the sequence hybridizing to the probe is linked to T-DNA.
Northern blot analysis of Arabidopsis wild type RNA reveals the presence of a hybridizing transcript in RNA extracted from seven-day-old wild type seedlings. No such hybridizing fragment is detected in the corresponding RNA of mkp1 seedlings.
A cDNA library (Elledge et al, 1991) and a genomic library (Stratagene) of wild type Arabidopsis thaliana ecotype Columbia is screened with the labelled SstI fragment mentioned above. Screening of the bacteriophage λ libraries is performed according to the protocols described in chapter 6 of Ausubel et al, 1994, "Current protocols in molecular biology", John Wiley & Sons, Inc. Hybridization is performed as described by Church and Gilbert, Proc Natl Acad Sci USA 81: 1991-1995, 1984. Bacteriophage clones hybridizing to SstI fragment are subjected to in vivo excision of plasmids according to Elledge et al, Proc Natl Acad Sci USA 88: 1731-1735, 1991, and Stratagene protocols. Inserts of the plasmids obtained are further analyzed by sequencing.
By partial sequencing and alignment of ten overlapping clones (pBN5.1 to pBN5.10) isolated from the genomic library a continuous genomic sequence of 6356 bp (see SEQ ID NO: 1) is decoded.
Ten cDNA clones representing the same gene, one of them a 3.0 kb full-length cDNA (SEQ ID NO: 2), are isolated from the cDNA library.
The 3 kb full-length cDNA clone of SEQ ID NO: 2 encodes an ORF with the start codon being defined by basepairs 298-300 and the stop codon by basepairs 2650-2652. The ORF encodes a protein consisting of 784 amino acids (SEQ ID NO: 3) and a predicted molecular mass of 86.0 kD. Alignment with the genomic sequence of SEQ ID NO: 1 reveals three introns. T-DNA is inserted within the coding sequence of the mkp1 mutant DNA before basepair position 502 according to the numbering of SEQ ID NO: 2. The sub-sequence VHCCQGVSRS (SEQ ID NO: 4) found in SEQ ID NO: 3 can be interpreted as corresponding to the mammalian sequence motif IHCXAGXXRS (SEQ ID NO: 5) defining the family of protein tyrosine phosphatases, wherein the lie at the first position can be replaced by Val and the Ser at the last position can be replaced by Thr (Van Vactor et al, Curr Opin Gen Dev 8:112-126, 1998). Therefore it is concluded that the wild type ORF encodes a protein tyrosine phosphatase with invariant aspartic acid, cysteine, and arginine residues (Fauman et al, Trends Biochem Sci 21: 413-417, 1996) in positions 204, 235 and 241 according to SEQ ID NO: 3.
A data base search using the TFASTA program (Wisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, Wis.) reveals that the encoded phosphatase has a significant similarity to dual specificity phosphatases. The closest homologue identified is Xenopus laevis MAP kinase phosphatase (MKP; Lewis et al, J Cell Sci 108: 2885-2896, 1995) showing 38.1% identity and 52.5% similarity in a 140 amino acid overlap region. The deduced AtMKP1 protein also has 36.0% identity and 52.5% similarity with a 140 amino acid overlap region encoded by the rat 3CH134/CL100 cDNA representing a rat MKP1. Essentially identical results are obtained when using the BLASTP 2.0.4 (Feb. 24, 1998) program of the NCBI BLAST family of programs which, allowing gapped alignment, compares an amino acid query sequence against a protein sequence database (Altschul et al, Nucleic Acids Res. 25: 3389-3402, 1997). No higher plant homologues are identified. The genomic position of the AtMKP1 gene is determined by hybridization to filters containing genomic YAC clones publicly available from the Arabidopsis Biological Resource Center (Ohio, USA). The AtMKP1 gene is found to map to chromosome 3 between markers ve022 and BGL1.
mkp1 mutant plants are transformed with DNA comprising the corresponding wild type genomic DNA including promoter and polyadenylation signal to find out whether the cloned wild type gene is able to complement the mutant mkp1 phenotype.
mkp1 mutant plants harbor T-DNA containing the NPTII and bar marker genes under the control of nos and CaMV35S promoters, respectively. Therefore, different marker genes are used for the transformation construct. The vector used is a derivative of p1′barbi which is highly efficient in Arabidopsis transformation (Mengiste et al, Plant J 12: 945-948, 1997). In p1′barbi the EcoRI fragment containing 1′promoter, bargene coding region, and CaMV 35S polyadenylation signal is inverted in respect to the T-DNA borders by EcoRI digestion and re-ligation. In the resulting plasmid the 1′promoter (Velten et al, EMBO J 3: 2723-2730, 1984) is directed towards the right border of the T-DNA. This plasmid is digested with BamHI and NheI, and the bar gene and CaMV 35S polyadenylation signal are replaced by a synthetic polylinker with the sites for the restriction enzymes BamHI, HpaI, ClaI, StuI and NheI. The resulting plasmid is digested with BamHI and HpaI and ligated to a BamHI-PvuII fragment of pROB1 (Bilang et al, Gene 100: 247-250, 1991) containing the hygromycin-B-resistance gene hph linked to the CaMV 35S polyadenylation signal. The T-DNA of the resulting binary vector p1′hygi contains the hygromycin resistance selectable marker gene under the control of the 1′promoter and unique cloning sites for the restriction enzymes ClaI, StuI and NheI located between the marker gene and the T-DNA right border. p1′hygi is used to insert the reconstructed AtMKP1 gene as follows. Plasmid pBN1 of example 1 is digested with PstI and MunI and dephosphorylated. The restriction fragment containing the 3′portion of the AtMKP1 gene and pBluescript-SK(+) is purified from the agarose gel and ligated to the PstI-MunI restriction fragment of the wild type genomic clone pBN5.2 (example 2) including the 5′ end of the coding sequence of the AtMKP1 gene and 2.4 kb of upstream sequences. The reconstructed AtMKP1 gene is excised by PstI and NotI and after filling the ends is inserted into the StuI site of p1′hygi. The construct is introduced by transformation into Agrobacterium tumefaciens strain C58ClRifR containing the non-oncogenic Ti plasmid pGV3101 (Van Larebeke et al, Nature 252: 169-170, 1974). T-DNA containing the reconstructed AtMKP1 gene is transferred to mutant plants by the method of in planta Agrobacterium mediated gene transfer (Bechtold et al, C R Acad Sci Paris, Life Sci 316: 1194-1199, 1993). Seeds of infiltrated plants are grown on hygromycin-containing medium to screen for transformants. The progeny of selfed hygromycin resistant plants is analyzed for the segregation of hygromycin resistance. The families in which a 3:1 segregation ratio is observed are used to isolate homozygous lines bearing the newly introduced T-DNA inserted at a single genetic locus. The obtained hygromycin resistant lines are analyzed by Northern blot analysis for the restoration of AtMKP1 expression. In these lines the restoration of transcription of the AtMKP1 gene can be observed as well as the restoration of the wild type level of MMS resistance and ABA mediated stress responses. Complementation is not observed in plants transformed with p1′hygi only.
Use of AtMKP1 cDNA as a probe for Southern hybridization with genomic DNA from other plant species such as Sinapis alba (mustard), Lycopersicum esculentum (tomato) and Zea mays (maize) is successful in the case of Sinapis alba which belongs to the same family as Arabidopsis (Brassicaceae).
Homologous sequences from the other species can be identified in a PCR approach using degenerate primers 1-3 below, wherein I is inosine, derived from the regions conserved between VH-PTP13 of Clamydomonas eugametos and AtMKP1 protein:
A PCR reaction is performed in a total volume of 50 μl containing 1�reaction buffer (Qiagen), 200 μM of each dNTP, 1.25 units of Taq polymerase (Qiagen), and 100 pmol of each primer.
Reaction 1 is performed with primers 1 and 2 using genomic DNA from Sinapis alba (200 ng), Lycopersicum esculentum (400 ng), or Zea mays (600 ng) as the original template DNA. Amplification is carried out after an initial denaturation step of 3 min at 94� C., followed by 30 cycles of 30 sec at 94� C., 30 sec at 40� C., and 3 min at 72� C. The resulting amplification mixture is diluted 103 fold.
Reaction 2 is performed using 2 μl of the above dilution to provide the necessary template DNA. This time primers 1 and 3 are used under the same conditions as specified for reaction 1. The resulting amplification products are cloned into the T/A vector pCR2.1 (Invitrogen) and further analyzed by nucleotide sequencing.
Using this PCR approach it is possible to amplify sequences homologous to the AtMKP1 gene from all the species mentioned above. Whereas the nucleotide sequence from Sinapis alba SaMKP1 ((SEQ ID NO: 15 encoding SEQ ID NO: 16) is 90.8% identical to the AtMKP1 sequence, the nucleotide sequence from Lycopersicum esculentum LeMKP1 (SEQ ID NO: 17 encoding SEQ ID NO: 18) is 72.3% and the Zea mays sequence ZmMKP1 (SEQ ID NO: 19 encoding SEQ ID NO: 20) 71.8% identical. The fragments hybridize to genomic DNA from corresponding species under the usual hybridization conditions for Southern blot analysis. The fragments can be used as probes to screen cDNA libraries for corresponding cDNA sequences.
The 243 bp ZmMKP1 fragment amplifying from maize DNA is used as a probe to screen a maize cDNA library (Clontech) made in the Lambda ZAP�II Vector (Clontech) from "Blizzard" hybrid etiolated shoots, which were treated with the herbicide safener Benoxacor. The titer of the library is determined as 3�109 pfu/ml.
Library screening is conducted as described in the Clontech Lambda Library Protocol Handbook, with some slight modifications. Briefly, a single colony of XL-1 Blue is picked and incubated overnight at 37� C. in LB medium, containing 10 mM MgSO4 and 0.2% maltose. 600 μl of stationary phase grown bacteria for each 150 mm plate is combined with 100 μl of phage library dilution in sterile 1�lambda dilution buffer (100 mM NaCl; 10 mM MgSo4; 35 mM Tris-HCl, pH7.5) to yield approximately 30,000 pfu per plate. This mixture is incubated at 37� C. for 15 minutes, subsequently 7 ml of melted LB soft top agarose (at 48� C.) is added to the cell suspension for each 150 mm plate, shortly mixed and then poured on two-day-old LBMgSO4 agar plates, which have been pre-warmed to 37� C. for four hours. The plates are then incubated at 37� C. until plaques reach appropriate sizes (after about 8 to 9 hours). After chilling the plates at 4� C. for one hour, phage particles are transferred to Hybond N nitrocellulose filters and the orientation of each filter to its plate is recorded with a waterproof pen. The filters are then treated by placing them on Whatman 3MM paper saturated with the appropriate solution. The treatments include denaturation solution (0.5M NaOH; 1.5M NaCl) for 2 minutes, followed by neutralization solution (0.5M Tris-HCl, pH 7.2; 1.5M NaCl; 1 mM EDTA) for 3 minutes and 2�SSC for 3 minutes. DNA is subsequently crosslinked to the filters by UV.
Filters are then pre-hybridized, hybridized with the radioactivelly labeled ZmMKP1 fragment and washed as described in Sambrook et al, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, chapters 9.47-9.57 and 11.45-11.49, 1989. An agar plug from the position of a positive plaque is then removed from the master plate and incubated overnight at 4� C. in 1 ml of 1�lambda dilution buffer, containing 20 μl of chloroform. Each titer is determined and the phages are re-plated to obtain approximately 200 to 500 plaques on a 150 mm plate for a secondary screen as described above. Single plaques of interest are collected from the agar plates and incubated over night at 4� C. in 500 μl of 1�lambda dilution buffer and 20 μl of chloroform.
The pBluescript phagemid is excised from the λZAP™ vector as described by the In Vivo Excision Protocol using the ExAssisVSOLR System in the Stratagene Uni-ZAP™ XR Library Instruction Manual (1993). A {fraction (1/100)} dilution is made of XL1-Blue MRF' and SOLR overnight cultures (at 30� C.) and incubated at 37� C. for 2-3 hours. XL1-Blue MRF' cells are then pelleted for 10 minutes at 1,500�g and re-suspended at an OD600=1.0 in 10 mM MgSO4. 200 μl of these XL1-Blue cells are then combined with 250 μl of phage stock and 1 μl of ExAssist helper phage in a 50 ml conical tube and incubated at 37� C. for 15 minutes. 3 ml of LB broth is added and incubated at 37� C. for 5 hours, after which the cells are pelleted for 15 minutes at 2,000�g and the supernatant transferred to a new tube. The tube is then heated at 70� C. for 15 minutes and centrifuged again for 15 minutes at 4,000�g. The supernatant, containing the excised phagemid pBluescript packaged as filamentous phage particles, is decanted into a new tube. 10 and 100 μl of this phage stock are then added to two tubes with 200 μl of SOLR cells that have been allowed to grow to OD600=0.5-1.0 before being removed from the incubator and further incubated at room temperature. The tubes are incubated at 37� C. for 15 minutes, followed by plating 10-50 μl from each tube on LBamp (50 μg/ml) and over night incubation at 37� C.
The positive clones are checked for insert size by EcoRl/Xhol double digestion and end-sequencing with T3 and T7 promoter primers (Promega).
Screening of 360,000 pfu of the library results in three identical clones of 2.2 kb containing the 3′ poly(A) tail, but lacking part of the 5′ end, including the translation initiation site. The gene corresponding to the identified partial cDNA clone is named ZmMKP2, as it is not identical with the ZmMKP1 fragment used as the probe (92.3% identity on the nucleotide level over the 196 bp fragment flanked by the primers 1 and 3). An additional 213 nucleotides are amplified and cloned by 5′ RACE (rapid amplification of cDNA ends) carried out following the instructions of the 5′/3′ RACE Kit (Boehringer Mannheim) resulting in a longer cDNA sequence of 2,452 bp but still not complete, judged by the predicted mRNA length from the RNA gel blot analysis and the absence of a possible translation initiation site. The sequence information gained from the ZmMKP2 cDNA including the additional 213 nucleotides obtained by 5′ RACE (SEQ ID NO: 21 encoding SEQ ID NO: 22) is used to design two additional backward oriented degenerate primers wherein I is inosine to 3′ regions conserved between the deduced peptide sequences of ZmMKP2 and AtMKP1:
These primers are used to PCR amplify a larger fragment of a MAP kinase phosphatase gene from tomato. The amplified and cloned 522 bp long fragment is not identical to LeMKP1. Therefore, its corresponding gene is named LeMKP2 (SEQ ID NO: 23 encoding SEQ NO: 24; 75% identity on the nucleotide level over the stretch of 196 bp of ZmMKP1 flanked by primers 1 and 3). The origins of all identified MAP kinase phosphatase homologous gene sequences are confirmed by Southern blot analysis.
The following Table shows an alignment of a continuous stretch of 312 amino acids of AtMKP1 with the related amino acid sequence of ZmMKP2.
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Rory McIlroy stars in the Dubai sun
Friday 23 November 2012
Rory McIlroy overcame what he thinks was a
touch of sunstroke to move into a share of the halfway lead at the DP World
Tour Championship in Dubai today.
The world number one shot a second round 67 and goes into the weekend 11 under par alongside world number two Luke Donald and Scot Marc Warren.
South Africans Louis Oosthuizen and Branden Grace are only a stroke behind.
"I got off the course yesterday and had a really sore head and a fever," said McIlroy. "I think it was a bit of sunstroke.
"I took a few things and felt better, but I'm not feeling great again. The sun's taking it out of me a bit."
He birdied two of the first three, but came up short of the green at the 245-yard fourth and bogeyed.
It was his only dropped stroke, however, and further birdies came at the seventh, 10th, 14th - he lipped out for eagle there - and 15th.
Overnight leader Donald had a 68, while playing partner Warren - 189th in the world - matched McIlroy's round.
Earlier Sergio Garcia shot one of golf's most remarkable course records - two eagles, nine birdies, two bogeys and a triple bogey seven.
It all added up to a second round 64, eight under par, and from 49th place after his initial 73 he was back in contention at seven under.
The score was all the more amazing with this being the 32-year-old Spaniard's first event since the Ryder Cup in September. He underwent laser surgery afterwards to correct his astigmatism.
"It was unbelievable, wasn't it?" he said after moving from one over par to seven under.
An 18-foot eagle putt from just off the back of the final green made Garcia the sixth player to hand in a 64 on the Greg Norman-designed Earth course, venue for the European Tour's season-ending showpiece for the past four years.
Garcia went for the surgery because he was finding contact lenses uncomfortable and said of his improved eyesight: "I'm still kind of getting used to it."
He had five birdies in an outward 31, started for home with two more, bogeyed the next two, then sank a 30-foot eagle putt on the long 14th.
The tee at the 371-yard 15th had been moved up and after driving the green he sank an eight-foot second putt for birdie, but then pushed an eight-iron into the water on the next.
With three putts a triple bogey went on his card, but he hit back with a six-iron to four feet and then had his closing eagle - after his drive had headed for the stream running down the fairway, but then hit the rocks and bounced out.
Moments earlier German Marcel Siem had hit his second shot into the water by the green and by taking a bogey six had to settle for a 65.
Welshman Jamie Donaldson also ran up a six and, having bogeyed the 16th as well, he dropped back from 10 under to eight under.
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You're a structural analyst trying to dodge crumbling buildings, corroded bridges and exploding gas lines.
ST. LOUIS--It is a rare breed that can work at great heights. And few of those stalwarts have the opportunity to share their dizzying view of daily life with us mortals who are outside that ...
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great interactive website.At cookie, child experts and educators design interactive online games for kids. Our learning games for kids help in building skills needed for success in school. While playing our fun educational games, kids learn to read with phonics, build skills in math, language, social studies, science and much more. Our educational videos encourage children to keep learning, even after school hours.
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Some awesome sites for kids Pinned by Penina Penina Rybak MA/CCC-SLP, TSHH CEO Socially Speaking LLC YouTube: socialslp Facebook: Socially Speaking LLC www.SociallySpeak... Socially Speaking™ App for iPad: itunes.apple.com/...
Calling all 1st Grade Teachers! Here's a great resource for you! Check out this page at Technology-Rich Learning Experiences for 1st Graders! You'll find tons of resources for technology integration that is sure to enhance the first grade curriculum! Check it out at eduscapes.com/....
I Heart Guts is the brainchild of an anatomically obsessed illustrator Wendy Bryan, who loves internal organs. there's everything from plush hearts to a soft and furry appendix.
Computer Choices. A list of paid and free sites for great resources which may help generate even better ideas for teaching in a classroom
Great pre-writing activities for preschool and kindergarten that focus on development not worksheets.
ABC, 123, 100 party, and Sight Words Worksheets for Activity Centers and More! You could use them with play doe, finger-painting, coloring, or just laminate them and use dry erase markers! Super fun for those little fingers they fit perfectly for tracing! $
Free learning sites to share with families: Great handout offered by "A Year of Many Firsts"!
SCIENCE Teaching Strategy: Allow students to see and create. Students can keep these study aids to refer back to. Students can recall the creation process, the activity process and the study process in multiple ways. (Grades 6 - 12) Found on Pinterest.
"30+ Sites for Students Who Finish Early" has been a pretty popular post. 10,000+ views! This tells me that teachers (including me) need enrichment sites for students who finish early or for their free computer time.http://www.kbkonnectedkids.com/#!home/mainPage


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Professional Services
Company Overview of KCS Limited
Company Overview
8th Floor
Gloucester Tower
The Landmark
15 Queen's Road Central
Central, 19808-6192
Hong Kong
Key Executives for KCS Limited
Chief Executive Officer
Head of Registration & Compliance and Director of Corporate Secretarial
Age: 56
Head of Shanghai Office
Head of Corporate Restructuring and Director of Corporate Secretarial
Head of Corporate Accounting Services and Director of Corporate Accounting
Compensation as of Fiscal Year 2015.
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LJWorld.com weblogs The Lawrence Crime Blotter
Woman injured in early morning car accident
A 20-year-old Lenexa woman taken to the hospital after being injured during an early morning accident outside a downtown bar.The woman was struck by a car in front of The Bottleneck, 737 N.H., at 1:52 a.m. Friday. When police arrived, the woman was lying in the center of the street. She reported that she was walking eastbound on New Hampshire, when she was hit by a northbound vehicle.Witnesses identified the driver, a 27-year-old Lawrence man, and said he had fled the scene. Police later contacted him, citing him failure to report an accident and leaving the scene of an accident. The 20-year-old woman was transported to Lawrence Memorial Hospital where she was being treated for non-life-threatening injuries.Further information about her condition was not immediately available.
geekin_topekan 8 years, 1 month ago
OO!OO!I wanna be the first! A Twenty year old walking near the bottleneck at 2am?
compmd 8 years, 1 month ago
When you walk in front of a car, you might get hit.
I do hope she comes out ok though.
erod0723 8 years, 1 month ago
i highly doubt she was intoxicated or otherwise indisposed at the time.
Sigmund 8 years, 1 month ago
I am more interested in the intoxication of the 27-year-old Lawrence man who stuck her and then fled the scene. Throw the book at him.
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Aziz Ansari is Coming to Dallas
Before he became the entrepreneurial Tom Haverford on Parks and Rec, he starred in the video sketch series Human Giant. And before both of those things, the major venue stand-up comedy tours, television specials and wildly enthusiastic reviews by music publications Rolling Stone and Pitchfork, Aziz Ansari was a gangly kid in New York with a super addictive website. He'd record heartfelt bits about falling in love with MIA. Tour New York City playing the world's shittiest mix tape on a boombox and generally make fun of indie hipster pretension.
There's always been something different about this one, and Dallas gets to see him on his own and all grown up when Aziz Ansari brings his third national tour of all new material to the Verizon Theater on May 31. Tickets went on sale Wednesday, but there are still seats available in nearly every section of Verizon, according to the site's online reservation system. Get 'em now and get 'em quick.
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Hometown: Los Angeles
January 21, 2014
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I consider myself a 'bad' gamer. I'm an ex-WoW raider and am too easily addicted to sim games. Here are some silly games I've been working on since I quit WoW: Mass Effect (I'm super late to the party!), Pokémon X, The Sims 3 (feeding the addiction), Papers Please, The Stanley Parable
Cards Against Humanity, Dune, 7 Wonders, Werewolf/Mafia
I'd love to meet other gamer girls! I do love game nights, but other meetups to just hang out, have fun, eat, drink, talk, etc. would be great, too!
I'm a huge nerd and proud of it! I'm a software engineer/web developer. I'm a casual gamer & ex-WoW raider. I frequently attend my friends' game nights.
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Ellen DeGeneres' celebrity pals promote her e-commerce site
Justin Bieber, Madonna and Kim Kardashian-West tweeted about the launch of EDbyEllen.com.
Ellen DeGeneres' celebrity pals promote her e-commerce site
Justin Bieber, Madonna and Kim Kardashian-West tweeted about the launch of EDbyEllen.com.
Shopify's revenue jumps 89% in its first public quarter
The maker of software for online retailers processed more than $1.6 billion in orders in the quarter.
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Airline Continues to Transform Travel Bookings with Visa Checkout Payment Technology, Celebrates With Bonus Launch Offer.
Posted 02/25/2015
Square has hired Kevin Burke to lead customer acquisition for the company.
Posted 12/10/2014
They shop 13 times more often on U.S. e-commerce sites than the average for 10 major markets, Visa says. U.S. consumers are the most frequent overseas online shoppers at e-commerce sites in China.
Posted 09/26/2014Frank TongPost a comment
The VISA agreement complements the Company's membership of MasterCard Europe.
Posted 08/26/2014
Consumers in the United States, Canada and Australia can use Visa Checkout.
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Stroller and car seat brand Uppababy says it's saving thousands of dollars each month after correcting the merchant fees it pays to credit card companies.
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Sales reps can process "chip-and-PIN" online payments on their smartphones when signing up new business or consumer customers in the field.
Posted 07/01/2014Paul DemeryPost a comment
The group will initially focus on the adoption of EMV chip technology in the United States, in addition to addressing other security-related topics, including tokenization, point-to-point encryption and broader needs of the region.
Posted 03/13/2014
Introduces new standards, tools and guidelines for Visa payWaveExtends Visa Ready Program to enable Visa payment functionality in the cloud
Posted 02/19/2014
Criminals also obtained the associated expiration dates, giving thieves the information they would need to make an online purchase on some e-commerce sites. E-retailers should pay careful attention to any early warning signs of fraud, says one security expert.
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Description Are you a rock star workforce management analyst who loves leading your team to deliver outstanding results to members? Are you working in a bank or financial services call center and craving to leave for a fast-paced startup that is poised to change the banking7/27/2015 Healdsburg, CA Save This Job
Overview --- Please submit resume with position inquiry --- In this new position, the Global Compensation Analyst shall be involved in the development of a new global compensation structure for a $500M global technology company. This key role will be involved in collaborating7/16/2015 Bridgewater, NJ Save This Job
New York, NY Full Time Finance Department Mid Level SHARE BetterCloud is looking for a sharp, data-loving critical thinker to join our Finance team. This person would analyze and present financial and operating performance, and identify and implement improvements in7/13/2015 New York, NY Save This Job
San Francisco, CA Department Sales Job Overview Pentaho is looking for a highly motivated and experienced Senior Sales Operations Analyst to join our Sales Operations team. The position will be based in our San Francisco office reporting to the VP of Sales Operations. The6/29/2015 San Francisco, CA Save This Job
San Francisco, United States As Airbnb continues to expand our business globally, we continue to look for the best and brightest to drive strategic business decisions. Financial Planning & Analysis team is looking for a highly qualified Senior Analyst to join the team to support5/12/2015 San Francisco, CA Save This Job
The Information Security Analyst must have 4 years of experience monitoring and reviewing security operations tools and systems used in Security Operations Centers. The Information Security Analyst will follow all processes and procedures in collaboration with the IT and Security7/13/2015 Salt Lake City, UT Save This Job
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Department Product Marketing Description Pentaho is actively looking for energetic and creative out-of-the-box thinking, solutions-oriented product marketing managers to help revolutionize a ten billion dollar big data and business analytics market place. The ideal candidate has6/29/2015 San Francisco, CA Save This Job
Basic Function: The Product Analyst is responsible for performing data analysis on telematics for client specific projects. The role will report directly to the Chief Operating Officer. Principle Accountabilities: Perform both data and behavioral analysis on telematics data6/15/2015 Glen Allen, VA Save This Job
Job Description Job Summary The Business Intelligence Analyst ensures the accuracy and consistency of reporting savings to HealthHelp?s clients. This employee also must be able to document the formulas used in the analysis and articulately communicate the savings to HealthHelp6/13/2015 Houston, TX Save This Job
Job ID 2015-2882 Location Pleasanton CA Posted Date 4/18/2015 Overview: MegaPath is a leading provider of voice, data, networking, security and cloud services for small, medium and enterprise businesses nationwide. Our Hosted Voice solution has won multiple 2014 Product of6/8/2015 Pleasanton, CA Save This Job
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at Houzz Palo Alto, CA Houzz is seeking a Principal Quantitative Analyst to assist our growth initiatives with data driven insights. You will work with engineering, marketing, and editorial to analyze what drives user behaviour and evaluate tests we are conducting. This work will5/30/2015 Palo Alto, CA Save This Job
Reporting to the Manager of Network and Security Operations, the Security Analyst will work with Information Systems Management, to implement Tripwire's information security systems and processes, ensure enforcement of security policies, manage the information security threat7/9/2015 Portland, OR Save This Job
Imagine yourself driving growth with the leader of IT?s hottest Technology: Hyper-Converged Infrastructure. Pivot3 is a fast growing innovator of Hyper-Converged Infrastructure (HCI), an exciting technology that is driving a profound transformation of IT. Analyst firm7/3/2015 Spring, TX Save This Job
Imagine yourself driving growth with the leader of IT?s hottest Technology: Hyper-Converged Infrastructure. Pivot3 is a fast growing innovator of Hyper-Converged Infrastructure (HCI), an exciting technology that is driving a profound transformation of IT. Analyst firm7/3/2015 Spring, TX Save This Job
Senior Consultant - North America Company Pentaho is the business analytics company providing power for technologist and rapid insight for users. Pentaho is growing rapidly, in the spotlight of leading analyst firms like Gartner, and racking up public wins over traditional6/29/2015 Orlando, FL Save This Job
COMPANY PROFILE: InstaMed is a rapidly growing healthcare technology company that has established itself as a leader in the emerging market of healthcare payments. InstaMed?s solutions are delivered via its proprietary cloud-based platform and network of healthcare providers and6/28/2015 Newport Beach, CA Save This Job