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Elixr Exports is founded and lead by Eli Taylor-Lemire, a sixteen-year-old filmmaker and DJ from Port Chester, NY.
In the summer of 2016, Eli's film career first began to takeoff. Starting with music videos, Eli's presence on the internet grew extremely quickly. A video he directed for Flawless and Leed titled "Pablo Escobar Flow", quickly garnered well over 7,000 views and Eli was able to work with a bigger group of musicians and producers.
At the same time, Eli began DJing at a local nightclub in White Plains and word got around about his talent with the decks. From sweet sixteens to a Homecoming, Eli was hired time and time again for his professionalism, connection with teenagers, and great taste in music.
In the past year, Eli has filmed for a large variety of Hip-Hop artists as well as directed his own films. He has also provided music for over thirty events, ranging from 10 to 800 people.
Most recently, Eli released the music video for Bravo's "Do Not Disturb Freestyle", which has already reached over 20,000 views on Facebook.


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Twelve South Bookarc For Mac Pro | Horizontal Desktop Stand For Mac Pro
Chrome metal stand for Mac ProHolds Mac Pro horizontally, reducing operating height requirementSoft silicone-lined insert cushions MacPro, while padded feet protect surface beneath standMirror finish reflects Mac Pros unique finishIncludes cable management ties and polishing cloth BookArc Pro is a chrome stand made exclusively to cradle Mac Pro. Crafted from metal, this elegant throne holds Mac Pro securely on its side to operate horizontally vs. the typical upright position. This trims the Mac Pro's height requirement in half, giving you more placement options — like a secure equipment rack or a free studio shelf. BookArc also gives your Mac Pro a radically different look that some who "think different" may actually prefer. It's a space saver that makes Mac Pro look like a jet engine… ready to launch your next Mac Pro-driven project into orbit. Your Mac Pro will perform just as well sideways in a BookArc as it will upright. In fact, an Apple Tech Note regarding horizontal placement was the inspiration for BookArc for Mac Pro. If you plan to set up two or more Mac Pros with BookArc Pro, Apple recommends placing the Mac side-by-side, instead of end-to-end. Keep the ends of Mac Pro off walls to ensure proper airflow and you're good to go. Sold as is, final sale not eligible for returnexchange or warranty Guaranteed to be an authentic Twelve South product sold by Twelve South. Item for sale is the BookArc only and does not include a Mac Pro.
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The "Classic" Mac OS is a graphical user interface-based operating system developed by Apple Inc. for its Macintosh line of personal computers from 1984 until 2001, the original member of the family of Macintosh operating systems. The Macintosh platform, which was introduced in the classic Mac OS, is credited with having popularized the early GUI concept. Mac OS was preinstalled on every Macintosh computer that was made during the era it was developed; it was also sold separately in retail stores. Apple released the original Macintosh on January 24, 1984. Its early system software was partially based on the Lisa OS, previously released by Apple for the Lisa computer in 1983; as part of an agreement allowing Xerox to buy shares in Apple at a favorable price, it also used concepts from the Xerox PARC Alto computer, which former Apple CEO Steve Jobs and other Macintosh team members had previewed. The operating system integral to the Macintosh was originally named System Software, or simply "System", and referred to by its major revision starting with System 6 and System 7. Apple rebranded the system as Mac OS in 1996, starting officially with version 7.6, due in part to its Macintosh clone program. That program ended after the release of Mac OS 8 in 1997. The last major release of the system was Mac OS 9 in 1999. Mac OS is characterized by its monolithic system. From its original release through System 4, it ran only one application at a time. Even so, it was noted for its ease of use. Mac OS gained cooperative multitasking with System 5, which ran on the Macintosh SE and Macintosh II. It was criticized for its very limited memory management, lack of protected memory, no access controls, and susceptibility to conflicts among extensions that provide additional functionality such as networking or support for a particular device. After a four-year development effort spearheaded by Steve Jobs' return to Apple in 1997, Apple replaced Mac OS with a new operating system in 2001 named Mac OS X; the "X" represented the tenth major revision of the Mac system software as well as its history as part of NeXT and its relation to Unix. Mac OS X was renamed "OS X" in 2012 and "macOS" in 2016. The general interface design of the current macOS shares its legacy with the classic Mac OS, and there was some overlap of application frameworks for compatibility, but the two systems have different origins and use deeply different architectures. The final updates to Mac OS 9 released in 2001 provided interoperability with Mac OS X. The name "Classic" that now signifies the historical Mac OS as a whole is a reference to the Classic Environment, a compatibility layer that helped ease the transition to Mac OS X. more...

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Are you looking for great places to go or parties where you can still have a great time but not feel like you are trying to kick it with the kids? Are your friends married with kids, but you still love to go out? But who with? I really love music but I don't want to go out alone, lets all get together and make new friends and have fun.
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The Brothers Bloom Movie Review
In The Brothers Bloom, Mark Ruffalo and Adrien Brody star as the title characters, con men who decide they're going to take advantage of a lonely but rich hermit, played by Rachel Weisz. But what they don't suspect is that she is more daring and clever than first imagined. Oh, and when one of them […]
No Love for Michael Moore's Capitalism: A Love Story
The economy. The economy. The economy. No subject is more talked about or important these days than the economy. So, it's only natural that liberal filmmaker Michael Moore, having already taken on gun control, Republicans and universal healthcare, select the economy as the topic of his latest movie, the aptly titled Capitalism: A Love Story. […]
Old Dogs Movie Review: Blu-Ray Won't Save This One
Friday was not a good day, and I blame Disney. Instead of partying my friends, I chose to watch Alice in Wonderland and Old Dogs, the latter of which arrives on DVD and Blu-Ray this week. Both movies are terrible, and the few beers I had between viewings weren't enough. Read FilmJabber's full Old Dogs […]
Ponyo Movie Review
Ponyo, the latest from acclaimed director Hayao Miyazaki (Spirited Away, My Neighbor Totoro), is now available on Disney DVD and Blu-Ray, and it is one that every parent should go out and purchase immediately. Easily one of the better movies of 2009, Ponyo is an imaginative, intoxicating adventure the whole family will enjoy. Read FilmJabber's […]
Food, Inc. Movie Review
Nominated for an Oscar for Best Documentary Feature, Food, Inc. provides a compelling look at the food industry and the consequences its actions have on American lifestyles. While the picture provides some insightful facts, it lacks the shooting gun to put it on the same plane as similar movies such as Super Size Me. Read […]
30th Annual Razzies Winners Announced: Sandra Bullock Accepts Hers in Person
The 30th Annual Raspberry Awards (the Razzies) have been announced, declaring the worst movies and performances of the year. Sandra Bullock, who is expected to win Best Actress at tonight's Oscars ceremony, attended the anti-awards show to accept trophies for her performance in the critically panned All About Steve – which she also produced – […]
2012 Blu-Ray: The End of the World?
2012 is one of those movies I approached with trepidation. After all, we've seen director Roland Emmerich attempt to destroy the world on several occasions in the past (Independence Day, The Day After Tomorrow), and 2012 – the destruction movie to end all destruction movies – was bound to be just a graphics-heavy onslaught of… […]
The Worst Movies of 2009
It's not always simple to narrow down the worst movies of any given year as, hopefully, one has enough restraint to not watch every bad movie that has been released. There was a time – before I worked 50 to 60 hours a week at my day job – where I did watch just about […]
My 'Precious' Blu-Ray Experience: Not As Good the Second Time
Back in November, there was a clear front runner for Best Picture. The movie was Precious, or more officially Precious: Based on the Novel Push by Sapphire, an utterly depressing tale of physical, mental and sexual abuse, teen pregnancy, down syndrome, AIDS, rape and obesity. After watching the movie in theaters, I described it as […]
Alice in Wonderland Movie Review
Friday was a good day, up until I left work. At 5 pm, I drove home, ate some food and did some dishes. At 8 pm, I finished watching the Disney movie Old Dogs, which was painfully bad. At 10 pm, I sat down in a sparsely populated theater to watch Tim Burton's new movie […]
The Crazies Movie Review: Crazy Is As Crazy Does
The world just wouldn't be the same without zombie movies. There's just something deliciously satisfying about them. And despite the fact that they routinely share the same plot and the characters experience similar fates, the last decade has provided audiences with a shockingly consistent level of quality – in fact, I'd go so far as […]
Whiteout Movie Review
With the advent of special effects and life-like cartoons, the question continues to be posed: is the value of major actors in film deteriorating? Further pushing the issue are such films as The Hangover and a string of action flicks that have gone on to dominate the box office despite the lack of demonstrable stars. […]
The Private Lives of Pippa Lee Movie Review
Robin Wright turns in her best performance since Forrest Gump in the quirky drama-comedy The Private Lives of Pippa Lee, but the movie itself fails to leave a lasting impression. Written and directed by Rebecca Miller, Pippa Lee also stars Alan Arkin, Keanu Reeves, Maria Bello, Winona Ryder, Julianne Moore and Blake Lively. Read FilmJabber's […]
Oscar Predictions 2010
The Academy Awards are next Saturday, March 7, and here are FilmJabber's Oscar predictions. The expansion from five to ten nominations for the coveted prize really hasn't done much to make things more competitive, as it appears that it's going to be a battle between Kathryn Bigelow and her former husband James Cameron for a […]
Cold Souls Movie Review
Paul Giamatti has sold his soul. Not to the Devil, mind you, but to a corporation. He hasn't actually sold it, either, but it has been stolen and sold on the Russian black market. This is the premise for Cold Souls, an imaginative though anticlimactic comedy-drama from director Sophie Barthes. Read FilmJabber's full Cold Souls […]
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Links to sites don't work
I have a problem with my wp multisite installation. It works fine but I have problems with the links in the admin bar. For example, I have 3 sites:
But in the admin bar, every link goes to test.com :(. Also the links in "sites" screen and the administration links (always test.com/wp-admin)
If I write the url, every thing works fine but the links in the admin side don't work.
I'm using wp 3.5.1 and nginx.
I'd really appreciate your help guys!
- The topic 'Links to sites don't work' is closed to new replies.


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Boaters in Port Washington always have this day marked on their calendars.
The Port Washington Marina officially opens for the season on Saturday, April 1, with some boaters expected to launch just after midnight.
"There's always someone who wants the bragging rights to be first in the water," said Dennis Cherny, harbor master for the marina.
One local boater wants to wait until the morning to launch his boat, and for good reason.
"I'm not usually awake that time of night," joked Danny Fox, who has been chartering boats in Port Washington for more than 40 years. Fox plans on launching on Saturday morning, with his first scheduled charter set for Sunday.
"Around here, it's a long winter and a short summer, and boats are made for floating. The sooner and longer I can be on the lake, the better," said Fox.
According to Cherny, a new patio on the north end and a fire pit for visitors are the only major additions to the marina, which was extensively renovated in 2010 to install floating piers and concrete decking.
"Those renovations have really held up well, and I think that visitors will really enjoy the new additions to the marina this summer," said Cherny.
Cherny expects another great year for boating and fishing in Port Washington, with the Department of Natural Resources planting more than 60,000 fish in the lake this year.
The Port Washington Marina is one of the most popular boating sites on the western side of Lake Michigan, boasting a 95-percent filled capacity for the docks that roughly 250 boats call home during the summer.


text: Fergus is a railway traction engine. Trivia Fergus is best friends with Featherweight. Add a photo to this gallery Retrieved from "http://poohadventures.wikia.com/wiki/Fergus?oldid=530413" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: HEROES Allies Non-Disney characters Non-Disney heroes Pooh's Adventures allies Ash's adventures allies Thomas and Twilight Sparkle's Adventures allies Trains Steam Locomotives Standard gauge engines Honored Guests to Canterlot Thomas & Friends Characters Weekenders Adventures allies


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Changing Lives One Ride At A Time
BEAMING Inc.
Phone: 920-851-6160
Partners in Riding
We envision continuing to benefit our elementary and
middle schools as an adaptive physical education
opportunity at the barn with riding and farm-related
This community program is a six week spring/fall
collaborative partnership with the Oshkosh, Neenah,
Menasha and Winneconnie school Districts in providing
Adaptive Physical education and other equine experiences
to serve their special needs students. If you would like
more information about our Partners in Riding program
please feel free to contact us at firstname.lastname@example.com.
Changing lives one ride at a time.
Did You Know:
Receiving equine therapy can be a great way to begin recovering from an injury or disability of any kind.
Donate Now
Copyright © 2017 BEAMING Inc. All Rights Reserved.
2692 County Road GG, Neenah, WI 54956
Office Phone: (920) 851-6160


text: 2 Texture Milkmaid Braids - TUTORIAL February 23, 2016 Get link Facebook Twitter Pinterest Google+ Email Today's tutorial are on this really cute 2 Texture Milkmaid Braids! I decided to take a different twist on a regular milkmaid braid and add one regular braid & one fishtail braid. I was inspired by a photo I saw on Amber Fillerup's instagram a while back and loved how it turned out! This look is also using some new products I received from John Frieda as part of the PreenMeVIP program! I used the Frizz Free Leave in Conditioner & The 6 Effect Serum to get my hair really soft and frizz free before doing this hairstyle! I love the products I've used already and can't wait to try out the rest! with love, ashley Get link Facebook Twitter Pinterest Google+ Email Comments Post a Comment


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5 out of 10
Release Date: 1st June 2014 (DVD Premiere)
Director: Martin Kemp (Stalker (2011))
Cast: Leo Gregory, Ricci Harnett, Lorraine Stanley, George Russo, Dannielle Brent, George Sweeney, Susan Penhaligon, Francis Pope, Johnny Palmiero, Gary Lawrence, Ryan Oliva with Jason Flemyng and Vincent Regan
Writer: Dougie Brimson
Trailer: TOP DOG
Martin Kemp's (STALKER) second feature film as director, whilst being an improvement on his debut, is a solid yet melodramatic drama that refuses to distinguish itself. It's about a small time criminal, Billy Evans (LEO GREGORY – WILD BILL) biting off more than he can chew. Bolstered by committed performances from a talented cast of familiar and unknown faces, this could be producer Jonathan Sothcott's most serious and grown up film to date. What stops Top Dog being one to remember and celebrate though is its reluctance to stand out from the crowd, a lack of originality and yet another 'tin eared' script. This is rescued from stinkerdom though by notably excellent performances from Leo Gregory (who does convince as a man out of his depth), Lorraine Stanley (THE HOOLIGAN FACTORY) (who seems to be the 'go-to' lady when producers need a powerhouse actress in their low-budget flick) and Ricci Harnett (TURNOUT) (it's always great to see this underused actor in all too rare leading roles).
The plot runs on rails. It's all very predictable and plays like a violent, sweary yet over melodramatic episode of Eastenders. The soap operatics near the end over tip the whole film into parody come it's misjudged ending, but for most of the film's running time it keeps it's cool and nerve. The plot also includes some football hooliganism. Yes, it's a film without the word Hooligans in the title or a big ass St George's flag on the front. Incredible! Based on a novella by Dougie Brimson – the predictable plot sees top boy Billy of the local firm Acton Casuals (AC! CHA CHA CHA!) go up against Harnett's local mr big when Billy's Auntie's (SUSAN PENHALIGON – SOLDIER OF ORANGE) pub begins getting squeezed for protection money. The whole thing escalates in a tit-for-tat war that continually ups the stakes until people begin to get kidnapped, killed and the whole skirmish gets extinguished by bigger fish further up the food chain.
There are flashes of visual brilliance in this other wise ordinary point and shoot affair. The film's opening shot of what appears to be snow covered mountains turns out to be something altogether different is powerful and difficult to forget. It's a shame the rest of the film is directed and lensed so anonymously, but these are small complaints when Kemp, Sothcott and the actors have gotten so much right this time. A reliable Jonathan Sothcott production has been a long time coming. After his solo calling card Vendetta proved, his brand isn't arsing around in the shallows anymore. He's on the way up. Vendetta was a huge success and with Top Dog, it's proven itself to be a hard act to follow. I'm happy to say that whilst Top Dog isn't great at least it feels like a real film. It doesn't look cheap and the plot is tight and makes sense throughout. It's also harder to critique a film that I don't like because it's down to my personal tastes, rather than not liking it because it's a slap dash piece of cheap crap. Whilst this comes across like a place holder before the bigger more flashier Jonathan Sothcott productions come down the line it still serves as the second best film he's put together in his short but very busy run. Here's to We Still Kill The Old Way and Age of Kill doing less fence sitting and more thrill making.
5 out of 10 – Solid yet ordinary gangster melodrama that is helped no end by a stellar cast committed to getting this Dog on (or near) the top. Middle Dog it is then.
Another review by Joe Pesci II below.
- Leo Gregory: Northmen, The Hooligan Factory, All Things To All Men, One In The Chamber, Payback Season, Wild Bill (2012), Big Fat Gypsy Gangster, The Big I Am, Cass, Goal 3, Stoned, Green Street
- Ricci Harnett: Rise of the Footsoldier 2, Vendetta, The Rise and Fall Of a White Collar Hooligan, Ill Manors, Turnout, Psychosis, Rise Of The Footsoldier, Joy Division, 28 Days Later
- Lorraine Stanley: No Reasons, A Hundred Streets, Suffragette, Legend (2015), The Guvnors, The Hooligan Factory, The Hooligan Wars, He Who Dares, Essex Boys Retribution, GBH, Made In Dagenham, Cass, London To Brighton
- George Russo: Trespass Against Us, Gunned Down, Bonded By Blood 2, I Am Soldier, Green Street 3, Turnout, Tony
- Dannielle Brent: Ill Manors, Shifty
- George Sweeney: Revolver, Citizen Smith (TV), The Bitch
- Susan Penhaligon: A Fine Romance (TV), Leopard In The Snow, Soldier of Orange, Nasty Habits, The Land Time Forgot, No Sex Please We're British, Under Milk Wood (1972)
- Francis Pope: Turnout, Tony
- Ryan Oliva: Montana, Vendetta, The Seasoning House
- Johnny Palmiero: Rise of the Footsoldier 2, Bonded By Blood 2, Age of Kill, We Still Kill The Old Way, Bonded By Blood, The Kid (2010), Green Street
- Jason Flemyng: Gemma Bovery, Sunshine On Leith, Welcome To The Punch, I Give It a Year, The Great Expectations (2012), Wild Bill (2012), X Men-First Class, Hanna, Solomon Kane, Kick Ass, Ironclad, Dead Cert, Jack Falls, The Riddle, Shifty, Clash Of The Titans, The Curious Case of Benjamin Button, Mirrors, Stardust, Rollin With The Nines, Transporter 2, Layer Cake, Below, Mean Machine, The Bunker, Snatch, Lock Stock & Two Smoking Barrels,The Red Violin, The Hollow Reed, Deep Rising, Spice World, Alive & Kicking (1996), Deep Rising, Stealing Beauty, The Jungle Book (1994)
- Vincent Regan: Vendetta 2, The London Firm, Vendetta, St. George's Day, Outside Bet, Snow White & The Huntsman, Lock Out, Ghost Rider 2, Bonded By Blood, The Holding, The Big I Am, 300, Hard Men


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Sunday, November 27, 2011
Cranberry Strippers
I was looking around online at recipes to use up Thanksgiving leftovers, and saw a photo of some thumbprint cookies that someone had made using leftover cranberry sauce. Sounded like a good idea to me, and here is my version. Those of you who have been reading this blog will recognize these cookies: I have posted the recipe for them before using apple butter instead of cranberry sauce. You can really use any type of jam-like product you like. They are also delicious with raspberry or apricot jam.
I have changed the glaze for this version a little bit by using orange juice instead of lemon juice. Orange is such a nice pairing for cranberry, and I thought the added sweetness would help offset the tartness of the cranberry sauce. After the fact I thought that Cointreau or Triple Sec might make a nice glaze, too. If you try that, let me know how it works out.
Cranberry Strippers
1/3 Cup Sugar
5 Tbsp Butter, softened
1 1/2 tsp Vanilla Extract
1 Large Egg White
1 Cup All Purpose Flour
2 Tbsp Cornstarch
1/4 tsp Baking Powder
1/4 tsp Salt
Cooking Spray or Parchment Paper
1/3 Cup Cranberry Sauce
1/3 Cup Powdered Sugar
2 - 4 tsp Orange Juice
1/4 tsp Vanilla Extract
Preheat Oven to 375
Beat sugar and butter with a mixer at medium speed until well blended, about 5 minutes. Add the vanilla and egg white, beat well. Combine the flour, cornstarch, baking powder and salt in a bowl. Add the flour mixture to the sugar mixture ans stir until well blended.
Turn the dough onto a lightly floured surface and divide it in two. Roll each half into a log about 12 inches long. Place the logs about 3 inches apart on a cookie sheet or baking pan coated with cooking spray (I used parchment paper instead). Using the handle of a wooden spoon, make a 1/2 inch indentation down the length of each log. Spoon the cranberry sauce into the center. Bake for 20 minutes or until lightly browned. Remove to a cutting board.
Combine the powdered sugar, orange juice, and vanilla extract. Stir well with a fork and drizzle over the logs. Cut each log diagonally into 12 slices, but don't pull the slices apart yet. Let rest on the cutting board for 10 minutes, then remove to a wire rack to cool.
For a printer-friendly version of this recipe, click here.
Posted by Hoofer at 2:00 PM 1 comment:
Labels: cookies, dessert, fall, Thanksgiving
Thursday, October 27, 2011
"Poached" Egg in Tomato Sauce
This one is more of a method than a recipe. Last night I made sauce with the last of the season's tomatoes. I know I need to save most of the leftover for my sweetie to enjoy with mozzarella sticks this weekend, but I have been wanting to try baking eggs in tomato sauce for a long time. However, our oven takes a really long time to heat up, and as I just wanted to cook one egg, that seemed excessive. In browsing recipes online I found this one, and decided to give this method a go.
It was a bit tricky since I was only cooking one egg, and therefore had hardly any sauce in the pot, maybe 1/2 a cup? Next time I will use a shallower pan rather than a pot. I also cooked my egg just a tad too long, but it was still very tasty. I will be trying this again.
"Poached Egg in Tomato Sauce"
Tomato or Marinara Sauce, about 1/2 cup per egg
Heat up the sauce in a pan or pot over medium-low heat. Once the sauce is simmering, crack in the egg(s). Continue cooking until the egg whites are set, but the yolk is still somewhat runny, or to your tastes, 5 minutes or so for 'medium'-ish eggs.
Serve with (or over) toasted bread.
Tuesday, October 11, 2011
Mustard-Rosemary Roasted Potatoes
I can tell the seasons are changing just by what I am wanting to cook. Last night I made meatloaf, and wanted something other than mashed potatoes as a side, so I made these yummy roasted potatoes instead.
1 Tbsp Olive Oil
1 Tbsp Dijon Mustard
1 tsp Paprika
1/2 tsp Dry Thyme
Salt and Pepper to Taste
2 Medium Yukon Gold Potatoes, cut into 1/2 - 3/4 inch chunks
1 - 2 Sprigs Fresh Rosemary, broken into 1 1/2 inch pieces
Preheat Oven to 400 Degrees
Spray an 8 inch baking dish with cooking spray.
Whisk olive oil, mustard, paprika, thyme and salt and pepper in a medium mixing bowl. Add the potatoes and stir to coat evenly. Spread the potatoes in the prepared baking dish and sprinkle with the fresh rosemary. Roast in the oven about 25 minutes until nicely brown on the outside and soft in the middle, stirring at least once during cooking.
For a printer-friendly version of this recipe, click here.
Monday, October 10, 2011
Pork Loin Roast with Apples
I bought a pork loin roast without any real plan for what to do with it, so I went browsing the web to see what I could find. This recipe from The Food Network caught my eye, so I decided to give it a go. Following is my version, with a few tweaks to account for my family's tastes and what I had on hand.
1 Two Pound Pork Loin Roast
Salt and Pepper
3/4 tsp Chopped Fresh Rosemary (or 1/2 tsp dry, crumbled)
2 Tbsp Canola Oil
1 Medium Onion, Thickly Sliced
1 Celery Root (Celeriac), Peeled. 1/2-inch Dice
2 Medium Carrots, cut into 1/2 inch chunks
3 Apples, Cored and cut into 1-inch chunks
3 Cloves Garlic, Minced or Pressed
3/4 tsp Dry Thyme (or fresh if you have it)
1 Sprig Fresh Rosemary (or 1/2 tsp dry)
2 Tbsp Cider Vinegar
1 Cup Apple Juice or Cider
2 Tbsp Grainy Mustard
2 Tbsp Cold Butter
Take the roast out of the fridge 30 minutes or so before you plan to start cooking. Sprinkle it all over with salt, pepper, and chopped rosemary and let it sit on the counter.
Preheat the oven to 400 degrees
Chop the veggies and apples.
Heat the canola oil in an oven-proof skillet over high heat. Add the roast and brown all over, about 3 minutes per side. Remove the roast to a plate.
Reduce the heat in the skillet to medium and add the onions, celery root, carrots and rosemary sprig. Cook for about 5 minutes, and add the garlic, thyme, and apples. Continue cooking for another 4 minutes or so, until the vegetables are beginning to brown. Push the apple mixture to the sides of the pan, and place the roast in the center. Put in the oven and roast to an internal temperature of 135 degrees. You can use an instant-read thermometer, or insert a probe thermometer before you put the roast in the oven.
Remove the pan from the oven and place the roast on a cutting board. Tent it loosely with foil. Put the apple mixture on a serving platter (discard any herb sprigs) and return the pan to high heat on the stove. Stir in the vinegar, stirring to get up any brown bits on the bottom of the pan. Cook until reduced by half, then add the apple juice or cider. Reduce the juice by half, then turn off the heat. Whisk in the mustard, then the butter.
Slice the roast about 1/4 thick or so and arrange it over the apple mixture. Drizzle the roast with the sauce, and pass remaining sauce at the table. Enjoy!
For a printer-friendly version of this recipe, click here.
Posted by Hoofer at 1:39 PM No comments:
Friday, October 7, 2011
How to Freeze Fresh Green Beans
This one is not so much a recipe as a how-to. We were blessed to receive lots and lots (and lots and lots) of green beans from our CSA farm this year. So many, in fact, that we could not possibly keep up with eating them all fresh.
So....I asked my son to research ways to freeze them for later use. He did me one better. He not only did the research, but then came downstairs and did the work as well. That was a great day!
The first thing he did was snap off the stem ends of the beans.
Next, he blanched them in boiling water for three minutes.
The next step was to take the beans out of the boiling water and put them in a bowl of ice water, also for three minutes.
After that, he put the beans on a cookie sheet covered with a clean towel so they could dry a bit.
One of the most important steps, according to him, is to make sure that you label the bag. I am sure this will seem more important to me in December when I am looking for these green beans in the freezer!
Next the green beans go into the bag, and then into the freezer.
Thanks to my son for freezing the green beans. I am sure we will enjoy them this winter, after we haven't had any in a while.
Tuesday, August 30, 2011
One Pot Creamy Pesto Pasta
Here is a quicky lunch or side dish to use up some of that summer basil you may still have around.
One Pot Creamy Pesto Pasta
1 Small Garlic Clove
2 TBSP Pine Nuts or Walnuts
2 TBSP Shredded Parmesan Cheese
1/3 Cup Fresh Basil Leaves
Drizzle of Olive Oil
1 Cup Pasta
2 Cups Veggie Broth (or Chicken Broth)
2 Tbsp Flour
1 1/2 Tbsp Butter
1/2 Cup Milk
Combine the garlic, pine nuts or walnuts, Parmesan cheese, and basil in a food processor and process until smooth. Slowly add in a drizzle of olive oil until desired consistency is reached. Or, feel free to use pre-made basil pesto in this recipe, up to about 1/3 cup or so.
This little food processor that came with my stick blender is perfect for this job.
Put the pasta and the broth in a medium sauce pan. Bring to a boil over high heat. Reduce the heat to a simmer, and simmer until the pasta is tender, about 10-12 minutes. There will still be some liquid left in the pot.
Sprinkle the flour over the pasta and cook and stir for about a minute. Add the butter and stir until melted. Cook and stir another minute or two, then add in the milk. Stir to incorporate everything and let cook for about 90 seconds, or until the milk is simmering. Stir in pesto sauce to taste, I like a lot, so I used all of it, but start with a few tablespoons and see how you like it. Season to taste with salt and pepper, serve and enjoy.
For a printer-friendly version of this recipe, click here.
Monday, August 29, 2011
Padron Peppers
This one is super quick and easy, not even a recipe, so I am not going to bother with a printer-friendly version. We got padron peppers in our CSA share 2 or 3 times this summer, and it was a new veggie to us. After researching online, I found out that they are originally from Galicia, Spain, and are commonly served as tapas. Or is that a tapa? Anyway, no matter. Also, they are mostly mild, but a few spicy ones turn up here and there. You can't tell which will be spicy, so it is a surprise! There is even a saying : "Pementos de Padrón: uns pican e outros non." (Padron peppers, some are spicy some are not.)
The preparation is super simple and delicious. Simply heat a cast iron skillet or griddle over high heat, and cook the peppers until they are blackening. Remove to a plate or platter, drizzle with good olive oil, and sprinkle with salt. That's it. Yum!
Sunday, August 28, 2011
Stir-Fry Noodles with Spicy Peanut Sauce and Veggies
Okay, it's been forever. I know. I have been sick ALL summer, but despite that did manage to cook a few things, just never got around to posting.
My sweetie is not a big fan of peanut sauce, so I made this one in July when he was on a business trip to the East Coast. I have tried many times to make peanut sauce, and this was by far my best effort to date. I listed the vegetables I used this time, but, as always, you can use whatever you have on hand. Just try to have about 3 cups or so of vegetables.
For the Sauce :
1/3 Cup plus 1 TBSP Creamy Peanut Butter
1 Clove Garlic, Minced or Pressed
1/2 tsp Fresh Ginger, Minced
1 TBSP Brown Sugar
1/4 Cup Rice Vinegar
1/2 tsp Chili Garlic Sauce
1 tsp Sesame Oil
For the Stir-Fry
2 tsp Canola or Peanut Oil
1 Cup Green Beans, about 1 inch long
1/2 Cup Sliced Summer Squash
1/2 Cup Sliced Radishes
1/2 Cup Sliced Carrots
1 Cup Chopped Cabbage
1/4 - 1/2 Cup Chopped Fresh Cilantro
One 9 oz. Package of Fresh Asian Noodles, or Dried Linguine or Spaghetti
To make the sauce, combine all of the sauce ingredients in a food processor and process until smooth. Set aside.
Cook the noodles according to the package directions and set aside.
To make the stir-fry, heat the oil in a wok over high heat, add the vegetables and stir-fry until they just begin to soften, about 3 or 4 minutes. Add the sauce and the cooked noodles. Stir to coat everything thoroughly and cook until hot, about another 3 minutes or so. Stir in the chopped cilantro, serve and enjoy!
For a printer-friendly version of this recipe, click here.
Posted by Hoofer at 11:01 AM No comments:
Labels: Asian, pasta, vegan, vegetables, vegetarian
Monday, June 27, 2011
Strawberry Puff Pastry Galette
Here is another quick and easy one. I received 2 baskets of strawberries from the CSA last week, and while we usually just eat them for breakfast until they are gone, for some reason we haven't been recently, so I had strawberries that needed to be used up ASAP. Luckily, there was also a lonely sheet of puff pastry in the freezer, and here you go!
1 Sheet Frozen Puff Pastry
1 Pint Strawberries
1 tsp - 1 Tbsp Sugar
One Egg
3 Tbsp Strawberry Preserves, Jam or Jelly
1 Tbsp Cointreau, Rum or Water
Remove Puff Pastry from the freezer and allow to thaw on the counter for 40 minutes
Preheat Oven to 400
Wash and slice strawberries into about 1/4 inch slices. Stir in sugar. If your berries are very ripe and sweet, 1 tsp should be enough. Use more sugar for tart berries. Allow to macerate while the oven comes up to temperature.
Beat the egg in a small bowl. Once the pastry is thawed, place it on a baking sheet lined with parchment paper. Fold in about 1 inch all the way around to allow the pastry to puff up around the strawberries. Brush the border with your egg wash.
Pile the strawberries into the center of the pastry, reaching all the way to the folded over border. Place the baking sheet in the oven and bake for 15 minutes.
Meanwhile, whisk the preserves, jam, or jelly with the liquid of choice. As soon as the galette comes out of the oven, brush the berries with the jam mixture. Remove to a cooling rack and cool to room temperature before slicing.
For a printer-friendly version of this recipe, click here.
Saturday, June 18, 2011
One Pot Stove Top Mac and Cheese (No collander required!)
I developed this recipe as an alternative to packaged pasta sides. I happen to love those things. Boxed macaroni and cheese, packets of noodles and sauce mix of every variety. I love them for both the taste and convenience, but they are expensive for what you get, and full of salt and chemical preservatives.
So I started playing around with one pot homemade versions, and this macaroni and cheese recipe was born. Mind you, I still like a good old-fashioned casserole with crunchy topping from the oven, but this one is all about getting it on the table fast and easy clean-up. And it's pretty tasty, to boot!
One Pot Stove Top Macaroni and Cheese.
1 Cup Macaroni
2 Cups Veggie Broth (or Chicken Broth)
2 Tbsp Flour
1 1/2 Tbsp Butter
1/2 Cup Milk
1/3 - 1/2 Cup Shredded Cheese (Cheddar or a combination of Cheddar and another cheese like Jack)
Fresh Ground Black Pepper
Put the macaroni and the broth in a medium sauce pan. Bring to a boil over high heat. Reduce the heat to a simmer, and simmer until the pasta is tender, about 10-12 minutes. There will still be some liquid left in the pot.
Sprinkle the flour over the macaroni and cook and stir for about a minute. Add the butter and stir until melted. Cook and stir another minute or two, then add in the milk. Stir to incorporate everything and let cook for about 90 seconds, or until the milk is simmering. Stir in the cheese using a figure eight pattern until melted. Spoon into bowls and serve with fresh ground black pepper to taste.
For a printer-friendly version of this recipe, click here.
Friday, June 17, 2011
Chard Tart
There is a bit of a funny story regarding this recipe. I found it in the Joy of Cooking several years back, but was convinced my husband wouldn't like it. Rather than serve it to him , I made it for one of my girlfriends for brunch one morning. We both really liked it, but she agreed with me that he likely would not. So I left it at that. Lo and behold, he tried a piece, and really, really liked it. Goes to show what I know about his tastes...Anyway, I am glad he likes it so much, because I make it often. Sometimes on the weekend for breakfast, sometimes for a light dinner. Either way, it is a big hit every time. This would also make a lovely appetizer course for a small dinner party.
Adapted from The Joy of Cooking
For the crust:
preheat oven to 425
One 9-inch tart pan with a removable bottom, lined with parchment paper
1 3/4 Cups All Purpose Flour
3/4 tsp Salt
1/2 Cup Extra Virgin Olive Oil
1/3 Cup Cold Milk
Whisk together the flour and salt. Stir the in the olive oil and milk using a fork. Press the dough into the pan, pushing up the sides. Prick with a fork a few times to prevent bubbling up in the oven.
Bake for about 10 minutes, until lightly browned.
Lower the oven to 375
For the filling:
1 Tbsp Olive Oil
1/2 Cup Finely Diced Onion, Red Onion, or Shallot
1 Pound Chard, Thick Stems Removed and Coarsely Chopped
2 Cloves of Garlic, Minced or Pressed
2 Tbsp Fresh Basil, Chopped OR 1 1/2 tsp Dried Basil
1/4 tsp Salt, or to Taste
1/8 tsp Black Pepper, or to Taste
3 Large Eggs
1/3 Cup Milk
1 Cup Grated Cheese of you choice (Cheddar, Jack, Parmesan, Mozzarella, Fontina, or any Combination)
Saute the onion or shallot in the olive oil over medium-low heat until very soft, about 10 minutes, add the garlic and cook for another one or two minutes. Add the chard, in batches if necessary, and cook until very tender, about 10 minutes more. Season with the basil and salt and pepper to taste.
In a medium bowl, slightly whisk the eggs. Stir in the milk and cheese. Add the chard mixture, then scrape the lot into the tart shell, and spread evenly.
Bake at 375 until the filling is lightly browned and firm, 25-35 minutes. Cool to room temperature and enjoy!
For a printer-friendly version of this recipe, click here.
Tuesday, June 14, 2011
Chocolate Beet Cake
Chocolate beet cake. Yes, you read that right. I admit it sounds odd, but the cake is actually quite delicious. I was suspicious of this recipe when I first read it in a Live Earth Farm newsletter years ago, but as the beets were piling up I decided to give it a go. I was glad I did. And who knows, if you don't tell anyone there are beets in the cake, they may never catch on!
I usually use regular red beets for this recipe, but this time I didn't have enough, so I used some chiogga beets as well. They are the stripey ones.
I chose to make cupcakes this time, because they freeze well in nice little individual servings, but you could use a bundt pan or 8-inch round or square pan as well.
Chocolate Beet Cake
1 1/2 pounds Beets (or enough to make 1 1/4 cups puree)
3 Eggs
1 1/2 Cups Sugar
1/2 Cup vegetable oil
1 tsp Vanilla
1/2 tsp Salt
1 1/2 Cups All Purpose Flour
3/4 Cup Cocoa Powder
1 1/2 tsp Baking Soda
Cook the beets either by boiling in water to cover by 1 1/2 inches until tender, or baking them in a 400 degree oven. Once they are cool enough to handle, slip off the skins (I like to do this under cold running water) and put them in a food processor. Process until a puree forms, adding water or beet cooking liquid if necessary to get a pureed consistency.
Preheat the oven to 350
Lightly grease and flour a bundt pan, 8-inch round or square baking dish, or line muffin pans with cupcake liners.
Whisk the eggs in a medium bowl. Whisk in the sugar, vegetable oil, vanilla, salt, and beet puree. In a separate bowl, combine the flour, cocoa powder, and baking soda. Add the wet ingredients to the dry a bit at a time until completely incorporated.
Pour the batter into your prepared pan and bake 45-50 minutes for a bundt pan, 35-40 minutes for an 8-inch square pan, and 15-20 minutes for cupcakes, or until a toothpick inserted in the cake comes out clean. Cool and enjoy.
For a printer-friendly version of this recipe, click here.
Posted by Hoofer at 8:54 AM No comments:
Labels: beets, cake, dessert, vegetarian
Monday, June 13, 2011
Bok Choy and Noodle Soup
I was looking for a way to use up some greens for breakfast, but was not in the mood for eggs at all. I thought I would try a simple Asian noodle soup, and it was quite satisfying on this foggy morning.
Bok Choy and Noodle Soup
2 tsp Peanut or Canola Oil
2 Green Onions, sliced, white parts and green separated
1 Clove Garlic, chopped or pressed
1 Bunch Bok Choy, sliced thinly, white stems and greens separated
4 Cups Veggie (or Chicken) Broth
2 oz. Noodles (Spaghetti, Angel Hair, Fettucine or Fresh Asian Noodles)
2 tsp Soy Sauce
2 tsp Sesame Oil
Heat the oil in a sauce pan over medium-low heat. Add the white part of the sliced green onion and saute for about 3 minutes. Add the chopped or pressed garlic and saute for one minute. Add the sliced white stems of bok choy and saute for 2 more minutes.
Add the broth and bring to a boil. Add the noodles and simmer until just cooked (anywhere from 2 - 10 minutes, depending on the noodles used). Add the bok choy greens and cook until tender, about 3 minutes. Stir in the soy sauce and sesame oil. Ladle into bowls and garnish with the sliced green onion tops.
For a printer-friendly version of this recipe, click here.
Posted by Hoofer at 11:35 AM No comments:
Thursday, June 9, 2011
I won a Smart Car!
Pretty exciting stuff! Many thanks to Smart Chicken and New Leaf Community Markets for this fantastic giveaway! I have already been having so much fun driving this little car around.
I also wanted to take this opportunity to let everyone know that I am planning to start posting recipes again this summer. This blog has lain fallow for far too long, so with the help of my great new photographer (aka my son) I will be posting new recipes starting next week. Hooray!
Saturday, March 12, 2011
Whole Wheat Dinner Rolls
I have made a variety of rolls in my time, but I really really like this one. The rolls are super soft and chewy, even with the whole wheat flour. These would be a great addition to any dinner.
2 1/4 tsp Active Dry Yeast
1 Cup Warm Water (105-115 degrees)
2 Cup All Purpose Flour
1 1/4 Cup Whole Wheat Flour
1/4 Cup Sugar
1 tsp Salt
2 Tbsp Melted Butter plus more for brushing on the rolls
Combine the yeast and about 1/4 cup of the warm water. Set aside until frothy, 5-10 minutes.
Meanwhile, combine the rest of the ingredients in the bowl of your stand mixer fitted with the dough hook. Add in the yeast and mix on low speed until well combined. Continue mixing on low - medium low until very smooth and elastic, about 10 minutes. If you don't have a stand mixer, mix the ingredients in a mixing bowl, then turn out onto a lightly-floured surface and knead until very smooth and elastic.
Place the dough in a clean bowl and coat all over with olive oil. Cover the bowl with a damp tea towel and let the dough rise until about doubled in size, approximately 60-90 minutes.
Punch the dough down and form into 12 balls about the size of a golf ball. Place the dough balls in a greased baking dish about 1 - 1 1/2 inches apart. Brush the dough balls with melted butter and cover with the tea towel and let rise again until doubled in size, 30-60 minutes.
Bake in a preheated 375 degree oven for 12-15 minutes.
For a printer-friendly version of this recipe, click here.
Monday, March 7, 2011
Pasta Sauce with Meatballs and Sausage
Okay, so I know it's been unreasonably long since I've posted any recipes. The irony is not lost on me that as soon as we got the fancy new kitchen I stopped publishing recipes. But that is how it has gone. It's not that I haven't been cooking, because I have! For some reason the inspiration to photo and write about my recipes has been lacking.
But there are a few loyal souls out there who keep commenting asking for more recipes, so here goes. I hope to start posting more regularly.
Please enjoy this recipe for pasta sauce with sausage and meatballs.
For the meatballs :
1 - 1 1/4 pound ground turkey
1/2 cup dry breadcrumbs or panko (measured in a glass or 'liquid' measuring cup)
milk (enough to cover the breadcrumbs)
Salt and Pepper
1/4 tsp Garlic Powder
Preheat oven to 350
Lightly grease a cookie sheet or baking pan
Pour the milk over the breadcrumbs in the glass measuring cup. Put the turkey in a medium mixing bowl. When the breadcrumbs have absorbed most of the milk (3 minutes or so) add salt and pepper to taste, and the garlic powder. Mix with a fork, then add the breadcrumb mixture to the meat in the bowl. Combine well, and form into roughly walnut-sized meatballs.
Place the meatballs on the prepared baking pan, about 1 1/2 inches apart. Bake in the preheated oven for 20 minutes.
For the Sausage:
1 pound Italian Sausage Links, mild or spicy
Brown the sausage in a non-stick pan over medium-low or medium heat until browned on all sides.
For the Sauce :
1 Tbsp Olive Oil
1 Large Onion, Diced (2 cups)
2-3 Cloves of Garlic, Minced or Pressed
1 24 ounce can Crushed Tomatoes
1 15 ounce Can Tomato Sauce
2 tsp Dried Basil
1 tsp Dried Oregano
1/2 tsp Dried Thyme
Heat the olive oil in a large pot or dutch oven over medium heat. Add the diced onion and saute until very soft, but not brown, about 8 minutes. Add the garlic and stir for one minute. Stir in the crushed tomatoes and tomato sauce. Add the dried herbs, crushing between your palm and fingers as you add them to the pot. Continue heating over medium heat until the sauce begins to simmer.
Add the meatballs and the sausage to the sauce. Lower heat to low, cover, and simmer for at least 30 minutes, up to several hours.
Serve with your favorite pasta, or spaghetti squash. Buon Apetito!
For a printer-friendly version of this recipe, click here.
Posted by Hoofer at 8:44 PM 2 comments:
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text:
Get Your Kids to Eat Their Vegetables...Or at least Tomatoes!
Posted by GoGo Goodie on September 13, 2011.
A few years ago, you would have never caught my children eating anything with a tomato on it. The closest thing to a tomato they would let enter their mouth was pizza sauce or ketchup...that was it. Now, fast forward a few years and not only do they eat them in salads and on sandwiches, but they even eat them by themselves!
How did we pull off this herculean feat you ask. Well, we started a tomato garden. Yes the kids enjoyed planting the tomatoes and watching them grow, but the change in their acceptance was purely due to flavor. Plain and simple, our organically grown and vine ripened tomatoes tasted good! My husband likes to say eating one of our tomatoes vs a store bought one is like "drinking coke out of a chilled glass bottle vs drinking a coke in a plastic cup with melted ice".
Want to try growing your own tomatoes for your family? If you live in colder climates, plan ahead and plot out a garden for next spring. If you work some organic matter deep into your garden bed and have plenty of sunshine, your plants can reach 7-8 feet tall, my do! For those of you in milder climates, it's still not too late to have one more round of tomatoes before the end of November. Granted the plants won't yield as much as a summer garden, but you'll have enough to add to sandwiches and salads. Just select a tomato plant that matures quickly such as Early Girl. If you are limited on space, tomato plants aren't picky, they will grow fine in containers, just not as tall.
One more tomato tip, if you ever taste a tomato in a restaurant that you like save a slice and throw it in some dirt and water it, it will grow. Soon you will have an abundance of the tasty tomatoes. If it's too cold to plant the slice, just save some of the seeds in a cool dry place and start them in the spring. We've done this several times and it not only results in tasty tomatoes, but it's fun too!
Happy gardening!
Keep watching GoGoGoodie.com for spring time gardening goodie bags / loot bags.Tweet


text:
Milk Oolong tea is undoubtedly one of the most popular Chinese teas outside China. What is the secret of such popularity?
Apparently it is in its brightness, easiness and positive! Its incredible milky aroma and flavor will definitely delight you and your guests from the first time!
This tea is flavored.
How to brew Milk Oolong tea:
Boil water and cool it to 90-95°C. Put 3-5 grams of tea into a cup, and add hot water. Infuse tea leaves for 1-2 minutes depending on taste preference. You can vary the quantity of tea depending on your tastes. Each portion of tea may be infused 3-5 additional times.
- Origin Place: Fujian province
- Harvest time: May, 2016.
- Strong milk fragrance
- Low caffeine (less than 10% of a cup of coffee)


text:
Foreclosure Lawyer
Find Foreclosure Lawyers and Foreclosure Law Firms. Now that you have selected a practice area for your attorney search, narrow your results to find Foreclosure attorneys for your state. Or to expand your search for more attorneys, search all Real Estate areas including Timeshare, Construction, Eminent Domain, Land Use & Zoning, Landlord-Tenant and Other Real Estate attorneys.


text:
Blocking an inbound number
Unanswered Question
User Badges:
Hi all,
I have a customer that wants to block a particular number from dialing into the company.
I know you can use h323 to do this but they are currently using MGCP though.
They are upgrading to 7.0 soon and I thought that there was an option in 7.0 where you could block an inbound call with mgcp on the call manager.
is this true?
I have this problem too.
0 votes


text:
Shop by Price
Add you Tags
Item specifics
Brand New!!!
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Color: Chrome, Black (please make a note which color you want or we send by random)
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Package includes:
A Pair of Brackets
1) We accept Apay, West Union, TT. All major credit cards are accepted through secure payment processor ESCROW.
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Product Description
Packaging Details Unit
Type: pair
Weight: 0.062kg (0.14lb.)
Package Size: 7cm x 2cm x 2cm (2.76in x 0.79in x 0.79in)
If you would like to pay by PayPal, you can pay to our PayPal account: firstname.lastname@example.com and email us. Please email us the name of the product, the quantity and other product attributes. The email address is email@example.com. No matter how many goods you buy in one order, the freight you need to pay is only 15 dollars
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text:
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text:
Forums: Index > Requested Themes > Postman Pat wiki/Theme
| Abandoned Request
This is an abandoned request. This wiki does not currently have a Theme update. Or it has been inactive for a while.
Please replace the
Words "Postman Pat Wiki" in Aplha Matter font, red backroud, yellow letters22.214.171.124 22:54, 5 May 2009 (UTC)


text:
01 Transaction Pro Export Wizard 6.0 - Export Transactions or Lists from QuickBooks to Excel
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Want an easy way to export QuickBooks transactions or lists to Excel? The Transaction Pro Exporter 6.0 is the solution you've been looking for. Easily search for transactions or lists, review or edit them in program grid, then save to Excel in CSV or TAB delimited text format. Choose the fields you want to export including their field names and sort order. Supports lists with custom fields. New in version 6.0 is advanced filtering and the ability to pull data from reports. Data is exported in industry standard flat file format with repeating header rows for each detail row. Use in tandem with 01 Transaction Pro Importer to move data from one QuickBooks file to another regardless of version.
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Active Ingredients** - Purpose*: Allium Cepa 5C HPUS - Relieves Runny Nose Associated with Allergies. Ambrosia Artemisiaefolia 5C HPUS - Relieves Spasmodic Cough Associated with Hay Fever. Euphrasia Officinalis 5C HPUS - Relieves Burning, Irritated Eyes. Histaminum Hydrochloricum 9C HPUS - Relieves Symptoms of Allergic Conditions. Sabadilla 5C HPUS - Relieves Sneezing and Itchy Throat Associated with Allergies. Solidago Virgaurea 5C HPUS - Relieves Abundant Nasal Discharge. Inactive Ingredients: Croscarmellose Sodium, Lactose, Magnesium Stearate.
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Inner medulla collecting duct (IMCD) cells are the key part for urinary concentration. Hypotonic stress may trigger apoptosis of IMCD cells and induce renal injury. Epoxyeicosatrienoic acids (EETs) play an important role in anti-apoptosis, but their roles in hypotonic-induced apoptosis of IMCD cells are still unclear. Here we found increasing exogenous 11, 12-EET or endogenous EETs with Ad-CMV-CYP2C23-EGFP transfection decreased apoptosis of IMCD cells induced by hypotonic stress. Moreover, up-regulation of γ-ENaC induced by hypotonic stress was abolished by elevation of exogenous or endogenous EETs. Collectively, this study illustrated that EETs attenuated hypotonic-induced apoptosis of IMCD cells, and that regulation of γ-ENAC may be a possible mechanism contributing to the anti-apoptotic effect of EETs in response to hypotonic stress.
Citation: Wang L, Liu Y, Wang H, Liu X, Chen J, Wang M-H, et al. (2014) Epoxyeicosatrienoic Acids Attenuating Hypotonic-Induced Apoptosis of IMCD Cells via γ-ENaC Inhibition. PLoS ONE 9(4): e94400. https://doi.org/10.1371/journal.pone.0094400
Editor: Jeff M. Sands, Emory University, United States of America
Received: January 21, 2014; Accepted: March 13, 2014; Published: April 8, 2014
Copyright: © 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by NSFC [91029742, 81170647 and 81370837 to HH], Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China [132030], Yat-sen Scholarship for Young Scientists, the Science & technology star of Zhujiang (Guangzhou)[2011016] to HH, NSFC [81370866 and 81070612] to XL, AHA Grant-in-Aid grant (AHASE0054)to MHW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The kidney maintains stabilized extracellular volume and osmolality in body predominantly through electrolyte and water reabsorption. Inner medulla collecting duct (IMCD) is the final part of collecting duct, together with cortical collecting duct, contributing to reabsorbing 4–5% of filtered sodium. Sodium enters principal cells of IMCD through epithelial sodium channel (ENaC) [1]. ENaC consists of three homologous subunits, α, β and γ, which share 35% homology in amino acid sequence, among these three subunits, γ-ENaC is the major subunit responsible for sodium absorption in IMCD [2], [3]. From diuresis to antidiuresis, IMCD cells are constantly subjected to varying osmolar conditions [1]. However, severe osmotic stress exceeding the compensative capacity of cells may trigger a self-destruction program to programmed cell death (apoptosis) [4]. ENaC, as a rate-limiting step in transepithelial sodium reabsorption, has been involved in the process [5]–[8].
Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) by cytochrome P450 (CYP) epoxygenases, including four regioisomers: 5, 6-, 8, 9-, 11, 12-, and 14, 15-EET. These EETs are synthesized mainly by epoxygenases of the CYP2C and CYP2J family [9]. It is now widely accepted that CYP2C23 is the predominant and functionally relevant epoxygenase isoform in rat kidney. It is mainly responsible for 11, 12-EET formation [10]. EETs exhibit diverse and critical biological effects in renal protection, including regulating renal circulation [11]–[13], anti-inflammation, anti-thrombosis [14], increasing cell survival and decreasing apoptosis [15]–[19].
Sustained hypotonic stress in luminal and interstitial surroundings challenges the survival of IMCD cells[20]. Although, previous researches demonstrated that EETs are anti-apoptotic in several cases [9], [19], [21], the role of EETs in IMCD cells is not clear. Do EETs protect IMCD cells from the challenges of sustained hypotonic stress? The present studies were designed to determine the effects of EETs on IMCD cells exposed to hypotonic stress. Furthermore, we determined whether the effects of EETs on the apoptosis of IMCD cells under hypotonic stress were mediated through γ-ENaC, as one of the possible mechanisms.
Hypotonic stress inhibited proliferation and induced apoptosis in IMCD cells
To evaluate the effects of hypotonic stress on IMCD cells, MTT and FACS analysis were performed. As shown in Fig. 1A, B, the proliferation of IMCD cells began to decrease after being exposed to hypotonic stress for 12 h (hours), but the decrease in proliferation rate was not significant until the 24th hour (0.6±0.08 vs. 0.84±0.04, P<0.05). Meanwhile, flow cytometry analysis (FACS) results showed that the apoptosis percentage increased significantly after being exposed to hypotonic stress 12 h compared to isotonic group (29.9%±1.8% vs. 0.9%±0.08%, P<0.05). The apoptosis percentage was further increased after extending exposure time to 24 h (31.6%±3.28% vs. 23.4%±1.24%, P<0.05). These results illustrated that hypotonic stress inhibited proliferation and induced apoptosis of IMCD cells in a time-dependent manner.
(A) MTT assay assessing the proliferation of IMCD cells exposed to hypotonic or isotonic stress for 0 h, 12 h and 24 h. (B) FACS assessing the apoptosis percentage of IMCD cells exposed to hypotonic or isotonic stress for 0 h, 12 h and 24 h. *, P<0.05 compared with isotonic group. #, P<0.05 compared with isotonic group. Error bars represent the standard deviations based on three independent replicates.
Overexpression of CYP2C23 by adenovirus in IMCD cells
To demonstrate the transfection efficiency of recombinant CYP2C23, we observed the green fluorescence in IMCD cells transfected with Ad-CMV-CYP2C23-EGFP and adenovirus vectors without CYP2C23-EGFP (vehicle vectors) at MOI of 100. The transfection yielded marked GFP protein expression (green fluorescence) in IMCD cells, but not in vehicle-treated cells (Fig. 2). The transfection efficiency was 80.35%±3.23% at 24 h. It indirectly indicated the overexpression of CYP2C23 and elevation of CYP2C23-mediated EETs in IMCD cells.
IMCD cells were transfected with vehicle adenovirus vectors and Ad-CMV-CYP2C23-EGFP (green) at MOI of 100. Fluorescence intensity was observed at 24 h post-transfection (×10). (A) Vehicle adenovirus vectors. (B) Ad-CMV-CYP2C23-EGFP.
EETs alleviated the inhibition of IMCD cells proliferation induced by hypotonic stress
To evaluate the effects of exogenous and endogenous EETs on IMCD cells proliferation exposed to hypotonic stress, we conducted experiments involving either addition of 11,12-EETs or elevation of endogenous EETs via overexpression of CYP2C23 by Ad-CMV-CYP2C23-EGFP, and then determined the proliferative effects of hypotonic stress on IMCD cells by MTT viability assays. As shown in Fig. 3, hypotonic stress caused a time-dependent decrease in the proliferation of IMCD cells compared with isotonic stress. Exogenous 11,12-EET (100 ng/ml) significantly attenuated the inhibition of cell proliferation induced by 24 h hypotonic stress exposure. Increasing endogenous EETs by Ad-CMV-CYP2C23-EGFP transfection showed similar effects on IMCD cells. These findings indicated that CYP epoxygenase-mediated EETs promoted the proliferation of IMCD cells.
The results of MTT assay on assessing the effect of 11,12-EETs (100 ng/ml) or Ad-CMV-CYP2C23-GFP (100 MOI) transfection on the proliferation of IMCD cells exposed to 0 h,12 h or 24 h hypotonic stress. *, P<0.05 compared with isotonic group. #, P<0.05 compared with hypotonic group. &, P<0.05 compared with hypotonic group. Error bars represent the standard deviations based on three independent replicates.
EETs prevented the apoptosis of IMCD cells induced by hypotonic stress
As mentioned above, treatment with 11,12-EET or Ad-CMV-CYP2C23-EGFP transfection in IMCD cells recovered the inhibition of proliferation induced by hypotonic stress, suggesting potential anti-apoptotic effect of EETs. For further investigation, apoptosis was determined by annexin-V-FITC-PI analysis of IMCD cells exposed to hypotonic stress using flow cytometry. Treatment with 11,12-EET resulted in a moderate decrease in the apoptosis percentage of IMCD cells exposed to 12 h of hypotonic stress (25.10%±2.49% vs. 29.90%±1.8%, P>0.05), indicating a possible anti-apoptotic effect of 11,12-EET. Similarly, Ad-CMV-CYP2C23-EGFP transfection also decreased apoptosis (27.30%±3.34% vs. 29.90%±1.8%, P>0.05) (Fig. 4A, C). The apoptosis percentage of IMCD cells exposed to hypotonic stress for 24 h was steadily alleviated by treatment with exogenous 11,12-EET or Ad-CMV-CYP2C23-EGFP transfection (22.90%±2.56%, 22.20%±3.13% vs. 31.60%±3.28%, P<0.05) (Fig. 4B, D). The results demonstrated that CYP epoxygenases and their eicosanoid metabolites EETs prevented the apoptosis of IMCD cells induced by hypotonic stress in a time-dependent manner.
Representative figures and results of FACS analysis on the effect of 11,12-EETs (100 ng/ml) or Ad-CMV-CYP2C23-GFP (100 MOI) transfection on IMCD cells apoptosis induced by 12 h or 24 h hypotonic stress. (A) (C) 11,12-EETs or Ad-CMV-CYP2C23-GFP transfection showed an inhibitory but non-significant effect on the apoptosis of IMCD cells induced by 12 h hypotonic stress. (B) (D) 11,12-EETs or Ad-CMV-CYP2C23-GFP transfection significantly inhibited the apoptosis of IMCD cells induced by 24 h hypotonic stress. *, P<0.05 compared with isotonic group. #, P<0.05 compared with hypotonic group. &, P<0.05 compared with hypotonic group. Error bars represent the standard deviations based on three independent replicates.
EETs inhibited the up-regulation of γ-ENaC induced by hypotonic stress
To explore whether γ-ENaC is involved in the anti-apoptotic effects of EETs, we determined the protein level of γ-ENaC in IMCD cells exposed to hypotonic stress in absence and presence of 11,12-EET or Ad-CMV-CYP2C23-EGFP transfection. We found that hypotonic stress increased γ-ENaC expression as well as the apoptosis in IMCD cells at 12 h and 24 h. At both time points, treatment with 11,12-EET attenuated the elevation of γ-ENaC protein expression while decreased the apoptosis of IMCD cells. Ad-CMV-CYP2C23-EGFP transfection produced similar results at both time points (Fig. 5A, B, C, D). These data indicated that up-regulation of γ-ENaC could be induced by hypotonic stress, and that increasing exogenous and endogenous EETs inhibited the up-regulation of γ-ENaC, which suggested the involvement of γ-ENaC in the anti-apoptotic role of EETs in IMCD cells exposed to hypotonic stress.
EETs and CYP2C23 transfection decreased apoptosis of IMCD cells induced by hypotonic stress via γ-ENaC. (A)(B) EETs or Ad-CMV-CYP2C23-EGFP transfection inhibited the up-regulation of γ-ENaC induced by 12 h hypotonic stress. (C) (D) EETs or Ad-CMV-CYP2C23-EGFP transfection inhibited the up-regulation of γ-ENaC induced by 24 h hypotonic stress. (A) (C) Representative blots. (B) (D) Densitometric analysis. *, P<0.05 compared with isotonic group. #, P<0.05 compared with hypotonic group. &, P<0.05 compared with hypotonic group. Error bars represent the standard deviations based on three independent replicates.
In the present study, we showed that hypotonic stress inhibited proliferation and induced apoptosis in IMCD cells. Endogenous or exogenous EETs exhibited anti-apoptotic and pro-proliferative effects on the IMCD cells exposed to hypotonic stress. Down-regulation of γ-ENaC may be one of the possible mechanisms contributing to the process.
In the present study, IMCD cells were exposed to a hypotonic culture medium (200 mOsmol/kgH2O) composed of NaCl for 12 h and 24 h. Extremely low concentration of sodium results in hypotonic stress, challenging the survival of IMCD cells. Previous work showed that hypotonic stress significantly lowered the viability of mIMCD3 cells (an established cell line of inner medulla collecting duct derived from murine) [22]. Here, we further demonstrated that hypotonic stress inhibited proliferation and induced apoptosis of IMCD cells in vitro.
EETs show anti-apoptotic effects in some cases. Overexpression of CYP2J2 gene by recombinant adeno-associated virus (rAAV) delivery protects remnant kidney against renal injury in 5/6-Nx rats by inhibiting apoptosis [19]. 14,15-EET inhibits the apoptosis in LLCPKcl4 (an established adherent proximal tubule-like epithelial cell line derived from pig kidney) induced by serum starvation, H2O2, or etoposide [9]. The sEH-deficient mice with an elevated level of endogenous EETs show decreased renal tubular apoptosis induced by diabetes [23]. In the present research, treatment with exogenous 11, 12-EET or increasing endogenous EETs by overexpression of CYP2C23 showed significant pro-proliferative and anti-apoptotic effects on the IMCD cells exposed to hypotonic stress. Our results added new evidences for the anti-apoptotic role of EETs in renal protection.
The mechanisms involved in the anti-apoptotic effects of EETs appear to differ depending on species, type of cell, and EET regioisomers [9], [15], [17], [19], [23]. EETs have been proven to inhibit the expression and activity of ENaC. In polarized M1 cells (derived from mouse lymphocytes), 14, 15-EET inhibits ENaC, which is associated with increased threonine phosphorylation of the ENaC β and γ subunits [24]. CYP-epoxygenase metabolites of AA, mainly 11,12-EET, inhibits ENaC in cortical collecting duct. Down-regulation of CYP2C44 abolishes AA-mediated inhibition of ENaC [10], [13], [25]. What's more, ENaC has been shown to be involved in the process of apoptosis. For example, α-ENaC is involved in the apoptosis of primary rat alveolar epithelial cells induced by hypoxia [6], [8], as well as the apoptosis of HepG2 (a perpetual cell line derived from hepatocellular carcinoma) [7]. In this study, we observed that hypotonic stress increased γ-ENaC expression in IMCD cells, which was abolished by endogenous and exogenous EETs.
Nevertheless, how ENaC regulated apoptosis is still unclear. Electrochemical gradients change of monovalent ions across cell membrane lead to cell volume perturbations, which ultimately result in cell death [26]. Marunaka, Y et al. reported that up-regulation of ENaC increased Na+ influx paralleling with Cl-, the cell volume would increase associated with an increase in cytosolic Cl- concentration[27]-[29]. Admittedly, contrasting cell volume behaviors (shrinkage vs. swelling) are features that commonly serve to distinguish apoptosis from necrosis, but cell type-specific rather than ubiquitous involvement of cell shrinkage results in cell apoptosis machinery triggering and progression. In some cases, apoptotic collapse may occur in the absence of cell volume changes and even follow cell swelling rather than shrinkage [30], [31]. Therefore, more convincing researches are needed to demonstrate how ENaC regulates cell volume and how cell volume perturbations induce apoptosis.
In the present study, we only detected the expression of γ-ENaC as one of the potent mechanisms. More solid evidences are needed, for example, we could silence the γ-ENaC gene or use some specific antagonists to block γ-ENaC for further verifying its involvement in the anti-apoptotic role of EETs and CYP2C23 in vitro. ENaC consists of three homologous subunits, α, β and γ. α-ENaC is mainly present at the apical domains of principal cells, whereas β- or γ-ENaC is associated with intracellular vesicles dispersed in entire cytoplasm [32]. This suggests that there may be some differences in the regulation of different ENaC subunits. Previous studies have shown discrepancies with regard to the three subunits [33], [34]. In the present study we chose γ-ENaC as the target protein because it is the predominant channel responsible for sodium absorption in rat IMCD [2], [3]. Admittedly, α- and β-ENaC may also be involved in the process. Future studies will be aimed at defining the role of other ENaC subunits in the proliferation and apoptosis of IMCD cells. Taken together our results indicated that inhibition of γ-ENaC may be one of the possible mechanisms contributing to the anti-apoptotic effects of EETs on IMCD cells exposed to hypotonic stress.
Furthermore, γ-ENaC has been described to be involved in the anti-apoptotic role of EETs, but the signaling mechanisms are still unclear. The data from our work and others indicated that EETs by CYP2C and/or CYP2J significantly promoted endothelial cell proliferation and attenuated apoptosis, which was associated with PI3K/AKT and MAPK signaling pathways [35]–[37]. Naomi Niisato and some other scientists have shown that three members of the MAPK family, p38, JNK and ERK, and the EGFR-JNK-PI3K pathway contribute to the stimulation of sodium reabsorption in response to hypotonic stress [38]–[40]. Therefore, MAPK family and PI3K/AKT pathway may be involved in the anti-apoptotic role of EETs via γ-ENaC in IMCD cells exposed to hypotonic stress. Further studies are required to illustrate the potent relationships between them.
In addition, as many other ion channels, ENaC is regulated by factors that alter gating, expression, subunit assembly, membrane translocation, phosphorylation/dephosphorylation, and residence time. Multiple mechanisms can alter one or more of these factors, including proteolysis, hormonal effects on translocation and membrane assembly, changes in ubiquitination, retrieval, and degradation, and protein kinase-mediated negative or positive effects [24]. In this study we did not probe into this aspect. Further studies are required to define the precise mechanisms on the role of ENaC in anti-apoptotic process.
In conclusion, our results demonstrated that hypotonic stress inhibited proliferation and induced significant apoptosis in IMCD cells. CYP epoxygenases-mediated EETs showed an anti-apoptotic effect on IMCD cells exposed to hypotonic stress. Down-regulation of γ-ENaC may be one of the possible mechanisms contributing to the process.
Materials and Methods
Experiment Reagents
All cell culture reagents were obtained from GibcoBRL (Life Technologies, Inc., Grand Island, NY). Rabbit β-actin mAb was from Cell Signaling Technology Inc (Beverly, MA). Rabbit polyclonal antibody to γ-ENaC was from Abcam Inc. (Cambridge, UK). Enhanced chemiluminescent substrate was from Pierce (Rockford, IL). PVDF membranes were from Merck Millipore (Billerica, MA). Annexin-V-FITC-PI apoptosis detection assay kits, 11,12-EET, NG-methyl-L-arginine,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Dnase I were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). All other chemicals and reagents were from Sigma-Aldrich Chemical Company unless otherwise specified.
Ethics Statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Sun Yat-sen University (Approval Number: SYSU-2013-23). Rats were anesthetized with ether and decapitated, and all efforts were made to minimize suffering.
IMCD Cells preparation
IMCD cells were isolated from male Sprague-Dawley rats using hypotonic lysis isolation method described previously [41]. Rats were anesthetized with ether and decapitated. The kidneys were removed and rinsed with D-Hanks solution containing antibiotics three times. The inner 1/3 medulla was dissected, minced, and incubated in 0.1% collagenase I at 37°C for 90 min. The IMCD cells were isolated by a further incubation with the above solution made hypotonic with distilled water containing 0.01% deoxyribonuclease I, and cells were digested on shaker at 37°C for 45 min. After filtering with 200 mesh filter and centrifugation, the cell pellets were washed and resuspended with medium containing 1∶1 mixture of Dulbecco's Modified Eagles Medium (DMEM) and Ham's F-12 Medium supplemented with 10% fetal bovine serum (FBS). In this way, we acquired IMCD cells with purity more than 90% [42]–[44]. Cells were seeded at a density of 1.0×106 cells/well in six well plates and culture flasks. After cells reached 80-90% confluence on the 3rd day, experiments were conducted on the 5th or 6th day [45].
Overexpression of CYP2C23 by adenovirus in IMCD cells
To achieve overexpression of CYP2C23 in vitro, we used adenoviral CYP2C23 vector, Ad-CMV-CYP2C23 (Vector Biolabs, Philadelphia, PA). In the Ad-CMV-CYP2C23 virus, expression of CYP2C23 is under the control of CMV promoter. After three rounds of amplification in QBI-293 cells, we were able to obtain a high titer of Ad-CMV-CYP2C23 (4×1010 VP/ml). By using the similar approach, we obtained enhanced green fluorescent protein (EGFP) vector (Ad-CMV-CYP2C23-EGFP) through a custom adenovirus service. The virus contains internal ribosome entry site of encephalomyocarditis virus between CYP2C23 and EGFP coding region, allowing both CYP2C23 and EGFP to be translated from a single bicistronic mRNA. To demonstrate the expression of recombinant CYP2C23, we transfected IMCD cells, which do not express endogenous GFP, with Ad-CMV-CYP2C23-EGFP at multiplicity of infection (MOI) of 100, and cultured the cells for 24 h. Recombinant adenovirus vectors without CYP2C23-EGFP (100 MOI) were added as control. The fluorescence intensity of GFP was observed by Inverted Flurescence Microscopy (Leica DM13000B, Germany) and quantitated by Image-Pro Plus 6.0 (Media Cybernetics) [46]–[49].
Effects of EETs and Ad-CMV-CYP2C23-EGFP transfection on IMCD cells proliferation under hypotonic stress
The effects of EETs and Ad-CMV-CYP2C23-EGFP transfection on IMCD cells proliferation under hypotonic stress were evaluated by MTT colorimetric assay. IMCD cells were seeded in triplicate in 96-well plates (1×104 cells/well) with 10% FBS DMEM/F12 medium for 72 h. When cells reached 80% confluent, the medium was changed to experimental medium with different osmotic pressure: isotonic (300 mOsmol/kgH2O, DMEM/F12) and hypotonic (200 mOsmol/kgH2O, DMEM/F12+3.4% (V/V) pure water). Simultaneously, the cells were stimulated with 11,12-EETs at 100 ng/ml or recombinant adenovirus Ad-CMV-CYP2C23-EGFP (100 MOI) [48], [49]. After 12 h or 24 h, viable cell numbers were estimated by MTT assay. Briefly, medium was removed and replaced with medium containing 5 mg/ml MTT and incubated for 4 h. The medium was aspirated, and the product was solubilized with dimethyl sulfoxide. Absorbance was measured at 490 nm using a microplate reader according to manufacturer's protocol.
Flow Cytometry Analysis
Annexin-V-FITC-PI apoptosis detection assay kits were used to determine the apoptotic rate of IMCD cells. Annexin-V-FITC and PI were used for staining following the manufacturer's staining procedure. Briefly, cells were seeded at a density of 1.0×106 cells/well in six well plates. After intervention described above, cells were harvested and washed with cold phosphate buffered saline (PBS), and then centrifuged. The cell pellets were resuspended to 5.0×105 cells/ml. Annexin V-FITC and PI were added to cell suspensions, and the mixture was incubated on ice for 10 min in the dark. After incubation, the samples were tested. Excitation and emission settings were 492 nm and 520 nm respectively for annexin V-FITC, and 550 nm and 560–680 nm for PI (FACStar-Plus flow cytometer, BD Biosciences, San Jose, CA) [50].
Protein Extraction and Western Blotting
The protein of IMCD cells was extracted as previously described [41]. Briefly, the media in 6-well plate was discarded and cells were gently washed three times with cooled PBS. Lysis buffer (RIPA and PMSF) was added to the cells. After incubation on ice for 30 minutes, the lysate was centrifuged at 12,000 g at 4°C for 10 minutes. The protein concentrations of the supernatant were determined. Lysate samples (25 μg protein/lane) were resolved by SDS-PAGE (10%), transferred to nitrocellulose membranes, and blocked with 5% nonfat dry milk in TBS-T (10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 0.1% Tween-20). The membranes were then incubated with rabbit polyclonal antibody to γ-ENaC (1∶1000 dilution) overnight at 4°C, followed by peroxidase-conjugated secondary antibody for 2–3 h. The ECL system was used to visualize the separated proteins. The expression of β-actin is used as loading control.
Statistical Analysis
All experiments were repeated at least three times. Data were analyzed with SPSS 17.0 statistical software package (SPSS, IL, USA) and presented as mean ± SD. Comparisons between groups were performed by a Student's paired two-tailed t test. Two-way analysis of variance was used to examine differences between groups, with post hoc analyses performed by Student-Newman-Keuls method. P<0.05 was considered to be statistically significant.
Author Contributions
Conceived and designed the experiments: HH. Performed the experiments: LYW YL HMW. Analyzed the data: XL JC. Contributed reagents/materials/analysis tools: LYW YL HMW. Wrote the paper: LYW YL HH. Helped to draft the manuscript: MHW. Reviewed the manuscript and made final changes: JFW.
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