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Bliss by the Bay
5 bedroom Holiday house for rent in Rosebud
$495.42 - $716.37*per dayprice range estimate for 12 guests*
This five bedroom beachfront paradise offers access to the Bay Trail and beach reserve. It is discretely located at the end of a little known lane yet is conveniently close to all amenities, shops and eateries.
Experience magical sunsets and the ever-changing moods of the bay in extreme close up. A private lift whisks you upstairs where a dramatic wall of angled glass perfectly frames the view of sea, sand and passing ships and yachts.
The space includes light infused living and dining, large rumpus room, 4 of the 5 bedrooms upstairs and 2 private covered balconies with a bay view.
With a fully functioning kitchen and dining upstairs and a kitchenette, BBQ and outdoor dining setting downstairs, entertaining is a breeze. If you're not at the beach or upstairs enjoying the view, there is also a large flat screen TV downstairs, along with a kids corner (including table tennis table) and a growing collection of books.
Bedding configuration:
- Bed 1 - Queen bed plus ensuite
- Bed 2 - Queen bed
- Bed 3 - Double bed
- Bed 4 - 2 x single bunks
- Bed 5 - Queen bed
Can sleep 12 adults
Distance to:
- Peninsula hot springs: 9.9 km
- Bay: 5m
- Golf club: Rosebud Country Club 4.5km
- Local shops: 290m
- Please note recent changes have been made:
- A fence was installed across the back for better security
- Free Wifi installed
- Motion cameras and sensor lights are intalled outside the property
Make a booking request now
Cancellation with 30 days or more notice is offered a full refund of any monies paid, minus an administration fee of $50 (for December/January bookings, this requires 60 days notice for a full refund). Any cancellation of less than 30 days receives no refund.
SECURITY BOND: The security bond that you pay is used to ensure the owner against damages, breakages, breaches of the below terms and conditions and any other unforeseen costs incurred by the owner resulting from your stay. Please report any of the above or any relevant matter to the property management via text or email within 4 hours or as soon as possible to prevent any costs being charged. The bond will be refunded to you within 3-7 days of your departure (pending a successful inspection). Please notify us of your bank details.
WHAT TO BRING: All premises are fully self-contained, however linen and personal requisites are not always provided. Unless otherwise stated, you will need to bring sheets, pillowcases, towels, bathmats, tea towels etc. Whilst some holiday accommodation properties have small quantities of washing powder, detergents, soap and toilet paper, it is expected that you will need to bring supplies depending upon your length of stay. Properties are not serviced. (Cleaners etc do not attend during your stay)
**FIREWOOD** properties featuring wood fires will supply firewood. Kindling & firelighters may need to be supplied by you.
NO SMOKING: Please respect the No Smoking policy. There is to be NO smoking inside the house, and the butts from any cigarettes smoked outside must be collected and removed from the property. If there is evidence of smoking inside, the bond may be forfeited or at the least we will charge a $200 fumigation fee. If our cleaners have to collect cigarette butts outside, this will be charged at $100 per hour.
RUBBISH REMOVAL: Please ensure all rubbish is placed in the outside bins provided. Bin collection day is Monday, so please ensure the bins are placed out in the street for collection the night before and/or on your departure (whichever comes first).
If the bins are not put out for collection, then the charge to have rubbish removed is $50 per bin. Any excess rubbish must be taken to local tip on Truemans Road, Rye or to the Normanby Road Sorrento Garbage Hopper (or taken home with you). Bins left overflowing with excess garbage and any garbage left behind will incur a charge of $35 per bag.
CLEANING: Extra cleaning charges occur if there are stains on furniture/linen/flooring/walls etc, dishes left to be cleaned, ovens or fridges left in a messy state, BBQs needed to be cleaned, or rubbish to be collected inside or outside property. Please ensure the property is left in the same condition as you found it. Extra cleaning charges start at $40 per hour.
PETS: Unless specifically stated as being 'pet friendly', no pets are allowed either onto the grounds of the Property, nor inside the holiday accommodation under any circumstances. Any pets found in holiday accommodation properties will result in a termination of your booking and additional cleaning charges will be debited to your Credit Card if this occurs. The agent is not responsible for the confinement of your pet.
TELEVISION AND STEREOS: The television and stereo sound system has been provided at the property in good working order. If you pull out cords and tamper with the equipment then the cost of our technician to reinstate the equipment to the original working order will be deducted from your bond. (The same applied to alarms, air conditioners, heaters and any other electrical appliance)
GUEST CAPACITY: The property is rented out exclusively to one group at a time. You must not exceed the number of people permitted to sleep at the property.
PARTIES: This property prohibits functions or parties of any sort. Functions are defined as being gatherings significantly over and above the resident guests. Functions also include the provision of external equipment like additional chairs, marquees and outdoor amplification equipment. If a gathering is taking place, please ensure you have discussed with property management beforehand for approval or your bond may be taken.
CHECK OUT: Check out time is by 10.00am unless a late extension has been organised prior to arrival. Late departure will result in a bond deduction of $100 for the 1st hour, then $150 per hour thereafter. Check in is 2pm. Late check outs cannot be authorised in the peak summer period.
NOISE RESTRICTIONS: Please note that this is a residential area and normal noise levels must be observed. The neighbours will not tolerate loud music past 11pm on Friday and Saturdays and 10pm on Sunday to Thursday (as per local EPA laws). If there are any complaints from the neighbours regarding the noise levels then we reserve the right to immediately terminate your booking and/or to deduct your entire bond. Please respect the area.
CAR PARKING: Cars must be parked in the driveway of the property and not on gardens or in residential streets nearby. Only paid guests cars' are permitted to be parked at the property.
NEIGHBOURHOOD NOISE: Life's a beach holiday rentals or the owner of the property will not be held responsible for the noise caused by neighbours, or noise coming from neighbouring premises.
KEYS & ALARMS: Guests are responsible for all keys and remote controls. Lost/damaged keys will result in charges applied. Call out for alarm monitoring will be charged to the guest.
SWIMMING POOLS: Guests are not to touch the pool pump or equipment. If there is an issue with the pool, you will need to contact the office. Pools have scheduled cleans before and after each guest, any call out for an extra clean or tampering with equipment may be charged to you. Bombing is not permitted.
BBQ: Most properties have a BBQ and provide 2 full gas bottles. It is your responsibility if more are required for your stay. BBQs must be cleaned after use. A $50 fee will be charged if the BBQ is left unclean.
DEPARTURE CHECKLIST: When departing the property, please ensure that you have read and followed the Departure Checklist. This would include turning off all lights (inside and outside), closing market umbrellas, ensuring all heating and cooling is turned off and that all windows and doors are locked. A bond deduction will be incurred if you do not leave the property fully secured/locked.
LOST PROPERTY: If you leave behind any items at the property please let us know immediately and we will endeavour to find them for you. We will post them to you at your own expense or hold them at our office for collection. Items will only be held for a period of no longer than 30 days. After this time they will be disposed of. We hold no responsibility for these items.
INDEMNITY: The guest indemnifies Life's a beach holiday rentals against any liability for loss or theft of property occurring during the stay, and against any liability for injury or loss being personal or proprietary occurring during the stay. Life's a beach holiday rentals do not compensate for any booking that is incorrect or insufficient. It is your responsibility to ensure all property details are correct.
TERMS AND CONDITIONS: Please note that it is imperative for you to read the Terms and Conditions and Departure Checklist that is clearly visible at the property, as this will ensure that you know all of the conditions that could possibly cause a bond deduction. If at any time during your stay these conditions have been breached, we reserve the right to terminate your stay immediately and/or your security bond will not be refunded.
As per industry standards, we recommend you take out travel insurance for your stay.


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New Year's Eve Celebration and Balloon Drop at Noon
Adventure Landing
Thursday, December 31, 2009 from 10:00 AM to 2:00 PM (EST)
Event Details
New Year's Eve Family Style!
Children under 12 can join us from 10am-2pm for a day filled with fun and a chance to win great prizes in our Balloon Drop at Noon. We will have three age groups and children under 4 are FREE! Join us at our Beach Blvd. and Blanding Blvd. location.
Children 12 & Under can enjoy unlimited go-karts, unlimited laser tag and unlimited mini golf for only $10! Children over 12 can also enjoy the same special for just $15!
Visit our website for more details at http://www.adventurelanding.com/jaxbeach/events.html
When & Where
Adventure Landing
1944 Beach Blvd.
Jacksonville Beach,
Thursday, December 31, 2009 from 10:00 AM to 2:00 PM (EST)
Add to my calendar
Adventure Landing
Adventure Landing is The Place to Play! We have something for everyone, from kids to adults so the whole family can enjoy all of the excitement that Adventure Landing has to offer. Stay cool this summer and come swim, splash and play at Shipwreck Island Waterpark, where you can enjoy our many rides from mild to wild! Practice your putting skills on our miniature golf courses or perfect your gaming skills in our mega arcade. Keep your eye on the ball in the batting cages or build a stuffed-animal friend at the Teddy Bear Factory. Adventure Landing is affordable family fun…so close to home!

subset: commoncrawl


text: Politics 14 January 2010 Payroll politics and Labour's London hope The woman hoping to win Bethnal Green and Bow back for Labour and how the payroll vote has grown und Sign up for our weekly email * One of the few seats that Labour is confident it can win back at this year's election is Bethnal Green and Bow. The woman hoping to reclaim the seat - in which Respect's George Galloway famously overturned a Labour majority of 10,000 in 2005 on an anti-war ticket - is Rushanara Ali, currently an associate director at the Young Foundation. The 34-year-old grew up in Tower Hamlets, having emigrated from Bangladesh at the age of seven. A confident and articulate activist, Ali was radicalised by the "Greed is good" mantra of the 1980s. She promises to tackle homelessness and unemployment if elected. Asked for her verdict on Galloway's performance as an MP, Ali says: "Talking to local people, the constant complaint that comes up is that he's never there; he's never responding to their concerns." On Iraq, the issue that cost Oona King the seat, Ali's stance is unambiguous. "I was against the war and marched against it," she says, with some pride. Central to her campaign is regaining the trust of the area's 45,000 Muslims, most of whom abandoned Labour at the last election in protest over the war. A Muslim herself, she defends the government's record on religious issues as a progressive one. "We have the most advanced legislation compared to any other country in Europe to protect religious groups. And that includes Muslims."She describes the Muslims of Bethnal Green and Bow as "incredibly anxious" about the prospect of a Tory government. When recently David Cameron accused two Islamic schools of promoting extremism, she says, it sent "shock waves" through the community. In a constituency that feels threatened by the Tories and neglected by Respect, Ali is likely to prove a popular alternative. Often overlooked in analyses of Gordon Brown's survival is the remarkable growth of the payroll vote - the number of MPs who hold official positions and are obliged to support the government. Over the past 30 years, the payroll vote has swelled from 106 MPs to 157 today (see graph, above). It includes 23 cabinet ministers, nine others who can attend cabinet, 87 ministers, whips and law officers, 36 parliamentary private secretaries and two special envoys. With 349 Labour MPs in total, they make up 45 per cent of the Parliamentary Labour Party. Some of these may be the sort of "minister for paper clips" posts mocked by the Labour MP Chris Mullin, but others see them as the essential first rung on the ministerial ladder. And given the unprecedented size of the present government, there are many MPs who will think twice before turning against Brown. The extraordinary political upheaval in Northern Ireland could turn the six counties into a key election battleground. Following the revelation that Iris Robinson borrowed £50,000 from two property dealers to help her teenage lover launch a café, the Ulster Unionist/Conservative alliance is confident it can eclipse the Democratic Unionist Party to become Westminster's largest party in Northern Ireland. Most of the DUP's nine Commons seats have majorities of between 4,000 and 5,000, leaving them vulnerable to a wave of protest votes against the party. Following the alliance agreed between the UUP and the Tories in 2009, any MP elected under the joint banner will take the Conservative whip and could serve in a Cameron government. With a hung parliament still feasible, the alliance could give the Tory leader the edge. George Eaton is political editor of the New Statesman. Subscribe from just £1 per issue This article first appeared in the 18 January 2010 issue of the New Statesman, Palin Power More


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Working or studying can be demanding, both physically and mentally at times, and it important to try to relax well during the times you are not working or studying.
Relaxation is a very important part of people's lives as it helps to keep reduce stress levels, and consequently improves their mental and physical health.
Relax well during work
It is important that you take a break from your desk during your day. Taking yourself away from work during your lunch break allows you to de-stress and will untimely improve work productivity. Here are a few ideas of what you could do to relax during your working day:
Walk the university mile – Fresh air is good for everyone, it increases the amount of oxygen that you inhale, which will ultimately have an effect on your serotonin levels, the hormone associated with mood and feelings of wellbeing. Simply walking part or all of the university mile at lunch time could help alleviate stress and make you feel happier
Mindfulness – Practicing mindfulness is a great way to relax the mind and body, have a look at our Think for Wellbeing page for more information on how to be more mindful
Labyrinth – The University Labyrinth is a perfect place to go to relax, it provides a walking meditation area in which to reflect and relax. For more information on the labyrinth please contact email@example.com
Socialise – Take time for lunch and have your break with a friend, to get away from the tasks that you are doing and remember to talk about things that are not work related.
If it is possible, avoid looking at work emails during the weekend. Taking time to switch off from work to enjoy your home life and the environment around you can help with maintaining a healthy work-life balance.
Annual Leave
Consider what you will be coming back to, try to:
- Ensure that any tasks that need completing the week you return are done before you go. This way you will not be tempted to do them whilst you are away
Remember to turn on your out of office
- This way people will see that you are not available and, if urgent will contact someone else involved.
- Try to provide contact details of a colleague who will be able to deal with the email on your out of office, this way it may be solved before you return.
Try not to check your emails when you are off
- The idea of Annual leave is that you have booked a time off work to relax or do other activities, therefore avoid checking your emails unless it is of the upmost importance. If you have used the above tips then there should be no reason that you will need to read emails.


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This lawyer has been disciplined by a state licensing authority.
Practice areas
Represent on every aspect from child custody, child support, spousal support, divorce, restraining orders, and all procedural motions.
11 years
Litigation in federal and state courts covering various cases.
10 years
Began working in both the prosecutors' and public defenders' office. I continued my criminal practice cases when I started my office.
11 years
I have extensive experience with domestic violence restraining orders and criminal defenses. I have represented clients in both state and federal courts.
10 years
Very passionate about civil rights. I have experience in both federal and state courts with various civil rights violations.
10 years
Languages spoken
This lawyer was disciplined by a state licensing authority.
This sanction means the attorney lost his or her license to practice law for a period of time. The attorney typically returns to practicing law when the suspension expires.
|Company name
|Mataele Law Offices
|2009 - Present
|School name
|University of Hawaii at Manoa - William S. Richardson School of Law
Posted by Kathy
I have had my case with Isi for over 4 years now for a simple Divorce. I can't get him to respond to me to tell me if I am finally divorced or not. He nor his assistant will return my emails. This is how we have communicated for the last 4 years (since September 2012).
I just want to know what needs to FINALLY get done or what I need to do. He will not respond.. It has been about a year since hearing from him. Very upsetting. I do not know how or what to do next. A simple response from him would help immensely.
I am stuck!!!!!!!!!!!!!!!!!!!!
Posted by records2013
Mr. Mataele was always very quick to respond to my communications. NEVER did I find he was unprepared or needed more briefing on my issue.
He represented my interests aggressively and professionally.
I have never recommended a lawyer in my life. I recommend Mr. Mataele.
Justin S / Beverly Hill
Posted by Dennis
he is on top of everything and communication lines are always open. I would definitely use Isi for my legal needs and would recommend him.
I like his no nonsense approach, facts are facts.
Douglas Borthwick, Family Attorney
Relationship: Fellow lawyer in community
I endorse this lawyer.
John Bohren, Contracts and agreements Attorney
Relationship: Fellow lawyer in community
Mr. Mataele knows what he's talking about when it comes to law. He's a hard worker and extremely passionate about his cases- I endorse this lawyer.
Cash, Check, Credit card
4660 La Jolla Village Drive
Suite 500
San Diego, CA, 92122


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Eight years ago Claire Marie Panke set out to make a film about the daily drama inside the intensive care unit for newborns at St. Vincents Hospital in Greenwich Village, where nurses and parents struggle to save and nurture sick, fragile and underweight babies.
Now, days before its premiere on national television, Ms. Panke's film has suddenly taken on a newly tragic dimension, as the nursing staff at that neonatal unit mourns the death of four of its own, killed in a car accident in Virginia.
''It is all so fragile,'' said Ms. Panke, who has worked as a nurse in the unit, full time and then part time, since 1989. ''Which in the end is what the film is all about.''
The nurses, who died when the car in which they were riding plunged off a parking garage roof in Virginia Beach, were not stars of Ms. Panke's film, although they appeared in the background in several scenes. But the four -- Mary Ann Dono, 60; Mary Ann Going, 60; Kathleen Mollick-Kelly, 63; and Joan Ellen Walsh, 56, who had worked at St. Vincents for a combined 158 years -- were a major influence on a unit that is known for professional and emotional cohesiveness.
The women, who were close friends, had gone to St. Vincents nursing school together and worked together throughout their careers. They served as a professional yardstick for others who worked with them. Ms. Walsh was Ms. Panke's boss for almost 10 years, and Ms. Going had worked at St. Vincents since 1961, the year after Ms. Panke was born.Continue reading the main story
''As one of the nurses said the other night, we have worked together longer than most marriages,'' said Ms. Panke, herself a child of St. Vincents, where her father worked as a doctor and where she was born. ''Attrition has historically been very low.''
So now the film, conceived as an account of three infants and their families as they made their traumatic journey in and out of the neonatal unit, is serving as a tribute to the nurses who dedicated their life's work to those babies and thousands like them.
The film, titled ''A Baby's Battle for Life,'' ( Ms. Panke's original title was a ''A Chance to Grow'') will be shown on the Discovery Channel on Saturday night at 9.
After graduating from Georgetown University with an degree in English, Ms. Panke found herself drawn to neonatal nursing. ''It is so intimate, so intense, because the patients can't speak for themselves, because the emotions swing so quickly from joy to despair and back again,'' she said. ''You are dealing with patients who stay with you for three months, even more. I thought that it was a good fit for me, intellectually and emotionally.''
But even as she worked full time at St. Vincents, Ms. Panke used her free time to return to a high school passion for film and photography. ''People keep asking me how I do nursing and filmmaking,'' she said. ''The connecting point is the human story.''
The film had a difficult start. At one point a nonprofit corporation overseeing money that Ms. Panke had collected for her film went bankrupt, and she had to halt production for almost a year. She was later able to recoup some of that money and received a grant from the New York State Council on the Arts. The Discovery Channel finally stepped in as a co-producer in return for television rights and helped finance the final editing.
As a nurse and as a filmmaker, Ms. Panke said she felt at times as if she were split in two. Once she was filming a scene when the unit was suddenly overwhelmed by the emergency delivery of quadruplets, who were then followed by triplets, all seven of them seriously underweight. ''I wanted to step back and get in there and help, but I was filming,'' said Ms. Panke.
Another time she caught a glimpse of an infant's father sitting by himself, clutching a stuffed animal, while the baby, sealed off in an isolette, was undergoing another in a series of medical crises. ''There was the father holding the teddy bear while 15 other people were hovering over his son,'' she said. ''And he couldn't do anything. At that moment he was helpless. He was a child. And I remember thinking, where is my camera?''
What continued to guide her was the melding of two instincts, one to observe the joy and anguish experienced by the families and the other to help them and shield them as they underwent the experience.
''There were moments when my cameraman would say, 'This is it, we should stay and film,' and I would say, 'No, this is the moment when we turn off the camera and go,' '' she said. ''In the end the families trusted me because they knew I was not going to exploit them. To borrow someone's else phrase, there is a difference between stealing someone else's story and sharing it.''
The result is a film that mixes tenderness and realism as it tracks a single mother and two couples coping with babies who were born so sick and weak that for weeks they could not be held or removed from the battery of tubes and machines that had become their lifelines.
In the film one father describes how he would duck the usual questions from friends and colleagues about his new baby's birth weight, which was less than two pounds. ''You don't want to know,'' he said. Later he describes how he decided to put aside his anxieties over his son's precarious health and indulge in the pure joy of fatherhood.
The nurses, too, are on a roller coaster. One minute they must make complex split-second decision about saving a life, knowing the baby could later be faced with a lifetime of physical and mental disabilities, and the next minute they may have to hold the hand of a mother who after weeks of struggle is watching her baby die.
Like the filmmaker, the nurse has to know when to stand back, Ms. Panke said. ''No one wants a sappy neonatal nurse,'' she said. ''You have to be both compassionate and competent.''
''We've found ourselves in a situation where technology can run away with us,'' she said. ''We have to slow down and get over to the low-tech side. And here both parents and nurses have a lot to teach each other.''Continue reading the main story


text: Property ID#: 17901Apartment 5 Bedrooms 2 Bathrooms Sleeps 14Living Room: 1 Sofa BedMaster Bedroom: 1 Queen BedSecond Bedroom: 2 Queen BedsThird Bedroom: 1 Queen BedFourth Bedroom: 2 Double BedsFifth Bedroom: 1 Double BedConverted from an office, this is a sprawling 1700 square foot 5 bedroom, 2 bathroom duplex apartment in a pre-war doorman building with elevator. Laundry is available in basement of building. 5 bedrooms feature queen or full size beds, air conditioning/heat, and storage space for clothes and personal belongings. 2 of the bedrooms have 2 full/queen size beds, ideal for children. There is a full bathroom on the 2nd level of the duplex. There is also a full bathroom on the first floor of the duplex- the shower and toilet are in separate rooms. Fully equipped kitchen with pots/pans, plates, cutlery, coffee maker, toaster, dishwasher, refrigerator/freezer. Living room features plenty of seating and a 42" plasma TV. This apartment building shows plenty of pre-war charm with original radiator style heating, crown moldings, and original windows. Just three blocks to the Empire State Building and Macys Herald Square, and only a 10 minute walk to Times Square, Grand Central, Bryant Park, New York Public Library.--- FREE dinner and drinks offer ---Get dinner and drinks for two per bedroom when booking this apartment!*Booking Window: Oct 15 - Nov 15, 2012Travel Window: Jan 1 - Mar 31, 2013* Applies only to NEW reservations. Length of stay must be 4 nights or longer. Following the booking, guest or agent must register on OnlineFrontDesk.com by November 30, 2012. Free dinner vouchers will be sent to agents/guests December 1 - 15, 2012- Dinner includes one entree (appetizers / dessert not included) - Drinks mean one "house drink" or one soda or one soft drink or one juice - Taxes and tip not included - Restaurant is closed Sundays - Reservations recommended------------Check In Time: 3:00-8:00 PM (15:00-20:00)Check Out Time: 11:00 AM (11:00)*** Important ***After booking, you MUST register at https://www.onlinefrontdesk.com to access your check-in instructions


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In the chocolate community, there's been so much made of going natural where hair is concerned. Sometimes it seems as if it's a movement! I have been a natural chica (chemical free) since the 90's. I just tend to wear my hair straight – a lot. For YEARS I've gone back and forth with my hair. Sometimes it's curly but most times it's straight. When it's freshly washed, it's full of little springy curls. I like that. The issue is, from flatironing my hair so much, the ends of my hair sometimes flat out refuse to curl anymore. Boo!
That being said, I have recently decided to make my hair do what I want because I said so. For me that means wearing my hair natural, heat free and in all it's curly glory more often. I figure the more it stays in it's natural state, it will eventually get with the program and give me all my curls back. Right? Maybe.
The cool thing for me is right when I made this decision, I was offered the chance to review some products by Pantene Pro-V for natural, African American hair. I was totally game! I had already used their "Relaxed and Natural" line before, so I was excited that they had now separated the two and have a Truly Relaxed line and Truly Natural line. I got these goodies in the mail and immediately went to work on my head.
Here's what my hair looked like before. Its was just blow dried. I hadn't flatironed it, so it was a little on the lion mane side of things.
I washed with the Clarifying Shampoo. I had a lot of oils in my hair from wearing it slicked back all week and this really worked to get my hair clean. Then, I followed that up with the Cowash Cleansing Conditioner. That one was new for me. I'd heard of co-washes but hadn't used one before. Once I rinsed that out my hair felt pretty soft. Then, I used the Deep Conditioner. I rinsed it out and began to detangle my hair. It suprisingly didn't take as long as I though it would. My hair was pretty soft after washing and conditioning so the detangle went pretty smooth. I followed with the Curling Custard. I had used some others before and it was kind of hit and miss. I watched a few YouTube videos to help me with my application method and got the hang of it immediately. I had to keep spraying my hair using my water bottle to keep it wet and keep the application smooth. I swear, my hair soaks up water like the Mojave desert! After that, I let it air dry.
That's the other thing about my hair. It sucks up water for the most part and dries pretty fast except for the center of my head. That part could stay wet for days, I tell you! My curls were really defined and being their cute, springy selves. I was excited to see how it would turn out after it was dry.
Here I am all dry and curly! I used a little of the Pantene Shine Serum to control any potential frizz and to soften a bit from using the custard. I wore my hair in a few styles with this wash and go. I just let it run free at times and then I'd pin some of it back or wear a bun. I was happy with how versatile my hair is.
It's been about three weeks of me using the products and it's true, the longer I wear it heat free, the curlier it gets. I guess that means I am the boss of my hair! Whoot!
The Pantene Pro-V line of Truly Natural products are all under $6. From what I've seen regarding products for natural hair, this is a deal! My only issue is that for me and others going natural is more than a hair style choice it often applies to other areas of our lives. For me, it's about what I put on my body and in my body. So, I wish these products for naturals included more natural ingredients.
That being said, the products worked for my hair and I have gotten LOTS of compliments.
I was selected for this opportunity as a member of Clever Girls Collective, and the content and opinions expressed here are all my own.


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|Message view
|« Date » · « Thread »
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|"Stephane Bailliez" <sbaill...@apache.org>
|Re: [embed] few changes in xml processing
|Mon, 14 Oct 2002 18:28:35 GMT
----- Original Message ----- From: "Steve Loughran" <firstname.lastname@example.com> > > 4. I added ${} processing to text body. I hate <echo>${prop}</echo> > > behaving differently from <echo message="${prop}"/>. I don't know > > if this will ever get into the main branch, but I intend to keep it > > in the proposal for a while, maybe I can convince people. > > I woorry about tasks that explicitly expect the conversion not to catch, > things like SQL, maybe. It could be all those tasks that dont expand > properties do so by accident of course. I think there is a bug/patch hanging around in bugzilla to enable property processing in SQLExec I really need to sit down and do my homework in Ant to fix bugs but I have many things to do and this should not improve in the next weeks. (At least I hope so for some reasons) Stephane -- To unsubscribe, e-mail: <mailto:email@example.com> For additional commands, e-mail: <mailto:email@example.com>
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text:
Transportation cannot be provided for birthday parties, sleepovers,
play dates, scouts or other activities. No bus transportation will be
provided to businesses or day care centers.
Rules for School Bus Safety
for Bus Safety are emphasized at school and each bus driver insists on
the cooperation of each rider. Please review these rules with your
child at home.
Students must remain seated and face the front of the bus at all times.
Students may not delay the bus schedule while loading/unloading at school and/or at home.
smoking, eating, loud talking, horseplay, selling items, using
profanity or refusing to obey school authorities or a bus driver while
waiting for, riding or leaving a school bus will not be tolerated.
Students may not change seats during the bus trip unless directed to do so by the bus driver.
Students must meet the bus at the designated stop and may not get off at any unassigned stop without permission from the office.
Vandalism and tampering with buses will not be permitted. A fee will be charged for damages to buses.
Students may not be under the influence of or have in their possession alcoholic beverages or illegal drugs.
Cellular phones or electronic games are not allowed on the bus.


text:
"Everybody is family," the 6-foot-7 small forward told Scout.com. "Everybody knows their role on the team. We are just trying to win."
Winning is something Oak Hill is used to. The Warriors are off to a 9-0 start and have won all nine games by more than 24 points. Smith, who went for 16 points in Oak Hill's win over Jackson (Miss.) Callaway on Saturday, has been a big part of the strong start.
While Smith has been focused on performing on the court, he's also put a lot of thought into his recruiting process. The four-star prospect said he's still weighing his options, but has narrowed his list down to three.
"I'm still picking out my pros and cons," Smith said. "I'm just trying to find the right one."
Smith is down to Georgetown, Connecticut and Duke. All three schools have their particular advantages, and Smith broke down the three and what he likes about each.
"With Georgetown I like everything basketball wise and outside of basketball," Smith said. "Even though they always talk about the Princeton offense, it gets it done and they win games."
"I like their history with all of the small forwards and guys that played my position and got to the league," Smith said about Connecticut. "Coach Calhoun knows what he's doing."
"I like the system at Duke," Smith said. "I like the freedom in the offense everybody can shoot the three ball. I like freedom."
He took visits to all three schools prior to the November signing period, but still decided to wait.
"I didn't feel comfortable because I feel as though I have good situations and couldn't go wrong with any of my top three schools," he said. "I called the coaches and told them and they respected my decision."
Smith said he doesn't currently have a timetable on when he plans to make his college decision.


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To try the thousands of add-ons available here, download Mozilla Firefox, a fast, free way to surf the Web!Close
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Aw, how cute! Soft and cuddly little chicks...adorable! =)
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Denis Laws (born "Frederick Goodwin") played Sam Hench, an evacuee from the Mark Brittain Warehouse fire on Coronation Street in October 1975.
Under his usual credit of Dennis Lawes he also appeared in Club Night, Play for Today , I Didn't Know You Cared and the television play A Little Outing.
Outside of these appearances he was, between the wars, a music hall comedian who made several appearance on BBC radio in the 1930s. His granddaughter is the crime novelist Elly Griffiths.


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The experts in products for business
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View the full range Steel Strapping Equipment
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Heavy duty mobile dispenser trolley for safe storage and retrieval of ribbon wound steel strapping.
The mobile dispenser trolley for wound steel strapping is easily portable and perfect for use in a warehouse or other workplace that has the need to use steel strapping for securing parcels for storage or for transit to other places. Weighing 13.2 kg, it can be moved using the rubber wheels and tubular steel handle, making it fine for use in almost any area of the workplace. Portability is one of the most important features of this unit as it means that items can be packaged at different locations in the workplace. This type of strapping is used mainly on pallets and wooden packing cases which need to be secured properly before being moved.
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Basketball Hoop Charm in 10K Gold
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She shoots, she scores! Create a look all about you by adding a whimsical charm to your necklace or bracelet. This basketball hoop is crafted of 10K gold with a diamond-cut and polished finish.
Typographic errors are subject to correction. Merchandise enlarged to show detail and may not always be exactly as pictured. Promotions, prices, styles and availability may vary by store and online.


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A drama of parricide and intense family rivalry, this book is Dostoyevsky`s acknowledged masterpiece.
It tells the story of the murder of a depraved landowner, Fyodor Karamazov, and the ensuing investigation and trial,concentrating on the parts played by Karamazov`s three sons, Mitya, Ivan and Alyosha. Ivan is a revolutionary intellectual, while the young novice Alyosha is, according to Dostoyevskys, the novel`s hero. It is Mitya`s passion for two women that contributes to disaster, and it is he who inwardly accepts the guilt of his father`s murderer. In unfolding the fates of the Karamazovs, Dostoyevsky creates a tragedy of Shakespearian force and vividness. A story that embodies the disintegration of a whole society, Russia in the 1870s. This book is the culmination of his work, enduring in its vitality, masterly inventiveness and vision.


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Blue Mountain Hickory Collection
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Set in Fanadix, this air-conditioned villa features a patio and a garden with a seasonal outdoor pool. The unit is 27 km from Benidorm.
A TV is featured. There is a private bathroom with a bath or shower.
Gandía is 39 km from Casa Dels Tossals, while Calpe is 7 km from the property. The nearest airport is Alicante Airport, 73 km from the property....


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Late in spring 2016 we sent our team of expert ski and snowboard testers to Kuhtai for the SIGB ski test and Kaunertal for the Snowboard Spring Break. They spent a week riding and rating over 150 skis and snowboards. Every major brand was represented on piste, powder and park, and for all abilities. The skis and snowboards were painstakingly whittled down and winners decided for every category…
Gear Guide 2016-2017
The ski test...
Kühtai, in the Austrain Tirol is perched at 2,020m above sea level, making it Austria's highest resort. With doorstep skiing and guaranteed snow, as well as a range of terrain easily accessible from the base, it's the perfect testing ground for skis.
To sort the wheat from the chaff, every one of the 100 pairs of skis we tested was ridden and rated for overall performance and value, and then scored against specific performance criteria for the category it was competing in: be it Piste, All-Mountain, Freeride, Freestyle or Touring. We then picked one winner in each category, plus a women's All-Mountain winner.
Snow's ski test team...
Rob Stewart is a BASI Level three instructor, former freestyle competitor turned ski writer with over 30 years skiing experience. This is Rob's fifth year testing skis.
Bruce Pope has been a full-time sports journalist for almost two decades and is also a qualified BASI Level Two ski instructor – giving a valuable insight to the test.
Heath Judson is a highly experienced ski instructor and coach who has worked in Austria, Scotland and even South Africa. He's currently an Assistant Buyer for Sport Etz in Kitzbühel.
Martin Hannaford is a keen skier whose experience includes several seasons in the 3 Valley's and regular trips to the Alps. Martin skis mostly on-piste, but enjoys the deeper stuff too.
Cazz george started skiing as an adult but was soon hooked. After three years managing children's clubs for a travel company, Cazz passed her BASI instructor qualifications and made the move to Zermatt.
Ellie Koyander is an Olympic skier who first skied at 18 months old and competed at the 2010 Olympics in Vancouver. We can't confirm whether she did backflips on any of the skis during the test…
Our ski test team were kindly hosted by Crystal Ski in the four star ski-in, ski-out Sport Hotel, located in Kuhtai village centre.
Crystal Ski offer packages to both the Sport Hotel and the Hotel Astoria.
The snowboard test...
Our snowboard test took place at the annual Spring Break Snowboard Test, at the glacier-blessed Austrian resort of Kaunertal. With guaranteed snow this skiing area is the perfect testing ground. Just two lifts give you access to powder runs, leading to wide-open pistes, and an extensive park. Finally the run flows into a long and feature-packed piste with natural hits at either side for tricks, jumps and general fun.
In total we rode and rated 35 boards and scored them in each category against specific criteria for that type of riding.
Snow's snowboard test team...
Jamie barrow
Britain's fastest snowboarder, Jamie holds three world records, including the Guinness World record for the fastest speed on a snowboard while being towed by a vehicle. Which was not how we tested these boards…
Mark barber
Embedded in the snowboarding world somewhere between professional riders and brand managers, Mark is an action sports journalist specialising in winter sports travel, snowboarding and gear reviews.
Maisie potter
Testing our women's boards is three times national snowboard champion Maisie, who (when not shredding new boards for us) represents Great Britain in international Boardercross competitions from her base in Morzine.


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Microloans for clean energy in the developing world
One-third of the world's population today has no access to any form of modern energy, relying instead on highly polluting fuels like wood and kerosene. Working on the premise that reliable energy is a key to fighting poverty — and that it needs to be clean — Energy in Common is a New York-based nonprofit dedicated to facilitating microloans to bring green energy to people in need.
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Add these lines to autocmd.vim:
:au FocusLost * if(&modified) " buffer changes...: * PENDING :au FocusLost * :hi Normal guibg='#330000' " window background: * ALARM :au FocusLost * else" " buffer changes...: o none :au FocusLost * :hi Normal guibg='#000033' " window background: o ok :au FocusLost * cclose " quickfix window..: o close :au FocusLost * endif :au FocusGained * :hi Normal guibg='#000000'The first line is about the current buffer only (the one from the window where the input focus is)
Replacing it with the line bellow will check up to 3 windows:
:au FocusLost * if (getbufvar(winbufnr(1), '&modified') != 0) || (getbufvar(winbufnr(2), '&modified') != 0) || (getbufvar(winbufnr(3), '&modified') != 0)
What is this about? Why does ":hi" have a colon? JohnBeckett (talk) 10:25, June 24, 2016 (UTC)
--- Answer to the request for some explanation from JohnBeckett ---
I'm totally confident about how vim syntax keywords do convey enough real-life-language semantics to be significant by themselves. If it is not the case for a reader, I think she,he is not on the right path of an efficient learning curve.
Still, for the sake of positive thinking that can't hurt:
Losing focus, in the context of using an editor, can only mean one thing: The user is switching his,her attention on something else - mostly some kind of next-step along the current process.
This auto-command is supposed to attract her,his attention on the fact that something may be missing as a "supposedly" to-be-committed previous step of that process by slightly changing the vim window background.
That's all there is to it.
As, for the colon of the :hilightight command,
1 - It comes from my personal intensive use of single line vim commands saved in project-specific scratchpad-files I use every day by hitting a single key. My F1 is mapped to execute whatever there is in the line under the cursor (hence the colon to enter command-mode):
:map <F1> "ayy@a
...that's: Yank this line and @xecute it as a command-line as if I was typing it myself
2 - This allows line-by-line execution while editing a script.
3 - And it does not hurt a bit to leave them there for further editing.
Instead of the hard-coded 3-buffer check, I'd suggest using a :for loop over numbers from 1 to bufnr('$'), check if the buffer exists with bufexists(), and then get the modified flag as you're doing. Then you can check all the buffers instead of only a few. There may even be a better way to loop over the buffers but this should work at least. --Fritzophrenic (talk) 16:53, July 14, 2016 (UTC)
Right, I knew that it would come to something more than a few easy trick-lines eventually... You are absolutely right when it comes to hard-coded values mixed up with hard-coded logic. As of now, values belong to user space, logic is not mature enough for dynamic mutation... Digression aside, here is my next-step working solution:
:augroup focus_buffer_check " (160715) {{{ "---------------- FOCUS GAINED: Set your working GUI BACKGROUND COLOR :au FocusGained * :hi Normal guibg='#000000' "---------------- FOCUS LOST: check the 'modified' option of all visible buffers :au FocusLost * let i = 0 :au FocusLost * while i < bufnr('$') :au FocusLost * if getbufvar(winbufnr(i), '&modified') :au FocusLost * break :au FocusLost * endif :au FocusLost * let i = i+1 :au FocusLost * endwhile "---------------- SET GUI_BACKGROUND_COLOR ACCORDINGLY :au FocusLost * if i < bufnr("$") " FOUND SOME 'modified' buffer :au FocusLost * :hi Normal guibg='#330000' " -> [ALARM-COLOR] :au FocusLost * else" " FOUND NO 'modified' buffer :au FocusLost * :hi Normal guibg='#000033' " -> [OK-COLOR] :au FocusLost * cclose " -> close quickfix window :au FocusLost * endif :augroup END " focus_buffer_check }}}
...I dropped the bufexists() check for now, until I stumble on a use case to solve
- Nice! Feel free to go back and edit rather than just leaving a comment.
- By the way, were you aware continuation lines work in an autocmd? For example for just one of your "if" statements:
:au FocusLost * if getbufvar(winbufnr(i), '&modified') | \ break | \ endif
- --Fritzophrenic (talk) 14:52, July 15, 2016 (UTC)
I do appreciate both remarks - I only left the initial "simplistic" version, so serve as a concise topic intro (at least that was the idea). - As for the continuation line, that's good to know. But that too is the result of long-term habits aimed at preserving line-swappability as much as possible. (Curiously enough, after 35 years using ed, vi, vim, gvim!, I still have no idea why I still have no real knowledge about vim-scripting... (much to learn for me there is, hehe)


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Chris Faulkner at Wikia
Welcome to the Chris Faulkner mini wiki at Scratchpad!
You can use the box below to create new pages for this mini-wiki. Make sure you type
[[Category:Chris Faulkner]] on the page before you save it to make it part of the Chris Faulkner wiki (preload can be enabled to automate this task, by clicking this link and saving that page. Afterwards, you may need to purge this page, if you still see this message).
This is my test page for my personal wiki. Ill have to look into it a bit more to see if I am allowed to do this


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From September 10 to October 29 2005
Opening: September 10 2005
"The cupboards are sketches, 3d sketching with old and new materials. Try to find the right balance for a new product, what is authentic and would work with that in new materials. One antique cupboard became after cutting and glue-ing several new cupboards, like a collage models. When does the new product visual flips over from old towards a new style, how strong is this old cupboards on our memory, what if the new parts have decoration integrated in an artificial material. This project is a form and material study with trial and error method, just a step for searching in a new language. Cut and past is a theme what I'm working on at the moment. (…)" Hella JONGERIUS, July 2005.
Press Release


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|Home | About | Journals | Submit | Contact Us | Français
The observation that Tcf3 (MGI name: Tcf7l1) bound the same genes as core stem cell transcription factors, Oct4 (MGI name:Pou5f1), Sox2 and Nanog, revealed a potentially important aspect of the poorly understood mechanism whereby Wnts stimulate self renewal of pluripotent mouse embryonic stem (ES) cells. Although the conventional view of Tcf proteins as the β-catenin-binding effectors of Wnt signaling suggested Tcf3-β-catenin mediated activation of target genes would stimulate ES cell self renewal, here we show that an antagonistic relationship between Wnt3a and Tcf3 on gene expression is important for regulating ES cell self renewal. Genetic ablation of Tcf3 replaced the requirement for exogenous Wnt3a or GSK3-inhibition for self renewal of ES cells, demonstrating that inhibition of Tcf3-repressor is the necessary downstream effect of Wnt signaling. Interestingly, the molecular mechanism underlying Wnt's effects required both Tcf3-β-catenin and Tcf1-β-catenin interactions, as they each contributed to Wnt stimulation of self renewal and gene expression. Finally, the combination of Tcf3 and Tcf1 was necessary to recruit Wnt-stabilized β-catenin to Oct4 binding sites in ES cell chromatin. These results elucidate the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network.
The Oct4/Sox2/Nanog core self renewal circuit was defined primarily by experiments showing physical interactions between proteins1, 2, substantially overlapping sites of chromatin occupancy3, 4, and similar effects in loss of function experiments5–9. Several data indicated that Tcf3 was an integral component of the Oct4/Sox2/Nanog self renewal circuit. ChIP-seq experiments showed that of the 1369 known genes identified as Tcf3-bound, 1173 were also bound by Oct4, and 942 were bound by all four factors (Tcf3, Oct4, Sox2, and Nanog)10. The co-occupancy of Tcf3 with Oct4, Sox2 and Nanog on a genome-wide scale was also independently determined with ChIP-chip experiments11, 12. Whereas knockdown of Nanog or Oct4 caused largely similar effects on gene expression4, the ablation of Tcf3 caused strongly opposite effects on gene expression compared to Nanog knockdown and Oct4 knockdown13.
One intriguing aspect of Tcf3's function in stem cells has been its potential role as a downstream effector of the canonical Wnt signaling pathway. An important consequence of Wnt ligand binding its receptor is the inhibition of GSK3 phosphorylation of β-catenin14. This stabilizes β-catenin from ubiquitin-mediated degradation and promotes signaling by allowing β-catenin to form transcription-activating complexes with DNA-binding proteins, most notably Tcf factors15–17. Treating cells with Wnts18 or blocking GSK3 activity by genetic ablation19 or small molecule inhibitors 18, 20, 21 all effectively stimulated ES cell self renewal. Indeed, inhibition of GSK3 combined with inhibition of ERK activity in so-called two-inhibitor (2i) conditions was sufficient to promote ES cell self renewal in the absence of serum and extrinsic cellular signals20, 21.
The role of Tcf-β-catenin complexes as downstream mediators of Wnt and GSK3 effects has been complicated by the facts that GSK3 affects many cellular activities in addition to destabilizing β-catenin, and it is difficult to experimentally control for the broad effects of GSK3 inhibition. As such, it is not clear what biochemical activities downstream of Wnt and GSK3-inhibtion were most important for ES cell self renewal; however, previously published examination suggested three intriguing hypotheses: either, 1. the effects of Wnt-stimulated β-catenin needed to function through an Oct4-β-catenin interaction and Tcf-β-catenin interactions only secondarily affected self renewal22, 2. Tcf3-β-catenin activator complexes directly stimulated self renewal and the expression of self renewal genes11, or 3. Tcf3 transcriptional-repressor activity inhibited self renewal and was suppressed by Wnt13, 23.
To test these hypotheses, a series of cell-based experiments were completed. First, the ability of Wnt signaling to replace LIF and to support self renewal in Tcf3+/+ cells was confirmed by showing that Oct4 promoter activity and protein levels were maintained in ES cells by adding recombinant Wnt3a to LIF deficient cultures (Supplementary Fig. S1a,b). This effect required an intact Oct4/Sox2/Nanog circuit, as Wnt3a was unable to rescue LIF deficiency in cells genetically lacking Nanog (Supplementary Fig. S1c,d). Since a similar experiment using Tcf3−/− would not be informative because these cells self renew in the absence of exogenous LIF even without Wnt stimulation13, we tested the possibility that forced overexpression of Tcf3 could inhibit self renewal by using a doxycycline-regulated system to increase Tcf3 levels in Tcf3+/+ cells (Supplementary Fig. S2a)24. If the hypothesis of a Tcf3-β-catenin transactivator complex was correct, one would expect that ectopic expression of Tcf3 would enhance ES cell self renewal upon Wnt3a stimulation. Instead, a twofold increase in Tcf3 protein efficiently blocked self renewal, as no alkaline phosphatase (AP)+ ES colonies were present four days after induction of Tcf3 in +LIF cultures (Fig. 1a). Flow cytometry of propidium iodide-stained cells showed a conversion from the high S-phase, ES cell cycle profile to a G1-enriched, somatic cell cycle profile within 72 hours of Tcf3 overexpression (Fig. 1a). Wnt3a sharply inhibited the differentiation-promoting effects of Tcf3 by restoring AP+ colonies and an ES cell cycle profile to Tcf3-overexpressing cells in both +LIF and LIF-deficient conditions (Fig. 1a,b); however, colony formation of Wnt3a-treated, Tcf3-overexpressing cells was diminished relative to the non-overexpressing controls. These results best support the hypothesis that Tcf3 represses self renewal and Wnt3a antagonizes Tcf3-repression.
Recently, Tcf-β-catenin-independent effects were suggested to provide a mechanism whereby inhibition of GSK3 stimulated the self renewal of ES cells21, 22. These conclusions were based primarily on the inability of heterologously expressed Lef1ΔN21 or Tcf4ΔN22 proteins to rescue differentiation defects caused by stable β-catenin. These observations suggested that the self-renewal stimulating effects of Wnt or GSK3-inhibition were independent of Tcf protein function21, 22, which appeared to be inconsistent with the ability of full length Tcf3 to inhibit self-renewal when overexpressed (Fig. 1a,b). To provide a better test of the role of endogenous Tcf proteins in ES cells, we performed experiments using loss of function techniques. First, we tested whether ablation of Tcf3 could replace the requirement for GSK3 inhibition for self renewal in serum free culture conditions21. After a single passage in serum free conditions, Tcf3−/− cells generated a high percentage of AP+ colonies when only PD0325901 (ERK Inh) or both PD0325901 and CHIR99021 (2i) were added to media (Fig. 1c). In contrast, Tcf3+/+ cells generated a high percentage of AP+ colonies only in 2i conditions and differentiated in ERK Inh only (Fig. 1c). While both Tcf3+/+ and Tcf3−/− cells generated AP+ colonies in the presence of CHIR99021 alone (GSK3 Inh) for a single passage (Fig. 1c), both cell lines lost viability and differentiated in GSK3 Inh conditions after being passaged (Fig. 1d). Importantly, Tcf3−/− cells were serially passaged in ERK Inh conditions and maintained a high level of proliferation and AP+ morphology (Fig. 1d). Thus, ablation of Tcf3 effectively replaced the need for GSK3-inhibition in serum free culture conditions, suggesting that antagonism of Tcf3 is a critical downstream effect of GSK3-inhibitors in 2i culture conditions.
To define better how Tcf3 and Wnt signaling functioned together in ES cells, we compared effects of recombinant Wnt3a-treatment (W) or control media (C) on gene expression in Tcf3+/+ and Tcf3−/− ES cells. Principal component analysis (PCA) was used to explore correlations among gene expression microarray datasets for three biological replicates for each of the four different conditions (Tcf3+/+(C), Tcf3−/−(C), Tcf3+/+(W), Tcf3−/−(W)) (see Methods)25. Plotting each replicate according to its principal component (PC)1 and PC2 values effectively separated each of the four conditions while grouping replicates according to their condition (Fig. 2a). Interestingly, Tcf3−/−(C) and Tcf3+/+(W) conditions shared similar PC2 scores, suggesting these two conditions shared a common set of variations that differed in the other conditions. Tcf3−/−(C) and Tcf3+/+(W) had different PC1 scores, suggesting that these two conditions differed from each other based on the variations comprising PC1. Thus, Tcf3-ablation and Wnt3a-treatment cause one set of common changes and one set of different changes.
Since Tcf3-ablation and Wnt-activation both stimulated self renewal of ES cells11, 18, 23, common effects were likely to be mediators of self renewal. To identify these genes, we focused on the 831 genes that were significantly increased (Benjamini-Hochberg adjusted FDR< 0.05; Supplementary Fig. S3a) in any of three comparisons: 1. Increased in Tcf3+/+(W) vs Tcf3+/+(C), 2. Increased in Tcf3−/−(C) vs Tcf3+/+(C), and 3. Increased in Tcf3−/−(W) vs Tcf3−/−(C) (Supplementary Table S1). Each gene was given three scores corresponding to their ranked fold-increase for each of the three comparisons (Supplementary Table S2). To populate a group of common changes, a gene was assigned to group 1 (Red; Fig. 2b,c) when the effect (i.e. ranked fold-increase) caused by Wnt3a in Tcf3+/+ cells (score #1) was closer to the effect caused by Tcf3 ablation (score #2) than to the effect of Wnt3a in Tcf3−/− cells (score #3). For example, Tbx3 was placed in group 1 because its ranked fold-increases for comparisons 1–3 were 29|20|336, respectively; Cdx1 was placed in group 2 with ranked fold-increases of 10|257|17 (Supplementary Table S2). Performing this relatively simple calculation separated all genes into two strikingly distinct groups, which was evident when ranked-fold increase values were used to plot individual genes (Fig. 2b). For group 1 genes, Wnt3a effects on Tcf3+/+ cells displayed a strong correlation (Pearson correlation coefficient ρ= 0.90) when compared to effects of ablating Tcf3 (Fig. 2b, bottom left). Wnt3a had different effects on group 1 genes in Tcf3+/+ cells compared to Tcf3−/− cells (ρ = −0.23) (Fig. 2b, top left). In contrast, for group 2 genes (Blue; Fig. 2b,c), Wnt3a effects on Tcf3+/+ cells displayed a strong correlation (ρ = 0.91) when compared to Wnt3a effects on Tcf3−/− cells (Fig. 2b; top right). Thus, Wnt3a stimulated one set of genes similarly to the ablation of Tcf3 (group 1), and stimulated a second set of genes regardless of Tcf3's presence (group 2).
When the effects of Wnt3a and Tcf3 were compared to the effects of Oct4 shRNA for the two groups of genes (Fig. 2c), a moderate to strong anti-correlation was observed only for the Tcf3-dependent group 1 genes (ρ = −0.65 for Wnt3a on Tcf3+/+ cells, and ρ= −0.72 for Tcf3 ablation). No relationship with Oct4 was apparent for the Tcf3-independent group 2 genes (ρ values from −0.40 to 0.32), or with the effects of Wnt3a on Tcf3-dependent group 1 genes in the absence of Tcf3 (i.e. in Tcf3−/− cells; ρ = 0.31). Thus, group 1 genes correlated well with regulation by Oct4, consistent with these genes being most relevant for self renewal. In addition, a separate, independent set of 311 genes was selected because they were decreased by Nanog and Oct4 shRNA4; most of these genes (81%) were increased in Tcf3−/− cells compared to Tcf3+/+ cells. Wnt3a stimulation of these genes frequently achieved a higher level (Fig. 2d, green bar, 44% of genes) or nearly the same level (Fig 2d, blue bar; 12% of genes) in Tcf3−/− cells compared to Tcf3+/+ cells. Wnt3a-stimulation of genes rarely achieved a higher level in Tcf3+/+ cells compared to Tcf3−/− cells (Fig. 2d, orange bar, 2% of genes; Supplementary Fig. S3b). The overlapping chromatin occupancy of Nanog and Tcf3 at common genes10, 11 and Wnt3a/Tcf3-regulation of Nanog levels (Fig. 3a,c; Supplementary Fig S2a,b)23 suggested the possibility that Wnt3a/Tcf3 effects could occur secondarily through deregulated Nanog. We tested this by reducing Nanog transiently in Tcf3+/+ cells by siRNA (Fig3a) or stably in Tcf3−/− cells by shRNA (Fig 3b) and by increasing Nanog in Tcf3+/+ cells by stable transfection of a Nanog cDNA (Fig. 3c). Measuring levels of group 1 gene products after either loss or gain of function experiments indicated that Wnt3a/Tcf3 effects did not correlate with increased Nanog levels (Fig. 3d), indicating that increased Nanog levels did not mediate Wnt3a/Tcf3 effects on gene expression. Taken together, these data show that Tcf3-inhibits whereas Wnt3a stimulates a common set of genes, and these common genes are most closely related to the effects of the core Oct4/Sox2/Nanog self renewal circuit.
We were intrigued by the observation that most Wnt3a-stimulated genes did not require Tcf3 for activation, and instead were stimulated to a higher level in Tcf3−/− cells than Tcf3+/+ cells. Consistent with results from Fig. 1, this observation suggested that Tcf3-mediated repression was only inhibited by Wnt3a, and that Tcf3 did not function as an activator of target genes. These observations suggested Tcf3-β-catenin may not be important for actively stimulating Nanog/Oct4/Sox2/Tcf3 co-occupied genes, as suggested by Cole et al 11. To test this possibility and to determine the role of Tcf3-β-catenin interaction in ES cells, we sought to block the Tcf3- β-catenin interaction in ES cells. Given the differentiation-inducing effects of twofold overexpression of Tcf3 (Fig. 1), we reasoned that experiments utilizing heterologous expression of Tcf3 mutants would be prone to neomorphic effects; therefore, we engineered Tcf3ΔN/ΔN ES cells in which nine residues necessary for β-catenin binding were deleted from the endogenous Tcf3 gene product by homologous recombination (Supplementary Fig. S4a,b). When grown in self renewal culture conditions (+LIF, +serum) without exogenous Wnt3a, Tcf3ΔN/ΔN ES cells were indistinguishable from Tcf3+/+ cells in terms of cell proliferation, cell cycle kinetics, and expression of Tcf3, Oct4, and Nanog (Supplementary Fig.S4c, S5c). Interestingly, Wnt3a stimulation of AP+ colony formation in the absence of LIF was reduced, but not eliminated, in Tcf3ΔN/ΔN cells (56% for Tcf3ΔN/ΔN compared to 77% for Tcf3+/+) (Fig. 4a) and, treating Tcf3−/− cells with Wnt3a also stimulated AP+ colony formation (from 70% to 86%) (Fig. 4a). Similar results were obtained with experiments performed using serum-free, 2i conditions (Supplementary Fig. S4d). Moreover, ablation of the Tcf3-β-catenin interaction diminished but did not eliminate the ability of cells to activate Tcf-β-catenin responsive promoter reporters, SuperTOPFlash, Nanog, and Nr5a2 (also called LRH-1) (Fig. 4b). These data are not consistent with a previously described mechanism11 suggesting a key role for Tcf3-β-catenin as an activator. Moreover, the only partial inhibition of Wnt3a stimulation of self renewal and target gene activation in Tcf3ΔN/ΔN cells revealed that Wnt3a stimulation of self renewal involves a combination of Tcf3-β-catenin dependent and previously uncharacterized Tcf3-independent mechanisms.
The possibility that another Tcf protein could contribute Tcf3-independent effects of Wnt3a was supported by several observations. First, the highly conserved HMG domain shared by Tcf proteins allows all Tcf proteins to bind to the same sequences as Tcf326, 27. The more severe phenotypes of double mutant mouse embryos (Tcf1−/−; Tcf4−/−, Tcf1−/−; Lef1−/− and Tcf3cKO, Tcf4−/−) compared to single mutants demonstrated that one Tcf protein can compensate for the absence of a family member, thus indicating the conserved HMG domains allow Tcf family members to affect an overlapping set of target genes28–30. Finally, since the SuperTOPFlash reporter is specific for Tcf proteins31, reporter activation in Tcf3−/− and Tcf3ΔN/ΔN cells showed that another endogenous Tcf protein was present in ES cells, and that this Tcf was capable of activating target genes.
To determine which Tcf protein was activating Tcf3-target genes, we first measured levels of Tcf gene products in ES cell lines. Tcf3 mRNA was the most abundant family member in Tcf3+/+ and Tcf3ΔN/ΔN cells (Fig. 5a), Tcf1 and Tcf4 were also detected at levels that we reasoned could have a biological effect (Fig. 5a). The ability of each Tcf protein to activate gene expression in response to stable β-catenin was tested by measuring the activation of SuperTOPFlash (Fig. 5b). Tcf1 and Lef1 displayed a similar activation of Tcf-β-catenin signaling, whereas Tcf3 and Tcf4 both repressed SuperTOPFlash and Nanog reporter activity in ES cells (Fig. 5b, Supplementary Fig. S6b). Since, Tcf1 was the best candidate to function endogenously in ES cells to activate Tcf3-repressed genes in response to Wnt-stabilized β-catenin, we used stable lentiviral shRNA knockdown to inhibit the Tcf1 gene product in each Tcf3+/+, Tcf3−/−, and Tcf3ΔN/ΔN cells. This approach was effective at reducing Tcf1 mRNA and protein levels (Fig. 5c), and it affected >90% of cells for each cell line as indicated by GFP reporter gene expressed from the lentiviral shRNA vector (Supplementary Fig. S5a). Control cell lines harboring a scrambled shRNA sequence (SCRM) were also generated for each Tcf3 line. In the absence of exogenous Wnt3a, each of the Tcf1 shRNA cell lines were similar to corresponding SCRM cell lines in terms of cell proliferation, cell cycle kinetics, and expression of Tcf3 and Oct4 (Fig. 5c, Supplementary Fig. S5b,c). Thus, Tcf1 knockdown did not cause an apparent cellular defect under normal self renewal conditions, without the addition of exogenous Wnt to the media. In contrast, Tcf1 shRNA substantially reduced transcriptional activation by stable β-catenin in each Tcf3+/+, Tcf3−/−, and Tcf3ΔN/ΔN ES cells (Fig. 5d). Note that promoter activity was higher in the absence of Tcf3 (in Tcf3−/− cells) (Fig. 5d) without an increase in Tcf1 or Lef1 mRNA or protein or β-catenin protein (Fig 5a, Supplementary Fig. S6a), which was consistent with Tcf3 functioning as a repressor and directly interfering with Tcf1-β-catenin mediated transcriptional activation. Interestingly, whereas Tcf3ΔN/ΔN; Tcf1 shRNA and Tcf3+/+; Tcf1 shRNA cells displayed very low levels of activity, the Tcf3−/−; Tcf1 shRNA cells displayed promoter activity comparable to Tcf3+/+; SCRM cells. These data suggest that even residual levels of Tcf1 remaining after shRNA knockdown (Fig. 5c), and possibly the low endogenous levels of Lef1 (Fig. 5a,b), were sufficient to activate SuperTOPFlash when Tcf3-repression is eliminated by genetic ablation.
A role for endogenous Tcf1 in Wnt-stimulation of self renewal was tested with colony formation in serum-containing media lacking LIF. Without exogenous Wnt3a, Tcf1 shRNA and SCRM cell lines differentiated similarly to form primarily AP-negative colonies in Tcf3+/+ and Tcf3ΔN/ΔN cells and primarily AP+ colonies in Tcf3−/− cells (Fig. 6a). When exogenous Wnt3a was added, Tcf1 shRNA decreased the generation of AP+ colonies by half for both Tcf3+/+ and Tcf3ΔN/ΔN cells (Fig. 6a). In contrast, Tcf1 shRNA had a less substantial effect on Wnt3a treated Tcf3−/− cells, which still generated AP+ colonies from 68% of Tcf1 shRNA-containing cells. To confirm that self renewal effects were caused by Tcf1 knockdown and not off target effects, transfection experiments using siRNA molecules targeting a different Tcf1 sequence were performed; Tcf1 siRNA caused a similar 50% reduction in AP+ colonies for Tcf3+/+ and Tcf3ΔN/ΔN cells compared to control (Supplementary Fig. S5d,e). Considering both the clonal nature of the assay using shRNA cell lines and that 9% of Tcf3ΔN/ΔN; Tcf1 shRNA cells plated were GFP-negative (Supplementary Fig. 5a), the 14% of Tcf3ΔN/ΔN; Tcf1 shRNA colonies displaying an AP+ ES colony morphology suggested that Wnt3a stimulation of self renewal primarily relies on the combination of Tcf1 and Tcf3-β-catenin interaction. These results show that endogenous Tcf1 is a key mediator of Wnt stimulation of self renewal in ES cells.
To determine if endogenous Tcf1 was important for Wnt-effects on Tcf3-regulated genes, we measured expression of several Wnt3a-stimulated and Tcf3-repressed genes by qPCR. Expression levels of endogenous genes were measured for 12 conditions (+/− Tcf1 shRNA and +/− Wnt3a treatment in each Tcf3+/+, Tcf3−/−, and Tcf3ΔN/ΔN ES cells). The relative level of each gene is shown by heat map (Fig. 6b); numerical values and standard deviations are shown in Supplementary Table S5. For previously characterized Wnt/Tcf target genes (T, Cdx1, Axin2), Wnt3a stimulated high expression levels in Tcf3+/+ and Tcf3−/− cells, and much lower levels in Tcf3ΔN/ΔN cells (Fig. 6b, top). In each Tcf3+/+, Tcf3−/− and Tcf3ΔN/ΔN cells, Tcf1 shRNA reduced the level of Wnt3a stimulation. These results were consistent with Axin2, T, and Cdx1 being predominantly regulated by Tcfs and Wnt signaling in ES cells. Moreover, they showed that Tcf1 was necessary for full Wnt3a-stimulation of gene expression. Genetically blocking Tcf3-β-catenin interaction (in Tcf3ΔN/ΔN ES cells) inhibited both Wnt3a-mediated suppression of Tcf3-repressor activity and Tcf1-activator activity.
To determine if Wnt3a-effects on self renewal (Fig. 1, ,4,4, ,6a)6a) were caused by the combined activities of Tcf1 and Tcf3 described above, expression of several genes previously implicated in ES cell self renewal was examined. These genes behaved in one of three manners with respect to the 12 conditions (Fig. 6b, bottom). First, Gpa33, Tcl1, Nanog, Prdm14, Jam2, Nr6a1, Nr5a2, Eomes, Kit, and Tnfrsf19 were increased both by Wnt3a and by Tcf3 ablation. Tcf1 shRNA decreased the Wnt3a-stimulation of these genes in each Tcf3+/+, Tcf3−/−, and Tcf3ΔN/ΔN cells, demonstrating a role for Tcf1 in Wnt3a activation of gene expression. Second, Esrrb, Tbx3, Klf4, Nodal, and Nfib were increased by Tcf3 ablation, but not increased by Wnt3a. Interestingly, Tcf1 shRNA reduced the levels of these genes, suggesting a Wnt3a-independent role for Tcf1. However, the effect of Tcf1 on expression was small and did not correspond to a cellular effect on self renewal (Fig. 6a). Third, Oct4, Sox2, Rif1 and Mycn gene products were only modestly (<1.5 fold) increased by any combination of treatments. As expected, Tcf1 shRNA did not substantially affect expression of these genes. Together, these data were consistent with Tcf3 repression of endogenous genes being inhibited by Wnt through a combination of Tcf3-β-catenin-interaction-dependent and Tcf1-dependent mechanisms, and full Wnt-stimulation of genes requires both mechanisms.
Finally, to determine if the mechanism for Wnt stimulation of self renewal involves β-catenin recruitment to sites of Tcf/Oct4/Sox2/Nanog co-occupancy in ES chromatin, a series of ChIP-qPCR experiments was performed. The genomic binding sites for analysis were selected from previously published datasets1, 4, 10–12. Criteria for choosing common sites (Tcfcp2l1, Mycn, Nodal, Oct4, Sox2, Tcf3, Zic5) included: multiple studies reporting a high ChIP signal for Oct4 and Nanog occupancy, a known relevance of the gene to ES cell self-renewal, and reproducible enrichment of the site in our ChIP-qPCR experiments. In addition, previously characterized Tcf-β-catenin responsive sites (Cdx1 and Axin2) were included as positive controls for Tcf3-binding1, 32. Tcf3 and Oct4 antibodies both enriched all common sites as well as the Tcf3-binding controls (Fig. 6c,d), confirming the high level of overlapping binding discovered with ChIP-chip and ChIP-seq techniques10–12. Interestingly, the relative amount of Oct4 occupancy on chromatin was increased for 8 of 10 sites in Tcf3−/− cells compared to Tcf3+/+ cells (Fig 6d), suggesting the possibility that Oct4 and Tcf3 compete for occupancy. This observation was important as it provided an excellent context to test the relative contribution of Oct4 and Tcf proteins to the recruitment of Wnt-stabilized β-catenin to target genes. If Oct4-β-catenin complexes mediate Wnt-stimulation of self renewal22, then the increased Oct4 occupancy should enhance β-catenin recruitment in Tcf3−/− cells. Instead, β-catenin occupancy at common sites was reduced in Tcf3−/− cells, and was further reduced by Tcf1 shRNA for all 10 sites (Fig 6e). Taken together, these ChIP data do not support a substantial role for Oct4 directly binding to and recruiting β-catenin to chromatin in Wnt-stimulated ES cells. Instead they show Tcf3-β-catenin and Tcf1-β-catenin interactions recruit Wnt-stabilized β-catenin to chromatin.
The data shown here are most consistent with Tcf proteins being the critical regulators of Wnt's stimulation of pluripotent cell self renewal. That said, shRNA knockdown of Tcf1 did not completely eliminate Wnt3a-stimulation of AP+ colony formation in Tcf3ΔN/ΔN cells, as a small percentage of these Wnt3a treated cells formed AP+ colonies (Fig. 6a). This may have been caused by incomplete Tcf1 knockdown or activity of endogenous Lef1; however, the low levels of reporter activity (Fig. 5d) and endogenous gene expression (Fig. 6b) in Tcf3ΔN/ΔN; Tcf1 shRNA cells makes this possibility appear unlikely. Alternatively, a Tcf-independent effect of Wnt-stabilized β-catenin22 may contribute to stimulating self renewal. A primary role for Tcf-independent effects of β-catenin was suggested by Kelly et al., as overexpression a ΔNTcf4 protein failed to rescue differentiation defects in GSK3 double knockout ES cells 22. While our results do not rule out the possibility of a secondary effect of Tcf-independent mechanisms, they strongly suggest that Tcf proteins play the primary role because: Overexpressing full length, wild-type Tcf3 blocked ES cell self renewal (Fig 1a,b). Self renewal was restored in Tcf3-overexpressing cells with exogenous Wnt3a (Fig. 1 a,b). Conversely, ablating Tcf3 replaced the need to inhibit GSK3 for ES cell self renewal in serum free conditions (Fig. 1c,d). The common effects of Wnt3a and Tcf3 ablation of gene expression correlated with Nanog/Oct4 regulation, whereas Tcf3-independent effects of Wnt3a did not (Fig. 2). The effect of Wnt3a on Tcf3ΔN/ΔN; Tcf1 shRNA AP colony formation was small compared to Tcf3 ablation (Fig 6a). Finally, the Tcf-independent mechanism of β-catenin was suggested to work through an Oct4-β-catenin interaction22, but Oct4 did not recruit β-catenin to chromatin in the absence of Tcf3 and Tcf1 (Fig. 6c–e). We conclude that Wnt stabilized β-catenin stimulates ES cell self renewal primarily by inhibiting Tcf3-mediated repression of target genes.
The role for Tcf3 as a limiter of ES cell self renewal appears to be unique among Tcf factors. Moreover, endogenous Tcf1 opposed Tcf3-repression and was necessary for the complete Wnt3a-stimulation of target genes and self renewal (Fig 6a,b). In light of the need to suppress Tcf3-repression for LIF-independent self renewal, it is interesting to consider why overexpression of ΔNLef121, ΔNTcf122 and ΔNTcf422 each failed to inhibit self renewal or failed to rescue differentiation defects caused by β-catenin stabilization. We believe the simplest explanation is that Tcf3 possesses a biochemical activity not shared by other Tcf/Lefs, and this transcriptional repressor activity inhibits ES cell self renewal. Expression of another Tcf/Lef would effectively inhibit Tcf3-repression by competing for DNA binding via virtually identical HMG domains shared by all Tcf/Lefs. As such, overexpression of Tcf1, Tcf4 or Lef1 could have a similar effect as downregulating or inhibiting Tcf3. This mechanism is consistent with the observation that endogenous Tcf1 was necessary for maximal Wnt3a-suppression of Tcf3-repressed target genes (Fig 6b). Comparing biochemical properties of Tcf/Lef proteins in ES cells has potential to reveal a unique co-repressor that limits activity of the self renewal circuitry in pluripotent cells.
Fragments of the murine Nanog promoter and 5' UTR encompassing genomic region chr6:122652598–122657800 were amplified by PCR from a mouse genomic BAC (clone RP23-406B15, BACPAC Resources, Oakland, CA) and cloned into a pGL3b luciferase reporter vector (Promega, Madison, WI).
One thousand ES cells were plated on each well of gelatinized 6-well plate and media were replaced daily. Colonies were stained for alkaline phosphatase (AP) activity after 4 days. Briefly, cells were washed once with PBS, fixed in citrate-acetate-formaldehyde (18mM NaCitrate, 9mM NaCl, pH 3.6, 65% acetone, 3.7% formaldehyde) for 45 seconds, washed three times with water, and stained with NBT/BCIP solution (Promega). For each well, 100 random colonies were scored as AP activity positive (AP+) or negative (AP−). Experiments were performed in duplicate and results were expressed as means +/− standard deviations.
Single cell suspensions were made by trypsin and resuspended in 100 µl PBS. Cells were fixed by drop-wise addition of 1ml cold (−20°C) 70% ethanol while vortexing. Cell cycle profile was checked by analyzing DNA content. Briefly, fixed cells were rehydrated and washed twice with 10 ml PBS before finally being suspended in 200 µl PBS. Cells were stained by incubating with of 5 µg/ml propidium iodide (Sigma) and 1 mg/ml RNase A for 20 min at room temperature and analyzed on Beckman-Counter Cell Lab Quanta SC MPL using Cell Lab Quanta Collection program (Beckman Coulter).
For each condition, three independent Tcf3+/+ and Tcf3−/− ESC cultures were treated with or without 50ng rmWnt3A (Recombinant mouse Wnt3A, #1324-WN, R&D System) for 24 hours. Total RNA were extracted by Trizol reagent (Invitrogen, Carlsbad, CA) and purified using RNeasy Mini Kit (Qiagen, Hilden, Germany). Quality of RNA sample was assessed by spectrophotometer (A260/A280 between 1.9 and 2.1). 10 µg (1 µg/µl) total RNA of each sample were used for microarray analysis. Labeling of the RNA, hybridization onto Affymetrix mouse GeneChip 430 2.0 microarrays (Affymetrix, Santa Clara, CA), and scanning of samples were performed by the Functional Genomics Facility, University of Chicago. Raw data were normalized by quantiles and summarized by robust multiarray average. Pairwise comparisons between groups of three replicates were performed, and the t test was used to rank each probe set on the basis of its likely statistical significance using the Comparative Marker Selection module of GenePattern software (Broad Institute, Cambridge, MA). Probe sets displaying a significant difference as defined by a Benjamini-Hochberg adjusted t test p value < 0.05 were used for subsequent analyses. Principal component analysis (PCA) was performed using the PCA module of GenePattern software. All microarray data points were subjected to PCA to create a set of principal experimental components to identify relevant patterns or variations across the four conditions: Tcf3+/+ (C), Tcf3−/− (C), Tcf3+/+ (W), Tcf3−/− (W). The analysis identifies patterns by finding principal components (PC) that account for variance in data points among all samples. Each PC thus represents a combination of gene expression values, and each PC is mutually uncorrelated and orthogonal. The first two PC scores (PC1 and PC2) resolved 99% of variance amongst all samples. A two dimension plot of PC1 and PC2 was generated using the PCAviewer module of GenePattern.
Total RNA was isolated with TRIZOL reagent (Invitrogen, Carlsbad, CA), and 3.0 µg total RNA of each sample was used as template for cDNA synthesis. Reverse transcription was carried out with oligo-dT primers (0.5 mg/ml) using SuperScript™ III First-Strand cDNA Synthesis kit (Invitrogen). Quantitative real-time PCR reactions were performed with the iTaq SYBR Green Supermix (BioRad) and an iCycler apparatus (BioRad). Amplification was achieved by the following protocol: 1× 95°C for 2 min; 40× 95°C for 30 sec; 60°C for 30 sec. To identify potential amplification of contaminating genomic DNA, control reactions using mock cDNA preparations lacking reverse transcriptase were run in parallel for each analysis. To ensure specificity of PCR, melt-curve analyses were performed at the end of all PCR reactions. The relative amount of target cDNA was determined from the appropriate standard curve and divided by the amount of GAPDH cDNA present in each sample for normalization. All PCRs had an efficiency of 85% or higher. Each sample was analyzed in duplicate, and results were expressed as means +/− standard deviations. Primer sequences used for qPCR are specified in Supplementary Table S5.
Transient knockdown of Nanog expression in wild-type ESC was achieved as previously described by transfection of siRNA targeting Nanog or SCRM siRNA23. Twelve hours after siRNA transfection, cells were treated with or without 50 ng/ml Wnt3a for 24 hours. Overexpression of Nanog in Tcf3+/+ ESC was achieved by transfection of the ppyCag-Nanog-IP expression vector8 or empty ppyCagIP vector control and G418 selection for transfectants. Nanog knockdown in Tcf3−/− ESC was performed by stable integration of a pSuper shRNA vector (OligoEngine) designed with a target sequence directed to the Nanog transcript (GAGACAGTGAGGTGCATAT) as previously described33 or empty pSuper vector control. The pSuper vectors were linearized and electroporated for stable insertion into the genome. After puromycin selection, colonies were picked and clonally expanded to generate individual cell lines. RNA isolation and quantitative real-time PCR were performed as described above. Verification of Nanog knockdown or overexpression was performed by SDS-Page gel electrophoresis and western blotting with Nanog specific antibodies (Bethyl Labs A300-397A, CosmoBio RCAB0002P-F, and Abcam ab21603) and by quantitative real-time PCR.
Cells were plated 24 hours before transfection on gelatinized 24-well plates at a density of 1×105 cells/ml. Transient transfection of DNA vectors were conducted by Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. 24 hours after transfection, cells were lysed by 1X passive lysis buffer (Promega). Luciferase activities were measured by Clarity luminometer (Bio-Tek) with Dual-luciferase reporter assay system (Promega). Each transfection was carried out in duplicate. Relative activity was calculated as the ratio of the reporter plasmid Firefly luciferase activity to Renilla luciferase activity (pRL-CMV), which was co-transfected as control.
Tcf1 shRNA sequence was subcloned into lentiviral vector pLVTHM. Lentivirus were produced following previously established procedures. Briefly, 293T cells were plated 3 days before transfection. Tcf1 shRNA pLVTHM plasmid were mixed with envelop plasmid (pMD2G) and packaging plasmid (psPAX2) in desired ratio and transfected 293T cells by calcium phosphate. Fresh media were changed 14–16 hours post-transfection and supernatant containing lentivirus were collected 12 hours later. Supernatants can be harvested 3 times every 12 hours. Titers of virus were determined using 293T and ES cells by FACS analysis for percentage of GFP expressing cells. Three targeting sequences for Tcf1 were used to generate shRNA lentivirus (#1 TCTCCACTCTACGAACATT; #2 GGAGCTGCAGCCATATGAT; #3 GCCAAGGTCATTGCTGAGT) and tested for Tcf1 knockdown. The #3 sequence (GCCAAGGTCATTGCTGAGT) had the highest Tcf1 knockdown efficiency and was used to generate stable Tcf1 ES cell lines in this study. A scramble shRNA sequence (GTCACGATAAGACAATGAT) was used to generate control lentivirus (SCRM). Clonal Tcf1 knockdown ES cell lines and SCRM control lines were established by single colony picking and following expansion. Efficiency of Tcf1 knockdown was confirmed by qPCR and Western blot.
Approximately 2 × 107 ES cells were cultured and processed for each ChIP experiment. Cells were chemically crosslinked by the addition of one-tenth volume of fresh formaldehyde solution (11% formaldehyde, 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes pH 8) for either 10 minutes at room temperature (Tcf3) or overnight at 4°C (Oct4, β-catenin). The reaction was inactivated by the addition of 2.5 M Glycine (0.125 M final concentration) and incubated at room temperature for 10 minutes. Cells were rinsed twice with cold 1xPBS, harvested using a silicon scraper, and flash frozen in liquid nitrogen. Cells were stored at −80°C until the day of use.
Cells were resuspended in lysis buffer LB1 containing protease inhibitors (50 mM Hepes pH 7.7, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Trtiton-X-100), incubated for 10 minutes on ice and centrifuged at 4°C for 10 minutes at 2500 rpm. The cell pellet was resuspended in LB2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 7.5), incubated for 10 minutes on ice and centrifuged at 4°C for 10 minutes at 2500 rpm. Finally the cell pellet was resuspend in sonication buffer LB3 (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Na-deoxycholate) and sonicated to solubilize and shear crosslinked DNA. Sonication was performed using a Branson sonicator for 20 µ 30 second pulses (60 second pause between pulses) at a 60% amplitude. Triton-X-100 was added to the lysates after sonication to a final concentration of 1%. Cellular debris was removed by centrifugation. Samples were kept on ice at all times.
The resulting chromatin extract containing DNA fragments with an average size of 500 bp were immunoprecipitated overnight at 4°C using magnetic beads (Protein G Dynabeads, Invitrogen) that had been pre-incubated with 5 µg of the appropriate antibody. The antibodies used for immunoprecipitation were Tcf3 (Merrill Lab), Oct4 (Santa Cruz sc8628) and β-catenin (Invitrogen 71–2700). Beads were washed 3 times with RIPA ChIP buffer (50 mM Hepes, 1mM EDTA, 0.7% Na-deoxycholate, 1% NP-40, 0.5 M LiCl) and 1 time with TBS (50 mM Tris, 150 mM NaCl, pH 7.6). Bound complexes were eluted from the beads by heating at 65°C with occasional vortexing, and crosslinks were reversed by overnight incubation at 65°C. Whole cell extract DNA (reserved from the sonication step) was also treated for crosslink reversal. Eluted protein-DNA complexes were treated with RNase A (0.2 µg/µl) and proteinase K (0.2 µg/µl). Following phenol-chloroform extraction and ethanol precipitation, DNA was dissolved in Tris-EDTA.
Stealth siRNA duplexes (Invitrogen) designed against mouse Tcf1 (AGAGACUGUCUACUCUGCCUUCAAU) or a control siRNA (GGAAGACUAGAGGCGGUCAUGAGUU) were used to specifically downregulate Tcf1 mRNA expression levels. For transfection, ES cells were trypsinized, pelleted by centrigugation and resuspended in media without antibiotics. For each phenotype, 100,000 cells per well were combined with Lipofectamine 2000 transfection complexes of control or Tcf1 siRNA (50 nM) following the manufacturer's protocol (Invitrogen) and plated on gelatin on a 6-well dish. Twelve hours after transfection cells were split onto new plates and used in a colony differentiation assay.
The complete microarray dataset was submitted to NCBI GEO repository and is available as GSE27455.
We thank Kathrin Plath for her helpful discussions and thoughtful comments on the manuscript, and Dr. Minoru Ko and Dr. Akira Nishiyama for the inducible Tcf3 overexpression cell line. The work was funded by grants from the American Cancer Society (RSG GGC 112994, B.J.M.), the National Institutes of Health (R01-CA128571, B.J.M. and R01-GM065400, D.R.L.) and the Howard Hughes Medical Institute (D.R.L.).
AUTHOR CONTRIBUTIONSAll authors designed and analysed experiments. F.Y, L.P. J.A.H, B.S. and C.M.Y. performed experiments. F.Y., L.P. and B.J.M. wrote the manuscript. B.J.M and D.R.L. supervised the project.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Makeup trends often change but the one trend that always makes a comeback is definitely the dark, vampy lip. Essence Cosmetics released a new longlasting lipstick: nr 18 Velvet Matt. It is part of a matte lipstick series Essence launched starting with longlasting lipsticks nr 14 and nr 15, followed by Velvet Matts nr 23, nr 24 and nude lipsticks nr 07 and 08.
Essence Lipstick nr 18 Velvet Matt Review
Friday, 12 December 2014
Posted by Jolien Nathalie at 08:30:00 No comments
Labels: Essence Cosmetics, Lipstick, Review
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text: Posted December 7, 2012 Umm, because our allegiance to the Falcons is questioned non-stop by window lickers like yourself. You shouldnt feel the need to justify yourselves to an opposing teams fans. Unless of course youre a lying, bandwagon ass troll. Share this post Link to post Share on other sites


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