text:
Thanks for redeeming.{{else}}
Success! Thanks for repurchasing.{{/if}} {{else}} {{#if Expired}}
Increase my visibility!{{else}} {{#if IsMultiPack}}
{{#if HasMultiPack}} You have {{MultiPackCount}} top spot(s) left. Now loading you into the search listings... {{else}} You have used all top spots. Now loading you into the search listings... {{/if}}{{else}}
We are loading you into the search listing pages...{{/if}} {{/if}} {{/if}} {{/if}}
{{Current}} just saw you!{{/if}} {{#if LastTopSpotPurchaseIsMulti }} {{#if HasAnyTopSpot}}
{{#if HasFreeTopSpot }} {{ UnclaimedTopSpotFree }} Free {{/if}} {{#if HasPaidTopSpot }} {{#if HasFreeTopSpot }} & {{/if}} {{ MultiPackCount }} {{/if}} Top Spot{{#if Plural}}s{{/if}} Left{{else}}
0 Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{else}} {{#if HasFreeTopSpot }}
{{ UnclaimedTopSpotFree }} Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{/if}} {{else}} {{#if HasResults}}
{{Current}} see you now!{{else}} {{/if}} {{/if}}
You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
33 year old woman | Denver, CO, USA | Seeking men 27-35 within 100 miles
Vibrant, vivacious woman looking for love!
Don't judge this book by it's cover. On the surface, I look like a mild-mannered girl. I know when to be a professional. But I very much have a wild side. I recently moved to Denver area from Pittsburgh. I would love to meet some people and get out to learn more about the city. I am a fun-loving, outgoing, caring woman who loves to laugh. I hope to find a mate who too is outgoing and enjoys the humor in life. My family is very important to me. We are all very close. I would like to find someone who also values his family. There are a few ways to this girl's heart. One very important way is on the dance floor. There is a lot you can learn about a person that way. I also love relaxing and socializing with my friends. I like being on the move, exploring new places and new things. I hope to find someone who can appreciate sharing these experiences with me. When it comes to love and relationships, I am a hopeless romantic. Still seeking my prince charming.
Alumni connections , Coffee and conversation , Business networking , Cooking , Dining out , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing sports , Religion/Spiritual , Shopping/Antiques , Travel/Sightseeing , Volunteering , Watching sports , Wine tasting
Aerobics, Bowling, Dancing, Soccer, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
I like Dogs
Disney World is my favorite place. I love to be out with people. love going to movies, restaurants, lounges.
University of Maryland, College Park, College Park, MD
Big fan of classic motown, but I like all types except heavy metal. Favorite shows are Scandal and How to Get Away with Murder. Favorite foods- seafood, pasta, and anything with cheese
I LOVE dancing, particularly salsa dancing. I also love movies, going out to dinner with friends. I love to travel. I have only been a few places but would love to see more of the world. I am pretty much up for trying anything once.
Yep! I read Fifty Shades of Gray and not ashamed to say I loved it!
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'0" (152cms)
|5'0" (152cms) to 8'0" (243cms)
|Body type:
|About average, Athletic and toned, A few extra pounds, Stocky
|Black, Blue, Brown, Grey, Green, Hazel
|Dark brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Salt and pepper, Grey, Bald
|No Way
|No Way
|Social Drinker
|Social Drinker
|Medical / Dental / Veterinary / Fitness
|No preference
I am a physician
|$50,001 to $75,000
|No preference
|Never Married
|Never Married
|Have kids:
|Yes, and they sometimes live at home, No, Yes, and they live away from home, Yes, and they live at home
|Wants kids:
|Black / African descent
|Asian, Black / African descent, East Indian, Latino / Hispanic, Middle Eastern, Native American, Pacific Islander, White / Caucasian, Other
|Christian / Other
|Spiritual but not religious, Christian / Other
|Graduate degree
|Bachelors degree


text:
Rahm Emanuel, chief of staff for President-elect Barack Obama, addresses a gathering of corporate CEOs at an economic conference sponsored by The Wall Street Journal, at the Four Seasons Hotel in Washington, Tuesday, Nov. 18, 2008. Emanuel was a senior advisor to President Bill Clinton and has represented the Fifth Congressional District of Illinois in the House of Representatives since 2002. (AP Photo/J. Scott Applewhite)} add_action( 'genesis_after_post_content', 'single_sub' ); function single_sub() { ?>
Rahm Emanuel
January 30, 2013 by nicholeh Leave a Comment
Filed Under:

subset: commoncrawl


text: Posted 10 Feb 2013 · Report post I was sent out to buy pretzels for my nieces and naturally I bought the Rold Golds Panthers edition. It got me thinking about all the absurd things I have bought just because they said Panthers on them. Chairs, decks of cards, beer, glasses, seat covers, blankets, you name it, I've bought it. So I open the floor up to you guys. Do you have any purchases you are particularly ashamed or proud of? EDIT: This probably doesn't deserve it's own new thread but I picked up some awesome new Panther loots for my birthday! [attachment=6243:Panthersgift.jpg] This is a toast/waffle/grilled cheese maker. It burns a Panther logo into the bread! [attachment=6244:panthersgift2.jpg] These are painting my nieces did for me. They are 2 and 5 years old and they painted these for me in Panthers colors. I will be hanging them in the cave! 2 people like this Share this post Link to post Share on other sites


text:
Former Inter Milan striker Zlatan Ibrahimovic can't believe the price Barcelona paid for him.
Nothing has been confirmed, but there are claims that along with Samuel Eto'o, Barca also parted with £45 million cash for the Swede.
"No player is worth the money clubs pay nowadays," he told Spanish newspapers. "The fault is of the media who put some remarkable price-tags on people, but not even Diego Maradona was worth that much.
"In any case, that is the way the market is now and we must accept that."
Ibra continued to rub salt in the wounds of his former Nerazzurri teammates in an interview with El Mundo Deportivo.
"By signing Eto'o, Inter have got a great champion, but Barcelona remain absolutely the best team there is.
"Since coming here I have been struck by the winning mentality and fact everyone trains to earn a first team shirt.
"Running alongside champions like Iniesta, Leo Messi and Xavi is wonderful. I can't wait to get on the field.
"I want to have fun with the ball along with my teammates."


text:
On Friday night, December 18th, Pat & I braved the traffic to join a couple dozen members of the Unusual Suspects Ham Club, an informal group of hams living in Central Texas, from as far north as Cedar Park north of Austin, down south to San Antonio. Los Generales Mexican Restaurant on Nacogdoches Road, just inside Loop 1604 was the chosen place. But when Greg N5XO scoped out the location in advance, they forget to tell him about the live entertainment with this really, really loud female singer who never took a break in the 2 1/2 hours were were there.
The dinner was scheduled for 6:30pm, fortunately some folks got there early enough to get the establishment to start clearing out an area big enough to hold all of us. It was nearly 7pm by the time we were seated, and the food finally got delivered to our tables about 40 minutes later.


text:
iPad, iPad 2, iPhone 3G, iPhone 3GS, iPhone 4 for AT&T, iPhone 4 for Verizon/Sprint, iPhone 4S, iPod Touch 2nd Generation, iPod Touch 3rd Generation, iPod Touch 4
If you want to:
6,000 + (and growing) factories in our existing network are eager to make it for you.
When it comes to selling in stock products, we are no difference from many other Internet retailers, but we take pride in going extra miles to do something special for you: Have some products custom made for you.
We cannot do everything, but we usually can accommodate a certain changes in product options such as color, size, material, or minor design twist. We primarily work with vendors or factories, not individual artist or craftsman. For custom made products, we usually only cater to bulk size order that meets the minimum quantity requirement of a single factory run. Don't be shy. Ask. What we can accomplish may surprise you!
Read this first: Copyrights & Trademarks


text:
How wireless carriers, machine-to-machine connections and new devices affect corporate productivity.
Featured Stories
Articles about Mobility
Samsung Flow takes on Apple's iOS Handoff, Continuity features
Easily picking up an app where you left off between phones, tablets and other devices is what Flow delivers. It's not quite as seamless as similar features in iOS 8 though.
Microsoft rolls out touch-first Office apps preview for Android phones
Microsoft is rolling out test versions of its standalone, touch-first Word, Excel and PowerPoint apps for Android phones.
YotaPhone 2 e-ink handset launches at $500 for U.S. early birds
Want a phone that lasts for days? The YotaPhone 2 and its innovative e-ink rear display might fit the bill for consumers and office workers alike.
Connected cars expected to be a $1B business for AT&T in 2015
With the U.S. smartphone market nearly saturated, tablets are growing mobile broadband subscriptions for carriers. Connected cars are in the rear-view mirror and gaining, however.
Intuit tightens PayPal integration with data synchronization app
Intuit says roughly 25 percent of its QuickBooks Online users report using PayPal in their accounts.
Mobile app development: The state of play
Recent predictions and reports from analysts paint a challenging picture for business-focused CIOs as they move to a 'mobile-first' app development model.
from TechProResearch »
Enterprise employees choose adult content, app downloads over security
Research suggests despite knowing otherwise, workers worldwide regularly ignore IT policies and place businesses at risk.
Apple, Google urge Obama to reject smartphone backdoor proposals
A multitude of technology firms hope to stop legislation which would allow law enforcement access to data stored in mobile devices.
LG unveils 5.7-inch and 5-inch budget siblings for the G4
LG has released cheaper smartphone options for consumers without the budget to splurge on the G4.
Starbucks and Spotify announce partnership to power your coffee store music experience
Starbucks is known for promoting music to customers so this new partnership with Spotify makes sense. Rewards program members may also earn drink stars for using Spotify Premium.
Asus ZenFone 2 with Intel inside launches May 19 in U.S. for $299
Is it finally Intel's time to shine in mobile devices with an inexpensive off-contract phone that has 4 GB of memory and a quad-core Atom processor?
Brazilian computer giant Positivo sees Q1 profits soar
Foreign exchange hedging mechanisms helped the company - but sales targets for 2015 were revised
Don't let the elements slow you down with Catalyst, LifeProof, and Seidio cases for the Apple iPhone 6 Plus
Many of us take our iPhones everywhere, including our outdoor adventures. Whether you run, cycle, ski, ride, boat, or take part in some other outdoor activity there are a few solid choices to protect your iPhone 6 Plus from the toughest elements.
5 travel gadgets and apps you need for peace of mind
Whether you're traveling on business or for summer vacation, having the right technology can help make your trip worry free.
Xiaomi's 'very limited' sales come to US and Europe for the first time this week
Xiaomi is kicking off its first online sales to Europe and the US, shipping goods directly from its factory in China.
Keeping up with an event-driven mobile workforce
For event management business Cattleyard Collective, success hinges on its ability to mobilise staff quickly and effectively on behalf of clients. The transition from traditional IT infrastructure to a robust cloud-centric setup was essential in keeping staff responsive and agile.
Protect your Galaxy Note Edge and iPhone 6 Plus with Urban Armor Gear (in pictures)
The Samsung Galaxy Note Edge has a unique form factor and the iPhone 6 Plus has a slippery long metal frame. UAG offers solid protection for both and Matthew has been testing them out.
Belkin QODE Ultimate Pro for iPad Air 2: One of the best keyboards
First Impressions: This keyboard case for the iPad Air 2 is a solid investment for those wanting to use the tablet for work. It features a unique ability that makes it one of the most versatile options currently available.
Six clicks: How to reduce your mobile data usage
Mobile data is probably more of a commodity than anything else in our tech lives. Here are six ways to drastically reduce your monthly consumption.
Logitech Type+ for the iPad Air 2 hands on: Getting down to business
This portfolio case with a keyboard is a good option for working with the iPad Air 2.
Hands-on with Microsoft's Lumia 735: Does this affordable device deliver more than selfies?
A mix of compromises and extras makes one of the last Nokia smartphones an intriguing proposition.
WaterField Designs Bolt Briefcase hands-on: High quality functional design for the business traveler
WaterField Designs continues to offer well-designed bags for the professional. The soft Bolt Briefcase looks great at meetings, conferences, and in the airport.
iBridge: Add external storage to your iPhone, iPad
Does your iPhone or iPad need more storage for photos, videos, or other files? This little gadget can add up to 256GB.
Nexus 9 Keyboard Folio and Cover hands-on: HTC's design shows in these accessories
The new Google Nexus 9 is made by HTC and the two official accessories have some cool HTC design elements, but at a rather high price.
iOS 8.1 tips and tricks
Want to get more out of your iPhone or iPad? Here are a selection of tips and tricks to help you unlock more from iOS 8.1.
Fitbit Charge review: Comfortable, easy, and powerful daily activity tracking
The Fitbit Charge is the best daily activity tracker for the masses and Fitbit sets the bar for its powerful ecosystem and reliable tracker lineup.
SoftMaker Office HD for Android hands-on: Desktop level functionality in the palm of your hands
Microsoft markets the power of Office in your hands with the Surface Pro 3, but with SoftMaker Office HD for Android you can get desktop-level functionality on your Android tablet too.
Six Clicks: The best Internet TV Services
Cable? Satellite? Who needs 'em! The top Internet video services can give you more than enough entertainment.
Sony SmartBand Talk review: Much more than just an activity tracker
Sony continues to launch unique mobile designs with a focus on helping you manage your life in any environmental condition.
Top 9 keyboards for the iPad Air hands on (Jan. 2015)
Finding a good keyboard case for the iPad Air can turn Apple's tablet into a great solution for work. After testing dozens of them, I've found these to be the best of the lot.
Dell Venue 8 7000 tablet: Ultra-thin, with Intel RealSense (review)
The premium Android tablet from Dell is thinner than the iPad Air 2 and lighter than the iPad mini.
Full Spectrum: the trouble with Telstra
You could say it is a bad week when you're forced to bring down email and online services as well as reset the passwords of 60,000 customers.
High-tech hide and seek
The holidays have come around again, and that means finding ways to entertain yourself. How about a game of high-tech hide and seek with your friends? Nerdcam shows you how.
Siri and Wolfram: friends with benefits
Once you've found out what the weather's like in Ukraine, and asked what the meaning of life is, Apple's Siri assistant can seem pretty useless — doubly so, if you're in Australia. But if you team it up with Wolfram Alpha, you can get some nice results to questions with substance.
Apple and Samsung's patent (thumb) war
The freight train of patent litigation that is Apple vs. Samsung in the Australian Federal Court continues to barrel along with no signs of slowing down anytime soon. Is there a chance that we could settle this case quickly, like adults?
Defining 'intelligence' in Singapore's iN2015
We take stock of Singapore government's Intelligent Nation 2015 and find out how organizations in the island-state have adopted ICT to drive business growth and innovation.
Tech integration in cars picking up speed
Find out if technology innovations in cars set to become a big trend--and hit--with drivers in the near future.
Microsoft's Ballmer mocks Android phone
At the Web 2.0 Summit in San Francisco, Microsoft CEO Steve Ballmer talks about why users should buy a Windows phone rather than an Android device: "You don't need to be a computer scientist to use a Windows phone and you do to use and Android phone."
The iPhone 4S(teve)
The iPhone that experts told us no one would buy has flown off of the proverbial pre-order shelves in the US. This week's Nerdcam looks at why this happened.
Remembering Steve Jobs: Launching iTunes on Windows
A pivotal moment in Apple's history, CEO Steve Jobs announces iTunes for Windows on October 16, 2003. Jobs stated at the time, "It's probably the best Windows app ever written." The release was also considered a good strategic move by Apple because, for the first time, Windows users could buy music from Apple's online store, giving them a feel for the Apple user experience.
Apple unveils iPhone 4S 'world phone'
Cupertino's latest mobile supports both GSM and CDMA networks.
Apple unveils the iPhone 4S 'world' phone
Apple's senior vice president of marketing, Philip Schiller, announces the latest version of the company's smartphone, the iPhone 4S. The new phone features an A5 chip, dual-core graphics, a longer battery life, better camera, and both CDMA and GSM.
iPod Nano gets new features
At Apple's headquarters, Apple's senior vice president of marketing, Philip Schiller, unveils new iPod Nano features, including a new multitouch icon screen, new sensors for runners, and the ability to wear the device as a watch. The Nano comes in 7 colors, in an 8GB version for $129 and a 16GB version for $149.
First hand from Foxconn
If you're reading this, you're probably one of the many people who's excited to see what's just around the corner for Apple, but have you ever stopped to think what went into making that product? Monologist Mike Daisey has, and he's here to share.
Amazon's Kindle Fire has content, price to compete
Every time a new tablet computer hits the market, experts speculate whether it could be the one to take down Apple's iPad. CNET's Kara Tsuboi explains why Amazon's Kindle Fire could be its stiffest competition yet.
Intel, Google partner on optimized Android phones
At the Intel Developer Forum, Google announced it is teaming up with the chipmaker to optimize Android for Intel-powered mobile devices. Google Senior Vice President Andy Rubin made an appearance during the keynote address with Intel CEO Paul Otellini to announce that all future versions of Android will use the x86 chip architecture.
HTC One (M8) review: The flagship smartphone to beat
The HTC One (M8) ticks most of our boxes: design, build and performance are all excellent, there's no app overload, and HTC Sense is improving with every iteration. Battery life could be better and some of the camera tools may seem unnecessary, but overall it's a winner.
Huawei Ascend Y530, First Take: Entry-level smartphone with 'simple' Android UI option
This affordable smartphone includes a Simple Android Interface, but it's only skin-deep, and to access it you need to be able to navigate the standard UI.
LG G Flex review: Interesting design, shame about the price
There's more to this phablet than its curved chassis, although the moderate screen resolution and lack of storage expansion are disappointing. On the plus side, battery life is good and LG's Android tweaks are largely successful. Overall though, the G Flex is simply way too expensive.
Prestigio MultiPhone 7600 Duo review: An affordable dual-SIM phablet
This phablet offers flexible dual SIM support, an 8-megapixel front camera, NFC and a screen/stand, all for an affordable price. On the minus side, there's an imbalance between the paltry 4GB of internal storage and the generous software bundle, and the screen resolution is moderate considering its size.
Samsung Galaxy NotePRO 12.2 review: Excellent tablet, but no notebook replacement
Compelling though the Galaxy NotePRO 12.2 is, the asking price will buy you a well-appointed notebook that offers more capability for the average mobile professional.
Xero Touch for Android, First Take: Much-improved native mobile Xero client
A quicker and slicker new app for the online Xero accounting service adds automatic reconciliation of bank accounts, allowing Android users to stay on top of their finances.
TIBCO Spotfire Mobile Metrics review: No-fuss KPI delivery to your mobile device
Spotfire Mobile Metrics can collate and analyse your BI data and deliver the key metrics needed to monitor business performance direct to a notebook, smartphone or tablet.
Dell Latitude E7240 review: A nearly-great ultrabook
The Latitude E7240 is solidly made and well connected, delivering impressive performance in a thoughtful design. It's let down by the keyboard and a lack of configuration options, turning what could have been a superb business ultrabook into merely a very good one.
Lenovo ThinkPad X240 review: All-round excellence
The ThinkPad X240 offers plentiful configuration options, tried-and-trusted design, solid build quality and a twin battery configuration that delivers long battery life. Overall, this is an excellent business ultrabook.
Motorola Moto G review: Well-built and affordable, but slow
The Moto G has its drawbacks, but it's well built, and if its features are sufficient for your needs, then it delivers superb value for money. If you're looking to equip a workforce with a basic Android phone, it's arguably the best choice currently available.
LG G2 review: Recommended, unless you need storage expansion
LG has done a great job with the 5.2-inch G2, whose battery life is a big plus. The power button placement is odd, but the G2's main drawback is its lack of MicroSD storage expansion. If don't need this, then it's well worth shortlisting.
Asus Transformer Pad TF701T review: A productive Android tablet/keyboard combo
The 10.1-inch Android-based Transformer Pad TF701T has a decent specification, including a quad-core processor, a high-resolution screen and good battery life from its tablet and keyboard batteries. The design could use a refresh though.
Google Nexus 5 review: Great value for money
Given its specification and performance, the Nexus 5 delivers great value for money — if you can live with its (few) drawbacks. The middling-quality camera and uninspiring chassis design don't worry us, but the lack of MicroSD card storage expansion and moderate battery life are more serious minus points.
Samsung Galaxy Note 3 review: Big, fast and feature-packed, but pricey
The Galaxy Note 3 is a clear step up from its predecessor, with a larger screen in a smaller, thinner, lighter chassis, plenty of usability enhancements, plus impressive performance and battery life. The price tag is the only serious drawback.
Microsoft Surface Pro 2 review: Better, but too heavy and too expensive
The Haswell-based Surface Pro 2 delivers improved performance and battery life, and its dual-angle kickstand is also welcome. However, the Pro 2's unchanged chassis design feels bulkier and heavier than ever, and it becomes very expensive when fully accessorised.


text: Posted 22 Mar 2013 · Report post [url="http://espn.go.com/blog/nfcsouth/post/_/id/46236/eight-in-the-box-fa-winners-and-losers#comment"]http://espn.go.com/b...-losers#comment[/url] "You have to call the Panthers losers in free agency so far, unless you want to give them credit for leading the league in signing mediocre defensive backs" So the Panthers are losers in FA, yet everyone else in the NFC South is a winner? Not saying we've done great, but considering what everyone else has gained/lossed.... we're right with them. No team has significantly improved or gotten worse. Share this post Link to post Share on other sites


text:
President Barack Obama's lead in Wisconsin has narrowed to 2 points over Republican challenger Mitt Romney in the latest Public Policy Poll released Saturday.
Obama led by 49% to 47% in the survey of 979 likely Wisconsin voters. The poll was conducted Thursday through Saturday and has a margin of error of plus or minus 3.1 percentage points.
Two weeks ago, PPP showed Obama leading Romney by 52% to 45%.
"Mitt Romney's received a significant bounce in Wisconsin after his big victory in the debate on Wednesday," said Dean Debnam, president of Public Policy Polling. "The big question now is whether the renewed closeness of the race will be permanent or just temporary."
By 61% to 25%, those polled thought Romney had won the debate.
The poll showed a bounce in Republican enthusiasm about Romney and the election, with Republicans backing Romney 92% to 7%, compared with 89% to 10% just two weeks earlier.
Obama held his own with independent voters, maintaining a 9% lead in the poll.
PPP is a Democratic firm that frequently polls in Wisconsin and others states.
Before Wednesday's debate, Obama led Romney 53% to 42% in a Marquette University Law School poll of 894 likely voters.


text:
Latest Social network service Stories
5 years ago Facebook replaced MySpace. 2 years ago the movie "Social Network" exposed Facebook's beginnings.
One of the biggest challenges that most companies will face is using social networking to reach out to customers.
If you Facebook fan page needs more credibility by adding between 100,000 to 1 million Facebook fans quickly, then the social media
EliNext Group, an alliance of software, mobile, smart TV app development companies, today held a special meeting devoted to the ways businesses can further leverage its expertise in gamification
Online identity verification service miiCard introduces Twitter and LinkedIn validation to create trust on social networks Edinburgh (PRWEB) May 29, 2012
If something happened to you who would tell your friends that you have had over a lifetime? http://www.MyObitList.com is the innovator of emails from the
Job Search Expert Paul Hill, author of The Panic Free Job Search is teaming up with HAPPEN to deliver the first "get hired acceleration workshop" of its kind based on his book.
Company.com, the enterprise social network catering to the small and medium sized business, will launch the next generation of the site, including a new user experience and feature sets, in August.
- To befool; deceive; balk; jilt.
- An illusion; a trick; a cheat.


text:
One worker has been killed and two others are injured in an accident at an under-construction nuclear submarine site in Visakhapatnam where India is building its first class of three nuclear-powered ballistic missile submarines.
The incident took place when the pressure of the hydraulic tank of the Arihant class submarine was being tested and the tank's lid fell on the workers at Building-5 of the shipbuilding cente, Defence Research Development Organisation (DRDO) chief Avinash Chander told reporters.
"The submarines are safe and the accident does not adversely affect the project," the statement from DRDO said.
"During testing of a pressure tank at a facility of Department of Defence R&D related to development of submarines at Vishakhapatnam, an accident took place while testing of a pressure tank at about 5 pm in the evening," a statement by the DRDO stated.
INS Arihant, which is likely to go on trials any day, has not been damaged.
An inquiry into the accident has reportedly been ordered by the DRDO chief.
This comes just a day after a gas leak on an under-construction ship at the Mazagaon dock in Mumbai killed one Navy officer. Carbon dioxide had leaked from a malfunctioning unit during machinery trials of a yet-to-be-commissioned Kolkata class ship.
Only last week, two officers were killed in a fire aboard a Navy submarine INS Sindhuratna, which had been refitted and was being sea-tested.
Also read: Navy orders inquiry as BJP seeks AK Antony's resignation


text:
Pronunciation: clavoillon
Added on: 2010-05-27 Listened: 83 timesin: Add categories to clavoillon
Pronunciation in French [fr]
- Pronunciation by jeromearnaud (Male from France)
0 votes
Could you do it better? different accent? Pronounce clavoillon in French
Accents & languages on maps
- Random word: chambrée
Not satisfied?
Modify word:
· Add languages
· Add categories
Report word:
Is there anything wrong with this word?
Please report it here.


text:
Originally written on Fox Sports Southwest | Last updated 11/12/14
PHILADELPHIA - OCTOBER 11: Andy Reid, head coach of the Philadelphia Eagles works on the sideline against the Tampa Bay Buccaneers at Lincoln Financial Field on October 11, 2009 in Philadelphia, Pennsylvania. (Photo by Jeff Zelevansky/Getty Images)
Back-to-back losses to the Dallas Cowboys in 2009 caused Andy Reid to completely revamp his roster. I recall walking into GM Howie Roseman's office in the 2010 offseason and seeing only two teams' depth charts on the wall: the Eagles and Cowboys. Head coach Andy Reid, who had final say in all personnel decisions at that point, was disgusted by not being able to match up with the Cowboys. The Eagles had bludgeoned Dallas 44-6 in a winner-take-all game at the end of the 2008 season. And they advanced to the NFC Championship Game, where they lost to the Arizona Cardinals. Reid would never say it publicly, but he took a lot of pride in compiling a 17-11 record against the Cowboys. He was a master at exploiting matchups. Eagles running backs Brian Westbrook and later LeSean McCoy gave the Cowboys fits because of their quickness. The Cowboys didn't have linebackers who could cover Westbrook out of the backfield. And the Giants signed linebacker Michael Boley to a lucrative free-agent c...
Comments are closed.
What do you think?To leave a comment, you can:
NFL News
Delivered to your inbox
You'll also receive Yardbarker's daily Top 10, featuring the best sports stories from around the web. Customize your newsletter to get articles on your favorite sports and teams. And the best part? It's free!
Follow us on Twitter
|Best of Yardbarker
|NFL News
|MLB News
Today's Best Stuff
For Bloggers
Join the Yardbarker Network for more promotion, traffic, and money.
Company Info
What is Yardbarker?
Yardbarker is the largest network of sports blogs and pro athlete blogs on the web. This site is the hub of the Yardbarker Network, where our editors and algorithms curate the best sports content from our network and beyond.
|© 2015 Yardbarker, Inc. All rights reserved. Use of this website (including any and all parts and components) constitutes your acceptance of these Terms of Use and Privacy Policy. (What's New?)


text:
Students bragged about it, and that's what ultimately led police to a Kennedale High School teacher who, they say, had sex with five students.
Police said the offenses took place inside the Arlington home of 27-year-old English teacher Brittni Colleps.
A student told News 8 that one of the teenage boys who was involved was bragging about what happened and another student put the scandalous claim in her diary.
When another teacher found the diary in a classroom, she says, the school notified police.
Brittni Colleps' Web site said she was excited about her first school year and looked forward to meeting everyone.
Her photo has now been pulled from the school's Web site. Now the public now knows her by a mugshot taken by Arlington police.
Colleps is accused of having consensual sex with five Kennedale High School students at her home on multiple occasions. At least four of the students involved are football players, and all were described by a friend as honor roll students.
According to an affidavit, Colleps sent sexually explicit photos and text messages, and one student's phone contained a video clip portraying sexual activity with the teacher.
Colleps' neighbors said they had no idea what was going on.
It's a surprise, of course. You never think it will happen in your neighborhood said Sherry Berthiaume. It's just so sad.
One of the biggest questions for those neighbors now is where Colleps' children were during the alleged offenses. She has, one neighbor told News 8, two little girls and a little boy.
When News 8 visited the home of Colleps' mother on Monday afternoon, she hustled a child and suitcase into a waiting car and then declined comment.
Colleps faces five felony counts of improper relationship with a student. The case will now go to the district attorney's office.
Colleps' husband has not yet made a public statement. He has reportedly served in the Army and, according to one friend, had recently been in Louisiana awaiting discharge.
E-mail firstname.lastname@example.com


text:
American Sniper LP: Memorial Edition (Large Print / Paperback)
A celebration of the remarkable life and legacy of fallen American hero Chris Kyle.
This commemorative memorial edition of Kyle's bestselling memoir features the full text of American Sniper, plus more than eighty pages of remembrances by those whose lives he touched personally including his wife, Taya; his parents, brother, and children; Marcus Luttrell and other fellow Navy SEALs; veterans and wounded warriors; lifelong friends; and many others.
He was the top American sniper of all time, called "the legend" by his navy seal brothers, and a hero by those he served with on the home front . . .
From 1999 to 2009, Chris Kyle recorded the most career sniper kills in United States military history. Gripping and unforgettable, Kyle's account of his extraordinary battlefield experiences ranks as one of the greatest war memoirs of all time. American Sniper also honors Kyle's fellow warriors, who raised hell on and off the battlefield. And in moving first-person accounts throughout, Kyle's wife, Taya, speaks openly about the strains of war on their marriage and children.
About the Author
SEAL Team 3 Chief Chris Kyle (1974-2013) was awarded two Silver Stars, five Bronze Stars with Valor, and numerous other citations. Following four combat tours in Iraq, he became chief instructor for training Naval Special Warfare sniper teams. He is the author of American Gun: A History of the U.S. in Ten Firearms. A native Texan, Kyle is survived by his wife, Taya, and their two children.
Scott McEwen is a trial lawyer in San Diego.
Jim DeFelice is the co-author of Chris Kyle s #1 New York Times bestseller American Sniper. He also is the author of Omar Bradley: General at War, the first in-depth critical biography of America s last five-star general, and the co-author of the New York Times bestseller Code Name: Johnny Walker: The Extraordinary Story of the Iraqi Who Risked Everything to Fight with the U.S. Navy SEALs. He writes acclaimed military thrillers, including the Rogue Warrior series from Richard Marcinko, founder of SEAL Team 6, and the novels in the Dreamland series with Dale Brown.


text:
The New York Times has reported that the Justice Department has released a newly declassified version of a May 2004 legal memo regarding warrantless surveillance and data collection activities that President George W. Bush secretly authorized after the terrorist attacks of Sept. 11, 2001.
According to the Times,
The Justice Department's conclusion that the email metadata program was illegal led to a March 2004 confrontation between White House and department officials in the hospital room of Attorney General John Ashcroft, after which nearly the entire top leadership of the department threatened to resign, prompting President Bush to agree to changes.
On May 6, 2004, Mr. Goldsmith, who had taken over as the head of the Office of Legal Counsel the previous year and had reached the conclusion that the Internet data program was illegal, completed a 108-page memo that fully reassessed all aspects of the program.
Mr. Goldsmith concluded that the president's wartime powers both as commander in chief and under Congress's authorization to use military force against Al Qaeda were sufficient to overcome requirements in theForeign Intelligence Surveillance Act, or FISA, for court approval for most of the program.
"The president has inherent constitutional authority as commander in chief and sole organ for the nation in foreign affairs to conduct warrantless surveillance of enemy forces for intelligence purposes to detect and disrupt armed attacks on the United States," he wrote. "Congress does not have the power to restrict that authority."
The theory proposed in the Memo still seem tenuous. The suggestion that a wartime footing — even if applicable — would permit widespread surveillance in violation of the Fourth Amendment finds no basis in the text or history of the Amendment. Moreover, the scope of the program would allow for broad searches unrelated to any individualized suspicion of wrong doing.


text: Publications Search Results Users may wish to consult What's Included in the Bibliography. Results 1 - 1 Results per page: 25 50 100 Sort by: Pub Date (newest) Title Recent Additions Author Clear All Pub. Year Citation 1977 Coleman, Richard P. Husbands, Wives, and Other Earners: Notes from the Family Income Assembly Line. Working Paper. 48, Cambridge Joint Center for Urban Studies. Export Options: RIS EndNote XML [1]


text:
SEE FD support for Windows 8
Created: 23 Oct 2013 • Updated: 24 Oct 2013 | 4 comments
This issue has been solved. See solution.
Hi All,
Can somebody provide me a KB that tells weather SEE FD will support Windows 8 together with their time line? Thanks.
Operating Systems:
Discussion Filed Under:
Comments 4 Comments • Jump to latest comment
Also check this articles.Symantec™ Encryption Desktop 10.3.1 is Now Available
|Created: 2013-08-16
|Updated: 2013-09-26
|Article URL http://www.symantec.com/docs/AL1473
Just to clarify, James' post refers to the renamed PGP product, not SEE-FD.
From the looks of the below article, there are no plans to bring Win8 support to SEE-FD (i.e. it doesn't even advise you to subscribe for further updates):


text:
Utah Jazz v Houston Rockets
Similar imagesView all
James Harden of the Houston Rockets shoots around before the game... News PhotoAIDS,Basketball - NBA Pro,Basketball - Sport,Exercising,Gulf Coast States,Houston - Texas,Houston Rockets,James Harden,Making a Basket,Match - Sport,Sport,Taking a Shot - Sport,Texas,Toyota Center - Houston,USA,Utah Jazz,Vertical,World AIDS DayPhotographer Bill BaptistCollection: National Basketball Association 2012 NBAEHOUSTON, TX - DECEMBER 1: James Harden #13 of the Houston Rockets shoots around before the game against the Utah Jazz supporting a World AIDS Day warm up on December 1, 2012 at the Toyota Center in Houston, Texas. NOTE TO USER: User expressly acknowledges and agrees that, by downloading and or using this photograph, User is consenting to the terms and conditions of the Getty Images License Agreement. Mandatory Copyright Notice: Copyright 2012 NBAE (Photo by Bill Baptist/NBAE via Getty Images)


text:
City of St. Louis, MO
St. Louis is located in St. Louis County in Missouri. The median income is $34,582 and the median home value is $119,200. The population is 42% White, 47% Black, 3% Asian and 0% Native American or Native Hawaiian. 5% identify as another race or ethnicity, or two or more races. 3% of residents are of Hispanic or Latino origin. For more on schools, real estate, business, and health care in St. Louis, see each of the tabs to the leftabove. Walkability can be a key driver of the overall livability of a community. St. Louis has a Walk Score® of 60.
Median Household Income$34,582
Median Home Price$119,200
SchoolsIn St. Louis, Missouri
The quality of a school district is often a top factor for parents considering where to live. Strong school systems attract new residents and help their cities prosper. See how St. Louis rates.Public Schools in St. Louis, Missouri
Private Schools in St. Louis, Missouri
BusinessIn St. Louis, Missouri
Being a great place to live and being a great place to work, often go hand-in-hand. Here's what the business and employment climate is like in St. Louis.Major industries in St. Louis, Missouri
Employment in St. Louis, Missouri
HealthIn St. Louis, Missouri
Access to quality hospitals, doctors and healthcare providers are just part of what make a healthy community. Here's how to get and stay healthy in St. Louis.Healthy lifestyles in St. Louis, Missouri
Doctors in St. Louis, Missouri
Hospitals in St. Louis, Missouri
Real EstateIn St. Louis, Missouri
Ready to move to St. Louis? There are a variety of housing options in the area. Click below to learn about the recent trends, average home values and even a few current real estate listings in St. Louis.Housing in St. Louis, Missouri
Home Values in St. Louis, Missouri


text:
SMS Composer
50,000 - 100,000 downloadsAdd this app to your listsTweets por @Appszoom
Non threaded SMS app.
Enables you to:
Save template messages and signatures
Send SMS to multiple recipients
SMS Forward, Reply, Resend, Share
View SMS messages in non threaded Inbox and Sent views
Voice recognition
Notifications can be turned on/off in settings menu on main screen.
Tags: sms composer, sms composer apk, sms composer 1.9 apk, composing text message bug, compose sms, sms composer in, sms composer.apk, smscomposer, sms compose, sms composer notification.
App redesign
New features
Bug Fixes
(64 stars)
Please enable Hebrew on this application.
(64 stars)
(64 stars)
(64 stars)
Android stock messaging does not have classic view like inbox/outbox/sent/draft in separate folders which is very much useful for accessing / partial deleting etc. SMS compose provides very easy classic view with inbox/outbox/sent only i.e it does not save as draft if you close or accidentally press back during composing an sms. It consumes very little power unlike easy sms/ pansi sms
(64 stars)
Please add an ad free app ... And also I don't get the recipient number when I reply using sms composer.... please look into this issue...
(64 stars)
- Using this app, I typed a message, and chose to send it to around 75 contacts. - It started sending, and continued for such a long time - The mobile operator charged me so much, telling I crossed 100 sms ! - my sms was just 140 characters. (and 75 such sms) - Looks like the app sends so much more in the background. - uninstalled immediately
(64 stars)
Need improvement... Lacking sms pop ups


text:
Benedict XVI greets faithful for last time from balcony of papal retreat
More News from Agence France-Presse, Associated Press
CASTEL GANDOLFO, Italy—Pope Benedict XVI has arrived at the papal estate in Castel Gandolfo, as crowds in the town square cheered and waved flags.
Earlier, as a helicopter whirred overhead, bells started ringing more furiously than before. The crowd in the Castel Gandolfo square started cheering and chanting, "Benedetto, Benedetto" in rhythm.
As Pope Benedict XVI appeared on the balcony at Castel Gandolfo, people in the crowd below started screaming his name. He must wait before he could even speak to them.
"I will no longer be pope but a simple pilgrim who is starting out on the last part of his pilgrimage on this Earth," he told thousands of well-wishers gathered in the square below.
Get Inquirer updates while on the go, add us on these apps:
Disclaimer: The comments uploaded on this site do not necessarily represent or reflect the views of management and owner of INQUIRER.net. We reserve the right to exclude comments that we deem to be inconsistent with our editorial standards.
To subscribe to the Philippine Daily Inquirer newspaper in the Philippines, call +63 2 896-6000 for Metro Manila and Metro Cebu or email your subscription request here.
Factual errors? Contact the Philippine Daily Inquirer's day desk. Believe this article violates journalistic ethics? Contact the Inquirer's Reader's Advocate. Or write The Readers' Advocate:
c/o Philippine Daily Inquirer Chino Roces Avenue corner Yague and Mascardo Streets, Makati City,Metro Manila, Philippines Or fax nos. +63 2 8974793 to 94


text:
ADB Wireless (no-root)
10,000 - 50,000 downloadsAdd this app to your lists
+ By Sherdle
✔ No-Root wireless ADB
✔ Easy to set up
✔ Portable & Tiny Desktop Application
✔ ADB functions the same as it would with a cable
Connect in seconds
1. Connect your phone to your desktop.
2. Click the "set up" button in the desktop app.
3. Unplug your phone and click "connect wireless".
For more information & the desktop app, go to http://sherdle.com/adb
-Only works on 4.0.4. or higher
-Windows pc with java is required
-This app will only work if both (Pc & Phone) are connected to the same wifi network.
-Setup with a cable is required
Note that this application is in an early beta stage, especially the desktop application is very unstable. Due to the limitations of having no-root access, the phone gets disconnected after a certain time, though we have a solution in mind and we will start working on it soon.
If you have any questions or suggestions, feel free to contact us.
Tags: adb wifi no root, adb wireless root, adb wireless no root, wireless adb connection, wireless adb no root, adb wireless without root, adb wirelesw no root, wifi adb, adb wireless authentication, no adb connection.
Play store Beta release
(34 stars)
(34 stars)
But takes patience to get this to work
(34 stars)
It allows me to debug while I have a peripheral connected to the USB port, great :) Setup is very straightforward. Thanks!
(34 stars)
As a newcomer to android studio, I wasted hours trying to run an app using the emulator: disgracefully slow; or with USB connection: unreliable, kept disconnecting. Once connected with this app, my nexus stays connected and I can concentrate on developing my app. Thanks guys!
(34 stars)
waiting for you
(34 stars)
Heartiest thanks for your efforts
(34 stars)
Its a showing only images.. Not an app


text:
Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce.
The toxin genes tcdA and tcdB were amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of both tcdA and tcdB genes in the vector have been verified by DNA sequencing. The constructs were transformed into B. megaterium protoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB) were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination.
We have generated the full length and active recombinant TcdA and TcdB in Bacillus megaterium.
Clostridium difficile is a Gram-positive, spore-forming anaerobic bacillus. It is the most common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of pseudomembranous colitis [1]. Antibiotic usage results in a reduction of commensal microflora in the gut, which permits C. difficile to proliferate more extensively, leading to the production of toxins [2]. C. difficile associated diarrhea (CDAD) include a range of symptoms varying from mild diarrhea to severe fulminate lethal disease [3]. Recent outbreaks of highly virulent C. difficile strains [4,5] have increased the urgency to devote greater resources towards the understanding of the molecular, genetic, and biochemical basis for the pathogenesis, with a view to use such information to develop novel preventative and treatment modalities.
Two exotoxins produced by toxigenic C. difficile, toxin A (TcdA) and toxin B (TcdB), are most extensively studied and thought to be major virulent factors of CDAD [6,7]. TcdA (308 kDa) and TcdB (269 kDa) belong to the large clostridial cytotoxin (LCT) family and share 49% amino acid identity [8]. The two toxins have a similar structure containing a putative receptor binding domain (RBD), a transmembrane domain (TMD), and a glucosyltransferase domain [8,9]. After receptor-mediated internalization and intracellular cleavage, the toxins glucosylate members of the Rho-Rac family of small GTPases at a specific threonine residue in host intestinal epithelial cells, leading to alterations in the actin cytoskeleton, massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa [7]. Purified TcdA possesses potent enterotoxic and pro-inflammatory activity, as determined in ligated intestinal loop studies in animals [10,11]. TcdA is also cytotoxic to cultured cells in a picomolar to nanomolar range. TcdB, more cytotoxic to cultured cells than TcdA, was previously reported to exhibit no enterotoxic activity in animals [11,12], but recent studies have found enterotoxic and proinflammatory activities in human intestinal xenografts in severe combined immunodeficient (SCID) mice [13]. Furthermore, the TcdA-B+ C. difficile strains are responsible for pseudomembranous colitis in some patients [14,15].
It is desirable to obtain relatively pure and biologically active TcdA and TcdB for studying the pathogenesis of CDAD and host immune response to the infection and for generating immunological tools for research and clinical diagnosis. The native toxins are usually purified from toxigenic C. difficile culture supernatant, which involves multiple steps and the purity is often unsatisfactory [16-18]. Attempts have been made to clone and express C. difficile toxins in Escherichia coli [19-21], but it is unclear whether or not purified toxins were obtained from the bacterial lysate in these studies. The Gram-positive Bacillus megaterium expression system may offer an alternative for the expression of C. difficile toxins due to several advantages over the E. coli system, including the lack of alkaline proteases activity and endotoxin LPS, capable of secreting expressed heterologous protein into the medium [22,23]. Although the expression level was low, Burger et al [24] were the first to successfully express and obtain the purified recombinant TcdA in B. megaterium. In this study, we have expressed the full-length of both TcdA and TcdB in B. megaterium. We were able to obtain an average of 5 – 10 mg of highly purified recombinant proteins from one liter of total bacterial culture. Both recombinant TcdA and TcdB were biologically active similar to their native purified toxins.
Cloning and expression of the recombinant toxins
After enzyme (BsrGI/KpnI) digestion of pHis1522 vector and PCR products, the ligation was set up and the mixture was incubated at 4°C for 2 days. The transformation was carried out following the standard protocol and more than 90 bacterial colonies were picked up. The plasmids (named pHis-TcdB) from these colonies were subjected to screening by digestion with a variety of enzymes. pHis-TcdB from multiple clones was used to transform B. megaterium protoplasts, and tetracycline-resistant clones were picked up. After induction with xylose, the bacterial crude extracts from approximately 30 different transformed clones were subjected to screening for activity causing rounding of cultured cells. The clones with high activities were selected and a Coomassie-staining gel showed expression of a protein of about 270 kDa when xylose was added for induction (Figure 1A Lane1). This protein was purified by Ni-affinity column from a total crude extract, resulting in a major band with molecular weight about 270 kDa and some weak contaminant bands of lower molecular weights (Figure 1A). A further step of ion-exchange purification resulted in a highly pure 270 kDa-protein without any visible contaminant bands on Coomassie-stained gels (Figure 1B). Western blot analysis using specific antibodies against His-tags (data not shown) and toxin B (Figure 1C) identified the 270 kDa protein from B. megaterium lysate as the recombinant His-tagged toxin B (rTcdB). From one liter of bacterial culture, approximately 10 mg of rTcdB was obtained. The pHis-TcdB used to transform the B. megaterium was subjected to DNA sequencing for the verification of tcdB gene, and no mutation was found (data not shown).
B. megaterium has a secretory pathway [22,23], which transports the expressed heterologous proteins into the medium. Because the native toxins are secreted into medium by toxigenic C. difficile, we attempted the expression of rTcdB in a secretory form. The DNA sequence encoding a 28-amino-acid signal peptide derived from B. megaterium extracellular esterase LipA [22] was synthesized and inserted at the 5' of tcdB. The resulting pHis-SP-TcdB was transformed into B. megaterium. Figure 1D shows the presence of a 270 kDa band from the concentrated bacterial culture supernatant on a Coomassie stained SDS-PAGE gel (lane 2). Western blot and cytotoxicity assays confirmed the expression of the secretory form of rTcdB. The expression level of this secretory form was, however, very low and we were unable to obtain a sufficient amount of purified rTcdB from the supernatant.
A similar strategy was utilized to clone and express recombinant TcdA. On a Coomassie stained SDS-PAGE gel of total bacterial lysate, a clear protein band of 308 kDa appeared only upon induction with xylose in pHis-TcdA-transformed B. megaterium (Figure 2A). His-tag combined with thyroglobulin affinity purifications resulted in a single strong band (Figure 2A Lane 5), which was confirmed to be TcdA by western blot analysis (Figure 2B). The pHis-TcdA used to transform the B. megaterium was subjected to DNA sequencing for the verification of tcdA gene, and no mutation was found (data not shown).
Figure 1. Expression and purification of recombinant TcdB. pHis-TcdB plasmid was transformed to B. megaterium protoplasts. Several transformed colonies were picked up and the expression of rTcdB was induced by xylose. (A) His-tag affinity purification of total lysate from B. megaterium. Lane 1: total bacterial lysate; Lane2: flow-through; Lane 3: wash; lane 4–6: elution fraction 1–3. (B) Anion-exchanging column fractionation after Ni-affinity chromatography. nTcdB: purified native TcdB from C. difficile culture supernatant. Lane 1: Elution fraction 2 from (A); Lane 2–8: fractions from a gradient salt elution. Fraction 5 and 6 contain purified rTcdB. (C) Western blot results of purified native TcdB and rTcdB of combined fraction 5 and 6 from (B). (D) Coomassie staining of SDS-PAGE from total sonication lysates a pHis-TcdB transformed clone (lane 1), concentrated supernatant from pHis-SP-TcdB transformed clone (lane 2) and purified native TcdB (lane 3). M indicates molecular weight marker and 250 kDa is showed.
Figure 2. Expression and purification of recombinant TcdA. pHis-TcdA plasmid was transformed into B. megaterium protoplasts. Over 20 transformed colonies were picked up and the expression of rTcdA was induced by xylose. (A) Coomassie staining of SDS-PAGE from crude bacterial extracts: Lane 1 shows no xylose induction; Lane 2 to 4 shows three different clones with xylose induction; Lane 5 shows purified rTcdA after Ni-affinity and thyroglobulin chromatograph. M shows molecular weight marker and the top band indicates 250 kDa. (B) Western blot results of the purified native TcdA (lane 1) and rTcdA (lane 2).
Cytopathic effect of recombinant toxins
We compared the cytopathic effects of recombinant and native toxins on cultured human epithelial HT-29 (data not shown) and mouse intestinal epithelial CT26 cells (Figure 3). rTcdA (Figure 3I and 3K) at 20 or 200 ng/ml caused CT26 cell rounding similarly to native TcdA (Figure 3J and 3L). Both rTcdB and native TcdB were more potent, capable of causing cell rounding at a much lower concentration than those of rTcdA or native TcdA (Figure 3E, F, G, and 3H). After xylose induction, both supernatant (Figure 3D) and total cell lysate (data not shown) from pHis-SP-TcdB transformed B. megaterium caused cell rounding, whereas the bacterial culture supernatants from pHis-TcdB transformed B. megaterium with (Figure 3C) or without (Figure 3B) xylose induction did not cause cell rounding. The same concentration of xylose in BHI medium did not cause any cell rounding (data not shown), suggesting the effects were specifically caused by the secreted toxin. Furthermore, the cytopathic effects of both rTcdA and native TcdA were blocked completely by a rabbit polyclonal antibody against TcdA (Figure 3M and 3N).
Figure 3. Cytopathic effect of recombinant toxins. Mouse colonic epithelial CT26 cells in a 96-well plate were untreated (A) or treated as following: 10 μl culture supernatant from pHis-TcdB-transformed B. megaterium without (B) or with (C) xylose induction; (D) 10 μl culture supernatant from pHis-SP-TcdB-transformed B. megaterium with xylose induction; (E) rTcdB (1 ng/ml); (F) native TcdB (1 ng/ml); (G) rTcdB (10 ng/ml); (H) native TcdB (10 ng/ml); (I) rTcdA (20 ng/ml); (J) native TcdA (20 ng/ml); (K) rTcdA (200 ng/ml); (L) native TcdA (200 ng/ml); (M) rTcdA (200 ng/ml) plus 1 μl/well of rabbit anti-TcdA serum; or (N) native TcdA (200 ng/ml) plus 1 μl/well of rabbit anti-TcdA serum. Cells were incubated overnight and the morphological changes were observed under a phase-contrast microscope.
Cytotoxic effect of recombinant toxins
It has been demonstrated that both TcdA and TcdB induce intestinal epithelial cells to undergo apoptosis [25-27]. We next compared the cytotoxicity of recombinant toxins to their native counterparts on cultured epithelial cells. As shown in Figure 4A, both native and recombinant TcdA induced a comparable and dose-dependent cell death of CT26 cells. Similarly, the exposure of CT26 cells to rTcdB induced a comparable cell death with nTcdB as determined by MTT assay (Figure 4B). These data indicate that the recombinant TcdA and TcdB have similar cytotoxic activity to their native counterparts in CT26 cells.
Figure 4. Cytotoxic effect of recombinant toxins. Mouse colonic epithelial CT26 cells in a 96-well plate were exposed to native (□) or recombinant (Δ) TcdA (A) or exposed to native (□) or recombinant (Δ) TcdB (B) for 72 hr. The MTT assay was performed and cell viability was expressed as the percentage of survival cells without exposure to toxins.
Glucosylation of Rac1 by recombinant toxins
C. difficile TcdA and TcdB target host cell Rho GTPase by glucosylating these proteins at the specific Thr37 (Rho A) or Thr35 (CDC42 and Rac1) residues [28,29]. To determine whether recombinant toxins can glucosylate Rac1 protein of host cells, mouse intestinal epithelial CT26 cells were treated with recombinant or native TcdA and TcdB for 5 hr. Western blot showed that both rTcdA and nTcdA treatment induced a dose-dependent, reduced recognition of Rac1 by monoclonal antibody (anti-Rac1 clone 102) that recognizes non-glucosylated Rac1 [30] (Figure 5A). The reduced recognition of Rac1 was not the results of protein degradation, because total Rac1 protein remained unchanged as determined by western blot with an antibody (clone 23A8) recognizing both glucosylated and unmodified Rac1 (data not shown). Both rTcdA and nTcdA at 40 ng/ml resulted in a complete glucosylation of Rac1 whereas 8 or 1.6 ng/ml of TcdA led to a partial glucosylation. rTcdB or nTcdB exposure resulted in a completely glucosylation of Rac1 at a dose range from 0.4 – 2 ng/ml (Figure 5B), indicating that the glucosyltransferase activity of TcdB was more potent than TcdA. These results were consistent with the activity of native C. difficile toxins in which TcdB is more toxic to cultured cells than TcdA. The treatment of rTcdA and rTcdB also led to the glucosylation of Rac1 in RAW 264.7 and CHO cells (data not shown).
Both TcdA and TcdB are thought to bind to specific cellular receptor(s), which mediate their cellular uptake through endocytosis [31-33]. Lysosomotropic agents such as chloroquine and NH4Cl can inhibit toxin-mediated cytotoxicity, suggesting that the endosomal acidification is involved in toxin intracellular trafficking [34,35]. To determine whether or not the cellular activity of the recombinant toxins also require endosomal acidification, CT26 cells were pre-incubated with NH4Cl for 30 min before the toxin exposure. While the exposure of CT26 cells to rTcdA or rTcdB induced the glucosylation of Rac1, pretreatment of cells with ammonium chloride completely blocked such an activity (Figure 5C).
Figure 5. Glucosylation of Rac1 by recombinant toxins. CT26 cells were either untreated or treated with the indicated amount of native or recombinant TcdA (A) and TcdB (B) for 5 hr. (C) CT26 cells were pretreated with ammonia chloride for 30 min before the exposure of the indicated amount of recombinant toxins for 5 hr. Cells were harvested and western blot was performed as described in the Materials and Methods. Monoclonal antibody clone 102 recognizes unglucosylated Rac1 and has a reduced affinity to glucosylated Rac1.
Disruption of tight junctions of intestinal epithelial cells by recombinant toxins
It is known that TcdA and TcdB alter the structure of intestinal epithelia by disrupting tight junctions [36]. We investigated whether or not the recombinant toxins we generated have similar activities. Human intestinal epithelial HCT-8 cells were cultured in a transwell for 10 to 14 days until transepithelial resistance (TER) reached 1000 Ω*cm2 and tight junctions were formed (Figure 6A). rTcdA did not alter cell-cell connections of HCT-8 cells within 4 hr of treatment (Figure 6B and 6C) but disrupted tight junctions after 6 hr of treatment (Figure 6D). rTcdB, at the same concentration with rTcdA, completely disrupted the tight junctions of HCT-8 within 2 hr of treatment (Figure 6E and 6F).
Figure 6. Disruption of tight junctions by recombinant toxins. HCT-8 cells on transwells were cultured untill the formation of tight junctions. The polarized monolayers were untreated (A) or treated with 300 ng/ml of rTcdA for 2 hr (B), 4 hr (C), and 6 hr (D); or with rTcdB 300 ng/ml for 2 hr (E), and 4 hr (F). Cells on the transwell membrane were fixed and stained with anti-oocludin and fluorochrome-conjugated secondary antibodies, and then visualized under a confocal microscope.
CDAD is largely attributed to the bacterial exotoxins TcdA and TcdB, although other virulence factors probably also play some roles [7]. Both toxins glucosylate Rho GTPase family proteins, leading to their inactivation [1,7]. To study the pathogenesis of CDAD and host immune response to the toxins, and to use the toxins as a research tool for signal transduction involving Rho GTPases, it is necessary to obtain large quantities of highly purified active TcdA and TcdB. Native toxins can be purified from toxigenic C. difficile culture supernatant. The purification process however usually involves multiple steps such as ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography [17,18]. In the case of TcdA, affinity purification through thyroglobulin is available and relatively pure TcdA can be obtained ([16] and unpublished data). TcdB however is often contaminated with some unknown substance even after extensive purification steps ([18] and unpublished data). Gene engineering to generate recombinant toxins seems to be a logical approach for obtaining large quantities of pure toxin proteins. The E. coli system is popular and is widely used for the expression of recombinant proteins. Several studies have reported to have cloned and expressed active C. difficile toxins in E. coli, but failed to demonstrate that the purified toxins were obtained from bacterial extracts [19-21]. Burger et al was the first to report a successful expression of recombinant TcdA using Bacillus megaterium system [24]. Recombinant TcdB, however, has not yet been stably expressed. In this study, we were successfully expressing a full-length recombinant TcdB in B. megaterium and greatly enhanced the expression level of recombinant TcdA.
Because E. coli expression system is widely used and easy-to-handle, we have utilized various E. coli expression vector systems to clone and express both toxins and found that both recombinant TcdA and TcdB were expressed in E. coli but we were unable to obtain the purified toxins from the bacterial lysate (unpublished data). The large size of the heterologous proteins may possibly be prone to protease digestion, and intrinsic protease activities of these toxins [37,38] may also contribute to the instability. Another possible reason for the inability to obtain purified recombinant toxins from E. coli is that the expression level was too low. In fact, large proteins are difficult to express at high levels in E. coli. Furthermore, an unusually high content of AT (~70%) in these clostridial gene sequences and a different codon -usage make them particularly difficult to express at a high level in E. coli [39,40].
There are several advantages associated with B. megaterium expression system including lack of alkaline phosphatase and endotoxin LPS. More importantly, B. megaterium has been successfully used to express rTcdA [24], we therefore decided to use this system. Initially, we followed the reported cloning strategy but we were unsuccessful. This might be due to the extremely low ligation efficiency [24] when putting several fragments together during the cloning. We then changed our strategy to a direct PCR amplification of whole toxin genes. However, this strategy was associated with a potential risk that PCR might introduce mutations due to the large size of the genes (7101 bp and 8133 bp for tcdB and tcdA respectively). Because of the large size of both genes, it was impossible to sequence all constructs in order to identify the correct clones. We therefore took a different approach, screening for recombinant protein expression and cytotoxicity. We screened as many colonies as possible (more than 90) after ligation (data not shown) and then transformed these constructed shuttle vectors into B. megaterium protoplasts. The resulting colonies were subjected to screening again by examining the cytotoxic activity in bacterial crude extracts. The specific expression of recombinant toxins from the positive colonies (with cytotoxic activity) was then determined by western blot. The plasmids encoding tcdA or tcdB respectively were finally subjected to DNA sequencing and no single mutation was found in either of the toxin genes.
The average yields of recombinant TcdA and TcdB were about 5 – 10 mg/liter of bacterial culture, which were much higher than that of native toxins from C. difficile culture, and up to 20-fold higher than the amount of rTcdA reported by Burger et al [24]. The fact that we used a different shuttle vector from that of Burger et al [24] might explain our higher yields of recombinant proteins. In addition, we have found out a twelve- to sixteen-hour period of induction with xylose yielded more recombinant proteins than five hours as suggested by the manufacturer. The other possible explanation was that the tcdA gene was fused into orf1 of the vector in the construct reported by Burger et al [24], resulting a modified N-terminus of recombinant TcdA. This modification might affect the efficiency of protein expression and/or its stability. We inserted the tcdA or tcdB gene into the BsrGI site and the protein translation directly started from toxin gene, resulting in recombinant toxins without any N-terminal modification.
To facilitate purification, a His6 tag was fused to the C-terminus of both toxins. One-step of Ni-affinity chromatography resulted in approximately 70–80% purity of recombinant toxins as determined by Coomassie staining of SDS-PAGE (Figure 1 and data not shown). To achieve a greater purity, an additional step was introduced after Ni-affinity chromatography. For rTcdA, thyroglobulin affinity purification was used. rTcdB was further purified by an anion-exchange fractionation (Figure 1B). These additional purification steps for rTcdA and rTcdB, respectively, led to highly pure recombinant toxins. No additional band was seen on a Coomassie-stained gel (Figure 1B for rTcdB, and Figure 2A for rTcdA). In addition, there were no detectable TLR2 or TLR4 stimulants within the purified recombinant toxins as determined by sensitive bioassays (unpublished data). This is important because any use of the impure proteins to study host immune response or signaling transduction would possibly result in unintended consequences.
Unlike E. coli, a protein secretory pathway is present in B. megaterium [22,23]. A 28-amino-acid signal peptide of B. megaterium extracellular esterase LipA can direct the transport of expressed heterologous proteins into medium and the sequence is removed by a protease after secretion [22]. Because of the nature of exotoxins of native toxins and relatively easy purification from supernatant without the need for sonication or other means of cell disruptions, we sought to express rTcdB extracellularly. After inserting the gene of the leader signaling peptide at the 5' of tcdB, we were able to generate active secretory rTcdB (Figure 3D) and Coomassie staining of concentrated supernatant on a SDS-PAGE gel and western blot showed rTcdB band (Figure 1D and data not shown). However, for some unknown reasons, the expression level of rTcdB was significantly decreased when the leader sequence was introduced to the construct. In addition, we found the secretion was very inefficient and the majority of toxins were still trapped inside of the bacteria, since the total bacterial lysate contained the majority of toxin activity (data not shown). This secretory pathway may not be efficient for such a large size of protein. The low amount of rTcdB in the medium might also be due to the inefficient refolding after their extracellular transport.
In this study, we have successfully expressed the full-length of recombinant C. difficile toxin A and B proteins in B. megaterium and greatly improved the yield of the recombinant toxins. We were able to obtain 5 – 10 mg of highly purified rTcdA or rTcdB from one liter of bacterial culture. These recombinant toxins had similar biological activities with their native counterparts. We believe the recombinant toxin proteins will provide an invaluable tool for studying the pathogenesis of C. difficile associated disease and host immune response to toxins, as well as tools for studying the signaling transduction involving Rho GTPase family proteins.
Cell lines
Human intestinal epithelial cell lines HCT-8 and mouse colonic epithelial cell CT26 were purchased from ATCC (Manassas, VA). All these cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA) containing 2 mM L-glutamine, 100 U/ml penicillin G, 50 μg/ml streptomycin sulfate, and 10% fetal bovine serum.
Purification of native toxins
Native TcdA and TcdB were purified from C. difficile strain VPI 10463 (kindly provided by Abraham L. Sonenshein, Tufts University School of Medicine) as described previously [41]. Briefly, a dialysis bag (100 kDa cutoff, Millpore, Billerica, MA) containing 100 ml of 0.9% NaCl immersing in a total volume of 1 liter of brain heart infusion (Difco, Lawrence, KS) was inoculated with 5 ml of an overnight culture of C. difficile, and the culture was grown in an anaerobic chamber (Bactron BACLITE-1, Sheldon Manufacturing Inc., Cornelius, OR) at 37°C for 72 hr. The supernatant collected from the dialysis bag was concentrated by ultrafiltration through an Amicon XM-100 membrane (Millipore). The concentrated supernatants were dialyzed against Tris-HCl (50 mM, pH 7.5) buffer overnight and loaded onto a HiTrap DEAE column (Amersham Biosciences, Piscataway, NJ). The fractions containing toxins were collected and then passed through a thyroglobulin column, and the flow-through was further passed through a Mono Q column (Amersham Biosciences). The elutions from thyroglobulin and mono Q yielded TcdA and TcdB respectively.
Constructs and cloning
The tcdB gene was amplified from C. difficile (VPI 10463) chromosomal DNA using forward primer 5'-GCGCTGTACAATGAGTTTAGTTAATAGAAAAC-3' and reverse primer 5'-ATATATGGTACCCTTCACTAATCACTAATTGAGC-3'. The PCR product was digested by BsrGI and KpnI enzymes, and then ligated to pHis1522 vector (MoBiTec, Goettingen, Germany). The full-length of tcdA gene was amplified using the primers 5'-GCGCTGTACAATGTCTTTAATATCTAAAGAAGAGTTAA-3' and 5'-ATATGCATGCCCATA TAT CCCAGGGGCTTTTA-3'. The PCR product was digested by BsrGI and SphI, and then inserted into pHis1522 vector. Both sequences of tcdA and tcdB genes in pHis 1522 vector have been confirmed by DNA sequence using a panel of primers (Table 1). The gene encoding a 28-amino-acid signal peptide of B. megaterium extracellular esterase LipA [22] that directs protein secretion in the secretory pathway of B. megaterium was synthesized by GeneArt (Regensburg, Germany) and inserted at the site of BsrGI of the tcdB construct. All restriction endonucleases were purchased from New England Biolabs (Cambridge, MA). All DNA cloning and plasmid construction were performed at Tufts University and approved by the Institutional Biosafety Committees and conformed with NIH Recombinant DNA technology guidelines.
Table 1. The sequences of DNA sequencing primers
Expression of recombinant toxins
The transformation of Bacillus megaterium protoplasts were performed according to the manufacturer's instruction (MoBiTec). The transformed B. megaterium colonies were picked up and grown overnight in an LB medium supplemented with 10 μg/ml tetracycline. The overnight cultures were diluted 1:30 in LB medium containing tetracycline and grown to an optical density (OD600) around 0.3 before the addition of xylose (0.5% w/v) for inducing protein expression. Bacteria were harvested by centrifugation after 12 to 16 hr of induction. In case of the secretory rTcdB, the culture supernatant was also collected.
Purification of recombinant toxins
Purification of recombinant His-tagged rTcdB from bacterial lysate was performed by Ni-affinity chromatography following an ion-exchange fractionation. Briefly, the B. megaterium culture pellet (from 100 ml of culture) was resuspended in 5 ml lysis buffer (300 mM NaCl, 20 mM imidazole, 20 mM NaH2PO4, 500 μM EDTA, protease inhibitor cocktail (Cat #P8849, Sigma), adjusted to pH 8.0). Cells were disrupted by sonication and the lysate was centrifuged at 14,000 g for 20 min. The supernatant was then passed through a nickel-charged HiTrap chelating HP column (Amersham Biosciences, Piscataway, NJ) and the bound His-tagged rTcdB was eluted with an elution buffer containing 250 mM imidazole, 300 mM NaCl, and 20 mM NaH2PO4, pH 8.0. The eluent was then desalted and applied to HiTrap Q column (Amersham Biosciences) and rTcdB was eluted by a gradient concentration of NaCl solution. The fraction containing rTcdB was combined and stored at -80°C until use. To purify rTcdB from supernatant, the bacterial supernatant was passed through a 0.45 mm filter first, and then concentrated through ultrafiltration with a 100 kDa cutoff membrane (Millipore). The concentrated supernatant was then performed with Ni-affinity chromatography following an ion-exchange fractionation as described above. rTcdA was purified by Ni-affinity chromatography following an thyroglobulin affinity column. The method for thyroglobulin purification has been described elsewhere [16].
SDS-PAGE and western blot
CT26 cells were exposed to toxins for 5 hr before they were harvested. In other experiments, ammonium chloride was pre-incubated with the cells for 30 min before the cells were exposed to toxins. The methods for SDS-PAGE and western blot have been described previously [42,43]. Briefly, the samples were boiled for 5 min in 1× NuPage SDS sample buffer (Invitrogen) and then loaded on a gradient (4 – 20%) sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (BioRad, Hercules, CA). After electrophoresis, the gels were stained with GelCode Blue (Pierce, Rockford, IL) according to the manufacturer's instructions. For western blot, TcdB-specific monoclonal antibody (clone 5A8-E11, Meridian Life Science, Inc., Memphis, TN), and TcdA-specific monoclonal antibodies (clone PCG4.1, Meridian Life Science, Inc.; or clone A1E6 generated in our laboratory) and HRP-conjugated anti-mouse IgG (Amersham Biochiences) were used as the primary and secondary antibodies respectively and the protein bands were visualized by an enhanced chemiluminescence assay (ECL, Amersham Biochiences). To determine Rac1 glucosylation, the antibody (clone 102, BD Biosciences, San Diego, CA) specific against non-glucosylated form of Rac1 was used. Anti-actin antibody (clone AC-40, Sigma, St. Louis, MO) was used to monitor an equal loading of samples.
Cytotoxicity assay
Subconfluent CT26 cells (2 ×104/well) seeded in 96-well plates were incubated with toxins, bacterial culture supernatants, or crude extracts. For blocking experiments, 1 μl/well of rabbit anti-serum against TcdA was added simultaneously with toxins. Cells were cultured overnight and the morphological changes of CT26 cells were observed by light microscopy with a CCD camera. For the MTT assay, after 3 days of incubation, 10 μL of MTT (5 mg/ml) were added to each well and the plate was further incubated at 37°C for 2 h. The formazan was solubilized with acidic isopropanol (0.4 N HCl in absolute isopropanol), and absorbance at 570 nm was measured using a 96-well ELISA reader. Cell viability was expressed as the percentage of survival of the control wells.
Disruption of tight junctions by recombinant toxins
HCT-8 cells were seeded into a 24-well plate with 3-μm pore transwells (Corning Inc., Wilkes Barre, PA) and cultured for 10 to 14 days. Transepithelial resistance (TER) was monitored daily until TER reached 1000 Ω/cm2. rTcdA (300 ng/ml) or rTcdB (300 ng/ml) was added into the upper chamber of transwells for the indicated time. The cells were fixed and stained with anti-occludin (clone OC-3F10, Invitrogen) and fluorochrome-conjugate secondary antibodies. The slides were examined under confocal microscope (Leica LSM TSC SP2 AOBS).
Authors' contributions
GY and BZ carried out the molecular cloning, design of constructs, DNA sequencing, and data preparation. JW carried out protein purification. XH established the methods for protein purification. XS and WN participated in molecular cloning. ST contributed intellectually to experimental design and text. HP initiated the project, conceived the whole study and experimental design, and drafted the manuscript. All authors read and approved the final manuscript.
The authors thank Dr. Abraham L. Sonenshein for a critical review of the manuscript. This work was supported by NIH N01AI30050, and in part by NIH K01DK076549 to HF and National High Technology Development Program of China (2007AA021702) to JW.
Cloud J, Kelly CP: Update on Clostridium difficile associated disease.
Curr Opin Gastroenterol 2007, 23(1):4-9. PubMed Abstract | Publisher Full Text
Owens JR, Donskey C, Gaynes R, Loo V, Muto C: Antimicrobial-Associated Risk Factors for Clostridium difficile Infection.
Kuijper EJ, van Dissel JT, Wilcox MH: Clostridium difficile: changing epidemiology and new treatment options.
Curr Opin Infect Dis 2007, 20(4):376-383. PubMed Abstract | Publisher Full Text
McDonald LC, Killgore GE, Thompson A, Owens RC Jr, Kazakova SV, Sambol SP, Johnson S, Gerding DN: An epidemic, toxin gene-variant strain of Clostridium difficile.
N Engl J Med 2005, 353(23):2433-2441. PubMed Abstract | Publisher Full Text
Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S, Bourgault AM, Nguyen T, Frenette C, Kelly M, et al.: A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.
N Engl J Med 2005, 353(23):2442-2449. PubMed Abstract | Publisher Full Text
Kelly CP, Pothoulakis C, LaMont JT: Clostridium difficile colitis.
N Engl J Med 1994, 330(4):257-262. PubMed Abstract | Publisher Full Text
Voth DE, Ballard JD: Clostridium difficile toxins: mechanism of action and role in disease.
Clin Microbiol Rev 2005, 18(2):247-263. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Just I, Gerhard R: Large clostridial cytotoxins.
Reviews of physiology, biochemistry and pharmacology 2004, 152:23-47. PubMed Abstract | Publisher Full Text
Schirmer J, Aktories K: Large clostridial cytotoxins: cellular biology of Rho/Ras-glucosylating toxins.
Biochimica et biophysica acta 2004, 1673(1–2):66-74. PubMed Abstract | Publisher Full Text
Kurtz CB, Cannon EP, Brezzani A, Pitruzzello M, Dinardo C, Rinard E, Acheson DW, Fitzpatrick R, Kelly P, Shackett K, et al.: GT160-246, a toxin binding polymer for treatment of Clostridium difficile colitis.
Antimicrobial agents and chemotherapy 2001, 45(8):2340-2347. PubMed Abstract
Lyerly DM, Lockwood DE, Richardson SH, Wilkins TD: Biological activities of toxins A and B of Clostridium difficile.
Infect Immun 1982, 35(3):1147-1150. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lyerly DM, Saum KE, MacDonald DK, Wilkins TD: Effects of Clostridium difficile toxins given intragastrically to animals.
Infect Immun 1985, 47(2):349-352. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Savidge TC, Pan WH, Newman P, O'Brien M, Anton PM, Pothoulakis C: Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine.
Gastroenterology 2003, 125(2):413-420. PubMed Abstract | Publisher Full Text
Shin BM, Kuak EY, Yoo SJ, Shin WC, Yoo HM: Emerging toxin A-B+ variant strain of Clostridium difficile responsible for pseudomembranous colitis at a tertiary care hospital in Korea.
Diagn Microbiol Infect Dis 2007. PubMed Abstract
Kikkawa H, Hitomi S, Watanabe M: Prevalence of toxin A-nonproducing/toxin-B-producing Clostridium difficile in the Tsukuba-Tsuchiura district, Japan.
J Infect Chemother 2007, 13(1):35-38. PubMed Abstract | Publisher Full Text
Krivan HC, Wilkins TD: Purification of Clostridium difficile toxin A by affinity chromatography on immobilized thyroglobulin.
Infect Immun 1987, 55(8):1873-1877. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sullivan NM, Pellett S, Wilkins TD: Purification and characterization of toxins A and B of Clostridium difficile.
Infect Immun 1982, 35(3):1032-1040. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Keel MK, Songer JG: The distribution and density of Clostridium difficile toxin receptors on the intestinal mucosa of neonatal pigs.
Veterinary pathology 2007, 44(6):814-822. PubMed Abstract | Publisher Full Text
Phelps CJ, Lyerly DL, Johnson JL, Wilkins TD: Construction and expression of the complete Clostridium difficile toxin A gene in Escherichia coli.
Infection and immunity 1991, 59(1):150-153. PubMed Abstract
Tang-Feldman YJ, Ackermann G, Henderson JP, Silva J Jr, Cohen SH: One-step cloning and expression of Clostridium difficile toxin B gene (tcdB).
Molecular and cellular probes 2002, 16(3):179-183. PubMed Abstract
Wren BW, Clayton CL, Mullany PP, Tabaqchali S: Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K12.
FEBS letters 1987, 225(1–2):82-86. PubMed Abstract | Publisher Full Text
Malten M, Biedendieck R, Gamer M, Drews AC, Stammen S, Buchholz K, Dijkhuizen L, Jahn D: A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium.
Applied and environmental microbiology 2006, 72(2):1677-1679. PubMed Abstract
Vary PS, Biedendieck R, Fuerch T, Meinhardt F, Rohde M, Deckwer WD, Jahn D: Bacillus megaterium–from simple soil bacterium to industrial protein production host.
Applied microbiology and biotechnology 2007, 76(5):957-967. PubMed Abstract
Burger S, Tatge H, Hofmann F, Genth H, Just I, Gerhard R: Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system.
Biochem Biophys Res Commun 2003, 307(3):584-588. PubMed Abstract | Publisher Full Text
Brito GA, Fujji J, Carneiro-Filho BA, Lima AA, Obrig T, Guerrant RL: Mechanism of Clostridium difficile toxin A-induced apoptosis in T84 cells.
J Infect Dis 2002, 186(10):1438-1447. PubMed Abstract | Publisher Full Text
Matarrese P, Falzano L, Fabbri A, Gambardella L, Frank C, Geny B, Popoff MR, Malorni W, Fiorentini C: Clostridium difficile toxin B causes apoptosis in epithelial cells by thrilling mitochondria: involvement of ATP-sensitive mitochondrial potassium channels.
J Biol Chem 2007. PubMed Abstract | Publisher Full Text
Kim H, Kokkotou E, Na X, Rhee SH, Moyer MP, Pothoulakis C, Lamont JT: Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase.
Gastroenterology 2005, 129(6):1875-1888. PubMed Abstract | Publisher Full Text
Just I, Selzer J, Wilm M, von Eichel-Streiber C, Mann M, Aktories K: Glucosylation of Rho proteins by Clostridium difficile toxin B.
Nature 1995, 375(6531):500-503. PubMed Abstract | Publisher Full Text
Just I, Wilm M, Selzer J, Rex G, von Eichel-Streiber C, Mann M, Aktories K: The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.
J Biol Chem 1995, 270(23):13932-13936. PubMed Abstract | Publisher Full Text
Genth H, Huelsenbeck J, Hartmann B, Hofmann F, Just I, Gerhard R: Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B.
FEBS letters 2006, 580(14):3565-3569. PubMed Abstract | Publisher Full Text
Na X, Kim H, Moyer MP, Pothoulakis C, LaMont JT: gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A.
Infect Immun 2008, 76(7):2862-2871. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pothoulakis C, Gilbert RJ, Cladaras C, Castagliuolo I, Semenza G, Hitti Y, Montcrief JS, Linevsky J, Kelly CP, Nikulasson S, et al.: Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A.
J Clin Invest 1996, 98(3):641-649. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Krivan HC, Clark GF, Smith DF, Wilkins TD: Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc.
Infect Immun 1986, 53(3):573-581. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Henriques B, Florin I, Thelestam M: Cellular internalisation of Clostridium difficile toxin A.
Microbial pathogenesis 1987, 2(6):455-463. PubMed Abstract | Publisher Full Text
Jefferson KK, Smith MF Jr, Bobak DA: Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes.
J Immunol 1999, 163(10):5183-5191. PubMed Abstract | Publisher Full Text
Nusrat A, von Eichel-Streiber C, Turner JR, Verkade P, Madara JL, Parkos CA: Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins.
Infect Immun 2001, 69(3):1329-1336. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Reineke J, Tenzer S, Rupnik M, Koschinski A, Hasselmayer O, Schrattenholz A, Schild H, von Eichel-Streiber C: Autocatalytic cleavage of Clostridium difficile toxin B.
Nature 2007, 446(7134):415-419. PubMed Abstract | Publisher Full Text
Egerer M, Giesemann T, Jank T, Satchell KJ, Aktories K: Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.
The Journal of biological chemistry 2007, 282(35):25314-25321. PubMed Abstract | Publisher Full Text
Makoff AJ, Oxer MD, Romanos MA, Fairweather NF, Ballantine S: Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons.
Nucleic acids research 1989, 17(24):10191-10202. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Makoff AJ, Ballantine S, Smallwood AE, Neil F: Expression of tetanus toxin fragment C in E. coli: its purification and potential use as a vaccine.
Gerhard R, Burger S, Tatge H, Genth H, Just I, Hofmann F: Comparison of wild type with recombinant Clostridium difficile toxin A.
Microb Pathog 2005, 38(2–3):77-83. PubMed Abstract | Publisher Full Text
Feng H, Zeng Y, Whitesell L, Katsanis E: Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity.
Blood 2001, 97(11):3505-3512. PubMed Abstract | Publisher Full Text
Feng H, Zhang D, Palliser D, Zhu P, Cai S, Schlesinger A, Maliszewski L, Lieberman J: Listeria-infected myeloid dendritic cells produce IFN-beta, priming T cell activation.
J Immunol 2005, 175(1):421-432. PubMed Abstract | Publisher Full Text


text:
Nine & the Gang - Spring 2013 Business Wire NEW YORK -- February 13, 2013 Come on! What are you waiting for? Get over to Nine West right now because the Gangs are all here and they can't wait for you to see what they've brought with them - an array of footwear as bright as a Moroccan sunrise, as cool as a spring breeze over Laguna Beach and as sensual as moonlight on Montego Bay. The Exotic Bohemians (Photo Business Wire) The Exotic Bohemians (Photo Business Wire) The internationally celebrated photographer Craig McDean has photographed three irresistible Nine West Gangs for you to choose from, each with their own dynamic style: THE EXOTIC BOHEMIANS These free spirits crave adventure, finding a hidden treasure, discovering the one-of-a-kind.They want spice in their life. So their shoes are awash with handcrafted beads caught in intricate weavings and ablaze with colors that give off their own heat. THE GETAWAY GIRLS The perfect summer weekend for this carefree pack must include a boardwalk, a pool, a beach party, or - you know what? - all three.The more reasons the better to slip into their sleekly streamlined wedges and candy-colored cross-pieced espadrilles. PRETTY LITTLE PRINT - CESSES They're the savvy ones who get showered in bouquets because they know how to flirt until it hurts so good. Whether stepping out for a garden party or outdoor wedding, their glorious floral-printed heels and platforms let you know that romance is never far from their minds. So which Nine West Gang do you choose? Well, here's the best part. YOU CAN JOIN ALL THREE because the only membership requirement is that you love shoes that make you happy! Our joy-filled Nine & the Gang campaign launches this month in the March issues of Vogue, InStyle and Glamour, The New York Post, in Nine West's ever-growing social media network and online portals (Facebook,Twitter, ninewest.com & Channel 9) and can be viewed in Nine West flagship store videos and on New York City's inescapable Taxi TV. Nine and the Gang is the next big step in expanding Nine West's presence on a global landscape as a brand that incites its customers to shop for inspiration, dream in fashion, and revel in their individuality. Nine West is a division of The Jones Group Inc. (NYSE: JNY). ABOUT NINE WEST: Founded in 1978, Nine West is a global footwear leader.With the mantra "We Dream in Fashion," Nine West is dedicated to leading the shoe and fashion conversation: • Become a fan of Nine West on Facebook for exclusive discounts & giveaways. • Follow @NineWest on Twitter for the latest news from Nine West. • Follow @NineWestPRGals on Twitter for updates from Nine West's PR team. • Subscribe to Channel 9 on YouTube to watch and shop the latest shoe and fashion videos. ABOUT THE JONES GROUP INC. The Jones Group Inc. (www.jonesgroupinc.com) is a leading global designer, marketer and wholesaler of over 35 brands with product expertise in apparel, footwear, jeanswear, jewelry and handbags.The Jones Group has a reputation for innovation, excellence in product quality and value, operational execution and talent. The Company also markets directly to consumers through branded specialty retail and outlet stores, through concessions at upscale department stores and through its e-commerce sites. Photos/Multimedia Gallery Available: http://www.businesswire.com/multimedia/home/20130213006520/en/ Multimedia Available:http://www.businesswire.com/cgi-bin/mmg.cgi?eid=50563739&lang=en Contact: Press: HL Group Julia Ruttner, 212-529-5533 firstname.lastname@example.com or Nine West Andrea Fodera, 212-536-9659 firstname.lastname@example.com
Nine & the Gang - Spring 2013
Press spacebar to pause and continue. Press esc to stop.


text:
Contact Info / Websites
Entry #2
I'm hoping to start moving up soon to freelance my art at some conventions. Hopefully I'll have a website right around that time so I don't look like a total hack hehehehahahahhhh....... ehh.
- 0 comments
You must be logged in to comment on this post.


text:
Lisa Rinna
Real Housewives of Beverly Hills
Yolanda Foster and the Real Housewives Spread Lyme Disease Awareness on Instagram
Originally posted 04/21/2015 07:20PM
This celeb social media campaign is way tastier than dumping ice on your head – and for a just as important cause.
Yolanda Foster is asking her famous friends to help spread awareness of Lyme disease by posting Instagram photos of them sucking on – you guessed it – actual limes.
"It takes a village, please pass it on," Foster, 51, captioned a picture of her and her husband, music mogul David Foster, biting into the fruit Sunday.
Kim Richards' RHOBH Costars Express Their Support Following Her Arrest
Originally posted 04/16/2015 06:05PM
Though relationships between fellow Housewives can sometimes be contentious, support for Kim Richards has been pouring in from her RHOBH costars following her arrest for public intoxication on Thursday.
"Kim, love having you as a BFF," Brandi Glanville wrote in a blog post for Bravo on Thursday. Though she did not specifically address Richards' arrest, she went on to write, "I'm always here for you, and I hope you are always here for me. You are a strong chica, and I know your family is better for it. You are hilarious, and I love our funny talks and text-offs. Laughter is the best medicine. I heart you."
Lisa Rinna Transforms Into a 'Kardashian' with Mega Hair Extensions
Originally posted 04/09/2015 02:50PM
See the reality star's bold new look
Real Housewives of Beverly Hills
Real Housewives of Beverly Hills Reunion: What Did Kim Say to Make Kyle Break Down?
Originally posted 04/08/2015 07:15AM
Real Housewives of Beverly Hills
Brandi Glanville Felt Bullied by Her Costars at RHOBH Reunion, Says Source
Originally posted 04/03/2015 11:55PM
She's known for dishing it out on The Real Housewives of Beverly Hills, but after a particularly brutal reunion show, Brandi Glanville felt the heat in a big way.
"Brandi really struggled," a source close to Glanville tells PEOPLE. "She felt bullied at the reunion."
Says the source of the single mom of two, whose ongoing feud with Kyle Richards, Lisa Rinna and Lisa Vanderpump came to boiling points at the taping. "She feels like everyone else in the cast can do whatever they want, but whatever she does gets criticized."
Lisa Rinna Flaunts Toned Bikini Bod (PHOTO)
Originally posted 04/01/2015 02:50PM
Lisa Rinna has proven once and for all that you can rock a bikini bod at any age.
Kylie Jenner's Scandalous Bikini Selfie, Lisa Rinna's Tiny Two-Piece and More Celeb Swimsuit News
Originally posted 03/30/2015 05:35PM
Catch up on all the bikini posts you missed from this weekend
Real Housewives of Beverly Hills
Watch The Real Housewives of Beverly Hills Stars Rip One Another Apart in Reunion Sneak Peek
Originally posted 03/19/2015 05:05PM
Let's get ready to rumble!
The first sneak peek of the The Real Housewives of Beverly Hills reunion show is here, and it's got all the smack talk and finger-pointing of a WWE showdown.
The three-part reunion is once again bravely hosted by Andy Cohen, who plays mediator (and sometimes instigator) for Kyle Richards, Lisa Vanderpump, Yolanda Foster, Brandi Glanville, Kim Richards, Lisa Rinna and Eileen Davidson.
Real Housewives of Beverly Hills
Real Housewives of Beverly Hills: Lisa Rinna Smashes a Glass at Kim Richards
Originally posted 03/04/2015 07:45AM
The Real Housewives of Beverly Hills' trip to Amsterdam was anything but a relaxing escape – and things went south the moment they arrived.
"If you poke a hornet's nest, you are going to get stung," Lisa Rinna exclusively told PEOPLE on Monday about her fight with Kim Richards, who threatened to reveal a secret about Rinna's husband, Harry Hamlin, on Tuesday.
RHOBH's Lisa Rinna on Kim Richards: 'If You Poke a Hornet's Nest, You Are Going to Get Stung'
Originally posted 03/02/2015 08:00PM
Glasses – and rumors – will be flying on Tuesday night's episode of The Real Housewives of Beverly Hills, when Kim Richards and Lisa Rinna go head-to-head over Rinna's husband, Harry Hamlin, during a drama-filled dinner.
Rinna, who is ever-protective of her husband of 17 years and their two daughters, explodes when Richards threatens to reveal a secret about Hamlin.
Top 5 Most Voted Stories
Father of 12 Allegedly Beat, Starved and Raped His Children: Police
The Bachelor's Chris Soules and Whitney Bischoff Have Split After 6 Months
19 Kids and Counting Spin-Off May Be in the Works at TLC: Source
Sherri Shepherd Makes Uncomfortable Joke About Children on The View, Blasts Ex-Husband in Wake of Custody Ruling
Pregnant Mom Kicked Off Flight Because Her Toddler Cried: 'I Don't Like to Make a Fuss'
Top 10 News Categories
The most buzzed about stars this minute!
Treat Yourself! 4 Preview Issues
More On PEOPLE.com
Got a News Tip?
Send it to our People.com editors!
News Archive


text:
Create 3D type in Photoshop, and learn about topics like extruding, inflating, beveling, and texturing type.
Use Photoshop's tools to create works of art from scratch.
Create complex, nondestructive transformations and speed up repetitive tasks.
Explore advanced photomontage techniques.
Explains the basic principles of perspective and how they apply to working in Photoshop.
"I'm learning stuff that I wouldn't necessarily have even considered before, due to time and cost. You guys rule." —Nate M.
more from our members »