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The following are a list of the Irish Kennel Club requirements to make up your dog as a Field Trial Champion:
(a) Championship Points valued 1st -
Championship Points valued 2nd -
Championship Points valued 3rd -
The Irish Kennel Club Ltd. At Championship, Open or Derby Stake.
(b) At least one Green Star won at Field Trials and 12 Championship Points, one of which must be valued 4 points and the remaining eight include either one valued 4 points or two valued 3 points at Field Trials held under the Licence of The Irish Kennel Club Ltd., having been cumulatively attained such dog shall be entitled to the prefix Field Trial Champion, provided that it complies with the provisions of Sections 2, 3, 4, and 5 of the Rules as appropriated and that:
(i) At least 8 dogs compete in each Stake.
(ii) Not more than five Championship Points towards the title of Field Trial Champion were awarded by the same judge.
(iii) Not more than five Championship Points gained at Field Trials confined to one breed shall count toward the title of Field Trial Champion.
(iv) It has won a Green Star or at least 1st, 2nd, 3rd or Reserve in a Graduate, Limit, Open or Field Trial Class at a recognised Championship Show where Green Stars were offered in the breed.
(v) As and from the introduction of these rules Green Stars won at Field Trials previously shall have the same value as Championship Points.
(vi) In addition and subject to the In addition and subject to the provision of Rule 52 (v), of these rules, the following dogs shall be entitled to be
(c) The winner of the Pointer and Setter Championship Stake
(d) The winner of the Retriever Championship Stake
(e) The winner of the Spaniel Championship Stake
(f) The winner of the Dual Purpose Gundog Championship Stake
* In the light of the recent changes to the Green Star System in Ireland the above may be subject to change.
Field Trails
To gain the title of Ch. (Champion) the dog must obtain a Show Gundog qualifying Certificate. A dog may enter a Field Trial to qualify for the title of Champion in accordance with the requirements laid down for the breed concerned.
For Golden Retrievers Judges must sign a Certificate of Competency for the dogs entered for a Qualifying Certificate to certify that the dog mentioned:
· Entered water and swam
· Faced cover and retrieved
· Is not gun shy
· Retrieved game tenderly
· Did not bark or whine while in line
· That there was adequate game

subset: commoncrawl


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Multicoloured Amber Hearts Bracelet
This beautiful bracelet consists of heart shaped sterling silver links set with lemon, cognac and green amber. Trigger style clasp.
Length: 7.5 inches
Size of each link: 10mm
Product Code: B13
Delivery Information
UK P&P is free Royal Mail Recorded Delivery (Not Insured),
International shipping is £15.00.
Have a Question?
If you have a question then please
call us on 01502 723394


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The School of Liturgy
Policies and guidelines for Liturgy in the Diocese of Wollongong.
Diocesan Choir Expression of Interest Form
The Diocesan Choir was formed in June 2013. The vision is for a choir of voices from all parts of the Diocese coming together for Diocesan celebrations. Its inaugural performance was in support of the 2013 Diocesan Significant Marriage Anniversaries Mass, under the baton of Robyn O'Dea.
Since then the Diocesan Choir has supported the major liturgies of the Diocese each year, including the annual Chrism Mass, Significant Marriage Anniversaries Mass, Ordinations and special Diocesan liturgical celebrations, such as the Lift Up Your Hearts Conference liturgies and Bishop Peter Ingham's 60th Anniversary of Ordination.
Annette Griffin has been appointed Director of the Diocesan Choir for 2014-2015. She is also Choir Director for the Lumen Christi Pastoral Region Choir and the St Francis Xavier Cathedral Choir.
If you would like to join the choir, please complete the below information to register your expression of interest.
Liturgy Office
Liturgy Handbook


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Lesson #2 of Famously Bad Estate Planning
Coldwater Daily Reporter (blog)
ESTATE MISTAKE: Joe did not plan for estate taxes, which ended up being $47 million. His family had to sell the Miami Dolphins football team, which Joe co-founded and helped build into a powerhouse that won two consecutive Super Bowls. Even if the …


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Today, a great deal of the younger generation spend a large amount of time uploading electronic photos into their social networking sites. This means that many photos wind up left on a video camera or a computer screen and also do not get printed. Right here in this article we shall consider the various means you can make the most out of your pictures, from image books via to canvases and also the different results you can add to make your pictures that little bit much more unique.
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The personification of the perfect Southern gentleman.
Lee may not have been the greatest of the generals but his charisma made him a hero to his armies and an American icon. In the War Between the States, both sides awarded him the highest regard.
Portrait by artist-historian George Stuart.
Courtesy Museum of Ventura County.
Personalites of the Lincoln Era
Images © copyright by Peter d'Aprix


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Chile Peanuts
Chile Peanuts is :
caveman , gluten free , dairy free , lacto ovo vegetarian , primal , fodmap friendly , veganBy Leites Culinaria
Prep Time: 5 minutes
Cook time: 30 minutes
Total time: 35 minutes
Yield: 4 Servings
Tags: Vegan
Health Score: 54 %
1 - 1/2 teaspoons
cayenne pepper
cayenne pepper
4 cups
dry roasted peanuts
dry roasted peanuts
2 - 1/2 tablespoons
lime juice
lime juice
2 tablespoons
olive oil
olive oil
2 teaspoons
1 tablespoon
sweet paprika
sweet paprika
Preheat the oven to 250°F (120°C).
Toss the peanuts with the remaining ingredients in a bowl or on a rimmed baking sheet until the nuts are evenly coated. The peanuts may look wet at this point �?? that�??s just fine.
Spread the peanuts in an even layer on a large rimmed baking sheet.
Bake until the coating is dry and the spices are lightly toasted, about 30 minutes.
Transfer the peanuts to a plate and let them cool completely. (The peanuts can be stored in an airtight container at room temperature for up to 3 weeks. If they lose their crispness, reheat them on a baking sheet in a 250°F (120°C) oven for 15 minutes before serving.)


text:
voir traduction français
|Where ?
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|North-South Relations & Sustainable development
|4 - 8 February 2019
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|Cross cultural dimensions in the city
|4 - 8 February 2019
|Liège (HELMo)
|All different but together in school : managing diversity
|25 February - 1 March 2019
|Valencia (UCV)
|4 - 8 March 2019
|Barcelona (Blanquerna)
|Education in the 21st Century
|4 - 8 March 2019
|4 - 8 March 2019
|Madrid (ESCUNI)
|11 - 15 March 2019
|Harmony in Education
|25 - 29 March 2019
|Mechelen (Thomas More)
|Drama in Education
|1 - 5 April 2019
|Outdoor Education
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|8 - 12 April 2019
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|22 - 26 April 2019
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|Music and Culture
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|Creating culture
|13 - 17 May 2019
Member institutions of the Comenius Association organize international weeks for staff and students that usually last for one or two weeks.
These international weeks can be implemented in different ways :
1. a week including school visits, discussions about practice, cultural visits, etc.
2. a thematic week focusing on a specific topic
These international weeks could also consist of
3. a school placement
4. courses in the host institution
5. a research week
The international weeks have several objectives :
above all to challenge their 'ethnocentric' views on culture by meeting other cultures, other ways of living, other families, other people.
to enhance the personal development of students and staff by enabling them to experience something different from their daily routine
to encourage their autonomy by giving them the opportunity to develop their own projects
to broaden their educational experiences by being exposed to other systems
to give them the opportunity to communicate in another language
Target groups : any student or any staff member from member institutions interested in the project
Deadline for students' nominations : 20 November. For the institutions who can inly host a limited number of participants this will be clearly mentioned in the description. Nominations can only be forwarded by the international office.
Cost : we try to keep it as low as possible to stimulate mobility. More precise informations will be communicated in the description of each international week.
Accommodation : to be negotiated with the host institution
ECTS credits : 1 or 2 credits for a week, dependent on the preparation and commitment
Certificate : each participant will receive a certificate.
Preparation : depending on the project, some preparation might be required.
During the project, participants have to be present at all activities involved in the project.
After the project, participants are encouraged to disseminate the results, the contents of the project in their home institution.


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I've got a little Irish snack for you today since St. Patty's is coming up.
Well actually, in the coming days I have two more St. Patrick's Day recipes scheduled before the 17th, but this one is actually Irish.
And it's dang delicious.
Toasties are one of my simple pleasures. I love a meal that comes together this quickly, and anything with bubbly-brown cheese on top tastes heavenly.
So put on your Irish hat and dig in! xx
- 2 tbsp salted butter
- 4 thick slices bread
- 2 large eggs
- 1 tbsp Dijon mustard
- 1 tsp Worcestershire sauce*
- Fresh cracked pepper, to taste
- Course salt, to taste
- 2 cups shredded Irish cheddar
- Preheat the oven to 400 degrees F.
- Generously butter one side of each slice of bread and place the pieces buttered-side down on a baking sheet. Bake for 5 minutes.
- Meanwhile, whisk together the eggs, mustard, Worcestershire sauce and pepper. Stir in the cheese and mix to evenly coat the shredded pieces.
- Remove the toasts from the oven and divide the cheese mixture over the top of the four pieces of bread, spreading it to the edges of each slice.
- Bake until puffed and golden brown, about 13 minutes. Serve warm.
Recipe adapted from Simple Bites
*Worcestershire sauce is not vegetarian, so please use vegetarian Worcestershire (or other substitute) if you need to.


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Executive Leadership Team
The Executive Leadership Team oversees the day to day operations of Family LifeCare. This highly experienced and committed group of professionals pledges personally to you and your loved ones that the Family LifeCare team will work closely with you to meet your goals of care. We encourage you to learn more about our compassionate team in the following biographies.
SueAnn M. Reynolds, BS, MA
President/Chief Executive Officer
Serving our organization since1999
Hospice administrator's certification (HACP), September 2002
Professional Involvement
- Geographic Area Representative, Great Lakes Region, since 2010
- Board of Directors, since 2010
- Council of States, since 2004
- Council of States Steering Committee, 2007-2008
- Council of States Nursing Facility Workgroup Chair
- Managed Care Advisory Council
- Nursing Facility Advocacy task Force
- Nursing Facility Workgroup Chair
- Research Committee
Indiana Hospice & Palliative Care Organization (IHPCO):
- Education Services Committee Chair, since 2003
- Membership Committee Past Chair, 2005
- Board of Trustees Chair, 2006-2008 and current
- Board of Trustees Chair -Elect, 2004-2005
- Board of Trustees Secretary, 2003
Indiana Hospice Veterans Partnership (IHVP):
- Board Chair, since 2010
Decatur Chamber of Commerce:
- Board of Directors, since 2010
Berne Rotary Club:
- Member, since 2007
State Representative (current):
- CMS, Region V
- Medicaid Workgroup
- Palmetto GBA Coalition
- Palmetto GBA Provider Outreach and Advisory Group
- Indiana Dually Eligible Members Advisory Council
National Hospice & Palliative Care Organization:
- Council of States, 2004-2008
- Steering Committee, Council of States, 2007-2008
Bernie Wiebe, MD
Chief Medical Officer
Serving our organization since 1996
Professional Involvement
Board Certifications:
- Hospice & Palliative Medicine
- Family Medicine
Professional Experience:
- Canadian native from the province of Saskatchewan
- Family Medicine, rural Canada, 15 years
- Moved to Bluffton, Indiana in 1995
- Physician, Markle Medical Center since 1995 now Bluffton Medical Group
- Member AAHPM
Community Involvement:
- Governing Board, Bluffton Regional Medical Center (BRMC)
- Past Co-leader, Physician Leadership Group (PLG), BRMC
- Chair, Ethics Committee, BRMC
Charles Caylor Founder's Society award for distinguished and meritorious service rendered to the community, 2002
Our Governing Body is committed to ensuring that you and your loved one receive optimal care and compassionate support from our organization.
|Keith Huffman
Dale, Huffman & Babcock
|SueAnn Reynolds
President & CEO, Family LifeCare
|Grant Porter
Thunderbird Products
|Paul Reiff
|Matt Lehman
Bixler Insurance and
Indiana State Representative
|Larry DeBolt
Bluffton Regional Medical Center
|Larry Reed


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IET offers customers with best of breed security solutions providing flexibility for the future while leveraging the latest advancements in IP Video Surveillance, Intrusion Detection and Electronic Access Control. IET designs, installs and integrates security systems within new and existing complex/campus, building, and IT infrastructure. Our solutions have provided users with the new capabilities to monitor object removal, people movement, vehicle movement, license plate recognition and more - all in real time. IET designs our security solutions to be 'future flexible' enabling customers to upgrade components that incorporate the latest hardware and software advancements. Our customers expect the best security in the most demanding environments and we deliver security solutions to support the entire process from data collection and identification to processing and analysis to storage and distribution. We understand security for local government and educational campuses to complex federal government and defense installations is a top priority to monitor daily routines and achieve mission objectives securely.
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Between the Rafters
Friday, May 2, 2014
The Power of Spray Paint Part II
I have a couple of large projects I am looking forward to sharing with you over the next few weeks. For now, a fast, simple little project. I want to showcase the power of a can of spray paint! I found this cutie at Goodwill for $6.
Here it is before. Not bad looking of course, but I wasn't a fan of the natural wood frame.
And here it is after.
A reminder to keep your eyes open at Goodwill for pieces with great "bones"!
Posted by
Ms. A
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New books for the week of September 16, 2017
Brief Illustrated Guide to Understanding Islam�
by I.A. Abrahim
Newly donated to the library this book is specifically written for non-Muslims to help you better understand Islam by providing general information and correcting some misconceptions about the religion.
by John Miller
Newly donated to the library this book offers a look inside the faith, the people and the conflicts of the world�s fastest growing religion.
Rights a Historical Perspective b"
by Dr. Abullah Kahtany
Newly donated to the library this book discusses the miserable state of millions of oppressed women all over the world, including in the West, and the doctrines and practices of some prominent religions.
Downloadable ebooks and Audiobooks
The library has just added hundreds of new titles! Everything from crafting to cooking to children�s books can be found on our website www.whitco.lib.wa.us. Just go to our downloadable ebook and audiobook page and click on the link.


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How to Work with Your Hair Type
Article taken from: http://www.mynhcg.com/how-to-work-with-your-hair-type.html
It doesn't matter what type of hair you have there are natural gentle style options for all types of hair types. The most import thing is to understand what styles will work with your hair's characteristics.
Below you will find some general ideas of styles that you can wear with your hair type at different lengths. In other place on this site you will find step by step instructions on how to do the styles below plus combinations and variations.
Coily Hair Styles
If your hair is coily it is best to look for a style that works with and complement your hairs natural tight coils and twists.
Short length coily hair (1 – 3 inches) can be worn in:
- Afro (TWA)
- Afro puff
- Comb Coils
- Finger Coils
- Double strand twists
- Braids
- Cornrows
Medium (4 – 8 inches) to long (8+ inches) length coily hair can be worn in all of the above styles plus:
- Flat twists
- Twist Out
- Braid Out
- Curled Twist out
- Curled Braid out
- Curled Twists
- Curled Braids
Some things to keep in mind when choosing a style for your coily hair:
- Styles that work with your coil will last longer than styles that try to loosen you coil or make your hair look straighter.
- Try to find a style that allows you to wash your hair without worry.
- If the style requires hours to achieve but the results only last a day or two it may be impractical.
- Styles that create other styles are double blessings.
- Try to find few styles that you can maintain yourself and that you think are nice. It is easy to fall back on chemicals when your hair is standing all over your head and you have no clue what to do with it and no one to do it for you.
Curly Hairstyles
If you have curly hair you have many options as well. If your curls are the diameters of a pen or larger and pretty uniform all over you hair you can probably do the wash gel shake and go as well as many of styles mentioned above.
Some things to keep in mind when choosing a style for your curly hair:
- Depending on the size of your curls braids and twist will look looser. That is fine that is just what your hair does.
- As your curls get bigger and the "s" shape gets longer. Some styles may not works as well but still can be done. Although a picked out afro could make your hair look like cotton candy a curly afro is very pretty.
- Extension may slip out of your hair easier, so if you want to get extension try to find someone who is accustomed working with curly hair or know different methods of anchoring hair.
- Your natural braided and twisted styles as well as styles with extension will start to get frizzy faster than coily hair. It is very important to tie your hair up at night as well as when you pull things over your head to keep your braided or twisted to keep you hair looking neater longer.
Wavy Hairstyles
Wavy hair was made for wash and go since it doesn't change shape when it dries. You can wear your hair braided and twisted but you will need to secure your ends with something to keep your hair from coming undone immediately.
Since your hair doesn't naturally coil and is usually resistant to curling, trying to make wavy hair behave like coily hair can be frustrating and will require a lot of time and products. In the end it is always better to enjoy the styles that work with your hair.
Some things to keep in mind when choosing a style for your wavy hair:
- Extension will slip out of your hair easily, so if you want to get extension try to find someone who is accustomed working with wavy hair or know different methods of anchoring hair.
- Also braids, extension and natural hair have a shorter style life you may want to think twice before investing a lot of money in a style that may only last a week or two.
- If a style requires your hair to behave like another type it may be better to select different style.
Mixed types on One Head
- Maybe you have found that you have more than one type of hair on your head. May be the back of your head is curly while the front is coily or maybe the back is coily and the top is wavy. Or maybe you are like me and have a little bit of everything.
- The hair at my crown is wavy and soft. I have curls a little smaller than a pencil in various spots around my head and the back of my head is garden of coils. When I would finish washing my hair the back would be close to my head and it would get longer and fuller as it went to the crown.
- Finger coils and twist would look nice in part of my hair but no amount of gel or twisting would make it work in others. When I locked my hair half would be almost there while the other half was just deciding to begin. You get the idea. So if you have mixed types of hair I can completely empathize with you.
- I have found that it best when choosing a hair style to try to keep in mind how the area that is most resistant to the style will react and then style may hair accordingly.
- For example when I would double twist my hair I would sometimes put black rubber bands on the ends of the areas of my hair that were wavy to keep them from coming undone. When I would wear a loose natural, braid out or twist out, I would part pin and gel the front in a way that made the fact that some of my hair was coming undone look like it was planned.
- If you have mixed type hair, you may find mapping your hair types helpful. If you can't feel the difference in textures have a friend look through your natural unprocessed hair and tell you what they find. It might give you an answerer as to why some styles don't come out the way that you were hoping that they would.
Again interesting article, just had to repost this on the blog. Hope you gained some knowledge and insight.
❤ Kinjee
Monday, February 11, 2013 12:37 AM
I also have wavy hair and I've to say that several in the things undoubtedly ring correct to me also. Nevertheless it is one thing that you simply get used to and attempt to make the best of. Ther are excellent days and poor days, but I do manage to get my hair to appear the way I want!
Latoya permalinkSaturday, November 7, 2009 10:15 PM
Very informative!


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We unite against SPPU malpractices. We want justice. Pass the students by grace mrks.
This petition had 340 supporters
Due to mistakes in revaluation process and reckless paper checking from the university,many engineering students are losing their one year just due to few marks in a single backlog.
Students of engineering who are having only a single critical paper are getting failed due to 3-4 marks. Even after the marks are increasing on the step marking basis,they didn't got marks for the attempted answers in subjects like Engineering Mathematics 3 and SOM and remaining subjects of all branches. And there are some rackets in the exam section who are taking a huge amount (65K) for increasing marks and students who are paying are getting passed. These people involved in rackets are freely working here, no investigation is done on them. They should be punished. No staff transfers has been done from several years.
there is a strategy in this, students who are genuinely getting marks are failed due to 3-4 marks on boundary and University is taking a huge amount from students for rechecking and revaluation.
This system is pressuring the studens and mainly their parents who are working hard each and evey miniute of their life for the betterment of their children. Students are not able to concentrate on BE projects, studies,extra courses and their future is being spoiled. Final year students had lost opportunity to fill GATE 2018 form. Parents are spending huge amount on studies by taking huge loans, and here the frustrated students are sufferig mentally.
Inspite of this,students who were having hopes that they would get clear in revaluation process had filled the examination forms under provisional admissions are still getting no change in the result (even if there are couting mistakes in the photocopy.still is hasn't changed in revaluation)and lot of money is being taken from students. In this way,due to careless paper checking students are being failed in a single subject and losing a whole year.
Today we all unite together to stand against these malpractices.
We demand justice.
our demand is to "Pass the students who are getting a year drop just because of 3-4 marks by carefully rechecking and applying grace marks properly."
"Grace should be given without consideration of that 5% increase rule of revaluation i.e if 2.5 marks increase then only there will be change in marks otherwise no change" this is mistake of university, if students would have got proper marks in first checking attempt only then why would their year should be wasted by getting no change in revaluation even if their 1-2 marks are increasing. Just because of 1-2 marks they are not eligible for grace after revaluation.University should not consider this rule and pass with proper grace by rechecking because it is a mistake from moderators and examiners.
Students should get their marks properly.!!!
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Today: Siddhaanth is counting on you
Siddhaanth Patil needs your help with "Students: Pune university should pass the students who are failing by 3-4 marks due to reckless paper checking. Pass the students dont spoil their future.". Join Siddhaanth and 339 supporters today.
Today: Siddhaanth is counting on you
Siddhaanth Patil needs your help with "Students: Pune university should pass the students who are failing by 3-4 marks due to reckless paper checking. Pass the students dont spoil their future.". Join Siddhaanth and 339 supporters today.


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If you are not yet a client but are interested in learning more about Digiliti Money's solutions and technical capabilities, please visit our Solutions page or Contact Us to discuss how our mobile banking support for financial institutions can meet your specific needs.


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I love the idea of interviewing kids—they often say the funniest things! I think it's even sweeter to do it each year around their birthday, taking note of a few of that year's favourite things (book, movie, breakfast food), not to mention important thoughts on friends, food, and family vacations.
While I didn't start this yearly birthday keepsake with my older boys, I fully intend on doing it with my youngest son. I think sometime around age three is a good place to start, although you can certainly begin the tradition whenever it suits you. If you keep a baby book on hand, or a box where you place special mementos, print off the questionnaire, ask the questions, attach a current photo of your child and put it away until the following year when you repeat the activity and read through the previous year's answers (you will love them—I promise).
Here is a list of possible questions for your questionnaire, but feel free to get creative and include a few of your own, if desired.
- My full name is:
- I like to be called:
- I am _____________ years old.
- My favourite food is:
- My favourite book is:
- My favourite movie is:
- If I could change my name it would be:
- When I grow up I want to become a:
- When I was little I used to:
- My best friend is:
- The snack I would eat every day if I could is:
- My best memory is:
- My favourite breakfast food is:
- I want to go on vacation to:
- My favourite toy is:
- My favourite game is:
- My favourite colour is:
- My shoe size is:
- My favourite thing to wear is:
- I like my house because:
- My signature looks like:
Tell us, do you do anything special to document your children's childhood?
Tagged under: kids,parties,Birthday,birthday party,survey,interview,questions,yearly birthday questionnaire,questionnaire
Category: partysavvy
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From the easiest summer dessert you'll ever make to 16 entertaining hacks you need to know about to tips for hosting picnic, pie, birthday, movie and ice cream parties, we've got everything you need to have the best summer ever.
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Sydney FC release Liam Reddy
Sydney FC have terminated Liam Reddy's playing contract effective immediately.
Sydney FC have terminated Liam Reddy's playing contract effective immediately, the result of an invesigation after he was found to be drinking on the flight to Wellington, before their lone finals appearance.
A statement from the club said "Sydney FC today terminated the employment and contract of Liam Reddy following incidents that took place before, during and after a flight to Wellington before the recent semi final."
"Those actions represented serious misconduct and do not reflect the actions we believe to be appropriate for a professional football player with Sydney FC."
Reddy is now without a club ahead of the next Hyundai A-League season and joins Bruno Cazarine, Juho Makela and Jamie Coyne on the list of players Sydney have released.


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White Paper: The What and Why of Regulatory Outsourcing
PAREXEL white paper outlining key considerations in the design of an outsourcing strategy. Click below to access content.
PAREXEL Regulatory Operations Partnerships takes the effort, uncertainty and overhead out of managing mature products by matching the requirements of each drug in your portfolio to the skills of our team. Having an experienced regulatory operations team working on your behalf to deliver the necessary regulatory outsourcing services ensures maximum readiness and data integrity for any regulatory inspections. It also positions you to take advantage of regulatory reforms as they happen.
Whether it's a few people to review and scrub data for consistency, a global network of experts schooled in emerging offshore regulatory concerns, or something in-between, our team is always ready to serve. PAREXEL only charges for the resources you need – and only when you need them. Should your needs change mid-stream, we will add, remove, or redeploy staff as required.
With PAREXEL, you'll always get the latest thinking and technological advances on your projects with the attention of experts in on-going licensing support. But more than that, you can streamline business processes. Switching from a full-time employee model to a unit transactions model will help you better understand the true effort put into meeting regulatory needs. When coupled with PAREXEL regulatory information management software that tracks regulatory effort over time, you will also be able to create more accurate forecasts, estimates and budgets – a huge win for any business.
PAREXEL white paper outlining key considerations in the design of an outsourcing strategy. Click below to access content.
PAREXEL white paper outlining key considerations for selecting an outsourcing partner.
This white paper outlines recommended governance structure and performance measurement plans for new regulatory outsourcing partnerships.
Our Regulatory Affairs Consulting Services group have an average of 14 years of real-world life sciences experience and are active in industry work groups like HL7, DIA, RAPS, TOPRA, and IRISS.


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Dhaka, Mon, 22 Apr, 2019
Hollywood debut of Priyanka
Priyanka looks so hot as she sports the perfect beach body in Miami (see pics)
13 May, 2017 20:28:00
Bollywood famous actress Priyanka Chopra is gearing up for the release of her Hollywood debut film - 'Baywatch', which is set to hit theatres on May 25.
The film, which has Priyanka playing the baddie, Victoria Leeds, also features Dwayne 'The Rock' Johnson and Zac Efron. The actress may not have been spotted wearing beachwear in the initial rushes of the film, but the actress stepped out in breathtaking bikinis and perfect swimwear in Miami.
Pictures of the former Miss World went viral on the internet where she could be seen posing with supermodel-actor Adriana Lima. The two ladies look like complete hot as they frolic in the sun and sand.


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The Los Angeles Times is reporting that Saturday's UFC 225 PPV in Chicago drew less than 150,000 buys on Pay Per View according to an industry official.
UFC 225 featured the return of former WWE star Phil "CM Punk" Brooks to the octagon. Punk lost to Mike Jackson via unanimous decision after going three rounds.
Just a week before the fight, the MMA community was criticizing the UFC for pushing Alistar Overeem vs. Curtis Blaydes to the prelims on FS1 and instead putting Punk vs. Jackson as the first fight on the main PPV card. Punk and Jackson were both 0-1 going into the fight.
UFC President Dana White shot back at critics for the decision, arguing that Punk was still a draw on Pay Per View for the company. White wasn't completely off in thinking a card with Punk could do repeat business for the company. Punk's fight fight for the promotion at UFC 205 in Cleveland drew an estimated 450,000 buys on Pay Per View.
Despite the low estimated buyrate, the UFC still did strong business with the fight card last weekend in Chicago. The show drew an estimated gate of $2.55 million with 18,117 fans in attendance. This was the most attended card and highest gate to date for UFC in Chicago. It was also the most attended non-basketball event for the United Center.


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build a taxi app
Ngân sách $250-750 USD
We would like to build an android and IOS app , the app should cover the basic taxi app features + our own specifications
more details available in the attached file
159 freelancer đang chào giá trung bình $1279 cho công việc này
Hi, We have worked on similar solution. Please check Taxi Booking apps: [login to view URL] (Customer App) [login to view URL] (Driver App) As per your brief we have prepared a brief document please che Thêm
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Little Mermaid DIGI STAMP Illustration
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Little MERMAID Digi Stamp Illustration by Alisa Ramirez 2009
© alisaramirez.com 2010
Downloadable DIGI STAMP ILLUSTRATION- perfect for your card making, scrapbooking or other craft/art projects.
After purchase please download zip file from your ACCOUNT or ORDER HISTORY on the website as you don't have to wait for email confirmation if you don't want to.
1. Little MERMAID digi stamp pdf (3 sizes on 8x10 page), png
2.Colored SAMPLE reference color guide pic.
3. ANGEL POLICY file
4. HOW TO USE DIGI STAMP illustrations
COLORED picture shown is a SAMPLE of the digi stamp image and use for watermark purposes.
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Page contents:
Scott Polar Research Institute Library - REES Collection, Cambridge University
Contact details
Website: http://www.spri.cam.ac.uk/library/ E-mail: email@example.com Tel: 01223 336552
Image copyright: SPRI
About the library
Copac does not contain the full SPRI catalogue but contains Russian and East European Studies (REES) records added as part of the COCOREES project and subsequent Cofor project to improve access to Russian and East European Studies material.
The Library of the Scott Polar Research Institute (SPRI Library offers the world's premier polar information centre. Sited within a department of the University of Cambridge, this is a resource of national and international importance consulted by governments, industry, scientists and scholars. The Library also houses World Data Centre for Glaciology, Cambridge, with special responsibilities for the provision of information to British and European glaciologists, and the SPRI Archives, containing the world's finest collection of unpublished material relating to the polar regions.
For scientists and scholars, SPRI Library offers a collection developed since the 1920s with over 700 current journals and over 150,000 volumes covering all subjects relating to the Arctic, the Antarctic, and to ice and snow wherever found. For industry, it is a prime information source on such subjects as exploration and exploitation of natural resources and on the environmental implications of such activities in the polar regions; on the design of ice-strengthened shipping and selection of sea routes; and on problems of construction and transportation in cold environments. For Government users, the Library offers an unrivalled resource for the needs of international relations and strategic defence.
The local library catalogue is available online.
Accessing materials
Can I see live document availability on Copac? No
Can I obtain items via Inter Library Loan? Items are not available for inter-library loan, but photocopies of material are available on request (subject to copyright). Please contact the library for details.
Can I visit the library to view materials? Yes - visit the library's web site for access details. If you wish to visit it is advisable to contact the library in advance to ensure the material you need will be available.
How do I find primary source material held at this institution? Visit the Scott Polar Research Institute's contributor page at the Archives Hub for information.
Scott Polar Research Institute Library data on Copac
Example record from the Scott Polar Research Institute Library REES collection:
Tile: An historical account of the British trade over the Caspian Sea : with the author's journal of travels from England through Russia into Persia; and back through Russia, Germany and Holand. To which are added, the revolutions of Persia during the present century, with the particular history of the great usurper Nadir Kouli. / [By: Hanway, Jonas.]
Published: London : T. Osborne, etc. 1762
You can browse all the titles on Copac starting with the above title.


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Games & Hobbies
Ep 88: Numenera & Main Characters
Taylor is back and the Nerdcast sullies forth! On episode 88 Taylor gives us an indiegamegasm with Monte Cook's epic, Numenera. Later in the show, the Nerdcasters discuss main characters at a LARP.
3:00 - Indiegamegasm Numenera18:34 - The Event Calendar21:00 - Main characters at a LARP
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Waxing is a great way to ensure hair removal lasts for several weeks, and leaves clients feeling confident and beautiful. It is important that my clients leave hair free, but have also enjoyed the experience at Revive. Leg waxing includes a complimentary massage to revitalise those tired legs.
Half leg – $28.00
Bikini line from – $28.00
Extended Bikini – $40.00
Brazilian (first time) – $58.00
Brazilian 4 week maintenance – $48.00
Underarms – $25.00
Backwax – $32.00
Upper Lip – $15.00
Full leg – $55.00
Full Leg, Regular Bikini or underarm – $75.00
Imagine lying on a comfy bed, listening to soft music and experiencing a massage after waxing… then call Nicky to book in and make it a reality!


text: We are listed among the leading Kristal Coat for Pavers manufacturers in Chennai, India. We develop the high quality kristal coats for pavers of all kinds. Usually, this coat is made up of good raw material and has made effective to give a glossy finish to the surface or paver tiles. We have made this coat useful for pavers and is blessed with air-drying property. This coating is easy to apply on the pavers and gives a perfect shine and look to the surface. Available in the crystal form, this coating is useful in enhancing the colour intensity of the pavers or tiles. We provide this quality Kristal Coat For Pavers in different quantity packets at the market leading prices.


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(REUTERS) U.S. Republican Senator Lindsey Graham said he wants to examine fellow Republican President Donald Trump's business dealings for any ties to Russia, CNN reported on Tuesday, one day after the issue was raised at a Senate hearing.
Graham said it would be helpful to see Trump's tax records but that it was too soon to say whether he would be willing to subpoena them, according to CNN.
Representatives for Graham could not be immediately reached to confirm the report or give more details. Representatives for the White House also could not be immediately reached


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- Research article
- Open Access
- Open Peer Review
Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells
- He Ailan†1, 2,
- Xiao Xiangwen†1, 2,
- Ren Daolong†1, 2,
- Gan Lu1, 2,
- Ding Xiaofeng1, 2,
- Qiao Xi1, 2,
- Hu Xingwang1, 2,
- Liu Rushi1, 2,
- Zhang Jian1, 2Email author and
- Xiang Shuanglin1, 2
© Ailan et al; licensee BioMed Central Ltd. 2009
- Received: 28 March 2008
- Accepted: 11 August 2009
- Published: 11 August 2009
Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis.
To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453.
20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them.
This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future.
- Human Breast Cancer Cell Line
- ChIP Assay
- Gene ErbB2
- Consensus Binding Site
- Salt Wash Buffer
The transcription factor activator protein-2 (AP-2) family consists of AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2ε; they are retinoic acid inducible and developmentally regulated [1–6]. The AP-2 protein forms a unique modular structure containing an N-terminal proline- and glutamine-rich transactivational domain and a complex C-terminal helix-span-helix motif necessary and sufficient for dimerization and site-specific DNA binding. Through binding to a GC-rich recognition sequence in the regulatory regions of many genes, AP-2 protein provides a fundamental mechanism for regulating gene expression and involving into lots of cellular processes [7, 8]. AP-2 proteins have been demonstrated to participate in the regulation of various signaling pathways during development, cell growth, differentiation and apoptosis by mouse knock-out studies [9–15]. AP-2 proteins are also essential for maintaining cellular homeostasis, and play a strict regulation role in the battle between normal cell growth and neoplasia [16, 17].
AP-2 proteins have an essential role in the regulation of normal breast development [18], and are also involved in carcinogenesis, especially in breast cancer [19]. AP-2γ has been shown to participate in the regulation of ErbB2 [1] and estrogen receptor (ER) α [20], both of which contain binding sequence of AP-2γ in their promoter region and can promote tumorigenesis and metastasis [21]. On the other hand, AP-2γ also induces p21 expression, arrests cell cycle, and inhibits the tumor growth of human breast carcinoma cells [22, 23]. These data suggest that there is a dual role for AP-2γ in different mammary tumorigenic stages: it initially delayed the appearance of tumors, however then accelerated tumor progression [23]. Taken together, these findings provide evidence that AP-2γ participates in a complex biologic dynamics, including cell cycle progression, apoptosis, and tumor formation.
To further understand the complicated roles of AP-2γ played in development and carcinogenesis, it is necessary to search more downstream target genes regulated by AP-2γ, and find out how they are regulated by AP-2γ. In this study, we isolated the binding sites of AP-2γ on the whole genomic DNA from human breast cancer cell line MDA-MB-453 using chromatin immunoprecipitation (ChIP) method. ChIP technique can identify the binding sites of native transcription factors in a given cellular context and reveal potential target genes. We found 20 novel binding sites of AP-2γ in the genomic DNA of MDA-MB-453 cells. These sites are associated with genes involved in the regulation of carcinogenesis, metabolism or others. A combination of sequence analysis, reporter gene assays, electrophoretic gel mobility shift assays, quantitative real-time PCR (Q-RT-PCR), and immunoblot analysis validated and further defined the binding sites on four target genes (ErbB2, CDH2, IGSF11 and HPSE) in carcinogenesis group. The expressions of these four genes were confirmed to be significantly altered by silencing or overexpression of AP-2γ. Moreover, Q-RT-PCR analysis revealed the effect of transcriptional enhancing or suppression by AP-2γ for different genes. The novel target genes of AP-2γ might be used as therapeutic targets against breast cancer in the future.
Cell culture
Human breast cancer cells MDA-MB-453, MDA-MB-231 and HBL-100 were cultured under conditions of 37°C and 5% CO2 in RPMI 1640 (Gibco, Rockville, MD, Carlsbad, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 5 μg/ml insulin.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed using the EZ ChIP™ Kit (Catalog # 17–371, Upstate Biotechnology, Boston, USA) according to the manufacturer's instruction. Briefly, cells were grown on 100 mm plates to 85% confluence; formaldehyde was added to a final concentration of 1%, and the plates were incubated for 10 min at 37°C; the cross-linking reaction was stopped by the addition of 100 mM glycine containing protease inhibitors (Boehringer cocktail, Ingelheim, Germany). Cells were then collected by centrifugation and lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM pH 8.0 Tris-HCl, and protease inhibitors); after sonication, DNA was sheared into 0.3–2 kb fragments; insoluble materials were removed by centrifugation. The extracts were diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM pH 8.1 Tris-HCl, and 167 mM NaCl) and precleared by incubation with Salmon Sperm DNA-Protein A coated agarose beads to reduce nonspecific interactions. The precleared supernatants were incubated with 2 μg of rabbit polyclonal anti-AP-2γ antibody (sc-8977, Santa Cruz Biotech, Delaware, USA), or 2 μg of normal rabbit IgG (Catalog # 12-371B, Upstate Biotechnology) overnight at 4°C; immune complexes were collected using Salmon Sperm DNA-Protein A coated agarose beads. The immunoprecipitated complexes were washed once in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM pH 8.1 Tris-HCl, and 150 mM NaCl), once in high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM pH 8.1 Tris-HCl, and 500 mM NaCl), once in LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% Sodium deoxycholate, 1 mM EDTA, and 10 mM pH 8.1 Tris-HCl) and twice in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Following extensive washing, the cross-linked DNA-protein complexes were eluted using 1% SDS, 0.1 M NaHCO3 and dissociated by incubation at 65°C for 4–5 hours. After treatment with RNase A (Roche Applied Science, Basel, Switzerland) and proteinase K (Roche Applied Science), DNA was purified using the Qiagen MinElute kit; the purified DNA fragments can be used directly for PCR amplification of the genomic fragments of interest, or cloned as pUC19-ChIP-DNA for sequencing and further experiments.
AP-2γ-ChIP-fragment luciferase vectors
Oligonucleotides used for site-directed mutagenesis and electrophoretic mobility shift assay (EMSA)
Sequence 5'-3'
Name pTAL-Luc vector
WtR3-1/ErbB2 (F)
WtR3-1/ErbB2 (R)
Transient transfections and reporter gene assays
In 100 mm culture dishes, HBL-100 and MDA-MB-231 cells were grown to 80% confluence and transfected with 12 μg of pCMV-Myc-AP-2γ or pCMV-Myc, MDA-MB-453 cells were grown to 85% confluence and transfected with 600 pmol of AP-2γ siRNA or scramble siRNA. Twenty four hours after transfection, the cells were collected to prepare total RNA and protein lysates for quantitative real-time PCR and immunoblot analysis of reporter genes. The siRNAs (purchased from Shanghai GenePharma) were as follows: AP-2γ siRNA (sense), 5'-GCUCUACGUCUAAAU ACAATT-3'; AP-2γ siRNA (antisense), 5'-UUGUAUUUAGACGUAGAGCTG-3'; scramble siRNA (sense), 5'-UUCUCCGAACGUGUCACGUTT-3'; scramble siRNA (antisense), 5'-ACGUGACACGUUCGGAGAATT-3'.
For luciferase assays, MDA-MB-453, MDA-MB-231 and HBL-100 cells were seeded at an initial density of 2 × 105 cells/well in 12-well plates, then transfected with 600 ng of the luciferase reporter constructs and 600 ng of pCMV-Myc-AP-2γ or pCMV-Myc. 400 ng of pCMV-LacZ were used as internal reference to monitor transfection efficiency. Twenty four hours after transfection, the cells were lysed, and luciferase assays were performed using the Promega Luciferase Assay system. All data were from duplicate of at least three independent experiments.
Electrophoretic gel mobility shift assays
The oligonucleotides containing wild-type or mutated binding sites of transcription factor AP-2γ were synthesized; and the wild-type consensus sequences of AP-2γ binding site on the promoter of human metallothionein IIa was used as competitor DNA (Table 1). The forward oligonucleotides were end-labeled with [γ-32P] ATP using T4 polynucleotide kinase, and combined with unlabeled reverse oligonucleotide after purification (QIAquick Nucleotide Removal Kit). The annealing mixture was heated to 95°C for 10 min, and cooled down slowly to room temperature to form probes. Vector pGEX-4T-2 (Amersham) was used to express GST-AP-2γ fusion proteins. 5 pmol of labeled probes were incubated with 50 pmol of purified recombinant GST-AP-2γ protein for 20 min at 37°C in 15 μl of reaction buffer: 10 mM HEPES (pH 8.0), 60 mM KCl, 4 mM MgCl2, 0.1 mM EDTA, 100 μg/ml BSA, 0.25 mM DTT, and 10% glycerol; then were resolved on 6% non-denaturation polyacrylamide gels. After drying, gels were exposed to Kodak X-ray film at -80°C, and signals were detected by autoradiography. Competition reactions were performed by pre-incubating the GST-AP-2γ protein with 10- or 50-fold of excess unlabeled competitor DNA prior to addition of the radioactive probes.
Quantitative real-time PCR (Q-RT-PCR)
Total RNA was isolated from mammary-derived cell lines with Trizol reagent (Invitrogen, Carlsbad, CA), and contaminated chromosomal DNA was removed by RNase-free DNase treatment. cDNA was synthesized from 2 μg of total RNA using Oligo (dT)18 Primer according to the manufacturer's instruction (Fermentas, Vilnius, Lithuania). Then gene expressions were analyzed by quantitative real-time PCR (Q-RT-PCR) using 5 ng of reverse-transcribed total RNA template. The amplified products were detected with SYBR Green (ABI, San Francisco, USA) in combination with a melting curve analyses to ensure specificity (7900 HT Fast Real-time PCR System), and Delta Ct Method was used for quantification. The primer sequences were as follows: AP-2γ sense, 5'-GCCATCTCATTTCGTCCTCC-3'; AP-2γ antisense, 5'-GCCATCTCATTTCGTCCTCC-3'; ErbB2 sense, 5'-GAGACCCGCTGAACAATACC-3'; ErbB2 antisense, 5'-TGGATCAAGACCCCTCCTTT-3'; IGSF11 sense, 5'-GACTTGGGCCAATCTTTCTC-3'; IGSF11 antisense, 5'-TGTGGTGATGACCCTCTGTT-3'; CDH2 sense, 5'-ACCAGGTTTGGAATGGGACA-3'; CDH2 antisense, 5'-ACATGTTGGGTGAAGGGGTG-3'; HPSE sense, 5'-TTGCTATCCGACACCTTTGC-3'; HPSE antisense, 5'-CACGCTTGCCATTAACACCT-3'; β-actin sense, 5'-GCGCGGCTACAGCTTCA-3'; β-actin antisense, 5'-CTTAATGTCACGCACGAT TTCC-3'. The reactions yielded a 433-bp PCR product for AP-2γ, a 109-bp PCR product for ErbB2, a 119-bp PCR product for IGSF11, a 156-bp PCR product for CDH2, a 210-bp PCR product for HPSE and a 60-bp PCR product for β-actin. All experiments were preceded in triplicate.
PCR amplification
Standard PCR was carried out to determine the specificity of AP-2γ binding DNA fragments derived from the AP-2γ-ChIP and normal rabbit IgG negative control. The ChIP-DNA fragments from MDA-MB-453 cells were amplified under conditions of 16.6 mM (NH4)2SO4, 0.67 mM Tris (pH8.8), 6.7 mM MgCl2, 10 mM β-mercaptoethanol, 10% dimethyl sulfoxide, 1.5 mM nucleotides, and 1.25 U of Taq polymerase. PCR products were resolved on a 1% agarose gel, and stained with ethidium bromide. The primer sequences were as follows: R4-28/CDH2-271 bp, (F) 5'-ATTTCCAAGTGGCTGAGGCA-3', (R) 5'-TGAACCATTGATTACGCCCA-3'; R1-15/IGSF11-291 bp, (F) 5'-GCATTCTGTCATTTTGGGTC-3', (R) 5'-CAACAACAATGGGTTCCTTC-3'; R3-36/HPSE- 260 bp, (F) 5'-GGCAGAAGGGCATAGTGTCT-3', (R) 5'-CAGGGATGTGGGCTAAGTGA-3'; R3-1/ErbB2-227 bp, (F) 5'-GTAAAA CGACGGCCAGT-3', (R) 5'-AGGAAACAGCTATGAC-3'.
Western blot
Cell pellets were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% (w/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) containing protease inhibitors (Boehringer cocktail). Then the proteins were subjected to electrophoresis on 10% SDS- polyacrylamide gel, and detected by western blot using rabbit polyclonal antibodies: anti-AP-2γ (sc-8977, 1:200), anti-ErbB-2 (sc-284, 1:200), anti-HPSE (sc-25825, 1:200), and anti-CDH2 (BA0673, 1:200). GAPDH (Mab-2005079, 1:2000) was used as loading control.
ChIP assay identifies novel genomic binding sites for AP-2γ associated with genes involving carcinogenesis in MDA-MB-453 breast cancer cells
So we used ChIP assay to isolate AP-2γ binding sites in its target genes from MDA-MB-453 cells. Protein/DNA complexes were immunoprecipitated using polyclonal anti-AP-2γ antibody; Normal rabbit IgG and without antibody were taken as negative controls. The immunoprecipitated DNA fragments were cloned into pUC19 vector for blue/white screening. Forty clones from AP-2γ-ChIP-derived DNA fragments were obtained and sequenced, whereas no clones were found from negative controls.
Categories of putative AP-2γ target genes
Fragment/Gene Carcinogenesis
GeneBank accession no
Chromosome location
Distance from gene (kb)
Intron 3
Catalytic activity/metabolism
Intron 2
Intron 1
Intron 1
Intron 3
The genes near to AP-2γ-ChIP fragments were considered as potential targets for AP-2γ binding and regulation. They were grouped into different functional categories (Table 2), which involved in the regulation of carcinogenesis, metabolism or others. Fragments appeared in multiple clones were R1-7/GALK1, R3-36/HPSE, R4-8/HIVEP1, R3-14/APOL3, R2-2/TMEM148, R2-7/LOC401864, R3-1/ErbB2 and R4-28/CDH2, in which R3-1/ErbB2 was well known to contain AP-2γ binding sites before [1, 25]. Genes involved in the regulation of carcinogenesis include IGSF11, HPSE, CDH2 and ErbB2 (Additional files 2, 3, 4 and 5). Considering the role of AP-2γ in carcinogenesis, we chose these four genes for further investigation to elucidate their functions in breast cancer.
AP-2γ upregulates the transcription activity through binding to ErbB2, CDH2, HPSE and IGSF11
Identification of putative AP-2γ binding sites on genes CDH2, HPSE and IGSF11
AP-2γ-dependent regulation of candidate target genes at mRNA and protein level
In MDA-MB-453 cells transfected with AP-2γ siRNA, AP-2γ mRNA levels showed an about 3-fold decrease (Figure 6A). Accordingly, the mRNA levels of ErbB2, CDH2, HPSE and IGSF11 also dramatically decreased 2~3-fold. In HBL-100 cells, while AP-2γ mRNA levels increased about 7.5-fold (Figure 6B), the HPSE mRNA level increased, but the ErbB2, CDH2 and IGSF11 mRNA levels remained unchanged. In MDA-MB-231 cells, while AP-2γ mRNA levels increased about 2.5-fold (Figure 6C), the mRNA levels of ErbB2, CDH2, HPSE and IGSF11 were then dramatically up-regulated about 4~8-fold.
In post-genome era, studying the gene expression and regulation in eukaryotic cells becomes more and more important. The genomic DNA exists in eukaryotic cells as chromatin, which is a dynamic structure could control the access of transcription factors to DNA binding sites, influences the interactions between different transcription factors, and mediates their subnuclear localization [27, 28]. So studying the interaction between proteins such as transcription factors and genomic DNA at its chromatin status is very critical for elucidating the regulation mechanism of gene expression. Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. This technique gives a picture of the protein-DNA interactions that occur inside the nucleus of living cells or tissues. Although there are more methods to study the interactions between transcription factors and genomic DNA, this is the only real in vivo approach. Using ChIP assay to isolate AP-2γ binding sites from accessible regions of native chromatin in MDA-MB-453 breast cancer cells allowed us to identify a number of AP-2γ target genes as well as to find out how AP-2γ affect these gene expressions in living cells.
In this study, we have isolated forty clones from AP-2γ-ChIP-derived genomic DNA fragments; we did not obtain any clones from the negative controls indicating a low rate of false positive clones. Among the forty clones, eight fragments were found within multiple clones, and one of these fragments (R3-1/ErbB2) has previously been shown to contain functional AP-2γ binding sites. The binding of AP-2γ to the isolated ChIP fragments was confirmed using independent ChIP assays (Figure 2), emphasizing the reliability of our findings. The four AP-2γ binding fragments examined in more detail (associated with genes ErbB2, CDH2 IGSF11 and HPSE) could all be stimulated by co-expression of AP-2γ in reporter assays (Figure 4), and were able to bind to recombinant protein GST-AP-2γ (Figure 5).
All four of the genes validated here were found to be positively regulated by AP-2γ. However, examination of the regulation of expression of the remaining 17 novel candidate genes, using Q-RT-PCR to examine both AP-2γ over-expressing MDA-MB-231 cells and AP-2γ silenced MDA-MB-453 cells, has shown that some of the genes (LOC401864, SEMA6D, STPA1L2, and FRY) identified in the screen can be negatively regulated by AP-2γ (Additional file 6). Thus, the ChIP-cloning assay described here has successfully identified both activated and repressed genes; however more work is still required to further define how AP-2γ regulates these 17 potential target genes.
AP-2γ has been reported to participate into the regulation of breast cancer initiation and progression [21, 29, 30]. And it has also been suggested to be a marker of testicular and germ cell malignancies [19, 31–33]. These findings show the biological importance of AP-2γ in human neoplasia. In our study, the AP-2γ-ChIP fragments were obtained from human breast cancer cell line MDA-MB-453 with high malignant degree; knock-down of AP-2γ expression in MDA-MB-453 cells by siRNA decreased the expressions of ErbB2, CDH2, IGSF11 and HPSE (Figures 6A and 7), but overexpression of AP-2γ in MDA-MB-231 cells elevated their mRNA and protein levels (Figures 6C and 7). They are the direct regulatory targets of AP-2γ. Current studies suggested that these four genes are all involved in carcinogenesis. In human breast cancer, ErbB-2 is overexpressed in 25–30% of all cases, and is representing a clinical marker of poor prognosis [34]; CDH2 promotes tumor cell survival, migration and invasion, and its high expression is often associated with poor prognosis [35]; IGSF11 expression is elevated in colorectal cancers, hepatocellular carcinomas and intestinal-type gastric cancers [36]; HPSE plays a key role in the metastasis of solid tumors as well as mediating tumor angiogenesis by both directly promoting endothelial cell migration and indirectly releasing proangiogenic molecules from the ECM [37–39]. They probably mediate the effects of AP-2γ on the tumor progression. In human mammary carcinoma, AP-2γ may also functionally interact with these genes to influence tumor progression besides transcriptionally activating their expressions.
Overexpression of AP-2γ in HBL-100 and MDA-MB-231 presented different results on regulating ErbB2, CDH2 and IGSF11 (Figures 6B and 6C), indicating that the transcriptional regulation network are complex. Similarly, although the regulation patterns for most of the other 17 identified genes were revealed identical using the two complementary approaches – overexpressing AP-2γ in MDA-MB-231 and silencing AP-2γ in MDA-MB-453 (Additional file 6), upon closer examination, there are some inconsistencies. In particular, for AAK1 (Adaptor protein 2-associated kinase 1), overexpression of AP-2γ results in a 4-fold increase in AAK1 expression, while there is no decrease in AAK1 expression in the siRNA experiment. This also implies the complexity of the regulation of AAK1 by AP-2γ. Transcription factors (TFs) directly control gene expressions by interacting with cis-elements, but transcription regulators (TRs) only assist to control gene expression through interacting with TFs, remodeling chromatin, or other mechanisms. Both types of proteins constitute master controllers of dynamic transcriptional networks. In different cell lines, transcriptional regulation networks might be different.
In summary, AP-2γ coordinates the expressions of a network of genes, involving in carcinogenesis, especially in breast cancers. Our data might contribute to better understanding the controversial findings concerning the role of transcription factor AP-2γ in human breast cancer. The novel AP-2γ target genes could serve as therapeutic targets for anticancer therapy.
This work was supported in part by the National Natural Science Foundation of China (Nos.20335020, 90608006) and the Science & Technology Department of Hunan Province (2006JT2008).
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Pre-publication history
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Uphill Battle for Belgians Clijsters and Henin on Grass
The grass season hasn't been very kind to Kim Clijsters and Justine Henin of Belgium. Both players face a real test at Wimbledon next week, especially with their past results on the surface. Clijsters, who lost to Victoria Azarenka in the Eastbourne quarterfinals faces a physical test. She did not play in the French Open due to injury. Henin, however, must pull through a mental barrier. Her coach, Carlos Rodriguez, warned the tennis world a couple days ago that the former world number one isn't ready to win the tournament yet. We'll see how their stories play out in the biggest stage of tennis shortly.
But even as results and words suggest otherwise, it's never wise to write off this duo. The talent and strong spirits in both exudes winning potential at Wimbledon. It won't be easy for either, even to make it to the second week. Clijsters' last showing was four years ago. While she made it to the semifinals then, it's currently a different tour. That same year, Henin made it to the finals of the event, but proved unable to win. In the semifinals the two Belgians faced off with Henin prevailing 6-4, 7-6. In the final — Amelie Mauresmo's breakthrough year — the Frenchwoman beat the Belgian in three sets, 2-6, 6-3, 6-4.
Of the two, I see Henin with the greater shot at pushing through past history at the event and winning. Her new game suites the speedy surface, and her grass season has already proven strong. Furthermore, I doubt the words of her coach mean much to Henin. In fact, they could be reverse psychology on his part — an attempt to psych out Henin's opponents and the media, while actually inspiring her to try her hardest. Clijsters, meanwhile, remains physically incapable at this stage. She will, however, be just fine at the US Open.
If any two players can surprise, it's these two.
Serena Takes Home Twelfth Title, Sends Henin Home in Three Superb Sets
Serena Williams has finally done it: breaking the curse that allowed her to only win the Australian Open singles championship every other year. Williams successfully defended her title against Belgium's surprise finalist, Justine Henin, in the first three-set final since Henin's Wimbledon final against Amelie Mauresmo at the 2006 Wimbledon with a score of 6-4, 3-6, 6-2.
The match, which had its ups and downs, including Henin's run of fourteen consecutive points to take it to a third set, proved that while Serena is the best player in the world, the Belgian is close behind.
With the win, Serena now matches Billie Jean King's record of twelve Slam titles, an amazing accomplishment. Serena has shown she has what it takes to keep winning in convincing fashion, and I have no doubt she will continue with a strong showing at the French Open in May. Yes, it's her worst surface, but after her talk with ESPN commentator Mary Joe Fernandez, Serena mentioned a desire to win on the red clay of Paris. We all know that when Serena has the desire, there is little that stands in her way.
Regarding Justine, it was a long, difficult road to the final, and for her string of points from the middle of the second set to the beginning of the third set, it looked like the comeback champion would match compatriot Kim Clijster's superb play at the US Open. However, it was not to be. By playing in her next tournament, though, Justine should have enough points to crack the top 50, which means her seeding will drastically improve by the time the French Open, her most successful Slam, rolls around.
It should also be noted that not only did the Williams sister win alone, but beat out the top doubles team with sister, Venus Williams, to take home their eleventh Grand Slam double title. Props to Serena for her physical stamina. It's truly impressive that she has once again strung together twelve wins, playing every day, to reign supreme.
While it's too soon to make any definitive predictions, and only time can truly tell, based on the form shown by these two chmapions, I see both making it to the semifinals of the French Open, and possibly even further, depending on the seeding. As of now, no players on the WTA tour hold the same confidence as Serena and Henin.