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Nova 3.0 released
I'm really happy to announce version 3.0 has been released of Nova Framework, (Nova is the new name, replacing Simple MVC Framework). The main focus of 3.0 was to place files above the document root.
This meant restructering the folders and there paths, normally assets such as css, js files and images are placed in the public folder so they can be ran normally, we went futher and placed everything except .htaccess and index.php above the root including all images, css and js files!.
This change results in a lot of other changes, documented in the docs and will be covered in future screencasts, here is the overall summary of the changes:Continue Reading


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Phishing: How to protect your business?
You no longer need to be a sophisticated hacker to commit fraud on the Internet. Anyone who is motivated can join in, thanks to the off-the-shelf phishing kits provided by a thriving cybercrime ecosystem. From virtual server hacking to mobile phone scams, phishing is a potent threat and can seriously impact your business.
This Symantec white paper explores the latest tactics and potential impacts of phishers, and how you can protect your brand and your business.
Provided by: Symantec Topic: Security Date Added: Oct 2012 Format: PDF

subset: commoncrawl


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Logi i Europa och världen
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Hotel Kraipaten Rai
Hotel Kraipaten Rai, Ul. Neofit Rilski 52, Banya
Hotel Kraipaten Rai is set in Banya, 6 km from Bansko and 42 km from Velingrad. Guests can enjoy the on-site restaurant. The rooms are fitted with a TV. Some units include views of the mountain or garden. Rooms have a private bathroom. Hotel Kraipaten Rai features free WiFi throughout the property. There is a 24-hour front desk at the property. Borovets is 43 km from Hotel Kraipaten..
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Luxury Villa Bansko
Luxury Villa Bansko, 22 A Second Str., Banya
Situated 4 km from the closest ski lifts, Luxury Villa Bansko is a very spacious and elegant house with amenities like 3 fireplaces, a fully equipped kitchen with a dishwasher, and a sauna. A balcony and a terrace with mountain views are provided and the closest supermarket is within 400 metres. The chalet comprises 5 bedrooms, a living area with a leather sofa, a flat-screen..
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Luxury Villa Bansko 2
Luxury Villa Bansko 2, 22 Second Str., Banya
Located 5 km from the Bansko Ski Area, Luxury Villa Bansko 2 offers mountain views, free WiFi, and a ski storage room with ski equipment on site. A bus stops 1 km away. This villa features a terrace with barbecue facilities, 2 bathrooms with a shower, a hairdryer, free toiletries and a separate toilet, a living room with a sofa and a flat-screen TV with satellite channels, a CD Player..
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Guest House Zhabokrek
Guest House Zhabokrek, Ulitsa Vtora №5, Banya
Offering a year-round outdoor pool and views of the pool, Guest House Zhabokrek is located in Banya in the Blagoevgrad Region, 6 km from Bansko. Free WiFi is featured throughout the property and free private parking is available on site. A TV with cable channels, as well as a CD player are available. Certain units feature a seating area where you can relax. Enjoy a cup of tea from..
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Villa Leventy
Villa Leventy, ul. Vtora no 18, Banya
Offering a garden, Villa Leventy is located in Banya. Bansko is 5 km away. Free WiFi is featured throughout the property. All units include a dining area and a seating area with a cable flat-screen TV. There is also a kitchen, fitted with a dishwasher and oven. A microwave and fridge are also available, as well as a kettle. There is a private bathroom with a shower and free toiletries..
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Hot Springs Medical and SPA
Hot Springs Medical and SPA, 1901 23, Banya
Set in Banya, 5 km from Bansko, Hot Springs Medical and SPA boasts indoor and outdoor pools with hydromassage jets, a children's pool, a kids club and a playground. Guests enjoy free access at the property's spa centre including sauna, sanarium - bio sauna, steam room, Turkish hammam and a salt room. Free, on-site parking is possible. Opening to terraces overlooking the surrounding..
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Guest House Gradina
Guest House Gradina, 15 Osma Str., Banya
Set in Banya in the Blagoevgrad region, Guest House Gradina is a holiday home featuring a terrace. Guests have a private balcony. Opening onto a patio, the holiday home consists of 3 bedrooms and a fully equipped kitchenette. The holiday home also provides a bathroom with a bath or shower. The holiday home offers a barbecue. Guests can also relax in the garden. Bansko is 6 km from..
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Studio Atani
Studio Atani, 23 Neofit Rilski street, Banya
Situated in Banya and 3 km away from Bansko, 500 metres from the Roman baths and the hot springs. Studio Atani offers self-catering accommodation with views of the ski runs. Free private parking is available on site. The kitchen is fitted with a toaster, coffee machine, electric kettle and a fridge. There is a private bathroom. A TV with satellite channels is featured. A mineral pool..
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Villa Victoria Thermal Spa
Villa Victoria Thermal Spa, 15 Treta street, Banya
Villa Victoria Thermal Spa is located in Banya village, famous for its mineral springs. The hotel offers free use of the outdoor pool with hydromassage nozzles and a children's pool. Each room has a flat-screen TV with cable channels, a seating area and a private bathroom with shower and free toiletries. Guests can start their day with a home-cooked breakfast. Guests enjoy free..
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Carpe Diem
Carpe Diem, 25 "8-th" Str., Banya
Set in Banya, a 10-minute walk away from the town centre and a 15-minute walk away from the public thermal beach, Guest House Carpe Diem offers free WiFi and a garden. An outdoor covered swimming pool with hot mineral water from the thermal spring with temperature around 36 degrees celsius is also at guests disposal. Free parking spaces are available on site and a bus stop is 80 metres..
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Guesthouse Trite Kambani
Guesthouse Trite Kambani, 27 26st street, Banya
At a distance of 5 km from the Bansko Ski Resort, Guesthouse Trite Kambani is centrally located in the Banya Village, just steps from the nearest restaurant and a supermarket. Free Wi-Fi and private parking are provided. Simply furnished rooms have wooden furniture and hardwood floors. Cable TV is provided in all rooms, while most rooms have a flat-screen TV. A private bathroom with..
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Spa Hotel Aspa Vila
Spa Hotel Aspa Vila, Banya Village, Banya
Set in Banya Village, famous for its more than 70 hot mineral springs, Spa Hotel Aspa Vila offers an outdoor pool, a sauna, a hot tub and a steam bath, all provided free of charge. Free WiFi is available throughout the hotel. Guests benefit from free private parking on site. Each room comes with a TV with cable channels and a CD player, a seating area and an en-suite bathroom with a..
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Thermal SPA Hotel Roman Bath
Thermal SPA Hotel Roman Bath, Vtora str. 58, Banya
In a quiet location on the edge of Banya, Spa Hotel Rimska Banya is 5 km from the Bansko Ski Resort and offers free shuttle to the ski lifts in winter. The spa centre includes free sauna, steam bath and swimming pool facilities. Wi-Fi is available in the entire hotel for free. All rooms are simply furnished and feature cable TV, a minibar and a kettle. Most of the rooms come with a..
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Hotel Pri Spaska
Hotel Pri Spaska, 22-nd Str No11, Banya
Housing a small grocery shop and a restaurant serving traditional Bulgarian cuisine, Hotel Pri Spaska offers accommodation with cable TV and free Wi-Fi. It is quietly located in the well-known Banya Spa Resort. All units feature hardwood floors and walls painted in light tones. The private bathroom comes with bath or shower and free toiletries. Pri Spaska Hotel is just steps away..
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Aquilon Residence & Spa
Aquilon Residence & Spa, 6 Sixth Str., Banya
Detta lägenhetshotell ligger i hjärtat av en fridfull och vacker dal bland bergen. Här erbjuds en utmärkt kombination av behandlingar med varmt mineralvatten och sportaktiviteter. Här bor du mycket nära den populära skidorten Bansko och kan njuta av en lugn småstadskänsla. Boendena har ett fint läge, både för dig som gillar vintersport och dig som söker avkoppling och..
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A Promotion Structure Built on Known Expectations
- July 19, 2011
- Job Hunting
- 0
Wouldn't it be great if you knew what it took to be promoted, how much you were going to make, and get that promotion quickly? That's the principle behind Zappos' Pipeline, a leadership-building philosophy built on the theory that people should be treated with respect and know when, why and how much they will be rewarded for their work.
At the Future of Web Apps Conference, I chatted with Ryan Carson, the show's producer and the owner of Carsonified, an online video tutorial training service for Web designers and developers. For his employees, Carson has fully adopted the Pipeline.
Carson fully understands he needs to treat his employees like adults. That's why he goes out of his way to treat them well by doing little things like feeding them and giving them four-day work weeks — but also to help his team grow their careers in ways that are predictable and exciting. He doesn't fall into the yearly review trap where no one pays attention or cares.
Employees at Carsonified set their goals and an agreed upon pay raise. If they don't achieve that goal they're given a timeline in which to achieve it. There's no ambiguity.
Each person has the power to control their career. Carson loves it and so do his employees. They actually know where their career is going.
Fascinating model. Sadly, conventional management theory requires the evaluation to be spent pointing out weaknesses and insisting that folk "get better" at those tasks. A better use of resources is coaching the employee into a position that plays to strengths.


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Features Works by Borodin, Gershwin, and Sibelius
SPOKANE, Wash. – The Gonzaga Symphony Orchestra presents its season-opening concert at 7:30 p.m., Monday, Oct. 22 at the Martin Woldson Theater at The Fox (1001 W. Sprague Ave.). Directed by Kevin Hekmatpanah, the orchestra will perform Borodin's "Polovetsian Dances," Gershwin's "An American In Paris," and Sibelius' Violin Concerto.
The concert will feature Kurt Nikkanen, one of America's most renowned violinists, during Sibelius' Violin Concerto. Nikkanen made his Carnegie Hall debut at age 12 while performing with the New York Symphony and debuted with the New York Philharmonic at age 14. Nikkanen went on to attend and graduate from the Juilliard School and has since performed with orchestras, symphonies, and philharmonics worldwide. Some notable ensembles he has performed with include the Los Angeles Philharmonic, the BBC Symphony and BBC Philharmonic, and the Perth Symphony Orchestra. Nikkanen also is concertmaster of the New York City Ballet Orchestra.
General admission is $12, $10 for students and seniors (free to GU faculty, students and staff). Tickets are available either by calling (509) 624-1200 or by visiting the ticket office at the Martin Woldson Theater at The Fox.
For more information about this or other Gonzaga music department concerts, please call (509) 313-6733 or visit the music department website [www.gonzaga.edu/music].
* A free shuttle service will take Gonzaga University faculty, staff and students to the Fox Theater, departing from St. Aloysius Church at 6:40 p.m. & 7:05 p.m. The shuttle also will take GU community members back to campus after the concert.


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* Prices may differ from that shown
I bought these last year as a newbie to binoculars but I wanted something lightweight and compact for travelling. The choice was a bit mind boggling but these were recommended to me by someone in the business and I've not been disappointed.
Minox is a German company and has a good reputation. I've read that it was set up by some ex Leica employees which means high quality lenses but a much lower price. The binoculars weigh less than 10 oz and come with their own carry case and strap. They are nearly 4.5 inches high and just under 4 inches wide and have soft rubber eye-cups which means that my husband who wears glasses can also use them easily. The body is very sturdy and according to the blurb waterproof.
As I say, I do not claim to be an expert or an avid bird-watcher but for the casual user I've been very impressed with my Minox which seems to offer very good value for money. I've struggled in the past to use binoculars successfully but have found these really easy to use and focus. I've used them for bird watching and general sight-seeing. I bought them with a USA road trip in mind when they came into their own for spotting wildlife - deer, etc (but sadly no bears!) - and identifying things on the horizon. My husband has used them for checking tiles on the roof and I used them at Wimbledon when I was lucky enough to get tickets on Centre Court but they were right at the back.
So all in all very practical for a variety of uses but I would imagine that a dedicated bird watcher might want something more.


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Stephen Kenny's first competitive match in charge of Dundalk will be the visit of his former club Shamrock Rovers as the fixture list for the 2013 Airtricity Premier Division was revealed today.
Kenny left the Hoops after an unsuccessful spell as manager with the club and will immediately face his former employers.
Champions Sligo Rovers will face a clash with Derry City in The Brandywell, while Limerick's first match in Thomond Park will be a derby with Cork City.
The format for the 2013 Airtricity League is similar to the 2012 season with the 12th placed team in the Premier Division at the end of the campaign being relegated and the champions of the First Division being promoted.
The second and third placed teams in the First Division will contest a two-legged play-off for the right to play the 11th placed team from the Premier Division over two legs for a place in the top flight in 2014.
The first round of the EA Sports Cup will take place on Monday, March 11 with the second round on May 20 and 21 and the quarter-finals on July 1. The semi-finals are scheduled for August 5 and the final is on September 21.
The first round of the FAI Ford Cup, featuring clubs from the intermediate and junior levels of the game, will take place on March 31 with the second round scheduled for June 2 and the third round on August 25.
The quarter-finals are on September 15 while the semi-finals will take place on the week-end of October 6 and the final is at the Aviva Stadium on November 3.
The mid-season break will take place from 17 June until 26 June inclusive.
Read the full fixture list here.
Opening day fixtures:
Fri. 8 March Bohemians v UCD, Dalymount Park, 7.45 pm
Fri. 8 March Bray Wanderers v Shelbourne, Carlisle Grounds, 7.45 pm
Fri. 8 March Derry City v Sligo Rovers, Brandywell Stadium, 7.45 pm
Fri. 8 March Dundalk v Shamrock Rovers, Oriel Park, 7.45 pm
Fri. 8 March Limerick v Cork City, Thomond Park, 7.45 pm
Fri. 8 March St. Patrick's Athletic v Drogheda United, Richmond Park, 7.45 pm


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1. On Monday, April 15 at 10 p.m. police reported a false reporting to law enforcement. A man near the 13000 block of South Wakial Loop initially reported his wife had been kidnapped, police said. An investigation indicated the wife was having drinks with another male subject, the man knew that and was trying to file a false report, police said.
2. On Monday, April 15 at 5:20 p.m. a man was cited for soliciting near the 500 block of East Mountain Sage Drive. The man had entered a gated community posted for no trespassing and no soliciting and was going door to door soliciting. After several complaints he was contacted by police and cited.
3. On Monday, April 15 at 9:55 p.m. near the 4600 block of East Chandler Boulevard an adult female was arrested on suspicion of shoplifting. About $122 worth of baby clothes were taken from a retail store.
4. On Thursday, April 18 police took report of a metal theft near the 3600 block of East Ray Road. Between March 19 and April 18 suspects unknown climbed over a 9-foot wall to gain access to a secured generator and stole an unknown amount of copper from the generator. Approximate value is $4,000 to $6,000 to repair the generator.
5. On Thursday, April 18 police took a report of shoplifting from a business near the 4700 block of East Ray Road. Two adult females took approximately four items of wall art from a store without paying for it. No arrests have been made.
6. On Thursday, April 18 police took a report of a burglary from a garage near the 13600 block of South 44th Street. Between April 14-18 unknown suspects gained entry to a detached garage. Suspects took numerous woodworking tools and a tool box containing precision tools. Police estimate about $6,000 worth of tools were taken.
7. On Thursday, April 18 around 10:20 p.m. police took a report of a burglary from a home near the 9400 block of South 43rd Place. The home was broken into sometime between noon on the 16th and 8 p.m. on the 18th. Suspects took a personal computer, tablet, Winchester rifle, and 12-gauge shotgun. Total value estimated to be $7,000.
8. On Thursday, April 18 around 10:50 p.m. police took a report of a theft at a business near the 4900 block of East Ray Road. Two adult females grabbed assorted beauty supplies and walked out without paying.
9. On Saturday, April 20 around 9 a.m. there was a drowning at a home near the 15000 block of 39th Place. An adult male was visiting and had spent the night at the residence. He was found deceased in the pool early in the morning by a family member. Alcohol may have been a factor.
10. Police took a report of an armed robbery on Sunday, April 21 at a business near the 3200 block of East Chandler Boulevard. A man walked into the store acting strangely and raised his shirt as if he might have a weapon, police said. The man left the store with liquor that he did not pay for. On April 23 officers spotted a man that matched the suspect's description. He was ID'd in a photo line up and charges are pending.
• Source: Phoenix Police Department.


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Nearing the summit of
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the Grassy Mountains HP, we had this view back west with a layer of fog and pollution laying in the valley below. Craner Mountain is the main peak you see in the distance. Salt Lake City lies about 75 to the east on this nice January day (our temps were in the 20's with just a little wind).
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text: Want to know the latest about celebrity couples? Who is dating whom? How do celebrities keep the romance alive? What do celebrity couples do on breaks? Are they single again? Celebrities who have long marriages, celebs who have married many times…Here's everything you need to know about the highs and lows of relationships in the celebrity world!


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A new TV spot for Gareth Edwards' Godzilla remake has arrived, giving us our clearest look yet at the titular beast.
The new spot spends most of its 30-second runtime focusing on the film's human players, as Bryan Cranston is torn away from his family and Elizabeth Olsen and Aaron Taylor-Johnson are tearfully parted.
However, the money-shot comes right at the end, with Godzilla rearing up in front of the camera and emitting an almighty roar.
Directed by Edwards and co-starring Juliette Binoche, Ken Watanabe and Sally Hawkins, Godzilla will open in the UK on 15 May 2014.
Source: Legendary


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Earlier this summer, rumors swirled that Rupp Arena may undergo a face-lift in the near future. This news came with some very mixed emotions to many, and that hasn't changed in the subsequent months.
Change can be good, but not to all in Big Blue Nation who feel that renovating Rupp Arena and potentially changing the name of the arena — gasp — would cause a divide among the fan base. In the latest update of this saga, mayor Jim Gray said he welcomes all kinds of second-guessing when it comes to renovating Rupp, according to KentuckySports.com: "It's been very informing to hear those voices, which illustrate just how much people care and the attachment they have to this shrine."
A major part of the renovation would be to add luxury suites, loge boxes and a private lounge. Suites and other lavish amenities — who wouldn't want this? On the surface it looks nice, but the primary issue is that ticket prices would almost assuredly increase, and that is reason for concern for much of Big Blue Nation. Fans whose wallets aren't quite as thick as some others may be forced to give up their seats.
University of Kentucky trustee Carol Martin "Bill" Gatton summed up his thoughts on the possibility of fans losing their seats due to a rise in prices: "That's not fair."
As for naming rights, Gray saw multiple possibilities: the Lexington Convention Center (Alltech and an undisclosed second firm have made bids), Rupp Arena, the entire entertainment district, an outdoor amphitheater and who knows what other elements. But he called adding a corporate name to Rupp Arena a "last resort."
Gatton also said he'd give "a few million" to Gray's project if the city agreed to a scaled-down renovation: Only upgrade the restrooms, plus install a scoreboard over center court and electronic ribbon signage at the bottom of the upper arena. After noting that a scoreboard over center court would require a "substantial" strengthening of Rupp Arena's roof, Gray showed little interest in a modest renovation. "To me, it's worth doing only if it's extraordinary and remarkable," he said.
Would Kentucky basketball have it not be "extraordinary" or "remarkable?" That's all they know.
President Trump fires back at LaVar Ball on Twitter
The never-ending war of words between President Donald Trump and LaVar Ball escalated to another level on Wednesday morning.
Starting his early-morning tweets with some messages aimed at Ball, President Trump continued to double down on his insistence that he helped play a role in the safe return of three UCLA players arrested in China for shoplifting. LiAngelo Ball, LaVar's middle son, was one of the three players involved in the international incident as fellow Bruins Jalen Hill and Cody Riley were also arrested. The trio returned to the United States last week after UCLA left China without them following a win over Georgia Tech in the Pac-12 China Game.
ACTUAL HOOPS COVERAGE: Scott Drew's coaching gem | Porter Jr. out | Toledo player's Thanksgiving feast
MORE HOOPS COVERAGE: Player of the Year Power Rankings
LaVar has drawn the ire of President Trump for downplaying the President's role in the return of the UCLA trio as Ball maintains that others had more to do with the release. All three UCLA players publicly thanked President Trump and the United States government during their return press conference on Nov. 15. The three players remain suspended indefinitely from all activities with the men's basketball team.
In an interview with CNN earlier this week, LaVar was critical of Trump's role in the whole ordeal while also questioning why the President would spend so much time bothering for a thank you from the father of one of those arrested.
No. 22 Baylor comes from 12 down to beat Creighton
It was another rough night for the Scott Drew Can't Coach crowd.
No. 22 Baylor got 15 points apiece from Jo Lual-Acuil and Terry Maston and closed the game on a 37-19 run as they knocked off Creighton, 65-59, in the title game of the Hall Of Fame Classic in Kansas City.
King McClure led the way for the Bears with 19 points, picking up the pieces for Manu Lecomte, who struggled to deal with the defense of Khyri Thomas.
Creighton jumped out to a 33-24 lead at the break and extended it to 40-28 with 18 minutes left in the game, but that's when Baylor turned the game around. A couple of tweaks to the way that they played their zone coupled with the Bluejays missing some shots that they were capable of making led to the comeback. Instead of simply writing another 'See, I told you Scott Drew can coach' column, I figured it would make more sense to show exactly what I mean when I say that.
Creighton had a smart, simple game-plan offensively on Tuesday night. Get the ball into the paint, whether it was via dribble penetration or finding one of their big guys near the foul line or at the short corner, and then find a shooter on the perimeter, a cutter going to the rim or, simply, score from 8-10 feet out. That's the best way to beat a zone, especially a zone that has the amount of length and athleticism that Baylor's does. Notice in the clip below how extended Baylor's guards are and, as a result, the space it creates:
Once Baylor got down by 12, their game-plan changed. Instead of extending, their defense became more compact. What is usually something of a 1-1-3 zone turned into more of a 2-3, with the focus seemingly being cutting off penetration. Baylor dared Creighton to let Ronnie Harrell be the guy that beat them, and it worked. The result was that the open threes dried up, and the jumpers that Creighton shot in down the stretch were much more contested than the looks they were getting earlier in the game:
That's coaching right there.
Game-planning is a part of coaching. Player development is, too, as is recruiting. But making in-game adjustments like that, figuring out how a team is beating you, devising a way to stop them from doing that and getting your players to execute those adjustments is arguably the most important part of being a coach.
Here's another example of what I mean.
Khyri Thomas might be the best on-ball defender in college basketball, and I don't say that lightly. He essentially eliminated Manu Lecomte from the game. He is to point guards what Darrelle Revis was to No. 1 receivers. Whoever he is guarding is on Khyri Island.
Lecomte is typically Baylor's closer, but Drew ran actions that allowed Lecomte to be a facilitator and a decoy, taking Khyri out of the play and taking advantage of matchups he thought his guys could win. That involved running a double-high ball-screen, which confused Harrell and Martin Krampelj defensively a couple of times, and resulted in a high-low action between Maston and Lual-Acuil on a number of possessions down the stretch.
But then there was also this set he drew up, using McClure as the ball-handler in that double-high ball-screen and while putting Lecomte in the same side corner. McClure refused the ball-screen, drove straight at the gap where Thomas was not going to help off Lecomte and got a bucket out of it:
That's coaching!
And I'm not trying to say McDermott got out-coached here. His game-plan worked. Drew's adjustment turned out to be just a bit better.
But Creighton also has players that can make the tough shots that they were forced into in the second half. If two more of them go down – if the Bluejays shoot 37.5 percent from the floor instead of 34.4 percent, if they go 7-for-30 from three instead of 5-for-30 – then they probably win this game.
Sometimes that's how basketball works.
It's why you always hear coaches refer to it as a 'make or miss game'.
The larger takeaway from this game should be this: Both Baylor and Creighton are good teams. Both landed good non-conference wins during this event. Both are likely headed to the NCAA tournament.
And both took part in a fun, tactical battle between head coaches on Tuesday night that one of them had to lose.
No. 13 Notre Dame drubs LSU 92-53 to reach Maui title game
LAHAINA, Hawaii (AP) — T.J. Gibbs scored 26 points, Matt Farrell added 17 and Notre Dame dominated LSU 92-53 on Tuesday night to reach the Maui Invitational championship game.
The Irish (5-0) expectedly breezed through their opener against Division II Chaminade did the same thing to LSU in their first game against a power program this season.
Notre Dame shot well, shut the Tigers down on defense and were in control from the opening tip in a superb all-around game.
Bonzie Colson had 12 points and 11 rebounds for the Irish, who shot 52 percent and hit 15 of 32 from 3-point range.
Next up: A top-of-the-marquee title game against No. 6 Wichita State on Wednesday night.
LSU (3-1) lost starting guard Brandon Sampson to an ankle injury in the game's opening minute and struggled without one of its top defensive players.
The Tigers had trouble slowing the Irish on defense and labored from the perimeter on offense, hitting 6 of 23 shots from the 3-point arc while shooting 36 percent overall. Duop Reath led LSU with 17 points.
LSU beat Michigan 77-75 in its Maui opener behind the stellar play of Tremont Waters. The talented freshman point guard had 21 points and set up the go-ahead basket with a spectacular over-the-shoulder, no-look assist from his knees.
Notre Dame had a much easier road to the semifinals, dominating Chaminade from the start of an 83-56 rout.
The Tigers had a tough break on their first possession of the semifinals, when Sampson came down on someone's foot and rolled his left ankle. He had to be helped off the court, leaving LSU without arguably its best defensive player.
The Irish took advantage, scoring at the rim and the 3-point arc during a 15-2 run that put them up 25-10. Farrell had the highlight-reel play of the spurt, bouncing a pass between the long legs of 6-foot-11 Reath to set up Martinas Geben for a dunk.
Notre Dame didn't let up, hitting seven 3-pointers, 15 of 31 shots overall and holding the Tigers to 1-for-8 shooting from the arc for a 40-24 halftime lead.
The Irish continued to stretch the lead in the second half, using a 6-0 burst midway through to go up 61-35.
Notre Dame turned its first game against a power program into a laugher with a strong effort on both ends of the court.
LSU was hurt by the loss of Sampson, but it may not have mattered the way the Irish played.
Notre Dame plays No. 6 Wichita State in Wednesday's championship game.
LSU gets Marquette in the third-place game on Wednesday.
For more AP college basketball coverage: http://collegebasketball.ap.org and http://twitter.com/AP_Top25
Hot shooting leads No. 3 Kansas past Texas Southern, 114-71
LAWRENCE, Kan. (AP) — Kansas shot the ball from 3-point range better than it ever has in its illustrious history.
Once the Jayhawks found their rhythm from deep, their offense was virtually impossible to stop. Texas Southern coach Mike Davis was in awe.
"I've never seen a team pass the ball and shoot the basketball as well as they do," Davis said.
Svi Mykhailiuk scored 21 points, Udoka Azubuike added 20 and No. 3 Kansas cruised to a resounding 114-71 victory over Texas Southern on Tuesday night in the Jayhawks' first game of the Hoophall Miami Invitational.
They got after it early, as with just under 5 minutes remaining in the first half Lagerald Vick hit the team's seventh 3 of the half — a program record. A similar feat was achieved in the second half, when Devonte' Graham hit No. 17, the record for 3s in a game.
"It's super fun," Graham said. "Being active, sharing the ball, it's contagious. Just making that extra pass, and when the ball's going through the hoop like that, it just feeds energy into us."
Graham, Vick and Marcus Garrett all finished with a double-double for Kansas, as Vick posted 19 points and 10 rebounds, Graham had 17 points and 11 assists, and Garrett logged 13 points and 11 boards.
Texas Southern's Demontrae Jefferson led all scorers with 24 points. Donte Clark added 19 and had a game-high 14 rebounds as well.
Davis has seen plenty of high-powered offenses run by Bill Self, as the pair used to meet regularly when they coached at Illinois and Indiana, respectively. After watching a performance like this, he has no doubts over his former rival's future chances.
"I've been around for a long time," Davis said. "If you play basketball like they play basketball, they'll be cutting the net down in April."
Kansas continues to thrive without freshman Billy Preston, who remains benched as the school investigates a single-car on-campus incident involving him earlier in the month. His absence has left Self with just two big men, but the lack of depth has yet to truly hurt the Jayhawks.
Texas Southern is still searching for its first win after facing a daunting schedule to start the season. Even though the Tigers have yet to find themselves in the win column, games against bigger schools like Kansas will continue to provide invaluable experience regardless of the score.
"It was a great opportunity for us," Davis said. "We leave tomorrow to go play Clemson on Friday, and this game right here will get us ready for our next game."
Azubuike earned a technical foul midway through the first half when he hung on the rim following a thunderous dunk.
"He deserved it," Self said of the technical. "I told the official — he said 'I hate calling that,' I said 'but you got to call it.' I mean, that's good for us … he has a bad habit of doing that, and I was glad they called it because that may end up not costing us where we really need it, in a close game."
While Self agreed that the Jayhawks shot the ball about as well as they possibly could have, he wasn't overtly enthused by the record, as per usual.
"I couldn't be happier. I think we should celebrate for a week," Self said. "My reaction is we made shots. That doesn't mean anything to me."
"Love the way they play," Davis said of the Jayhawks. "That's the way I want my team to play. When we get to January and play in our conference, that's the way we want to be playing basketball."
Kansas will continue Hoophall Miami Invitational play Friday night with another home game against Oakland, which has already dropped its first two games of the tournament.
Texas Southern will once again face an uphill battle for its first victory as it travels to Clemson on Friday.
For more AP college basketball coverage: http://collegebasketball.ap.org and http://twitter.com/AP_Top25
No. 12 Cincinnati uses strong start to defeat Richmond 75-48
GEORGE TOWN, Cayman Islands (AP) — Unlike the previous night, Cincinnati didn't need a hero on Tuesday night.
Instead, the 12th-ranked Bearcats relied on their defense, smothering Richmond in the first half and cruising into the championship game of the Cayman Islands Classic with a 75-48 win over the Spiders.
Cane Broome led a balanced attack with 13 points and Jacob Evans added 12 for the Bearcats (5-0).
Jarron Cumberland opened the scoring with 3-pointers on consecutive possessions and Evans hit another before the Spiders scored and when Justin Jenifer hit a 3 just beyond the five-minute mark Cincinnati was up 14-4.
The Bearcats hit 8 of 14 3-pointers and shot 54 percent overall to race to a 40-14 lead at the half and Wyoming's opponent for Wednesday's championship game was never in doubt. Five different Cincinnati players had six to nine points while forcing the Spiders (1-3) into 13 turnovers and 4-of-18 shooting.
"The first half, we played great defensively and on offense, when we did make the right pass, we found open shooters," Cincinnati coach Mick Cronin said. "We try to start the best defensive lineup. We want to focus on things that win games, defense, rebounding, physicality, and guys that play together."
Jenifer went 3 of 4 from behind the arc, scoring all nine of his points in the first half. Gary Clark, the leader in a hard-fought 73-68 win over Buffalo in the opener on Monday night with 24 points and 14 rebounds, had his eight points — all in the first half — and finished with eight rebounds and seven assists.
Richmond made five 3-pointers and went 12 of 24 from the field but never challenged in the second half.
"Obviously Richmond has a young team," Cronin said. "I thought Buffalo made us better last night. We were able to jump out to an early lead and put the game away with our defense.
Freshman Jacob Gilyard led the Spiders with 12 points.
Richmond: Gilyard was leading the nation's true freshmen in playing time, averaging 37.3 minutes a game. He played 32 in the loss. … Grant Golden, a redshirt freshman, had 26 points in a win over UAB in the opener, the most by a Spider freshman in 10 years and with his seven rebounds was the first UR frosh with a 20 and 7 line since 1999. The Bearcats held him to four points and one rebound on Tuesday. … Khwan Fore, Richmond's leading scorer last year, made his debut after missing the first three games recovering from a stress reaction in his left shin. He went 1 for 1 from the foul line with a rebound and an assist and three fouls. … The Spiders have one senior and five juniors.
Cincinnati: Freshman Sam Martin scored his first points, making a pair of free throws. … The Bearcats had 20 assists on 25 baskets but they also had 20 turnovers, 11 in the second half. But in that first half had a 16-0 advantage in points off turnovers. … With reserves seeing plenty of time, Cincinnati's bench outscored Richmond's 38-17, 28-15 in the second half.
Richmond will face Louisiana-Lafayette in the third place game Wednesday night.
Cincinnati meets Wyoming in the championship game Wednesday night.
For more AP college basketball coverage: http://collegebasketball.ap.org and http://twitter.com/AP_Top25


text:
A great sequel to a very accomplished tee. Grats on this piece, the composition, is killer. Interesting use of color selection
the jimiyo never fails … 1
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How intrincate design… Though beautiful.
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beautiful details! +1
Hi, hope you can help me too on my submission on the link below
Click on the link below, register and vote for Medulla by INODORO! Feel free to express your thoughts – it will go a long way. Please pass this on to family, friends and everyone you know. Each vote counts. Thanks for the support!
Yummy ! killer details ever, ever ! V+
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love this design in the ALTERNATE colors!
Still got the first Shirt in my wardrobe, I hope this one get's printed too! Love your designs, here & on threadless! +1
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This is boss, I'm picking one up.


text:
I know my fellow Greenvillians were saddened by the news of an armed robbery at Min Sang Grocery Store, while relieved to hear that the Sue brothers, though wounded, are fine.
What is troubling is how long the store on Alexander Street has operated even as the neighborhoods around it have declined, and the blow it would be to the area if it closed.
My grandmother's house was just a couple of blocks from Min Sang, and I saw first hand as a little boy how hard the people worked to keep the store running. It is the epitome of the American Dream.
Here's the bottom line — if Min Sang decided to close its doors today, odds are nobody is going to come in behind them. The area loses a grocer that has been one of its icons for years and years. Businesses, including the grocer Stareka, which was once located a few blocks from Min Sang, have closed or relocated, but Min Sang's owners have stayed. Hats off to them.
At least it appears justice will be served.
Two men are being held on bonds of $5 million each related to the robbery that occurred at closing time on Jan. 3.
Assistant Police Chief Andrew Kaho told The Delta Democrat Times reports that 19-year-old Elton Harper Jr. and 26-year-old Korderro Robinson are each charged with armed robbery, conspiracy to commit armed robbery and attempted murder. They are being held in the Washington County jail.
Kaho said police are looking for a third suspect.
Police said 66-year-old Harry Seu was struck over the head with a handgun. He was treated and released at the hospital. His brother, 70-year-old Jerome Seu was shot. Jerome Seu was treated at Delta Regional Medical Center and held overnight for observation and released the next day.
Min Sang is not a large store. The economic impact to the city would be negligible if it closed. But simply put, Greenville cannot afford to lose anything. It is less about money and more about quality of life and opportunity.
Here's hoping for a speedy recovery for the Seus and the continued success of Min Sang.


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Home Toss Pillow
Perfect for someone moving into a new home or a new couple, this pillow describes the importance of home. With a cotton cover and polyester fill, the 20" square pillow has a natural color scheme to match any room.


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Libertate economică şi performanţă. Analiză comparată a diferiţilor indicatori statistici ai dezvoltării
Approaching economic freedom from an institutional perspective provides realistic economic insights. This study emphasizes the correlation between extending economic freedom and real GDP per capita, human development, global competitiveness, and the diminution of corruption, by comparing the index of economic freedom against various statistical indicators of development.
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Volume (Year): (2010)
Issue (Month): 02 (June)
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The Rev. Al Sharpton is the latest to applaud President Barack Obama's rendition of Al Green's "Let's Stay Together." Leaving his radio show in New York Friday, Sharpton caught up with TMZ to explain why the president's smooth vocals could help his campaign:
President Barack Obama's kick ass rendition of Al Green's "Let's Stay Together" at The Apollo last night was GREAT for his campaign … because it humanizes him to voters … this according to Al Sharpton.
The Rev — who ALSO killed it on stage at The Apollo a few years ago with a GREAT rendition of James Brown's "I Feel Good" — was out in NY today when we asked for his expert opinion.
Click here to read the rest of this story.


text:
Type 2 diabetes (T2D)-associated SNPs are more likely to be expression quantitative trait loci (eQTLs). The allelic expression imbalance (AEI) analysis is the measure of relative expression between two allelic transcripts and is the most sensitive measurement to detect cis-regulatory effects. We performed AEI screening to detect cis-regulators for genes expressed in transformed lymphocytes of 190 Caucasian (CA) and African American (AA) subjects to identify functional variants for T2D susceptibility in the chromosome 1q21–24 region of linkage. Among transcribed SNPs studied in 115 genes, significant AEI (P < 0.001) occurred in 28 and 30 genes in CA and AA subjects, respectively. Analysis of the effect of selected AEI-SNPs (≥10% mean AEI) on total gene expression further established the cis-eQTLs in thioesterase superfamily member-4 (THEM4) (rs13320, P = 0.027), and IGSF8 (rs1131891, P = 0.02). Examination of published genome-wide association data identified significant associations (P < 0.01) of three AEI-SNPs with T2D in the DIAGRAM-v3 dataset. Six AEI single nucleotide polymorphisms, including rs13320 (P = 1.35E-04) in THEM4, were associated with glucose homeostasis traits in the MAGIC dataset. Evaluation of AEI-SNPs for association with glucose homeostasis traits in 611 nondiabetic subjects showed lower AIRG (P = 0.005) in those with TT/TC genotype for rs13320. THEM4 expression in adipose was higher (P = 0.005) in subjects carrying the T allele; in vitro analysis with luciferase construct confirmed the higher expression of the T allele. Resequencing of THEM4 exons in 192 CA subjects revealed four coding nonsynonymous variants, but did not explain transmission of T2D in 718 subjects from 67 Caucasian pedigrees. Our study indicates the role of a cis-regulatory SNP in THEM4 that may influence T2D predisposition by modulating glucose homeostasis.
- type 2 diabetes
- eQTL
- allelic expression imbalance
type 2 diabetes [T2D (MIM 125853)] is a complex metabolic disease for which a genetic contribution is well recognized (2). Since 2007, global efforts to map T2D genetic susceptibility factors have mostly focused on genome-wide association strategies (33). Despite the significant success of these genome-wide association studies (GWAS), based on most recent analyses, the 63 T2D-associated loci discovered so far in Caucasian populations together account only for 5.7% of the liability-scale variance in disease susceptibility, and sibling relative risk (λs) attributed jointly by these variants is 1.104 (19). Thus, targeted analysis of previously identified genomic intervals representing well-replicated genome-wide significant linkage signals for T2D can be considered as an alternative strategy. Chromosome 1q21–24 is one such region of linkage for T2D that has been replicated in multiple studies in Caucasian and non-Caucasian families (3). However, fine mapping studies have not identified a common variant that explains linkage in this region, and no genome-wide significant association signal was discovered in this region in recent GWAS (29).
Recent studies have indicated that most single nucleotide polymorphisms (SNPs) associated with complex diseases are non-coding and more likely to be expression quantitative trait loci (eQTL) (20). Thus, identifying SNPs in chromosome 1q21–24 that regulate expression of transcripts as eQTL may be an alternative strategy to prioritize T2D susceptibility loci in this region. Most heritable gene expression traits are complex and are modulated by a combination of cis- and trans-acting loci. Allelic expression imbalance (AEI) analysis is a measure of relative expression between two alleles of a transcript (35). In AEI analysis, allele-specific mRNA levels are measured within individual samples. Thus, this approach is a very sensitive and specific method to query the effects of genetic and epigenetic differences on cis-regulatory control in a diploid genome, and it is unaffected by sources of variation such as environmental and trans-acting genetic factors (26).
We hypothesized that the linkage signal in the chromosome 1q21–24 region results from multiple common regulatory variants, many of which act in cis to regulate expression of adjacent transcripts. In this study, we performed pyrosequencing (PSQ)-based AEI screening to detect cis-regulators for genes in the chromosome 1q21–24 region that are expressed in transformed lymphocytes (TLs) of 190 Caucasians (CA) and African Americans (AA). We used data from GWAS to find associations of AEI-SNPs or proxies with T2D and glucose homeostasis traits. We further analyzed AEI in TLs from HapMap Caucasian (CEU) subjects to determine if any T2D-associated SNPs in linkage disequilibrium with tested AEI-SNPs modulate allelic expression and explain AEI variance. We identified a 3′-untranslated region (UTR) SNP in the thioesterase superfamily member-4 (THEM4) gene that showed a strong AEI and was associated with glucose homeostasis traits. Furthermore, functional analysis utilizing 3′-UTR luciferase construct and expression analysis in other tissues validated the regulatory role of this SNP. We resequenced coding and other putative functional domains of the THEM4 gene in 192 subjects to identify additional functional variants in this gene. Our results provide evidence that THEM4 is likely a susceptibility gene at the chromosome 1q21–24 locus and may influence T2D predisposition by modulating glucose homeostasis.
Genes in a 21.4 Mb region flanking SNP rs1056847 and rs859637 in chromosome 1q21–24 were screened for allelic expression imbalance. A schematic workflow for this project is described in Fig. 1.
Human Subjects
We used TLs from 95 CA and 95 AA from Utah and Arkansas, and 95 HapMap II-CEU subjects for AEI and total gene expression analysis (31). Of 95 CA individuals selected for AEI analysis, 50 are unrelated members from pedigrees showing linkage with T2D at chromosome 1q21–24 region. We also used RNA isolated from subcutaneous adipose and skeletal muscle tissue of 168 metabolically characterized subjects (31). Genomic DNA samples from 611 metabolically characterized nondiabetic CA and AA subjects (5) and 718 subjects from 67 CA T2D pedigrees (4, 10) were used for sequencing and genotyping studies. Sample characteristics were published elsewhere (4, 5, 10, 31). All study participants provided written, informed consent under protocols approved by the University of Arkansas for Medical Sciences. Our current study is approved by the Institutional Review Board of Wake Forest University Health Sciences.
Cell Culture
Allelic expression imbalance was primarily screened in total RNA and genomic DNA isolated from TLs. The TLs from 95 CA and 95 AA from Utah and Arkansas and 95 HapMap II-CEU subjects were grown under normoglycemic (5.6 mM glucose) conditions in RPMI-1640 culture media (Omega Scientific, Tarzana, CA) supplemented with 10% heat-inactivated fetal bovine serum (Omega Scientific), l-glutamine, and antibiotics (31). The cells were grown at 37°C and 5% CO2. Cell growth was monitored by taking cell counts with a Beckman Coulter counter, and all cells were harvested at a density of 0.5–1 × 106 cells/ml. Harvested cell pellets were immediately used for isolation of RNA and DNA.
Isolation of DNA and RNA
Genomic DNA from TLs was isolated by using a Puregene DNA extraction kit (Qiagen, Valencia, CA), and total RNA was extracted with an RNeasy Mini Kit (Qiagen). We also used RNA samples isolated from subcutaneous adipose and skeletal muscle biopsy samples to validate selected gene expression findings. We extracted total RNA from subcutaneous adipose by using an RNeasy Lipid Tissue Mini kit (Qiagen) and from muscle using the Ultraspec RNA kit (Biotecx Laboratories, Houston, TX) (31). The quantity and quality of the isolated nucleic acids were determined by ultraviolet spectrophotometry and electrophoresis, respectively.
cDNA Synthesis
Total RNA (1 μg) was reverse transcribed using Qiagen QuantiTect RT kit (Qiagen) using the manufacturer's protocol, which includes a DNase digestion step to remove genomic DNA (gDNA) contamination. cDNA from TLs were used as template for AEI and total transcript expression analysis. Additional cDNAs were synthesized similarly from adipose and muscle RNA and were used for total transcript expression analysis (31).
Selection of SNP and PSQ Assay Design for AEI Analysis
The T2D linkage peak at chromosome 1q21–24 (chr1:151288172–172711000, build Hg19/GRCh37) includes 412 genes (represented by 755 RefSeq IDs). We searched our in-house genome-wide gene expression data (Agilent 4X44K array, n = 16 TLs and Illumina HT-12 V4 array, n = 160 TLs) (6, 15) and identified 238 expressed genes in this region in TLs. We searched the dbSNP database (v.128) to identify validated transcribed SNPs (cSNP) located in cDNA of expressed genes. We identified 592 cSNPs with minor allele frequency (MAF) ≥0.05 included in 176 genes. Similar to our published studies (35), we designed modified PSQ assays that used a biotin-labeled universal M13-tail primer to evaluate AEI of cSNPs. Initial PCR amplification primers were designed by using Primer 3 software (http://frodo.wi.mit.edu/primer3/), and PSQ assays were designed by SNP Primer Design software (Pyrosequencing, version 1.0.1). We attempted to design PSQ genotyping assays that can perform allele-specific quantitation for a cSNP using same amplification and sequencing primers in both cDNA and gDNA samples. However, AEI analyses for cSNPs located near an exon-intron boundary or in a very short exon necessitated designing separate PSQ assays for cDNA and gDNA. Each of these customized assays was tested for successful amplification and allele-specific quantification using gDNA and cDNA templates. Assays that passed appropriate quality control and optimization were used for further analysis (see Supplementary Table S1 for details of successful assays).1
We first genotyped DNA from 95 CA and 95 AA subjects by using PyroMark Gold Q96 reagents (Qiagene) on a PSQ96 pyrosequencer (Qiagene), according to the manufacturer's instructions. Any cSNPs for which we found >4 individuals with heterozygous genotypes were investigated further. cDNAs from individuals heterozygous for respective cSNPs were then selected and analyzed similarly by PSQ to quantify the expression of both alleles. Additional cDNA samples from major and minor allele homozygotes were included as positive controls. We used a 20 ng cDNA template in each PSQ reaction. We successfully evaluated AEI of 115 cSNP in 115 genes; most (52.2%) were located in 3′-UTR of the gene. cSNPs showing strong AEI (>10%) were also analyzed in cDNA isolated from HapMap-CEU samples for confirmation.
Real-time PCR
Transcripts showing significantly strong AEI (>10% mean difference in allelic expression in both CA and AA samples) were measured by quantitative real-time PCR using SYBR green Master Mix (Applied Biosystems, Foster City, CA) on an ABI 7500 Fast RT-PCR system (Applied Biosystems). We analyzed the effect of genotype on total expression of these transcripts. cDNAs from TLs of 84 CA subjects were used for this analysis. The standard curves were generated by using pooled cDNA from the samples assayed (31). Transcript-specific oligonucleotide primers for real-time PCR were designed to capture most known splice variants (see Supplementary Table S2 for details). Target genes were normalized to 18S ribosomal RNA and ratios were logarithmically transformed and used in all calculations, as described previously (31). We similarly analyzed the expression of a transcript (THEM4) in subcutaneous adipose and skeletal muscle tissue cDNA of 168 metabolically characterized subjects by qRT-PCR.
Identifying Associations of AEI-cSNPs with T2D and Glucose Homeostasis Traits
To investigate whether the cSNPs showing significant AEI in our study were associated with T2D and glucose homeostasis traits, we examined data from large GWAS in CA subjects. We used meta-analyses of association data for T2D from the DIAGRAM (http://diagram-consortium.org/) consortium. We initially used DIAGRAM-V2 meta-analysis data (8,130 cases and 38,987 controls) but finalized our analysis using DIAGRAM-V3 data. Publicly accessible DIAGRAMv3 GWAS meta-analysis (19) includes association summary statistics (risk allele, P value and odds ratio) for 19,285 SNPs in the chr1:151288172–172711000 region from an analysis of 12,171 T2D cases and 56,862 controls across 12 GWAS in populations of European descent. Data for glucose homeostasis traits were taken from the MAGIC dataset (http://www.magicinvestigators.org) (8, 30). In MAGIC, fasting glucose and insulin resistance [homeostatic model assessment-insulin resistance (HOMA-IR)] data sets were generated by performing a meta-analysis of up to 21 GWAS in up to 46,186 nondiabetic CA participants (summary statistics including effect allele, effect size, and P values were available). Two-hour glucose data sets were generated by a meta-analysis of nine GWAS in 15,234 nondiabetic CA individuals.
We further selected trait-associated cSNPs and significant rSNPs to genotype in 611 CA and AA subjects to identify the role of these SNPs in glucose homeostasis (5). Subjects in this cohort was evaluated by insulin modified frequently sampled intravenous glucose tolerance test (FSIVGT) and minimal model analysis to measure insulin sensitivity index (SI) and beta-cell function (acute insulin response to glucose, AIRG) (1). Genomic DNA isolated from TLs or total blood samples was used for genotyping by Sequenom MassARRAY system (Sequenom San Diego, CA) or PSQ (PSQ96 pyrosequencer, Qiagen). Sequenom genotyping (SEQ) procedures were performed according to the manufacturer's iPLEX application guide at the genotyping laboratory of the WFUHS Center for Human Genomics and Personalized Medicine. Details of all selected SNPs and genotyping assays are described in Supplementary Table S3.
Luciferase Reporter Gene Assay
Our strategy for functional evaluation of the SNP rs13320 located in the 3′-UTR of THEM4 gene was similar to that of Innocenti et al. (14). We first cloned a 453 bp region around the SNP. Inserts representing the T and C alleles of rs13320 were generated by PCR amplification of genomic DNA from three individuals homozygous for each allele. The forward and reverse primers used for this amplification were 5′-ATGGTGGGAAACACACAGG-3′ and 5′-GATCCTCGAGGGAGACACACAGAGGGAATG-3′, respectively. The reverse primer included a site for the XhoI restriction enzyme. Purified PCR products were digested with NheI and XhoI restriction enzymes and cloned into the pLightSwitch-3′-UTR vector (Switchgear Genomics, Menlo Park, CA). This vector includes an optimized Renilla luminescent reporter gene (RenSP) whose expression is driven by the RPL10 constitutive promoter. The inserts were cloned into the 3′-end of the RenSP gene in the vector. The ligated vectors were transformed into DH5α Escherichia coli-competent cells, positive clones selected by antibiotic resistance and further confirmed by PCR amplification and restriction digestion of plasmids. Plasmid constructs were verified by DNA sequencing prior to use in transfection.
HEK293 cells maintained in antibiotic-free DMEM supplemented with 6% FBS were used for transient transfection reporter assays. We have selected HEK293 cells for high transfection efficiency and reported (16, 28) endogenous expression of THEM4/carboxy-terminal modulator protein (CTMP). In brief, 25,000 cells were seeded per well in a 96-well cell culture plate before transfection. The next day, used culture medium was replaced with 180 μl fresh DMEM with serum. We prepared a transfection mixture by adding plasmid DNA and CaCl2. We added 20 μl transfection mix, which contains 100 ng plasmid DNA and 1.2 μl of CaCl2 (2 M), in each well. After 24 h, we fed cells with 200 μl of fresh media after removing old media and incubated them for an additional 24 h. At 48 h posttransfection, we removed old media and added 100 μl of fresh media to each well before freezing at −80°C overnight. For luminescence analyses, plates were thawed for 45 min at room temperature, and 100 μl cell lysate was transferred to wells of a white 96-well plate. Then 100 μl of LightSwitch Luciferase assay reagent (LS010, Switchgear Genomics) was added and incubated for 30 min in the dark at room temperature. Luminescence was read for 2 s per well on a 96-well compatible plate luminometer (LUMI Star Optima; BMG Labtech, Ortenberg, Germany). Wells with untransfected cells were used as controls for luminescence background correction. We performed three independent experiments, and each experiment included three to six biological replicates for each allele.
A Student's t-test was performed on normalized luminescence values to compare the effects of the T and C alleles of SNP rs13320. A two-tailed P value of ≤ 0.05 was considered significant.
Sequencing the THEM4 gene and Genotyping in Pedigrees
We sequenced a 1,661 bp region of the THEM4 gene in 96 T2D and 96 nondiabetic CA subjects. We selected 96 T2D subjects from our published genome-wide linkage study, which included members of CA pedigrees from Utah and showed linkage with T2D at chromosome 1q21–24 region (4, 10). Nondiabetic subjects were metabolically characterized by oral glucose tolerance test (OGTT) and FSIVGT. Our sequencing region included all six coding exons, part of 5′-UTR, and part of 3′-UTR around the SNP rs13320 to identify functional variants in the THEM4 gene. Sequencing was performed by a two-step "boost/nest" PCR strategy. Seven different nested PCR products (see Supplementary Table S4a for sequencing primer details) were sequenced by utilizing BigDye Terminator 3.1 kits using the ABI 3730XL DNA sequencers. Data were analyzed by a customized version of Automated GENotyping Tool (AGENT) software by Paracel, which utilizes advanced TraceTuner MX base calling and quality value assignment algorithms to identify polymorphisms and assign genotypes. To ensure accuracy and sensitivity, only data from the high-resolution, high-quality region of the DNA sequencing runs were used. SNPs were called only when confirmed on both strands. Sequencing and data analyses were performed by Polymorphic DNA Technologies (Alameda, CA).
We further genotyped two less-common (MAF < 0.05) coding-nonsynonymous variants (observed in T2D subjects of our sequencing experiment) in 718 subjects from 67 CA pedigrees (4, 10). SNPs were genotyped by pyrosequencing as described above (see Supplementary Table S4b for genotyping assay details).
Data Analysis
AEI data analysis.
Allele-specific quantification of both cDNA and gDNA samples were determined from pyrogram peak heights with PyroMarkID V1 software (Qiagene). Ratios observed between two alleles of a SNP in a heterozygous subject were assay specific and may deviate from the expected ratio of 1 (50:50) due to uneven amplification of alleles unrelated to uneven transcription. We normalized our allele-specific expression data by the methods of Fogarty et al. (11). In brief, we normalized the observed gDNA or cDNA %B/%b ratio (B and b are two alleles of a cSNP) by dividing it by the mean %B/%b ratio of gDNA from individuals heterozygous for the cSNP. The normalized %B allele (%Bn) was used in all AEI tests. In each test, we compared the %Bn allele in gDNA to the %Bn allele in cDNA in samples heterozygous for the cSNP. We used paired sample t-tests (2-sided) and Wilcoxon signed-rank tests (nonparametric) for statistical evaluation. A nominal P < 0.05 was considered as a statistically significant difference.
Evaluation of 15 cSNPs was performed by using separate assays for gDNA and cDNA. We performed normalization of allele-specific expression for these SNPs by constructing a standard curve, mixing purified PCR products from minor and major allele homozygous samples in known ratios. Similar to our published protocol (35), we used mixed samples of five B/b ratios: 80:20, 60:40, 50:50, 40:60, and 20:80 in duplicates as known standards. We used linear regression to fit a standard curve using observed and expected values of these standard samples, and the %B alleles of each unknown sample were interpolated from the standard curve. Further data analysis using %B data was performed as described above.
The AEI can be directly tested only at the transcribed or cSNP. However, the pattern of AEI observed at the cSNP can be influenced by a regulatory SNP (rSNP). We hypothesized that patterns of AEI observed at tested cSNPs would depend on linkage disequilibriums with a trait-associated rSNP. In some tested cSNPs, we observed significant and unidirectional AEI (that is, all heterozygous subjects showed AEI in the same allelic direction). We searched association of these cSNPs (or a SNP in strong linkage disequilibrium at r2 > 0.8 located within ± 500 kb of the cSNP) with T2D and glucose homeostasis traits. A set of cSNPs showed large variations in allele-specific expression (>15% standard deviation in %Bn gDNA − %Bn cDNA in heterozygous subjects) in heterozygotes. To understand the possible causes of this result, we measured allele-specific expression in cDNA isolated from TLs for 95 HapMap-CEU subjects. Utilizing publicly available genotype data of these subjects, we further investigated whether trait-associated rSNPs in linkage disequilibrium with these cSNPs may account for the observed variance in allelic expression. As suggested by Fogerty et al. (2010), depending on the linkage disequilibrium relationship between cSNPs and rSNPs, we used either two-sided t-tests (comparing %Bn in cDNA of subjects homozygous vs. heterozygous at rSNP) or one-sided F-tests (comparing variance of %Bn in subjects heterozygous for rSNP vs. subjects homozygous for rSNP) (11).
Genotypic association.
Genotypic effects on glucose homeostasis traits (SI and AIRG) were tested by univariate analysis of variance using general linear regression models (GLM) implemented in SPSS v.12 for Windows (SPSS, Chicago, IL). Age, body mass index (BMI), sex, ethnicity, genotype, and protocol (tolbutamide or insulin) were included as fixed factors and covariates, as appropriate (9). Skewed variables were ln-transformed to normality before analyses. Genotypic effects on total transcript expressions in adipose and muscle were tested similarly using GLM with age, BMI, sex, ethnicity, and genotype included as fixed factors and covariates, as appropriate.
Family-based associations with T2D were tested in 67 families using a modification of the transmission disequilibrium test, as implemented in the Pedigree Analysis Package (jPAP, http://hasstedt.genetics.utah.edu/jpap/). The minor allele was tested for overtransmission to T2D cases with a one degree of freedom mixture χ2.
Genes in Chromosome 1q21–24 Region Shows AEI
Among cSNPs studied in 115 genes, we observed significant AEI (P < 0.001, Wilcoxon signed-rank test) in 28 genes among TL cDNA of CA subjects and in 30 genes among AA subjects (Supplementary Table S5). Among 109 genes studied in both CA and AA subjects, 12 showed significant AEI (at P < 0.001) in both populations. Six genes in our study showed strong allelic expression imbalance (>10% mean difference in expression of an allele in cDNA compared with gDNA) in both CA and AA populations at a nominal significance level of P < 0.05 (Table 1). These six genes were THEM4, IGSF8 (immunoglobulin superfamily, member 8), IVL (involucrin), FCRL3 (Fc receptor-like 3), XCL1 (chemokine C motif ligand 1), and TMCO1 (transmembrane and coiled-coil domains 1). The AEI analysis of a 3′-UTR SNP rs13320 in THEM4 showed significant (P = 1.68 × 10−7) overexpression of the T allele in cDNA, while a 3′-UTR SNP rs6660601 in TMCO1 showed significant (P = 6.55 × 10−4) underexpression of the C allele in cDNA (Fig. 2).
We further tested the effect of genotype of these six SNPs on total transcript expression in TLs. In concordance with the AEI results, we found significantly higher expression of the THEM4 gene in T/T homozygotes for SNP rs13320 compared with T/C and C/C subjects (P = 0.027). Individuals with a C/C genotype for SNP rs1131891 also showed significantly higher expression of the IGSF8 gene compared with TC and TT subjects (P = 0.02) (Fig. 3). However, we observed no significant association of cSNP genotype with total expression of IVL, XCL1, and TMCO1. We also searched publicly available eQTL datasets (in Genevar database, http://www.sanger.ac.uk/resources/software/genevar/) and found that some AEI-cSNPs also may affect total gene expression as cis-regulators (Supplementary Table S6). Irrespective of the data source, rs13320 in THEM4 was identified as a strong cis-eSNP.
SNPs Showing AEI are Associated With T2D and Glucose Homeostasis Traits
To identify associations of AEI cSNPs with T2D, we utilized meta-analysis of GWAS in CA populations by the DIAGRAM consortium. In the DIAGRAM-V3 dataset, association results for 19,285 SNPs were available for the chromosome 1q21–24 linkage peak region (between SNP rs1056847 and rs859637). In this region, we identified 60 cSNPs that showed nominally significant AEI (P ≤ 0.05) in CA subjects. We tested associations of 55 of these cSNPs with T2D directly (for 38 cSNP) or by a proxy SNP in strong linkage disequilibrium (r2 ≥ 0.8) with tested cSNP (includes a total of 926 SNPs). At the significance threshold of P ≤ 0.01, we found associations of cSNP rs7345 [in SNAPIN, P = 0.00061, odds ratio (OR) = 1.06, 95% confidence interval (CI) of OR for risk allele T = 1.02–1.09] and rs3813609 (in ARHGAP30, P = 0.0071, OR = 1.05, 95% CI of OR for risk allele C = 1.01–1.09) with T2D. A cSNP in SLAMF8 (rs7418910) also showed marginal association with T2D (P = 0.016) (Table 2).
Using the MAGIC dataset, we tested associations of 57 of these AEI-cSNPs with glucose homeostasis traits, either directly (for 49 cSNP) or by a proxy SNP in strong linkage disequilibrium (r2 ≥ 0.8) with tested cSNPs (includes a total of 1,022 SNPs). In the MAGIC dataset, the A allele of AEI-cSNP rs2301286 in the POPX gene was significantly associated with increased fasting glucose (P = 0.0082), decreased fasting insulin (P = 0.0057), and decreased HOMA-B (P = 8.35 × 10−5). An AEI-cSNP in SNAPIN (rs7345) that showed association with T2D in the DIAGRAM dataset also showed significant association with increased HOMA-IR (P = 0.0058) and fasting insulin (P = 0.0066). Among the cSNPs that showed strong AEI, rs13320 in THEM4 showed association with 2 h glucose values in the MAGIC dataset (Table 2). The T allele for this SNP showed increased expression and was associated with increases in 2 h glucose values during OGTT (P = 1.35 × 10−4).
Three cSNPs (rs982143, rs2282283 and rs913996) showed high variations in allelic expression (SD > 10%). Thus, despite strong AEI (mean gDNA-cDNA %B is >10%), these SNPs reached marginal significance (P > 0.001) in CA. We hypothesized that this pattern of AEI is caused by rSNPs in linkage disequilibrium with the tested cSNP. We considered any T2D-associated SNPs within ± 500 kb of the tested cSNPs as rSNPs. We measured allele-specific expression in cDNA from TLs of HapMap-CEU subjects heterozygous for these SNPs. Utilizing DIAGRAM-V2 GWAS data, we identified the closest T2D-associated SNP and the most significantly T2D-associated SNP within ± 500 kb of these cSNPs. Depending on the linkage disequilibrium relationship between rSNP and the tested cSNP in HapMap-CEU genotype data, we implemented statistical tests to evaluate the role of genotype at rSNP in modulating AEI patterns at cSNPs. The strongest T2D-associated SNP within ± 500 kb of the FCRL3 cSNP rs2282283 is rs12123141 (P = 0.0036, OR = 0.84 in DIAGRAM-V2 dataset), which is located at a distance of ∼22.4 Kb and shows low linkage disequilibrium (r2 = 0.023 and D′ = 1). Genotypes of the SNP rs12123141 were unable to explain the variation in allelic expression observed at rs2282283. However, among the T2D-associated SNPs, rs3811024 (P = 0.049 in DIAGRAM-V2 dataset) shows the strongest linkage disequilibrium with cSNP rs2282283 (r2 = 0.479 and D′ = 0.803). The cDNA t-test showed that, compared with homozygotes, HapMap-CEU subjects heterozygous for rs3811024 had significantly lower expression of the T allele of cSNP rs2282283 (P = 8.9 × 10−5, Fig. 4). Thus, the T2D-associated SNP rs3811024 may act as an rSNP to modulate the allele-specific expression of the FCRL3 gene and may explain the variation in allelic expression observed at cSNP rs2282283. However, the association of T2D with rs3811024 was marginal in the DIAGRAM-V2 dataset and was not significant in the larger DIAGRAM-V3 dataset. We found no T2D-associated rSNP that explained variation in allelic expression observed at other two tested cSNPs (rs982143 in XCL1 and rs913996 in IVL).
Association of AEI cSNPs with FSIVGT-derived Glucose Homeostasis Phenotypes in Nondiabetic Subjects
We genotyped eight SNPs in a cohort of 611 metabolically characterized CA and AA nondiabetic subjects. We selected two cSNPs with very strong unidirectional AEI (rs13320 in THEM4 and rs6660601 in TMCO1). Additionally, we selected cSNPs for rs7345 (SNAPIN), rs7418910 (SLAMF8), rs2301286 (PPOX), rs2282283 (FCRL3 and rSNP rs3811024), and rs3813609 (ARHGAP30), which showed significant AEI in CA and significant associations with T2D or glucose homeostasis traits in the DIAGRAM and MAGIC datasets, respectively. No SNPs showed significant associations with SI in our cohort. Among the tested SNPs, only rs13320 (in THEM4) showed a significant association with AIRG. In the CA subset of our cohort, individuals with a TT genotype (estimated marginal mean of AIRG = 1,851 ± 6, n = 163) showed significantly lower AIRG compared with TC or CC genotypes (AIRG = 2,236 ± 6, n = 246) (P = 0.021). Associations of the rs13320 genotype with AIRG also remained significant in the combined CA and AA cohort (P = 0.005).
3′-UTR SNP rs13320 is a Functional Regulator of the THEM4 Gene.
Utilizing cDNA from TLs, we identified strong AEI in THEM4 gene as detected at SNP rs13320. A study by Maira et al. (16) identified that THEM4 (also known as CTMP) is a negative regulator of protein kinase B/Akt phosphorylation and thus may play a critical role in insulin-induced glucose metabolism. Thus, we further analyzed the association of rs13320 genotype with total THEM4 mRNA expression in subcutaneous adipose and skeletal muscle of human subjects. We found a significant association of rs13320 genotype with THEM4 expression in adipose tissue (P = 0.005, n = 142) but not in the muscle (P > 0.05, n = 145). In adipose, individuals with TT or TC genotype showed significantly higher expression of THEM4 compared with individuals with CC genotype (Fig. 5).
We subsequently sought experimental evidence for the functional nature of the SNP rs13320. We identified that luciferase reporter constructs of the 453 bp region around rs13320 modulate reporter activity (luminescence). Constructs with the T allele had significantly higher reporter activity (∼30%, P = 7.15 × 10−7) compared with constructs with the C allele of rs13320 (Fig. 6). This effect was in the same direction as the AEI and total expression measurements in TL and adipose tissue.
In silico analysis of transcription factor binding in and around rs13320 by Matinspector (http://www.genomatix.de/online_help/help_matinspector/matinspector_help.html) showed binding of MYBL on allele C, whereas binding of SOX5 was observed on the T allele. We searched the Encyclopedia of DNA Elements (ENCODE) Consortium and Roadmap Epigenome Mapping Consortium data to identify additional functional role of SNP rs13320. A search using RegulomeDB (http://regulome.stanford.edu/) identified "minimal binding evidence" (score 6) for this SNP. Similarly, a search using HaploReg-V2 tool (http://www.broadinstitute.org/mammals/haploreg/haploreg.php) was unable to identify additional regulatory role of this SNP. Interestingly, a search for the effect of this SNP on microRNA target binding (http://www.bioguo.org/miRNASNP/) showed that the rs13320 G allele in THEM4 mRNA is recognized by hsa-miR-101, while the A allele may cause a loss in miRNA binding (indicated by an energy change from −16.80 kcal/mol to −13.10 kcal/mol).
Search for Other Functional Variants in THEM4
Sequencing of coding exons and the UTR in 192 subjects identified 11 variants, including six coding variants, in THEM4 (Table 3). Among the identified SNPs, three are novel (not in dbSNPv137). None of the identified variants shows significant linkage disequilibrium (r2 < 0.2) with the 3′-UTR SNP rs13320. All coding nonsynonymous variants were present in nondiabetic subjects of our sequencing cohort and unlikely to be functional variants with high penetrance. We genotyped two rare (MAF < 0.05) coding nonsynonymous variants in 718 subjects from 67 CA T2D pedigrees. The coding variant S72P (rs144757240) was present in nine subjects of a pedigree, while the coding variant S38C (rs144257719) was present in 27 subjects from six pedigrees. We observed only heterozygotes for these coding variants that did not explain transmission of T2D in pedigrees (rs144757240, transmission probability = 66.10, P = 0.216 and rs144257719, transmission probability = 61.78, P = 0.302).
Genomic regions representing replicated linkage signals are likely to be driven by causal variants. Thus, our search for regulatory variants within the chromosome 1q21–24 region utilizing AEI analysis is a reasonable approach to prioritize and identify functional genetic susceptibility variants for T2D. In our study, AEI analysis by PSQ identified cis-regulatory polymorphisms for 28 of 115 genes (24.3%, P < 0.001) in CA subjects. This proportion is comparable with that reported in previously published studies. Genome-wide surveys in TLs from 53 Hapmap-CEU subjects utilizing hybridization-based methods (on Illumina Human1M BeadChips) identified AEI in 30% of the 9,751 tested genes (12). This study used both exonic and intronic SNPs of unspliced primary transcripts (heteronuclear RNA) and reported 13 high-confidence full-transcript AEI in the chromosome 1q21–24 region we studied (12). Our study replicated (P < 0.05) AEI in four of these transcripts, including FCRL3, CD244, NDUFS2, and TMCO1. We have not tested AEI in non-RefSeq transcripts; because of technical differences, we could not replicate AEI in genes that were detected by intronic regulatory SNPs.
A recent study successfully used AEI analysis to identify genes influenced by cis-acting regulatory SNPs and to prioritize functional gene candidates for high-density lipoprotein cholesterol from a GWAS locus (11). The goal of our study was to prioritize T2D susceptibility genes in the chromosome 1q21–24 region via AEI analysis. Successful AEI experiments require that genes in the tested region are expressed in an available tissue "relevant" to the associated trait. Current understanding of T2D pathophysiology indicates a complex interplay of different tissues (including pancreas, adipose, muscle, liver, brain, and intestine) and cell types within each tissue (e.g., adipocytes, macrophages, and T cells in adipose). Recent studies have shown diverse and distinct roles of the TCF7L2 gene (the gene showing the strongest genetic association with T2D susceptibility) in tissues other than pancreatic β-cells (17, 21). Thus, selecting a relevant tissue for each gene in the region based on available experimental evidence or bioinformatics will be difficult. We selected expressed genes from available genome-wide expression array data and used RNA from transformed lymphocytes to identify AEI. A limitation of our study is the lack of RNA from different tissues of human subjects for AEI analysis.
One major requirement to test AEI is that a gene contains at least one common transcribed SNP, so that sufficient heterozygous samples are available for analysis. In this study we selected common transcribed SNPs (MAF > 0.05) to obtain at least >4 heterozygous individuals in our CA or AA DNA samples. AEI can be studied for intronic regulatory SNPs using hnRNA as a template (26). However, because of technical limitations of our method, we selected transcribed SNPs that are present in mature (spliced) mRNA. Most published studies have used BigDye Terminator-based normalized sequencing methods or Taqman allelic discrimination assays to evaluate allele-specific expression (11, 27). The PSQ methods used in our study were optimized and validated for AEI analysis by our laboratory and represent a more sensitive and comparatively high-throughput method (35). Recently developed technologies based on cDNA sequencing by second-generation sequencing methods (RNA-Seq) provide an in-depth and high-throughput detection of allele-specific expression. However, sensitivity of RNA-Seq in AEI analysis depends on numbers of sequence read coverage (18, 7) and is currently an expensive method.
Comparisons of allelic expression in our study showed a strong correlation (r = 0.81) in the degree of AEI and the direction of allelic expression in CA and AA. We identified 12 loci that showed significant AEI in both CA and AA (P < 0.001, Wilcoxon signed-rank test). Among SNPs showing high allelic expression imbalance (>10% mean % difference gDNA-cDNA), only rs2241108 (in SLC25A44) showed higher expression of "G" allele (11.08 ± 5.18%, P = 1.11 × 10E-09) in cDNA from AA subjects, while in CA subjects it showed marginally lower expression (1.91 ± 4.59%). cSNPs in PRUNE (rs11204762) and IL6R (rs2229238) showed similarity in the direction of allelic expression in both populations but did not reach statistical significance in either AA or in CA samples, respectively. Differences in regional linkage disequilibrium (between tested cSNPs and untested rSNPs) may cause these differences in allelic expression among populations.
Expression of genes at the transcript level is a complex biochemical quantitative trait and is influenced by a combination of cis- and trans-acting genetic variations, epigenetic factors, and environmental and other nongenetic factors. Allelic expression imbalance analysis compares allelic expression within individuals (in heterozygotes) and, in principle, eliminates the effect of trans-acting and nongenetic factors. Thus, compared with total transcript-based analysis (performed across individuals), the AEI analysis provides higher sensitivity to uncover cis-regulatory elements (26). Thus, cSNPs showing AEI as well as genotypic associations with total transcript expression (e.g., rs13320 in THEM4) may represent a strong cis-regulatory element. Genes that showed AEI but not genotypic associations with total gene expression in our study (or in publicly available cis-eQTL data) are possibly under control of stronger trans-regulatory or nongenetic factors.
Meta-analyses of large genome-wide studies in the DIAGRAM dataset failed to identify any T2D susceptibility loci in the chromosome 1q21–24 region that reached a genome-wide significance level (P < 5 × 10−8) (19). Similar meta-analyses of glucose homeostasis traits by MAGIC also found no loci of genome-wide significance in this region(8, 30). Among the cSNPs showing significant AEI in CA samples in our cohort, the SNP rs7345 in SNAP-associated protein (SNAPIN) gene showed the strongest association with T2D (P = 6.1 × 10−4, OR = 1.06) in the DIAGRAM-V3 dataset. SNP rs7345 exhibits significant but minor AEI. As part of the SNARE complex, the SNAPIN gene is required for vesicle docking and fusion and regulates neurotransmitter release (13). The existing literature does not describe its possible role in altering glucose homeostasis or T2D pathogenesis. A similar search in the MAGIC dataset for glucose homeostasis traits identified the strongest association of AEI cSNP rs2301286 in the protoporphyrinogen oxidase (PPOX) gene with HOMA-B. The A allele of this SNP shows moderately low expression compared with the C allele and is also associated with decreased HOMA-B (P = 8.4 × 10−5, β = −0.013). The PPOX enzyme is located in the inner membrane of mitochondria from various tissues and is involved in the heme biosynthetic pathway (34). No direct evidence exists to support a role for the PPOX gene in T2D pathogenesis. However, recent studies indicate that altered iron metabolism may be a factor in T2D pathogenesis, and involvement of PPOX in iron metabolism may support it as a candidate gene (32).
The SNP rs13320 in the 3′-UTR of THEM4 (or CTMP) gene showed the strongest AEI in our study. Interestingly, the T allele of this SNP showed significantly high expression compared with the C allele and was associated with increased 2 h glucose values (P = 1.35 × 10−4, β =0.073) in the MAGIC dataset. In a cohort of 611 nondiabetic subjects from Arkansas, this SNP also showed significant associations with an FSIVGT-derived index of beta cell function. Individuals with the TT genotype showed significantly low AIRG. However, this SNP was not associated with T2D in DIAGRAM-v3 [P = 0.67, OR = 1.01 (95% CI = 0.97–1.04), case n = 12,171, control n = 56,862] or AA [P = 0.70, OR = 1.02 (95% CI = 0.9–1.16), case n = 966, control n = 1,029] GWAS (19, 23). Higher expression of the T allele was confirmed in publicly available lymphoblastoid cell eQTL data (Supplementary Table S7), in adipose tissue of nondiabetic subjects from our study and by luciferase reporter assay. Thus, SNP rs13320 may act as a strong cis-regulator of the THEM4 gene and modulate its expression at transcript level. Alternatively, based on in silico analysis, SNP rs13320 alters the binding of microRNA (hsa-miR-101) and may modulate THEM4 expression posttranscriptionally. Further functional studies will be required to elucidate the complex mechanism involved in modulating THEM4 expression and altered glucose homeostasis.
The THEM4 gene was first described as CTMP and was shown as a negative regulator of Akt/protein kinase B-α (PKB) (16). Akt phosphorylation plays a major role in insulin-stimulated glucose uptake; thus, the THEM4 gene can be considered as a strong candidate for T2D pathogenesis. Since binding of CTMP to Akt reduces its activity by inhibiting phosphorylation on Ser473 and Thr308 (16), higher CTMP/THEM4 expression may cause insulin resistance. Studies by Zhou et al. (38, 39) show that the expression of CTMP is increased by fatty acid, tumor necrosis factor-α, or in mice with diet-induced obesity. Genetic data from our study indicate the association of a high-expressing allele of THEM4 with increased 2 h glucose values and low AIRG, which parallels the proposed role of this gene from the in vitro study by Maira et al. (16). A study by Zhao et al. (36) demonstrates direct CTMP-Akt1 binding by immunoprecipitation and by inhibition of Akt1 kinase activity, thus providing independent evidence that CTMP is an Akt1 negative regulator. However, a study by Ono et al. (22) indicates that CTMP also can act as activator of Akt phosphorylation under certain experimental conditions. Nonetheless, both studies indicate the involvement of CTMP in Akt activity and downstream response. Further studies will be required to confirm the role of CTMP in modulating Akt function at the organismal or tissue-specific level.
Several studies indicated that CTMP has a major function in mitochondria-mediated apoptosis via inhibition of PKB (24, 25, 28). Interestingly, studies by Zhao et al. (37) demonstrate that in vitro, the CTMP protein exhibits highly efficient thioesterase activity. The CTMP/THEM4 catalyzes the hydrolysis of acyl-CoA thioesters to release free fatty acids. Together, these studies provide support for multiple functions of THEM4 protein. Further functional studies will be required to understand the regulatory mechanisms involved in modulating THEM4 expression in different tissues and its connection with altered glucose homeostasis and T2D pathogenesis.
In summary, our AEI analysis identified several regulatory variants in the chromosome 1q21–24 region of T2D linkage. A strong regulatory variant in this genomic region regulates expression of the THEM4 gene and may increase susceptibility to T2D predisposition by modulating glucose homeostasis.
This work was supported by National Institutes of Health (NIH) Grant R01 DK-039311. Clinical studies were supported by NIH General Clinical Research Center Grant M01RR-14288 to the University of Arkansas for Medical Sciences.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: A.K.M., N.K.S., S.C.E., and S.K.D. performed experiments; A.K.M., N.K.S., S.C.E., and S.K.D. analyzed data; A.K.M., N.K.S., S.C.E., and S.K.D. interpreted results of experiments; A.K.M. and S.K.D. prepared figures; A.K.M. and S.K.D. drafted manuscript; A.K.M., N.K.S., and S.K.D. edited and revised manuscript; A.K.M., N.K.S., and S.K.D. approved final version of manuscript; S.C.E. and S.K.D. conception and design of research.
The authors are grateful to DIAGRAM, MAGIC, and HapMap Project investigators for generously making genetic association and genotype data publicly available. We thank Dr. Sandra J Hasstedt (University of Utah) for performing family-based association analysis. We also thank Karen Klein (Office of Research, Wake Forest University Health Sciences) for critical reading and editing of our manuscript.
↵1 The online version of this article contains supplemental material.
- Copyright © 2013 the American Physiological Society


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Organizational Behavior Skills
There are four key barriers to communication: process barriers, personal barriers, physical barriers and semantic barriers.
Describe how you, as a manager, would overcome each of these barriers.
Identify and describe the guidelines, tools, practices or procedures that you would use to overcome each type of communication barrier.
Solution Preview
Process barriers: Make sure the other person gets the message. The sender must choose the appropriate format for sending a message and the correct words. The message is then formulated and sent to the receiver who must then put it in the proper format for understanding. The correct form of delivery is a key to this barrier's success. Using a blackboard to convey messages only works if people check the board. Using email is a good avenue for message sending and receiving when the recipient must be reached quickly. Sometime communication should be more graphical, such as instructions for working a machine. Representation of what the buttons mean is important. Process barriers are used in appropriate settings and are dependent on the situation. Wanting a class of people to understand Maslow's theory of needs? Do not write it out, use a pyramid graph to show the different levels and the contents ...
Solution Summary
This solution discusses the four key barriers to communication: process barriers, personal barriers, physical barriers and semantic barriers.


text:
CALGARY - Through the first nine games of the CFL season, the Calgary Stampeders had uncertainty at the left-tackle position.
The return of Edwin Harrison changed that in one week, and now the 26-year-old plans on holding the spot for the foreseeable future.
Although Harrison could have tested free agency this off-season, just like Ben Archibald this year, the second-year Stampeders lineman has found a home in Calgary. He has inked a contract extension, keeping him with the team through the 2014 campaign.
Stability is huge, especially for an offensive lineman, said Harrison, who spent parts of two season with the Kansas City Chiefs before coming to Calgary.
Ive enjoyed my time here, and I really enjoy the team. Im happy and grateful to be here.
Last year, I was getting used to being in Canada. I wanted to make sure this was a place I wanted to be. Last year was my best time playing football that Ive had in quite some time. We had a great team, and we did a lot of great things.
The Stamps suffered a blow before training camp started when Harrison tore a pectoral muscle. The sophomore was put on a nine-game injured list while doing his rehab, and he debuted last Friday in Edmonton.
It just so happened to coincide with a rejuvenated rushing attack from the Stamps, who had struggled in that department the previous few weeks.
Gerald Cadogan started the season at left tackle, but he was recently replaced by rookie Tony Washington, who struggled on Labour Day against the Eskimos, taking three penalties and blowing coverage on a crucial forced fumble.
Last week, while Harrison took over at his position, the Stamps also switched centres from Tim ONeill to Jon Gott. As the Stamps get ready to face the B.C. Lions Saturday, they are keeping the same five guys as the starting offensive line for the first time in a month. Cadogan was released with Harrison back.
As last seasons team nominee for top rookie, having Harrison back meant there was a presence on the offensive line again.
Eds done an admirable job since hes been here, said Stamps quarterback Henry Burris. He was a cornerstone at right tackle for us, but he was a left-tackle waiting to happen.
Ben made the move, and Ed was ready to make that transition. Hes as sound as can be but one thing he brings is leadership. When he speaks, the offensive line listens.
Heading into this off-season, the Stamps didnt make a free-agent offer to Archibald, despite the fact he won the CFLs most outstanding lineman award in 2010, and he signed with the Lions.
Believing Harrison would make the transition over to left tackle, the Stamps were comfortable with who they had, as fellow import Stanley Bryant was penciled in at right tackle.
Spending a year with Archibald, Harrison tried to model his game after the Lions new left tackle.
Hes not only a great player but a great person, a great family man, Harrison said. Anybody could learn a whole lot from Ben, not only on the football field but off, as well.
He comes to work every day. Hes a true professional. Theres nothing more to say than that. Ben always comes to work.
Being out for three months meant that Harrison still needs to work on his conditioning, and head coach John Hufnagel said as much this week.
Harrison cant disagree with that statement, but hes pleased with the comeback so far.
Its hard to simulate game speed at practice, Harrison said. You dont want to get any of your own guys hurt. Its a matter of getting used to the game and getting up to the same speed as everyone else.
Its coming every day. Im feeling more and more comfortable.


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Gender: F Meaning of Jamie: "supplanter" Origin of Jamie: Diminutive of James Jamie's Popularity in 2016: #499
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