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From a string of bad harvests to last winter's international export-fraud scandal and the European Union's recent decision to remove tariffs on the Tunisian competition, the Italian olive oil industry has faced its share of setbacks over the past couple of years.
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Nevertheless, the 24th edition of the Ercole Olivario — a prestigious national olive oil competition held in March in Perugia, Italy (which I attended as a guest of the Italian Trade Commission) — was a bright spot amid the gloom, going to show that the ancient art of harvesting and crushing olives into liquid gold (or green, as the case may be) remains alive and well.
Granted, very few of the 100 Ercole finalists export their bottlings to the United States. Such is the nature of craft production; farms like one I visited in Spoleto, Azienda Agricola Antonio Bachetoni, still prefer to sell their oil the old-fashioned way — on site and in local markets. A few, though, are available in the U.S., albeit in more or less limited supply. Good sources include Olio2Go, City Olive, Eataly, Famous Foods and Market Hall Foods.
Five olive oils to try
Located in Puglia, Italy's top-producing region, this acclaimed mill relies on the softer Ogliarola variety and the more intense Coratina for bittersweet oils that show grassy, nutty and vegetal characteristics.
Frantoi Cutrera
The most widely imported brand on this list comes from Sicily, where the Cutrera family makes a full range of DOP (protected designation of origin), organic and infused oils.
Paolo Cassini
This Ligurian producer makes monovarietal oils from the richly fruity Taggiasca olive. Its Extremum CRU — golden and multilayered with herbal and vegetal notes — took home the top prize at the Ercole Olivario for non-DOP extra virgin oils in the "fruttato leggero" category (essentially "light-flavored").
Pasquini makes IGP (protected geographical indication) and other extra virgin oils from the Frantoio and Moraiolo cultivars, which contribute to the green, savory, peppery qualities for which Tuscany is famous.
In the Sicilian DOP zone of Valle Trapanesi, this organic farm produces an extra virgin oil from a blend of three olives: Nocellara, Cerasuola and Biancolilla. Redolent of tomato plant and herbs like basil and mint, it's known for its staying power — use sparingly.
'Preserving Italy': The book
Besides cooking and dressing salads with quality Italian olive oils like these, what else can you do?
Plenty. During the trip, I had the pleasure of getting to know fellow journalist Domenica Marchetti, who wrote the book on preserving Italy's olive oil heritage — literally. Just released by Houghton Mifflin Harcourt, "Preserving Italy: Canning, Curing, Infusing, and Bottling Italian Flavors and Traditions" is chock-full of recipes for pickles, relishes, sauces, jams, liqueurs and other Italian pantry staples; verdure sott'olio — or vegetables under oil — play a key role. I asked her for an introduction to the technique.
Ruth Tobias: What are some common uses for verdure sott'olio?
Domenica Marchetti: I use oil-preserved vegetables all the time. Because they tend to be vinegary (in spite of the fact that they're submerged in oil), they make a great side to roasted or grilled meat — pork, beef, chicken, lamb, sausages. I like to fold oil-preserved asparagus, garlic scapes, peppers or zucchini into frittatas. Oil-preserved mushrooms, peppers and eggplant also make great toppings for pizza, because they counter the richness of the cheese. I dice them up and put them in insalata di riso (rice salad) and farro salad [as well as] egg or tuna salad. And of course they are great as part of an antipasto platter, with cheese and salumi.
RT: Can any vegetable be preserved in olive oil, or do some work better than others?
DM: Most vegetables can be preserved in oil, but the proper technique requires several steps: salting or semi-drying the vegetable to remove excess moisture, bathing it in a vinegar brine, draining and letting it dry out a bit more, and then submerging it in oil. These steps together make the vegetables safe for long-term keeping — though, just FYI, the USDA does not provide guidelines for preserving in oil and so doesn't recommend it. For this reason, I store any homemade oil-preserved food in the fridge and use within three months.
Certain dense, watery vegetables, such as onions, carrots and celery, are better preserved in a vinegar brine to maintain their crunchy character; some, such as zucchini and peppers, are suited to both methods.
RT: Does the quality and/or character of the oil make a difference to the final product?
DM: Generally, the freshest oil is not used for preserving, because it's not the best use of the oil. Rather, it's reserved for drizzling on bread or grilled vegetables and meat. Older, mellower or milder oil is better for preserving, because it doesn't overwhelm the flavor of whatever is being preserved. I used good (but not break-the-bank expensive) extra virgin olive oil for nearly all the oil-preserved recipes in my book.
Main image: Samples from a tasting of oils submitted to the Ercole Olivario competition. Credit: Copyright 2016 Ruth Tobias
In the past few years, Denver — joined by its deluxe alter ego, Boulder, Colorado — has been at or near the top of so many national rankings, it would probably top the list ranking the lists themselves. It has consistently been named among the best (and fastest-growing) cities for millennials, for singles, for entrepreneurs, for outdoors enthusiasts, for beer lovers, you name it — and now that Denver is in the spotlight, its long-underrated dynamo of a dining scene is finally getting a chance to shine.
Here are just some of the kicks awaiting visitors in search of a Mile High culinary adventure. Or at least a cure for the munchies. Let's face it: Marijuana legalization might have something to do with Colorado's soaring profile.
Union Station
It has stood at the edge of what's now known as LoDo (Lower Downtown) since the fin de siècle — and Union Station's grand reopening in 2014 after a multimillion-dollar renovation marks the apotheosis of the neighborhood's own comeback from late-20th century Skid Row into prime real estate.
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Some of the city's most celebrated restaurateurs have set up shop on all sides of the magnificent Great Hall. At the casual end, there's funky daytime franchise Snooze — where buttered-popcorn pancakes meet Thai-chili Bloodies — and Next Door, an ethicurean pub known for its beet burgers and kale chips. At the splashier end, Stoic & Genuine revels in a seafood repertoire that skews both wildly original — think miso-cured uni over kimchi granita — and classic, from clam rolls to caviar. Anchored by a gleaming deli and exhibition kitchen, Mercantile Dining & Provision turns out an ever-changing array of contemporary creations: highlights include exquisite pastas and anything featuring products from co-owner Alex Seidel's Fruition Farms. And The Cooper Lounge, overlooking all the action, is as swanky a setting for cocktails as you'll find in this dressed-down town.
The Source
Think of it as Union Station's flip side: a gritty-chic urban marketplace that opened in 2013 along a still-gentrifying stretch of Brighton Boulevard with a roster of rising culinary stars and cult vendors. Now arguably the hub of the RiNo (River North) district, The Source is a one-stop shop for extraordinary beans (Boxcar Coffee Roasters), breads (Babettes) and beef (Western Daughters Butcher Shoppe), among other goods, that prove the word "artisanal" hasn't lost all meaning just yet. It's home to two beloved restaurants — the globally inspired Acorn and Comida, a modern taqueria/cantina — and ultra-cool cocktail bar RiNo Yacht Club. Capping it all off is the taproom of Crooked Stave, founded by a brewer whose experiments with brettanomyces and barrel aging have put it at the forefront of Denver's world-class beer scene.
Breweries, breweries and more breweries
Speaking of beer: If ever there were proof that statistics don't tell the whole truth, consider that Colorado, with about 250 craft breweries (or 6 per 100,000 adults), ranks "only" third in the nation. After all, that figure comes from the Brewers Association, which happens to be located not in California (first) or Washington (second), but in Boulder — the outgrowth of an earlier organization started by association president Charlie Papazian, aka the godfather of American home brewing. Papazian also founded the nation's largest craft-beer showcase and competition, the Great American Beer Festival, held annually in Denver. It's worth noting, too, that a fellow local microbrewing pioneer, Wynkoop co-founder John Hickenlooper, is now governor.
Of course, the ultimate metric of achievement becomes evident to anyone who spends even a short time here: the presence of a taproom on every other street corner, each with its own niche. For the most up-to-date and comprehensive information on breweries large and small, check out Westword's Beer Man column and the Fermentedly Challenged blog. But some of my favorites include Diebolt and Prost for traditional (read: Eurocentric) styles, Former Future and Coda for adventurous tastes, and Renegade and Station 26 for sheer high-energy atmosphere.
One of Colorado's most renowned (and widely distributed) envelope-pushing brands, Avery Brewing Co., recently opened a state-of-the-art, city-block-sized facility complete with sit-down restaurant and gift shop at the northern edge of Boulder. It's a must for any suds buff — as are much smaller but no less superb breweries such as the chef-run BRU and the locals' secret, J Wells — but it's just the tip of the Berkeley of the Rockies' gastronomic iceberg. To name some solid candidates for the connoisseurs' to-do list: splendid sandwiches and specialty goods at gourmet shop Cured. Tea at the jaw-dropping Dushanbe Teahouse, an architectural masterpiece built by Tajikstani craftsmen. Genuine farm-to-table feasts at Blackbelly or Black Cat Bistro — both labors of love by chefs who really do run their own farms. Exquisite Japanese bites at the twinkling izakaya called Amu, wood-fired pies at the mod-rustic Basta, displays of Old World oenophilia at PMG. And as for Frasca Food and Wine — suffice it to say that chef Lachlan Mackinnon-Patterson and master sommelier Bobby Stuckey's mecca of Friulian cuisine continues to earn the accolades it rakes in nationwide (and beyond).
Aurora, Colorado
If it's teeming diversity you crave — admittedly not Boulder's strongest suit — Aurora, on Denver's eastern border, is your destiny (along with Federal Boulevard, thronged with Vietnamese and Mexican kitchens). Though it feels like a suburb, it's actually Colorado's third-largest city and a center of immigrant life. Here, sharp-eyed explorers will find Korean, Thai, Middle Eastern, Indian, Sudanese and Hawaiian restaurants lined up in strip malls one after the other; they'll find ramen and barbecue and tacos galore — and they'll encounter the most random of surprises to boot, like soul fast food (Kirk's Soul Kitchen) and a biker bar that serves Chinese eats (Piper Inn).
Dive bars
On that note, long before Denver had a culinary leg to stand on, it boasted watering holes whose potent mix of Wild West grit and urban grime earned them a place in, variously, Jack Kerouac novels, Tom Waits songs and one particularly infamous Playboy article. It still does. And although a whirlwind tour isn't for everyone, here's the itinerary any counterculturalist at heart should follow. Start at Charlie Brown's Bar & Grill or My Brother's Bar to hang out where Kerouac, Neal Cassady and other Beat legends once drank. Catch some live jazz at El Chapultapec, the 80-year-old remnant of an era when the Five Points neighborhood was known as the Harlem of the West. Or simply cruise East Colfax Avenue: Though in the throes of change, it's still an embarrassment of divey riches. There you'll find Pete's Satire Lounge, where an as-yet-undiscovered Bob Dylan used to perform, as did the Smothers Brothers; not far away, PS Lounge illustrates the power of kitsch to bring all walks of life together. Meanwhile, situated at the western end of the 26-mile-long avenue, Casa Bonita may not be a dive, but it's got cliff divers, among other carnival attractions parodied in a famous "South Park" episode.
Local ingredients
Casa Bonita is strictly a sightseers' stop, but you'll have no trouble finding terrific Mexican eateries on just about every corner of this city (to pinpoint just a few admittedly downscale gems: El Taco de Mexico, El Original Tacos Jalisco, Tarasco's, Chili Verde and La Calle Taqueria y Carnitas on West Alameda Avenue). Many of them will offer green chile; the sauce/stew is as traditional here as it is in New Mexico, though the Colorado style is thicker and often includes tomatoes with the chiles, pork, onions, garlic and so on. Cruise down Federal Boulevard in summer and you'll see the roadside roasting stands hawking Pueblo (as well as Hatch) chiles by the bushel. Of course, Colorado lamb and beef are even more famous, as is Rocky Mountain trout — but locals equally covet Olathe corn, Palisade peaches and Rocky Ford melons in season. For a taste of the bounty, head to farm-centric fixtures such as Beast + Bottle, The Kitchen and Old Major.
Worldly palates
Where a local focus and a cosmopolitan outlook come together, you'll find Denver's most distinctive dining and drinking spots. Take Beatrice & Woodsley, combining eye-popping decor designed to evoke a mountain cabin with a fascinating menu that simultaneously reflects the agrarian past and a global future. At Lower48 Kitchen, up-and-coming chef-partner Alex Figura takes a similar approach to yield some of the most exciting food around. Visionary restaurateur Justin Cucci is building an empire on extraordinary ambiance as well as consciously sourced contemporary cuisine, with venues housed in a former gas station, mortuary and brothel, respectively; the latter, Ophelia's Electric Soapbox, emits a dazzlingly risqué vibe. Jim Pittenger of Biker Jim's Gourmet Dogs has rightly garnered national attention for his reindeer, rattlesnake and other wild sausages, with wacky toppings to match. Same goes for Sean Kenyon, bartender-owner of Williams & Graham, a celebrated rendezvous for cocktail aficionados. And then there's Work & Class: its exuberant yet intimate atmosphere and Latin-influenced comfort food will linger in your mind long after your visit.
The great outdoors
With its enviable high-desert climate (not to mention the Rocky Mountains in its backyard), Denver is an obvious draw for outdoors enthusiasts — and an ideal site for seasonal festivals and markets of all kinds. Food-truck chasers mustn't miss Civic Center EATS, where mobile specialists in everything from pierogi to Popsicles gather in the namesake park twice a week from May through October. The Big Wonderful is its even-hipper counterpart, bringing to a vacant lot in RiNo not only trucks but also stalls selling gourmet pantry products and household goods, a live-music lineup and a full bar. The Denver Flea hosts similarly massive bashes with food, booze and arts-and-crafts vendors a couple of times a year. In a Larimer Square courtyard, the pop-up Le Jardin Secret proves as charmingly chichi as it sounds. And — to return once again to Denverites' favorite subject — themed beer festivals are a near-weekly occurrence. But be warned: They often sell out in no time.
Main photo: Stoic & Genuine at Denver's Union Station. Credit: Copyright 2015 June Cochran
In 2005, a group of seven Pinot Noir specialists from the West Coast banded together at the behest of their importer/distributor in Colorado to do a promotional tour of the state. The events they hosted under the irreverent banner of the Pinot Posse — motto: "No stinkin' badges and no Cabs" — were such a smash the tour has become an annual tradition.
Granted, their success may have gotten a boost from a little movie called "Sideways," released nationwide in 2005. Before it, mainstream America knew little about domestic production of the Burgundian grape; today Pacific Pinot Noir needs no introduction. But that doesn't mean even savvy wine drinkers know everything there is to know.
After a dinner at Denver's Table 6, I asked some of the producers to explain what they think consumers should understand about their signature grape.
How did you come to recognize the potential for Pinot Noir in your area?
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Dan Kosta, Kosta Browne, Sonoma County, California: I've been drinking Burgundy since I was 5 years old, so Pinot Noir has always played a big part in my life. However, in the late 1980s, I really started discovering the potential of Russian River Pinots, particularly in the wines made by Joseph Swan, Dehlinger, Rochioli, Williams Selyem and others. These were not the lean, soulless wines that many California producers were making at the time. There was complexity and elegance, and they inspired us to focus on Sonoma County Pinot.
Peter Cargasacchi, Cargasacchi and Point Concepción, Santa Barbara County, California: Since the late 1980s, I'd been drinking Pinots from Sanford Winery here in the Sta. Rita Hills. Richard Sanford was a neighbor, and over a period of 10 years, he convinced me that my high-pH, calcareous soils were similar to the great sites in Burgundy. So in 1998 I started planting Pinot Noir for him, Siduri and Babcock.
Ed Kurtzman, August West, Russian River Valley, California: When I started making Pinot Noir, in 1994, I was working with well-established vineyards: Bien Nacido, planted in 1973, and Chalone, planted in 1946. So I kind of walked into Pinot as it was already in motion. Then I began working with Santa Lucia Highlands in 1999, when it was still a young appellation with first-crop vineyards. Same thing with the Russian River Valley/Sonoma Coast: So many of the vineyards there were planted between 1997 and 2004. It has been interesting to have worked with both old vines and young vines. I wasn't someone who recognized the potential for Pinot Noir so much as someone who gladly participated in its popularity.
Jenne Lee Bonaccorsi, Bonaccorsi Wine Co., Santa Barbara County, California: When my late husband, Michael, and I started making wine in 1999, our goal was always to go to the Russian River. But we had to keep our day jobs, and the closest wine region was Santa Barbara. We planned to learn there and move on at some point. Then we tasted some of Greg Brewer's wines. They were phenomenal, and we could not figure out how this region was so overlooked. We made a very specific choice to stay. Of course, back then, Cabernet was king, and Pinot Noir was not very popular. We had to really talk restaurants and wine stores into purchasing it.
What don't a lot of American drinkers know or understand about Pinot Noir that you wish they did?
Jim Prosser, J.K. Carriere, Willamette Valley, Oregon: I wish more people understood it from a classic, historical perspective: It's basically a connoisseur's wine due to its subtlety, complexity and movement. It's the antithesis of an in-your-face wine; it's more come hither, more about enticement, and its acid combines with, rather than overwhelms, food.
Kurtzman: That color is only important when it comes to high points from Robert Parker or Wine Spectator. Pinot Noir is naturally a thin-skinned, light-colored red wine. People will hold one of my Pinots up to the light, in their glass, and they'll say, "Hey, what a light wine." All they have to do is taste it — it's full of aromas and flavors.
Bonaccorsi: I think we as Americans drink wine too young, especially Pinot. People understand about aging varietals like Cabernet, but they tend to drink American Pinots upon release. They may not need to be aged as long as Burgundies, but they can definitely benefit from a few years. At this point, my 2010 Pinot and 2009 Syrah are tasting well.
What's your personal favorite food to serve with Pinot Noir and why?
Kosta: This, of course, depends on seasonality, but I tend to lean toward lighter meats and earthy vegetables. In the spring, rack of lamb with morel mushrooms is perfect. Chicken, salmon and pork dishes usually work great. One hint that I offer in the kitchen is to pay attention to the quantity and quality of salt — don't be afraid to use it! Good salt can really bring out great flavors in Pinot Noir.
Kurtzman: Anything made with duck goes well with Pinot: duck breast, duck confit, duck burritos, duck with scrambled eggs, duck-bacon pizza.
Prosser: Maybe it's because I'm from Oregon, but charcoal-grilled Columbia River salmon and Pinot is ridiculously good.
Bonaccorsi: The idea of white wine with fish and red wine with meat is very tired; food has changed in leaps and bounds since the days of beef Wellington, and the same is true of pairings. With that said, there is nothing better than a grilled steak and a glass of Pinot Noir.
What are domestic Pinot's most distinctive qualities — what sensory clues should a wine drinker look for? How best to serve it to show off those qualities?
David O'Reilly, Owen Roe, Willamette Valley, Oregon, and Yakima Valley, Washington: In evaluating American Pinot Noir, I tend first to note the deeper ruby color. It is also more intensely aromatic and flavored than Burgundy, with a lovely, silky richness. The expression of domestic Pinot Noir varies along with the diverse growing areas: wines from the cooler Russian River sites, Santa Rita Hills and Oregon tend to be more fresh-fruited, while wines from the warmer Santa Lucia Highlands and Sonoma will have more power and richness.
However, one defining feature of all is immediate approachability, so I tend not to decant younger domestic Pinots. Only after some cellaring, when the wine is throwing a little sediment, do I decant. The perfect serving temperature is the same as for Old World Pinots: 55 to 60 F.
Craig Strehlow, Camlow Cellars, Russian River Valley, California:
Most domestic Pinots are very fruit-forward, with good acidity and softer tannins. Look for bright red fruit like cherry and raspberry and darker fruit such as currant and plum, as well as some spiciness and a cola finish. The use of new oak is judicious, to keep the wine in balance. Here in California, we don't need to worry about ripeness, so the winemaker can pick at the time that's ideal for them to get the flavor profile they ultimately want.
I believe that decanting all wines is a good idea. If they are young, this will open them up and encourage them to evolve in the decanter. For me the ideal drinking temperature is between 65 and 70 F.
What can consumers expect with respect to the wines you're releasing this year?
Cargasacchi: Our wines will be ripe and dark, balanced by acidity, and will increase people's vocabulary and singing ability!
Prosser: Well, from J.K. Carriere, you can always expect higher-acid, classic wines that are made for food and built for age. The wines from the 2012 vintage that are out now are gorgeous and will cellar as well as or better than any domestic Pinot. But I don't make much, so it tends to move pretty fast!
Kurtzman: The 2013s from August West will be on the riper side, similar to the 2009s. Right now, they're a little closed down, since they're so young — but as the year progresses, they'll open up to reveal the incredible growing season that was 2013.
Kosta: The 2013 wines are complex and luscious. Our Pinot Noirs are fruit-driven, with depth and structure that remind me of the intensity of 2005 and 2007. Hold them for a year, if possible.
Main photo: Pinot Posse members Ed Kurtzman, left, and David O'Reilly during an event at Table 6 in Denver. Credit: Copyright 2015 Ryan Olsen
Take it from this staunchly indoorsy Coloradan: You don't have to ski here to drink as though you do. The following cocktails, all featuring local products, come straight from the bars of some of this state's most beloved wintertime destinations. Just whip them up, serve them before a crackling fireplace and — voilà — your living room may as well be a resort lodge overlooking the snow-capped Rockies.
The Bachelor
Courtesy of Bachelors Lounge, The Ritz-Carlton Bachelor Gulch, Beaver Creek, Colo.
The bartenders use bourbon made exclusively for the Ritz by Breckenridge Distillery, but any label will do.
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Prep time: 3 minutes
Total time: 3 minutes
Yield: 1 cocktail
2 ounces bourbon
1 ounce blood-orange liqueur (for example, the Solerno brand)
3 to 4 dashes aromatic bitters
1 sprig rosemary for garnish
2 blackberries for garnish
Add the first three ingredients to a cocktail shaker. Stir and strain into a rocks glass over ice. Garnish with rosemary and blackberries.
Feisty Winter Warmer
Courtesy of The BARLey, Steamboat Springs, Colo.
This one's for the serious home bartender, as it requires a 3-liter mini-barrel for small-batch aging. You can purchase one online, but be sure to cure it according to the manufacturer's instructions first. Feisty Rye, made in Fort Collins, may be hard to come by outside of Colorado, so feel free to experiment with brands you like. You can purchase the DRAM bitters used in this recipe on the Silver Plume company's website.
Prep time: 3 to 4 minutes, plus 3 to 4 weeks for aging
Total time: Less than 5 minutes, once aging is complete
Yield: About 26 servings
2 bottles rye
1 bottle spiced-apple liqueur
2 ounces honey chamomile bitters
1 ounce sage bitters
Cinnamon sticks for garnish
1. Add all the liquid ingredients to your aging barrel and let sit for at least three weeks, sampling the rye mixture daily thereafter to taste. (Kara Kahn, assistant manager at The BARLey, finds that "it's like dessert" after about four weeks.)
2. Once it has mellowed to your liking, store in a Mason jar. When ready to use, add a large ice cube to a toddy glass, measure in 3 ounces of the cocktail and garnish with a cinnamon stick.
Snow on the Fruits of Fall
Courtesy of Frost at The Sebastian, Vail, Colo. The CapRock Organic Pear Eau-de-Vie used here comes from Peak Spirits in Hotchkiss, Colo., which has some out-of-state distribution. If you can't find it, though, many substitutes exist.
Prep time: 5 to 6 minutes
Total time: 5 to 6 minutes
Yield: 1 cocktail
4 1/2 ounces apple cider
1 1/4 ounces whipped cream-flavored vodka
1 1/4 ounces spiced rum
1/3 ounce pear eau-de-vie
1/3 ounce butterscotch schnapps
Pinch of ground cinnamon
Whipped cream for garnish
1 thin slice of pear for garnish
1 cinnamon stick for garnish
1. Combine the cider, vodka, rum, eau-de-vie, schnapps and cinnamon in a small saucepan; set it over low heat until warm.
2. Use a small dab of whipped cream to adhere the pear slice to the cinnamon stick. Pour the cider mixture into an Irish coffee glass and carefully place the stick inside the drink so the cream does not touch the liquid (the garnish is more for visual and aromatic effect than flavor). Serve.
Ullr's Nightcap
Courtesy of Modis, Breckenridge, Colo., which showcases Spring 44 vodka.
Prep time: 3 minutes
Total time: 3 minutes
Yield: 1 cocktail
1 ounce vodka
1/2 ounce Branca Menta
1/2 ounce coffee liqueur
2 dashes chocolate bitters (for example, Fee Brothers, Scrappy's or The Bitter Truth)
Heavy cream
Candy cane for garnish
Combine the first four ingredients in a mixing tin over ice and shake. Pour over ice into a double rocks glass, add a splash of cream and serve with a candy cane for stirring.
Courtesy of St. Regis, Aspen, Colo. The resort has featured wine from Paonia's Azura Cellars, but your favorite Cabernet will work just as well.
Prep time: 5 minutes
Total time: 45 to 50 minutes
Yield: 8 servings
1 cup orange juice
1 1/2 cups sugar
2 cinnamon sticks
8 whole allspice berries
1 star anise pod
2 oranges
10 cloves, whole
8 juniper berries
1 lemon
1 1/2 bottles Cabernet Sauvignon
Orange twists for garnish
1. Combine orange juice, sugar, cinnamon sticks, allspice and star anise in a pot with 2 cups water over high heat. Bring the mixture to a boil, then lower to a mild simmer.
2. Cut the oranges in half and squeeze juice into simmering liquid. Stud the squeezed halves with the cloves and gently place into the pot. Add juniper berries. Cut the lemon in half and squeeze the juice into the simmering liquid, then place the halves in the pot.
3. Reduce mixture to half of its original volume. Add the wine and heat until just below simmering. Ladle into glass mugs and garnish each with an orange twist.
Main photo: The Bachelor from Bachelors Lounge in Beaver Creek, Colo. Credit: Courtesy of Bachelors Lounge
For the past couple of years now, sherry has begun making a slow but steady comeback among wine drinkers in the know — as well as it should. Its long-suffering reputation as the cheap, cloying tipple of grandmothers notwithstanding, good sherry, made in a range of styles from dry to sweet, couldn't be more elegantly versatile, especially when paired with food.
Just ask McLain Hedges and Mary-Allison Wright. As owners of The Proper Pour and RiNo Yacht Club, a high-end liquor shop and adjacent craft bar at celebrated Denver artisan marketplace The Source, they sing the praises of the Spanish fortified wine to anyone who will listen. And they practice what they preach; in fact, their own plans for showcasing it at Thanksgiving dinner are ones you'd do well to follow step by step if you're looking to shake up the old Champagne-and-a-bottle-of-red routine.
Fino with appetizers
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"When your family's arriving and you've got your snacks set out," dry, chilled Fino Sherry — including the Manzanilla of Sanlucár de Barrameda — is the way to go, marked as it is by "bright acidity but also briny minerality and almond notes," Hedges said.
In his view, Fino proper is a natural with salumi, salted nuts, crudités, even pickled vegetables, while Manzanilla "has a little more relationship with the sea" — think shrimp cocktail, oysters, smoked salmon and the like.
Two pet picks: Bodegas Grant Fino La Garrocha from the "really cool microclimate" of Spain's El Puerto de Santa Maria Fino and La Cigarrera Manzanilla, which is "a little smokier, with a brinier effect, made so close to the ocean. And it's fantastically approachable — about 16 bucks on the shelf."
Amontillado and Oloroso with the main meal
"Look at Amontillado as the next step. You're going from the flor stage, which protects oxidization, to the point where that starts to die. So you still get amazing notes of salted almond, but you're also starting to see walnuts and hazelnuts — richer, earthier development," Hedges explained. "It's perfect with chestnuts, perfect with game birds — anything that flies, really: quail, duck, turkey. And mushroom soup is a super-classic pairing."
He calls El Maestro Sierra's 12-year bottling "phenomenal — it's got depth, complexity and richness, but it's also really elegant."
Whereas Amontillado complements "anything that flies," Oloroso is your best bet for "anything that's walking on land." Fully oxidized, "it's gorgeous and luscious and almost comes off as sweet, but it's bone dry, with enough acidity to cut through the fat of the meat and those rich sauces," Hedges noted.
So if you're eschewing the turkey in favor of "pork loin or rib roast or lamb or whatever it may be," consider something like Hedges' pet pick, Gutiérrez Colosia Sangre y Trabijadero.
Pedro Ximénez and Moscatel with sweets
"Moving into dessert, your choices are pretty obvious," Hedges said. "First, you've got your PX (Pedro Ximénez). Sticky-sweet, with notes of dates and figs, it is dessert in a glass — though you could have sticky toffee pudding." But for his money, Moscatel is a fascinating alternative. "The grapes are sun-dried for a few weeks, so it's condensed and extracted, yet still vibrant, with honeyed and floral notes, as well as plums, pepper and a bit of citrus."
He recommends serving it alongside poached fruit with a soft and creamy or blue cheese, again naming La Cigarrera as a producer of note.
To throw you one final curveball: How about kicking off your holiday celebration with a sherry cocktail or two? Both Hedges and Alexandra Flower — a bartender at Acorn, a nationally acclaimed contemporary restaurant in The Source — have whipped up just the things.
Acorn's Smoked Sherry
Prep time: 5 minutes
Total time: 5 minutes
Yield: 1 cocktail
3/4 ounce Laphroaig 10-year Scotch
1 ounce Lustau Pedro Ximénez San Emilio Solera Reserva
3/4 ounce grapefruit juice
1/4 ounce lime juice
2 dashes Angostura bitters
1 disk cut from the peel of a grapefruit for garnish
1. Place all ingredients but the grapefruit peel in a cocktail shaker and shake hard and long. Strain into a coupe glass. Hold the grapefruit disk skin side down over the glass and squeeze it to express the oils.
RiNo Yacht Club's Fino & Dandy
Prep time: 4 minutes
Total time: 4 minutes
Yield: 1 cocktail
1 ounce Barsol Pisco
1/2 ounce Calvados
1/2 ounce Fino sherry
1/2 ounce lemon juice
1/4 ounce simple syrup
2 tablespoons spiced pear butter (available at gourmet shops, or make your own)
1 lemon wheel for garnish
1. Add all ingredients but the lemon wheel to a shaker tin; add ice. Shake and strain into an ice-filled double old-fashioned glass. Garnish with the lemon wheel.
Main photo: Sherry will complement every course at your Thanksgiving meal. Credit: Courtesy of RiNo Yacht Club/The Proper Pour
Jennifer Jasinski is a James Beard Award-winning chef with four acclaimed Denver restaurants, a cookbook and an impressive stint on "Top Chef Masters" to her name. Now she's about to tackle a whole new challenge: cooking Thanksgiving dinner for American expats in Paris.
Jasinski knows a thing or two about the cravings that come with homesickness. The Santa Barbara, Calif., native admits to missing Mexican food mightily while training in France as a young Wolfgang Puck protégée. So the opportunity to treat the guests at Auberge Flora to a good, old-fashioned turkey feast isn't one she's taking lightly.
More from Zester Daily
» Fry bread and corn soup for Thanksgiving
» Forget sweet potatoes, bring on kabocha soup
Granted, "old-fashioned" doesn't, in this case, mean the same thing as "down home." Take this wonderfully rich and elegant chestnut soup, which I first sampled a few years ago at one of Jasinski's Denver restaurants, Rioja, where it came in a tiny pumpkin "lidded" with a foie gras-topped slice of brioche. Though you can serve it in plain old bowls, squash vessels do make for an impressive flourish. In fact, having just reprised the appetizer at a charity event in New York City, Jasinski acknowledges, "I'd forgotten what a really cool dish it is, but people were freaking out about it!"
Though you'll find the recipe in her cookbook, "The Perfect Bite," she graciously allowed us to reprint it here as well. And she adds that gourds serve as equally lovely containers for soufflés, wild-rice salads and the like.
Savory Chestnut Soup
Prep Time: 10 to 25 minutes
Cooking Time: 1 1/2 to 2 hours
Total Time: 1 3/4 to 2 1/2 hours, including time for the optional step of making the bowls.
Yield: 8 servings
For the soup:
1/4 cup duck fat (preferred) or pure olive oil
1 1/4 cups sliced onion
1/4 cup garlic cloves, peeled
1 1/2 cups domestic mushrooms, sliced
10 sage leaves, destemmed
1 teaspoon whole black peppercorns
1 bay leaf
12 to 14 ounces whole, fresh, peeled chestnuts
1 1/4 cups white wine
6 cups chicken stock
1-inch cinnamon stick
1/2 teaspoon finely ground cardamom
1 1/4 cups heavy cream
1 to 2 teaspoons kosher salt, or to taste
1/2 teaspoon black pepper, or to taste
1 tablespoon sugar
For the assembly (optional):
8 mini pumpkins, 8 small butternut squash or 1 large pumpkin
Duck fat (preferred) or extra virgin olive oil, as needed
Kosher salt and black pepper, to taste
For the soup:
1. In a large stockpot over medium heat, melt the duck fat, then add the onions and garlic and sauté until translucent — do not let them color. Add the mushrooms, sage, peppercorns and bay leaf. Sauté a few minutes, until the mushrooms have softened.
2. Add the chestnuts to the pot and deglaze with the wine. Let cook until the wine has reduced completely, then add the chicken stock. Raising the heat as needed, bring the soup to a boil and then reduce heat and simmer on very low for about 45 minutes.
3. Add the cinnamon stick, cardamom and cream and continue to simmer for 10 minutes. The chestnuts should be very soft by now. Removing the cinnamon stick, take the soup off of the heat, transfer it to a blender and blend until smooth. Season with the salt, pepper and sugar, then strain the soup through a china cap. If you will not be serving it immediately, store it in the refrigerator.
For the assembly:
1. Preheat oven to 350 F.
2. If you are using mini pumpkins as serving bowls, cut off the tops, clean out the insides and then brush the hollows with the duck fat and season them with salt and pepper. Place the pumpkins on a sheet pan and roast in the oven about 20 minutes — until the flesh can be removed easily with a fork or spoon, but not so long that the skin becomes weak and fragile, as this will make it difficult to use as a serving bowl.
If you are using butternut squash as serving bowls, remove the tops so you are left only with the bulbous bases, then follow the instructions for the mini pumpkins.
If you are using a large pumpkin as a soup terrine, cut the top off and clean out the insides. Boil some water and ladle it into the pumpkin to warm the flesh, then pour it out.
3. Ladle the soup into the squash(es), or regular bowls if you prefer, and serve.
Main photo: Chestnut soup. Credit: Courtesy of Jennifer Jasinski


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Uniform Solutions for You offers a unique idea-based solution to buying uniforms online. The company has updated its information page on the waiter uniform online category.
Location independent workers (i.e. "digital nomads") can live and work in 12 countries over 12 months, with all travel arrangements, as well as living and work accommodations, included. This...
Other Links:
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subset: commoncrawl


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Songs Of Innocence - Illustrated By Honor C. Appletonby William Blake, Honor C. Appleton
View All Available Formats & Editions
Pook Press celebrates the great Golden Age of Illustration in children's literature. Many of the earliest children's books, particularly those dating back to the 1850s and before, are now extremely scarce and increasingly expensive. Pook Press are working to republish these classic works in affordable, high quality, colour editions, using the original text and artwork so these works can delight another generation of children. Honor Charlotte Appleton (1879-1951) was known for her delicate watercolour illustrations influenced by Kate Greenaway and Arthur Rackham. She illustrated more than 100 children's books during her career including Alice in Wonderland, Aladdin, and Black Beauty.
Product Details
- 9781447449447
- Publisher:
- Read Books Design
- Publication date:
- 04/23/2012
- Pages:
- 94
- Product dimensions:
- 7.00(w) x 10.00(h) x 0.25(d)
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37 People Who Are Worse At Cooking Than You
It takes real effort to screw food up this badly. Respect.
1. The person who thought this was a fun idea.
2. The person who tried to make these s'more cookies.
3. This guy.
4. The person who tried to cook pasta in a coffee maker.
5. The person who tried to bake this pizza.
Gotta use protection.
6. And this pizza.
7. And this pizza.
8. And this pizza.
9. The person who forgot that cheese melts.
10. The person who baked these cupcakes.
11. And these cupcakes.
12. And these cupcakes.
13. And these cupcakes.
14. And these cupcakes.
15. The person who made whatever this is.
16. And…this.
17. The person who ruined this waffle iron.
18. The person who decided to use this as a colander.
Well…this is arguably a win. Kind of brilliant.
19. The person who forgot how to use an electric kettle.
20. The person who set this pasta on fire.
21. The person who frosted this cake.
22. And this cake.
23. And this cake.
24. The person who tried to make these kiwi pops.
25. The person who tried to cook eggs this way.
26. The person who came up with this great spaghetti squash "hack."
27. The person who accomplished this.
Truly impressive.
28. The person who cooked this rice.
29. The person who sliced this cabbage.
30. The person who made whatever these are.
31. The person who tried to bake this this "single-serving cookie."
32. The person who cooked this pasta.
33. The person who fried these eggs.
34. The person who baked these cookies.
You know what melts? Plastic!
35. The person who did this to an innocent doughnut.
36. The person who thought Goldfish ham salad sounded like a great idea.
That does NOT look like a healthy aquatic environment for those fishies.
37. And last, but NOT least: Steven Reed.
Host of Weber Cooks, the saddest cooking show in the universe. Have you seen the video? You should see the video.
- Hillary Clinton's campaign was reportedly hacked as part of what appears to be a broad cyber attack on Democrats.
- Two brothers suspected of planning terror attacks have been arrested in Belgium, prosecutors say.
- Four people in Florida are likely the first to contract the Zika virus from mosquitos in the US, the state's governor says.


text:
Devs prefer PS4 over Xbox One, but PC is still king, survey says
A survey conducted by GDC Europe event organisers polled some 400, Europe-based game developers about their platform preference.
The survey found that 33% of developers are expecting to build PS4 games while 23% are looking to develop on Xbox One. 58% of surveyed developers said that they're working on PC titles, and 60% said that they're releasing their next project on PC.
It's not all rosy in the land of high-end gaming however, as 65% of those developers are currently working on mobile titles.
You can head through the link if you want to take a look at the full thing.
Thanks, CVG.
Sometimes we include links to online retail stores. If you click on one and make a purchase we may receive a small commission. For more information, go here.


text:
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For Personal Loan up to $5,000 - Apply Here
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Can you imagine a directive coming down for our soldiers not to criticize the enemy and their atrocities during WWII?
Talk about undercutting morale! Â If they're not evil and don't have evil intentions, why are we even at war against them?? Â Taliban terrorists and their actions are NOT morally equivalent to our troops, and our soldiers shouldn't be forced to pretend otherwise!
Here is a strong indicator that the Obama Administration's crusade to appease Islam has gone too far; a  new U.S. military handbook for troops deployed to the Middle East orders soldiers not to make derogatory comments about the Taliban or criticize pedophilia, among other outrageous things.
It gets better; the new manual, which is around 75 pages, suggests that Western ignorance of Afghan culture— not Taliban infiltration—is responsible for the increase in deadly attacks by Afghan soldiers against the coalition forces.
The soon-to-be-released Army handbook is still being drafted, but a mainstream newspaper got a sneak preview and published an article that should infuriate the American taxpayers funding the never-ending war on terror. The manual is being created because someone with authority bought the theory that cultural insensitivity is driving insider attacks on U.S. troops in Afghanistan.
More than three dozen insider attacks have killed 63 members of the U.S.-led coalition this year, according to the article, and some blame "American cultural ignorance.†The bottom line is that troops may experience social-cultural shock and/or discomfort when interacting with Afghan security forces, the new military handbook says. "Better situational awareness/understanding of Afghan culture will help better prepare [troops] to more effectively partner and to avoid cultural conflict that can lead toward green-on-blue violence.â€
The draft leaked to the newspaper offers a list of "taboo conversation topics†that soldiers should avoid, including "making derogatory comments about the Taliban,†"advocating women's rights,†"any criticism of pedophilia,†"directing any criticism towards Afghans,†"mentioning homosexuality and homosexual conduct†or "anything related to Islam.â€
Obama Admin Secretly Releasing Taliban Prisoners Back Onto Battlefield
Vice President Biden: Taliban Is Not The Enemy
Incomprehensibly Stupid Army Regulation Killing Americans in Afghanistan
Taliban Suicide Bomber Strikes US Base Hours After Visit By Defense Secretary Panetta
Taliban Cut Off Nursing Woman's Breasts, Tell Other Women to Eat the Pieces
Taliban Assassinate Afghan Schoolgirl For Working On Polio Eradication Campaign
Pakistani Taliban Attempt To Assassinate Second Schoolgirl
Taliban Training 5-Year-Olds to be Terrorists in Secret Camp
Afghanistan: Taliban Using Children as Young as Nine as Suicide Bombers
Obama to Hand Afghanistan Over to Taliban While Declaring 'Success'
Obama's Servile Negotiations With the Taliban are an Untold Outrage
U.S. money is Taliban's second highest source of revenue, after opium


text:
Previous partners say that they become more orgasmic (all the time) just from hanging around me.
Teaching and coaching powerful sexy (mostly) women in shifting to change the world.
tech gadgets ;)
Intelligence and deep conversation
That said, my 'alone time' is quite valuable to me and I do take a lot of it.
And are at least slightly interested in surrender.
"There are two sources of conflict in relating. One is 'how much do you love me?' The other is 'who is in charge'?"


text:
Today we've got twenty amazing WordPress themes united by a similar topic: media. So, if you are browsing the Internet for those, this list will surely help you in your quest. Also, these themes might be of some help for those who don't have the inspiration, time or skills to create a custom design. We've chosen only…Continue Reading "20 Stunning WordPress Themes for Media Websites"
If you are looking for a bundle that will make your font library fontastic, then this deal is definitely something for you! The guys at The Hungry Jpeg have an amazing deal. In this amazing deal you get 45 absolutely fontastic fonts, this bundle is guaranteed to get your creative juices flowing! Hurry though as the bundle will only…Continue Reading "Get 45 amazing fonts in this fontastic bundle for only $29"
The guys at Dealjumbo are at it again, with their amazing Mega Bundle with awesome custom fonts. If you're looking looking to beef up your custom font library then this Jumbo Deal is just for you! With this offer, you will save almost 95% on the original price, so there really shouldn't be anything to consider, you…Continue Reading "Save 95% on these 30 awesome custom fonts"
If you are looking for a bundle that will spruce up your spring designs then this is definitely something for you. The guys at The Hungry Jpeg have an amazing deal. This HUGE floral bundle includes over 1900 illustrations and graphics, as well as 17 beautiful fonts. With this offer, you will save 96% on the…Continue Reading "Floral super bundle to spruce up your spring – save 96%"
The guys at Dealjumbo are at it again, with their amazing 5in1 Mega Bundle volume 15. Can you believe its been 15 amazing bundles already, we can't wait to see what the future brings. If you're looking for some illustrations, Shapes, Graphics & Textures in one mega bundle, then this Jumbo Deal is just for you! With this offer, you…Continue Reading "5in1 mega bundle v.15: vintage graphics"
The guys at HeyBundle have an amazing deal to share with us today. If you're looking for unique and amazing resources from different authors of Creative Market, then look no further. This awesome bundle includes With 26 items and bundles from 8 different authors you'll get Fonts, Logos, Labels and Badges, Graphics, Add-Ons and more! With this…Continue Reading "HeyBundle, a treasure trove for creatives, for 94% off"


text:
Skechers Persistent Slip-On Sneaker
Compare at $65.00
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Item # 313906
Experience all day comfort and ease with the Skechers Persistent men's slip-on shoe! The memory foam removable insole on this shoe will have your feet feeling like they're walking on clouds!
- Breathable mesh and synthetic upper
- Slip-on design
- Side stretch panels for secure fit
- Padded collar for comfort
- Removable memory foam cushioned insole
- Lightweight molded FlexSole midsole provides lightweight cushioning and shock absorption
- Synthetic outsole
- Flex grooves provide increased flexibility
- Imported
- View more Skechers
By: ShopperShel
Location: Phoenix, AZ
July 12, 2016
Exactly what I was looking for...
"I purchased these shoes for my Mom who is blind and disabled. It's important for her to have something that is easy to put on, comfortable, slip-resistant, and matches many ensembles. This was the perfect pair of shoes!"
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text:
1 item from 2012
13 February 2012 10:00 AM, PST | Shadowlocked | See recent Shadowlocked news »
If there is one famous literary character that has made such an impact on film and television, it has to be Sir Arthur Conan-Doyle's immortal detective. Holmes has generated such a fascination for filmmakers; he is probably more popular, and certainly more prolific, than Dracula and James Bond put together. The number of Holmes films produced since the pioneering days of the silent era is so extensive it's unlikely the Great Detective will ever be absent from our screens for very long.
Within the last couple of years, Holmes has become fashionable again thanks to Robert Downey Jr's cinematic reinvention of the role in two successful Guy Ritchie movies and the excellent TV series Sherlock, which effectively transports Holmes (brilliantly played by Benedict Cumberbatch) to modern day London. Oddly enough the concept is not a new one considering Holmes, like Dracula, is a man of his time »
1 item from 2012
IMDb.com, Inc. takes no responsibility for the content or accuracy of the above news articles, Tweets, or blog posts. This content is published for the entertainment of our users only. The news articles, Tweets, and blog posts do not represent IMDb's opinions nor can we guarantee that the reporting therein is completely factual. Please visit the source responsible for the item in question to report any concerns you may have regarding content or accuracy.See our NewsDesk partners


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Bilal Ag Acherif
From Wikipedia, the free encyclopedia
(Redirected from Bilal ag Acherif)
|Bilal Ag Acherif
|President of the Transitional Council of the State of Azawad
|In office
6 April 2012 – 12 July 2012
|Vice President
|Mahamadou Djeri Maïga
|Preceded by
|Position established
|Succeeded by
|Position abolished
|General Secretary of the MNLA
|Assumed office
October 2011
|Preceded by
|Position established
|Personal details
Bilal Ag Acherif (born 1977,[1] last name alternatively spelled Cherif)[2] is the Secretary-General of the National Movement for the Liberation of Azawad (MNLA) and president of a briefly independent Azawad.[3][4]
On 26 June 2012, he was wounded in clashes between MNLA fighters and the Islamist Movement for Oneness and Jihad in West Africa during the northern Mali conflict. According to an MNLA spokesperson, he was taken to Burkina Faso for medical care.[5][6]
- ^ Mali's shaky rebel alliance and a looming war Archived copy at the Stanford Web Archive (July 6, 2012).
- ^ Elhadj Ould Brahim (19 February 2012), Conflict in Northern Mali: Internal Facts and Regional Impacts (PDF), Al Jazeera Centre for Studies, p. 3
- ^ "Tuareg rebels announce 'end of military operations' in Mali". Channel NewsAsia. Agence France-Presse. 5 April 2012. Retrieved 6 April 2012.
- ^ "Explainer: Tuareg-led rebellion in north Mali - Africa". Al Jazeera English. 3 April 2012. Retrieved 6 April 2012.
- ^ "Mali: Islamists seize Gao from Tuareg rebels". BBC News. June 27, 2012. Archived from the original on November 23, 2012. Retrieved June 27, 2012.
- ^ Serge Daniel (June 27, 2012). "Islamists seize north Mali town, at least 21 dead in clashes". Google News. Agence France-Presse. Retrieved June 27, 2012.
|This article about a Malian politician is a stub. You can help Wikipedia by expanding it.


text:
|Home | About | Journals | Submit | Contact Us | Français
Autism spectrum disorders (ASD) represent a group of neurodevelopmental disorders characterized by a core set of social-communicative and behavioral impairments. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting primarily via the GABA receptors (GABR). Multiple lines of evidence, including altered GABA and GABA receptor expression in autistic patients, indicate that the GABAergic system may be involved in the etiology of autism.
As copy number variations (CNVs), particularly rare and de novo CNVs, have now been implicated in ASD risk, we examined the GABA receptors and genes in related pathways for structural variation that may be associated with autism. We further extended our candidate gene set to include 19 genes and regions that had either been directly implicated in the autism literature or were directly related (via function or ancestry) to these primary candidates. For the high resolution CNV screen we employed custom-designed 244 k comparative genomic hybridization (CGH) arrays. Collectively, our probes spanned a total of 11 Mb of GABA-related and additional candidate regions with a density of approximately one probe every 200 nucleotides, allowing a theoretical resolution for detection of CNVs of approximately 1 kb or greater on average. One hundred and sixty-eight autism cases and 149 control individuals were screened for structural variants. Prioritized CNV events were confirmed using quantitative PCR, and confirmed loci were evaluated on an additional set of 170 cases and 170 control individuals that were not included in the original discovery set. Loci that remained interesting were subsequently screened via quantitative PCR on an additional set of 755 cases and 1,809 unaffected family members.
Results include rare deletions in autistic individuals at JAKMIP1, NRXN1, Neuroligin4Y, OXTR, and ABAT. Common insertion/deletion polymorphisms were detected at several loci, including GABBR2 and NRXN3. Overall, statistically significant enrichment in affected vs. unaffected individuals was observed for NRXN1 deletions.
These results provide additional support for the role of rare structural variation in ASD.
Autism spectrum disorders (ASD) represent a group of neurodevelopmental disorders characterized by a core set of social-communicative and behavioral impairments. There is abundant evidence for a strong genetic contribution to ASD, with concordance rates among MZ twins ranging from 60% to 90% [1]. It is now evident that the underlying genetic architecture of autism is highly complex, with numerous genomic loci contributing to overall risk. Efforts to identify specific risk loci have met with some success, and variants in several genes and genomic regions having been implicated to date. These loci include SHANK3, CNTNAP2, NLGN4X, pCDH10, 16p11.2, NRXNa1, SYNGAP, SEMA5A, and AVPR1A, among others [2-6]. Evidence for the role of common variation in ASD risk was reported within the CDH9/CDH10 region of chr5 on risk [7]. Despite these important advances, phenotypic and genetic heterogeneity have made it challenging to unravel the underlying causes of ASD, and a substantial component of this disorder's genetic etiology remains to be discovered.
A number of studies have implicated copy number variations (CNVs) in ASD [8-15]. In addition to specific structural mutations, global enrichment in rare and/or de novo CNVs has been reported within affected vs. unaffected individuals (for example [5,7-15]). To date, studies examining copy number variation in ASD have relied heavily on array technologies that were originally designed for the purpose of conducting SNP-based genome-wide association studies (GWAS). This is primarily due to the fact these data already exist (that is they were obtained for genotyping purposes). These GWAS arrays typically place a limit on the resolution at which CNVs can be reliably detected at 10 to 50 kb or larger (contingent upon the array and analysis parameters), although smaller events can be detected in certain circumstances. The use of custom designed comparative genomic hybridization (CGH) arrays, combined with a targeted, candidate-gene oriented strategy, can extend the ability to reliably detect variation below 10 kb. As it is evident that structural DNA variation with potential relevance to pathological phenotypes can and does exist at sub-10 kb scales (for example [16]), we sought to examine a subset of autism candidate regions at high resolution. The GABA receptor family of genes and genes within biological pathways associated with GABA are attractive candidates for autism and ASD. GABA is the primary inhibitory neurotransmitter in the human brain, and it is known to assume both excitatory and inhibitory roles during early development stages [17,18]. Previously our group has reported evidence of both linkage and association with ASD at the GABA receptor genes in the 15q11-13 region [19,20]. In addition, differential expression of GABA receptors has been observed in ASD cases [21-23], and rare coding variants in GABRB3 have been found that segregate with ASD [24]. In light of the above evidence, we chose to examine 24 GABA receptors and 19 additional GABA-related genes for evidence of sub-5 kb structural variation that might contribute to the autism/ASD risk. We further extended our candidate gene set to include an additional 19 genes and regions that had been previously implicated in autism (Table (Table1).1). These loci were either recognized ASD candidates or directly related to ASD candidates through biological function or sequence homology. Targeted genes include members of the Neurexin and Neuroligin gene families, the CDH9/CHD10 region on chromosome 5p, the 16p11.2 regions, CNTNAP2, OXT, OXTR, MECP2, and SHANK3. While most of these candidate genes and regions have been previously examined for structural variation in the context of GWAS array analysis, these loci have not been systematically targeted with custom designed high resolution aCGH in a population of ASD and control individuals to survey fine-scale structural variation.
This research was carried out in accordance with the World Medical Association's Declaration of Helsinki. Participants in the study were ascertained under applicable IRB protocols of the John P. Hussman Institute for Human Genetics (HIHG), University of Miami, Miller School of Medicine, Vanderbilt University Medical Center, and University of California, San Francisco (UCSF) Medical School. Following a description of the study, informed consent was obtained for each individual or, where appropriate, their guardian.
Individuals were recruited as part of our ongoing family-based recruitment via support groups, advertisements, and clinical and educational settings. Written informed consent was obtained from parents for all minor children and those who were unable to give consent. In addition, we obtained assent from all participants of the appropriate developmental and chronological age. Core inclusion criteria were as follows: (1) between 3 and 21 years of age, (2) a clinical diagnosis of ASD, (3) an expert clinical determination of ASD diagnosis using DSM-IV criteria supported by the Autism Diagnostic Interview (ADI-R) [25,26], and (4) an IQ equivalent > 35 or developmental level >18 months as determined by the Vineland Adaptive Behavior Scale (VABS) [27]. A best estimate diagnostic determination was based on review by a three-person panel, including experienced clinical psychologists and a pediatric medical geneticist. This determination included ADI-R results. In those instances where an ADI-R was not available (n = 86; six were parents, 67 were lost to follow-up, refused to complete the interview, or withdrew, and 13 are in progress), a best-estimate diagnosis was assigned by this panel using all available clinical information including clinician summaries, caregiver report, and medical records. Following review of case materials and discussion, panel members reached a consensus. We excluded participants with severe sensory problems (for example visual impairment or hearing loss), significant motor impairments (for example failure to sit by 12 months or walk by 24 months), or identified metabolic, genetic, or progressive neurological disorders. Family history and pedigree information was collected in a standard semi-structured interview from a knowledgeable informant, frequently the mother. Additional clinical data were also collected by reviewing available medical and psychiatric records of affected individuals.
The majority of control participants were recruited by the HIHG at the University of Miami and included 565 children between the ages of 3 and 21 years. Participants with developmental, behavioral, or neurological conditions were excluded, as well as those with first-degree relatives with such disorders. We obtained consent from all participants or, in the case of minors, their parents. Participants meeting these criteria provided a saliva sample. A knowledgeable informant, usually the mother, completed the Social Communication Questionnaire to screen for potential ASDs [28]. The above controls were supplemented with additional set of 65 individuals obtained from the UCSF School of Medicine. Blood samples were obtained from self-reported Non-white Hispanics, ranging from 15 to 37 years of age and reporting no history of chronic disease.
For the CNV discovery stage of the project, DNA from whole blood and/or saliva (Oragene kit) was collected from 173 Caucasian non-Hispanic and Caucasian Hispanic autism cases and 185 racially matched control individuals (≤37 years of age) with no known signs of ASD (matched backgrounds were confirmed with Eigenstrat clustering [29]). As described below, data from some samples were excluded from further analysis based on laboratory QC metrics, resulting in a final sample set of 168 cases and 149 controls. CNV loci that were molecularly confirmed and trended towards significance were subsequently tested within a larger sample set of 2,564 individuals, consisting of 755 affected (728 Caucasian/non-Hispanic, 17 Asian, 7 Hispanics, 3 African-Americans) and 1,809 unaffected individuals who were family members of the 755 cases.
DNA from both blood and saliva sources was extracted using the Autopure (Gentra) system according to the manufacturer's standard protocols. For a subset of six control individuals for which DNA extracted from both blood and saliva sources was available, we ran replicate CGH arrays on both blood and saliva DNA in order to look for any systematic differences evident between the two sources. Although batch effects attributable to numerous experimental factors have been previously noted for CNV studies, quality metrics and total CNV events called indicated no significant DNA-source effects within this CGH-based study. Specifically, Nexus 4.1 (Biodiscovery) noise metrics and CNV copy call numbers showed no systematic differences between blood and saliva groups (Additional file 1: Table S1).
In addition to the set of GABA-related gene targets forming the primary focus of this study, a set of 19 additional ASD candidates were selected based on several criteria, including a recently completed GWAS [7]. The list included both a set of genes previously implicated in the ASD literature, as well as genes known to interact biologically and/or exhibit significant homology to those genes. As not all putative and/or established ASD loci could be accommodated on the array, the final prioritized list was arrived at by consensus among study collaborators.
We designed a custom 244 k Agilent comparative genomic hybridization (CGH) array targeting our regions of interest. Array hybridization probes were chosen using Agilent earray software https://earray.chem.agilent.com/earray/, with a preference for catalogue probes, where available. Probe design was based on coordinates from the hg18 build of the human reference sequence. Collectively, our probes spanned a total of approximately 11 Mb with an average probe density of one unique probe every 200 nucleotides. Given that most standard CNV calling approaches require four to five consecutive significant probes to identify an aberrant region, our coverage theoretically allows for the detection (on average) of CNVs 800 bp or larger. The GABA family of receptors poses particular challenges for hybridization-based copy number assays. Due to the level of sequence homology resulting from the ancestral relationships among GABA genes, increased background probe noise within conserved regions was anticipated. In an effort to mitigate the influence of assay noise on the CNV calling algorithm, we employed triplication of all internal experimental probes as well as running technical array replicates of a subset of 40 individuals to aid in identifying and excluding problematic regions.
Genomic DNA was fragmented, enzymatically labeled, hybridized to arrays, and washed according to the manufacturer's (Agilent) standard protocols. Briefly, 0.75 μg of extracted DNA from a single hybridization reference individual (sample NA10851, available from the US National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository) was labeled with Cy3 dye (following recommendation of [30]). An equal amount of extracted DNA from experimental samples was enzymatically labeled with Cy5 dye. Labeled samples for each experimental individual was combined with labeled reference DNA, hybridized to the CGH array for 40 h at 65°C, and washed to remove non-hybridized fragments prior to imaging.
All arrays from this study were imaged using a single Molecular Devices GenePix 4000B microarray scanner, processed with Agilent Feature Extraction 9.5 and analyzed with Nexus (Biosdiscovery) software version 4.1. Preliminary analysis was conducted using Rank Segmentation (an extension of the circular binary segmentation algorithm [31] as implemented in Nexus 4.1. Final results presented in this manuscript are based on the Nexus Rank segmentation algorithm with the gain/loss LogR threshold set to 0.25, maximum inter-probe distance at 25 kb, and the (default) minimum of five probes required per called CNV event.
As indicated above, each probe was replicated three times (each at a randomly distributed location) on the CGH array and fluorescent signal intensities were averaged prior to subsequent analysis. For detection of common sample handling errors, markers on the X chromosome were examined to ensure observed sample sex was consistent with the original sample manifest. In addition, sex chromosome markers, as a proxy for true-positive controls, a set of five highly polymorphic CNV locations reported in the Database of Genomic Variation http://projects.tcag.ca/variation/ were used to evaluate the ability of our array design (and associated processing technique) to detect structural DNA variation at scales of 5 kb and below (Table (Table11 bottom). Because the selected loci harbored reported minor allele frequencies of 15% and above, each of the minor structural alleles was expected to be observed, with high probability, one or more times within our sample of 317 individuals. All five of the targeted CNVs were readily detected within our sample population. Although it was observed as variant in our sample set, one of the five targets chosen from the database, hg18 locus chr16:4710525-4715494, was found upon further examination to harbor extreme high repetitive content making the reliability of hybridization results questionable. We therefore do not recommend its use as a positive control locus in future experiments.
To identify potential outlier samples (with respect to sample quality) total CNV calls per individual were rank sorted. A clear demarcation point was observed at approximately 60 total CNV calls per individual, with a relatively small number of samples exhibiting significantly higher numbers of CNV calls. We suspected that the excess number of calls associated with these arrays was attributable to poor sample quality or processing issues; as evidence of this, the Nexus 4.1 quality metric (a measure of probe-to-probe variation across contiguous probes) among these individuals was elevated compared to that of the non-excluded set. Collectively, our quality criteria resulted in a subset of 41 samples being excluded from all subsequent analysis.
Initial CNV calls output from Nexus 4.1 rank segmentation algorithm were converted to compatible format for loading into PLINK [32]. For the purpose of analysis, PLINK provides the option of handling heterogeneous CNV calls at the same genomic locus by treating each unique CNV start and stop coordinate as separate markers. Any copy number gain (or loss) that overlaps the position of a marker assumes a value (0, 1, 2, 3, or 4-plus copies) for the marker. After initial processing in PLINK to produce a .ped file with CNV markers, logistical regression analysis was conducted via R scripting. Results of logistic regression analysis are provided in Additional file 2: Table S2. Regression results corresponding to -LOG10 (P value) ≥ 3 were evaluated as candidates for additional analysis. For analysis of the larger family-based dataset, the GENMOD procedure (SAS vers8.1) was run on the dataset and a generalized estimating equation (GEE) was employed to account for the relationships between the parents and siblings (unaffected) and cases. GEE allows for specification of a correlation matrix of the between-subject relatedness of participants belonging to the same pedigree [33] and is commonly used in the context of genetic epidemiology studies to produce robust variance estimates that otherwise would likely be too small/conservative in the presence of relatedness between observations [34]. For each of the candidate genes evaluated by GEE at the validation stage of the study, Bonferroni correction was used to correct for the number CNV positions examined within the candidate. Global CNV burden was assessed using the mperm option in PLINK to perform a global permutation test (10,000 permutations) on segment number, total kilobase difference, and average kilobase difference.
Prioritized loci were validated by real-time quantitative PCR using TaqMan chemistry (a full list of target locations are provided in Additional file 3: Table S3). Assays were performed in quadruplicate, as recommended by the manufacturer (Applied Biosystems {Life Technologies, Carlsbad, CA}), reactions were run on the ABI-7900HT real-time PCR system and analyzed using CopyCaller software v.1.0 (Life Technologies, Carlsbad, CA). Regions were chosen for validation based on several criteria, including their potential for biological relevance and logistic regression scores (Additional file 2: Table S2). CNVs spanning known exons were given the highest priority, followed by those including regions of evolutionary conservation, as evidenced by interspecies sequence homology (per UCSC Genome Browser, build 36.1, March 2006, conservation tracks). Some predicted copy number variable regions that exhibited statistically significant scores were nevertheless excluded from further analysis because data from technical replicates indicated poor replicability for calls at these regions. Examination of these loci indicated that they tended to exhibit extreme GC content, repetitive element content, or extensive homology to other areas of the genome likely due to common ancestry and subsequent evolutionary conservation (data not shown). In addition to the above criteria, CNV regions were excluded from follow-up if they exibited 75% or greater overlap with known copy number variable regions reported in DGV http://projects.tcag.ca/variation/; however, exceptions were made in cases where the variants encompassed exons within candidate genes and instances where gained or loss alleles were discordant in case/controls. All loci meeting these criteria subjected to TaqMan CNV assays (Applied Biosystems) for further interrogation in additional cases and controls.
A total of 24 GABA receptors, 19 GABA-associated coding genes, and 19 additional autism candidates were targeted for aCGH assessment (Table (Table1).1). After exclusion of outlier samples, a total of 168 case and 149 control individuals were examined by CGH arrays in the initial discovery set. In all, 6,143 CNV events calls were identified across our tested population, representing 1,223 potentially unique variations. The overall number of CNV events did not exhibit significant differences between ASD affected and unaffected individuals, although both the average and total kilobases impacted did show statistical significance (perm P < 0.001; 10,000 permutations; one-sided), consistent with previous reports indicating pathogenic CNVs tend to be larger in size and over-represented in affected individuals (for example [35]). The full list of CNV calls from the Nexus Rank Segmentation algorithm is provided in Additional file 4: Table S4. The logistic regression analysis of case and control allele frequencies were used, in conjunction with additional biological annotation criteria, to prioritize a subset of regions for confirmation via a second molecular platform, TaqMan copy number quantitative PCR assays (Applied Biosystems). In addition to the ASD candidate loci prioritized for follow-up, five control genomic regions, representing known polymorphic locations with minor allele frequencies >10%, were also targeted with TaqMan assays to test the sensitivity of our laboratory technique. The 29 targeted regions and five control loci targeted for follow-up are detailed in Additional file 3: Table S3, along with confirmation results. A diagram of the work flow for discovery and validation is provided in Additional file 5: Figure S1. The sample set used for molecular confirmation and additional population screening was comprised of the individuals in which the CNVs were initially observed (for confirmation), along with an additional 170 Caucasian cases and 170 unaffected ethnically-matched controls that were not present in the original discovery set (Table (Table2).2). A subset of independently confirmed loci that remained interesting was run on a larger set of 755 case and 1,809 unaffected family members. GEE analysis results for these data, accounting for relationships between individuals, are provided in Table Table33.
Targeted CNV call events were confirmed with quantitative PCR at 13 of 29 loci, 45%. This suggests a minimum false-positive rate (at the locus level) of 55% (addressed below).
Medical records were examined for a subset of individuals harboring rare CNV events at loci of interest (ABAT, NRXN1, JAKMIP1, GABBR2, NLGN2, OXTR). We examined salient phenotypic and medical history characteristics for these individuals to look for commonalities among carriers of CNVs at particular loci. Characteristics examined included developmental abnormalities in speech and motor development, learning disabilities, and evidence of dysmorphologies. Additional co-morbidities (for example irritable bowel syndrome, asthma) and neuropsychiatric features were also noted. No obvious correspondence between aberrant CNV state and specific phenotypic traits was observed. A summary of available phenotypic and medical characteristics for the individuals examined is provided in Additional file 6: Table S5.
In addition to these rare variants, common structural polymorphisms were observed and validated at multiple loci, including NRXN3, CNTNAP2, GABBR2, SLC38A3, and NLGN2. Of these more frequent events, only the GABBR2 locus approached nominal significance (GEE; P = 0.029; OR = 1.16; 95% CI: 0.7-1.93) in our larger validation set, but the result was not significant at α = 0.025 after correction for multiple testing (Table (Table33).
The role of structural variation, particularly rare and de novo CNVs, in autism has been established in a number of previous studies. Here we examined a set of autism candidate loci using high-resolution aCGH analysis. Our principal objective was to detect structural variants at these loci that may have been potentially missed by other screening platforms, such as SNP-based arrays. Among the limitations impacting our study was the presence of a high false-positive rate (in excess of 50%) in our initial aCGH discovery set. The most likely explanation for this elevated false-positive rate is that the majority of genes in our study, including both GABA and non-GABA-related targets (for example MBD1-MBD4), belong to families of homologous genes which can lead to aberrant probe hybridization. As the majority of loci in our study belonged to homology groups, we could not formally test for this effect. In addition, the modest overall sample numbers used in this study limited our ability to accurately estimate population allele frequencies for rarer events. Even with these complicating factors, several promising events were detected and independently confirmed by quantitative PCR. Rare structural variations were detected at ABAT, NLGN4-Y, NRXN1, and JAKMIP1 (Table (Table2).2). Most notable among these results, statistically significant enrichment of deletions (GEE; P = 0.0215; OR = 2.75; 95% CI: 1.161-6.5; α = 0.025) in affected vs. unaffected individuals was observed at NRXN1 within our larger family-based validation set with after correction for multiple testing (Table (Table33).
We observed and confirmed an approximate 4 kb deletion in a female autistic indvidual within the JAKMIP1 gene that removes two exons. JAKMIP1, a member of a family of microtubule interacting proteins, is known to interact with GABBR1 protein [36] and knockdown of its expression via siRNA results in increased GABBR2 expression, suggesting a possible role for JAKMIP1 in GABBR2 regulation. Misregulation of JAKMIP1 has also been observed in lymphoblastoid cell lines from autistic individuals [37]. We note, however, that no significant enrichment for the deletion was observed in our larger validation set. More detailed characterization of these deletions in the affected and unaffected individual is warranted before further inference can be made, as the precise boundaries of the deletion events may be different in case and control individuals.
A large (approximately 70 kb) deletion encompassing two exons of the NRXN1 genes was detected in an autistic male (Figure (Figure1).1). NRXN1 is a neuronal adhesion molecule that has been implicated in a number of neurological disorders, including autism (for example [38-41]. Suggestive SNP association results at the NRXN1 locus were reported in a large collaborative study, but genome-wide significance was not reached [7]. Significant enrichment was previously reported for rare structural events [42]. The removal of this exonic region within an autistic male in our dataset, along with the detection of significant enrichment of NRXN1 deletions in cases vs. unaffected individuals in this study, provides further evidence for NRXN1 as an ASD risk locus. Furthermore, the location of our CNV, along with the placement of previously reported NRXN1 events (Figure (Figure1),1), suggest that disruptions of the alpha form of NRXN1. The beta form of NRXN1, which is transcribed from a secondary downstream promoter, is less frequently observed to be disrupted by CNV in ASD cases. Combined with the aforementioned genetic association results, our data suggests that both rare and common variants may be contributing to ASD risk at this genomic location.
A deletion of the entire Neuroligin 4Y gene within an autistic male was also observed and confirmed. In the larger dataset, the deletion was observed in the original individual's unaffected father as well as within an unaffected male sibling. Two additional unaffected carriers (unrelated) were also observed in the larger dataset. Interestingly, however, we observed two duplication events within the larger validation set, both of which were present in unrelated ASD individuals. Neuroligin 4Y, which resides on the non-recombining portion of the Y chromosome in modern humans was, in the evolutionary past, allelic with Neuroligin 4X (NLGN4X) gene. Several studies have previously implicated NLGN4X in autism (for example [50-52]). Nucleotide mutations in NLGN4Y have been reported in an autistic male and his father, who possessed learning disabilities [53]. While there is currently no data regarding the functional status and possible role of NLGN4Y, EST evidence from the NCBI database suggests that NLGN4Y retains a molecularly active promoter and is expressed in the brain. A recent report demonstrated a potential role for some pseudogenes in regulating the expression of their parent or source genes by providing decoy targets for miRNAs [54]. It is possible that, through this or some other mechanism, NLGN4Y is involved in regulating the expression of the NLGN4X locus in males. Our finding of copy number aberrations of this gene in autistic individuals suggests that the locus warrants further investigation.
We observed a deletion at the ABAT gene locus within multiple ASD individuals, one in our discovery dataset and a second case ASD case in the validation set. Further examination of the locus within the larger, family based set revealed 7 of 755 affected individuals carrying the deletion and five (of 1,809) unaffected individuals (unrelated to the seven affected individuals). Although we observed a trend of enrichment in affected vs. unaffected individuals in our dataset, statistical significance was not met at α = 0.05. The observed trend, however, suggests that ABAT locus, and the GABA system as a whole, warrant further scrutiny for their potential role in ASD risk. ABAT is involved in the catabolism of GABA, has previously been implicated in autism by genetic association, although replication of the association was not observed in the small validation dataset (n = 91 autism trios) [55]. In the one deleted individual from our dataset for which CGH array data was available at the locus, the ABAT deletion removes exon 1 of the gene (Additional file 7: Figure S2). We note that the nearby A2BP1 and GRIN2A loci, which have also been implicated in autism [55], were not included in our deleted region (Additional file 8: Figure S3).
We also observed a previously reported deletion in the OXTR receptor in a single male ASD individual [56]. A smaller duplication region in OXTR was also detected in a separate ASD case.
In addition to the rare copy number events described above, we also observed and confirmed common copy number polymorphisms at SLC38A3, GABBR2, NRXN3, SHANK3, and NLGN2 (Table (Table2).2). Among these, only duplication events in GABBR2 achieved nominal statistical significance at α = 0.05, but this result failed to hold up after multiple correction for multiple testing. Furthermore, the relatively high level of background noise of assays targeting this locus makes us approach the GABBR2 finding with caution. Additional examination of the locus through alternative platforms (for example long-read high throughput sequence technologies) will be necessary before further conclusions can be drawn.
These data further indicate the importance of structural variation in ASD risk and provide additional evidence that rare variants at multiple genomic loci are potentially contributing to this common neurodevelopmental disorder. In particular, we report statistically significant enrichment of rare exonic deletions in NRXN1 in autistic vs. non-autistic individuals. The detection and confirmation of structural variants below the 10-50 kb typically afforded by SNP-based GWAS arrays indicate that aCGH remains an important complementary method for CNV detection.
The dataset(s) supporting the results of this article are included within additional supplemental files.
aCGH: Array-based Comparative Genomic Hybridization; ADI-R: Autism Diagnostic Interview; ASD: Autism Spectrum Disorders; BP: Base Pair(s); CGH: Comparative Genomic Hybridization; CNV: Copy Number Variation; DNA: Deoxyribonucleic Acid; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders IV; GABA: Gamma-aminobutyric acid; GABR: Gamma-aminobutyric acid receptors; GEE: Generalized Estimating Equation; GWAS: Genome Wide Association Study; HIHG: Hussman Institute of Human Genomics; IQ: Intelligence Quotient; MZ: Monozygotic; NIGMS: National Institute of General Medical Sciences; PCR: Polymerase Chain Reaction; UCSF: University of California, San Francisco; VABS: Vineland Adaptive Behavior Scale.
The authors declare that they have no competing interests.
DJH, MPV, JLH, and JRG conceived study. ZMK, BLB, HNC, PLW, and LN performed experiments. DJH, KLH, GWB, DM, and JMJ analyzed experimental data. SJS, LN, JML, and MLC evaluated clinical data. SLH, JRO, and MPV contributed samples and reviewed manuscript. DJH wrote the manuscript. All authors read and approved the final manuscript.
Table S1. Examination of Noise metric and call rates of blood and spit derived DNA extractions from same individuals.
Table S2.
Table S3. Validation Targets.
Table S4.
Figure S1. Outline of experimental work flow.
Table S5.
Figure S2. A 40 kb deletion removing the first exon of the ABAT gene.
Figure S3. Larger genomic region of ABAT exonic deletion. Although the start 5' flank point of the deletion has not been localized, intervening probes between ABAT and A2BP1 genes indicates that the deletion terminates a significant distance away from either A2BP1 or GRIN2A, both which have been implicated in autism.
We appreciate the contributions made to this work by the families of individuals with autism and the research staff at collaborating universities. This study was supported by funding from the National Institute of Neurological Disorders and Stroke (P01NS026630) and the National Institute for Mental Health (5R01MH080647-14), the National Alliance of Autism Research, and the Hussman Foundation. A subset of the participants was ascertained while Margaret A. Pericak-Vance, Michael A. Cuccaro, and John R. Gilbert were faculty members at Duke University.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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While relaxation of geographical restrictions on bank expansion permitted banking organizations to expand across state lines, it allowed states to erect barriers to branch expansion. These differences in states' branching restrictions affect credit supply. In states more open to branching, small firms borrow at interest rates 80 to 100 basis points lower than firms operating in less open states. Firms in open states also are more likely to borrow from banks. Despite this evidence that interstate branch openness expands credit supply, we find no effect of variation in state restrictions on branching on the amount that small firms borrow.
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