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Latest Drama ReleasesMore
Oh My Ghostess
1.24K Views0 Comments39 Likes
Na Bong-sun has an extremely timid personality and low self-esteem, doesn't have any close friends, and is constantly getting reprimanded at her job as a restaurant sous chef. She also occasionally sees ghosts, thanks to a shaman grandmother. One day, Bong-sun gets possessed by a lustful virgin ghost named Shin Soon-ae. To make up for the lack of romance in her short life, Soon-ae is determined to seduce as many men as she can by possessing various women, and she finds the perfect vessel in Bong-sun. Bong-sun's boss is arrogant star chef Kang Sun-woo, whom she secretly has a crush on. Sun-woo still hasn't gotten over an old girlfriend, but when Bong-sun seemingly gets rid of her shyness and suddenly changes into a confident, dynamic woman, she finally catches his eye.
My Beautiful Bride
1.98K Views0 Comments37 Likes
This drama is about a husband, Kim Do Hyung (Kim Moo Yul) who desperately searches for his wife, Yoon Joo Young (Go Sung Hee) who suddenly disappears. Meanwhile, Cha Yoon Mi (Lee Si Young) is the detective on the case who helps him along the way. "My soon-to-be bride all of sudden disappeared. And I turned into a monster to find her". The drama depicts the heartbreaking, pure hearted love by a man, who drives himself to the extreme situation in order to find his only love, his bride.
The Time I Loved You
3.38K Views0 Comments37 Likes
Something that's right in front of your eyes is sometimes the hardest for you to see. Jang Ha Na (Ha Ji Won) and Choi Won (Lee Jin Wook) have been lifelong friends. Through thick and thin, they've been through many things together in the nearly 20 years of friendship, including relationships that each has had with other people. Approaching her 30th birthday, Ha Na receives an email from Won that jokes about the "symptoms of premature aging." Angry about the widely held belief that men are like grapes that turn into a fine bottle of wine as they age while women are like grapes that shrivel up into raisins, Ha Na makes a bet with Won that she can get married first before they turn 35. Will Ha Na and Won be able to find their true loves or have they been blind to what's been there all along? "The Time I've Loved You (aka The Time We Were Not in Love)" is a 2015 Korean dramas directed by Jo Soo Won. It is a remake of the popular 2011 Taiwanese drama "In Time With You."
I Remember You
3.55K Views0 Comments36 Likes
All different personalities come together in the police department's Special Crime Investigation Team. Lee Hyun (Seo In Guk) is a genius criminal profiler with a degree in criminal psychology whose tongue is as sharp as his wit. He is forced to work with fellow colleague Cha Ji An (Jang Na Ra), an elite detective who has an ulterior motive for being in her profession and becoming a member of the team. Can the two different investigators learn to work together as they go after dangerous criminals? "I Remember You" is a 2015 South Korean drama series.
Make A Woman Cry
3.90K Views0 Comments36 Likes
Jung Duk In was a former detective who resigned after her son died. She then opens a food stall in front of her son's school to preserves the memory of him and to protects the other students. Along the way she will struggle to forgive people who were responsible for her son's death.
Hidden Identity
3.10K Views0 Comments37 Likes
The heat is on in Investigation 5, an undercover police unit specializing in thwarting violent crimes. Led by Superintendent Jang Moon Won (Park Sung Woong), this team of young and elite officers, including Cha Gun Woo (Kim Bum), Jang Min Joo (Yoon So Yi), and Choi Tae Pyung (Lee Won Jong), assume new identities and infiltrate dangerous crime syndicates. With only their wits and poker faces at their disposal, can the team complete their missions without blowing their covers in hostile territory?
High Society
6.62K Views0 Comments39 Likes
Jang Yoon-ha is the youngest daughter of a chaebol family. Despite her wealth, she works part-time at a food market and hides her true identity as an heiress because all she wants is to find a man who'll love her for herself and not her money. Choi Joon-ki grew up in extreme poverty, and has a huge chip on his shoulder about his poor parents being belittled. Now working as a middle manager for a conglomerate, Joon-ki is smart and talented but also jaded and calculating, believing that love is a luxury only for the wealthy.
My Love Eun Dong
5.58K Views0 Comments35 Likes
A major star has achieved the success he has always wanted and wants to fill the emptiness he has had in his heart for the past 20 years. In 1995, Park Hyun Soo (Junior) is an energetic 17-year-old boy when he meets 13-year-old Ji Eun Dong (Lee Ja In), an orphan who still has a positive outlook on life. Hyun Soo's heart is awakened by Eun Dong but she suddenly disappears before he could really get to know her. In 2005, Hyun Soo now goes by the stage name Ji Eun Ho (Baek Sung Hyun) and aspires to become an actor. When he bumps into Eun Dong (Yoon So Hee) again after 10 years, the two fall deeply in love. But again without warning, Eun Dong disappears without a trace. In 2015, Eun Ho (Joo Jin Mo) is now a top actor who has the fame and wealth he has always wanted. But what he doesn't have is the love of his life to share his success with. So Eun Ho decides to write an autobiography that details his love for Eun Dong over the past 20 years in the hopes of locating her. He hires a ghostwriter, Seo Jung Eun (Kim Sa Rang), to help him write the book. After he dictates his stories of Eun Dong, he sends them to Jung Eun to transcribe and incorporate into his book. But why do these stories sound vaguely familiar to Jung Eun? "My Love Eun Dong (aka This Is My Love)" is a 2015 South Korean drama series directed by Lee Tae Gon.
6.64K Views0 Comments36 Likes
Byun Ji Sook has always had a hard life with her father constantly going into debt and being chased by loan sharks. She desperately wants to know what life is like if she was born into a better family and didn't have to worry about money all the time. When a series of circumstances brings Ji Sook to Eun Ha, a woman from a wealthy family who strongly resembles Ji Sook, she grabs hold of the chance for a better life and assumes Eun Ha's identity and begins to live her elite life. She meets Min Woo, a chaebol heir who is mistrusting of the people around him. But when he realizes that Ji Sook is not like the other women he has met, he is drawn to her. How long can Ji Sook keep up the charade and hide her true identity?
Masked Prosecutor
5.49K Views0 Comments37 Likes
The story is about a man, played by Joo Sang Wook with a double identity. He's a normal prosecutor during the day and a masked vigilante at night. Kim Sun Ah will play a role of detective chief in the violent crimes unit.
Orange Marmalade
9.39K Views0 Comments37 Likes
Despite a 200-year-old treaty between humans and vampires, both races still don't get along. Amidst the looming tension, Jung Jae Min (Yeo Jin Goo), a posh yet kindhearted high school student, quickly falls for the mysterious new girl Baek Ma Ri (Seol Hyun). But can Ma Ri conceal her true vampire identity and give love an honest chance? If these star-crossed lovers can find the courage to make the leap, Jae Min and Ma Ri may just be the key to a final resolution between humans and vampires…
8.75K Views0 Comments36 Likes
A group of young television producers — Ra Jun Mo (Cha Tae Hyun), Baek Sung Chan (Kim Soo Hyun), Tak Ye Ji (Gong Hyo Jin), and Cindy (IU) — work tirelessly around the clock within the entertainment division of a major broadcasting network. Amidst their dynamic and challenging workloads, serious drama reaches new heights in this backstage glimpse of the people who make television magic happen.
My Love From Another Star
106.28K Views0 Comments20 Likes
Do Min-Joon is an alien who landed on Earth 400 years ago during the Joseon Dynasty period. Do Min-Joon possesses a near perfect appearance and enhanced physical abilities involving his vision, hearing and speed. Do Min-Joon holds a cynical view to human beings, but he falls in love with actress Cheon Song-Yi.
The Heirs
47.60K Views0 Comments12 Likes
A romantic comedy about high school students living in top 1% high society. Things get turned topsy turvy when the students end up getting tangled with a girl from lower class who is the heir of poverty, and romance unfolds.
Glorious Day
45.58K Views0 Comments16 Likes
Hong Song-jung is a novelist and single mother. She raised three daughters on her own and is determined to marry them off to decent men. But in the end, Song-jung finds herself getting married as well. Jung Da-ae is the eldest daughter; she is the perfect bride material — not only beautiful and smart, she also helped raise her younger sisters with loving care. Jung Da-jung is the middle daughter, a girl overflowing with warmth and boundless optimism, who cheers up anyone around her. Jung Da-in is the youngest daughter. The family next door runs a rice cake store, and their eldest grandson is Seo Jae-woo. Jae-woo earns a steady income as a salaryman at a large company, and has some brusque and conservative traits he inherited from his grandfather, but also some sweet gentlemanly traits from his father. It makes him rather stuffy and old-fashioned, but he's a good guy, and becomes romantically involved with Da-jung.
Doctor Stranger
41.20K Views0 Comments18 Likes
The series will revolve around Park Hoon, a South Korean who was captured with his father and grows up in North Korea. He is trained to become a genius doctor, and though Park Hoon lands a job at the best South Korean hospital after defecting, he is treated as a foreigner. To find his lost North Korean first love, Song Jae Hee, he does anything to make money.
A Well-Raised Daughter
38.34K Views0 Comments10 Likes
Fated to Love You (Korean)
37.64K Views0 Comments18 Likes
A remake of the hit 2008 Taiwanese drama "Fated to Love You". Lee Gun (Jang Hyuk) is the president of a chemical company that produces household items. He has a girlfriend dancer who is deeply in love. Meanwhile, Kim Mi Young (Jang Na Ra) works as a secretary in a law firm. Her nickname is "Post-it" (sticky note). She earned his nickname because of his inability to say no to what they ask their colleagues. She collects task because they take advantage of their kindness and little character. One night, both of them are drunk and passes a night together. Then, she becomes pregnant….
37.57K Views0 Comments10 Likes
Eleven celebrities live together in close quarters as they share the cooking, cleaning and everyday chores around the house. Will they become close friends? Develop deeper relationships? Or find themselves fighting each other and making enemies? The celebrities include Park Chan Yeol (EXO), Hong Soo Hyun, Jo Se Ho, Lee Dong Wook, Lee So Ra, Na Na (After School), Park Bom (2NE1), Park Min Woo, Seo Kang Joon (5urprise), Shin Sung Woo, and Song Ga Yeon.
32.36K Views0 Comments30 Likes
The drama will be about young adults' first step into society, and the harried process by which they become journalists. When confronted with the truth they'll cry, get wounded, and be discouraged, but as they grow up they'll come to realize that protecting the truth is their job.
Marriage Without Dating
30.59K Views0 Comments17 Likes
This drama is about a man who doesn't want to get married and a woman who has no luck in marriage prospect. Gong Ki Tae (Yun Woo Jin) is a successful and happy bachelor who does not want to find himself a wife. He believes that his life is perfectly planned, and being single is the only option he accept. However, his family is badgering him to marry, and to get them to give up on their efforts, he then comes up with a plan to introduce Joo Jang Mi (Han Groo), whom he thinks will never be approved by his family…
Hotel King
29.97K Views0 Comments7 Likes
A love story between an heiress who tries to save her father's legacy, the one and only seven-star hotel in the nation and the hotel manager who becomes an enemy to his own father who abandoned him in order to help the heiress.
You're All Surrounded
27.47K Views0 Comments20 Likes
A drama about four newly recruited policemen in their 20s who have never thought about becoming detectives, but end up entering the violent crime unit at the Gangnam police station. The drama will follow their journey to become good detectives with romance, comedy, and action!
My Lovable Girl
27.33K Views0 Comments10 Likes
Hyun Wook (Bi) is the CEO of a talent agency that scouts and trains idols. He is a brilliant composer-producer who appears cold and indifferent on the outside but is sensitive and kind-hearted. Hyun Wook lives in great difficulty as he unable to let go of his ex girlfriend who dies in an accident he witnesses first hand. Said ex-girlfriend's sister, Se Na (Krystal Jung), moves to Seoul in order to pursue her music-composing career and meets Hyun Wook who helps her achieve it. In the midst of healing through music, they fall in love.
It's Okay It's Love
26.67K Views0 Comments21 Likes
Jang Jae Yul (Jo In Sung) is a mystery writer and radio DJ. He suffers from a obsession. He then meets a psychiatrist named Ji Hae Soo. Ji Hae Soo (Gong Hyo Jin) is going through her first year fellowship in psychiatry at a University Hospital. She chose psychiatry because she doesn't want to perform surgeries. After she meets Jang Jae Yul, her life goes through big changes. They end up becoming home-mates and things turn romantic.
Birth of a Beauty
25.33K Views0 Comments20 Likes
Han Tae Hee is the heir of a large company. He is suffering from a broken heart syndrome due to a psychological shock. To get the woman he loves back, he completely changes an unattractive and overweight Sa Geum Ran to beautiful Sara. In the process, Han Tae Hee falls in love with Sara.
King of High School
24.21K Views0 Comments14 Likes
A romantic comedy between a high school student who enters a major company who then becomes part of a management team and a woman who is a temporary employee at that same company. Lee Min Suk (Seo In Guk) is a high school student who plays ice hockey for his school. One day, he suddenly begins works as a company director through his resemblance to his older brother. At work, he meets Jung Soo Young (Lee Ha Na) who is a temporary employee there.
Surplus Princess
24.12K Views0 Comments23 Likes
Surplus Princess is a story about a mermaid who attempts to become a human being so that she can stay forever with the man she loves. Ha Ni (Jo Bo Ah) is a mermaid princess in the underwater empire but she loves for the human world when she sees and falls in love with a man. She transforms into a human and follows Hyun Myung (Ohn Joo Wan) to live in a temporary house for people who are preparing for employment. The pragmatic Hyun Myung is trying to find a good-paying job to provide him with a comfortable life. But will he be distracted by Ha Ni, who has just 100 days to make Hyun Myung fall in love with her so that she can permanently remain as a human being?


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We are turning things up at the 2015 Health, Labor, and Employment Law Institute at the Wilderness Hotel and Golf Resort in Wisconsin Dells.
One hour seminars provide lawyers with the business perspective to succeed as law practice owners.
Save 20% when you purchase 3 PINNACLE books in print; 25% on 4 PINNACLE books; and save 30% on 5 or more.

subset: commoncrawl


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Tag Archives: upgrades
Recommended ocPortal Addons
I wanted to talk about some of the addons for ocPortal which I install on a regular basis depending on which type of site I am creating. Some of them …
Tags: addon, addons, best, favorite, ocPortal, plugin, plugins, recommended, upgrades Posted under ocPortal Leave a comment
ocPortal 9.0.14 Released
The latest version of ocPortal has recently been released. This version is 9.0.14 and is primarily a bug fix update so it shouldn't create too many theming issues. I do …
Tags: 9.0.14, new version, ocPortal, release, updating, upgrade, upgrades, upgrading Posted under ocPortal Leave a comment
How to Create User Upgrades in Xenforo
Hello everyone. Today we will be discussing how to create User Upgrades via Xenforo. First, go ahead and log into your admin panel and click the Users tab at the …
Tags: add, create, creating, how, how to, to, upgrade, upgrades, user, users, Xenforo Posted under 3rd Party Software Leave a comment


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The perfect weight-loss article would include only four words: Eat less. Exercise more.
Of course, if it were really that simple, I'd be out of work and the R.D. after my name would stand for Really Desperate (for a job).
Unfortunately for you (and, I guess, fortunately for me), it's a lot more complicated than that. Some guys need to eat more to lose weight. And some who are already exercising need to do less, but at a higher intensity.
My goal here is to show you 10 ways that you can manipulate your diet -- adding in some places, subtracting in others -- to start losing weight the next time you open your mouth with the intention of putting something in.
I'm a mother, but I'm not your mother. So I can't order you to eat breakfast. But if you don't, you're putting your number-one fat-burning tool in time-out. It takes calories to digest calories, so eating in the morning fires up your metabolism.
Perfect breakfasts: Try a cup of whole-grain cereal with 1 percent low-fat milk and a banana, or two pieces of whole-wheat toast with two tablespoons of peanut butter and an apple. If you hate breakfast food, or have to eat on the fly, take a turkey sandwich (going easy on the mayo) or a slice of leftover cheese pizza and go.
Here's where I disagree with your mother. She discouraged you from eating between meals-probably so you'd eat more of her delicious boiled-liver casserole-but I want you to have a snack handy at all times.
Here's why: If breakfast stokes up your metabolic fires, then snacks keep them going between meals. And planning your snacks in advance keeps you from making a mid-morning Chips Ahoy run.
Perfect snacks: Include one of the following two or three times a day: an ounce of nuts, pretzels, or string cheese; a medium piece of fruit; six graham cracker squares; or six cups of low-fat microwave popcorn.
Latest Weight Loss Stories
How Eating One Apple Could Help You Eat Healthier for a Week
A new study discovered a fascinating snowball effect when you eat the fruit at the right time
Why Food Tastes Weird When You're on an Airplane
Cabin clatter could be the secret ingredient that throws off your taste buds
Here's the Actual Reason Why You Snack More at Night
Discover the science behind your late-night refrigerator raid
The Truth behind 5 Food Myths
Think sweet potatoes are superior to regular potatoes, and red meat will kill you? You may want to read this
4 Weird Side Effects of Extreme Weight Loss
Dropping pounds can lead to some strange consequences. Here's how to avoid them so you can enjoy your new physique
How Counting Calories Can Drain Your Brain
Make a Sugar Craving Disappear Instantly
Why Dwayne "The Rock" Johnson Is Technically Obese
You might be surprised who the government considers too fat, too skinny, and just right
How a Men's Health Editor Lost 30 Pounds of Fat
Has your success gone to your gut? It did for our fashion director, but then he made a change
20 Reasons to Lose 20 Pounds
Need a little motivation to keep losing weight? Read this for a few reminders of what's waiting for you on the other side
How Listening to Your Mom Can Make You Fat
20 Healthy, Protein-Packed Smoothie Recipes
6 Treadmill Workouts That Will Kick Your Ass
Stuck inside for your run? These routines are anything but run-of-the-mill
Why You Must Add This Explosive Medicine-Ball Move to Your Workout
The med-ball short slam will help you build a better-performing, more athletic body
The Workout That Can Help You Lose 2 Inches of Belly Fat
Why the Body You Want Won't Happen Overnight
The sensible man's approach to fitness that lasts a lifetime
The 2-Minute Body-Fat Detonator
The Best Cardio Workout for People Who Hate Cardio
Sizzle fat and rev your metabolism for hours after you leave the gym


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Match Well Made, LLC filed as a Florida Limited Liability in the State of Florida and is no longer active. This corporate entity was filed approximately two years ago on Wednesday, May 8, 2013 , according to public records filed with Florida Department of State.
Pamela Reiner served as the Managing Member and has interests in other corporate entities including Home Health Care LLC, My Visiting Nurse LLC , and Truluv, LLC .
Pamela M. Reiner is also the registered agent for the company. Also known as a statutory or resident agent, the registered agent is responsible for receiving legal notifications regarding court summons, lawsuits, and other legal actions involving the corporate entity.
Key roles for Match Well Made, LLC
Pamela M. Reiner
Managing Member
|Filing Type:
|Florida Limited Liability
|State ID:
|Date Filed:
|Wednesday, May 8, 2013
|Registered Agent
|Pamela M. Reiner
Comprehensive Insight Plus Report
|Learn More
|The D&B Comprehensive Insight Plus Report for Match Well Made, LLC combines D&B's proprietary statistical scoring with business, payment and financial information all in one report.
US Company Profile Report
|Learn More
|The D&B U.S. Company Profile contains basic information such as address and telephone number, executive names and titles, line of business and SIC codes, D-U-N-S Numbers, organization status, and year and location of incorporation for Match Well Made, LLC.


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User and Group Authentication
Office administrators and even some home users will appreciate the option to setup user groups and access policies for individual users.
To start down the path of managed access, you must first setup and enable ADS.
After enabling support for the function you can start by adding local users. If at some point you need to edit users access or remove them entirely, the User area is where those options are found.
It is also possible to make group policies and add users to a specific group.
One very nice feature is the ability to upload or download a backup file that holds the user policy data.
Application Server
Using your NAS as an application server is quite easy and the Application Server area is where you configure your applications. Later in this article we will discuss the Thecus User Communities and how they have managed to build custom applications that add to the functionality of Thecus NAS servers.
Out of the box Thecus includes two applications. The first is the print server. You can attach a USB printer to one of the many USB ports on a Thecus NAS.
The second application included with the NAS handles iTunes. I have to admit that I am not the biggest Apple fan so you will have to explore this one on your own.
PRICING: You can find products similar to this one for sale below.
United States: Find other tech and computer products like this over at Amazon's website.
Australia: Find other tech and computer products like this over at PLE Computer's website.
Canada: Find other tech and computer products like this over at Amazon Canada's website.


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Christmas Appetizers
These appetizer recipes are perfect for family and friends to gather during Christmas. Find party-ready options for easy appetizers, finger foods, dips, spreads plus more Christmas appetizers and hors d'oeurves.
Recipe Spotlight
This creamy topping has a subtle tang that pairs perfectly with crisp rye bread. "We enjoy this simple recipe at our summer deck parties," says Missie Klei from Cincinnati, Ohio. "It's ideal for unexpected guests or a quick snack."View Mozzarella Rye Snacks Recipe


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Should your preferred language not be available, contents will be displayed in English.
« Back to overview
Last modified On 11 March 2015
Extended desktop functionality is not available with ClickShare if you are using Windows XP/Vista or Mac OS X. If your machine is using AMD Catalyst, ensure your catalyst driver is version 13.8 or higher if you want to use extended desktop. If your machine is using nVidia control panel, ensure that the nVidia driver is 320.18 or higher if you want to use extended desktop.
To fix the problem, update your AMD catalyst drivers to 13.8 or higher
Extended desktop doesn't work on systems with:
We have seen problems with:
To verify if your system should be able to share extended desktop:
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30 Jun 2015
Amsterdam RAI installs ClickShare to facilitate great meetings
06 Oct 2015 - 08 Oct 2015Mässans Gata/Korsvägen - SWEDEN


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XUL Tutorial
A guided tutorial that will help you get started with XUL, originally from XULPlanet.
A guided tutorial that will help you get started with XUL, originally from XULPlanet.
XUL (XML User Interface Language) is Mozilla's XML-based language for building user interfaces of applications like Firefox. The term XUL is sometimes used to refer to the whole Mozilla platform (e.g. XUL applications are applications using XUL and other components of the platform).
XUL Controls lists some of the common controls provided by XUL.
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Document Tags and Contributors
Contributors to this page: Sheppy, Jorge.villalobos, trevorh, Brettz9, Nukeador, BenjaminSmedberg, Verruckt, Sevenspade, Josh, Jürgen Jeka, jrmuizel, Nickolay, Enn, MarkFinkle, Tonymec, Shimono, Ptak82, ethertank, Mgjbot, Dria, Blakeross, JhonnyX2003, Johnsonne, Pizdorvan, Pizdorvanec, Chbok, Mathieu Deaudelin, Andreas Wuest, Eric Suen, Goodwill, Justin Shreve, Anonymous, Charles, PWeilbacher, Pmash, Ben, Federico, Readams, another_sam, VladVukicevic, Suguni, Kynilyator, Mrolappe, Caston, osaier, Kangell, Kwinto, Kastor, f.malina
Last updated by: Sheppy,


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The world's largest food company Nestle has found itself in trouble – for bottling water in drought striken California. The giant's permit expired almost three decades ago.
Tags » Profiting
Homo Sapiens Enchained
We stand today, blessed in a modern era of technological and industrial advancement never dreamed of by any of our ancestors, even mere generations before us. 533 more words
The idea of "For profit" prisons is utterly insane.
I remember when I first learned that there is such a thing as a for profit prison. I laughed at the person who told me about them, because I thought they were being sarcastic and that the lunacy of it all was funny. 577 more words
Creating Poverty
As we have learnt from Keynes explanation and the LOC that the real method of creating poverty is the method of creating inflation. In short, inflation means giving somebody more money. 1,036 more words
Profiting From The New MobileNet
How can you communicate with your customers in a way that is relevant and turn communication into cash on a dime. Is your business website mobile ready? 17 more words
HQ PLR Store
If you are ready to get a brand new, unique and High Converting Turn-Key PLR "Business in a Box" every single month, ready to plug in and start profiting like never before, you have come to the right place. 27 more words


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If you are viewing this calendar on a mobile device and cannot see all the days of the week turn your device to landscape view. If this does not work, scroll to the bottom of the page and click "View as Desktop." You should then be able to each of the days of the week as you move or zoom around the screen.
If you see a blank white page, please access via a different browser. We are aware of some browser dependent issues that intermittently affect the calendar. The issue is related to the end user and not the StoaUSA website.
All Stoa tournaments and NITOC Modeled tournaments are open to all Stoa members.
The National Stoa Organization hosts only one tournament per year (NITOC), but local, regional, and national tournaments are run throughout the country as "Stoa Tournaments." This means they utilize Stoa membership, tournament aids, ballots, rules, orientation slides, and other resources. Tournaments that meet the requirements to be posted on Speechranks can help students earn an invitation to NITOC.
Tournaments will need to be completed by April 25 with result uploaded to Speechranks by April 26 for NITOC consideration.
This page contains the evolving tournament schedule.
New tournament information is posted as it becomes available.
Please include tournament:


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katie couric
#4018007 Katie Couric was honored by the University of Chicago Cancer Research Center this evening at the Four Seasons Hotel in Chicago for her tireless work in cancer research and awareness. Her husband, Jay Monahan, died 12 years ago from colon cancer; her sister Emily did from a form of cancer also. Much of Katie's charitable work (including her founding the Entertainment Industry Foundation's National Colorectal Research Alliance in 2000 and helping to launch The Jay Monahan Center for Gastrointestinal Health at New York-Presbyterian Hospital/Weill Cornell Medical Center in 2004) is inspired by them. She is seen here posing for pictures on November 14, 2009 in Chicago, IL before the start of the ceremony.
Fame Pictures, Inc – Santa Monica, CA, USA – +1 (310) 395-0500


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I recently updated all plugins and wordpress to latest versions.
used to have polylang 0.8.7 + wordpress 3.4.1.
there was a language filter in the back-end's 'pages' page, it defaulted to 'all languages'... now there is no such filter. it used to be beside 'show all dates'.
do others have it or is this missing just for me?


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The Brain & Spine Institute of North Houston specializes in minimally invasive brain and spine surgery and non-surgical treatments for neck, back and spine pain. Serving patients in The Woodlands, Conroe, Spring, Kingwood and North Houston. Contact our office for a consultation.
Thank you from the bottom of my heart for the wonderful care and attention you provided my brother."
Margee H.
Thank you for the excellent care given our loved one while a patient in Houston Northwest. Please accept our deepest gratitude."
Jackson R.
In most U.S. schools, students receive three routine medical screening tests: vision, hearing, and scoliosis. The first two need no explanation, but many people aren't familiar with scoliosis, or abnormal spinal curvature. ...
Strokes can happen at any age, and it's important to understand what they do to the brain and the measures you can take to prevent them. Approximately 7 million Americans above the age of 20 ...


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I've been wanting to visit Shatila Bakery for ages, after seeing so many Instagram friends post pictures of their incredible looking pastries and baklava. Shatila Bakery is a Middle Eastern bakery with locations in both Dearborn and West Bloomfield. A couple weekends ago, the huz and I found ourselves in the relative vicinity of West … Continue reading
Filed under Baking and Desserts …
Blank Slate Creamery
I've now made two visits to Ann Arbor's newest ice cream shop, Blank Slate Creamery. Apparently I was not the only one who got the memo stating that they opened! My first visit was back in August, about one month after they opened, but there was still a line out the door on a Friday … Continue reading
Holiday Cookies Class at Sweet Heather Anne
On North Main Street in Ann Arbor resides the bakery Sweet Heather Anne. This boutique cake studio offers not only stunning wedding cakes, but also creates custom baked goods for corporate and special events, and offers a rotating dessert counter with things like French Macarons and gift-able 'Cakes in a Jar." Classes are also offered … Continue reading
Chocolate Chai Snickerdoodles
You guys know I LOVE baking cookies! Today I have a unique twist on classic Snickerdoodles to share with you (courtesy of Taste of Home's recipe). These warm, aromatic spices – which are all typically found in Chai tea – add a little extra zest to the cookies. Traditionally, Snickerdoodles are rolled in cinnamon sugar … Continue reading
Easy Peach Crisp
Who can resist a big, juicy, late-summer peach? Not this girl. Why not incorporate nature's candy into a summer dessert? Using Cooking Light's Peach Crisp recipe, I placed two sliced peaches into melted butter in a skillet over medium heat. Peel fruit before slicing, if you prefer. This recipe is easily cut in half to … Continue reading
Easter Musings
I find myself dreaming of vacation lately. Where should we go for our "big" vacay this summer? Current contenders are Central Europe (i.e., Czech Republic, Germany, Croatia, and/or Austria), Costa Rica, all-inclusive resort in Caribbean, Quebec, Western US National Parks trip… Yeah. Seeking a budget-minded trip with lots of culture and adventures. And food. In … Continue reading
Cake Nouveau Valentine's Dessert Tasting 2013
Valentine's Day 2013 was much like Valentine's Day 2012! And that's a good thing. My valentine and I first went to Jolly Pumpkin for a couple beers and some salty truffle fries. A fellow Instagramer commented that these fries are like "kryptonite" and I completely agree. My funny valentine. <3 I sported pink for the … Continue reading
Beer Bread
Any other beer drankers out there? I love many different styles of beer, but the dark beers are my very favorite. Love those creamy stouts and roasty porters. I would probably say that Great Lakes Brewing Company's Edmund Fitzgerald Porter (affectionately dubbed "Eddy Fitz," by many) is my favorite beer. I bring up the topic … Continue reading
Blondies with Brown Butter
Have you ever browned butter? It is a task that relies on one's sense of smell, more than anything else. Place butter over medium heat. Let it melt. It will start to get foamy. Once the foaming calms down, gently swirl the butter around the pan, to keep it moving. Keep it moving! Eventually, after … Continue reading
Everything But The Kitchen Sink Cookies
Check out this little guy. This is Buckwheat. He is always scurrying around our backyard, and has a special spot in one of our trees where he likes to lay and rest. I captured this perfect candid shot of him last week on our deck. In other news, I've been baking! It's no secret that … Continue reading


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Presented By Marium Bilal Atif Masood Presented To Mr. Amir Sidiqi WHAT IS FRANCHISING? Franchising is one of three business strategies Franchising is a network of interdependent business relationships Allows a number of people to share: A brand identification A successful method of doing business A proven marketing and distribution system *BENEFITS OF FRANCHISING* FINANCIAL ASPECT OF THE BUSINESS. Depending on the amount of capital you have available, there are two financial routes to become a McDonald's Franchisee: i. Conventional Franchise: ii. Business Facilities Lease Franchise: McDonald's One of the world's largest chains of fast food restaurants. *HISTORY* This company began in 1940 with a restaurant opened by Dick and Mac MacDonald, but it was their introduction of the "Speedee Service System" in 1948 that established the principles of the fast food restauratn. American businessman Ray Kroc first established McDonald's in 1955. *TERMS & CONDITIONS* *TRAINING & SUPPORT* We want our Franchisees to be successful; therefore we have a thorough initial training period..." *FINANCIAL INFORMATION* "Depending on the amount of capital you have available, there are two financial routes to become a McDonald's Franchisee" i. Conventional Franchise: ii.Business Facilities Lease Franchise: *WHO IS IDEAL FOR McDonald's* McDonald's continues to look for people who can help develop the global brand locally We seek to give our successful Franchisees the opportunity to operate multiple units. Currently 74% run multiple restaurants. This normally happens within 3-4 years. *SELECTION PROCESS* When selecting new franchisees, with great care we make sure that we - and you - feel you're absolutely right for the business THANK YOU FOR THE ATTENTION! ANY QUESTION PLEASE!!! *~*~*~*~*~*~*~*
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Celtics-Hawks Live: Rajon Rondo Meets Up With Good Friend Josh Smith As Two Teams Search for Consistency
Originally written on NESN.com | Last updated 1/25/13
8 a.m. ET: Maybe Josh Smith can give his old high school teammate some pointers on getting through a rough stretch of losses. Smith and the Hawks are 5-8 in January, although they have won their last two by taking care of two sub-.500 clubs. As you might have noticed, that is exactly what the Celtics are at the moment. Rondo's triple-double was not enough Thursday to prevent the Celtics (20-22) from losing their fifth straight game. One of the few factors working in Boston's favor is that the Hawks (24-18) are dealing with a sea of injury issues, most notably Lou Williams' season-ending right knee injury. Al Horford, Zaza Pachulia and Devin Harris are all uncertain in their statuses for this game. The Hawks still have Smith, though, even if the Oak Hill graduate's numbers are down this season. They are also one of the most dangerous long-range shooting teams in the NBA, and contrary to their shoot-first, pass-seldom reputation, they are actually fourth in the league in assists. Join us for updates and analysis during the game, which tips off at 7:30 p.m. ET.
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The Calgary Stampede, thronged on Saturday, was pelted by marble-sized hail and heavy downpours in the afternoon, sending Stampede-goers scrambling for cover.
Pollen Forecast
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Secret Garden | Art Walk Experience
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fax: 507-284-1767.
Liver Biology/Pathobiology
A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver†
Article first published online: 7 JAN 2008
DOI: 10.1002/hep.22195
Copyright © 2008 American Association for the Study of Liver Diseases
Volume 47, Issue 5, pages 1714–1724, May 2008
Additional Information
How to Cite
Wangensteen, K. J., Wilber, A., Keng, V. W., He, Z., Matise, I., Wangensteen, L., Carson, C. M., Chen, Y., Steer, C. J., McIvor, R. S., Largaespada, D. A., Wang, X. and Ekker, S. C. (2008), A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver. Hepatology, 47: 1714–1724. doi: 10.1002/hep.22195
- †
Potential conflict of interest: Nothing to report.
Publication History
- Issue published online: 24 APR 2008
- Article first published online: 7 JAN 2008
- Accepted manuscript online: 7 JAN 2008 12:00AM EST
- Manuscript Accepted: 14 DEC 2007
- Manuscript Received: 12 AUG 2007
Funded by
- Arnold and Mabel Beckman Foundation
- Alpha1 foundation
- National Cancer Institute. Grant Numbers: R01CA113636, P30CA077598
- University of Minnesota Startup funding
- Chinese National 863 Project. Grant Number: 2006AA02Z474
- N.I.H. Grant Number: DK074561
- Medical Scientist Training Program
- National Institute of General Medical Sciences
- University of Minnesota Doctoral Dissertation Fellowship
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Current techniques for the alteration of gene expression in the liver have a number of limitations, including the lack of stable somatic gene transfer and the technical challenges of germline transgenesis. Rapid and stable genetic engineering of the liver would allow systematic, in vivo testing of contributions by many genes to disease. After fumaryl acetoacetate hydrolase (Fah) gene transfer to hepatocytes, selective repopulation of the liver occurs in FAH-deficient mice. This genetic correction is readily mediated with transposons. Using this approach, we show that genes with biological utility can be linked to a selectable Fah transposon cassette. First, net conversion of Fah−/− liver tissue to transgenic tissue, and its outgrowth, was monitored by bioluminescence in vivo from a luciferase gene linked to the FAH gene. Second, coexpressed short hairpin RNAs (shRNAs) stably reduced target gene expression, indicating the potential for loss-of-function assays. Third, a mutant allele of human α1-antitrypsin (hAAT) was linked to Fah and resulted in protein inclusions within hepatocytes, which are the histopathological hallmark of hAAT deficiency disorder. Finally, oncogenes linked to Fah resulted in transformation of transduced hepatocytes. Conclusion: Coexpression with FAH is an effective technique for lifelong expression of transgenes in adult hepatocytes with applicability to a wide variety of genetic studies in the liver. (HEPATOLOGY 2008;47:1714–1724.)
Microarray and cancer genome sequencing projects have produced an emergent catalog of genes implicated in liver regeneration, metabolism, cancer, and inherited diseases.1, 2 In turn, there is increasing need for in vivo assessment of these genes in a meaningful context. One method for assessing cancer genes is to express putative oncogenes in primordial liver cells, transplant these cells into recipient mice, and measure tumor outgrowth.3 An easier method, in which genes could be directly and stably expressed or suppressed in most hepatocytes in vivo, would facilitate the validation and testing of genes thought to affect liver biology.
Current techniques for gene expression in the liver have limitations. Germline transgenesis establishes stable gene expression, but its expense constrains scale-up or the ready study of genetic interplay. For example, genetic modifiers are thought to affect α1-antitrypsin disease severity in humans,4 yet transgenic mouse models are on a syngeneic background.5–7 A method to selectively and easily express transgenes in hepatocytes would be useful for the study of this and other liver diseases.
Integration and expression of multiple genes is possible using transposon plasmids. The Sleeping Beauty (SB) transposon enables long-term gene expression on gene transfer to the mouse liver.8, 9 Delivery is accomplished by a rapid, large-volume injection via the tail vein called "hydrodynamic injection."10, 11 The major shortcoming of this system is that stable integration and expression are restricted to fewer than 1%-2% of hepatocytes after a single delivery.9 One notable exception is with Fah gene delivery to the hepatocytes of FAH-deficient mice, where the initial integration rate may be the same as in wild-type animals but selective repopulation leads to stable Fah gene expression in more than 50% of hepatocytes.12–14 Our results demonstrate that genes encoding luciferase, a short hairpin RNA (shRNA) that targets luciferase, a mutant allele of human α1-antitrypsin (hAAT), the NRAS oncogene, and a p53 shRNA, were all stably expressed in the liver on fumaryl acetoacetate hydrolase (FAH)-mediated repopulation. Thus, we present a facile method for somatic, lifelong manipulation of multiple genes in the mouse liver.
Materials and Methods
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Plasmid Construction.
A single amino acid substitution, K33A, shown previously to increase transpositional activity by 400% in vitro,15 was incorporated into plasmid pPGK-SB11.16 The resultant PGK-SB-K33A transgene was inserted into pKT2/FAHIL17 outside of the transposon on the 3′ end to generate pKT2/FAHIL//SB.
IRES and luciferase sequences of pKT2/FAHIL were removed to generate pKT2/CaFAH. A pGL3-Promoter vector fragment (Promega Corp.) containing SV40 promoter-luciferase was inserted into pKT2/CaFAH to generate pKT2/Luc-FAH. The U6 promoter-luciferase shRNA fragment of plasmid pSHAG-Ff118 was placed in pKT2/Luc-FAH to generate plasmid pKT2/siLucFAH. Site-directed mutagenesis of the luciferase sequence of pKT2/siLucFAH was performed by 3-step polymerase chain reaction (PCR) mutagenesis to generate pKT2/si*Luc*FAH.
Luciferase complementary DNA (cDNA) from pGL3-Promoter vector was replaced with Fah cDNA to generate pSV40-FAH. The size-minimized Caggs promoter (miniCaggs) from pKT2/mCAG (A. Wilber, unpublished) was excised and combined with an SB-containing fragment of pKT2/FAHIL//SB to make pKT2/CA//SB. The SV40promoter-FAH transgene was inserted into pKT2/CA/SB to make pKT2/FAH-CA//SB. This plasmid contains an EcoRI site in front of the miniCaggs promoter that can be used to insert any cDNA of interest for coexpression with FAH.
The cDNA for hAAT was removed from plasmid pT/hAAT.PGK-SB8 and was inserted into pT2/Caggs19 to generate pT2/Caggs-hAAT. The Z allele of hAAT was made by three-step PCR mutagenesis and was cloned into pT2/Caggs to generate pT2/Caggs-Z.
Complementary DNA encoding wild-type and Z hAAT were inserted into pKT2/FAH-Ca-SB to make pKT2/FAH-hAAT//SB and pKT2/FAH-Z//SB.
The bidirectional promoter from plasmid pKT2/GFP-PGK-CLP-Luc (A. Wilber, unpublished) contains PGK and cytomegalovirus enhancer/EF1α promoters in juxtaposition. The promoter was removed and assembled with FAHIL and NRAS cDNA sequences to generate pKT2/FAHIL-PGK-EF1α-NRAS. The vector MLMS-p53.1224 contains an shRNA that targets mouse p53 and a green fluorescent protein (GFP) transgene.20 The PvuII fragment containing the shRNA and GFP was transferred to an SB transposon plasmid to generate pT2/sh.p53-GFP.
Cell Culture.
HT1080 human intestinal carcinoma cells and HeLa human cervical carcinoma cells were maintained on Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1× penicillin/streptomycin. Transfections were performed using FuGene 6 reagent (Roche Diagnostics) according to the manufacturer's directions. Luciferase assays were performed using Luciferase Assay System (Promega) according to the manufacturer's directions.
Mouse Experiments.
All animal work was conducted under an institutionally approved animal welfare protocol.
Fah-deficient mice21 were maintained on 7.5 μg /mL 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC)22 until injection with the Fah transposon plasmids. Mice between 18 and 25 weeks of age were injected with plasmid DNA by hydrodynamics as described.16 The next day, NTBC was removed from the drinking water. The amount of DNA injected for luciferase expression was 25 μg pKT2/FAHIL//SB; for shRNA expression, 20 μg pKT2/siLucFAH or pKT2/si*Luc*FAH plus 10 μg pPGK-SB11; for hAAT expression, 25 μg pKT2/FAH-hAAT//SB or pKT2/FAH-Z//SB plus 1 μg pKT2/Caggs-Luc23; and for oncogene expression, 1 group of mice was injected with 20 μg pKT2/FAHIL-PGK-EF1α-Nras plus 10 μg pPGK-SB11 and a second group received these plus a third plasmid, 20 μg pT2/sh.p53-GFP. In vivo luciferase imaging was performed at selected time points as described.16 Hepatocytes were isolated for transplantation by collagenase perfusion as described.24
Liver tissue was fixed with 10% formalin and paraffinized. Three-micron sections were hydrated and antigen retrieval was performed by 5 minutes of microwaving in 10 mM sodium citrate, pH 6.0 + 0.1% triton X-100. Slides were washed with phosphate-buffered saline (PBS) plus 0.05% triton X-100 and 0.05% Tween 20, then blocked for 1 hour at room temperature with wash solution plus 10% fetal lamb serum and 2% bovine serum albumin. The slides were then incubated for 1 hour at room temperature with chicken anti-mouse FAH (generated by Dr. Xin Wang) and rabbit anti-hAAT (Dako) at a 1:500 dilution in the block solution. The slides were immersed 3 times in wash solution, and a 1:500 dilution of Alexa Fluor 555 donkey anti-rabbit immunoglobulin G (IgG) (Invitrogen) and Alexa Fluor 488 goat anti-chicken IgG (Invitrogen) in block solution was added for 1 hour at room temperature. Slides were then washed 3 times, with 1 μg/mL 4′,6-diuamidino-2-phenylindole included in the second wash, and mounted with Fluoromount-G (Southern Biotech).
Immunohistochemical staining for FAH was conducted as described.17 NRAS staining was performed with monoclonal mouse-anti-NRAS antibody (Santa Cruz Biotechnology) and Ki67 staining with monoclonal mouse anti-Ki67 antibody (Novocastra). These antibodies were applied to tissue sections using the Mouse on Mouse immunodetection kit (Vector Laboratories) according to the manufacturer's instructions.
Periodic Acid-Schiff-D Staining.
Diastase digestion of tissue sections was performed at 37°C for 15 minutes with 6.7 g/L Amylase Type VI-B (Sigma) in distilled water. The slides were then stained with periodic acid and Schiff's reagent, counterstained with hematoxylin, dehydrated, and mounted.
Microscopic Imaging.
Immunofluorescent images were captured at 100× magnification using a Zeiss Axioplan 2 (Zeiss Inc.) microscope and Zeiss Axiocam MRm or Canon PowerShot G6 cameras. Multiple optical sections were acquired in each fluorescent channel, and the sections were processed using AxioVision software (Zeiss Inc.). Adjacent sections (fluorescent/brightfield) were aligned optically using visible tissue landmarks.
Western Blotting.
Liver protein was extracted in 1% Triton X-100, 0.5% deoxycholic acid, 10 mM ethylenediamine tetraacetic acid, and 2 mM phenylmethane sulfonylfluoride by grinding and sonication. The lysate was cleared by centrifugation, and 50 μg total protein was boiled for 5 minutes, separated on an acrylamide gel, and transferred onto a nitrocellulose membrane. Blots were blocked with blotto (5% nonfat milk powder in PBS plus 0.1% Tween 20) for 1 hour at room temperature or overnight at 4°C. Primary antibodies were added in blotto solution for 1 hour at room temperature. Blots were washed three times with PBS plus 0.1% Tween 20 before secondary antibody was added then washed again and immersed for 1 minute in chemiluminescent substrate (Pierce). Radiographic paper was then exposed to the blots. For detection of hAAT, rabbit anti-human α1-antitrypsin (Dako) was used at a dilution of 1:5000 in combination with goat anti-rabbit IgG, horseradish peroxidase (HRP) (Sigma) at 1:10,000, or goat anti-hAAT (1:3000, Diasorin) was used with donkey anti-goat IgG, HRP (1:6000; Promega). Albumin was detected with rabbit anti-mouse albumin (1:10,000; Accurate Chemical and Scientific Corp.) in combination with goat anti-rabbit IgG, HRP (1:10,000; Sigma). FAH was detected with rabbit anti-mouse FAH (1:3000; Dr. Xin Wang) in combination with goat anti-rabbit IgG, HRP (1:10,000; Sigma). Luciferase was detected with affinity-purified rabbit anti-firefly luciferase (1:200; Cortex Biochem.). Purified hAAT (Sigma) was used as a standard.
Reverse Transcription PCR.
Total RNA was isolated using the TRIzol Reagent (Invitrogen) according to the method provided by the manufacturer. One microgram total RNA was reversed transcribed with random hexamer primers using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. Subsequent PCR analysis was carried out with 1 μL synthesized cDNA template and various primer sets using standard conditions for nonsaturated PCR.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Rapid and Quantifiable Establishment of Luciferase Expression in Mouse Liver.
FAH enzyme deficiency causes neonatal lethality in mice from hepatic toxicity.21 The drug NTBC blocks neonatal lethality in Fah−/− mice by inhibiting 4-hydrozyphenylpyruvate diogenease, an enzyme upstream of FAH in the tyrosine catabolic pathway, enabling colony propagation of Fah−/− mice.22 Correction of FAH deficiency using transfer of the Fah gene results in therapeutic liver repopulation by the FAH gene–corrected Fah−/− hepatocytes, such that the mice no longer require NTBC.12–14
To stably coexpress the reporter luciferase with FAH in repopulating liver cells, the construct pKT2/FAHIL//SB was injected into two Fah−/− mice. NTBC was removed to select for FAH-expressing hepatocytes, and at 1 month postinjection their weights surpassed pretreatment level, indicating therapeutic correction of the metabolic defect (Supplementary Fig. 1A). Immunohistochemistry of liver tissue using an antibody against FAH revealed nodules of liver cells with positive staining surrounded by unstained cells (Fig. 1B). Western analysis of liver lysates indicated that FAH and luciferase were both expressed in the pKT2/FAHIL//SB-repopulated mouse (Fig. 1C).
In vivo bioluminescent imaging was used to track the luciferase expression over time in live animals. The expression was highest in the area of the liver, as seen in previously using hydrodynamic injection16, 25 (Fig. 1D). An image taken at postinjection day 26 likely represented an early point in repopulation, as the mice were on NTBC treatment for tyrosinemia-induced weight loss. Luciferase activities measured at 42 and 55 days postinjection were approximately 10-fold higher than the first measurement, indicating that the expression expanded with time. Expansion of transgene expression generally is not seen with standard gene transfer techniques; instead, diminution of gene expression is the norm.16 Therefore, this result demonstrates the ability to link transgene expression with liver repopulation. Sequencing of insertion sites revealed that the transposons integrated into TA dinucleotides, which are characteristic of SB transposase-mediated events (Supplementary Table 1).
To test the stability of expression in dividing hepatocytes, we performed the following transplantation experiment. The hepatocytes from one of the Fah−/− mice repopulated with pKT2/FAHIL//SB were isolated at 55 days postinjection, and suspensions of 800, 1500, 15,000, 30,000, and 150,000 hepatocytes were injected into the spleens of new Fah−/− recipient mice. Nine days later the mice were imaged in vivo for luciferase expression (Fig. 1E). The amount of light emitted was found to have a statistically significant, linear correlation with the number of transplanted hepatocytes (r2 = 0.95; P = 0.005), indicating that luciferase expression can be used as a marker for the relative number of injected hepatocytes (Fig. 1F). NTBC was removed from transplant recipients, and luciferase expression increased by an average of more than 100-fold during 5 weeks of repopulation with the transplanted cells (Fig. 1G).
Transposon-Based Engineering for Gene Knockdown in the Liver.
As a proof-of-principle that RNA interference can stably reduce target-gene expression after gene transfer to the mouse liver, we coexpressed, with FAH, a previously reported shRNA that targets the reporter luciferase.18 We constructed a transposon vector, pKT2/siLucFAH, with 3 separate transcriptional units: (1) shRNA; (2) luciferase; and (3) FAH. A control construct was made, pKT2/si*Luc*FAH, with 11 silent mutations in the shRNA-targeted region of luciferase, such that binding is unlikely (Fig. 2A). The energy required for hybridization of the shRNA to the luciferase RNA is increased by more than 26 kcal/mol with the substitutions.26 The plasmids were transiently transfected into tissue culture cells, and luciferase assays indicated a sequence-specific reduction in luciferase expression when the shRNA is combined with the original luciferase sequence (Fig. 2B).
Each of the shRNA expression vectors was mixed with Sleeping Beauty transposase plasmid and coinjected into Fah−/− mice. Bioluminescence was measured in vivo at regular intervals, and the mice that received the codon-modified luciferase construct, pKT2/si*Luc*FAH, had 10-fold more robust luciferase expression than the native luciferase construct, pKT2/siLucFAH (Fig. 2C). Overall, the results indicate that shRNA suppressed the target luciferase throughout repopulation.
Modeling of a Gain-of-Function Disease, Human α1-Antitrypsin.
We elected to study α1-antitrypsin deficiency, the most common genetic disease of the liver.27 Whereas wild-type hAAT is normally secreted by hepatocytes, the protein expressed by the Z allele (E342K) is abnormally folded and retained in the endoplasmic reticulum, resulting in characteristic protein inclusions within hepatocytes.28 We constructed transposon vectors that contain either wild-type or Z alleles of hAAT together with Fah, with a transposase gene outside of the transposon (Fig. 3A). The wild-type construct (pKT2/FAH-hAAT//SB) and the Z construct (pKT2/FAH-Z//SB) were introduced into FAH-deficient mice by hydrodynamic injection, and NTBC was withdrawn. With each injection mixture, a luciferase expression plasmid (pT2/Caggs-Luc) was included to monitor gene transfer. The luciferase expression levels of the mice determined at 1 day and 1 month postinjection were not different, indicating that gene transfer was the same for both groups (Fig. 3B). The average animal weight of both treatment groups normalized to pretreatment level by 3 weeks postinjection (Supplementary Fig. 1B).
Six months after plasmid injection, hepatocytes were collected from one mouse in each treatment group for transplantation. For each of the two treatments, 4000 or 400,000 hepatocytes were transplanted into two female and two male FAH-deficient recipient mice. NTBC was withdrawn to select for the transplanted hepatocytes, and the weights of recipient mice were measured (Supplementary Fig 1C). In each group, the male recipients of 4000 hepatocytes died, presumably from the stress of tyrosinemia. The remaining three mice per group exhibited a similar course of weight loss and gain after treatment. Based on the stability of the mouse weight after treatment, there was no apparent difference between Z mutant or wild-type hAAT coexpression in the ability of mice to repopulate. Insertion sites recovered by inverse PCR from DNA-injected mice are consistent with SB transposase-mediated integration (Supplementary Table 1).
Western analysis of the serum of treated mice was performed to determine whether the linked genes are secreted. Wild-type hAAT was detected at 1, 30, and 180 days after injection, at a concentration slightly above the normal range for humans with wild-type hAAT phenotype (0.85–2.15 mg/mL)27 (Fig. 3C). The Z protein was not detectable in the serum. Westerns were carried out on liver extracts to examine transgene expression within the hepatocytes, and both hAAT and FAH proteins were detected with pKT2/FAH-Z//SB and pKT2/FAH-hAAT//SB (Fig. 3D). The results indicate that wild-type hAAT could be secreted, but the Z protein was retained in the liver.
The histopathological hallmark of α1-antitrypsin deficiency in liver is the formation of protein inclusions within hepatocytes that are visible after periodic acid-Schiff staining with diastase pretreatment (PAS-D staining).27 A histological analysis was conducted on livers 10 months after repopulation with pKT2/FAH-Z//SB and pKT2/FAH-hAAT//SB (Fig. 3E). The FAH staining was cytoplasmic for both, and positive cells were in clusters, suggesting clonal outgrowth. FAH staining was noted in approximately 30% to 50% of hepatocytes, a range consistent with previous results with nonviral gene therapy in the FAH model.13, 14, 17 In mice repopulated with pKT2/FAH-hAAT//SB, the staining with hAAT antibody was fine and granular and was stronger in the area that coincided with FAH expression. Staining of wild-type hAAT was also visible throughout the section in areas around the cells (the interstitium), which likely corresponds to secreted hAAT. In mice repopulated with pKT2/FAH-hAAT//SB, the PAS-D stain showed normal-appearing liver tissue without PAS-D–positive intracytoplasmic inclusions (Fig. 3E).
In liver tissue repopulated using pKT2/FAH-Z//SB, the Z protein was confined within cells and always corresponded to FAH staining (Fig. 3E). Three unique features were found only in mice 10 months after treatment with pKT2/FAH-Z//SB: (1) clusters of cells that stained for FAH but not hAAT, suggesting that there was outgrowth of cells with attenuated expression of the mutant protein (Fig. 3E, double arrowheads); (2) inflammation corresponding to scant Z protein expression and PAS-D–positive material, suggesting an immune reaction to some of the cells with transgenes (Fig. 3E, thick arrows); and (3) large hAAT-positive, FAH-positive, PAS-D–positive globules (Fig. 3E, narrow arrows). Liver tissue samples from Z-homozygous human patients were stained as a control, and displayed PAS-D–positive globules as described in the literature (data not shown).
Expression of Oncogenes Using Somatic Transgenesis Results in Hepatic Tumors.
Hepatocellular carcinoma (HCC) results from genetic changes that activate oncogenes or cause loss of tumor suppressors.29 HCC is known to arise in FAH−/− mice that are discontinuously treated with NTBC,22 but no HCC is detected at higher doses of NTBC at 13 months.30 HCC is also detected in two-thirds of mice 1 year after repopulation using retrovirally delivered FAH transgenes.30 The HCC has its origins in FAH-negative, nontransduced cells30 and may result from accumulation of the tyrosine metabolite fumarylacetoacetate, a known mutagen, within these cells.31
To test whether HCC could be induced by expressing oncogenes, a vector was generated that coexpresses FAH, luciferase, and a constitutively activated NRAS oncogene.32 This was administered to two FAH-deficient mice together with a transposase construct. Two other mice received an additional transposon plasmid that expresses an shRNA that targets p53 to promote cancer.20 On withdrawal of NTBC, the mice underwent liver repopulation, as evidenced by stable weight gain (Supplementary Fig. 1D) and increasing luciferase expression (Fig. 4B). At day 43 after injection, liver tissue from mice in both groups was removed by partial hepatectomy and sectioned, and clusters of cells with large nuclei and bizarre mitotic figures were present (data not shown). By 70 days after injection, the mouse with p53 shRNA that had undergone partial hepatectomy developed a massively enlarged abdomen that colocalized with expansive luciferase in vivo expression (Fig. 4B). The second mouse in this group developed an enlarged abdomen by day 112. On necropsy, both mice had massive, expansile, multifocally cystic liver tumors that extended from the diaphragm to the posterior abdomen, filling most of the abdomen (Fig. 4C). In contrast, the mice repopulated with the NRAS construct, but without p53 shRNA, had discrete, nodular tumors in a liver that was otherwise normal in appearance. Transposon insertion sites were recovered and they were consistent with SB-mediated integration (Supplementary Table 1).
The p53 shRNA construct has a linked GFP transgene, and the tumor tissue from mice injected with both transposons was found to be strongly GFP positive, demonstrating the presence of the DNA construct encoding the p53 shRNA (Fig. 4C). Microscopic examination of the liver showed that 80% of the tissue consisted of cords of large, pleomorphic, anaplastic hepatocytes arranged in trabeculae and sheets forming nodules multifocally disrupted by variably sized cystic structures (glandular pattern) indicative of hepatocellular carcinoma (Fig. 4D). Small portions of compressed nonneoplastic liver parenchyma remained at the periphery of the tumor. Immunohistological analysis showed that the tumor cells are positive for FAH, N-ras, and Ki67, a marker of cell proliferation, providing further evidence that tumor formation is linked to the transgene (Fig. 4D).
Reverse transcription PCR was conducted to further assess gene expression in the repopulated liver tissue (Fig. 4E). The analysis confirmed expression of transgenes that were codelivered, including FAH, N-ras, and GFP. Furthermore, p53 expression was reduced in the liver tissue of the mice receiving the shRNA. Two markers of liver cancer were examined by reverse transcription PCR; alpha-fetoprotein33 and osteopontin34 messenger RNA levels were increased in the mice receiving NRAS plus p53 shRNA oncogenes, and alpha-fetoprotein was increased in mice receiving NRAS alone. Taken together, the data indicate a causal relationship between FAH-mediated expansion of cells harboring an oncogene and tumor formation.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Many approaches are used for the genetic manipulation of mammalian cells and tissues. Viral and nonviral vectors are capable of mediating gene transfer and expression in somatic tissues. Currently, however, only a relatively low proportion of genetically modified cells can be achieved after somatic modification of mammalian tissues, and most studies have focused on biological systems in which a limited frequency of gene transfer is sufficient to achieve an informative outcome. Germline transgenesis achieves genetic modification of all tissues in the test animal, but evaluation of tissue-specific effects demands a high degree of tissue specificity in regulating expression of the gene of interest.
We have devised a system that allows for the specific genetic modification of a high proportion of cells in the liver after somatic gene transfer. Our results demonstrate that transgenes linked to Fah can be stably expressed in mouse liver after repopulation of the FAH-deficient mouse liver by the initially transfected cells. Genes encoding luciferase, shRNAs, a human dominant-negative gene, and oncogenes were all expressed after proliferation of hepatocytes that were selected by the expression of the codelivered Fah gene. Our method of expression facilitates the stable genetic manipulation of the adult liver.
Luciferase coexpression enabled the tracking, in vivo, of liver cells as they repopulate. The correlation between number of cells transplanted and luciferase signal suggests that luciferase signal can be used as a surrogate marker of the number of cells with established FAH expression. Indeed, we have successfully applied FAH and luciferase coexpression constructs to measure the efficacy of two novel gene transfer methodologies in correcting the FAH mouse.12, 17
Recent data suggest that shRNAs expressed in liver can saturate the endogenous RNA interference pathway and cause liver damage and fatality from inability of cells to produce microRNAs.35 Results presented here indicate that an shRNA targeting luciferase was expressed long-term throughout liver repopulation. Furthermore, both experimental and codon-modified constructs used the same shRNA sequences, which rules out the possibility that the observed 90% reduction in expression of the target gene was due to toxicity of the shRNA. Therefore, this result indicates that an shRNA can potently reduce target gene expression in vivo in regenerating and quiescent liver cells without causing lethality.
Liver repopulation of FAH-deficient mice occurred with coexpression of wild-type and Z alleles of hAAT. The lack of difference in the kinetics of repopulation may be a reflection that the disease progression in hAAT is a slow process; only a subset of humans homozygous for the Z allele develop liver disease by their fourth decade.27 Mice transgenic for the Z allele develop pathological inclusions of the mutant protein, but they also develop sheets of hAAT-negative tissue, suggesting selective outgrowth of this tissue.7 In transgenic mice, hepatocytes that contain protein inclusions have a seven-fold decreased proliferation compared with inclusion-devoid hepatocytes.36 In our model, FAH-mediated repopulation occurred with either Z or wild-type coexpression, indicating that any counterselection due to Z expression was not as strong as the positive selection of due to the FAH. Our FAH-mediated expression model does demonstrate, however, full manifestation of the pathological development of hepatocytes inclusions and also demonstrates FAH-positive cells that do not express the Z allele. Previous work indicates unknown genetic modifiers to liver disease progression.4 The FAH coexpression method would be ideal for examining candidate disease-modifying loci by knockdown or expression, for modulation of the disease severity or latency.
There is heightened interest in cancer genome projects that can identify somatic mutations in tumor cells; the result is a list of hundreds of putative oncogenes and tumor suppressors that will have to be characterized for function and significance in biologically relevant models.2 Here, we somatically express an oncogene and target a tumor suppressor gene to cause HCC. Although Fah−/− hepatocytes have an intrinsic propensity to develop HCC from accumulation of fumarylacetoacetate in FAH-devoid cells,30 the HCC observed in our study is linked to FAH expression, has a pronounced onset, and has distinct morphology. Thus, our method of gene expression presents a tool for screening of putative oncogenes for aggressiveness and for cancer phenotype in vivo.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
We thank Perry B. Hackett for manuscript editing.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
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Supporting Information
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Supplementary material for this article can be found on the H EPATOLOGY Web site ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).
|Supporting Information file hep22195-SupplementaryFig.1.pdf
|Supporting Information file hep22195-SupplementaryTable1.pdf
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REALLY GOOD POTATO SALAD: House made with chopped celery, onion, eggs and skin on red potatoes all mixed up with real mayonnaise with a touch of Dijon. THAI PEANUT PASTA SALAD: Organic whole wheat pasta, scallions, honey roasted peanuts and cilantro combined with a spicy sesame honey dressing. SPINACH ORZO SALAD: Feta, spinach and orzo in light white balsamic vinaigrette. SPRING PASTA SALAD: Basil, tomato, cucumber, mozzarella and parmesan cheese mixed with our Italian vinaigrette. TOMATO, BASIL & MOZZARELLA SALAD: Drizzled in garlic balsamic vinaigrette. Includes Bistro house made cookies and gourmet bar cookies. Our chef selects from lemon bars, peanut butter chocolate bars, brownies, cream filled globs, raspberry bars, chocolate chips and macadamia white chocolate, chocolate dipped biscotti garnished with strawberries. SIMPLE BISTRO BUFFET One entrée with pasta: $26.95 Two Entrees with pasta: $29.95 Exceptional food, reasonably priced. Lunch or dinner Includes: appetizer, salad, rolls and butter, pasta, two side dishes, one or two delicious entrees: INCLUDES APPETIZER: GOURMET CHEESE AND VEGETABLE TRAY WITH DIPS INCLUDES SALAD: SIMPLE TOSSED SALAD (add a specialty salad listed above $1.00) ROLLS & BUTTER CHOOSE ONE DELICIOUS PASTA DISH BAKED PENNE PASTA WITH MELTED MOZZARELLA (with or without meat) PASTA IN A PARMESAN CREAM SAUCE FRESH TOMATO BASIL PASTA CHOOSE YOUR TWO HOT OR COLD SIDE DISHES: RED BLISS POTATOES BABY YUKON GOLD POTATOES WILD RICE GARLIC MASHED POTATOES BAHAMA VEGETABLES GREEN BEAN ALMANDINE CALIFORNIA MEDLEY CHOOSE YOUR GOUMET ENTREES One entrée with pasta: $26.95 Two Entrees with pasta: $29.95 HONEY BBQ CHICKEN OVEN BAKED CHICKEN CRISPY CHICKEN OVEN FRIED CHICKEN ITALIAN BRUSCHETTA CHICKEN TERIYAKI PINEAPPLE GLAZED CHICKEN BROWN SUGAR BAKED HAM BBQ or ROAST PORK COUNTRY MEATLOAF (BBQ sauce or country gravy) BEEF AND RICE STUFFED CABBAGE 3 CLASSIC BISTRO BUFFET $37.95 per person Traditional chef prepared Bistro fare Includes: An EXTRAORDINARY OCCASIONS hors d' oeuvres, one of our specialty salads, one pasta dish, Three Bistro side dishes and two delicious entrees CHOOSE ONE: EXTRAORDINARY OCCASIONS hors d' oeuvres presentation CHOOSE ONE FRESH SPECIALTY SALAD CHOOSE ONE DELICIOUS PASTA DISH BAKED PENNE PASTA WITH MELTED MOZZARELLA PASTA IN A PARMESAN CREAM SAUCE FRESH TOMATO BASIL PASTA ITALIAN CHEESE AND MEAT LASGANA PASTA BAKE VEGETARIAN LASAGNA BAKE (white béchamel or marinara sauce) CHOOSE THREE HOT or COLD SIDE DISHES RED BLISS POTATOES WHIPPED CREAM POTATOES CHEEZY BACON POTATOES SWEET POTATO SUGARED PECAN CASSEROLE BABY YUKON GOLD POTATOES WILD RICE WHITE RICE GARLIC MASHED POTATOES BAHAMA VEGETABLES MACARONI CHEESE GREEN BEAN ALMANDINE GREEN BEANS WITH SMOKED TURKEY CALIFORNIA MEDLEY SAUTED SUMMER SQUASH MEDLEY CHOOSE TWO HOT GOURMET ENTREES HONEY BBQ CHICKEN BAKED CHICKEN CRISPY CHICKEN CRUNCHY OVEN FRIED CHICKEN CHICKEN PARMESAN CHICKEN MARSALA COUNTRY CHICKEN PULLED BBQ PORK ITALIAN BRUSCHETTA CHICKEN BROWN SUGAR BAKED HAM TERIYAKI PINEAPPLE GLAZED CHICKEN BEEF AND RICE STUFFED CABBAGE BISTRO POT ROAST (traditional with carrots and gravy or BBQ) BBQ or ROAST ROAST PORK BAKED COD WITH LEMON CRUNCH MAPLE GLAZED TURKEY COUNTRY CHICKEN SMOTHERED PORK CHOPS BEEF TIPS IN MUSHROOM GRAVY Served with noodles CAJUN CHICKEN COUNTRY MEATLOAF (BBQ sauce or country gravy) 4 LEMON GINGER CHICKEN HONEY BBQ COUNTRY RIBS BEEF TIPS IN MUSHROOM GRAVY Served with noodles CAJUN CHICKEN BEST OF THE BISTRO BUFFET $52.95 per person The most glamorous buffet we offer! One EXTRAORDINARY OCCASIONS hors d' oeuvres, two specialty salads, three side dishes, one pasta selection, carving station plus two additional exceptional entrees: Traditional chef prepared Bistro fare Includes: An EXTRAORDINARY OCCASIONS hors d' oeuvres, one of our specialty salads, one pasta dish, Three Bistro side dishes and two delicious entrees CHOOSE ONE: EXTRAORDINARY OCCASIONS hors d' oeuvres presentation CHOOSE TWO FRESH SPECIALTY SALAD CHOOSE ONE DELICIOUS PASTA DISH BAKED PENNE PASTA WITH MELTED MOZZARELLA PASTA IN A PARMESAN CREAM SAUCE FRESH TOMATO BASIL PASTA ITALIAN CHEESE AND MEAT LASGANA PASTA BAKE RAVIOLI (Select one: butternut squash in a sage butter sauce, cheese filled with fresh basil tomato Bruschetta or wild mushroom in marsala cream) VEGETARIAN LASAGNA BAKE (white béchamel or marinara sauce) CHOOSE THREE HOT OR COLD SIDE DISHES RED BLISS POTATOES WHIPPED CREAM POTATOES CHEEZY BACON POTATOES SWEET POTATO SUGARED PECAN CASSEROLE BABY YUKON GOLD POTATOES WILD RICE WHITE RICE GARLIC MASHED POTATOES BAHAMA VEGETABLES MACARONI CHEESE GREEN BEAN ALMANDINE GREEN BEANS WITH SMOKED TURKEY CALIFORNIA MEDLEY SAUTEED SUMMER SQUASH MEDLEY INCLUDES A BISTRO CARVING STATION SUGAR BAKED HAM WITH PINEAPPLE SAUCE MAPLE GLAZED ROAST TURKEY CHOOSE TWO PREMIUM ENTREES (or any entrée from any menu) GARLIC AND HERB TENDERLOIN OF BEEF HONEY MUSTARD SALMON FLORENTINE SALMON STUFFED PORKCHOPS RICOTTA AND SPINACH STUFFED CHICKEN LEMON PARMESAN CHICKEN GRLLED PRIME RIB with aujus CAJUN RIB EYE STEAK HONEY BBQ BABY BACK RIBS 5 HAM AND SWISS STUFFED CHICKEN IN BECHAMEL SAUCE TRADITIONAL STUFFED CHICKEN COD ALMANDINE: Baked with lemon butter and toasted almonds NEW ORLEANS CHICKEN, CRAB & SHRIMP GUMBO over rice CRAB & SHRIMP ETTOUFFEE over rice SALMON CROQUETTES SEAFOOD LASAGNA (special order advance notice market price) CRAB STUFFED TILAPIA IN A SHRIMP NEWBURG SAUCE SHRIMP FETTUCINI ALFREDO: jumbo shrimp sautéed in butter and white wine tossed fettuccini and a light parmesan cream sauce CAJUN SPICED AHI TUNA: Lightly spiced served with Pico de gallo and spicy ranch CHICKEN ON A STICK: Skewers of juicy grilled chicken served with a Thai peanut sauce. $4.50 VEGETARIAN DELITES: Substitute any entrée for a vegan or vegetarian selection. Served with garlic breadsticks Ricotta mozzarella cheese stuffed portabella mushroom topped with seasoned lemon crunch breadcrumbs & Bruschetta served with marinara (vegetarian) Organic wheat pasta with roasted teriyaki vegetables garnished with honey roasted peanuts and scallions (Vegan) Spicy chick pea croquettes served with garden wild rice & garnished with Pico de gallo and spicy ranch (Vegan) Roasted vegetable lasagna Served with béchamel sauce or basil marinara (vegetarian) DESSERTS Always accompanying a delicious meal CHOCOLATE HO HO CAKE: A Bistro favorite! Three layers of rich chocolate cake with whipped and lightly sugared cream, drizzled with more chocolate. $2.75 pp CARROT CAKE: Two layers of bliss. Moist and flavorful cake. Just count it as a 'vegetable!" $2.75 pp COCONUT CRÈME CAKE $2.75 pp GOURMET CHEESE CAKES: advance notice ordered by request $3.00 pp COOKIES- $1.50 pp (2 each) GOURMET BAR COOKIES: BROWNIES, PEANUT BUTTER CHOCOLATE< RASPBERRY & LEMON BARS $2.25 pp CUP CAKES, vanilla sugar $1.75 pp PETITE PASTRY BUFFET: assorted specially sized desserts. Vanilla sugar cupcakes, gourmet bar cookies, mini cream puffs and chocolate éclairs garnished with chocolate covered strawberries $3.50 pp GOURMET DESSERT BUFFET: A little bit of everything!$4.25 pp BISTRO PACKAGED BUFFET MENUS- Need a little help putting it all together? THE SUNDAY DINNER: $28.00 Stuffed chicken breast with pan gravy, Brown sugar baked ham, green bean almandine or greens, garlic mashed potatoes, tossed simple salad, rolls and butter THE BISTRO'S COOKIN: $29.00 Crispy chicken with honey mustard, Honey BBQ roasted pork loin, macaroni and three cheese, Green beans with smoked turkey, coleslaw, Bistro rolls and butter. A LITTLE SOUTHERN SOUL one entrée $26.00 –two entrees $29.00 Choice an entrée or two! Country Chicken, Honey BBQ country ribs, Bistro pot roast, southern collard greens or green beans with smoked turkey, Yukon gold mashed potatoes and gravy or macaroni cheese, minted watermelon salad, cornbread with honey and butter TRADITIONAL BISTRO: $29.00 Maple glazed turkey with stuffing, Carved roast beef n gravy, Yukon gold mashed potatoes, green bean almandine, simple salad, rolls and butter A SUMMER PARTY UNDER THE STARS:$38.00 Roasted garlic & herb tenderloin of beef, honey mustard grilled salmon, Summer squash medley, wild rice, strawberry sugared pecan salad, Bistro rolls and butter. 6 BISTRO GARDEN PARTY-LITE: $27.00 Baked lemon crunch cod, Italian grilled Bruschetta chicken, Summer squash medley, wild rice blend, summer simple salad, Rolls and butter. THE SOUTHERN FAMILY PICNIC: $24.00 Honey oven fried chicken, and four sides with rolls and butter. THE BISTRO GIRLY GIRL SHOWER: $29.00 Bistro gourmet sandwich assortment, fresh fruit cascade, alfresco salsa and dip tray, strawberry sugar pecan salad, broccoli cheddar bacon, Bistro gourmet bar cookies garnished with petite cream puffs and chocolate dipped strawberries. THE BOARD ROOM: $38.00 Cheese display, butler passed stuffed mushrooms, tossed simple salad, Grilled Prime rib, Crab Stuffed Tilapia with shrimp newburg sauce, Roasted red skin potatoes, Bahama vegetables, 7
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"Bistro Catering Menu for Olympia Banquet Hall"Please download to view full document


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This tree has made friend with a rock sculpture in Centre de la Nature park, Laval, Québec, North America.
Arbre et sculpture au Centre de la Nature, Laval, Québec
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Photo taken in Centre de la Nature, 901 avenue du Parc, Laval, QC H7E 2T7
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