text: Scientific Writing Jobs 4 were found based on your criteria Post a job. It's free! Academic Writer / Research Assistant - Psychology and Consumer Behaviour Hourly – More than 6 months – 10-30 hrs/week – Posted March 8 2015 I am a researcher at the University of Cambridge and need someone who can help me complete a series of academic articles. I have a list of articles which contain the main content, references and data. I need you to: - Re-write and re-format the articles, using your knowledge to improve them. - Searching for additional materials/references where needed. - Format the paper and tables into an appropriate format - Reference the article in APA format - Work with very limited direction. You need ... academic-writing article-writing content-writing data-entry research-papers scientific-writing technical-writing ICP chemistry publications Hourly – Less than 1 month – 10-30 hrs/week – Posted Feb. 24 2015 I am looking for help in publishing 2 papers from my dissertation research. I work full time and am having difficulty finding the time to finish writing them. Both papers are on ICP-MS of biological samples. I have the majority of each paper done and need you to put them into ACS format and find suitable journals. Bonus to be paid upon acceptance. Please attach a journal you have published in the past when applying. proofreading research-papers scientific-writing Write a small summary of a Psychology study Hourly – More than 6 months – 10-30 hrs/week – Posted Feb. 9 2015 Hello, I am looking for someone who can read a study and write a summary on the findings. This is an immediate position and depending on how well the work is done, can be a long term position up to 2 more years. This position offers bonuses for better performance. Preferring someone who can begin working with my team immediately and can work on their own without much supervision. The most important thing is meeting with deadlines. If you feel ... academic-writing article-writing business-writing content-writing english internet-research research-papers scientific-writing technical-writing virtual-assistant ESports / Competitive gaming writer - New online magazine Hourly – More than 6 months – Less than 10 hrs/week – Posted Jan. 16 2015 Hi, We're looking for 2x writers to come on board and write for Competition Gamer Magazine as we're expanding faster than we expected we would. We're moving from a monthly to a weekly format because of prelaunch demand and so there will be multiple writing jobs per week available - however turnaround times will be relatively tight. The roles responsibilities include - - Writing 700-1400 word articles about competitive gaming - Researching ways to improve gaming performance - reaction times, strategy - Keeping ... academic-writing content-writing scientific-writing technical-writing writing


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Eve's Sorrow
Credits & Info
- Date
- 09/06/2012
- File Info
- Song
- 7 min 48 sec
- Score
- 4.36 / 5.00
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Licensing Terms
- Attribution:
- You must give credit to the artist.
- Noncommercial:
- You may not use this work for commercial purposes unless you make specific arrangements with the artist.
- Share Alike:
- If you alter, transform, or build upon this image, you may distribute the resulting creation only under a license identical to this one.
- Score:
- Rated 4.36 / 5 stars
- Plays & Downloads:
- 7,443 Plays | 465 Downloads
- Share Links:
- Genres:
- Easy Listening - Classical
- classic
- cover
- eve
Author Comments
Rated 5 / 5 stars
Finally, someone pays tribute to Parasite Eve. Which of the three games is your favorite, Shanath? I like 1 and 2 best. I liked the storyline to some degree in 3 but I wasn't overly fond of the gameplay.
You did this song much justice.
1 and 2 were the best entries for sure. The third game wasn't bad but the story was pretty much a lot of nonsense and didn't seem to reach the level of storytelling the first two games had. If they make a fourth game I hope they go back to the game's roots.
Anyway, glad you liked.
Rated 5 / 5 stars
That's very good! Thumbs up! 5/5
Rated 3.5 / 5 stars
Me liketh.
Rated 4.5 / 5 stars
This song reminds me of a good Final Fantasy game. It brings out the old emotions in me when i first discovered Final Fantasy years ago. If this could be played on Youtube it would be famous. You my friend are a silent symphony readied to be played
Haha, I don't know about that! Thanks for the compliment, though. :3
Rated 5 / 5 stars
That was truly amazing. All I need to say.

subset: commoncrawl


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The sale of the Dodgers sent shock waves through the industry. The sale price is now supposedly 2.15 billion dollars. Magic Johnson's group is open for business and they obviously targeted this coming offseason as a key to their resurgence. Players like Cole Hamels, Zack Greinke, Matt Cain, and Joey Votto were set to become free agents. Now, you can wipe two names off of that list. Furthermore, there are rumblings that the Brewers and Phillies are working on extensions for their star pitchers.
I've never made any bones about the fact that I have never been a big Brian Sabean fan. The Matt Cain contract is yet another example of why. The right-hander is due to be the highest paid right-handed pitcher in history. Yesterday, he agreed to a six year 127.5 million dollar extension. As best I can figure, that amounts to a little more than 21 million a season over the life of the deal. Add in Barry Zito's salary and Tim Lincecum's salary and you can see that three fifth's of the rotation is making well over 50 percent of the total team payroll.
- Matt Cain: 21.3 million
- Tim Lincecum: 18.0 million (22.o million in 2013)
- Barry Zito: 19.0 million (27.0 million in 2013 if you count 7 million buyout)
So, if we do the math for this season we'll see that the three pitchers total to earn 58.3 million. In 2013 that could jump to over 70 million if you count the seven million buyout for Zito's deal on the 2013 books. That's an insane amount of money to pay to three pitchers and while Zito will eventually go off the books, Madison Bumgarner will eventually be added to the list of high priced pitchers.
One thing I will give to Cain is that he has been remarkably consistent in his six full seasons. After his first full season (where he had 190 innings), he's managed to go 200 or more innings in each season. That's pretty remarkable and his ERA has never gone above 3.76 in the past five seasons. Unfortunately, consistence and dominance often get confused. His TRI scores have ranged between 17.7 and 19.0 during those five years. That puts him squarely between the likes of Trevor Cahill, Matt Garza, and Carl Pavano on the low end and Colby Lewis, Matt Harrison, and Ricky Romero on the high end. Those are good pitchers, but there isn't a 20 million dollar one in the bunch.
So, the long and short of it is that the Giants just gave superstar money to a pitcher that has always been good and sometimes very good, but never great. Maybe I'm not up with the cost of inflation, but if I'm going to pay a pitcher 100 million dollars or more they better be a legitimate staff ace.
Joey Votto is a different story entirely. That doesn't make his contract any better necessarily, but at least the Reds are making hay in the short-term. This year will be his age 28 season which means that his ten year 225 million dollar contract will stretch him through his age 38 season. What are the odds that he will be productive enough to justify that money at that point? Still, according to TRI, Votto is the best first baseman in the game right now and they effectively got him for the midpoint between Albert Pujols contract and Prince Fielder's contract. In essence this is what we call the cost of doing business.
If you want to keep your stud players you have to pay market value for their services. Now, the Reds are in a bind going into the next offseason. They have Brandon Phillips needing an extension and pitchers like Mat Latos may become arbitration eligible. For the Giants, they will have to find someone to scrape up enough nickels to build an offensive attack. The hangover usually follows binge spending and this case will be no different.
Filed under: MLB, MLB Transactions


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It was mostly just ok...I like Jennifer Beals so it was good enoughPublished 4 months ago by Paul Murany
Very good movie with Jennifer Beals one hot lady. Good story JB is great in most everything she does. YesPublished 19 months ago by Judy Eckroth
'Troubled Waters' turns into an increasingly interesting thriller with a highly unexpected ending. It involves a persistent FBI agent who somehow has clairvoyance, and three... Read morePublished 22 months ago by Loyd E. Eskildson
20 minutes into the viewing of this weak production I came to my trusted Amazon to check the reviews and remain astounded at the overall 4* rating... Read morePublished on February 18, 2013 by Desert Dog


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Shocking CO2 statistics 09/18/10
Take a look at an infographic that gives a depressing overview of the amount of CO2 emissions the human race creates.
We all know that we are slowly and surely destroying the planet… what is interesting is that the mere fact of chosing to eat a vegetarian diet can make a significant difference to global carbon emission numbers.
As this very interesting infographic shows, which I came across on www.infographicshowcase.com, if American citizens ate a vegetarian diet for only 7 days, it would be the equivalent of removing all the cars on the roads for that time. While I am not sure where exactly these statistics are derived from, it is still food for thought.
Related posts:
Get ready for the first Gaia Food Market - dedicated to plant-based cuisine...
A 1960 news report about a sturdy, cross-country hiking vegetarian.
How to cook cheap, fast and vegan. For simple, tasty and lovely recipes, ch...
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We have an app on Facebook called TouchActive
We provide ways for user to post content to Facebook, either from web (via AddThis) or native Android/iOS app.
When we post from say Android we identify the app and the post appears correctly, showing "... via TouchActive" all fine.
When we post from web, we post a URL that Facebook queries, and we provide an OpenGraph response from our server with all the og: meta tags, including the facebook app_id.
The content appears correctly (it's parsing the OG response OK), but it doesn't show as posted via our app, and the posts don't appear in our Insight statistics for the app.
We are assuming this can be done.
Anyone know what we could be doing wrong in our open graph response that causes this to not work?
I have tried the Facebook debug tool https://developers.facebook.com/tools/debug and it shows the correctly parsed OG response and no errors, only a warning about og:url.
Is og:url recommended or a MUST for this to work?


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Our blogs
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So I found Help installing cx_Oracle but am still stuck. I downloaded the latest instantclient from oracle, and set ORACLE_HOME to the location of the extracted files (both direct and with a bin folder between the ORACLE_HOME value and the files), but easy_install is popping an error when running setup.py saying it can't locate the Oracle include files. I did notice that only the 11g dll is in the folder, do I need all 3 drivers present for setup to complete? If so, where do I even get them?
Take the 2-minute tour ×
Stack Overflow is a question and answer site for professional and enthusiast programmers. It's 100% free, no registration required.
Honestly it is a hell of a lot easier to install cx_Oracle from one of the binary installers they have, than from source.
EDIT (HOWTO for *nix):
You might need to install libaio1.
HOWTO Install cx_Oracle
Assuming we have installed Oracle Instant Client 10,
To test,
For windows, run one of these.


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Why Tablets Haven't Taken Off In Business 449
from the try-this-electric-notepad-bill dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
>> why tablets have never taken off in business
No cupholders.
no, but In soviet america, Jobs has Hipsters!
>> why tablets have never taken off in business
No cupholders.
The reason tablets have never taken off is because you have to press that stylus really, really hard to cut into the stone.
Spoken by somebody who didn't take very good care of their body. There is no reason that you have to lose dexterity or have bad eyesight just because you get older.
and not enough thrust.
Wow. Just... Wow. If a prerequisite to being old and wise is to first be young and stupid, you are destined to become Methuselah.
Yes, they're called "hipsters", we get it.
Must be from all the American semen pumped into your great grandmother during WWI.
Time-sharing is the junk-mail part of the computer business. -- H.R.J. Grosch (attributed)


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Wed March 27, 2013
Did Team USA 'Win' Its 0-0 Match With Mexico?
Originally published on Wed March 27, 2013 12:16 pm
Soccer fans, lend us your expertise.
The 0-0 tie Tuesday night at Azteca Stadium in Mexico City is being hailed this morning as basically a victory for Team USA over its archrival, Mexico's national team.
The Americans have never actually won an important game in Mexico. But Tuesday's tie means they come away with a much-needed point in the standings among teams trying to qualify for the 2014 World Cup tournament. It's only the second time Team USA has earned a point from a game at Mexico City's Azteca Stadium.
As NPR's Carrie Kahn explained on Morning Edition, that stadium is a very tough place to play for Team USA. It's an understatement to say that the 100,000-plus fans are loud and raucous. And they aren't shy about expressing themselves. When the game ended, Carrie said, "a rain of beer cups and soda cans came crashing down" on the 500 or so U.S. fans who had been brave enough to attend. (They were protected by about 100 police in riot gear.)
Sports Illustrated's Grant Wahl told Carrie that Azteca Stadium "has been a house of horrors for the U.S. over the years." To tie Mexico there, he added, "is a huge moment for U.S. soccer."
Wahl writes today that the Americans got "the result they needed vs. Mexico." And he adds that:
"Last week, when the U.S. national team was beset by injuries and doubts over whether coach Jurgen Klinsmann had lost the locker room, the range of possible public opinions after the two World Cup qualifiers this week was fascinating. On one end was 'Fire Klinsmann Now' if the U.S. lost both games to Costa Rica and Mexico. And at the other end was 'Klinsmann is a genius' if the U.S. somehow found a way to win both games.
"Well, look what happened. The U.S. grabbed four points in two games, beating Costa Rica and earning a point in Estadio Azteca for the second time in history on Tuesday in a taut 0-0 tie against a frustrated (and now winless) Mexico team. While Klinsmann didn't quite end up on the extreme 'genius' end of the scale, he was closer than just about anyone would have expected 10 days ago."
Our question for soccer fans: How big a deal is this tie with Mexico?


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Alexandra "Sasha" McHale, the daughter of former Celtics great Kevin McHale, passed away on Saturday at the age of 22.
Remember ETwaun Moore? The player viewed by many as the other rookie in the Boston Celtics multi-team deal with the Houston Rockets to land Courtney Lee?
Doc Rivers poked his head into his son's bedroom on Saturday night after arriving home in Florida for a quick visit following the Boston Celtics win over the Washington Wizards.
From Comcast SportsNet
From Comcast SportsNetHOUSTON (AP) -- Jeremy Lin is finally getting his own bed.The 6-foot-3 point guard, who became an international phenomenon during one dazzling month with the Knicks last seaso


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A major task for clinical and routine toxicology screening is the reliable, fast and comprehensive identification of drugs and drugs of abuse (DOA). LC-MS/MS is an emerging technology that is more specific than immunoassays, provides more information than LC-UV/DAD detection, and covers a broader range of compounds than GC-MS. This application note describes a robust and automated screening solution based on the latest LC-MS/MS ion trap technology that promises to provide a combination of the highest performance LC-MS/MS and easy-to-use screening with the greatest transferability of results from lab to lab.
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New Delhi: As the stage is set for Bharatiya Janata Party`s prime ministerial candidate Narendra Modi`s first rally in the national capital, Janata Dal-United leader Sabir Ali on Sunday accused the opposition party workers for luring people to attend the rally with food, money and alcohol.
"BJP party workers are luring people to attend the rally with food, money and alcohol," Ali said.
"Modi has no factor for himself; but only money factor. He has lots of money and delivers money across the state. People like him will become PM only to run country with money," he added.
Modi, who was recently declared as the BJP`s prime ministerial candidate, will address a mega rally in Delhi today.
The BJP says that it expects at least five lakh people to attend the event.
According to television reports, an 80x40 ft stage has been erected at the sprawling Japanese Park in Rohini in north-west Delhi where Modi will be delivering a 45-minute-long speech and will later unveil his 100-feet-long portrait.
Meanwhile, security has been tightened in and around the venue and arrangements have been made for medical and fire services.
Around 3,000 policemen and paramilitary personnel will also provide security in and around the venue.
Special buses have been arranged to bring in students and supporters from far-flung areas of Delhi.
Diplomats from 40 different nations are likely to participate in the event. Sportspersons, ex-service men, former senior bureaucrats have also been invited.


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Rick Brand is a longtime Newsday reporter who writes about politics and government on Long Island.
Nassau Democratic Party chairman Jay Jacobs went on a delicate political mission last week in an attempt to defuse a potential primary fight he feels could hurt the party's slate in November.
Jacobs sat down with former county Comptroller Howard Weitzman on Thursday to try to dissuade him from his comeback bid against Republican George Maragos, who beat him in 2009.
Jacobs and other party leaders are planning to back Democratic county Legis. Wayne Wink, 46, of Roslyn, at the Democrats' May 28 convention. To get a rematch with Maragos, Weitzman, 67, would have to beat Wink, whom Republicans redistricted out of his seat, in a primary in September.
"I'd say it was a good meeting and we had an open and comfortable conversation," Jacobs said after the hourlong talk. "Both of us agreed that a primary in the comptroller's race is not ideal and we're working on a way to avoid that possibility."
Jacobs offered Weitzman a possible alternative -- a chance to run for the seat held by county Legis. Judy Bosworth (D-Great Neck). Democrats are eyeing Bosworth as a candidate for North Hempstead Town supervisor should veteran Democratic incumbent Jon Kaiman not run. Kaiman has yet to disclose his plans.
"We had some frank and open discussions," Weitzman said. But he said voters would be "best served" by his running for comptroller, and that he is "expecting to be in a primary." However, he acknowledged discussing the legislative race with Jacobs and noted, "the convention is still three weeks away."
Jacobs argued that a legislative nomination could put him back into elected office, yet provide him with a more manageable, less costly campaign. If the Democrats regain a legislative majority, Weitzman could head the legislature's finance committee, Jacobs said. Republicans control the legislature 10-9.
Jacobs wants to avoid a costly primary that would eat up scarce resources and create a second front, given that former County Executive Thomas Suozzi already is facing a Democratic primary from businessman Adam Haber.
A comptroller's primary could leave less for Democrats to spend in key legislative races in November. It also could leave the winner weakened as he faces Maragos, 64, who founded a financial services firm and has largely self-funded his comptrollers' campaigns and two unsuccessful runs for U.S. Senate.
Weitzman, a certified public accountant, lost four years ago by 644 votes; he noted that he had barely campaigned after being stricken with a rare blood disorder, from which he says he's recovered.
Republicans could attack a Suozzi-Weitzman slate as a political rerun of 2009.
But backers say Weitzman during his eight years in office implemented a prescription program that saved residents millions of dollars and conducted audits that highlighted abuse and fraud.
Weitzman said he has raised $400,000 for` the 2013 race. State Comptroller Thomas DiNapoli has endorsed him, and his campaign chairman is former Oyster Bay supervisor and Assemb. Lew Yevoli.
"After being out of the public eye, a primary puts me in a position to remind people what I was able to accomplish," Weitzman said. "And after the primary I believe I'll be able to raise the resources necessary to be competitive."
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House races bringing in big bucks
ST. PAUL — Everyone knew that U.S. Sen. Al Franken and Mike McFadden will run rich campaigns if they face off in November, as expected, but a couple of mostly rural U.S. House races involve more money than usual.
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Eric Chan | Encinitas, CA | Male (59)
Skill Level: 4.0 Competitor
Best time to play: Anytime Weekdays morning
Diane Smith | Encinitas, CA | Female (46)
Skill Level: 3.0 Intermediate
More info: Hi Everyone. I've dusted off my racket - haven't played… see more
More info: Hi Everyone. I've dusted off my racket - haven't played in a while but am serious about getting back into it. Looking for a couple of people to play with evenings and weekends. I'm a 3.0 and started taking lessons to progress exponentially. I'm very athletic so it won't be long before I plan on being past a 4.0. Eventually want to join a league.


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Fate of the 50p income tax rate
During the past week or so, the AccountingWEB editors' in-box has been swamped with messages and press statements putting forward arguments for and against the 50% tax rate band.
Is this all an artificial bit of political froth generated to fill the silly season vacuum, or is it a vital debate in which the views of AccountingWEB's members need to be brought to the Chancellor and business secretary's attention?
I'm concerned that in this instance my habitual cynicism may have clouded my news judgement and I've ignored the comments from the likes of Ernst & Young, UK200Group and Richard Murphy to concentrate on other issues. Do many people here feel strongly about the issue and want to go into it in more detail?
As a quick overview, here are some of the points pro & con:
Srap the 50% band
- "There's not much point in having taxes that are very economically inefficient." Chancellor George Osborne, Radio 4 Today programme, 16 August. The chancellor has reportedly asked HMRC to evaluate the impact of the rate on Self Assesment takings after its first year. The findings should be ready in time for the Budget.
- "It punishes wealth creation by imposing on entrepreneurs and business people a marginal tax rate in excess of 50% once national insurance contributions are added in... This is particularly damaging when the UK needs to create new businesses in new industries and promote growth by small companies, which can grow fast. It applies to just 1% of taxpayers, who already pay 24% of all income taxes."
- "A significant disincentive to doing business in the UK, providing a barrier to new business owners and executives from coming here, whilst also driving existing higher earners to foreign shores." Patrick Stevens, Ernst & Young
- "If something is not bringing in a profit and it cannot be made to do so, there is no point in continuing... In business and economic terms, if the 50% tax rate is not covering its costs then it should be scrapped but politically that's a totally different question!" Jonathan Russell, UK200Group
- "With National Insurance, top taxpayers pay over 50 per cent which means they are working more for the taxman than themselves... These high earners are often the creators of businesses and therefore jobs for those who do not have the same talent, management skills or qualifications. Do we really want to punish them for their successes and risk losing this hot-bed of talent?" John Kelly, Square One Financial Planning
Keep the 50% band
- "By definition most small businesses aren't going to get in the top 1% of income earners – there are about 4m small businesses in the UK right now, at least (over 1m companies and 3m or so self employed). All small businesses that will grow fast will be limited companies – enjoying effective tax rates of around 20% or so, utterly undermining this argument." Richard Murphy, TaxResearch blog.
- "According to recent articles, a 1% cut in income tax would cost the Exchequer £4.5bn. So these economists think that putting £10bn into the hands of 300,000 already-very-wealthy people is a better recipe for the well-being of the country than (say) cutting EVERYONE's income tax rates by 2p…" ChrisM, on Murphy's blog.
- "The government is committed to a competitive tax system, but in reducing the deficit, we have always been clear that those with the broadest shoulders should carry the greatest burden." A Treasury spokesman reaffirming the government's commitment to the 50p tax rate on Tuesday 7 September.
Let me know if you're interested in this topic and what you think. If I've been mistaken about the significance, I'll make amends with a more detailed study of the arguments, and perhaps put it to a community poll. As always, your guidance is appreciated.
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Change Decorating Style to Match House?
I would definitely put myself in the "cottage style" of the decorating spectrum. I've always been drawn to pictures and furniture that match this style. I love the soft colours, it mixes in old pieces, it's casual and family friendly. I feel that with this style, nobody is afraid to sit on your couch when they enter your house. This worked great for us in our cute little (1000 sqft) cape cod. It was like the structure of the house dictated the style it wanted to be on the inside.
Now we're moving into a 75 year old, 2200 sqft dutch colonial with 10 ft ceilings. I'm drawn to dark wood floors and gray tones for the walls. I picture an elegant dining room with upholstered chairs and a more traditional living room. As much as I love cottage style, I just feel like it would clash with the style of this house. With this house we are definitely leaning towards a more traditional, classic look. The problem is, a lot of the funiture we own is cottage style; lots of antique wood painted white, wrought iron beds, etc. Actually it was the wrought iron bed that brought me here to ask this question. In the new house, I'm picturing an upholstered head board for the master bedroom, but I really don't want to get rid of the wrought iron bed. I remember falling in love with it the second I saw it and spending more money than I had to get it because I just HAD to have it. So I guess what I'm asking is, has anyone ever seen a wrought iron bed incorporated into a more traditional looking master bedroom? I think I just need some reassurance and inspiration to make me realize that we could still make it work.
Has anyone else's style evolved into something completely different depending upon the house you are living in, or have you stayed true to your decorating style no matter what?
Yes -- my style was more cottage and switched to more tradtional -- well -- in my case -- more Brit Col Cottage!
It is wonderful to hear that you have truly listened to your house -- and will work towards making it your own home ...
Post photos here -- and I'm sure we will all help to slightly switch/mix your styles!
I think you can achieve what you want with most of your own things.
All that white painted furniture can be painted in more traditional tones, like deep green or Chinese red or chocolate or black.
You can still use comfortable upholstered things - if you use rumpled slipcovers now, you might like tighter more tailored ones, but keep your relaxed style.
Use heavy linen instead of cotton twill. Replace ticking stripes with more traditional stripes. Use Jacobean or William Morris or simple lattice, leaf, or floral prints in place of gingham checks. Things like that.
That sort of colonial outpost style that you see in Bermuda, and in films like "Out of Africa," can work wonderfully in a more traditional architectural style.
I can see using it fine if you do the above suggestions with the bedding etc. I'm hoping it has clean lines, if it's curly lines it won't work. FYI my husband and I made a six foot upholstered headboard with plywood, lots of foam and a soft chenille fabric. It was very tall and we loved it. I'm now using that in my lake house which is cottage style and it works well so why not try it and see how it works out. Then you can make a decision after it's completed.
Bronwynsmom has given you some terrific ideas to transition your cottage style to a more traditional version. I imagine that your wrought iron bed might work, too, depending on your choices of bed linens, wall color, bedside tables and lamps. However, if you don't love it in your new bedroom, you might consider moving it to a guest room and gifting yourself with a different bed.
Go to Houzz and search for wrought iron bed - you'll find them in every type home and some great inspiration photos of how it can work.
Here is a link that might be useful: Houzz - wrought iron beds
This house would have been filled with a lot of vernacular furniture when new, anyway, especially the bedrooms. You may want to go a bit more formal in the LR and DR, but I don't think it's quite as big a shift as it would be in moving from somes house styles to others.
My tastes always tend toward the eclectic so I can see using your cottage style furniture in a colonial home. I don't think you have to go strictly colonial, especially in the non public spaces. Cottage and colonial mix very well and you can express your own style in between. And I don't think you have to repaint a lot of stuff...you can go more feminine with light furniture and add the more colonial look in the fabrics and window treatments.
Oh, and let me recommend Minwax "English Chestnut" for your floors. It is the most perfect, beautiful deep brown I've ever used - no red like mahogany, no orangeyness like oak and some pines, no grayness like the provincial ones, no heavy blackishness like the jacobean ones. The color looks great with everything else, too. Give it a look.
I made the move from a Traditional house in Virginia to a more relaxed style in Florida. So, now I thought I would redo everything I had in Virginia, we sold a lot of the furniture and now I have just the few pieces I need to accomplish a more relaxed style.
The trade off? The stuff I kept needs refinishing or repurposing or getting something similar but smaller or larger. It will take me a while to reuse my things. If I had it to do over, I would have taken nothing with me, that way I could furnish the downsized house with what really fits here. Harder to take things with you and repurpose than it is to measure and fit what actually goes into the newer home.
I've stayed true to my traditional/transitional style of decorating. Our house is a craftsman style tract home but I didn't care for the outside facade to begin with so I just do what I want on the inside. DH liked the look of the house and it had all we needed on the inside, in case one wonders why the heck we even bought it to begin with.
Bronwynsmom, thanks for the suggestion for the floors! We were thinking about Minwax "dark walnut", but I will definitely check out the "english chestnut". I really appreciate your description of how the stain looks in real life.
I'm wondering if anyone has any similar comments regarding "dark walnut"...?


text:
Its a woman that looks like a man baby!
Your mom
Your Mom
That lady is a beast
Thats What Your Mom Said
Jump out of a closet, throw something on your dad, fart on your mom, we don...
In This Picture: Photo of kids outside with computers
Are You My Mom?
I'm on the TV!
Looks like I'm doing well.
Another weed-related death. So sad.
Well played, mom. Nice burn.
She's sort of a celebrity. Shut up
Is that you?
me and your mom


text:
2003 Chevrolet Silverado 2500HD User Reviews
Average Ratings from 23 reviews
- Performance:
- Appearance:
- Build Quality:
- Overall:
Review Highlights Click to filter reviews below
User Reviews
Displaying 1 - 10 of 23 2003 Chevrolet Silverado 2500HD reviews.
bpy writes:
2003 Chevrolet Silverado 2500HD 4 Dr LT 4WD Crew Cab LB HD Review
Very dependable
Not the greatest on gas
Strong And Reliable — This truck is loaded with everything on it that you could order. I bought it new in 2003. this is a great riding reliable comfortable truck. I've maintained it perfectly since it was new. I have had very little problems with it. I hope every truck I own from chevrolet in the future is as good as this one. Even with 250,000 miles on it I'm looking for many more miles.
Primary Use: Commuting to work
Looking for a Used Silverado 2500HD in your area?
CarGurus has 32,485 nationwide Silverado 2500HD listings starting at $3,000.
Philip writes:
2003 Chevrolet Silverado 2500HD 4 Dr LS Extended Cab SB HD Review
Safe and Reliable
Watch for Engine problems
Good For Family Decent On Diesel — This truck is great for off-road, and pulling anything, the engine quality is good minus the injector problems chevy didnt tell anyone about, its build quality is fair, sqeaks, intirior falls apart too easy, it looks decent but better with modifications, really fun to drive when performance parts are on it.
Primary Use: Family transportation
Frank writes:
2003 Chevrolet Silverado 2500HD 4 Dr LS 4WD Extended Cab SB HD Review
The Truck
The Cost of Diesel
2003 Chev 2500 Hd 4wd Duramax Diesel — This pickup is powered by the 6.6L Duramax Diesel with the 5 speed Allison Transmission. A number of modifications made to drivetrain giving it about 360HP and 620 lbs torque. On the outside threw on a couple of chrome accents to make it perdy too. At normal highway speeds gets 20-22 mpg. Not bad for a 7,000 lb truck! Currently has 120K miles and still running strong. Too bad the price of diesel got so high. It was my everyday driver.
Ryan writes:
2003 Chevrolet Silverado 2500HD 4 Dr LS 4WD Extended Cab LB HD Review
The truck is a great truck. 4x4 is great and can pull decent weight.
gas mileage is not the best. but this is the 6.0 gas engine and not the duramax.
6.0 Gas Not A Bad Truck I Just Want More. — I love the look of the 03-early 07 silverados. The gas 6.0 engine has plenty of power for my uses. I was able to pull a 10000 lbs load through the mountains realitively pain free. dont get me wrong the truck was screamin but it got it done. A little too heavy to be taken trough soft mud but handles fine off road. I love the truck.
CHEVY03 writes:
2003 Chevrolet Silverado 2500HD 2 Dr LS Standard Cab LB HD Review
Runs great and very reliable has never let me down nor left me stranded.
has a small dent on driver side of bed under gas cap. easily fixable.
Very Dependable Truck!! — I love that there is so many possibilities with a truck, and this truck is very dependable. Not necessarily a family vehicle but it has PLENTY of room for a single family. And it has the fifth wheel for a goose neck.
Primary Use: Commuting to work
Chris writes:
2003 Chevrolet Silverado 2500HD 4 Dr LS Extended Cab SB HD Review
Gas guzzler
Great American Made Truck. — Very powerful truck, great for towing. Strong six liter engine pulls anything. Looks nice and rides even better. It sucks down the fuel but it is worth it. Now i live in a three truck family. Just need some rims and tires and Ill be set.
Primary Use: Utility (towing boats, transporting cargo, etc.)
Mike writes:
2003 Chevrolet Silverado 2500HD 4 Dr STD 4WD Extended Cab SB HD Review
Great Work Truck — Build Quality: Havent had it too long, but so far no problems. My dad also has one, and has had no problems with his Car Comparisons: Considered the 2500HD and the Chevy 1500. Picked the HD because of the durability and how well they hold value Performance: Obviously not a race car. Has incredible torque however. Doesnt like going fast (
Chad writes:
2003 Chevrolet Silverado 2500HD 4 Dr LS Extended Cab LB HD Review
Because the exhaust rocks
496 V-8 does kind of drink the gas
It's Mine And I Like It — It's roomy plenty of space for the 3 of us and all the crap we carry, it has lots of power, gets pretty good gas mileage. and it is fun to drive since it has no muffler :) the 7" tv and subs also help.
Primary Use: Commuting to work
David writes:
2003 Chevrolet Silverado 2500HD 4 Dr LS 4WD Extended Cab LB HD Review
Could tow anything and everything; Had tons of cargo room; never had any problems
Gas guzzler; Too big for the mirrors; no special addons
hyrysk writes:
2003 Chevrolet Silverado 2500HD 4 Dr LS Extended Cab LB HD Review
Hauls everything
turns right at every gas station
Displaying 1 - 10 of 23 2003 Chevrolet Silverado 2500HD reviews.
Reviews From Other Years
2001 Chevrolet Silverado 2500HD Reviews
Pretty Sweet
good ole 6L farm/hunting truck Drags 2000lb bales through the snow without a grunt Can chase down a deer through a feild with ease. Have a 10000lb winch on the front with a big steel bumper to scar... Read More
Performance- great aceleration for a truck, it stops, and turns well. Build Quality- No repairs yet Appearance- looks good inside and out Cost of Ownership- costs a fortune to fill up and maintain Fun... Read More
2002 Chevrolet Silverado 2500HD Reviews
drove it from missouri to california, no problems. great truck. would def get another one. the interior is spacious and has plenty of room for you and the boys, or you and the family, whatever youre l... Read More
Been A Great Truck That's Served Its Purpose For Me.
I liked everything about this truck except the gas mileage. I now have 3 cars and one parking spot. I am forced to part ways with the truck I've owned for 10 years. If I were in the market for this... Read More
2004 Chevrolet Silverado 2500HD Reviews
For What I Use It For (hauling A Boat,camper, Utility Trailer) Can't Ask For More.
I've owned both the 2500HD and the 1500 series Chevy pickup's and feel the 2500HD's have it all over the rest. This is a nice compromise truck. Lots of power but still a comfortable ride, but if you ... Read More
Hard Running Truck
great for long trips, pulls the camper well & hauls things excellent. have used it for lots of hauling and towing and always gotten along well with it the only problem we have had with it is the 4wd n... Read More
2005 Chevrolet Silverado 2500HD Reviews
Great Family, Work, Toy, Hauler
Very easy to drive for a large vehicle, that being said, it is a large truck so plan accordingly when parking. Very comfortable inside and rides well for pick up. Duramax diesel is very quiet and pret... Read More
Great Work Truck
Overall this was a great truck. The Duramax Diesel was powerful and responsive. I put about 130K miles on it before I traded it in for a small sedan. I missed having the trucks utility but not the cos... Read More
2006 Chevrolet Silverado 2500HD Reviews
Great Truck Highly Recomended
A+++++ This truck has been absolutely gret to me, I have owned it for about 2 years and it has been great the mileage kind of sucks about 12 but I didnt buy it for good mileage I bought it to use it a... Read More
Work Truck
6.0 liter pulls our gooseneck horse trailer with no problem. Great transmission which does not change up and down while going up grades. Owned the truck for one year now and have had no maintenance ... Read More


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Hotel Kamila is the most beautiful hotel in Bratislava. There are a lot of polite personnel who always want to help. The room was very clean and very luxurious. In the price there is a swimming pool, of course in the summer season. You can also try golf. I can say that I was very satisfied with everything, with food, with staff, with room. It was one beautiful family hotel where you will feel very comfortable. I hope I will go there again.
- Reservation Options:
- TripAdvisor is proud to partner with Prestigia, Orbitz, Despegar.com and TripOnline SA so you can book your Hotel Kamila reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Kamila Hotel Bratislava


text:
commercial property for rent or lease in Talmage (PA), Industrial Space For Lease.
Talmage commercial real estate for lease, rent, and leased investments.
Find Talmage industrial buildings for lease, retail rentals, & Talmage shops. Locate a Talmage warehouse lease and any real estate for lease / investment, including industrial properties to rent and sale.
Negotiating a Talmage commercial lease can be a complicated procedure and its best to retain an experienced broker to work with early in the process. The brokers fee is typically paid for by the owner and you probably would not be able to negotiate a better position than the broker would be able to. There should be no reason why a broker should not be used to secure the transaction whilst there are numerous reasons as to why a broker should be used. Engage an attorney to review the terms of the lease contract.
commercial space listings
Williamsport (Lycoming), Strasburg (Lancaster), Cedar Run, Witmer, Hughesville, Picture Rocks
in Talmage Pennsylvania (PA) (Lancaster)


text:
Many women in today's top leadership roles are spread thin as they strive to complete their normal job duties, plus serve as the company's de facto "c ...
The infamous glass ceiling does exist — but often not in the way some may think.
Get vital information about some of the nation's top private employers, as defined by employee size.
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Book your room at Heritage Avenida Liberdade
Superior Double or Twin RoomMax Occupancy: 2 guests (up to 1 infant)
Standard Single RoomMax Occupancy: 1 guest
Heritage RoomMax Occupancy: 2 guests (up to 1 infant)
Standard Double or Twin RoomMax Occupancy: 2 guests (up to 1 infant)
Superior Double Room (Golden Age, Over 55 Years)Max Occupancy: 2 guests
4-star historic hotel in Lisbon (Avenida da Liberdade) near Castle of Sao Jorge
Collect 200 points
Simply enter your Nectar card number when prompted. Your points will be credited 30 days after you return from your trip.*Offers may apply
This historic hotel is located in Lisbon (Avenida da Liberdade), close to Ascensor da Gloria, Castle of Sao Jorge, and Rossio Square. Also nearby are Santa Justa Elevator and Lisbon Cathedral.
Hotel Features
At Heritage Avenida Liberdade, recreational amenities include an indoor pool and a fitness center. The hotel also features a bar/lounge and complimentary wireless Internet access.
Room Amenities
LCD televisions in guestrooms come with cable channels. Bathrooms feature shower/tub combinations, designer toiletries, and bidets.Information missing or incorrect? Tell us!
Hotel Amenities
Hotel Amenities
Heritage Avenida Liberdade features an indoor pool and a fitness center. A bar/lounge is on site where guests can unwind with a drink. A 24-hour business center is on site at this 4-star property.
Public areas are equipped with complimentary high-speed wired and wireless Internet access. This historic hotel also offers a library, tour/ticket assistance, and multilingual staff. For a surcharge, a roundtrip airport shuttle (available on request) is offered to guests.
Heritage Avenida Liberdade is a smoke-free property.
- 24-hour front desk
- Airport transportation (surcharge)
- Total number of rooms - 42
- Number of floors - 6
- Babysitting or childcare
- Bar/lounge
- Breakfast available (surcharge)
- Free newspapers in lobby
- Concierge services
- Dry cleaning/laundry service
- Elevator/lift
- Fitness facilities
- Tours/ticket assistance
- Free WiFi
- Library
- Multilingual staff
- Porter/bellhop
- Luggage storage
- Safe-deposit box at front desk
- Year Built 1789
- 24-hour business center
- Smoke-free property
- Number of buildings/towers - 1
- Use of nearby fitness center (discount)
- Free wired high-speed Internet
- Indoor pool
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi , Free wired high-speed Internet
Room Amenities
- DVD player
- Hypo-allergenic bedding available
- Air conditioning
- Soundproofed rooms
- Blackout drapes/curtains
- Minibar
- Daily housekeeping
- Private bathroom
- Designer toiletries
- Bathrobes
- Free toiletries
- Hair dryer
- Slippers
- Shower/tub combination
- Bidet
- In-room safe
- Rollaway/extra beds available
- Cribs/infant beds available
- Climate control
- Free WiFi
- Room service (24 hours)
- Pillow menu
- Connecting/adjoining rooms available
- Desk
- Cable TV service
Where to Eat
Breakfast is available for a surcharge and served each morning between 7:30 AM and noon.
24-hour room service is available.
Nearby Things to Do
Recreational amenities at the hotel include an indoor pool and a fitness center.
The recreational activities listed below are available either on site or nearby; fees may apply.
- Bicycle rentals nearby
- Scooter/moped rentals nearby
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
- Accessible bathroom
- Accessible path of travel
- Accessible parking
- In-room accessibility
- Roll-in shower
Hotel Policies
Minimum check-in age is 18
Check-in time starts at 3 PM
Check-out time is noon
Payment types
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Guests booked in a "Golden Age" rate plan must provide proof of age (55 or older) at check-in to receive the discounted rate. Without proof, and additional fee will be charged at check-in.
- Breakfast fee: EUR 14 per person (approximately)
- Self parking fee: EUR 15 per day
We should mention
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Awards & Affiliations
- The property is a member of Historic Hotels of Europe.


text:
WPPI Panel: Photographers Test Canon's 5DS and 5DSR
Canon's recent announcement of it new 50-megapixel 5Ds and 5Ds R cameras excited a lot of comments, and a lot of speculation. The cameras won't be on the market until June, but at the WPPI trade show yesterday, attendees got to see sample images shot with the new cameras. Four photographers–all members of the Canon Explorers of Light program– shared their experiences shooting with the new models. They were joined by photographer Peter Hurley, who typically shoots medium-format, and PDN's Technology Editor Greg Scoblete for a panel on the new cameras: Photo Forward, the blog of PDN's sister publication, Rangefinder, has a recap of the panel.
It includes comments by Hurley as well as photographers George Lepp, Hanson Fong, Ken Sklute and photographer/cinematographer Bruce Dorn about the cameras' resolution, speed and performance when shooting stills and video.
To read more, check out the Photo Forward blog.
Canon 5Ds Takes Aim at Medium Format with a 50-Megapixel Sensor


text:
I bought a new aerator and I see diagrams of the tube going through the bung and down into the carboy. How do you do this and nor create pressure for there is no where for it.to escape
If your talking about those attachments that go on the end of the siphon tube. You want to point side up so when wort hits that it makes it splash causing air by spreading it out more consistantly when transferring.
Pressure???? No comprende. YOu taliking one of those bubblers?,?Never really thought of that though but,now Im seing what you are making sense of ,like air in a balloon? Im interested in some other answers myself,now. HOw does that work for 30 min with an airtight seal? Wouldnt it be like pumping a bike tire up? It must not create enough pressure in the time frame.
Well the picture was of the aerator with a tube going into a carboy bung through where a airlock would normally sit . So can I just put it in my primary bucket and aerate away not worry about anything?
The most dramatic thing that could happen is the bung could be slowly pushed out, you should be fine!


text: Web exclusive! This clinically accurate blood pressure arm monitor can store up to 120 readings for two users and features oversized digits for easy-to-read convenience. Original: $70.00 Sale: $49.99 Includes: monitor, adult arm nylon cuff, zippered storage case, AC adapter port, 4 AA batteries, detailed English/Spanish guidebook, quick start guide, and blood pressure reading log 2-person memory bank stores up to 120 readings, 60 readings for each user with average of last 3 readings Hypertension Indicator provides an instant comparison to standards set by the World Health Organization Irregular Heartbeat Detection alerts the user to an irregular heartbeat during measurement Displays systolic, diastolic and pulse readings simultaneously with date and time stamp Adult size cuff fits arm circumference: 11¾" – 16½" Oversize, easy-to-read backlit display Latex-free design Imported More


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Page 1
Structural insights into triple-helical collagen cleavage
by matrix metalloproteinase 1
Szymon W. Mankaa, Federico Carafolib, Robert Vissea, Dominique Bihanc, Nicolas Raynalc, Richard W. Farndalec,
Gillian Murphyd, Jan J. Enghilde, Erhard Hohenesterb,1, and Hideaki Nagasea,1
aNuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford, London W6
8LH, United Kingdom;bDepartment of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom;cDepartment of Biochemistry, University of
Cambridge, Cambridge CB2 1QW, United Kingdom;dDepartment of Oncology, Cancer Research UK Cambridge Research Institute, University of Cambridge,
Cambridge CB2 0RE, United Kingdom; andeDepartment of Molecular Biology and Genetics, University of Aarhus, DK-8000 Aarhus, Denmark
Edited by Robert Huber, Max Planck Institute of Biochemistry, Planegg-Martinsried, Germany, and approved June 11, 2012 (received for review March
23, 2012)
Collagenases of the matrix metalloproteinase (MMP) family play
major roles in morphogenesis, tissue repair, and human diseases,
but how they recognize and cleave the collagen triple helix is not
fully understood. Here, we report temperature-dependent binding
the cooperative action of its catalytic and hemopexin domains.
Contact between the two molecules was mapped by screening the
Collagen Toolkit peptide library and by hydrogen/deuterium ex-
change. The crystal structure of MMP-1(E200A) bound to a triple-
helical collagen peptide revealed extensive interactions of the 115-
Å–long triple helix with both MMP-1 domains. An exosite in the
hemopexin domain, which binds the leucine 10 residues C-terminal
to the scissile bond, is critical for collagenolysis and represents a
unique target for inhibitor development. The scissile bond is not
correctlypositionedfor hydrolysisinthecrystallizedcomplex.Apro-
1 structure or the exosite interactions, by axial rotation of the colla-
gen homotrimer. Interdomain flexing of the enzyme and a localized
excursion of the collagen chain closest to the active site, facilitated
state of collagenolysis.
extracellular matrix|X-ray crystallography|protease
tendon, and blood vessels (1). They consist of three α chains with
repeating Gly-X-Y triplets (X and Y are often proline and
hydroxyproline, respectively) that intertwine each other to form
a triple helix of ∼300 nm in length (2). Interstitial collagens are
resistant to most proteolytic enzymes, but vertebrate collage-
nases cleave them at a single site approximately three-quarters of
the way from the N terminus of the triple helix, thus initiating
collagenolysis (3). Owing to this unique activity, collagenases
play important roles in embryo development, morphogenesis,
tissue remodeling, wound healing, and human diseases, such as
arthritis, cancer, and atherosclerosis (4, 5).
Matrix metalloproteinase 1 (MMP-1) is a typical vertebrate
collagenase (3). It consists of an N-terminal catalytic (Cat) do-
main containing an active-site zinc ion and a C-terminal hemo-
pexin (Hpx) domain comprised of a four-bladed β-propeller,
which are connected by a linker region (6, 7). Although the Cat
heat-denatured collagen (gelatin), its activity on native triple-
helicalcollagen isnegligible. Thecombination ofthe CatandHpx
domains is required for MMP-1 to be able to degrade native
collagen, and the same is true for all other collagenolytic MMPs,
namely MMP-2, MMP-8, MMP-13, and MMP-14 (3). How col-
lagenases interact with collagen and how the Hpx domain endows
these enzymes with collagenolytic activity is not clearly un-
derstood. Another enigma of collagenolysis became apparent
when the crystal structures of MMP-1Cat (8–10), MMP-8Cat (11,
12),andfull-length pig MMP-1(6)weresolved: Thecatalytic cleft
he interstitial collagens I, II, and III are the major structural
proteins in connective tissues such as skin, bone, cartilage,
of these enzymes is too narrow to accommodate collagen in its
native triple-helical conformation. A number of hypotheses have
been proposed to explain how collagenases may destabilize and
cleavetriple-helical collagen (13–16),andwehave experimentally
demonstrated that MMP-1 unwinds triple-helical collagen locally
before peptide bond hydrolysis (17, 18). A recent study mapped
the interaction sites in MMP-1 and in triple-helical collagen by
using NMR and proposed a model of collagen binding and un-
winding (19). However, because the interactions between MMP-1
andcollagen were not observed directly, a number ofassumptions
had to be made to derive a mechanism of collagenolysis.
In this study, we have combined biochemical experiments with
crystallographic structure determination to show how MMP-1
recognizes its unique cleavage site in interstitial collagens. Our
results suggest that collagenolysis relies on multiple exosite
interactions, which not only serve to position the scissile bond
near the active site, but also assist in the local unfolding of the
collagen triple helix that is required for cleavage to occur.
Cat and Hpx Domains of MMP-1 Cooperate in Collagen Binding. We
first examined a catalytically inactive MMP-1(E200A) mutant
(residue numbering based on the proMMP-1 sequence) and its
individual domains for binding to native collagen I. Full-length
MMP-1(E200A) showed saturable collagen binding with an ap-
parent KDof 0.4 μM, whereas the catalytic domain Cat(E200A)
showed no detectable binding and the Hpx domain bound only
weakly (Fig. 1A). The Cat(E200A) domain did not bind to
collagen I even when it was added together with increasing con-
centrations of the Hpx domain (SI Appendix, Fig. S1A), suggest-
ing that the Cat and Hpx domains bind collagen cooperatively
only when they are linked together.
The binding of MMP-1(E200A) to collagen increased with
temperature until collagen denatured above 37 °C (Fig. 1B),
indicating that MMP-1 prefers a looser triple helix but not
denatured collagen. The temperature-dependent enhancement
of MMP-1(E200A) binding to collagen was substantially reduced
by the active-site inhibitor GM6001 (Fig. 1C). This effect was
Author contributions: S.W.M., R.W.F., G.M., J.J.E., E.H., and H.N. designed research; S.W.M.,
F.C., R.V., D.B., and E.H. performed research; D.B., N.R., R.W.F., G.M., and J.J.E. contributed
new reagents/analytic tools; S.W.M., F.C., R.V., R.W.F., G.M., J.J.E., E.H., and H.N. analyzed
data; and S.W.M., E.H., and H.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID code 4AUO).
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.com
or email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 31, 2012
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| 12461–12466
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not observed below 10 °C, where little collagen cleavage occurs
(17). No significant temperature-dependent increase in collagen
binding was observed with either the Cat(E200A) or Hpx domain
alone (SI Appendix, Fig. S1 B and C). These results suggest that
the active site cleft of MMP-1 participates in collagen binding
at body temperature by stabilizing and/or inducing a looser
collagen conformation.
MMP-1–Binding Motif in Interstitial Collagens. To identify the col-
lagen residues interacting with MMP-1, we screened the Colla-
gen Toolkit library of triple-helical peptides encompassing the
entire collagen II sequence (20) for binding to MMP-1(E200A).
Only one peptide, Col II-44, starting at the collagenase cleavage
site G∼LAGQR... (∼ indicates the scissile bond) was recog-
nized by the enzyme (Fig. 2). Col II-43, which also contains the
cleavage site but with fewer C-terminal residues, did not bind
MMP-1(E200A). Further experiments with truncated and ala-
nine-substituted peptides identified the leucines at the P1′ and
P10′ positions (nomenclature after Schechter and Berger; ref.
21) of collagen as important for MMP-1(E200A) binding (SI
Appendix, Fig. S2A). A sequence alignment showed that the
leucines at these two positions are conserved in all α chains of
collagens I, II, and III from different species (SI Appendix, Fig.
S2B). The consensus sequence for collagenase binding is G∼(L/
I)(A/L)-GXY-GXY-GL(O/A) (P1′ and P10′ positions are bold;
O, hydroxyproline). This motif is unique and not found else-
where in collagens I, II, and III.
Mapping of the Collagen-Binding Regions of MMP-1 in Solution. We
then used hydrogen/deuterium exchange mass spectrometry (H/
DXMS) to map the regions in MMP-1 involved in collagen
binding. Five MMP-1 regions showed delayed deuterium in-
corporation in the presence of collagen I (Fig. 3A and SI
Appendix, Fig. S3 and Table S1): Residues 145–161 (site 1) and
205–215 (site 3) in the Cat domain and residues 266–277 (site 4),
283–297 (site 5), and 330–346 (site 6) in the Hpx domain. Sites
4 and 5 were reported to reduce deuterium exchange upon
binding to a triple-helical peptide (22). Residues 179–193 (site 2)
showed increased deuterium incorporation in the presence of
collagen, suggesting that this region, which includes the loop
between the fifth β-strand and the second α-helix of the Cat
domain, is more exposed to the solvent in the collagen-bound
state. This loop contains a collagenase-characteristic cis-peptide
bond (23, 24), and it was identified as important for the colla-
genolytic activity of MMP-1 (23). Our data suggest that this loop
participates in collagenolysis dynamically. The combined H/
DXMS results indicate the general orientation of the collagen
triple helix binding to MMP-1 (Fig. 3, dashed line).
S10′ Exosite in the Hpx Domain. Considering the H/DXMS results
and the importance for MMP-1(E200A) binding of the two
leucines at the P1′ and P10′ positions of collagen, we searched
for hydrophobic sites in MMP-1 that could accept these residues.
For MMP-1 to cleave the Gly(P1)-Leu(P1′) bond, the P1′ Leu
must be located close to the S1′ pocket of the Cat domain. Using
this constraint, we docked a modeled triple-helical peptide to
MMP-1 and identified candidate residues in the Hpx domain
that might be involved in binding the P10′ Leu (Fig. 3A). They
were mutated and the mutant proteins tested for their collage-
nolytic activity (Fig. 3B). The most dramatic reduction of colla-
genase activity was observed with the F301Y single mutant and
the I271A/R272A double mutant, which exhibited 10% and 4%
of wild-type activity, respectively. Together, Phe301, Ile271, and
Arg272 in the Hpx domain participate in forming the hydro-
phobic S10′ binding pocket, or exosite. Ile271 and Arg272 were
identified in the H/DXMS experiment as part of site 4. Phe301 is
located between H/DXMS sites 5 and 6. It was not detected by
H/DXMS analysis most likely because its backbone amide is
buried in the Hpx domain.
Crystal Structure of an MMP-1(E200A)–Collagen Peptide Complex.
Based on the results of the collagen peptide binding studies,
we designed a triple-helical collagen peptide for cocrystallization
with MMP-1(E200A). The peptide spans positions P7-P17′ of
the collagen II cleavage site and is capped by two and three
nonnative Gly-Pro-Hyp triplets at the N and C termini, re-
spectively, to enhance triple helicity (Fig. 4A). The melting
temperature of this peptide was 31 °C (SI Appendix, Fig. S4). The
peptide formed a stable complex with MMP-1(E200A) in solu-
tion (SI Appendix, Fig. S5). Crystals of the complex were grown
Fig. 1.
I. (A) Binding of full-length MMP-1(E200A) and its in-
dividual Cat(E200A) and Hpx domains at 20 °C. (B) Binding
of MMP-1(E200A) at different temperatures. (C) Binding of
1 μM MMP-1(E200A) in the presence of the active-site in-
hibitor GM6001 at different temperatures.
Binding of MMP-1(E200A) to immobilized collagen
Fig. 2.
collagen II with proMMP-1(E200A), MMP-1(E200A),
Hpx domain, and Cat(E200A) domain. Error bars show
SDs from three repeats. Critical Leu and Ile residues at
P1′ and P10′ subsites are highlighted in the collagen II
portion of the peptide sequences surrounding the
collagenase cleavage site. ∼, bond cleaved by colla-
Screening of a triple-helical peptide library of
| www.pnas.org/cgi/doi/10.1073/pnas.1204991109Manka et al.
Page 3
at room temperature and used to determine the structure at 3.0-
Å resolution (SI Appendix, Table S2).
The overall structure of human MMP-1(E200A) in complex
with collagen is similar to those reported for full-length pig (6)
and human (7) MMP-1 without collagen. The collagen peptide in
the complex with MMP-1(E200A) is an uninterrupted triple
helix ∼115 Å in length and bent by ∼10° near the Cat-Hpx
junction (Fig. 4B and SI Appendix, Fig. S6). The three chains of
the collagen triple helix are arranged with the characteristic one
residue stagger, resulting in a leading (L), middle (M), and trailing
(T) chain (25). All three chains contribute to MMP-1(E200A)
binding, creating an extensive interaction surface spanning nearly
60 Å in length. Complex formation buries 480 Å2of solvent-
accessible surface of the L chain, 440 Å2of the M chain, and
370 Å2of the T chain.
Interactions Between MMP-1(E200A) and the Collagen Peptide. The
majority of the interactions between the collagen peptide and the
Cat domain are of a polar nature (Fig. 4C and SI Appendix, Fig.
S7). Of the three collagen chains, the L chain is closest to the
active site of MMP-1, but the S1′ pocket remains empty. The
residue closest to the S1′ pocket is Gln(P4′L) (residues are
identified by their position relative to the scissile bond and
a subscript designating the collagen chain). The side chain of Gln
(P4′L) forms a hydrogen bond with the backbone amide of Tyr221
at the entrance of the S1′ pocket, and the backbone amide of Gln
(P4′L) forms a hydrogen bond with the side chain of Asn161. Leu
(P1′L) is 9 Å away from the S1′ pocket and stabilized by a hy-
drophobic contact with the zinc ligand His203. The Arg(P5′L)
side chain and backbone amide form hydrogen bonds with the side
chain of Tyr218 and the carbonyl oxygen of Pro219, respectively.
The M chain makes only a single contact with the Cat domain,
between the side chain of Gln(P4′M) and the side chain of Tyr218.
The T chain, however, makes extensive interactions with the upper
part of the substrate binding cleft, including five hydrogen bonds
(Fig. 4C and SI Appendix, Fig. S7). Some of these hydrogen
bonds are identical to those formed with a cysteine-switch sequence
(26–29) in proMMPs and with a predicted peptide substrate (30).
The interactions between the collagen peptide and the Hpx
domain are a combination of polar and apolar contacts, and are
mediated mainly by the M chain (Fig. 4D and SI Appendix, Fig.
S7). Consistent with our mutagenesis results, the side chain of
Leu(P10′M) is bound in a hydrophobic S10′ exosite pocket de-
lineated by Ile271, Met276, Phe301, Trp302, and the alkyl portion
of Arg272 (Fig. 4D). The S10′ pocket is surrounded by polar
residues (Arg272, Glu274, Arg285, and Gln335) that make a total
of five hydrogen bonds with the collagen peptide (SI Appendix,
Fig. S7). The interactions at the S10′ exosite are critical for col-
lagen recognition and collagenolysis by MMP-1 (Fig. 3B). In
addition, Ile(P7′M) forms hydrophobic interactions with Val300
and Phe301. The M and T chains make van der Waals contacts
with Phe289 and Tyr290, but these interactions evidently are
not critical, because the triple mutant MMP-1(F289A/Y290A/
P291A) retains 76% of wild-type collagenase activity (Fig. 3B).
Implications for Collagenolysis. The way in which the collagen
peptide is bound to the active-site cleft of MMP-1(E200A) is
more compatible with cleavage of the Gly(P3′L)-Gln(P4′L) bond
instead of the canonical Gly(P1)-Leu(P1′) bond (31). To test
whether MMP-1 can cleave also before Gln(P4′), we performed
N-terminal sequencing of the products generated by active
MMP-1 at 25 °C and 37 °C. We found that only the Gly(P1)-Leu
(P1′) bond was cleaved in the peptide, as in native collagen II,
and concluded that the mode of collagen binding in the crys-
tallized complex is unproductive. A productive mode could be
achieved by a substantial movement of the Cat domain relative
to the Hpx domain, but this necessitates the breaking of the Cat-
Hpx interface (19). We favor an alternative mechanism that
leaves the interface intact. The helical symmetry of homotrimeric
collagen dictates that axial rotation of the collagen homotrimer
(in other words, binding of MMP-1 to a different set of collagen
chains) changes the register of the chain passing through the
active site cleft, but leaves all other interactions unchanged (Fig.
5 and SI Appendix, Fig. S8). If Leu(P10′L) instead of Leu(P10′M)
is made to occupy the hydrophobic S10′ exosite, the T chain
becomes the chain that interacts most closely with the active site,
and now Leu(P1′T) is situated near the S1′ pocket. Notably, this
productive binding mode does not alter any of the extensive
interactions with the upper part of the substrate binding cleft
(now made by the M chain) or with the S10′ exosite in the Hpx
domain (now made by the L chain) (Fig. 5 and SI Appendix, Fig.
S8). The energy difference between the observed unproductive
and putative productive complex is likely to be small, and the two
forms may coexist in solution. The unproductive complex may be
favored by the E200A mutant of MMP-1 or it may have crys-
tallized preferentially in our experiment.
Starting from the modeled productive complex (SI Appendix,
Fig. S8), it proved straightforward to achieve the first transition
state of collagenolysis, in which one collagen chain is inserted
into the active site (Fig. 6). By stretching the P1′-P6′ region of
the T chain and reorienting three residues preceding the scissile
bond, it was possible to insert Leu(P1′T) into the S1′ pocket in
a manner that fully agrees with the observed binding mode of
single-chain peptidomimetic MMP inhibitors (8, 32). The Cα
atom of Leu(P1′T) moves by less than 4 Å during this localized
excursion from the regular triple-helical structure, and only four
collagen interchain hydrogen bonds are broken in the transition
state. We propose that the energy penalty for this local unfolding
event is compensated by the favorable interactions that the en-
zyme forms with the looped-out collagen chain.
Our study has revealed an unprecedented proteinase–substrate
interaction. The most striking feature of the MMP-1–collagen
complex is the extensive interaction of all three collagen chains
with the two domains of MMP-1, which explains the strict re-
quirement of the Hpx domain for efficient collagenolysis (3). The
individual Cat or Hpx domains of MMP-1 showed very little
collagen binding in our experiments, but when linked together,
they acted cooperatively in a temperature-dependent manner.
The affinity for collagen I increased up to 37 °C, a temperature
that would not cause collagen to denature within the time frame
of the experiment (∼2 h) (33). Above this temperature the
binding sharply decreased. This temperature dependence, which
is also unusually pronounced in the rate of collagen cleavage by
Fig. 3.
mutagenesis. (A) Sites 1 and 3–6 are protected from deuterium incorporation,
and site 2 shows enhanced deuterium incorporation upon collagen binding.
The sites are mapped onto the crystal structure of MMP-1(E200A) (7). Resi-
dues potentially involved in collagenolysis that were mutated are indicated.
Dashed line, predicted collagen binding direction. (B) Relative initial velocities
of collagen I cleavage by MMP-1 and its variants normalized to WT.
Collagen I footprint on MMP-1(E200A) determined by H/DXMS and
Manka et al.PNAS
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| 12463
Page 4
MMP-1 (34), can be explained by the relative thermoflexibility of
the triple helix at the collagenase cleavage site (35–38) and the
contribution of interdomain flexibility of MMP-1. The co-
operative binding is therefore energetically most favored close to
body temperature, when the triple helix is locally looser but the
overall fold of the substrate is still maintained. A complete loss
of triple helicity makes collagen bind less tightly to MMP-1, as
evidenced from a Kmvalue of ∼1 μM for collagen I compared
with 4–7 μM for gelatin (39). The small active-site inhibitor
GM6001 abolished the temperature-dependent increase of col-
lagen binding, indicating that the catalytic site of MMP-1 is not
only involved in the final hydrolysis step, but also in the unfolding
machinery, which uses the active site to extract one collagen
chain to form the first transition state of collagenolysis (Fig. 6).
Apart from providing a favorable environment for the loose
triple helix at the cleavage site, what other mechanism(s) might
contribute to collagenolysis? Comparison of the crystal structure
of proMMP-1 (29) with the structure of the activated form of
MMP-1(E200A) (6, 7) suggested that the Cat-Hpx linker region
confers interdomain flexibility. Such flexing was further demon-
strated for MMP-1 by NMR and small angle X-ray scattering
studies (40, 41) and seems to be a common property of MMPs
(42, 43). Our crystal structure of MMP-1 bound to collagen has
a more closed conformation than free MMP-1 (SI Appendix, Fig.
S9). This change in interdomain angle is accompanied by the
bending of the collagen peptide at a point halfway between the
Cat and Hpx domains. Thus, domain motions in MMP-1 may
facilitate structural changes in the triple helix. Our structural
results show how the two domains of MMP-1 anchor two of the
collagen chains (M and L in the modeled productive complex)
and position the third (T) chain near the active site. Stretching of
the T chain by interdomain flexing of the enzyme may assist in
local collagen unfolding. Because the P1′ residue of the trailing
chain is poised above the S1′ pocket, transient excursions of the
Fig. 4.
triple helical collagen peptide complex. (A) Se-
quence of the collagen peptide used for coc-
within 4 Å distance of the enzyme in the com-
plex are colored. The subsite designation is in-
dicated for the leading chain. (B) Stereoview of
the MMP-1(E200A)–collagen peptide complex.
The collagen chains are colored cyan (L), green
(M), and red (T), and the enzyme is shown as
a gray surface with areas within 4 Å distance of
the L, M, and T chains colored correspondingly.
Magenta sphere, active-site zinc ion. (C) Ster-
eoview of the interactions between collagen
chains (colored as in B) and the active site cleft
of MMP-1(E200A). Dashed lines indicate hydro-
gen bonds. (D) Stereoview of the interactions of
the collagen chains with the Hpx domain. Se-
lected residues making enzyme-substrate con-
tacts are labeled with respective colors and
shown in stick representation (N, dark blue;
O, red).
Crystal structure of the MMP-1(E200A)–
| www.pnas.org/cgi/doi/10.1073/pnas.1204991109Manka et al.
Page 5
scissile bond can be efficiently captured by the enzyme, leading
to the stabilization of the first transition state of collagenolysis
(Fig. 6). Although thermal relaxation of the cleavage site is im-
portant for MMP-1 binding, this alone is not sufficient for effi-
cient collagenolysis (17, 37). We propose that mammalian
collagenases do not serve merely as passive acceptors of spon-
taneously occurring unfolded collagen states (44, 45), but they
promote a local perturbation of the triple helix that facilitates
collagenolysis. The recently reported NMR study supports this
notion by demonstrating a weakening of the collagen interchain
contacts in the complex with MMP-1 (19).
Our mechanism of collagenolysis (Fig. 6) differs from that
derived from NMR experiments (19) in that it does not require
a separation of the Cat and Hpx domains. Bertini et al. (19), who
also used the E200A mutant of MMP-1, found that the Cat
domain interacted mainly with the L chain and the Hpx domain
mainly with the L and M chains. These results agree with our
crystal structure of an unproductive complex. In their modeling,
Bertini et al. (19) imposed a productive interaction of the
L chain with the S1′ pocket, which forced the breaking of the
Cat-Hpx interface. Our interpretation requires only small con-
formational changes in the enzyme but postulates the co-
existence of productive and unproductive binding modes.
The crystal structure of a bacterial collagenase comprising of a N-
terminal activator domain, a glycine-rich linker, and a peptidase
domain has been reported (46). Like MMPs, this bacterial colla-
genase requires an ancillary activator domain (unrelated to the Hpx
domain of MMPs) to cleave triple-helical collagen. The authors
proposed that the enzyme clamps collagen between the activator
and peptidase domains of the saddle-shaped structure. Our MMP-
collagen structure shows that MMP-1 binds collagen in an extended
manner, rather than surrounding it. These differences notwith-
standing, interdomain plasticity seems to be a common feature of
enzymes that can cleave the intractable collagen triple helix.
Most MMPs are believed to have specific substrate recognition
sites distant from the catalytic site, which are referred to as
exosites. Apart from the Hpx domains of collagenases, examples
include the fibronectin type II repeats in MMP-2 and MMP-9,
which contribute to the binding and degradation of elastin, gel-
atin, and type IV collagen (47). Recent NMR and mutagenesis
studies of the Cat domain of MMP-12 characterized several
exosites for elastin (48, 49). Similar studies were carried out with
the isolated Hpx domain of MMP-1 and a triple-helical collagen
peptide (40) and suggested a role for Phe282 (Phe301 in the
numbering used in ref. 40) in binding of collagen. However, the
exact modes of interaction between the enzyme and the target
substrate have not been elucidated in these studies. Our crystal
structure has revealed in detail the interactions that occur be-
tween full-length MMP-1 and a triple-helical peptide long
enough to define all relevant contacts. Phe282 is buried in the
Cat-Hpx interface in our structure and not involved in collagen
binding. Defining the exosites in proteolytic enzymes has im-
portant implications for the development of specific inhibitors.
The hydrophobic S10′ exosite in MMP-1 is a particularly at-
tractive target because it is essential for collagenolysis. Recent
studies of Robichaud et al. (50) showed the importance of Leu at
the P10′ subsite of collagen III for the cleavage by MMP-1.
Noncollagenous extracellular matrix and nonmatrix substrates
are cleaved by the Cat domain of MMP-1 (16). Therefore, typical
inhibitors targeting the S1′ pocket block all MMP-1 activities,
whereas inhibitors directed against the S10′ exosite are predicted
to selectively inhibit collagenolysis. This exosite of MMP-1 is
largely conserved in other MMPs, but to our knowledge it is
specific for collagenolytic activity. We propose that molecules
specifically binding the S10′ exosite be therapeutically useful in
diseases associated with accelerated collagen breakdown such as
arthritis, cancer, aneurysm, and atherosclerosis.
Materials and Methods
Proteins and Peptides, Binding Assays, H/DXMS Analysis, and Enzyme Activity
Assay. Recombinant proMMP-1(E200A), its Cat(E200A) and Hpx domains, and
MMP-1 variants were produced in Escherichia coli. Collagen I was purified
from Guinea pig skin and treated with pepsin. The library of triple-helical
peptides of human collagen II (Toolkit II) and all other collagen peptides
were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Col-
lagen and peptide binding assays were carried out in ELISA format by using
biotinylated proteins and detected by streptavidin-horseradish peroxidase
system. Local hydrogen/deuterium exchange kinetics of MMP-1(E200A) free
and bound to collagen I were analyzed by digestion with pepsin and mass
spectrometryand quantifiedby usingHX-Express software.The
Fig. 5.
sequence alignment on the left, the trailing chain is shown twice to emphasize the circular nature of the chain arrangement. The P1′ and P10′ residues are
boxed, and residues interacting with MMP-1 are in bold. The structural models on the right show that the unproductive and productive binding modes are
related by a simple rotation/translation of the collagen triple helix. Note that only the interactions with one collagen chain are changed (L in the unproductive
mode, T in the productive mode); the interactions with the other two chains are the same in both modes.
Modes of collagen binding to MMP-1. Shown is relationship between the unproductive and productive collagen binding modes (see text). In the
Fig. 6.
productive complex. The upper rim of the catalytic site cleft anchors the
M chain (green), and the Hpx domain anchors the L chain (cyan). These
interactions position the T chain above the active site. Interdomain flexing of
the enzyme bends the collagen triple helix and facilitates the insertion of
Leu(P1′) of the T chain into the S1′ pocket.
Model of the first transition state of collagenolysis based on the
Manka et al.PNAS
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collagenolytic activity of MMP-1 variants was determined by SDS/PAGE and
densitometric analyses of collagen I cleavage products. Detailed methods
are described in SI Appendix.
Crystallization, Data Collection, and Structure Determination. MMP-1(E200A)
purified by size exclusion chromatography. Crystals of the complex were
obtained at pH 8.5 and diffracted to 3.0 Å resolution. The structure was solved
by molecular replacement and refined to a free R-factor of 0.273. Detailed
methods are described in SI Appendix. Data processing and refinement statistics
are listed in SI Appendix, Table S2. The coordinates of the MMP-1(E200A)–
collagen complex have been deposited in the Protein Data Bank (ID code 4AUO).
ACKNOWLEDGMENTS. We thank Noriko Ito for construction of MMP-1
mutants, Ida B. Thøgersen for peptide sequencing, the staff at Diamond
beamline I24 for help with X-ray data collection, and Peter Brick for many
helpful discussions. Protein visualization and modeling was done in PyMOL
(www.pymol.org). The work was supported by Wellcome Trust Grants
068724, 075473, 094470, and 083942; the Medical Research Council, Arthritis
Research UK; and Cancer Research UK. E.H. is a Wellcome Trust Senior
Research Fellow.
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text:
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Written on: 22/07/2013 by shirleycampbell (0 reviews written)
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text:
Town Square
Palo Alto reaches CCS soccer finals again
Original post made on Feb 21, 2008
Read the full story here Web Link posted Thursday, February 21, 2008, 9:43 AM
Comments (2)
a resident of Midtown
on Feb 21, 2008 at 9:06 pm
Note: 5 seniors experienced last season's title loss: Nguyen, Kelly, Wilson, Paguirigan, and Ostrau.
a resident of Crescent Park
on Feb 22, 2008 at 11:22 am
Thanks Nguyen. You're the best! The other seniors try, but can't seem to perform when they really need to. I'm glad you're on the team. Good luck.
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