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- 4.79%November 2015504 20-Year Term
SBA 504 Loan Program benefits from economic stimulus act
The passage of H.R. 1, the American Recovery and Reinvestment Act of 2009 in February included several enhancements for the SBA 504 loan program.
Two fees have temporarily been eliminated on SBA 504 loans. The lender participation fee and the Certified Development Company (CDC) processing fee will now be paid directly by the federal government. CDCs will be reimbursed for their servicing fees by the SBA. The temporary elimination of these fees will make obtaining an SBA 504 loan more affordable for small business borrowers in the coming months.
[3/19/09 update: The fee reduction is now in effect and retroactive to February 17, 2009.]
The Stimulus Act provided $375 million for the elimination of fees for both the 7(a) and 504 programs through September 30, 2010. It remains to be seen how much of the money will be allocated between the two loan programs and SBA regulation will provide this information soon.
Also, refinancing of existing debt including government guaranteed debt such as SBA 7a & 504 loans will be allowed. This is a permanent addition to the Small Business Investment Act. The statute provides the following guidelines:
(A) Any 504 project "may include a limited amount of debt refinancing."
(B) If the project involves expansion of the small business, an amount not to exceed 50% of the project cost may be refinanced, if:
Proceeds will be used to acquire land, to construct or expand building or to purchase equipment.
- Existing debt is collateralized by fixed assets.
- Existing debt was incurred for benefit of small business.
- Proceeds will be used only for refinancing existing debt or costs related to the project.
- It will provide a substantial benefit when prepayment penalties, financing fees and other financing costs are accounted for.
- Borrower is current on all payments of the existing debt for not less than one year.
- New financing will provide better terms or interest rate.
[3/19/09 update: Refinancing is not yet available.]
Another permanent change enacted by this legislation is amending the job creation goal for SBA 504 loans. The job creation ratio has been increased from one job per $50,000 to one job per $65,000.
[3/19/09 update: The new job ratio is now in effect.]
Additionally, the Act established a program to provide up to $3 billion in guarantees for SBA 504 first mortgage pools that are to be sold to third-party investors. It appears these may be existing pools of loans, or new loans that may be pooled after the date of enactment.
The specifics on this new program are as follows:
- The seller of these pools must retain not less than 5% of the pool amount to be sold to investors.
- The seller must absorb all losses resulting from a shortage of monthly cash flows, but gets to keep any overage in monthly cash flows.
- The fee paid to the SBA must be not more than 50 bp of the outstanding guaranteed balance per year (assumed bill change).
- The program ends two years after date of enactment.
[3/19/09 update: Implementation of Secondary Market Guarantee Authority is expected in 90 days.]


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ASUS outs Thunderbolt-powered monitor, expansion card
updated 07:07 am EDT, Thu June 7, 2012
Expansion adds Thunderbolt to Asus 7-series motherboards
ASUS has demonstrated its first Thunderbolt-based monitor at Computex. It is the first PC display to use the technology outside of Apple's display range suggesting that the new high-speed I/O standard is starting to gain momentum. The 27-inch screen using the connection was shown at the same time it revealed an adapter for ASUS 7-series motherboards that added the port as an extra connectivity option.
The Acer Thunderbolt monitor has 1920x1080 resolution and includes DisplayPort, HDMI, and DVI connections. These are supplemented by a standard headphone jack, but no speakers. Other specifications were unavailable, while pricing and availability also remain unknown at this time. However, ASUS did confirm to Engadget that the display will be headed to North America without pinning down a timeframe.
Users of some ASUS 7-series motherboards will have the option to use a "unique system link connector" to enable a Thunderbolt EX expansion card. Taking up a PCIex4 slot and a DisplayPort connection, it acts as a pass-through via a Thunderbolt port. Again, ASUS has declined to say when it will be on sale nor how much it would cost. [via AnandTech]

subset: commoncrawl


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- Filed Under
Colgate exploded for four goals in the third period and rolled to a 6-1 victory against Cornell in ECAC Hockey men's action Saturday night at Starr Rink, snapping the Big Red's nine-game unbeaten streak.
The Raiders (14-11-3 overall, 10-5-1 ECAC Hockey) leapfrogged past Cornell and into third place in the conference standings with six games to play. The Big Red fell to 12-5-5 overall and 8-4-4 in the league with its first loss since it was beaten 3-2 by Boston University in the "Red Hot Hockey" clash on Nov. 30 at Madison Square Garden.
Mike McCann and Darcy Murphy scored first-period goals for ...


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Staying Home is a Smart Move for Athlete
Star basketball athlete – Courtney Ekmark – made a smart move using a home-based charter school to finish up her high school chapter, as reported by the Hartford Courant. The CT Post also noted her appreciation of time at home.
UConn recruit Ekmark: Home schooling 'a smart move' By Rich Elliott (more…)


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"You never want a serious crisis to go to waste."
These are the words of Rahm Emanuel. The former chief of staff to President Barack Obama made the remarks when he was the incoming chief of staff, during a 2008 Wall Street Journal business conference.
From there it was clear the administration's agenda would be bold. America was in crisis mode. The incoming chief of staff was clear: the Obama administration was going to support policy measures that addressed the crises America faced.
For the economic mess, we got the stimulus package, and a promise for some Wall Street regulatory reform down the road. But before Congress would get its chance to sound off at Goldman Sachs, there was a crisis called healthcare to deal with.
Emanuel had been hesitant to take on the issue. He knew it would drain energy from the administration, but energy was something the president had at the time. While the stimulus vote in Congress had revealed the beginnings of deep partisan war on Capitol Hill, the Obama gas tank was full.
Healthcare became an energy hog. It transformed the Obama administration from a Toyota Prius into a Hummer going up hill full of sandbags. Through reconciliation, healthcare made it across the finish line, but the damage had been done.
The Obama tank ran out of gas just in time to tackle energy. As oil spewed into the Gulf of Mexico, the world's attention focused on stopping the leak. On Capitol Hill, attention narrowed to offshore drilling regulation. Despite Interior Secretary Ken Salazar's attempts to clean up the Minerals Management Service (MMS), the department remained a mess by the time the disaster hit. The Obama administration had a perfect storm of a crisis on its hand.
The Emanuel team crafted an Oval Office speech for the president. But as Obama pleaded with Americans to support a new philosophy on energy policy, nothing happened. The engine simply couldn't take anymore.
So with the announcement that Rahm is out and Pete Rouse is in as chief of staff, will the Obama administration continue playing such a high-stakes game of political poker? The game has come with legislative victories in healthcare and financial reform, but those high points have come at the expense of energy and other issues. It's hard to tell where this is all going, but the tank is empty.
The opinions expressed by MNN Bloggers and those providing comments are theirs alone, and do not reflect the opinions of MNN.com. While we have reviewed their content to make sure it complies with our Terms and Conditions, MNN is not responsible for the accuracy of any of their information.


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November 29, 2015
A suspect in a shooting incident has been arrested in Troy. Twenty-three-year-old Brian Guice faces an assault charge.
He's accused of shooting another man earlier this month.
The victim was treated for a leg wound at a Troy hospital.
Guice was booked into the Pike County Jail.
Bond in the case has been set at $2,500.
Click Here to access the online Public Inspection File
Viewers with disabilities can get assistance accessing this station's FCC Public Inspection File by contacting the station with the information listed below. Questions or concerns relating to the accessibility of the FCC's online public file system should be directed to the FCC at 888-225-5322, 888-835-5322 (TTY), or firstname.lastname@example.com.


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Event Name- OR - Select an option below
The roaring fire at the back of O'Malley's on Main Street adds that welcoming Irish-pub warmth, both literal and figurative. The bar offers a full complement of whiskeys, vodkas and Scotches, as well as a selection of beers on tap. Try a pint of the Smithwicks, a tasty Irish number rumored to be the oldest ale produced by the Emerald Isle. O'Malley's also covers all the bases of pub grub, with nachos, quesadillas and chicken wings on one side and Irish sausage rolls, corned beef, and bangers and mash on the other. The shepherd's pie comes with a slagheap of tasty mashed potatoes on top that utterly hides the stew from the light (and oxygen) of day.


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48, 44.1 khz, why no and/or not 40 khz, Only for 2 channel audio...........
48, 44.1 khz, why no and/or not 40 khz, Only for 2 channel audio...........
Dec 18 2002, 04:11
Post #1
Group: Members
Posts: 657
Joined: 4-December 02
Member No.: 3989
Look at this Poll
How high can you hear (with music & lowpass)
The poll starter didn't put frequency above 20 khz in the poll
So why don't just use a 40 khz PCM instead of 44.1 khz as human
can't listen to frequency above 20 khz?
16 x 44100 x2 = 1141.2 kbit/s
16 x 40000 x 2 = 1280 kbit/s
It does help to save some space!
Dec 18 2002, 09:15
Post #2
Group: Members
Posts: 671
Joined: 21-November 01
From: California, US
Member No.: 514
QUOTE (/\/ephaestous @ Dec 17 2002 - 11:24 PM)
From John Watkinson, The Art of Digital Audio, 2nd edition, pg. 104:
The 44.1 Khz sample rate is used in Mp3s and such because most of them are ripped from CDs and resampling is was not as good as it is today (Not too good still)..
Fascinating! Thanks for the quote.
I remember Frank Klemm mentioned all this a while back (here's the post, which contains some other neat tidbits), but at the time I couldn't quite understand what he meant (a common problem when I try to communicate with electrical engineers ).
|Lo-Fi Version
|Time is now: 26th November 2015 - 00:07


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NBC Fires Producer of Misleading Zimmerman Tape
They forgot to fire the rest of NBC news.
Dershowitz unloads over Zimmerman arrest photo:
"The arrest affidavit did not mention the photograph, or the bleeding, gashes, and bruises on Zimmermans' head. Professor Alan Dershowitz of Harvard Law School stated upon release of the arrest affidavit that it was "so thin that it won't make it past a judge on a second degree murder charge … everything in the affidavit is completely consistent with a defense of self-defense."
After the release of the photo, however, Dershowitz went much further, telling Breitbart News that if the prosecutors did have the photo and didn't mention it in the affidavit, that would constitute a "grave ethical violation," since affidavits are supposed to contain "all relevant information."
Boy the media really dropped this case after the new information came out. I wonder why?
Yeah, a big mystery there, hmm? And of course, they are just all over the prosecutor withholding evidence and charging Zimmerman in bad faith. No way he gets convicted of Murder 2.


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Crime and the U.S. Justice System
Eric Holder, Jr spoke about crime and violence in America and the impact of crime on the federal judicial system.
People in this video
- Eric H. Holder Jr. U.S. Attorney District of Columbia
- Pauline Schneider Chairwoman
- Barbara Zelenko President Woman's National Democratic Club
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How Many Blog Posts Do I Need in Reserve?
One of the things I really love about the modern Internet is how easy it has become to find high-quality information on just about any topic. One of the things that really irritates me is how confusing it sometimes gets.
A common source of confusion that I encounter is the same term is used for different things, and "blogging" is an example of this.
The article I linked to below is thought-provoking however it can appear to contradict some of the things that I teach my clients so I need to explain.
The article below is about those of you who are putting large amounts of high-quality content onto the Internet, and it is the quality of that content that is drawing your customers to you.
One of the ways many of my clients work is to use blogging to create lots of short posts, highly related to their business, or their geographical area. The short posts then can be fed out through social media and additionally they give extra hooks for the search engines to find the sites. This short post is an example of this type, I need not spend hours crafting words on a topic, all I do is research what's happening out there and if I find an article that I believe will be beneficial to many of my clients then I put up a short post like this one.
Have a read of the article How Many Blog Posts Do I Need in Reserve?


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The look of his plated desserts, with their free-flowing natural look and feel, just breeds creativity.
There was an empty, haunting self-portrait by the Serbian performance artist Marina Abramovic, her mouth ringed with plated gold.
There was nothing to lay hold of; every point of vantage was plated with smooth ice.
I led him down to the court, where I'd laid out the plated tinware to dry.
She might have her mild preferences, but if Newell could give her muslin dresses and plated pins, he would suit her excellently.
All the fittings, inside and out, were either machine-turned or plated.
Some vessels were plated only about the centre, so as to protect the boiler and engines.
It is "plated" if it is made of some cheaper metal and covered with silver.
From the plated door-knob to the grained railing round the garret stairs, he had 'made lies his refuge.'
Your feeble spear ever pierce that slimy paunch or plated back?
mid-13c., "flat sheet of gold or silver," also "flat, round coin," from Old French plate "thin piece of metal" (late 12c.), from Medieval Latin plata "plate, piece of metal," perhaps via Vulgar Latin *plattus, formed on model of Greek platys "flat, broad" (see plaice (n.)). The cognate in Spanish (plata) and Portuguese (prata) has become the usual word for "silver," superseding argento via shortening of *plata d'argento "plate of silver, coin." Meaning "table utensils" (originally of silver or gold only) is from Middle English. Meaning "shallow dish for food," now usually of china or earthenware, originally of metal or wood, is from mid-15c. Baseball sense is from 1857. Geological sense is first attested 1904; plate tectonics first recorded 1969. Plate-glass first recorded 1727.
late 14c., from plate (n.). Related: Plated; plating.
plate (plāt)
A smooth, flat, relatively thin, rigid body of uniform thickness.
A thin flat layer, part, or structure.
A thin metallic or plastic support fitted to the gums to anchor artificial teeth.
A metal bar applied to a fractured bone in order to maintain the ends in apposition.
The agar layer within a Petri dish or similar vessel.
A sheet of glass or metal that is light-sensitive and on which a photographic image can be recorded.
Verb To coat or cover with a thin layer of metal.
Related Terms


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Giffords, husband pen gun laws bookComment on this story
Phoenix - Former US Representative Gabrielle Giffords, who was gravely wounded in a 2011 shooting rampage, has written a book with her husband "to rouse the long-overdue conversation" for gun control, a cause she has focused on since leaving office, her publisher said on Tuesday.
The book, "Enough: Our Fight to Keep America Safe From Gun Violence," builds on an opinion piece by Giffords published last April in The New York Times after gun control legislation stalled in the US Senate, according to a statement from Scribner, the publisher.
Giffords in the piece criticised senators for failing to enact the measures, which she argued could have prevented future mass shootings, and she accused them of being beholden to the gun rights lobby.
The legislation had been proposed in the aftermath of the December 2012 shooting at an elementary school in Newtown, Connecticut, that left 20 first-graders and six staff members dead.
"'Enough' was one of the first words Gabby said to me after we learned about the Newtown, Connecticut, massacre," Giffords' husband, retired astronaut Mark Kelly, said in a statement announcing the book, to be released in June by Scribner.
"As Second Amendment supporters and gun owners ourselves, we hope our book rouses the long-overdue conversation our country needs to make responsible changes to our gun laws so that no more precious lives are lost," he added.
Giffords was shot in the head on January 8, 2011, by gunman Jared Loughner, who opened fire during a "Congress on Your Corner" event at a Tucson, Arizona, supermarket. Six people were killed and 12 others were wounded.
Loughner, a college dropout with a history of mental illness, pleaded guilty in 2012 and was sentenced to life in prison.
Giffords, an Arizona Democrat who resigned from Congress two years ago to focus on her recovery, has been lobbying along with Kelly for tougher gun control laws, including expanded background checks and barring stalkers and domestic abusers from buying guns.
Giffords and Kelly formed a group aimed at curbing gun violence, Americans for Responsible Solutions, and have spent heavily on media advertisements and political campaigns in select states to press their efforts.
Susan Moldow, president of Scribner Publishing Group, said in a statement that the new book will offer "a rare look at Gabby's astonishing progress" since the mass shooting in Arizona that helped spark a national debate on gun control.
Giffords's recovery has been widely chronicled since the shooting, most recently when she went skydiving last month in southern Arizona to mark the third anniversary of the shooting.
In another opinion piece earlier this year in the New York Times, she wrote that she still struggles to speak, has vision problems and that her right arm and leg have been "paralysed." But she said there is progress in moving the arm.
Giffords told NBC's "Today" show she is working on her Spanish and again playing the French horn.
This is the second book by Giffords and Kelly. The first, published by Scribner in 2011, detailed the couple's lives together and her life and recovery after being shot. - Reuters
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Summit extends Twilight into virtual world Habbo
Summit Entertainment is looking to extend its vampire franchise Twilight online through a new deal with social networking site, Habbo.
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text:
Smoky Mountain Area Links
Gatlinburg, TN
Ripley's Aquarium of the Smokies - America's most visited aquarium in Gatlinburg featuring over 8,000 exotic sea creatures with exhibits, petting shows, lectures, hourly dive shows, gift shop and 2 restaurants. For additional information call 888-240-1358.
Ripley's Believe or Not Museum -The world's most unusual museum. Every year millions of people visit these museums to take part in an adventure, one in which they experience first hand the incredible world of Robert Ripley! For additional information call 865-436-5096.
Southland Car and Jeep Rentals -Serious Toys for Big Boys. We offer variety of vehicle rentals for you and your family. Leave the family car behind and jump into a mountain adventure. We have Jeep Wranglers, Hummer H2's to get you started. For additional information call 1-865-436-9811.
Ober Gatlinburg Ski Resort - America's largest aerial tram up to Ober Gatlinburg featuring go-karts, alpine slide, indoor ice skating, black bear habitat, water rides, chair lift, mini-golf, arcades, restaurant and lounge. For additional information call 865-436-5423.
Great Smoky Arts & Crafts Community - more than 70 artists' studios, shops and homes on an 8.5-mile loop, located 3 1/2 miles east of downtown. For additional information call 865-436-9214 or 865-436-7921.
Arrowmont School and Shop - Outlet for the sale of the arts, crafts, displays and exhibits in Gatlinburg. Handmade items such as woven placemats, baskets and clothes. For additional information call 865-436-5860.
Hollywood Wax Museum - Open year round, the Hollywood Wax Museum lies in the heart of downtown Gatlinburg and features the likenesses of such Hollywood celebrities as Marilyn Monroe, Angelina Jolie and much more. For additional information call 865-430-1800.
Smoky Mountain Fun - Want to have all of your family fun at one low price? Gatlinburg's Camp Thunder Fun Center and Reagan Terrace Attractions combine to offer Mini Golf, Bumper Cars, Laser Tag, Go Carts and more! For additional information call 1-865-430-7223.
Earthquake the Ride - Experience catastrophic disasters with high-tech special effects in Gatlinburg. Fun for the entire family. For additional information call 865-436-9765.
Guinness World Record Museum - Features the greatest records of all time, mind numbing feats and activities. For additional information call 865-436-9100.
A Walk in the Woods - Our tenth year offering spectacular trips into the Smokies. Our experienced guides will take you to incredible vistas, gentle cascades, waterfalls, majestic ancient forests, crystal clear mountain streams, old home sites, graveyards and hidden trails. For additional information call 1-865-436-8283.
Smoky Mountain Vacation Guide - Read blogs about Gatlinburg events, attractions, restaurants, and other things to do in Gatlinburg, Tennessee.
Gatlinburg TN Guide - Get Gatlinburg coupons, tips, and reviews for numerous things to do and places to eat in Gatlinburg, TN.
Pigeon Forge, TN
SeePigeonForge.com - SeePigeonForge.com has just what you are looking for! Not only do we have discounted tickets to the hottest shows but we also have the BEST DEALS IN TOWN! We can customize a package to fit your needs, or you may select one of our many pre-built packages. Imagine a great family vacation, or a quick weekend getaway with quality accommodations and top notch entertainment! No need to imagine, just call us or click here to have the best getaway in the Smokies!
Dollywood - Dolly Parton's theme park featuring live performances daily, rides, attractions, dining, shopping and exhibits. For additional information call 865-428-9488.
Splash Country - Dolly Parton's water theme park featuring
water adventure rides, dining and shopping. For additional information
call 865-428-9488.
Dixie Stampede - Dolly Parton's entertainment and dinner show featuring a spectacular four-course dinner and show with horses and performers. For additional information call 1-800-356-1676.
Hatfield and McCoys - Family, Food, Feudin? and Fun- that?s the theme of the newest show in town - The Hatfield & McCoy Dinner Feud!
Comedy Barn - The award winning family comedy variety show in the Smokey Mountains featureing magicians, fire-eaters, jugglers, ventriloquists, clean family comedians, live country and gospel music as well as fun barnyard animals.
Flyaway Indoor Skydiving - America's first vertical wind tunnel. Test your skills of human flight during a single flight session or as a participant in a personalized coaching program. For additional information call 877-293-0639.
USA Raft - We would like to take you on the whitewater rafting trip of your life! With 6 different choices, we will take you rafting with the best guides and staff on the East Coast. We offer whitewater rafting in WV, NC and TN. For additional information call 1-800-USA-RAFT.
Duffs Smorgasbord - We have enjoyed serving our homestyle cooking to the people of the Smoky Mountains for over 25 years. We offer great family value and something for everyone on our 70 item buffet & dessert bar. For additional information call 1-860-453-6443.
Tennessee Shindig Get ready for a spectacular theatrical production at the Tennessee Shindig! Featuring an all new two-hour show with fifteen fabulous entertainers. Tennessee Shindig celebrates the Volunteer State's musical legacy, and much more! From Graceland to Music Row to Rocky Top, enjoy fast-paced dancing, knee-slappin'laughs, lively country, patriotic, gospel, Elvis, bluegrass, and more! The second half presents "Song-sation, a jaw dropping exhibition of the award winning cast in the music of the decades. It's the most entertaining show the Smokies have ever seen! For Additional information Call 1-888-908-3327
Fishing East Tennessee - Enjoy East Tennessee fishing adventures like no other! Smokies Angling Adventures offers year round guided fishing trips on some of East Tennessee's premier lakes & streams with professional guides. For additional information call 865-933-3140.
Magic Beyond Belief - Escape the ordinary with a visit to Magic Beyond Belief TM Theater. Your family will witness stunning illusions, mind-boggling special effects and more. Enjoy two hours of nonstop illusions and a masterful performance by international star Terry Evanswood. For additional information call 1-865-428-5600.
Miracle Theater - Bring your entire family to The Miracle and witness a musical spectacular like no other. The Miracle is a stunning musical recreation of the life of Christ told in epic proportion and featuring an enormous cast of actors, singers, dancers, technicians and live animals. For additional information call 1-865-428-7469.
Wonderworks - Tennessee's only upside-down attraction. An amusement park for the mind, featuring over 150 interactive exhibits. It's 5500 sq feet of fun. For additional information call 865-868-1800.
The Old Mill - The Old Mill Square complex of restaurants, bakery, pottery and craft shops is more of a total destination than a single stop. Visitors can still watch meal and flour being ground the same way by the huge water powered granite French Buhrs in the mill, or pottery being thrown, or bread and candy being made. It's a day's worth or more of delight and wonder. For additional information call 888-453-6455.
Comedy Barn Theater - Voted the funniest show 6 years in a row. Featuring clean comedians, magicians, jugglers, fire-eaters, country music and barnyard animals. For additional information call 865-428-5222.
Country Tonite Theater - Voted the 2003 Show of the Year. Featuring quality entertainment with singing, dancing, comedy, gospel, patriotic, and more. For additional information call 800-792-4308.
Memories Theater - The ultimate Legends show featuring Elvis and Friends, tributes to Tom Jones, Patsy Cline, The Judds, Neil Diamond, Rod Stewart and more. For additional information call 865-428-7852.
ZORBÂ - Located at the base of the Great Smoky Mountains National Park and just down the road from Dollywood, ZORB? Smoky Mountains is the newsest and most unique Pigeon Forge adventure activity!Â
Pigeon Forge TN Guide - Get Pigeon Forge coupons, tips, and reviews for numerous things to do and places to eat in Pigeon Forge, TN.
Sevierville, TN
Caverns -Ancient mysteries, Indian legends and natural
wonders, with special lighting and sound presentations and guided tours.
Always 58 degrees. For additional information call 865-453-5972.
Bass Pro Shops - The Bass Pro Shops retail store in Sevierville is a leader among Tennessee hunting outfitters, with seminars and classes on everything from fishing and hunting to learning about the outdoors. Whether you need gear for fishing in Tennessee, or simply want to maximize your outdoor fun, shop Bass Pro.
Floyd Garrett's Muscle Car Museum - View over 90 of the largest collection of American Muscle cars. For additional information call 865-908-0882.
Nascar Speedpark - 25 acres of interactive family fun with 8 race tracks and state of the art arcade games. For additional information call 865-908-5500.
Reel Theaters - Local movie complex. For additional information call 865-453-9055.
Scenic Helicopter Tours - Aerial photography and sightseeing tours. For additional information call 865-428-6929.
Rainforest Adventures Discovery Zoo - Located in Sevierville featuring over 120 species. Educational live exhibits, hourly handling shows, and tropical rainforests. For additional information call 865-428-4091.
Smoky Mountain Deer Farm - Over 150 deer and exotic animals including zebras, tigers, birds and monkeys. For additional information call 865-428-3337.
Tennessee Museum of Aviation - 50,000 square feet of Warbirds Aircrafts, memorabilia, gifts and galleries. For additional information call 865-908-0171.
Dandridge, TN
Mountain H20 Sports - Offering Jet Skiing & Sea
Doos as well as leisurely rides on a pontoon boat. Your pontoon becomes
your island while your friends or family "Jet" to and from your location
on Douglas Lake. Douglas Lake is a beautiful, quiet lake and offers some
of the best fishing in the area. For additional information call 423-608-0726.
Smoky Mountain Baloon Adventures - Our flights originate near Sevierville, Tennessee at the foothills of the Great Smoky Mountains National Park. The topography of the area is 80% rolling farm land, and offers stunning 360? scenic views. For additional information call 1-865-484-0330.
Cherokee, NC
Harrah's Casino - North Carolina's biggest attraction, where you can experience 80,000 square feet of 24-hour casino action. Harrah's Cherokee Casino & Hotel features over 3,500 video gaming machines, five great restaurants, live entertainment, two gift shops, an arcade and friendly, first class service. For additional information call (828) 497-7777.


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Celebrity Style: Hilary Duff
Like us on Facebook
Hilary Duff grabbed coffee over the weekend, presumably on her way to a shopping excursion. I'm a big advocate of white jeans and Hil's fit like a glove. Smart girl for wearing tan pumps to elongate her legs. On top, she wore one of Monrow's watercolor tanks in gray and blue. Add a chunky metallic bangle and her signature hoop earrings, and you have the formula for a cute weekend ensemble.


text: I'm a postdoc in the Interactive Robotics Group (IRG) at MIT's CSAIL, working with Professor Julie A Shah. My primary research goal is to develop innovative models and tools to enable effective multi-agent coordination and human-agent collaboration, allowing a group of individuals to accomplish tasks of higher complexity. My research interests include multi-agent learning, reasoning under uncertainty, dynamic resource/task allocation, and stochastic modeling and optimization. Prior to joining IRG, I completed my PhD in computer science and worked as a post-doc at UMass Amherst, supervised by Professor Victor Lesser.


text:
Calliope's $16 Lunch (With Wine) Is a Fantastic Deal
It's inspired by little French bistros.
It's inspired by little French bistros.
"I didn't expect him to start debating with me."
Your server at Eleven Madison Park likely went to a better school than you did.
Waiters have pet peeves, too.
But he won't explicitly identify the East 40s sushi bar that has a no-tip policy.
Do we need maître'ds after all?
Restaurants are watching.
Do you really want to know what's happening in the back of the house?
The latest installment in a restaurateur's list of service no-no's.
A waiter claims he was axed because of an interaction with Kevin Spacey
No, we DON'T want to hear about the specials.
• Street food may have finally jumped the shark with Brix's $35 "unpaved" dinner of faux vendors in the garden. [SFoodie] • Bussers, like servers, must practice a bit of clairvoyance when it comes to properly disposing of dirty plates....
A blogger offers "40 Tips on How to Be a Good Waiter."
Bloggers are upset that Bruni advanced dining stereotypes and that Josh Ozersky is promising columns from 'the top men in the field.'
"Actually, it wasn't that great."


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|Staphylococcus aureus - a cross sectional study of prevalence and risk factors in one general practice.
MedLine Citation:
|PMID: 22558625 Owner: NLM Status: In-Data-Review
|Background Infection control and antibiotic resistant organisms are a community health concern. This article presents findings of a cross sectional study of 100 users of the Thirroul Medical Practice clinical treatment room, in Thirroul, New South Wales. Methods Nasal Staphylococcus aureus colonisation rates and risk factors were investigated. Results Twenty-six percent of participants (n=26) were found to have S. aureus; 11.5% (n=3) of cases were community acquired methicillin resistant S. aureus. Methicillin resistant S. aureus was significantly correlated with older age (p=0.02) and skin infection within the preceding year (p=0.03). Clinical staff (n=15) had low rates of S. aureus at 6.6% (n=1) and no methicillin resistant S. aureus. Discussion Overall, S. aureus rates were unremarkable, but methicillin resistant S. aureus rates were higher than elsewhere with older patients most at risk. General practice staff developing infection control strategies should consider the vulnerable nature and cross-contamination risks in this group of patients. Encouragingly, clinical staff showed low levels of S. aureus and no methicillin resistant S. aureus.
|Rebecca Warren
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Publication Detail:
|Type: Journal Article
Journal Detail:
|Title: Australian family physician Volume: 41 ISSN: 0300-8495 ISO Abbreviation: Aust Fam Physician Publication Date: 2012 May
Date Detail:
|Created Date: 2012-05-04 Completed Date: - Revised Date: -
Medline Journal Info:
|Nlm Unique ID: 0326701 Medline TA: Aust Fam Physician Country: Australia
Other Details:
|Languages: eng Pagination: 325-8 Citation Subset: IM
|MBBS, LLB, MEd, is an intern, Shoalhaven District Memorial Hospital, Nowra, New South Wales.
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|APA/MLA Format Download EndNote Download BibTex
|MeSH Terms
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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Autism comprises a spectrum of behavioral and cognitive disturbances of childhood development and is known to be highly heritable. Although numerous approaches have been used to identify genes implicated in the development of autism, less than 10% of autism cases have been attributed to single gene disorders.
We describe the use of high-resolution genome-wide tilepath microarrays and comparative genomic hybridization to identify copy number variants within 119 probands from multiplex autism families. We next carried out DNA methylation analysis by bisulfite sequencing in a proband and his family, expanding this analysis to methylation analysis of peripheral blood and temporal cortex DNA of autism cases and matched controls from independent datasets. We also assessed oxytocin receptor (OXTR) gene expression within the temporal cortex tissue by quantitative real-time polymerase chain reaction (PCR).
Our analysis revealed a genomic deletion containing the oxytocin receptor gene, OXTR (MIM accession no.: 167055), previously implicated in autism, was present in an autism proband and his mother who exhibits symptoms of obsessive-compulsive disorder. The proband's affected sibling did not harbor this deletion but instead may exhibit epigenetic misregulation of this gene through aberrant gene silencing by DNA methylation. Further DNA methylation analysis of the CpG island known to regulate OXTR expression identified several CpG dinucleotides that show independent statistically significant increases in the DNA methylation status in the peripheral blood cells and temporal cortex in independent datasets of individuals with autism as compared to control samples. Associated with the increase in methylation of these CpG dinucleotides is our finding that OXTR mRNA showed decreased expression in the temporal cortex tissue of autism cases matched for age and sex compared to controls.
Together, these data provide further evidence for the role of OXTR and the oxytocin signaling pathway in the etiology of autism and, for the first time, implicate the epigenetic regulation of OXTR in the development of the disorder.
See the related commentary by Gurrieri and Neri: http://www.biomedcentral.com/1741-7015/7/63 webcite
Classic autism comprises a spectrum of behavioral and cognitive disturbances of childhood development. The core autism phenotype includes deficits in social interaction, language development and patterns of repetitive behaviors and/or restricted interests. The population prevalence of the spectrum of autism disorders is estimated to range between 1/300 [1] to 1/100 http://www.nschdata.org/ webcite, with a male: female ratio of 4:1 [2,3]. The disorder has been shown to be highly heritable with the relative risk for siblings being approximately 2% to 8%, much higher than that of the general population [4]. To date, only a small percentage of autism cases (<10%) have been ascribed to single gene disorders such as fragile X syndrome, tuberous sclerosis [5] and Rett syndrome [6]. Numerous approaches including genetic linkage, genome-wide association, candidate gene association and gene expression analysis have been used to identify the additional genes implicated in the development of autism [7,8]. However, the heterogeneous nature of autism and autism spectrum disorders has limited their success.
An additional approach to identify genes involved in autism is to characterize copy number variants (CNVs), that is, chromosomal deletions and duplications, that are known to be present within at least 5% of individuals with idiopathic autism [9]. Autism CNVs have been shown to involve almost all chromosomes [10,11], with the most frequently observed alteration localizing to chromosome 15q11-13 [12-23]. A number of different methods have been used to characterize autism related CNVs, including but not limited to, cytogenetic G-banding [14,23,24], metaphase fluorescence in situ hybridization (FISH) [22], Southern blotting [18], loss of heterozygosity (LOH) analysis [15-17,19], quantitative polymerase chain reaction (PCR) [25] and, more recently, genotyping and representational oligonucleotide microarray analysis (ROMA) [26].
Here we describe the use of genome-wide tilepath microarrays and array comparative genomic hybridization (CGH) to identify CNVs in a dataset of 119 unrelated probands from multiplex autism families [27]. The genomic profiles of our autism dataset were compared to the array CGH profiles of 54 phenotypically normal individuals, to previously published CNVs present within the database of genomic variants [28] and to the Autism Chromosome Rearrangement Database http://projects.tcag.ca/autism/ webcite. The most significant finding thus far from our analysis is a heterozygous deletion of the oxytocin receptor gene (OXTR) (MIM accession no.: 167055) in an individual with autism and his mother with putative obsessive-compulsive disorder (OCD). We further investigated the relationship between OXTR and autism by carrying out epigenetic analysis of the promoter region of OXTR. We show that the gene is hypermethylated in independent cohorts with autism as compared to controls, in both peripheral blood mononuclear cells (PBMCs) and the temporal cortex. Additionally, our analysis of expression levels of OXTR in the temporal cortex shows decreased levels of expression in individuals with autism as compared to controls matched for age and sex.
These data suggest that OXTR and the oxytocin signaling pathway play an important role in the etiology of autism and autism spectrum disorders and implicate epigenetic misregulation of OXTR in this complex disease.
Diagnostic and Statistical Manual of Mental Disorders criteria
Research diagnostic classification entailed the collection of detailed family, developmental, and medical history and systematic screening of each child with autism before proceeding with clinical assessments. The clinical diagnosis of autism was confirmed by standardized diagnostic assessments once a family had completed the screening process and was considered eligible. For research classification, a clinical diagnosis that conforms to Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria was made based on all available medical, clinical, and behavioral data. The clinical diagnosis was established as a research diagnosis on the basis of scores that exceed established cut-offs for autism on the Autism Diagnostic Interview-Revised (ADI-R) [29,30]. In addition, all individuals demonstrated a minimum IQ equivalent of 35 or Vineland Adaptive Behavior Scale age equivalent of 18 months. This ensured a sufficient developmental level for reliable classification on diagnostic measures. All instruments were administered by clinical staff that had been formally trained on all measures and, as appropriate, had demonstrated reliability within the research group as well as at clinical centers. All individuals are qualified only after review of materials by a licensed child psychologist (MC). Parents/caregivers were informed of the purposes, risks, and benefits of participating in this project and provided informed consent.
Peripheral blood collection
All aspects of the research study were approved by the Institutional Review Boards of the participating centers. DNA for copy number and methylation analyses was obtained from the peripheral blood of autism probands from multiplex families, the extended family of the proband with the OXTR deletion, and phenotypically normal controls. Genomic DNA was extracted from whole blood using the Pure Gene method and standard protocols, as described previously [31]. DNA for the methylation analysis was collected from 20 autism cases and controls, all individuals were Caucasian, and the male to female ratio was 1:1.
Autism brain tissue
Postmortem studies were performed using frozen brain tissue samples dissected from the superior temporal gyrus (BA 41/42) of eight patient-control pairs, obtained through the Autism Tissue Program from the Maryland National Institute of Child Health and Human Development (NICHD) Brain Tissue Center and the Harvard Brain Tissue Resource Center (Table 1). This neocortical region was chosen because it hosts well documented structural and functional abnormalities in autism [32-35]. These tissue samples largely overlap with those employed in our recent studies of the MET and PRKCB1 genes [36,37]. Clinical and demographic information, family history, and autopsy reports were obtained from the Autism Tissue Program web site http://www.atpportal.org webcite and are summarized in Table 1. The presence of mental retardation was defined on the basis of a full-scale IQ <70. Autism spectrum disorder cases fulfill DSM-IV diagnostic criteria [38], confirmed using the ADI-R [29,30]. Although the methylation study was not planned according to a matched design, all controls were selected to match patients on sex, age (± 2 years, wherever possible) and postmortem interval (PMI), where possible (Table 1).
Table 1. Description of clinical details associated with the temporal cortex tissue used in the methylation and expression analyses
Array hybridization
High-resolution genome tilepath microarrays were constructed using large bacterial clones (bacterial artificial chromosome (BAC) clones) as previously described [39]. Briefly, BAC DNA was purified by alkaline lysis and used as the template for three different, degenerate oligonucleotide primed PCR reactions, prior to a second round of amino linking PCR and printing onto Codelink slides (GE Bioscience, Piscataway, NJ, USA) using a Genetix Qarray2 (Genetix, Boston, MA, USA). A total of 450 ng of autism proband and control DNA, from peripheral blood, were differentially labeled with Cy3-CTP and Cy5-CTP (BioPrime Labeling Kit; Invitrogen, Carlsbad, CA, USA), purified (Qiagen, Hilden, Germany) and hybridized to the tilepath arrays using the MAUI hybridization station (BioMicro Systems Inc, Salt Lake City, UT, USA). Image capture of the hybridized arrays for fluorescent intensity extraction was performed using a Genepix 4100A scanner (Molecular Devices, Sunnyvale, CA, USA) and Bluefuse microarray software (BlueGnome http://www.cambridgebluegnome.com/bluefuse_micro webcite) was used for data processing of the scanned images prior to porting into Nexus (BioDiscovery http://www.biodiscovery.com/index/nexus webcite) for analysis.
Array CGH analysis
Microarray data was preprocessed in Nexus by removing poor quality flagged spots (confidence <0.3 as defined by Bluefuse software), removing background by Lowess correction before normalization of log2 ratios and combining BAC replicates. BioDiscovery's rank segmentation algorithm (RSA), which is similar to circular binary segmentation (CBS) [40], was used to identify genomic rearrangements. Briefly, the algorithm uses a normal distribution function for testing for change points as opposed to the non-parametric permutation based statistics used in the original CBS algorithm. It also uses the log ratio rank of each probe as oppose the log ratio value itself. The calling algorithm used cluster values and defined log2 thresholds of ± 1.4 and 0.38 for two and one copy gain/loss, respectively. We applied a conservative cut-off of three BAC clones showing the same copy number change trend to define genomic deletion or duplication with the significance threshold set at 0.005. Array CGH dye-swap experiments were carried out on 21 samples to confirm 21 genomic changes, including novel and known CNVs within the database of genomic variants.
Quantitative real-time PCR validation
DNA copy number was validated using the Applied Biosystems 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers and probes were designed to a region spanning exon 2 in OXTR: forward 5'-GCTGAACATCCCGAGGAACTG-3' and reverse 5'-GCAAATGAGCGGGAATCCTCTA-3'. DNA samples from autistic probands and their family members were used to determine if the CNV was familial or de novo. DNA isolated from tumor samples with known deletions at the regions containing OXTR were used as 1 × copy number controls and an unaffected individual identified in our normal-normal CGH experiment served as our 2 × copy number control at these loci. A primer and probe set designed to the single copy gene that encodes the RNA moiety for the RNase P enzyme, RPPH1 (MIM accession no.: 608513), a region of stable copy number (2 ×), was used to normalize each sample for equal input. Copy number was assessed in quadruplicate for each sample and the 2-ΔΔCT method was used for analysis [41].
Microsatellite screening
Microsatellite markers were analyzed in the family carrying the deletion of OXTR to determine maternal or paternal contributions. Five markers on chromosome 3 (D3S1489, D3S18, D3S4539, D3S4163, D3S3691) were fluorescently labeled using PCR (primers indicated in Additional file 1) and size fractionated on an Applied Biosystems 3730 capillary sequencer (Applied Biosystems, Foster City, CA, USA). Then, 1 μl of the PCR product was diluted 1/70 and run on an agarose gel to confirm amplification, and 1 μl of the diluted PCR product was added to 19 μl of HiDye Formamide/LIZ 500 Standard solution and loaded on to the capillary sequencer and analyzed using standard software.
Additional file 1. Cloned bisulfite sequencing results for each peripheral blood mononuclear cell (PBMC) sample.
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Methylation analysis
DNA isolated from blood or brain samples was bisulfite converted using a methylSEQr Bisulfite Conversion Kit (Applied Biosystems, Foster City, CA, USA). Primers were designed to bisulfite-converted regions of CpG islands in the promoter region of the OXTR gene and in the third intron that had previously been shown to exhibit different patterns of methylation among individuals [42] (Additional file 2). PCR was performed on bisulfite treated DNA using the following reaction conditions, 1 cycle: 95°C for 3 min, 55 cycles: 95°C for 1 min followed by 56.8°C for 1 min followed by 72°C for 3 min, and a final extension at 72°C for 5 min. The reaction was run on an agarose gel and the bands were gel extracted (Qiagen, Valencia, CA, USA) and TOPO TA cloned (Invitrogen, Carlsbad, CA, USA). Single clones were isolated and plasmids purified (Qiagen, Valencia, CA, USA). Sequencing was performed using the M13 forward primer provided with the TOPO TA cloning kit. On average, 10 individual clones were sequenced per amplicon in order to calculate percentage methylation at each of the CpG sites (note: 8 clones were used in OXTR family analysis). Bisulfite conversion was verified for each clone by assessing the C to T conversion of non-CpG sites. The average methylation level (%) was determined for each individual and these values were used to generate an average percentage methylation for each group, autism cases and normal controls, respectively (Additional file 1). The Welch-Satterthwaite t test was employed to adjust for unequal variances between groups and to compare the mean methylation level (%) at each site between the autism cases versus control groups. A nominal significance of P ≤ 0.05 indicated a significant change in the methylation state between the two groups. The direction of the change was determined by comparing the average methylation level at each site between the two groups.
Additional file 2. Primer pairs used for this study.
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Expression analysis by quantitative PCR (qPCR)
Total RNA was extracted from four case-control pairs matched for age, sex and PMI (see Table 1) using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to standard methods, and RNA quality was checked using a Bioanalyzer (Agilent, Santa Clara, CA, USA). Reverse transcription was performed with the QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA, USA), using random hexamers as primers and a starting RNA quantity of 1 μg in a 20 μl final volume. OXTR cDNA was quantified using an iQ5 Multicolour Real-time PCR Apparatus (Bio-Rad, Hercules, CA, USA), according to a standard ΔΔCt Syber Green protocol [43]. PPIA (MIM accession no.: 123840) cDNA was measured in parallel and used as a standard for normalization. Due to relatively low neocortical OXTR gene expression levels compared to PPIA, PCR reactions were performed using 1:5 and 1:25 cDNA dilutions, respectively. OXTR primers were targeted to exon 2, which is shared by all four alternative mRNA transcripts, and designed as follows: F2, 5'-CTGAACATCCCGAGGAACTG-3', and R2, 5'-CTCTGAGCCACTGCAAATGA-3'. To ensure specificity, PCR products were initially sequenced using a CEQ8000 DNA sequencer (Beckman-Coulter, Fullerton, CA, USA), and afterwards checked by running a melting curve at the end of each experiment. Analysis was performed using the 2-ΔΔCT method [41] and expression level from the group of cases and the group of controls were compared using a one-tailed paired t test.
Normal-normal analysis
Array CGH data was generated for 54 phenotypically normal individuals in 27 sex-mismatched hybridizations to establish the performance of our large insert bacterial clones on the genomic tilepath microarray. Sex mismatching was used to establish the baseline of heterozygous loss using the X chromosome as a reference. Analysis of the 27 normal-normal array CGH experiments identified 12 CNVs within 2 or more of the array CGH hybridizations. In all, 10 CNVs were contained within previously published CNVs from the database of genomic variants [28], while 2 CNVs extended the size of the known CNV regions (Additional file 3).
Additional file 3. Genomic duplication and deletion events within 54 control and 119 autistic individuals identified by whole genome tilepath array comparative genomic hybridization (CGH).
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Autism CNVs
The genomic profiles of 119 idiopathic autism probands (93 male and 26 female) were generated by sex-mismatch array CGH experiments using controls from our normal-normal analysis. A total of 113 loci within 111 autistic individuals contained at least 1 genomic deletion or duplication, with a range of 1-6 CNVs per individual (Additional file 3). Approximately half of these, 52, were wholly contained within known copy number variants from the Database of Genomic Variants [28], 57 loci overlapped with 1 or more known CNVs regardless of size (CNV regions), and 5 were novel regions of loss (n = 2) or gain (n = 3) that contained 21 genes (Additional file 3). According to Fable http://fable.chop.edu/ webcite only one gene within these novel regions, ring finger protein 19A (RNF19A, also known as p38) in a 0.6 Mb deletion on chromosome 8q22.2, has been previously implicated in autism spectrum disorders (ASDs) via an observation in Rett syndrome [44], and none fell within previous regions of linkage (as reviewed in [7]). We chose to focus on 19 of the 113 putative deletions or duplications identified in the first screen to validate by array CGH dye swap experiments using select individuals (Additional file 3, top) or, in the case of the OXTR deletion described below, by microsatellite and quantitative real-time PCR analysis. Genomic rearrangements of note included a 12.5 Mb deletion within 2q24.1-2q24.3, a region previously linked to autism [45,46] and containing 41 known genes; a 4.6 Mb duplication and a 3.4 Mb deletion within 15q11-13 that contains a complex arrangement of CNVs and has previously been associated with autism by numerous genetic linkage [47-49] and genome copy number studies [12-23]; and a 0.7 Mb deletion in 3p25.3 that contains 5 genes: Lim and cysteine-rich domains 1, LMCD1 (MIM accession no.: 604859); C3orf32; Caveolin 3, CAV3 (MIM accession no.: 601253); oxytocin receptor, OXTR (MIM accession no.: 167055); and RAD18 (MIM accession no.: 605256) (Figure 1). Though this region contains known smaller copy number variants, none of these correspond to a deletion of 0.7 Mb identified by this study or contain the genes mentioned above. Additionally, OXTR is a candidate autism gene whose levels may be important for social development of offspring [50].
Figure 1. Graphical representation of the 0.7 Mb heterozygous deletion in 3p25.3 (8,231,927-8,985,513 base pairs) within an individual with autism identified by array comparative genomic hybridization (CGH). The horizontal ideogram represents the chromosome 3 region of interest with the log2 plot below it of array CGH results from the tilepath clones (individual blue circles) on the genomic array. The dotted line at '0' represents copy neutral log2 plot between the cohybridized samples, while the green line represents single copy gain and the red line single copy loss. OXTR and four adjacent genes are contained within a deletion called in Nexus by circular binary segmentation (CBS) [40] (bold black line). Known copy number variants from the Database of Genomic Variants http://projects.tcag.ca/variation/ webcite are denoted by horizontal pink bars.
Copy number analysis of OXTR
Based on the strength of the novel deletion of OXTR and the previous data implicating OXTR in the development of autism [51-53], we focused further copy number analysis on the 3p25.3 deletion by screening other members of the proband's family. Quantitative real-time PCR (qRT-PCR) analysis confirmed that the deletion containing OXTR was not a de novo event (Figure 2a) and microsatellite analysis confirmed that the deletion was inherited from the mother (Figure 2b), who has possible OCD (not clinically evaluated). OCD cases have been reported to be more common among first-degree relatives of autistic probands, which may hint at a common genetic mechanism or pathway shared between these two disorders [54,55]. Curiously, the proband's affected brother did not inherit the allele containing the genomic deletion (Figure 2). Microsatellite analysis of the deletion revealed that the proband's alternative allele was inherited from the father, therefore excluding the prospect of uniparental disomy at the OXTR locus contributing to the phenotype (Figure 2).
Figure 2. Microsatellite and quantitative real-time polymerase chain reaction (PCR) validation of OXTR deletion. (a) Microsatellite markers within and adjacent to the copy variable region in the OXTR deletion family were assayed to confirm homozygosity and parental transfer of alleles. (b) Normalized real-time PCR results confirm the loss of a single copy of OXTR in both the mother and the proband (marked by a black triangle). Copy number was normalized to RNAseP and controls were used to verify deletion. The 1 × control is a tumor sample with a known chromosome 3 deletion. The 2 × control is an unaffected sample from the copy number variant (CNV) study in this paper that is not copy variable at OXTR.
Methylation analysis of the OXTR promoter region
Based on the CNV data within the OXTR deletion family, we hypothesized that a hemizygous deletion of OXTR may result in a reduction in the available levels of OXTR in utero and during development, ultimately leading to the development of autism. However, if this is true, it is unclear how this deletion confers autism in the proband when the proband's affected sibling does not carry the deletion. To investigate the possibility that a mechanism other than a genomic deletion may play a role in decreasing OXTR levels in the affected sibling we investigated the DNA methylation status, a form of epigenetic silencing, of two regions of OXTR previously shown to be methylated and associated with differential expression of the gene in liver and myometrium [56,57]. We carried out bisulfite sequencing (BSS) analysis of cloned alleles of two OXTR CpG islands in the peripheral blood mononuclear cells (PBMCs) of all four family members (Figure 3). The first CpG island overlaps exons 1, 2, and 3 of OXTR and is located on chromosome 3 at 8,783,962-8,786,280 base pairs (Ensembl Homo sapiens version 54.36p, NCBI36). Kusui et al. had shown that the methylation status of a specific 405 base-pair fragment of this CpG island (termed 'MT2' by Kusui et al.) is important for tissue-specific expression of OXTR (Figure 3 depicts MT2 position). The second CpG island is located within the third intron of OXTR on chromosome 3 at 8,775,160-8,776,624 base pairs (Ensembl Homo sapiens version 54.36p, NCBI36). This island has previously been experimentally defined as an EcoRI-KpnI fragment that was less methylated in myometrial DNA than in the DNA from peripheral blood leukocytes [42]. All CpG dinucleotides tested within the intron 3 CpG island were heavily methylated in all family members (data not shown). However, several of the 33 CpG dinucleotides tested within the region overlapping exons 1, 2, and 3 of OXTR differed in methylation level between family members. Specifically, CpG dinucleotides at positions -959, -934, -924, -901 and -860 (relative to translation start site) showed the most dramatic differences in methylation status, with the proband's affected sibling being heavily methylated at sites -934, -924 and -901. These specific residues fall within the critical region (MT2) in the CpG island of OXTR shown by Kusui et al. to regulate the expression of OXTR in liver [56]. Importantly, in keeping with the hypothesis that increases in DNA methylation (hypermethylation) lead to gene silencing, CpG sites -934 and -924 showed the largest increase in methylation, 37.5%, between the autistic sibling and the non-autistic father (62.5% methylated versus 25% methylated, 100% methylated versus 62.5% methylated, respectively), both lacking the OXTR deletion. The 100% methylation of a specific CpG could indicate maintenance of an aberrant methylation mark, making this a more dramatic change.
Figure 3. CpG dinucleotides analyzed within a predicted CpG island in the promoter region of OXTR. Untranslated regions are represented as gray boxes and protein coding exons are white boxes, the site of translation is denoted by a black arrow. Predicted CpG islands are horizontal black bars beneath the gene. The region of interest within the 5' CpG island, MT2 as reported by Kusui et al. [56], is denoted by a dashed line (NCBI36 coordinates). Groups of CpGs sequenced per clone are indicated with brackets. Clear circles with diagonal lines denote untested CpG dinucleotides, filled circles represent region of interest. CpGs of interest are numbered according to translation initiation site of +1. Clear boxes denote CpG dinucleotides that showed no methylation in bisulfite sequencing of subclones; black within boxes denotes methylation of the CpG dinucleotide; F = Father, M = Mother, P = Proband, S = Sibling.
Hypermethylation of residues critical for OXTR gene silencing are associated with autism
Given the data observed within the OXTR deletion family, we hypothesized that DNA methylation of residues found within the critical region of OXTR regulation may extend beyond this family and represent a more general epigenetic contribution to autism spectrum disorders. We extended our BSS analysis of the 5 differentiallly methylated CpG dinucleotides to PBMCs from 20 phenotypically normal control individuals (10 males and 10 females) and 20 individuals with autism (10 males and 10 females). BSS data showed significantly increased methylation in the PBMCs of CpG -860 (P = 0.0009), -934 (P = 0.0081) and -959 (P = 0.0026) in individuals with autism compared to controls (Table 2). When the data are stratified by sex, the associated significance of -860 and -934 can be ascribed only to males (Table 3) and that of -959 to females (Additional file 4).
Additional file 4. OXTR promoter CpG methylation status in peripheral blood mononuclear cells (PBMCs) of 10 autistic females and 10 age-matched controls.
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Table 2. OXTR promoter CpG methylation levels in peripheral blood mononuclear cells
Table 3. OXTR promoter CpG methylation status in peripheral blood mononuclear cells (PBMCs) and cortex samples of males
Hypermethylation of OXTR CpG sites in temporal cortex tissue of autistic individuals
To determine if differential methylation of the five putative regulatory OXTR CpG dinucleotides may be important in a tissue implicated in the etiology of autism, we analyzed temporal cortex tissue from eight individuals with autism and eight controls matched for age and sex (see clinical data in Table 1), six male and two female. The BSS results in the cortex again showed statistically significant hypermethylation in the autism cases versus normal controls. There was significant increased methylation at CpG -860 (P = 0.0251), CpG -901 (P = 0.0149), CpG -924 (P = 0.0448) and CpG -934 (P = 0.0233) between autism cortex tissue compared to the normal cortex controls (Additional file 5). We performed a stratified analysis to increase the homogeneity within the sample using six male samples matched for age and sex, and to validate our sex-specific findings in blood. This analysis reproduced the significance at sites -924 and -934 (Table 3).
Additional file 5. OXTR promoter CpG methylation status in the cortex of eight autism cases and controls matched for age and sex.
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Expression analysis of OXTR in temporal cortex tissue of OXTR
To functionally correlate DNA methylation levels within the temporal cortex DNA and expression of OXTR from which the DNA was derived, we carried out quantitative PCR analysis of mRNA levels from the cortex tissue of four autism cases matched for age and sex and four controls. Assays were designed to exon 2 of the gene. Although OXTR expression levels were low, we detected a sex-specific decrease in expression of OXTR for each of the autistic male control pairs tested (Figure 4a), and in two of the three cases expression levels correlated with an increase in methylation at site -934. As a group, autistic males showed a 20% decrease in expression compared to controls that was significant at P = 0.0389 (Figure 4b). These data suggest that increased methylation of the promoter in OXTR correlates with decreased expression of the gene in a tissue type relevant to the development of autism.
Figure 4. Expression of OXTR in the cortex of male autistic brains is decreased compared to controls. OXTR expression in the cortex was quantitated using quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to PPIA in four cases (autism) and controls matched for age and sex. (a) The expression level of OXTR is decreased in male cases compared to controls and the methylation of site -934 (Me-CpG) correlates with expression in two of the male individuals. (b) As a group, the expression of OXTR in the cortex of the male autistic brain is significantly lower than in controls matched for age and sex. †Paired t test.
For several years the characterization of CNVs in autism patients has identified genes that potentially contribute to the etiology of the disorder [9,58-60]. In this study we used high-resolution genomic tilepath arrays with comparative genomic hybridization to identify 113 CNVs within 119 unrelated autistic individuals. Of note were five novel CNVs that do not map to prior regions of linkage and which, according to Fable http://fable.chop.edu/ webcite, contain only one gene in 8q22.2 (RNF19A) that has previously been implicated in autism spectrum disorders [44]. CNVs observed in known autism regions included a 12.5 Mb deletion within 2q24.1-2q24.3 [45,46], two rearrangements in 15q11-13 [12-23,47-49], and a 0.7 Mb deletion in 3p25.3. We chose to focus on the deletion in chromosome 3p25.3 because of previous reported linkage to the region [61,62], association data implicating the oxytocin receptor (OXTR) that is contained within the deletion to the etiology of autism [51-53]. More recently, the DECIPHER database https://decipher.sanger.ac.uk/ webcite has had copy number data deposited from a syndromic patient who has developmental delay and cognitive impairment presumably emanating from a novel 1.1 Mb deletion that contains OXTR, amongst other genes. Detailed DNA methylation analysis of the promoter region of OXTR, prompted by the observation that the proband's autistic brother did not carry the novel deletion, identified a single CpG dinucleotide within a known OXTR control region [56] that, independently, showed a statistically significant increase in methylation in PBMCs and the temporal cortex tissue of autism cases compared to controls. The identification of similar methylation profiles in the temporal cortex and peripheral blood may be indicative of an early developmental event during which these profiles are differentially established, prior to germ layer specification. If this locus is epigenetically labile, then it might be vulnerable to (unknown) exposures in utero during the first few weeks of pregnancy that lead to shifts in methylation status of OXTR. Similar influences have been seen in IGF2 methylation based on in utero exposures such as smoking and folic acid intake in humans [63]. Since epigenetic profiles undergo reprogramming during very early gestation (fertilization to implantation), it is not surprising that the profiles in these two disparate tissue types of ectodermal and mesodermal origin are similar and furthermore provides a potential means for detection in multiple, more accessible tissues.
The impact of this epigenetic mark on OXTR expression was supported by analysis of the corresponding mRNA from the temporal cortex of autism cases matched for age and sex and controls. As one would hypothesize given their increased methylation of OXTR, the autism cases also showed statistically lower transcript levels of OXTR expression compared to the normal controls. Because deletions encompassing OXTR have not been observed in other studies characterizing structural variation in autism [58,64,65] such events appear to be rare. At the same time our findings suggest that the hypermethylated status of OXTR in autism, resulting in a decrease in OXTR expression, may more commonly contribute to the disorder. While the mechanism for the development of autism from a germ line deletion of OXTR versus epigenetic silencing of the gene in the temporal cortex may clearly be different, the effect on oxytocin signaling could be the same. Together, these data provide strong evidence for the role of OXTR and oxytocin signaling pathway in the etiology of autism and, for the first time, show that the epigenetic regulation of OXTR may be an important factor in the development of the disorder.
Epigenetic control of autism susceptibility is a recent concept and most certainly a topic of great interest in the field. Over the past decade, researchers have uncovered suggestive links between epigenetics and autism, for example, autism is associated with duplications of 15q11-13 (especially maternally inherited), an imprinted region in the genome where DNA methylation status has been linked to Prader-Willi syndrome (PWS) and Angelman syndrome (AS) [66]; mutation within a gene that encodes a methyl-DNA-binding protein (MECP2, (MIM accession no.: 300005)) is the causative agent of Rett syndrome [67]; and mutation of this same gene has been associated with both autism and AS populations [55]. Nagarajan et al have shown that 79% of autism cases have a decrease in MECP2 expression in the frontal cortex and that an increase in aberrant DNA methylation correlates with this decrease in MECP2 expression [68]. These data implicate epigenetic dysregulation as a mechanism for the development of autism and justify the examination of DNA methylation of autism candidate genes, such as OXTR identified in this study.
The oxytocin receptor, OXTR, is spatially and temporally regulated by a complex combination of sex hormones, inflammatory cytokines, oxytocin (OXT) feedback and epigenetic mechanisms [56,57]. It is a high affinity G-protein coupled receptor for OXT, a nine amino acid hormone that has important and complex roles in animal physiology and behavior [69], including parturition and neurotransmission. A putative role for OXT in the development of ASDs was first highlighted by Insel and colleagues [70] who showed that signaling of oxytocin and the closely related molecule vasopressin play important 'prosocial' roles influencing social behavior and cognitive function in a species-specific manner [71-73]. Subsequent studies in mice revealed that Oxt [74] and Oxtr [75] knockouts largely resulted in normal parturition, however, social memory was impaired. Recently, Lee and colleagues [76] showed that only male mice with a targeted forebrain knock out of Oxtr exhibited social impairment by failure to recognize individuals of their own species. It has been suggested that the normal social behavior of the female knockout mice may be attributed to the compensatory function of vasopressin or developmental compensation [69,77]. In an excellent review by Carter [50] that describes the complex sexually dimorphic roles of vasopressin and oxytocin in brain function and behavior, it is apparent that aberrant oxytocin and/or vasopressin signaling is likely to elicit sex-specific outcomes, which is of particular relevance to autism because of disproportionate number of affected males. Our family analysis identified an OXTR deletion in the mother who does not present with autism. Sex-specific effects of OXTR function from animal models, together with statistical data that suggest dominantly acting de novo mutations may have reduced penetrance in females [78], may explain why the heterozygous deletion of OXTR in the mother from our study does not result in autism while the male proband displays the disorder.
We identified a single CpG dinucleotide within a predicted CpG island that showed statistically significant hypermethylation in PBMCs and the temporal cortex of individuals with autism compared to controls. Although the dominant paradigm for regulation of transcription by DNA methylation suggests that clusters of CpGs are necessary for silencing, recent evidence has indicated that methylation of single CpG dinucleotides may also lead to transcriptional changes via interference of transcription factor binding or recruitment of silencing factors [79]. This is the most likely explanation for the findings presented here as CpG site -934 falls within a predicted c-Rel, ZHX2, and LGALS4 binding domains (T. Wang, personal communication). However, further molecular work is required to dissect the likely complex transcriptional regulation of OXTR.
In this study, we focused on the temporal cortex (Brodmann areas 41/42 and 22) because studies have shown that localized temporal dysfunction is linked with clinical symptoms in autism, such as perceptive, emotional and cognitive deficits [32-35]. Although we were not able to determine the methylation status of cortex tissue from the family in which we observed the OXTR deletion, the data we collected from the affected sibling shows hypermethylation of the CpG dinucleotide (-934) in the predicted CpG island upstream of OXTR. Given our results suggesting that DNA methylation of OXTR in PBMCs may be a marker of methylation status in the temporal cortex, it follows that the affected sibling may also exhibit a decrease in OXTR expression due to methylation instead of deletion; thus implicating two separate mechanisms of transcriptional regulation with the same outcome. These results suggest that because the methylation status of site -934 is significantly different in the temporal cortex and PBMCs between autism cases and controls, measuring the methylation status of OXTR from the blood for autism cases could act as a surrogate for methylation status in the temporal cortex and be diagnostic of affection status, together with traditional diagnostic criteria.
In summary, the data presented suggest that genomic deletion or aberrant methylation of OXTR may contribute to the development of autism. We acknowledge that screening for these genomic changes in a large, well controlled autism sample is necessary to determine the prevalence of these changes and their relationship to the disease. However, regardless of the sample size, we provide strong evidence that the epigenetic regulation of OXTR may be an important factor in the development of the disorder and suggest that further studies begin to focus on this regulatory event and its relationship to disease.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SGG conceived the experimental design and wrote the paper; JJC assisted with experimental design, carried out experiments and wrote the paper; AT, JJ, DB, DB, CM and CL carried out the experiments; RKA, HHW, GW ascertained autism families for the paper; PE and CFL assisted with experimental design, SKM assisted with the writing of the paper; MLC helped ascertain autism families and assisted with clinical assessment of the study population; AP and MAP-V assisted with experimental design and in the writing of the paper.
We thank all the patients and families who generously participated in this study, including the donors and their families of the cortex tissue, and the collaborating clinics and physicians for referring individuals to the study. We would also like to thank Joshua Virgadamo, Christina Sheedy and Rachel Cote for their laboratory assistance, Dr Elizabeth Hauser for evaluating our statistical techniques, Dr Ty Wang for transcription factor binding analysis, and Sarah Nelson for comments on the draft of this manuscript. This research was supported by the JP Hussman Foundation and National Institutes of Health grants P01-NS026630 and R01-NIH080647, and by MIUR PRIN 2006058195 (AMP). We also gratefully acknowledge the Autism Tissue Program, the Maryland NICHD Brain Tissue Center and the Harvard Brain Tissue Resource Center for providing brain tissues for this study (SGG and AMP).
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