text: Trump's Autographed Bibles, Guitars, and Boxing Gloves Are on eBay If That's Your Kind of ThingMatt Novak3/14/16 2:49pmFiled to: donald trumpautographsebay30EditPromoteShare to KinjaToggle Conversation toolsGo to permalink Donald Trump signing a camo hat that's now on eBay on October 23, 2015 in Doral, Florida (Getty Images) Donald Trump rallies almost always devolve into autograph-signing sessions. And many of those signed items are showing up on eBay. A wide variety of Trump-signed items are just a click away, if that's the kind of kinky shit you're into. Like this Bible, autographed by Trump, which is going for $3,500: Advertisement The only bible signed by Trump I have ever seen. Ths bible is unused and has new crisp pages and is unmarked. Signed by Mr. Trump in the historic Exeter NH town hall. No box for bible. Leather bonded NIV study bible. Or this camo hat, which is currently priced at $2,000: Advertisement This is a one of a kind rare iconic autographed Official Donald Trump "Make America Great Again, Cap". This was signed on the campaign trail by Donald Trump himself on his first political campaign rally in the state of Florida At his exclusive Doral property.The photo at the top of this post is provided in the listing as proof that this hat was actually signed by Trump himself. And then there this guitar, which is currently being offered for 1,274 American dollars, and is quite a find. Sponsored Here we have 2017 Presidential candidate DONALD TRUMP Autographed Brand New Full Size AMERICAN FLAG Telecaster Guitar!!! You will not find another Trump autographed guitar like this one!! This is a must have for any Trump fan! This makes a great gift for a birthday or the holidays!! Check out the picture of Trump signing!!The funny part? Yes, that's Trump's signature. But it's not a Telecaster. Fender owns the trademark on the Telecaster. That's something called an Eleca guitar—a knock-off made to look like a Telecaster body. Want to wallop the deficit? This country doesn't punch anymore. Well, at least they don't outside of Trump rallies. But you can change all that with this autographed boxing glove, currently going for the low low price of $1,200. Advertisement Advertisement People are offering all kinds of certificates of authenticity, but the most common authenticator on eBay are the press photos that show up afterward. The listing for this boxing glove tells quite a story:MR. PRESIDENT DONALD TRUMPHE WAS IN JACKSONVILLE,FLORIDA SATURDAY OCT.24 2015YOU WILL GET THE AUTHENTIC EVERLAST LEFT HAND GLOVE SIZE 14SIGNEDTHE JACKSONVILLE PAPER,THE WALL STREET JOURNALAND ALL OF THE PHOTO'STHE GLOVE IS SIGNED BY HIS BLUE SHARPIELOOK AT PHOTO # 4 AT THE FAR RIGHT HAND SIDE I'AM STANDING NEXT TO THE NICE OLDER LADY WHICH IS IN THE NEWSPAPER PHOTO,YOU CAN SEE THE RED GLOVE THERE I'AM HOLDING IT IN MY RIGHT HANDPHOTO # 7 SHOWS MR. TRUMP COMING TOWARDS ME WHILE I'AM HOLDING THE BOXING GLOVEPHOTO # 8 MR.TRUMP IS GETTING CLOSERPHOTO # 9 MR.TRUMP IS IN FRONT OF ME SIGNING THE GLOVE YOU CAN HIS BLUE SHARPIE AND NEXT PHOTO SHOWS JUST A LITTLE OF THE GLOVEPHOTO # 10 SHOWS ME AND THE NICER OLDER LADY STANDING NEXT TO MEPHOTO # 11 IS THE WALL STREET JORUNAL WHICH YOU WILL GETPHOTO # 6 SHOWS THE WALL STREET JOURNAL IN FRONT OF MR.TRUMP AS HE'S SPEAKING AT THE PODIUMHE SPOKE ABOUT THE WALL STREET JOURNAL AND THREW IT DOWNAND I HAVE THAT PAPER,ITS ALSO MENTION IN THE JACKSONVILLE PAPER THAT HE THREW IT DOWN ON THE CONCRETETHIS IS A NICE COLLECTION OF MR.TRUMPS MEMORABILIA WHICH ALL IS WELL DOCUMENTED.And there are countless signed copies of Playboy, Time and other magazines. The $100 bills are also asking for a pretty penny, including this one which can be yours for just $895: Advertisement DONALD TRUMP SIGNED 100 DOLLAR BILL WAS SIGNED IN JACKSONVILLE FLORIDA DURING A RALLY STOP. WILL PASS ANY AUTHINCATION We obtain all autographs in person. We stand by all our items.Of course, there are some Trump signatures that you just can't put up on eBay: Donald Trump signs a woman's chest at a December 2015 rally in Manassas, Virginia (Associated Press) But if Donald Trump isn't your guy, there's currently a fire sale of Jeb Bush-autographed items on eBay right now. Like this baseball signed by Jeb himself, currently on offer for $13.49. Poor Jeb[!].Contact the author at firstname.lastname@example.com repliesLeave a reply


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- Upgrade your look with this sleek New York Islanders Duster mesh back flex hat from Old Time Hockey. This hat is the definitive example of Old Time Hockey swag, with a comfortable stretch fit and breathable, mesh side and rear panels for extra comfort. It also features authentic New York Islanders graphics that set this hat apart from all the others in your collection!
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Svchost Process Analyzer 1.3
The Svchost.exe process is a Windows system process for services that run from dynamic-link libraries (DLLs). The original svchost.exe file is located in C:\Windows\System32, but numerous malwares and trojans use the same file and process name to hide their activities. The free Svchost Process Analyzer lists all svchost instances and checks the services they contain. This makes it easy to uncover Svchost worms like the infamous Conficker worm.
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If we're going to show our faces, well, here I am.
I'd like to think I look like I sound. (okay, but if I'm disgruntled, and about to puke on your shoes, I'll try to miss, because that would be the polite thing to do).
Edited to add: Let's all get Ann Margret – V(d)iva Las Vegas!Powered by Sidelines


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|Enabling URL-based read access to the search index
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I am planning to upgrade from Lucene 1.0 to Jakarta Lucene 1.2. My current implementation uses Jason Pell's URLDirectory class so that Lucene can access the search index while running in an applet. I modified IndexReader.java to use URLDirectory instead of FSDirectory. Is there a more appropriate way to allow Lucene to function using URL-based file access? I would prefer to extend classes or interfaces instead of modifying them. I have no requirement to update the search index from the applet. Thanks, David David Schaeffer Computer Associates Programmer tel: +1 856 914-8385 email@example.com
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Need to Know: Will the Redskins be interested in C Mangold, RB Murray?
Need to Know: Will the Redskins be interested in C Mangold, RB Murray?
Here is what you need to know on this Monday, February 27, 2 days before the March 1 NFL franchise tag deadline.
Days until:
—NFL Combine (3/2) 3
—Start of NFL free agency (3/9) 10
—Redskins offseason workouts start (4/17) 49
—NFL Draft (4/27) 59
—First Sunday of 2017 season (9/10) 195
Monday morning Redskins musings as free agency approaches
1. It appears that the Raiders are going to let Latavius Murray, who rushed for over 1,800 yards combined in the last two seasons, test free agency.
I'm told #OAK expects to let RB Latavius Murray go into FA. Source: "The door will remain open for a posb return after he tests his market."— Josina Anderson (@JosinaAnderson) February 26, 2017
I'd be surprised if the Redskins showed any interest. I'm not sure that they will be jumping into the RB free agent pool, at least not into the deep (expensive) end of it. They might draft a back to compete with Rob Kelley, Matt Jones, and Mack Brown. Chris Thompson will remain the third-down back.
2. Do you wonder why the Redskins have been reluctant to draft a safety?
It's a tough eval because it's such a scheme-specific position now in the NFL. Moneybacker, center fielder, big nickel box guy. https://t.co/TUxVO8GD4r— Doug Farrar (@BR_DougFarrar) February 26, 2017
Eventually, someone will need to figure out the safety position in the NFL. With all the spread offenses that safeties face in college the safeties have become more like additional corner. Also, the better defensive backs in college want to be cornerbacks because it pays better in the pros. The pool coming out is generally small to begin with and then it shrinks even further for each team due to the scheme factors that Farrar discusses. Su'a Cravens could be the solution to one safety spot for the Redskins but they continue to be in search of someone to pair with him.
RELATED: NFL Mock Draft Version 4.0
3. The Jets allowed C Nick Mangold to announce his own release. He was one of the best centers of the past decade; Mangold went to seven Pro Bowls and was named first-team All-Pro twice. But I don't see him being of interest to the Redskins. They are happy with Spencer Long and even if they weren't, Mangold is 33 and he missed half of last season with an ankle injury. Those factors likely will make the Redskins steer clear.
4. Maybe some fans don't care if they are getting information from the team through the media at the combine. Or at the Senior Bowl. Or anywhere. And it is the team's option to have someone available or not during most of the offseason (a press conference prior to the draft is mandatory). But they are missing out on a chance to give their side of the story.
One of the problems with changing what has been a longstanding practice and going into radio silence is that it leaves people speculating. If the team doesn't want to put any information out there that is the organization's option. But if you choose not to fill in the blanks, the fans and media will.
More Redskins: #RedskinsTalk podcast: Is Kirk too nice for his own good?
5. We have another D-lineman signed as the Panthers retained DE Mario Addison.
The #Panthers signed DE Mario Addison to a 3-year deal worth $22.5M, source said. At 29 years old, he had 9.5 sacks in 2016.— Ian Rapoport (@RapSheet) February 26, 2017
Addison, who was with the Redskins for five games in 2012, also had six and 6.5 sacks the previous two year, not bad for a guy who has started just one game in the last three seasons. That average of just over $7 million per year sets another data point for the Redskins to use to try to retain Chris Baker. Addison is a 4-3 end while Baker is a 3-4 end so it's not an apples to apples comparison but it will help figuring out what a reasonable contract is. Without seeing the details, it looks like Addison gave the Panthers something of a hometown discount as that's a good price for a guy who was half a sack away from double digits.
In case you missed it
- A Redskin fan's guide to the NFL Combine
- Why won't Redskins brass talk to the media at the NFL Combine?
- Sunday six pack—Cousins' next step and a positive step for Doctson
Stay up to date on the Redskins. Rich Tandler covers the team 365 days a year. Like his Facebook page Facebook.com/TandlerCSN and follow him on Twitter @Rich_TandlerCSN.
Quick Links
A Redskin fan's guide to the NFL Draft Combine
A Redskin fan's guide to the NFL Draft Combine
This week in Indianapolis the NFL world will converge at the Scouting Combine to watch college football players work out, sprint and lift weights in anticipation of the upcoming draft. For the Redskins front office, this draft needs to be a win.
The 2016 Draft could still yield strong results for Washington, but overall the class did not play particularly well as rookies. This year, Scot McCloughan has nine picks at his disposal, with the extra picks late in the draft in the fourth, fifth and sixth rounds.
It's no secret that the 'Skins need help along the defensive line, a lot of help. That should be a major area of focus for the Redskins scouts and coaches, and that will make next Sunday arguably the most important of the week in Indianapolis.
The combine divides players into 11 position groups, but Groups 7, 8 and 9 will matter most. Groups 7 and 8 represent defensive linemen and 9 are the linebackers. That group officially arrives on Thursday but won't work out on the field until Sunday. The days in between include interviews, psychological testing and the bench press.
Obviously the Redskins won't spend all nine picks on only defensive linemen. The team will likely invest in the offensive line as well, and that group will arrive earlier in the week and work out on Friday. Cornerbacks and safeties are the last to work out on Monday, March 6.
With the likely departure of at least one of DeSean Jackson or Pierre Garçon, and the possible departure of both, it would make sense for the 'Skins to bring in another receiver via the draft. They work out on Saturday, and should the Redskins decide to take a quarterback in the draft, the passers will work out that day too.
Running back could be another spot the 'Skins invest. Jay Gruden said that Robert Kelley is locked into the RB1 role, but still the team might want increased competition at the position. The backs will work out Friday.
Want more Redskins? Check out @JPFinlayCSN for live updates or click here for the #RedskinsTalk Podcast on iTunes, here for Google Play or press play below. Don't forget to subscribe!


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Train Cologne → DüsseldorfFrom HK$122
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Overview The average travel time between Cologne and Düsseldorf is 23 minutes. The quickest route is 21 minutes. The first train leaving Cologne is at 04:29, the last at 19:10. There is an average of 7 trains a day between Cologne and Düsseldorf, leaving approximately every 1h02 minutes.
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"Dusseldorf - Koeln trip" Alisa s. 08/01/2014
Very nice silent train, with free Wi-Fi access (with promo-code on your ticket), nice seats
Very nice silent train, with free Wi-Fi access (with promo-code on your ticket), nice seats
"timing" Phongchai s. 21/10/2013
It convenience , on time
It convenience , on time


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When it comes to French movies, I am a complete sucker. The merest glimpse of neon-lit rain on Parisian cobbles, the brusque cadences of the language (which I don't speak a word of, by the way), or the false-memory nostalgia of yearning accordion music, rumbles through my soul like the night train to Nice.
Yet despite an almost genetic predisposition to all things Gallic, by the end of director Luc Besson's latest movie, Angel-A, I was left wanting to hurl myself from the nearest bridge — exactly the situation in which the two central characters find themselves when we first meet them.
After defaulting on payments to the mob and with a contract on his head, Andre (the grizzled-looking Jamel Debbouze) decides to throw himself into the Seine. To his surprise, further along the ledge there's a statuesque blonde (Rie Rasmussen), long of leg, high of heel, and, this being a French movie, wearing the skimpiest of black cocktail dresses, who also wants to end it all. Unable to accept that someone so beautiful doesn't have a reason to live, Andre jumps in and saves her.
After which Andre discovers that the woman, Angela, happens to be an angel. Not just a metaphorical angel but the real deal with wings who happens to be down on her luck, feeling jaded about the sordid business of saving souls.
The sharp contrast between their attitudes and appearances (light/dark, male/female, short/tall, good/bad, happy/sad, human/supernatural being!) coupled with Angela's less than angelic behaviour when it comes to other men, and Andre's unexpected morality should provide us with a screwball rom-com set against the noir-ish world of a subterranean Paris.
Instead we get a too-predictable retread of It's A Wonderful Life or Wings Of Desire but plucked of their respective humour, warmth, and pathos and oozing cheap sentimentality. This aspect has been a recurring difficulty in Besson's work, tarnishing his otherwise classic Leon, although thankfully the saccharine was effectively staunched by Jean Reno's sour yet vulnerable aura in the title role.
Here however, the absence of comparable talent is telling. Debbouze may be fine in small parts (he makes a fleeting but effective appearance in Jean-Pierre Jeunet's Amelie as the greengrocer's son who gets revenge on his tyrant father), but like Rasmussen lacks the range of skills or old-fashioned "presence" required to carry it off. Charisma is more than being able to pull off the ramshackle loser look or pouting sexily as your mascara runs.
The blame lays not so much with the hapless cast punching above their weight as with the lead-lined script which clouts us with clunkers such as "You may not have a past but at least let me give you a future."
Even accepting that Besson's dialogue may have lost something in translation when it comes to the subtitles, there seems little excuse for such poor writing from a director as experienced as this. This would-be parable about how heaven is in the most unexpected of places if we open our eyes and ourselves probably looked great in storyboard but falls to earth with a resounding thump.Powered by Sidelines


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Gorgeous 1BR located in Murray Hill area of NYC. Located in high rise, full service doorman building featuring elevator, garage, laundry room and bicycle room. Home features sparkling wood floors,spacious bedroom, gourmet kitchen, and classic ceramic bath. Huge windows throughout home provide beautiful lighting! You can also enjoy amazing river → Read More
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Subaru COO Thomas Doll Discusses Building Cars in U.S. (Audio)
Dec 5, 2012
Thomas Doll, executive vice president and chief operating officer at Subaru of America Inc., discusses the Indiana plant where 50% of the company's vehicles are being built. Doll talks with Bloomberg's Pimm Fox and Vonnie Quinn on Bloomberg Radio's "Taking Stock."
(Source: Bloomberg)
Running time 06:15


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32 year old woman treated with Power Assisted Liposuction
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Mac and cheese
Macaulay Culkin must be feeling a little lactose intolerant right now … After making his stage debut with his pizza-themed Velvet Underground tribute band The Pizza Underground (sample song: "I'm Beginning to Eat the Slice"), he came under attack for treading on the (toma)toes of a San Francisco-based pizza-punk act called Personal and the Pizzas, whose lead singer threatened: "First we're gonna kill 'em, then we're gonna sue 'em." To top it all (top it all? Oh, forget it), Culkin's film Home Alone has just been voted second in a poll of the "Cheesiest Christmas Films of All Time", conducted by Port Salut. That's got to grate.
Still hazy after all these years
He turns 90 today, so no one could deny former Sunday Telegraph editor Sir Peregrine Worsthorne the occasional "senior moment". But while Harry Mount was quick to point out in his Spectator profile last week that Perry's brain was still "highly functional", no one (least of all the magazine's fact-checkers) seemed to notice that his anecdote about Lord Reith being sacked as director-general of the BBC in 1938 contained a glaring error: "He was at dinner when we turned on the radio to find Churchill had sacked him," Worsthorne recalled. "He was absolutely furious, beside himself with rage. Churchill hated Reith because …" blah blah blah, and so on. The ugly fact that ruins the beautiful story? It wasn't Churchill, but Neville Chamberlain who sacked Reith. Churchill would, as everyone with a passing interest in history knows, not be in a position to do so until 1940.
Hairy encounter
"David Burns is a first-class communicator and hustler. I would not hesitate to use him again." So says the actor and writer Steven Berkoff on the website of said entertainment publicist. And it turns out Berkoff's not wrong – with Burns using last week's death of Ronnie Biggs as the perfect opportunity to tout his client's 1991 book A Prisoner in Rio around again. The book, Berkoff's diary of the making of the 1988 film Prisoner of Rio, gives this account of the actor and train robber's dinner together: "He gives me a brilliant description of his escape from the Scrubs which ended with 'paying two mates £500 each to hold the two screws down while I nipped over'. He tells me about the plastic surgery which not only altered his features but at the same time seems as a bonus to have rendered him more youthful. Now he's quite a ladies' man, even if he does have to shave behind the ears."
Endless love
The web is often perceived as a place for all the worst traits of human nature, with trolls and "haterz" running amok. So it is with some pleasure that I might turn your attention to The Best Thing on the Internet Ever. It's an ongoing project by Milwaukee's answer to Banksy, a young artist and blogger called Molly Evans, whose stated aim is to create "unusual, temporary public art designed to make folks stop and appreciate the good thing they have going". Evans's work-in-progress involves embroidering random lyrics from Lionel Ritchie songs on to discarded furniture left out on the streets of her home town. A recliner chair emblazoned with "That's why I'm easy". A mattress bearing the immortal words "All night long". And, best of all, a sofa with a missing seat that reads "Hello?" and the cushion carefully positioned nearby with "Is it me you're looking for?" lovingly crafted into its weft. The project's name? Lionel Stitchie, of course. Find it at mollyeeeee.com
Be careful what you tweet for
Spare a thought for the Tottenham and England footballer Andros Townsend. The speedy winger found himself trending on Twitter last Wednesday night. The reason? After his club had lost to West Ham in the League, they had drawn them in the quarter finals of the Capital One Cup and Townsend had tweeted: "The perfect draw #revenge". Sadly, that didn't turn out to be the case, as West Ham came back from a goal down to win the tie. To add injury to insult, Townsend limped off the field before the end of the game after pulling a hamstring. His Twitter account has stayed strangely silent since. But let Townsend's experience be a warning to all of us who use social media – especially now that Facebook allows your "friends" to see original versions of "edited" status updates.
Keeping it real
It pays to be discreet if you are dating a member of the Middleton family, so it's no surprise that TV presenter and actress Donna Air – who has had an agent since she was 10 years old – knows how to play the game. Asked recently about her alleged new beau, Kate and Pippa's brother James, she told our man that she was reluctant to discuss royal matters. Had she seen the new addition to the family? "Well," she said with wink, "I did DJ at the George and Dragon in east London the other day."
Life in plastic, it's fantastic
A 21st-century reworking of a popular Christmas carol:
There's a rink, made of plastic
And the Santa, is sarcastic
For such wintery scenes
Come to Milton Keynes
Day out at the Winter Wonderland
Gone away, is the public
Here to stay, is a sludge pit
The whole thing's a dud
Unless you love mud
Day out at the Winter Wonderland
(Quick, let's scarper before readers ask for their money back.)Reuse content


text:
Google Analytics Ecommerce
- Status Closed
- Budget $30 - $250 AUD
- Total Bids 5
Project Description
We need a Google Analytics specialist to set up and optimize our Google Analytics account for our ecommerce website. Looking to have expert implementation of funnel tracking, events and goals. I need the analytics to show me where my traffic and conversions are coming from.
We are looking for somebody who can consult and set up the best methods of tracking. Setup and monitor key performance indicators and site goals. Also experienced with assigning dollar values etc
Include "GA Expert" in your reply to show you have read this.
After completed tracking installation I want to know:
- at which stage of the order process customer will cancel their order
- which Adwords campaigns or keywords will convert and which one not
- which entry page and other pages leads to conversions
Link to Google AdWords: Monitor our revenue, conversions, and ROI.
Link to Google Merchant Centre: Monitor our revenue, conversions, and ROI.
We have 75 Products.Get free quotes for a project like this
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- Forbes
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text:
Articles tagged peter facinelli
8 Jun 2009 // 2:38 AM
Edie Falco is surely dynamic, but the strength of Nurse Jackie lies in its smart, respectful examination of a nurse's life.READ more
While Meyers' books tend toward formulaic characterizations, Hardwicke takes the material and successfully transforms it into a subtle, layered atmospheric film.READ more
18 Mar 2009 // 6:38 AM
After months of hype and screaming fangirls and barrels of newspaper ink dumped on Robert Pattinson and "Twilight," there was bound to be some backlash.READ more
7 Jan 2009 // 7:59 PM
The film is a successful adaptation of the book not only because Pattinson is so talented and dreamy, but also because Hardwicke knows a thing or two about filming adolescents.READ more
Fantasy rules in Twilight, fantasy simultaneously delicate and ravishing, chaste and utterly bloody.READ more
19 Nov 2008 // 8:52 PM
Blame Anne Rice. Blame her for being the literary stake in the original vampire's heart. If it wasn't for her spinster prose takeREAD more
10 Sep 2008 // 9:59 PM
Like the sainted sigh of relief that comes after another shriek-filled All Hallow's Eve, November usually means the start of the 'nominate me' process for the proposed prestige pictures of 2008.READ more
27 Jun 2008 // 12:58 AM
Too much of Peter Tolan's movie takes up Taylor's self-absorption as if it's actually interesting.READ more
6 Apr 2008 // 9:59 PM
A superbly acted, complex serial story, this show is well-suited to the DVD medium as it can followed at your own pace without fear of missing some of the story.READ more
3 Oct 2002 // 12:00 AM
The Rock, director Russell says, has a 'deep commitment' to acting, bringing as much passion to the role as he does to his athletic endeavors -- good to know.READ more
The shots are close (is that her hand in his pants?), the cutting is speedy, the soundtrack is kicking: just above the din of the engine, the Vines wail.READ more
25 Apr 2002 // 12:00 AM
The Rock is a star, straight up.READ more
1 Jan 1995 // 12:03 AM
"Congratulations, Benjamin," sighs Supernova's shipboard computer. "Your strategy was both subtle and forceful. You can play with me whenever you want." This sure isREAD more
1 Jan 1995 // 12:03 AM
The story of the angst-ridden salesman struggling with his past, coping with regret and searching for meaning in life is a tale that audiences haveREAD more
Depeche Mode "Where's the Revolution" (Singles Going Steady) // Music
Beth Bombara "Sweet Time" (audio) (premiere) // Music
Oceans Are Zeroes "Inside" (audio) (premiere) // Music
'Fire Emblem Heroes' Is a Bad Crossover
// Moving Pixels
"Fire Emblem Heroes desperately and shamelessly wants to monetize our love for these characters, yet it has no idea why we came to love them in the first place.READ the article


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For the past five years, Tony Sparano has been a staple in the AFC East. He spent four seasons as the head coach of the Miami Dolphins and one as offensive coordinator of the New York Jets.
However, Sparano was fired by the Dolphins and Jets in back-to-back years. Now, he's taking his talents to the AFC West.
Sparano was hired Wednesday to be the next offensive line coach of the Oakland Raiders, ESPN's Adam Schefter reports. This is a position where Sparano flourished before becoming head coach in Miami.
Sparano failed as a head coach and offensive coordinator. But he is a likeable person who is well-respected in NFL circles. Sparano should do well in Oakland as a position coach, which is more in his comfort zone.


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jasonh300 wrote:Kandralla wrote:The user contacted me this morning... I'll leave it at that for now.
As for state and country, in Erie, PA I've noticed new segments randomly defaulting to things near the border (New York, Ohio, and occasionally the country goes to Canada) but never something completely in a different area.
There are border issues in some spots where WME doesn't seem to know what state you're in. The Louisiana border extends east into MIssissippi almost 50 miles at a couple of points. I was drawing in a new subdivision in Gulfport, MS the other day and about half of the segments defaulted to Louisiana and half defaulted to MIssissippi, so I guess I was right on the line. They all would've allowed me to select either state, but if I tried to save with Louisiana on there, it gave an error that I was too far from the state.
They must really hate Mississippi, then, because I had the same thing happen when I was doing some editing in the Grenada area. Most roads there were saved or defaulted to Arkansas. That's ~70 miles from the state line.


text:
Round Top Waste Receptacle
Your review: For the most part, these trash cans have worked excellently for us. They're well-made, sleek, and reliable. The only complaint I have is that when I first ordered a batch of 6 of them (in both the 12 and 15-gallon sizes), two of them arrived with significant dents rendering them either unusable or unpresentable. Otherwise, they've been great!


text: i have a pair of pikes bonded from day 1 got them at 9" now 12" each and only fish in the tank. Will not ship but willing to travel a little. I live in northwest Arkansas and asking 150$ for the pair. Perfectly healthy no lateral or hth problems. Can send pics to any interested party


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Steven D. Lear, a partner in the Corporate and Securities department, is the Recording Secretary for the task force of the Florida Bar Business Section that is drafting the new Florida LLC Act.
This is the first in a series of blog posts on the new Florida Limited Liability Company Act. Future posts will discuss issues relating to the Act and its ramifications on Miami (and Florida) businesses.
Florida is in the process of totally revising its Limited Liability Company (LLC) Act (Chapter 608 of the Florida Statutes). LLCs are the most common form of business entity in Miami (and all of Florida), and are commonly formed to acquire and develop real estate by both foreign and domestic investors and businesses. More LLCs are formed in Florida than in any other state. In 1982 Florida became the second state in the U.S. (after Wyoming) to have an LLC statute. But thirty years later our LLC act is a hodgepodge, quilted together from multiple sources that are not always in synch. Glitches abound. The time has come to update and modernize the Florida LLC Act, and I am part of a task force of the Florida Bar Business Law Section that has been working on a new LLC Act for more than three years. The new Florida LLC Act is almost finished and is expected to go to the legislature later this year for enactment in 2013.
The new Florida LLC Act will look nothing like the existing statute. It generally will be based on the Revised Uniform Limited Liability Company Act ("RULLCA") prepared in 2006 and amended in 2011 by the National Conference of Commissioners on Uniform State Laws. RULLCA has been adopted in some form by five states and the District of Columbia and also is expected to be enacted this year in California and New Jersey. Eight other states currently are studying it for possible passage. While modeled on RULLCA, we have incorporated desirable provisions from the Delaware LLC Act, the ABA Prototype LLC Act and other leading state LLC acts, as well as a number of provisions from existing Florida law. There will be a transition period for existing LLC agreements to be revised to comport with the new Florida LLC Act. Existing Florida LLCs and their attorneys (assuming they have them) will need to carefully review their operating agreements (assuming they have them) and compare them to the new Act (the current draft is approximately 125 pages, without commentary to be added) to avoid unintended consequences as a result of the major statutory revisions.
The new LLC Act will contain a number of material changes from existing Florida LLC law. For example, the new Act will eliminate the concept of a "managing member," which is fairly unique to Florida and has resulted in confusion as to whether this means an LLC is manager managed or member managed. In addition, Florida LLCs will now be able to have a non-economic member (i.e., one without a capital account), which should facilitate debt financing. And while the new Act, like Delaware, is expected to codify the principle that freedom of contract is king and that any and all remedies in the operating agreement for breach by a member are enforceable, Florida will not go so far as Delaware and allow a complete waiver of fiduciary duties or unlimited indemnification of members and managers.
However, the new LLC Act will not change the recently revised and controversial charging order section of the Florida LLC Act (§ 608.433). Enacted in response to the 2010 Florida Supreme Court's watershed Olmstead decision, these provisions are the first of any state to expressly provide a right of foreclosure of an LLC membership interest by an unsecured judgment creditor of the sole member of a single member LLC.
See more »
DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
© Bilzin Sumberg Baena Price & Axelrod LLP
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HP 78. Printing colours: cyan, magenta, yellow, Capacity: 19 ml, Page yield: 560 pages. Width: 15.7 cm, Depth: 11.8 cm, Height: 4.2 cm
Clear, vivid color images every time you print. Patented, dye-based HP color inks and print cartridge technology are designed together with the printer to make professional-quality printing easy and reliable for an excellent value.


text:
Looks like O2 could output about 500-600mW at 38ohms. If 1W is optimal for HE-500, then in theory I could get only 3dB queiter than that with O2, because double power equals to double sound intensity, which is 3dB difference.
If I will feel that O2 is not sufficient, how much would I have to spend on transparent solid-state amp that could power these (and maybe also even more power hungry headphones like HE-6)? I could chain it with my O2 to reuse ODAC.
Your reasoning is correct but it's not just as simple as volume output. You can get the 500 and even the 6 louder than what your ears can take with an O2. But quality won't be good. Every amp is a compromise and pricing is all across the board, as is sound preference. Set yourself a budget and then audition, audition, audition is the best advice I can give. For some people a low end Emotiva will be 'good enough', others end up with a >10k DAC/amp combo. Diminishing returns apply fiercely and it will primarily be your finances that determine how much those last couple % are worth.


text:
• Washington Whispers: Trent Lott found his refrigerator three blocks from his house and inside were two sentimental bottles of champagne. . . D.C. television viewers are sick of Barack Obama. . . George H.W. Bush's presidential library plans to add mock press briefing room. . . Robert Bork jokes, "How do you pronounce Souter in Spanish? Gonzales.". . . News-addicted actor Joe Pantoliano likes "the girls" on MSNBC, especially Natalie Morales. . . [USN&WR]
• Lloyd Grove's Lowdown: Brian Lamb marries someone named Victoria. Spokeswoman: "They were married in a small, private ceremony in the Washington area and Brian was in the office all week". . . Hillary supporters hope "Inside the Bubble," a new documentary about the Kerry-Edwards campaign, will end his chances in '08. Kerry flack David Wade: "The 20 poor souls subjected to this movie will be reaching for caffeine and begging for old Lamar Alexander tapes on C-SPAN2. Michael Moore has nothing to fear. I think the working title was 'The Snore Room.'" [NYDN]
• Inside the Beltway: Diane Rehm, Cokie Roberts, John Cochran, Pat Buchanan, Matt Cooper, John Dickerson, Bill Plante, Mark Shields among the attendees of Congressional Quarterly's 60th anniversary party. [WT]
• Rush & Molloy: Roger Ailes, Paul Begala, Judy Woodruff, Katrina vanden Heuvel will debate at a Harvard forum moderated by Tom Brokaw to honor John Kennedy Jr. and George magazine. [NYDN]
• Cindy Adams: Fidel Castro wears Armani. [NYP]
$5.00 Add to cart


text:
Highly Selective Tuning of a Silkworm Olfactory Receptor to a Key Mulberry Leaf Volatile
- DOI: 10.1016/j.cub.2009.04.035
- PubMed: 19427209
Background: The olfactory system plays an important role in the recognition of leaf volatiles during the search of folivore insects for a suitable plant host. For example, volatiles emitted by mulberry leaves trigger chemotaxis behavior in the silkworms Bombyx mori, and as a consequence, they preferentially reside on and consume mulberry leaves. Here, we aimed to identify natural chemoattractants and their corresponding olfactory receptors (Ors) involved in silkworm behavior to mulberry leaves. Results: Chemotaxis behavioral assays for headspace volatiles detected by gas chromatography-mass spectroscopy analysis revealed that among the volatiles that were emitted by mulberry leaves, cis-jasmone was the most potent attractant for silkworms, working at a threshold of 0.3 pg from??a 20??cm distance. Among a total of 66 Ors identified in the B.??mori genome, we found that 23 were expressed in the olfactory organs during larval stages. Functional analysis of all the larvae-expressed Ors in Xenopus oocytes revealed that one Or, termed BmOr-56, showed a high sensitivity to cis-jasmone. In addition, the ligand-receptor activity of BmOr-56 reflected the chemotaxis behavioral response of silkworms. Conclusions: We identified cis-jasmone as a potent attractant in mulberry leaves for silkworms and provide evidence that a highly tuned receptor, BmOr-56, may mediate this behavioral attraction. The current study sheds light on the mechanism of the correlation between olfactory perception in folivore insects and chemotaxis behavior to a natural volatile emitted by green leaves. ?? 2009 Elsevier Ltd. All rights reserved.
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Lot 424: Equipment
An auction of London Underground, Bus, Tram, & Trolleybus Collectables and Railwayana
by London Transport Auctions
October 22, 2016
Weybridge , United KingdomLive Auction
Looking for the realized and estimated price?
Description: Set of Underground tools / keys on a presentation board.
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Making rooms safe for users to share sensitive information
Making rooms safe for users to share sensitive information
We see a world where your team isn't afraid of clicking on the wrong link and logging into the wrong website.
MetaCert monitors chat rooms for web links, warning your team about malicious & inappropriate content before harm is done to their phone or computer.
We provide you with a security operations analytics platform, enabling you to have an automated incident response workflow for monitoring files & URLS.
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MetaCert helps every member of your team have peace of mind knowing they can collaborate safely and share sensistive information inside of Hipchat!
User reviews(1)Sign in to write a review »
Paul Walsh supports Atlassian Room Security by MetaCert. You can visit the support site to get help.Get support
HipChat Cloud • Released 2017-01-09
Minor version update
DetailsMinor version update
Click on Get it now to be redirected to your HipChat instance to install this add-on. Then follow on-screen instructions. If your add-on can only be installed by an administrator, you will see a prompt. Otherwise, choose a room you own to install the add-on.


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Compare Real Estate attorneys near you. Read reviews, see Avvo Ratings, and contact them directly.
5.0 stars 3 reviews
Avvo Rating: 7.1
Allow me to introduce myself to those of you I have not already met. I am an estate planning and small business attorney in League City, Texas. I graduated from the University of Houston in 2000 and Harvard Law School in 2003. I have been practici...
Practice areas & skills: Real Estate, Estate Planning, Corporate & Incorporation
5.0 stars 5 reviews
Avvo Rating: 6.8
Review: "I am an out-of-state attorney that uses Mr. Montalbano's services in the state of Texas on a regular basis. He is an extremely reliable, responsive and thorough attorney."
Practice areas & skills: Real Estate, Wills & Living Wills, Debt Collection, Business, Probate
5.0 stars 3 reviews
Avvo Rating: 6.7
Review: "Mr. Brown handled the real estate matters in my divorce and rental property issues with extraordinary care and skill. He has been very professional and polite in dealing with all of the different matters that he has handl...
Practice areas & skills: Real Estate, Business, Probate, Construction & Development, Elder Law
Not yet reviewed
Avvo Rating: 7.2
Mr. Graham is a Texas attorney, financial advisor, and developer with strong expertise in the apartment business, having financed, owned, operated and developed apartment properties in Texas. Finance & Property Experience · Finance...
Practice areas & skills: Real Estate
Get the legal help you need at a fixed price. No hidden fees or long-term commitment.
Real estate attorneys deal with the many laws and practice areas associated with land ownership issues. For example, a commercial real estate attorney knows which laws apply to commercial transactions for construction/development projects (such as condo construction), or land use/zoning projects (such as subdividing a parcel of land for development), or real property issues (such as financing a large land purchase). Likewise, residential real estate lawyers help you buy a home, or prevent you from losing your home in foreclosure. They also know how residential tenancy laws work and can advise you of your rights as a tenant. With so many types of real estate attorneys, it is important to find a specialist who does the work you need.


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Glad Jack 3.6
You will direct a strange creature named Jack, and help him overcome a lot of obstacles and traps, while all the while not getting lost. Climb the walls and rocks to give his big brother gifts. Bright, beautiful graphics, good music and interesting gameplay will entertain for hours!
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Aqua Arkanoid 4.0
Aqua Arkanoid is an arcade game of the Arkanoid genre, but in a new, original style. There is a fish bowl, which will harbor marine life even in the most difficult situations! Game features fun music, a wide variety of levels and bright modern graphics. Aqua Arkanoid appeals to all players, regardless of their age.
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Kubonoid 4.6
Do you like Minecraft? Then this game is for you. Fascinating like Arkanoid, and will consume even the most demanding gamer. Upbeat music, colorful effects and of course the bonuses, sooooooo many bonuses! Online table that will allow you to share your high scores with friends and unique levels are features. Go through the forests and swamps, show your skills in the forest and Cave of Pokhara. Prove that you have the quickest reflexes!
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Aiseesoft Screen Recorder helps to capture almost any video played on your computer, including 4K videos. You can use this screen capture software to record various kinds of videos, like online movies, video conference, video tutorials, game play and other videos you want to save. When you record the screen, you can choose to record the sound of the video itself or record your own voice through microphone. Key features 1. Record any videos on your computer Aiseesoft Screen Recorder is a professional video recording APP. With this recording software, you donˇŻt have to buy another downloader because it will save more time and bandwidth to record rather than download. It is more powerful than downloader because it can save more, including game play process, video meetings and online streaming videos, etc. You can record any formatˇŻs video on your computer, even 4K videos. 2. Highlight the mouse cursor as you wish This is especially useful when you make video presentations. You can highlight the mous ...
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Rec&ts2mpg converter 2.9.55
Most digital video recorders, including satellite, (Topfield, DreamBox and so on) use the MPEG format for compressing the digitized video signals. But you cannot play back the recordings with a regular MPEG player on your computer (or create DVD from this MPEG) because they are formatted differently from the standard MPEG2 format. This program converts .rec and .ts formats (from DVR's) to the standard MPEG2 format.
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Orden Of Scarabey 4.6
Do you think that "betrayal"is just a big word? Do you think if you're the best, you will always stay on top? Do you really think if you know the secrets and you alone, then you will be left alone? If only.. I took part in the raid to seize nukes ... and was betrayed. Now I'm somewhere in the catacombs, lost in the desert with next to no visibility. But ... perhaps, you can get out of this jam? - A mysterious shooter with puzzle elements and ...
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Legend Of The Seeker 4.8
First off let's meet our seeker. His name is Edward. The essence of the game is to help him to become a legendary seeker. For the first time he performed the task and, of course, came home and played like most modern men would. After opening up new characters he meets and makes new friends. The game has a lot of objects, effects, and mini-games.
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RasterVect Free Edition 16.3.36
RasterVect can convert any raster images (BMP, GIF, TIFF, JPEG, PNG, and so on) to a vector format. Raster images can be imported by scanning original paper images. The target vector formats (DXF,WMF,EMF,EPS and AI) are supported by most CAD applications that use vector graphics, such as AutoCAD, Adobe Illustrator, Corel Draw. There are viewing tools like scrolling, color selection, zooming and tools for vector and raster editing.
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3D racing game. You need to use nitro to win this crazy racing. Turn it on and your car will gain incredible speed. Win competitions and get points to gain access to more powered cars. ULTRA version features: -new car physics; -new game mode - Time Attack (you compete with your best result ('ghost car')); -options menu to meet your PC power; -fullscreen and windowed mode;
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Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis.
We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.
As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins.
This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.
To understand complex cell systems, functional analysis of proteins has become the main focus of growing research fields of biology in the post-genome era; however, the roles of many proteins in cellular events remain to be elucidated. Among various methods to elucidate protein functions, the approach of chemical genetics is notable, with small molecular compounds used as probes to elucidate protein functions within signal pathways [1,2]. Indeed, several bioactive compounds have led to breakthroughs in understanding the functional roles of proteins [3-11]; however, one significant hurdle to developing new chemical probes of biological systems is identifying the target proteins of bioactive compounds, discovered using cell-based small-molecule screening.
A variety of methods and technologies for identifying target proteins have been reported [12]. Among them, affinity chromatography is often used for identifying biological targets of multiple small molecules of interest; however, it is usually very difficult to identify compound-targeted protein with low expression because of the low sensitivity of detection using coomassie brilliant blue (CBB) staining and MS/MS analysis. Thus, target identification of small molecules using affinity chromatography is severely limited. To overcome the limitations of affinity chromatography, we propose a new protocol combining in silico screening and experimental verification for identification of target proteins.
In our previous work, we developed an in-silico screening system, called "COPICAT" (Comprehensive Predictor of Interactions between Chemical compounds And Target proteins), to predict the comprehensive interaction between small molecules and target proteins [13]. If a target protein is input in the system, a list of chemical compounds which are likely to interact with the protein is predicted. In our previous work, several potential ligands for the androgen receptor were predicted by this system, these predictions were experimentally verified, and a novel antagonist was found [14]. On the other hand, if a chemical compound is input in the system, a list of proteins which are likely to interact with the compound is predicted by the system.
Previously, we isolated the natural product incednine from the fermentation broth of Streptomyces sp. ML694-90F3, which consists of a novel skeletal structure, enol-ether amide in the 24-membered macrolactam core, with two aminosugars. In addition, it was reported that incednine induced apoptosis in Bcl-xL-overexpressing human small cell lung carcinoma Ms-1 cells when combined with several anti-tumor drugs including adriamycin, camptothecin, cisplatin, inostamycin, taxol, and vinblastine [15]. Because this compound inhibits the anti-apoptotic function of Bcl-2/Bcl-xL without affecting its binding to pro-apoptotic Bcl-2 family proteins, it may target other proteins associated with the Bcl-2/Bcl-xL-regulated apoptotic pathway. To address the mode of action of incednine underlying its interesting function, we first synthesized affinity-tagged incednine which is biologically active (data not shown), and proteins bound to incednine were separated by SDS-PAGE followed by CBB staining, and each protein band was directly identified using liquid chromatography-tandem mass (LC-MS/MS) spectrometry analysis. Fifty-three proteins were identified as listed in Table1, and some of which, such as eukaryotic initiation factor 4A3(eIF4A3), prolyl 4-hydroxylase, beta subunit (PDI), heat shock protein 70 (HSP70), and protein phosphatase 2A (PP2A) were reported to relate to cancer cell survival[16-19]. Therefore these were knocked down by siRNA or inhibited by a specific inhibitor, and assessed for their ability to modulate Bcl-2/Bcl-xL anti-apoptotic function, as does incednine. However, the candidate proteins tested did not appear to be the target responsible for modulating Bcl-2/Bcl-xL anti-apoptotic function (Additional file 1). Therefore, the target protein of incednine responsible for modulating Bcl-2/Bcl-xL anti-apoptotic function has not yet been determined, and further candidate proteins as targets of incednine are expected to emerge.
In this context, we propose a new protocol combining in silico screening and experimental verification for the identification of target proteins. We first predicted the candidate proteins likely binding to the input compound by applying the COPICAT system, and then employed western blotting to detect the binding of predicted proteins to the input compound. This method solves the problem of the low sensitivity of the traditional method (as illustrated in Figure Figure11).
We set the chemical compound "incednine" as the binding ligand, and candidate proteins for the targets of incednine were computationally predicted from the KEGG database by using the statistical prediction method for protein-chemical interaction. The training dataset of protein-chemical interactions to construct the SVM-based statistical learning model was collected from the approved DrugCards data in the DrugBank database [20], and 53 interactions with incednine obtained from our previous binding experiments using affinity chromatography (see Table1 and Methods) because the prediction accuracy was increased when more training samples of protein-chemical interactions were given to the SVM-based statistical learning model. Among 24,245 human proteins in the KEGG repository, 182 proteins were newly predicted as positive, that is, to interact with incednine with high probability greater than the 0.5 threshold (the default threshold value).
The 182 proteins that were computationally predicted to bind to incednine were clustered by the hierarchical clustering method using 199-dimentional feature vector that was used for encoding amino acid sequences to construct the SVM-based statistical learning model (See Methods section for the details). Note that the similarity based on this 199-dimentional feature vector is different from the sequence similarity, and this similarity measure based on the 199-dimentional vector was proven to work well for protein-chemical interaction predictions in our previous work [13]. For example, 5HTT and AR α-1A showed only about 10% sequence similarity although both were reported to interact with the MDMA drug and successfully predicted by our SVM-based statistical learning method. A cutoff threshold on the constructed clustering tree was determined so that the proteins were clustered into 11 clusters and each cluster had a statistically significant number of members. The proteins predicted to bind to incednine are listed in Additional file 2.
Next, to examine whether incednine can bind to the proteins, an in vitro biotinylated incednine pull-down assay using the lysate of Bcl-xL expressing Ms-1 cells was performed. We tested 16 proteins as pilot experiments, which are selected from each cluster by one or two based on antibody availability. Negative candidates that were predicted not to bind to incednine were extracted for experimental verification. These proteins, positive candidates and negative candidates, are listed in Table2. Among positive candidate proteins, 2 positive candidates PIK3CG and ACACA were found to bind to incednine, and 5 positive candidates DAPK1, PIK3C2B, PIP5K3, CHD4, GTF2IRD2 did not bind to incednine. Among negative candidate proteins, 2 negative candidates BECN1 and KIF5B did not bind to incednine, and 1 negative candidate PARP1 did bind to incednine (Figure (Figure2).2). On the other hand, ITPR1, PARP14, PLCB1, KIF1A, KIF21B, and RGPD5, listed as positive candidates in Table2, were not well expressed and were not detected in Bcl-xL-expressing Ms-1 cells; therefore, accuracy of 40% (4/10), sensitivity of 66.7% (2/3) and precision of 28.6% (2/7) were achieved.
For target identification using affinity chromatography, conventional method requires multiple steps as follows; SDS-PAGE, CBB staining, excision of gel, destaining, reduction, trypsinization, and application to LC-MS/MS system (7 steps); these steps can be cumbersome, time-consuming and require expensive installation. Furthermore, CBB staining used in conventional method can detect proteins over nanogram order. In contrast, our proposed protocol for predicting target protein allows us to use western blotting to detect proteins in picogram order. Indeed, we found two incednine-binding proteins by this prediction. Additionally, we can enhance the precision of COPICAT by feeding back the experimental results to the system.
In this work, PIK3CG, PARP1, and ACACA were revealed to bind to incednine by applying our protocol to identify potential target proteins of chemical compounds. These proteins are potential targets of incednine because it has been reported that these proteins are related to cancer survival and drug resistance, as follows.
PI3KCG encodes p110 catalytic subunit isoform p110γ and heterodimerizes with regulatory subunit p101, composing class IB PI3K in the PI3K family [21,22]. Although PIK3CG and PIK3C2B are distant homologous with 20% sequence identity, incednine selectively binds to PIK3CG but not PIK3C2B (Figure (Figure2).2). In contrast to class IA, class IB PI3K acts downstream of G-protein coupled receptors (GPCR). It has been reported that p110γ was upregulated and activated by the chimeric oncogene Bcr-Abl expression to contribute to cell proliferation and drug resistance in chronic myelogenous leukemia [23], and was found to be highly and specifically expressed among the PI3K family in human pancreatic cancer [24], suggesting that class IB PI3K might relate to cell survival and drug resistance. Product of enzymatic activation of class IB PI3K as class IA, phosphatidylinositol-3,4,5-trisphosphate, makes BAD dissociate from Bcl-xL and promotes cell survival via Akt activation [22]. Therefore class IB PI3K might contribute cell survival in Bcl-xL-overexpressing cells.
PARP1 is a member of the PARP protein superfamily that catalyzes the polymerization of ADP-ribose moieties onto target proteins, using NAD+ as a substrate and releasing nicotine amide in the process [25]. PARP1 activity is important for the regulation of homeostasis and the maintenance of genomic stability, participating in DNA repair, the regulation of transcription, DNA replication, cell differentiation, proliferation and cell death [26-28]. Many in vitro and in vivo experiments demonstrated that inhibition of PARP1 potentiates the cytotoxicity of anti-cancer drugs and ionizing radiation [29-32]. Therefore, incednine could bind to PARP1 and could function as antagonist of anti-apoptotic PARP1 protein. Alternatively, PARP1 is emerging as an important activator of caspase-independent cell death. It has been previously reported that PARP1 mediates the release of apoptosis-inducing factor (AIF), one of the initiators of caspase-independent cell death, possibly due to enzymatic over-activation [33-35]. We also observed that co-treatment of Bcl-xL-overexpressing Ms-1 cells with incednine and ant-tumor drugs induced AIF release and subsequent caspase-independent cell death (unpublished data); therefore, we can not exclude the possibility that incednine binds to PARP1 and functions as PARP1 agonist by accerelating AIF release.
However, the most likely candidate of an incednine target protein is ACACA (acetyl-CoA carboxylase-α), which was classified in cluster 9. ACACA is the rate-limiting enzyme for long-chain fatty acid synthesis that catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA, playing a critical role in cellular energy storage and lipid synthesis [36]. There is strong evidence that cancer cell proliferation and survival are dependent on de novo fatty acid synthesis [37-40]. Additionally, ACACA is upregulated in multiple types of human cancers [41,42]; therefore, ACACA may also contribute to cell survival in Bcl-xL-overexpressing tumor cells. Indeed, our preliminary experiments suggested that chemical inhibition of ACACA using TOFA (5-tetradecyloxy-2-furoic acid, ACACA antagonist) or small interfering RNA-mediated ACACA silencing results in the induction of apoptosis in Bcl-xL-overexpressing human small cell lung carcinoma Ms-1 cells when combined with anti-tumor drugs as does incednine (unpublished observation), suggesting that ACACA might be a molecular target of incednine. The possibility that incednine targets ACACA is being actively investigated.
While our experimental verification implied the relatively low precision value 28.6% (2/7), new detections of two incednine-binding proteins in addition to previously identified 53 proteins are significant. On the other hand, while we selected 7 candidates by clustering 182 predicted proteins for experimental verification, more comprehensive verification experiments for the 182 predicted proteins are needed.
The application of our method to incednine resulted in 28.6% (2/7) precision according to in vitro pull-down assay. However, this relatively low precision value does not represent the true statistical significance of the method and is not comparable to the benchmark performances (including 98.4% precision) by 10-fold cross-validation for COPICAT system.
This 28.6% precision can be evaluated by using the following P-value.
Here, N is the number of human proteins, M is the number of proteins potentially binding to the incednine, t is the number of tested proteins, and p is the number of true positives. With N =24,245, which is the number of human proteins in the KEGG repository, and M=N×1%243, which is based on the overestimated assumption that 1% of all proteins could be regarded as potential binding proteins for the incednine. This P-value defines the probability that the prediction precision can be obtained by random selection of proteins. Then, P-value of 0.002 was obtained for the prediction precision 28.6%. This small P-value means that 28.6% (2/7) precision can be obtained with very small chance by random selection, and therefore, this small P-value proves the validity of our method.
Although further study is required for complete determination of the target protein of incednine, this study demonstrated that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.
The DrugBank dataset was constructed from Approved DrugCards data, which were downloaded from the DrugBank database [20]. These data consist of 964 approved drugs and their 456 associated target proteins, constituting 1,731 interacting pairs or positives. Additional data about 53 interactions with incednine, listed in Table1, were obtained from our previous binding experiments.
An amino acid sequence of protein is divided into trimers (three amino acid residues), and all of the 8,000 trimers are clustered into 199 groups according to physical-chemical properties. Then, an amino acid sequence is converted to a 199-dimensional feature vector based on the frequencies of 199 clusters (See for [13] the details of this procedure). A chemical compound is also converted to another feature vector of 199 dimension representing substructure statistics extracted from the structural formula of a chemical compound. The size of the dimensions, that is, 199 dimensions, was determined based on the variance of each dimension. The top 199 dimensions with significantly diverse variances in statistical classification were selected.
We developed a comprehensively applicable statistical prediction method for interactions between any proteins and chemical compounds, which requires only protein sequence data and chemical structure data and utilizes the statistical learning method of Support Vector Machines (SVM)[13,14].
We consider the problem as the binary classification of protein-chemical pairs whose abstractive identities are represented numerically by the 199 dimensional feature vectors defined above. We obtained a "positive" sample set, i.e., a set of protein-chemical pairs that have been proven to interact with each other via biological assays, from the DrugBank database [20]. Along with the positive sample set, SVM-based classifiers require a "negative" sample set, i.e., a set of protein-chemical pairs that do not interact with each other. Such a negative sample set can be extracted randomly from the whole complement set of the positive sample set. Though we used random pairs of drugs and proteins as negative samples in constructing a model, the lack of reliable negative samples is always a problem when applying the statistical learning methods. In our current study, it is assumed that drugs in the DrugBank dataset rarely interact with proteins other than their known targets because they are approved drugs. Using the resultant positive and negative protein-chemical pair sets, we trained two-layer SVMs. First, we trained each multiple first-layer SVM with small sample sets designed with different criteria. Next, using another larger sample set, we trained a second-layer SVM whose input is a set of probabilities output from the firstlayer SVMs. The prediction performances were evaluated by 10-fold cross-validation using the DrugBank dataset. The sensitivity, specificity, precision, and accuracy were 0.954, 0.999, 0.984, and 0.997, respectively, in cross-validation. The details of the algorithms and their prediction accuracy are described in our previous reports [13,14].
Given n samples, each of which has an m-dimensional feature vector and one of two classes, such as binding and non-binding , an SVM produces the classifier
where x is any new object which needs to be classified, K (·,·) is a kernel function which indicates that the similarity between two vectors and (α1,,αn) are the learned parameters. The RBF kernel was utilized for the SVM classifier. In our study, the LIBSVM program [43] was employed to construct the SVM model.
Bcl-xL-overexpressing human SCLC Ms-1 cells [15] were maintained in Rosewell Park Memorial Institute media (Nissui, Japan) supplemented with 5% fetal bovine serum, 100 U/ml penicillin G, and 0.1mg/mL kanamycin at 37°C in a humidified 5% CO2 atmosphere.
Mouse monoclonal anti-DAPK1 (DAPK-55), rabbit monoclonal anti-PIK3CG (Y388), rabbit monoclonal anti-ACACA (EP687Y), mouse monoclonal anti-PIK3C2B, rabbit polyclonal anti-ITPR1, mouse monoclonal anti-PIP5K3, mouse monoclonal anti-CHD4, mouse polyclonal anti-GTF2IRD2, mouse polyclonal anti-PLCB1 antibodies were purchased from Abcam (Cambridge, MA). Rabbit polyclonal anti-KIF21B and mouse monoclonal anti-KIF5B (clone H2) antibodies were purchased from Millipore (Bedford, MA). Goat polyclonal anti-PARP14 and goat polyclonal anti-KIF1A were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-Beclin (clone 20) antibody was purchased from BD Transduction Laboratories (San Diego, CA). Rabbit polyclonal anti-PARP1 antibody was purchased from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal anti-RGPD5 antibody was purchased from Lifespan Biosciences (Seattle, WA). Mouse monoclonal anti-Flag (M2) antibody was purchased form Sigma (St. Louis, MO).
Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from GE Healthcare (Little Chalfont, UK). Horseradish peroxidase-conjugated anti-goat IgG was purchased from Santa Cruz Biotechnology.
Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore) by electroblotting. After the membranes had been incubated with primary and secondary antibodies, the immune complexes were detected with an Immobilon Western kit (Millipore), and luminescence was detected with a LAS-1000 mini (Fujifilm, Tokyo, Japan).
Incednine was isolated from the culture broth of Streptomyces sp. ML694-90F3 [15]. To obtain biotinylated incednine (see Additional file 3), incednine (137.0mg) and the amine-reactive biotin-X (100.0mg; Invitrogen) were dissolved in 13.0mL CHCl3:MeOH (10:1). After stirring at 40°C for 20h, the reaction mixture was concentrated to dryness. The residue was resolved in 50mL CHCl3:MeOH:H2O (5:6:4) and partitioned three times under basic conditions. The lower layer of CHCl3:MeOH:H2O (5:6:4) was evaporated in vacuo to yield a brown residue. The residue was purified by HPLC (Senshu Pak Pegasil ODS 30 x 250mm) and eluted with MeOH:40mM KH2PO4 aq. (70:30) to give 19.4mg biotinylated incednine.
Bcl-xL-overexpressiong Ms-1 cells were collected and sonicated twice in IP buffer (50mM HEPES (pH 7.5), 150mM NaCl, 2.5mM EGTA, 1mM EDTA, 1mM DTT, and a protease inhibitor cocktail (Roche, Mannheim, Germany)) for 10s. The cell lysates were centrifuged at 10,000g for 15min at 4°C. The resulting supernatants were incubated with biotin (50nmol) or biotinylated incednine (50nmol) and avidin beads at 4°C for 3h. The beads were washed three times with phosphate-buffered saline (PBS). The bound proteins were eluted with 2mM biotin in PBS, and concentrated by a centrifugal filter device (Ultracel (YM-10); Millipore). The resulting proteins were boiled in SDS sample buffer for 5min and subjected to western blotting.
Incednine binding proteins purified using biotinylated incednine / avidin beads, and flag-tagged incednine (see Additional file 4) / anti-Flag antibody were anaylzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) system as previously described, respectively [44,45].
YS and MI designed the study and analyzed the data. HK, HH, MN and YF performed the experiments. YS, MI and HK wrote the paper. YF synthesized biotinylated incednine. AI, MY, SI, KS, TD, TT, and TN performed MS/MS analysis. All authors read and approved the final manuscript.
Validation work for eIF4A3, PDI, PP2A and Hsp70.
Proteins computationally predicted to bind to incednine (grouped into 11 clusters).
A stucture of biotinylated incednine.
Preparation of Flag-tagged Incednine[46,47].
This work was supported in part by a Grant program for bioinformatics research and development from the Japan Science and Technology Agency.
This work was also supported by Grant-in-Aid for Scientific Research (A) No.23241066 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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