text:
Rated 3.5 out of 5 by 1865 reviewers.
Rated 4 out of 5 by enel3635 The staff were very responsive While the fabric of this hotel has some deficiencies because of its age, the staff were very efficient and cordial when responding to the needs of our group. Many of us could be considered seniors or "golden agers." Therefore, some found it difficult to raise themselves up from the low toilet seats without the presence of a grab bar. The staff responded with the suggestion of installing a temporary seat riser. And, maintenance person arrived almost immediately to install it. We also had a problem with the room chair being too low. The staff very quickly provided conference room chair. Someone always followed up to insure that our needs had been met. The breakfast buffet provided a wonderful selection of food, and the wait staff were, for the most part, very cordial and observant of our needs,such as coffee and water refills without our having to flag them down. The banked elevator service was excellent, and the direct access to underground shopping was certainly a plus. November 14, 2012
Rated 5 out of 5 by Monzie101 Best stay ever Loved the hotel and the staff were so friendly. Thank you for making this a great stay. November 13, 2012
Rated 4 out of 5 by gcheng Hotel has great location and friendly staff This hotel has great location right in front of Toronto City Hall and surrounded by great restaurants. Staff are very friendly. The only I'm not fond of is the noise level of the vents. If you are close to the fire exit you can hear constant noise level through your vent. Something to consider for light sleepers. This is the 3rd time I got a room in this site with this issue. November 13, 2012
Rated 1 out of 5 by piacan68 a nightmare stay Went for a conference for 4 days. I was placed in Richmond Tower where the walls are thin like a sheet of paper. Had to call security twice because some very loud partying was going on at 11:00 at night and then at 3:00 AM. Guests in the next room could be heard talking as if they were in the room and they were not that loud. Avoid this place. November 13, 2012
Rated 4 out of 5 by PC11 Great Stay We had a great stay. Dylan at the front desk was very helpful when we needed to change our room to accommodate an extra child when we arrived. The view from the Club Lounge both at breakfast and at night was fabulous. The only negative during our stay was the backlog in getting the car out with the valet service. Great location and definitely return. November 13, 2012
Rated 4 out of 5 by Neuron No free Internet Extortionate room wifi charges are absurd in this day and age!!! Service I lobby very poor. Everyone gives free Internet these days. You shout be ashamed. November 13, 2012
Rated 5 out of 5 by Medi Great Club Lounge Excellent staff and facilities make the Sheraton Centre Toronto one of the best I have ever been to, compared to other Sheratons across the world. A big thank you to all the staff. November 12, 2012
Rated 5 out of 5 by Consultant33 Excellent location and amenities People at reception, at the launge on the 43rd floor, at restaurants are all very nice and helpful. When you add the great location and awesome heated pool and other amenitoes, this hotel is a anbolute pleasure to stay at. November 12, 2012
Rated 2 out of 5 by val17 Terrible Experience I was very unhappy at this hotel. The staff was rude and did little to make my stay enjoyable. The dining room staff was nasty. I will never stay here again, would not recommend it to anyone and it has provided me with a bad impression of the Sheraton name. November 12, 2012
Rated 5 out of 5 by toomuchwork Great Location/Friendly staff Wonderful location downtown Toronto. Easy access to Eaton Center for holiday shopping and access to subways. November 11, 2012
Rated 5 out of 5 by Lori33 Best Hotel Ever! After two years of staying at the Sheraton Centre Toronto, I am sad to leave and would like to recognize all of the people that work at the property that have made such a difference in my life and have become my family over the years. Everyone that is at the Sheraton is amazing, and makes my day every single day with all the wonderful things that they do. I always receive the warmest welcome and everyone is so thoughtful and generous. I couldn't imagine staying anywhere else, and I know that I will never find another hotel and staff like it anywhere in the world. I would recommend this hotel to anyone. 1. Valet and Bellmen – I honestly don't know how I will live without these guys in my life. The valet and bellmen have seriously become my family and I get choked up just thinking about not seeing them every day. They are so sincere, genuine and helpful. They give me the warmest welcome every morning and night. George, George, Fetsum, Alwin, Jeffrey, Long and the rest of the gang are so special to me. 2. Front Desk – Everyone at the front desk always makes me feel so special. From already having my keys made up and giving me the warmest welcome ever, to making sure that I have an extra special room and that my preferences are taken care of, they make a difference every day. Lily, Ronnie, Max, Steven and the everyone else on the team make my stay extra special every single time. 3. Concierge – The concierge staff has always been so helpful to me when I have needed help the most. Of course, with staying in the area for the last couple of years it is rare that I have an easy request. And they always are happy to help. When I was new to the hotel and didn't know my way around the city, they were extremely helpful with their recommendations and directions to make me feel comfortable and at home. 4. Room / Maid Service – Room service at the hotel is exceptional. From the little things like making sure that a flower is sent up with my meal, and ensuring that it is delivered quickly, all the difference is made. The hotel always makes my day by sending a little something special up to the room each week, and on special occasions such as my birthday they have been sweet enough to send something extra special. 5. Lounge / Restaurants – I love going up to the lounge every day to see Blesilda, John and the rest of the crew. I am going to especially miss Blesilda who is so special to me and one of the sweetest people in the world. She is even sweet enough to send me a note when they have something special in the lounge that I should definitely stop in for in the evening. Everyone in the restaurants and the bar are amazing too! 6. Programs – In addition to the wonderful programs that the hotel offers such as the Green Choice Program (which I always love to take advantage of), the management at the hotel has always been amazing from the day to day things that make my stays so special to the events that they so kindly host and I have received personal invitations to. Most of all, I will miss Rose and Clinton, who are near and dear to my heart. November 11, 2012
Rated 5 out of 5 by williemcjc SHERATON - HOTEL LIKE HOME 1. WATER CLOSETS SHOULD BE EQUIPPED WITH SPAY WASHERS. 2. ROOM REFRIGERATORS ARE RECOMMENDED. November 10, 2012
Rated 2 out of 5 by Guinness ROOM 1044 As frequent guests at Sheraton, Queen St. West, we were extremely disappointed with the standard of Room 1044. A vent located close to the ceiling was partly covered with masking tape. The surrounding wall and ceiling areas were black. A substandard room and not at all worthy of price paid. November 9, 2012
Rated 4 out of 5 by enna room Beautiful hotel, air venting was annoying, noisy all night and like a whistle if u opened the window, Pillows were lousy, like a big puff ball, no support. other then that the hotel was great! November 9, 2012
Rated 1 out of 5 by amre Not to the par For the price and the name, the quality of the hotel room and service was poor. Dirty room and appliances (for e.g. Hair dryer). God hotel, but the management needs to take more care of the details, lie cleanliness. November 9, 2012
Rated 5 out of 5 by Brat Great time had by all we thoroughly enjoyed our girls weekend in Toronto. Mother, daughter and grandaughter had a beautiful weekend, saw War Horse and attended the Royal Winter Fair. Your rooms are lovely and met all our needs. We were close to the theater and a ten minute ride to the fair. We shopped at the mall at the hotel and spent too much money. Your staff are extremely helpful and very friendly. We are already looking forward to next year. See you November 8, 2012
Rated 5 out of 5 by Lucille location great location, friendly staff. Thanks November 8, 2012
Rated 2 out of 5 by disappointed222 You must be upset for someone to help I tried the Sheraton for a second time, after a very disappointing stay in the summer. The location and pool is wonder however as usual the room was a disappointment, I had booked a King suite and was given the worst one in the hotel. My room had an ice machine to the right and an emergency exit to the left. The employee who checked us out was helpful and understanding, however the GM could use some customer service training. November 7, 2012
Rated 4 out of 5 by Sunshine1 Great Location For an early Christmas present I brought my Mom to Toronto to stay at your hotel, go to dinner and see War Horse. Also there was the nostalgia side b/c my Mom lived in the area when she was a teenager and she could point out what used to be there versus what is there today. It was a great getaway although short, and will have good memories of the fun we had here! Your location is within walking distance to yummy restaurants, amazing theatrical productions and a safe feelting while out and about. Thank you! November 7, 2012
Rated 5 out of 5 by mailman quality hotel This hotel met all our needs. Front desk staff were excellent. One minor problem. The menu on the TV did not work. I wanted to review my folio and was unable to do so. The front desk printed out a copy for me upon my departure. November 6, 2012
Rated 5 out of 5 by stevelikesrush Perfect! I couldn't have made a better mistake. I had originally wanted to book a hotel closer to the Air Canada Centre for a concert I was going to, but I waited too long and had to look for something else. The Centre Toronto Hotel was recomended to me, and even though I was at first disappointed in the location, on arrival realized I couldn't have asked for a better place. It lives up to it's name. CENTRE TORONTO. All I had to do was walk out of the lobby and everywhere I wanted to be was just a short walk away. No taxis or busses. The Eaton Centre just around the corner. Historic buildings all around. The Air Canada Centre, The CN Tower and The Rogers Stadium (did I get that name right?) just a few blocks away. (Saw a great RUSH concert at the ACC) Great restaurants and shops everywhere. Didn't even have to go outside, all right under the streets and buildings in the causeway. Boy! did I get lost! Had fun though. And, Oh Yeah! The hotel was pretty good too. Excellent staff, great room with an awsome view. Very comfortable bed. Can't wait to come back. November 5, 2012
Rated 5 out of 5 by EJAY Divinely comfortable bed.... We were very pleased with our stay at the Sheraton Centre. Clean, comfortable, quiet and within walking distance of everything we wanted to see. My only negative would be coffee cups and water glasses. I think a hotel on the scale of the Sheraton should spring for ceramic and glass, not cardboard and plastic. EJay November 5, 2012
Rated 5 out of 5 by Brenda62 Great location Clean quiet rooms, great location November 2, 2012
Rated 4 out of 5 by TriplePlatinum great workout, great stay as a swimmer who stays in hotels for half of the year, i have *never* stayed at a hotel with such a great pool. the workout facility is also quite large (~10 treadmills...extrapolate from there) and never crowded. the staff is friendly and the rooms are comfortable. amazing view from the club lounge and super friendly staff as well. only issue is that i wish there were more power outlets in the room. November 1, 2012
Rated 5 out of 5 by Miles3855 I love the Sheraton Centre The Sheraton Centre has a great location in Toronto, convenient to the subway and to most downtown attractions. The hotel and the rooms are very attractive, and the staff could not have been friendlier. November 1, 2012
Rated 1 out of 5 by PeterJ2012 Never sent me my folio Always, always get your folio printed before you leave with this hotel. I have asked them repeatedly to send me my folio but they never do and no one ever calls me back on my messages to the voice box there that is responsible for it. So, months later, I am still out over $600 b/c my company won't reimburse w/o a receipt. Appalling service from a Starwood property. November 1, 2012
Rated 2 out of 5 by bjt703 room below expectations I stayed one night in a juniour suite and was disappointed at many levels. The heating in the room was not functioning a fact that was known by hotel staff as external heaters were behind the couch and I was not informed of this. The bathtub was blocked and did not drain properly. The shower was not functioning well. As my fault I did not complain as I was staying one night and was quite busy but I do have higher expectations from the Sheraton. November 1, 2012
Rated 4 out of 5 by John49 Toronto in Occtober I stayed for 6 nights & enjoyed an excellent level of service from friendly attentive staff who were very responsive to my questions & requests including supplying a bathrobe and extending checkout time as I had an evening flight back to the UK. Clubroom on floor 43 very good & again excellent service morning & evening. Couldn't quite get used to the walk from the men's locker rooms down corridor to gym/pool - why no direct access? Other than that a brilliant stay - thankyou! November 1, 2012
Rated 3 out of 5 by Golden2012 not happay The bed was in bad shape, needs change November 1, 2012
Rated 5 out of 5 by Tina23 This hotel has exceptional staff I stay at this hotel weekly and cannot say enough about the amazing customer service provided by the staff. From the front desk, to guest services to club lounge, they are all very professional and eager to make my stay as comfortable as possible. The location cannot be beat and the services always meet my needs. October 31, 2012


text:
Within City
Within 1 mile
Within 2 miles
Within 5 miles
Within 10 miles
Within 20 miles
Sort by
highest rated first
closest first
most reviewed first
alphabetical order
best match
"Buddy is absolutely the best in the area. He upgraded virtually every room in our house as well as building a beautiful attached garage addition. Along the way ..."
"Sunrise is a beautiful assisted living facility located in Fairfield, NJ, right on the border of North Caldwell. The staff are very nice and the placeis meticulous. The ..."
This doctor matches your preferences for Specialty.

subset: commoncrawl


text:
Currently I work shifts, so every other week it's difficult to go out as I'm working over 70 hours. The rest of my time is reasonably free, though especially during the day.
I'm a part time goth, a lover of most types of games, enjoy reading and am generally a massive geek. I even do LARP. However, most of my time is spent procrastinating.
In principle I'm polyamorous, but shyness tends to get in the way. I've been single for a while recovering from the breakup of a long lasting primary relationship, but now I'm ready to try for a new one.


text:
Raised in Huntington Beach, CA, Mary Beth describes herself as a "California beach girl."
User Score: 158
User Score: 4
CBS Entertainment | About TV.com | About CBS Interactive | Jobs | Advertise
© CBS Interactive Inc. All rights reserved. | Privacy Policy | Ad Choice | Terms of Use


text: July 2013 SAS named a Leader in Magic Quadrant for Data Integration Tools 29 iulie 2013 With SAS®, CESCE reduces costs, improves investment decisions 29 iulie 2013 SAS helps publishers manage mobile channel to increase revenue 24 iulie 2013 SAS named best place to work for work/life balance by Glassdoor 24 iulie 2013 SAS ranked top in data quality by industry analysts 22 iulie 2013 SAS® Anti-Money Laundering named best compliance solution 22 iulie 2013 SAS recognized for data management and analytics innovation 17 iulie 2013 Bank of North Carolina expands data access with SAS to be a "better banker" 15 iulie 2013 DirectPay to optimize risk management with SAS® Visual Analytics 09 iulie 2013 American Red Cross names SAS blood drive sponsor of the year 09 iulie 2013 SAS No. 4 among Best Workplaces in Europe 08 iulie 2013 SAS again ranked No. 1 advanced analytics provider, market share continues growth, says industry research firm 08 iulie 2013 SAS India featured in the Best Places to Work Rankings 03 iulie 2013


text:
Woodcote House Hotel3
Hooton Road Hooton, Wirral, CH66 1QU
Price FromView Rooms
- Excellent
- 92 guest reviews
"Return visit. Most welcoming. Staff and facilities delightful. Thoroughly recommend. Short walk from room to main property for meals COULD b..."
"We enjoyed every minute of our b&b overnight stay. John was the perfect reception host, showing us to a delightfully furnished comfortable b..."
"A second visit to this gem of a hotel. From our arrival, to departure, the staff couldn't have been more welcoming, friendly and professiona..."
"It was a wonderful experience, very relaxed and peaceful our room was lovely, a really good size clean and tidy. The owner of the business J..."
"The room was clean, spacious and had the necessities needed for a weekend stay. The staff were extremely polite, friendly and only too happy..."
About the hotel
Ideally located for a shopping! Cheshire Oaks Retail Outlet Village with over 145 stores is only 5 miles from the hotel, and of course Liverpool, famous for its superb shopping is only a 20 minute drive. Hooton train station (10 minutes' walk) can get you into Liverpool, Chester and Wales. Liverpool Airport is a 40 minute drive (about 27 miles).
The hotel is 2 minutes from the M53, which gives you easy access to Cheshire Oaks, The Boat museum, Ellesmere Port, Chester, North Wales, the beautiful Wirral peninsula with all its beaches and many more attractions including Ness Gardens, Chester Zoo, New Brighton. The Woodcote House Hotel is newly refurbished to a very high standard, modern, all rooms have en-suite with superb showers, mostly king sized beds, flat screen TVs, tea and coffee making facilities. Bar and restaurant with superb freshly cooked food. Breakfast is bookable on your arrival for a supplement if you require. You will never run out of places to go and places to see when you visit us, and with there being a fantastic railway link from Hooton Station (just a 10 minute walk away from the hotel) you can hop aboard a train to Chester only 10 minutes away and Liverpool 15 minutes away. Perfect for exploring the area without worrying about driving or parking. The Bar and Restaurant is not open on Sunday Nights.
Read MoreLess
Room Availability
Showing Tuesday 7 July 2015 to Wednesday 8 July 2015Change Search
We currently have no rooms available for this hotel that match your search criteria. Try changing your search or select an alternative hotel.
Facilities Show/hide
- Main
- Hotel Non-Smoking Throughout, Disabled Facilities
- Family friendly
- Cots available
- Parking
- Car parking available onsite
- Business
- Wi-Fi Available, Meeting Facilities, Wi-Fi Available In Rooms
- Dining
- Restaurant, Lounge Menu, Bar
- In room entertainment
- In room facilities
- Tea/Coffee making facilities in each room, Telephone In Each Room
- Hotel services
- Wake Up Call Available
- Other
- Ground floor bedrooms
- Accessibility
- Disabled Access Public Areas, Disabled Access Bedrooms, Disabled toilets on ground floor, Wheelchair Access
Useful Info Show/hide
- From 14:00 hours
- Until 11:00 hours
- 19/08/2014 to 31/12/2014 - Important: from 27 of December 2014 until the 30th there will be no restaurant or bar facilities at the hotel. The LateRooms.com rates for Woodcote House Hotel in Wirral are the total price of the room and not the 'per person' rate. All prices are inclusive of all local taxes.
- Pets are not allowed
Customer Reviews
Our reviews are from genuine LateRooms.com customers. We verify that the customer has stayed before leaving a review.
98% would recommend it to a friend
Customer service
Food quality
Room quality
Woodcote House Hotel: one night, one breakfast
Return visit.
Most welcoming. Staff and facilities delightful. Thoroughly recommend. Short walk from room to main property for meals COULD be a problem if raining - perhaps mention in information. Would suggest clarification re Breakfast not included in price and quotation of price for Breakfast. Needs brighter/higher levels of lighting to facilitate reading in comfort.
Read More Less
How soon can we stay again?
We enjoyed every minute of our b&b overnight stay.
John was the perfect reception host, showing us to a delightfully furnished comfortable bedroom (a photo of which could easily grace the pages of a glossy magazine) with a spotlessly clean en suite bathroom. The evening meal was most enjoyable with good quality ingredients being used, well presented and at reasonable pricing. Breakfast was also served using good quality ingredients of which there was plenty of choice. Only sorry we had to travel on to another area. On the basis of "if you can't say anything nice......" I have dismissed writing reviews on other hotels. So you have read that this one really got it right for us. Don't think you could be disappointed either.
Read More Less


text:
you are viewing a single comment's thread.
On the other hand, had you purchased on 1-2-99 you would have paid $40.31/share and that is my point. I hate to admit to it but I did buy some at $33 but have sold recently in preparation for the next merger where the stock will go back down and allow me to rebuy more shares for the same money and go through the cycle again.


text:
Birth or Butchery?
Life is cheap, where moonbats who believe in abortion are concerned. Should you let your child live, or kill it? You could flip a coin to decide. Or you could leave it up to strangers on an Internet poll, as Pete and Alisha Arnold of Minnesota are doing:
"Voting is such an important part of who we are as a people," Pete told Gawker. "Here's a chance where people can be heard about whether they are pro-choice or whether they are pro-life, and it makes a difference in the real world." The possible papa says he wants to use this position of uncertainty to engage citizens in the ongoing abortion debate or use this fetus (a boy they call "Wiggles") to teach us how every vote counts or blah blah blah.
Whatever this is, "it's definitely not a pro-life campaign," maybe-mama Alisha assured Gawker. "I believe in a woman's right to choose." Apparently, she also believes in the Internet's right to choose for her.
Fortunately voters have been in favor of showing mercy:
As of this post, the vote is approximately 78 percent "give birth" to 22 percent "have an abortion," with more than 17,000 votes total. This is a distinct upset from earlier today when "have an abortion" was firmly in the lead, thanks to a strong voter turnout from the 4chan demographic, those Internet pranksters that once skewed a vote to make North Korea a winning country on Justin Bieber's tour schedule.
Pete has no problem with 4chan freakazoids encouraging people to vote to abort his son out of sheer wickedness.
"I see 4chan as encouraging people to vote in the poll," he writes on the blog. "How they do it is beyond my control and should not influence decisions about what we are doing here." In the end, he wrote, "we don't see a difference between this and what politicians or supporters do in regards to campaigning."
The whole tasteless stunt is apparently a Balloon Boy style bid for attention. The Arnolds admit that the poll results won't be binding. At least it has drawn some attention to how frivolously we have come to regard human life since the Supremes imposed a "right" to abortion.
Just in case it helps save a life, you can vote at Birth or Not.
On a tip from Ianto. Cross-posted at Moonbattery.
Also see...
Facebook62.5kTwitter76Email1 Considering how often we see horrific stories of animals being abused and murdered by sadistic monsters, it's a nice break to see the often unnoticed acts of kindness towards...Read More
Dave Blount
Related Articles
Self-Mutilating Inmate Demands Free Sex Change
FacebookTwitterEmail Remember Stanley Thornton, the "adult baby" who spends his days wallowing in an oversized crib and soiling his diapers
Gruesome: Family Severely Beats 14-Yr-Old Girl to Force Her Baby to Abort
FacebookTwitterEmail A 14-year-old African American girl in Dallas was cruelly beaten by four members of her own family in order
[SHOCKING VIDEO] A Donor Calls Planned Parenthood to Fund Black Abortions ONLY – and They are THRILLED!
FacebookTwitterEmail Black abortions outnumber black births in the US. There is a government funded genocide of blacks in America. This


text:
Update: Hanrahan (hamstring) was activated from the disabled list Tuesday, the MassLive.com reports.
Recommendation: While Hanrahan's role is up in the air upon his return, manager John Farrell relayed that Andrew Bailey is likely to hang onto the closing gig for the time being. While he didn't impress in his rehab assignment at Triple-A Pawtucket, allowing two runs in two innings, a couple of crisp outings back with the Red Sox could throw a wrench in Farrell's plans.
- Sports & Recreation
- Baseball


text:
|Home | About | Journals | Submit | Contact Us | Français
Conceived and designed the experiments: LP. Performed the experiments: EC LD LV FM AM ME. Analyzed the data: GG FC LP. Contributed reagents/materials/analysis tools: SWW FC LP. Wrote the paper: FC LP.
tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt−Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.
The formation of the mature vertebrate heart with separated chambers and valves involves a complex orchestration of gene expression. Numerous genes are critical for cardiac morphogenesis, although their exact functions and their integration with other cardiac regulators is poorly understood [1].
The T-box gene tbx5 encodes a key transcription factor for vertebrate heart development [2], [3]. Tbx5 function in the heart is gene dosage sensitive, as both haploinsufficiency and gene duplication give rise to Holt−Oram syndrome (HOS). HOS is a highly penetrant autosomal dominant disease characterized by congenital malformations of the heart and upper limbs, which are two sites of Tbx5 expression [4]–[7]. Nonetheless, the molecular mechanisms accounting for gene dosage sensitivity are not known. Mice heterozygous for mutations in Tbx5 display many of the phenotypic abnormalities of individuals with HOS [8], [9]. Comparable defects are seen in the zebrafish Tbx5 mutant heartstrings, suggesting that Tbx5 expression and function have been conserved throughout vertebrate evolution [10], [11]. In the murine model of HOS, gene expression profiling of a tbx5 allelic series demonstrated that Tbx5 could regulate hundreds of downstream genes [8]. Examination of the expression dynamics of mouse genes regulated by Tbx5 indicates that Tbx5 can act via an array of independent mechanisms, some of which include direct DNA binding as has been shown for Gja5 [9], and others via indirect mechanisms that may involve complex regulatory networks.
Several genes regulated by mouse Tbx5 encode transcription factors (TFs), or are involved in transcriptional regulation suggesting that HOS might in part be the consequence of "misregulation of regulators". Besides TFs, microRNAs (miRNAs) play key roles in heart development and cardiac diseases [12]–[14]. TFs and miRNAs comprise two major layers of gene regulatory networks with strictly interconnected activities: TFs control miRNA expression and many miRNA targets are TFs. There is increasing evidence that TFs and miRNAs can work cooperatively through mutual cross-regulation [15]. Starting from these considerations, we performed an in silico screening to identify miRNAs embedded in genes highly sensitive to Tbx5 dosage. We focused our attention on miR-218-1 that, together with its host gene slit2, is involved in heart development [16]. We confirmed a correlation between tbx5 and miR-218 expression and showed that alterations of miR-218 expression have a significant impact on zebrafish heart development. Interestingly, down-regulation of miR-218 is able to rescue most of the defects generated by Tbx5 over-expression, demonstrating the pivotal role of miR-218 in mediating the effects of Tbx5 dosage on heart development. These data support the idea that a miRNAs/Tbx5 regulatory circuit is crucial in cardiac morphogenesis.
To identify miRNAs modulated by Tbx5, we developed a bioinformatic tool to search for miRNAs within introns of Tbx5-controlled genes (Fig. S1). As a source of Tbx5 targets, we used genes identified by microarray analysis of a conditional mouse allelic series of tbx5 [8] and of a mouse 1H cell line infected with adenovirus expressing tbx5 [17]. Four miRNAs were identified: miR-218-1, miR-678, mir-719 and miR-335 (Table 1). We focused our attention on miR-218 since: i) it is conserved from human to zebrafish (www.mirbase.org); ii) the miR-218-1 host gene, slit2, is highly sensitive to Tbx5 mis-expression [8]; iii) the secreted Slit ligands, together with their Robo receptors, contribute to the control of oriented cell tissue growth during chamber morphogenesis of the mammalian heart [18]; iv) Slit/miR-218/Robo are part of a regulatory loop required during heart tube formation in zebrafish [16].
We compared tbx5, slit2 and miR-218 expression in newborn mouse lung, liver, brain, aorta, skeletal muscle and heart. slit3 expression was also analyzed since its host miRNA, miR-218-2, cannot be separately quantified because it is identical to miR-218-1. In agreement with the literature [19], we observed tight co-expression of slit2 and miR-218, and a general correlation among tbx5, slit2, slit3 and miR-218 expression (Fig. S2).
To assess whether there are functional regulatory interactions among Tbx5, Slit2 and miR-218, we first examined these genes in an in vitro model for cardiomyocyte differentiation. P19CL6 cells proliferate in growth medium (GM) and differentiate into beating cardiomyocytes in differentiation medium (DM) [20], [21]. P19CL6 increased the expression of cardiac differentiation markers such as GATA4, α-MHC, CX40 and decreased the expression of the marker of pluripotency Oct4 after a few days of culture in DM, compared to cells maintained in GM (Fig. 1A). A progressive increase in tbx5 expression was also observed (Fig. 1B), which was paralleled by an increase in slit2, slit3 and miR-218 transcripts. To show that the slit/miR-218 increase was at least partially dependent on Tbx5, tbx5 was up- or down-regulated by transfecting P19CL6 cells with a tbx5-carrying expression vector (CMV-Tbx5), or with a siRNA mix directed against tbx5, respectively. Tbx5 over-expression tripled slit2 expression, almost doubled miR-218 expression and had no effect on slit3 expression (Fig. 1C). On the other hand, tbx5 silencing, the effect of which was highest 2 days after silencing (6th day in culture, see Methods), caused significant reduction of slit2 and miR-218 expression 4 days after transfection (8th day in culture, Fig. 1D). Pre-miRNA 218-1 expression paralleled the increase in miR-218 level during cardiomyocyte differentiation (Fig. S3A) and after Tbx5 modulation (Fig. S3B). Moreover, the transfection of a siRNA mix against Slit2 cut the level of Slit2 by half without affecting miR-218 expression, supporting the idea that miR-218 expression depends on the regulation of Slit2 transcription rather than on its translation. Overall these results suggest that the expression of Slit2 and its embedded microRNA miR-218-1 are modulated by Tbx5 during cardiomyocyte differentiation.
To analyze the role of miR-218 in heart development, we decided to use zebrafish since this model is particularly informative for studying cardiac early patterning networks due to its relatively simple two-chambered heart coupled with its ability to develop even in the absence of a functioning heart. Moreover, various data derived from Tbx5 knock-down experiments in zebrafish [10], [11] have revealed developmental defects of the heart and limbs comparable to the defects observed in human with Tbx5 mutations [4], [22] or in Tbx5 knock-down mice [9], [23], suggesting that the functional role of this crucial transcription factor is evolutionarily conserved.
In zebrafish, as in mammals, two isoforms of miR-218, miR-218a-1 and miR-218a-2, are embedded in slit3 and slit2 genes, respectively. A third genomic copy of this miRNA, miR-218b, is present in fish. However miR-218b, an intergenic miRNA, has very low expression, suggesting that its contribution to the global miR-218 level might be irrelevant [16]. miR-218a1/2, slit2 and slit3 are highly expressed in zebrafish neural tissue (Fig. 2A, [24], [25]) and Fig. 2B [26]–[28]. In cardiac tissue, slit2 and slit3 are clearly detectable at 48 hpf (Fig. 2B), while the expression of miR-218a1/2 is barely detectable by in situ hybridization (ISH) up to 48 hpf (not shown) but is visible around 72 hpf (Fig. 2A).
To over-express miR-218a1/2, we injected double stranded RNA oligonucleotide with a miR-218a sequence (miR-218a mimic) in Tg(cmlc2:eGFP) embryos. Injection of 260, 135 and 35 pg of miR-218a mimic generated embryos with cardiac defects in a dose-dependent way (Fig. 2C). At the highest dose (2 ng in Fig. 2C), we observed a slight decrease in embryos with cardiac defects, which was balanced by the increased number of dead embryos and, to a lesser extent, of embryos with different morphological defects. As specificity controls, 2 ng of either miR-492, a control miRNA, that is not annotated in zebrafish, or miR-214, which is not heart-specific, were injected without generating embryos with cardiac defects in significant percentages (Fig. 2C). Conversely, in miR-218a over-expressing embryos we found different cardiac defects accounting for the ratios shown in Fig. 2C: hearts failing to complete looping, ventricles showing very irregular walls, and atria that were strongly reduced and sometimes stretched to a thin "string-like" morphology (Fig. 2F,G). To down regulate miR-218a, we injected either a morpholino targeting the mature form of miR-218a (MOM-218), or a longer morpholino also targeting the Drosha cleavage site of pre-miR-218a (MOD-218, see Methods and [16]). The knockdown efficiency of these morpholinos was confirmed by their ability to down-regulate mature miR-218 (Fig. S4A), and to rescue the phenotype caused by miR-218a over-expression (Fig. S4B). Nonetheless, injection of either MOD-218 or MOM-218 (not shown) generated no cardiac edema or cardiac defects even at very high dose (12 ng) in any of the genetic backgrounds we tested (Fig. 2D,H). This is consistent with the extremely low level of miR-218a during early stages of development (data not shown and [16], [29]).
Injection of miR-218a mimic or MOs-218 in Tg(flk1:eGFP), which express GFP driven by the endothelial-specific enhancer of flk1, allowed a direct visualization of vascular integrity. As previously reported [16], MO-218 microinjection did not cause gross alteration in vascular structures (Fig. S5C) nor the hemorrhagic events described in mice after miR-218a knock down [19]. miR-218a over-expression did not affect vessel morphology either (Fig. S5B), supporting the idea that in zebrafish miR-218a does not overtly influence the organization of blood vessels during embryogenesis.
Finally, we analyzed the expression of some cardiac markers in embryos over-expressing miR-218a. Despite the strong cardiac morphological alterations, miR-218a over-expressing embryos showed normal expression of the ventricular myosin heavy chain and the atrial myosin heavy chain (Fig. S6), as previously described in Tbx5 (hst) mutants [10]. It has been shown that Tbx5 activity is crucial for the specification of the AV boundary and valve formation [30]. To verify whether miR-218 over-expression can affect cardiac valve development, we analyzed the expression of the tie-2 gene, a member of the Tie family of tyrosine kinase receptors, which is expressed mainly in endothelial cells [31] and is up regulated during atrio-ventricular canal differentiation [32], [33]. In hemizygous Tg(tie-2:GFP) embryos, the transgene has low expression [34] and it is easy detectable only in the atrio-ventricular canal thus allowing to visualize the valve tissues (Fig. 3A, a,a′). Hemizygous Tg(tie-2:GFP) embryos injected with miR-218a showed an increase in Tie-2 expressing cells in the ventricle and atrium (Fig. 3A, b,b′ and Fig. 3B,C). A similar Tie-2 dysregulation was observed in hemizygous Tg(tie-2:GFP) embryos after Tbx5 misexpression (Fig. 3A, c,c′,d,d′ and Fig. 3B).
All together these data indicate that correct expression of miR-218 is crucial for proper cardiac development.
Recent data have shown that, in zebrafish, delayed heart field migration was caused by either miR-218a reduction or by silencing of Robo1, an established target of miR-218a [16], [19]. In line with data showing robo1 as target of miR-218a, over-expression of miR-218a significantly reduces the translational rate of a reporter construct (sensor), carrying GFP coding sequence upstream of robo1 3′UTR (Fig. S7). Therefore, we decided to verify whether miR-218a over-expression might modify the migration of bilateral heart field cells to the midline. miR-218a mimic was injected in Tg(cmlc2:eGFP) embryos and the migration of the GFP-expressing cells was followed by confocal analysis. Myocardial cells migrated more slowly in miR-218a than in miR-Ct injected embryos (Fig. 4). Even after injection of high miR-218 doses, cardia bifida was never observed, suggesting that miR-218 over-expression slows down but does not arrest the migration of cardiomyocytes to the midline. On the contrary, MOD-218 injection did not affect cmlc2-positive cells migration (Fig. 4C), further confirming that reducing the level of this miRNA during the first hours of development has no overt effects on heart development.
These data show that the cardiac defects observed in miR-218a over-expressing embryos might be at least in part related to defects in heart field migration.
To further investigate the functional interaction between Tbx5 and miR-218a, we assessed whether the morphological alterations generated by Tbx5 misexpression might be compensated for the modulation of miR-218a. We reasoned that if the phenotype induced by Tbx5 over-expression was due, at least in part, to increased expression of miR-218, the co-injection of MO-218 should rescue the Tbx5 gain of function phenotype. To test this hypothesis, Tbx5a was over-expressed by injecting its mRNA into the cytoplasm of one–cell-stage embryos. Tbx5 over-expression affects heart development in humans [35], [36],mice [37] and chicks [5], while no reports describe the effect of Tbx5 over-expression in zebrafish. Over-expression of tbx5a mRNA induced a range of cardiac defects, from cardiac edema in 25% of embryos injected with low doses of mRNA (35 pg), to mild cases of looping defects or absence of looping and alteration of chamber morphology in 60% of embryos injected with higher doses (100 pg, Fig. 5A and C, panels b′–d′). Pectoral fins were occasionally asymmetric in embryos injected with 200 pg of tbx5a transcript (Fig. 5C, panel d). tbx5a mRNA injection also caused eye morphology malformations ranging from asymmetrically positioned eyes, fusion of eyes or reduction/absence of eyes (Fig. 5C, panels b,c). None of these defects was observed at significant percentages in hundreds of embryos injected with comparable doses of GFP mRNA (not shown). Increased miR-218a expression was observed as a consequence of Tbx5 up regulation (Fig. 5B). Co-injection of 8 ng of either MOD-218a or MOM-218a along with 100 pg of tbx5a mRNA restored normal heart morphology and looping in a high percentage of injected embryos and reduced the number of embryos with eye defects as compared to control-injected embryos (Fig. 6A and B, panels b,b′). Co-injection of MO-218 was also able to counteract the tie-2 expansion observed in Tbx5 dysregulated embryos (Fig. 6 C,D). This result strengthened our hypothesis that the effect of Tbx5 over-expression on heart development might, at least in part, be be mediated by miR-218.
The apparent lack of phenotype that we observed after MO-218a injection (Fig. 2D,I) and the very low expression of miR-218 at early developmental stages, suggest that decreased miR-218a should not contribute to the phenotype generated by Tbx5 knockdown. To verify this assumption we downregulated Tbx5 using a morpholino directed against the tbx5a translational start site (MO-Tbx5a). The effectiveness of this morpholino was confirmed by the injection of GFP-carrying a chimeric target (Fig. S8A,B) and its specificity was confirmed by rescuing the MO-Tbx5a phenotype co-injecting in vitro transcribed Tbx5a-mRNA (not shown). Tg(cmlc2:GFP) embryos injected with MO-Tbx5a showed the heart and limb defects characteristic of the well described heartstrings phenotype (Fig. S8E,G and [10], [11]). In addition to tbx5a, a second tbx5 isoform has been recently identified in zebrafish, tbx5b, which has lost the characteristic forelimb/pectoral fin expression of tbx5 genes [38]. Consequently, we also designed a morpholino against this tbx5 isoform, MO-Tbx5b (Fig. S8C,D). Similarly to MO-Tbx5a injection, MO-Tbx5b injection caused looping defects and stretched cardiac chambers, but it did not induce Tie-2 mis-expression and lack of fins (Fig. S8F). For these reasons and since the penetrance of the MO-Tbx5b phenotype was much lower compared to MO-Tbx5a (compare Fig. S8G,H) we chose MO-Tbx5a for further analysis. Co-injection of mimic miR-218a did not rescue the cardiac edema and looping defects generated by MO-Tbx5a (Fig. S9A). Instead, the co-injection increased both the frequency and the severity of the mutant phenotype, doubling the number of heartstring-like embryos (Fig. S9A) and the number of embryos that showed a particularly extended edema at 48 hpf (Fig. S9B). This synergism was further demonstrated by MO-Tbx5a and miR-218a mimic co-injection in sub-phenotypic doses. In fact, zebrafish embryos tolerated the injection of either 35 pg of miR-218a or 0,5 ng of MO-Tbx5a well and only rarely revealed heart looping defects as compared to un-injected siblings (Fig. 2A and Fig. S9C,D). However, the injection of both MO-Tbx5a and miR-218a mimic at these doses dramatically increased the number of embryos with looping defects (Fig. S9C,E).
As a whole, these data support a functional interaction between Tbx5 and miR-218a in heart morphogenesis.
Our data show that miR-218 is part of a regulatory circuit through which Tbx5 controls heart morphogenesis. Previous studies in mice identified slit2, which encodes miR-218-1 within one of its introns, as one of the genes highly sensitive to Tbx5 dosage [8]. Moreover, the coordinate expression of miR-218-1 and its host genes has been largely documented both in physiological (mouse development [19]) and pathological (cancer progression [39], [40]) conditions. We showed a functional relation between Tbx5, Slit2 and miR-218 in P19CL6 cells in which a progressive increase of Tbx5, Slit2 and miR-218 expression was observed during cardiomyocyte differentiation. Moreover, we showed that Tbx5 deregulation affects miR-218 expression. Tbx5 might regulate slit2 expression directly or indirectly. However the presence of T-box consensus sequences upstream of both mouse and fish slit2 transcription start site, as identified by the Transfac program (http://www.biobase-international.com; not shown), supports the hypothesis that Tbx5 might directly bind to and activate the slit2 promoter. To demonstrate that the Tbx5/miR-218 regulatory circuit is also functional during development, we used the zebrafish as a model system. Through functional assays in zebrafish, we showed that either over-expression or down-regulation of Tbx5 affects heart morphogenesis. In line with the hypothesis that miR-218 might be a Tbx5 effector, we demonstrated that miR-218a deregulation generates cardiac defects (Fig. 2A). As in zebrafish the expression level of miR-218a is extremely low during the first stages of development (our observation is supported also by the data of Fish et al. [16] and by data regarding miRNA microarray of Thatcher et al. [29], the introduction of very low amounts of miR-218a mimic into embryos at the one- or two-cell stage generated a severe cardiac phenotype. miR-218a over-expressing hearts were not able to complete the looping process, showed marked alteration of the cardiac chamber morphology and mis-expression of a marker of valve cardiac tissues. A large pericardial edema was visible after 48 hpf. The frequency and severity of all these cardiac phenotypes were dose-dependent (Figs. 2 and and3).3). Our data suggest that at least part of these morphological anomalies may be due to a reduced migration rate of myocardial cells (Fig. 4). Conversely, miR-218a down regulation, even through injection of high doses of two different morpholinos, did not affect heart development.
Since Tbx5 and Slit2 are both expressed in myocardial cells [16] we hypothesized that early Tbx5 over-expression might cause heart malformation through early activation of miR-218a and silencing of the target genes of this miRNA. This hypothesis is supported by the observation that miR-218 down-regulation by MO-218a injection rescues the effects of Tbx5 over-expression (Fig. 6). robo1 has been identified as a target of miR-218 in many different organs and tissues [19], [39], [41]. Fish et al [16] showed that robo1 is a miR-218a direct target and that early robo1 down-regulation by morpholino injection in zebrafish embryos induces severe pericardial edema and heart defects caused by reduced migration rate of endocardial cells. Thus, robo1 is a candidate gene through which Tbx5 and miR-218a early over-expression affects heart development. Fish et al [16]reported that miR-218a down regulation, performed by the microinjection of one of the two morpholinos that we used in our study, causes severe cardiac defects and cardiac edema through reduced migration of endocardial and myocardial cells. This is inconsistent with both our data and with the results of Fish et al. [16] who observed that Robo1 knock-down generates the same phenotype, because the same authors also showed that Robo1 is targeted by miR-218. In fact, an effect of miR-218a on the migration of endocardial and myocardial cells is not expected, as miR-218a seems not to be substantially expressed before 24 hpf [16], [29], when the fusion of migrating cardiac cells is about to be completed. We speculate that the timing of miR-218a upregulation during heart development is crucial for heart morphogenesis. miR-218 and Robo1 are supposed to be upregulated and downregulated, respectively, when myocardial cell migration is about to end. In this view, Tbx5 mis-expression by mRNA microinjection at the one-cell stage might speed up the up-regulation of miR-218 and reduce the migration of myocardial cells precociously, which in turn might affect heart morphogenesis by impairing the correct interaction between myocardial and endocardial cells.
A negative role of miR-218 on cell migration has been highlighted in different biological contexts. In mice, it has been shown that miR-218 regulates vascular patterning by modulating Slit-Robo signaling [19]. The authors showed that miR-218-driven repression of Robo1/2 and of heparan sulfate proteoglycans (HSPGs), which are proteins essential for Slit/Robo signaling, negatively affects endothelial cell (EC) migration. miR-218 has also been shown to affect cancer progression by inhibiting tumor cell migration and metastasis via the repression of the Slit2/Robo1 pathway in gastric [40] and in nasopharyngeal [39] tumors, respectively. Therefore our data showing a migration delay of cmlc2-positive cardiac precursors in embryos over-expressing miR-218 (Fig. 4), but not in embryos in which miR-218 was down-regulated by MO-218 injection (Fig. 4C), are in line with these findings.
In the human heart, Tbx5 is expressed not only in the myocardium, but also throughout the embryonic epicardium and coronary vasculature. Using chick development as a model system, Hatcher et al. [42] showed that, in vivo, over-expression of biologically active Tbx5 inhibits proepicardial cell migration. Although we do not know whether the slit2 is expressed in proepicardial cells, it is tempting to speculate that the up regulation of the Tbx5-miR-218 circuit might also impact the proepicardial cell migration by targeting robo1 or other cell migration regulators such as Semaphorins, some members of this large class of molecules being predicted targets of miR-218 (not shown).
Tbx5 over-expression affects heart development in different organisms. In humans, tbx5 gene duplication produces cardiac abnormalities [35], [36]; in mice, persistent cardiac Tbx5 over-expression results in heart looping defects and abnormalities of early chamber development [37]. In chicks, Tbx5 over-expression determines a significant decrease in heart size and a marked decrease in ventricular trabeculation [5]. We also observed looping and cardiac chamber alterations in zebrafish after injection of tbx5a mRNA in embryos at one-cell stage. Unexpectedly, the severity of cardiac morphology defects was paralleled by the severity of eye defects (Fig. 5). Tbx5 is expressed in optic primordia from zebrafish to humans [43], [44] and its mis-expression has been shown to affect eye morphogenesis and the visual projection in chicks [45]. Moreover, ophthalmological examination of HOS patients revealed alteration of dorso-ventral polarity in developing eye vesicles [46]. However, our Tbx5 over-expressing embryos showed particularly severe eye defects such as asymmetrically positioned eyes, fusion of eyes, and even unilateral anophthalmia (Fig. 5). Comparable eye defects are observed in Brg1 over-expressing zebrafish embryos [47]. Recently the importance of the balance between Brg1, a member of the SWI/SNF chromatin-remodeling complex, and several cardiac transcription factors including Tbx5, has been demonstrated [48]. Since Brg1 is maternally expressed [47], the over-expression of Tbx5 might generate a strong imbalance between these two factors during the first hours of development, and this imbalance might be at the root of eye defects. At the moment we do not know how miR-218 might also partially rescue eye defects. It is interesting to know that Pax2, that is negatively controlled by Tbx5 [45], is a predicted target of miR-218. Overall, these observations suggest that Tbx5 over-expression affects heart and eye development and that this might be at least partially mediated by miR-218. Our observation that MO-218 co-injection is able to ameliorate both heart and eye defects caused by Tbx5 injection is consistent with this model.
Our data suggest that the haplo-insufficiency of the Tbx5 gene, at the moment the most significant cause of HOS, does not impact heart and upper limb formation through miR-218 misregulation. The simplest explanation for this might be that other key RNAs controlled by Tbx5 than miR-218 might be necessary for heart morphogenesis by regulating mechanisms other than myocardial cell migration. This would still be consistent with the requirement of a tight tbx5 gene dosage regulation in space and time for proper heart morphogenesis. More rarely, HOS has been associated with increased Tbx5 expression by partial chromosome duplication [6], [28] or mutation resulting in Tbx5 gain of function [49]. However advances in DNA sequencing technology also highlighted the potential role of non-coding variants in congenital malformations. Recent studies in mice uncovered three enhancers that collectively recapitulate the endogenous expression pattern of tbx5 but that singularly have specific and compartmentalized expression in the heart and forelimbs [50]. Interestingly, a mutation in one of these enhancer sequences was identified in a cohort of non-syndromic patients and it has been shown to affect the enhancer activity in mice and zebrafish transgenic models. The population-wide frequency of this variant suggests that a significant number of congenital heart defects (CHD) associated with Tbx5 dysregulation might arise from non-coding mutations in Tbx5 heart enhancers effectively decoupling the heart and hand phenotypes of HOS syndrome. Therefore it is likely that modulation of Tbx5 in general, and over-expression of miR-218 as a consequence of Tbx5 up-regulation in particular, might have a higher impact on CHD population than previously hypothesized.
Finally, our results highlight the potential advantage of using miRNAs as target molecules for heart disease therapies. Their potential to restore the expression of hundreds of dysregulated mRNAs to their pre-pathological level in one shot and, in so doing possibly reverse the disease, places miRNAs among the most exciting molecules for the development of new therapeutic strategies.
Ensembl (http://www.ensembl.org/index.html), was used to obtain information about chromosome location, position and segment of the selected genes. Ensembl is a joint scientific project between the European Bioinformatics Institute (EBI; http://www.ebi.ac.uk/clustalw/) and the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/). EBI provides a centralized resource with annotations on genomes of sequenced species and the Ensembl Perl API (Application Programming Interface) models for access to biological objects, such as genes and proteins. Moreover EBI allows the execution of Perl programs for retrieving data from a public database MySQL (http://www.mysql.it/). We generated two local databases, one for genes, and one for microRNAs. By applying the Perl program that uses Ensembl API, we compared the gene databases with the microRNA database using a standard database interface module for Perl.
ReagentsMature miRNA mimics (mmu-miR-218a, mmu-miR-492, mmu-miR-214 and miR-Ct) were synthesized by GenePharma (Shanghai, China), morpholinos (Gene Tools, LLC USA.), Lipofectamine 2000 TRIzol reagent, DNaseI amplification grade, SuperScript II reverse transcriptase, RNAse out, α-minimal Essential Medium (Invitrogen), HyPerFect Transfection Reagent, miRNeasy mini kit, miScript Reverse Transcription kit Quantitec Reverse Transcription kit and Quantifast SYBR Green PCR kit (QIAGEN, Milan, Italy), LightCycler 480 SYBR Green I Master, anti-DIG antibody-alkaline phosphatase Fab fragment, Blocking reagent, BM Purple and DIG-RNA labeling kit (Roche Diagnostic, Mannheim, Germany), DIG-labeled miRCURY LNA microRNA detectin probe (Exiqon), SP6 RNA polymerase, RNAse free DNAse I (Fermentas International Inc), Herculase DNA polymerase, (Stratagene), pGEM Teasy vector, (PROMEGA), Fetal Bovine Serum (Lonza), zebrafish diet (SDS, Dietex, France), Tetramisole (Sigma), mMESSAGE mMACHINE® Kit (Applied Biosystems).
Current italian national rules: no approval needs to be given for research on zebrafish embryos. Wild-type AB and transgenic Tg(flk1:eGFP), Tg(cmlc2:eGFP), Tg(cmlc2:eRFP) and Tg(tie-2:GFP)s849 transgenic lines were used in these studies. Zebrafish were raised and maintained under standard laboratory conditions (Westerfileld M zebrafish book) in a ZEBTEC Zebrafish Housing System (Tecniplast, Varese, Italy).
P19CL6 cells were obtained from Dr.Antonio Baldini (Telethon Institute of Genetics and Medicine, Napoli, Italy). Cells were cultured in growth conditions as previously described [20], [21]. For differentiation conditions, growth medium was supplemented with 1% DMSO and the medium was replaceded every two days. Cells were grown at 37°C in a humidified atmosphere containing 6% CO2. The pCMV-Tbx5 plasmid expressing mouse Tbx5 (Kindly provided by Prof. Mona Nemer, Universitè d'Ottawa) or the pCMV empty vector as control were transfected using Lipofectamine 2000. To downregulate mouse Tbx5 the following siRNAs were designed according to previously identified criteria [51]: siRNA-Tbx5-1 (5′-CUGG ACCCGUUUGGACACAUU-3′/5′-UGUGUCCAAACGGGUCCAGUU-3′) and siRNA-Tbx5-2 (5′-GCCC CGAUUACACAUCGUGUU-3′/5′-CACGAUGUGUAAUCGGGGCUU-3′). To downregulate mouse slit2 the following siRNAs were designed: siRNA-Slit2-1 (5′-GCUCACUCUGAACAAUAACUU-3′/5′-GU UAUUGUUCAGAGUGAGCUU-3′) and siRNA-Slit2-2 (5′-GGCUCAGAAGGAAAAGAAUUU-3′/5′-AUUCUUUUCCUUCUGAGCCUU-3′). For the silencing experiments in P19CL6, cells were transfected with control siRNA or a siRNA-Tbx5-1 and siRNA-Tbx5-2 mix in GM. 6 hrs after transfection the GM was substituted with the DM. Four days after the first round of transfection, the cells were transfected with the respective siRNA again. Cells were pelletted 6 h after the first transfection, 48 h and 96 h after the second round of transfection. SiRNAs were transfected using HiPerFect Transfection reagent.
Total RNA was extracted from ~30 embryos or from P19CL6 cells cultured in 6-well plates to 80-90% confluence at using the RNeasy mini kit. After DNase treatment, 1 µg of total RNA was retro-transcribed using miScript Reverse Transcription kit (for miRNA analysis) and Quantitec Reverse Transcription kit (for gene analysis) following the manufacturer's instruction. Real-time PCR (qRT-PCR) was carried out using either QuantiFast SYBR Green kit with Rotor gene (Quiagen) or LightCycler 480 SYBR Green I Master with LightCycler 480 (Roche) following the manufacturer's instructions. Primers used for mRNA analysis were as follows: for mmu-Tbx5 F 5′-CCACTGGATGCGACAACTT-3′, R5′-GCATGGAGTTCAGGATA ATGTG-3′; for mmu-Slit2 F 5′-GCCACCTCAGTGAGAACCTC-3), R 5′-TGTCCCTCAAAGCCCAGA-3′; for mmu-Slit3 F 5′-GCCACCTCAGTGAGAACCTC-3′, R 5′-TGTCCCTCAAAGCCCAGA-3′; for mmu-Cx40 F 5′-GAGCCTGAAGAAGCCAACT-3′, R 5′-AGCTCCAGTCACCCATCTTG-3′; for mmu-αMHC F 5′-CAAGACTGTCCGGAATGACA-3′, R 5′-GGCTTCTTGTTGGACAGGAT-3′; for mmu-GATA4 F 5′-GGAAGACACCCCAATCTCG-3′, R 5′-CATGGCCCCACAATTGAC-3′; for β III-Tubulin F 5′-TTCTGGTGGACTTGGAACCT-3′, R5′-ACTCTTTCCGCACGACATCT-3′; for mmu-Oct4 F 5′-TCAG CTTGGGCTAGAGAAGG-3′, R 5′-GGCAGAGGAAAGGATACAGC-3′and for mmu-PAX6 F 5′-CCT CCTTCACATCAGGTTCC-3′, R 5′-CATAACTCCGCCCATTCACT-3′. For dre-Tbx5a F 5′-AACCATCT GGATCCCTTCG-3′, R5′-TGTTTTCATCCGCCTTGAC-3′; for dre-Slit2 F 5′-TCCTTTCAAGGTCTCCG TTC-3′, R 5′-AGGCCTTTGGGAAGTTCAGT-3′. Transcript values were normalized with those obtained from the amplification of mmu-α-actin with the following primers: F (5′-CGAGCTGTCTTCCCATCCA-3′), R (5′-TCACCAACGTAGCTGTCTTTCTG-3′) for P19CL6 analysis and from the amplification of dre-α-actin or dre-EF1 respectively with the following primers F 5′-CGAGCTGTCTT CCCATCCA-3′, R 5′-TCA CCAACGTAGCTGTCTTTCTG-3′ and F 5′-CTGGAGGCCAGCTCAAACAT-3′, R 5′-ATCAAGAAGAG TAGTACCGCTAGCATTAC-3′ for zebrafish embryos analysis. The primers for mature miR-218a, mmu- or dre-U6 (as standard) quantification were respectively: 5′-TTGTGCTTGATCTAACCATGT-3′, 5′-CGCAAG GATGACACGCAAATTC-3′, 5′-ATGACACGCAAATCCGTGAAG-3′. To amplify both pre-miRNAs of 218 in mouse, the following primers were used: for pre-miR-218-1 F 5′-GATAATGGAGCGAGATTTTCT G-3′, R 5′-TAGAAAGCTGCGTGACGTTC-3′ and for pre-miR-218-2 F 5′-AGTTGCCGCGGGGCTTTC-3′, R 5′-AGGAGAGAGCGATGCTTTC-3′.
All reactions were performed in triplicate. Relative quantification of gene expression was calculated as described [52].
Cardiac tissue-enriched total RNA extracted from 5 day old embryos was used to clone zebrafish tbx5a. dre-tbx5a cDNA was directely amplified by RT-PCR (forward primer 5′-AGATCTAGAC ATCGTACAGGC-3′, reverse primer 5′-CTGCATGTTAGCTGGCTTCGT-3′) The tbx5a PCR fragment was cloned into the pGEM-T Easy Vector (Promega) and sequenced by Genechron C.R.ENEA (Roma, Italy). For zebrafish tbx5a mRNA production the entire open reading frame was subcloned in pCS2-GFP vector using BamHI and XbaI restriction sites. The construct was linearized with NotI and the capped mRNA was synthetized with mMESSAGE mMACHINE® Kit (Applied Biosystems). As control, capped mRNA of GFP obtained from pCS2-GFP linearized template was used.
The morpholino antisense oligonucleotide MO-Tbx5a (5′-GAAAGGTGTCTTCACTGTCCGCCAT-3′) and MO-Tbx5b (5′-GGATTCGCCATATTCCCGTCTGAGT-3′) were designed against the translational start sites of tbx5a and tbx5b respectively. To test the specificity of the two MOs two plasmids pCS2-Tbx5a-GFP and pCS2-Tbx5b-GFP were generated by fusing a 180-bp fragment of tbx5a and a 219-bp fragment of tbx5b, both including the morpholino target sides, into GFP producing vector. The primers used for fragment amplifications were: for Tbx5a F 5′-AAGCTTCAACCGCTAGTGCTGGAAG-3′, R 5′-GGATCC TTCGCTGTCACTGGGAGAG-3′ for Tbx5b F 5′-AAGCTTGAGAGCCGGAGAAGACTG-3′, R 5′-GGATCCCATTGGATTCGCCATATTC-3′ where the underlined sequences represent anchored Hind III (F primers) and Bam HI (R primers) restriction sites used for subcloning in pCS2 vector. tbx5a and b PCR fragments were cloned in pGEM-T Easy Vector, sequenced and subcloned in pCS2-GFP vector.
The morpholino antisense oligonucleotide MOM-218 (5′-GCACATGGTTAGATCAAGCACAACA-3′) and MOD-218 (5′-TGCATGGTTAGATCAAGCACAAGGG-3′) were designed against the mature form of miR-218a and the Drosha cleavage site of pre-miR-218a respectively. The sequence of the control MO was 5′-CCTCTTACCTCAGTTACAATTTATA-3′.
The one- and two-cell stage embryos were injected with a constant injection volume (~1 nl, confirmed by volume analysis) using a microinjector (Tritech Research, Los Angeles CA USA).
Optical microscopy was performed with Olympus SZH microscope, images were acquired with Nikon DS-Fi1 camera and NIS-Elements F 3.0 software. Images were processed with Gimp-2.6 software. For confocal analysis embryos were fixed in 4% PFA for 1 h at room temperature under slow agitation and embedded in 2% low-melt agarose. Images were acquired with a Leica DM IRE 2 confocal microscope. Image stacks were processed with FIJI-WIN32 by projection. For migration analysis, embryos were injected with the indicated miRNA mimics or morpholinos and allowed to develop at 26°C until the 14-somite stage. Then they were embedded in 1% low-melting soft agarose and imaged at 26°C with Leica DM IRE 2 confocal microscope to obtain stacks. To calculate cell velocity stacks were processed with MATLAB.
Zebrafish Tg(tie-2:GFP-cmlc2:eRFP) embryos were injected and raised in standard conditions until 72 hpf. Next embryos (n=15−25 embryos for each analysis) were collected and treated with trypsin 0,125 mg/l at 37°C. After a 30 min incubation, trypsin was inactivated, samples were filtered with 50 µm cell-strainer and processed with FACScalibur BD.
slit2 and slit3 clones for in situ hybridization were kindly provided by Dr. Hitoshi Okamoto (Laboratory for Development of Gene Regulation, RIKEN, Brain Science Institute Japan).
Whole mount in situ hybridization was performed as previously described [53] with some modification: pre-hybridization temperature was 62°C; hybridization temperature was 62°C for gene probes and 52°C for miR-218 probe. 500 ng of antisense DIG labelled RNA probe was added to the hybridization mix. The anti-DIG antibody-alkaline phosphatase Fab fragment was diluted 14.000 in MABlock buffer (2% Blocking reagent in 100 mM Maleic acid and 150 mM NaCl) and incubation was performed at +4°C for gene probes and at room temperature for miR-218 probe. After incubation with the anti-DIG antiserum, embryos were washed in PBT and then incubated in the alkaline Tris buffer solution containing 2 mM Tetramisole (Sigma). The final staining step was performed in BM Purple AP-Substrate, precipitating buffer according to the manufacturer's recommendations. Labeled embryos older than 24 h were bleached as follows: embryos were washed at room temperature under slow agitation in PBS changed 2x at 5 min. intervals. PBS was removed and embryos were incubated for 10 minutes in pre-bleaching buffer (SSC 0,5X and 6% Formamide). After discarding the pre-bleaching buffer, bleaching buffer (pre-bleaching buffer containing 33% H2O2) was added to the embryos which were subsequently exposed to a bright source of light for 15 minutes. Afterwards embryos were transferred back to 87% glycerol in PBS incubated at room temperature under slow agitation for 4 h and then stored at –20°C.
Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA USA). Statistical differences were determined by unpaired t-test, with values of P<0.05 considered statistically significant. Each experimental point in the graph represents the mean ± SE of at least three independent experiments. In the graph of Fig. 6A comparisons between groups were performed by one-way analysis of variance, followed by Bonferroni's post-hoc for multiple comparisons.
Schematic representation of the bioinformatic tool developed to identify in silico Tbx5-controlled miRNAs. Ensembl was used to obtain information about chromosome location, position and segment of the selected genes. Ensembl is a joint scientific project between the European Bioinformatics Institute (EBI; http://www.ebi.ac.uk/clustalw/) and the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/). EBI provides a centralized resource with annotations on genomes of sequenced species and the Ensembl Perl API (Application Programming Interface) models for access to biological objects, such as genes and proteins. Moreover EBI allows the execution of Perl programs for retrieving data from a public database MySQL (http://www.mysql.it/). We generated two local databases, one for genes, and one for microRNAs. By applying the Perl program that uses Ensembl API, we compared the gene databases with the microRNA database using a standard database interface module for Perl.
tbx5 and miR-218 are co-expressed in mouse tissues. A-D, relative expression of tbx5, slit2, slit3 and miR-218 as evaluated by q-RT-PCR in different newborn mouse tissues. Results are standardized against GAPDH for genes, and against U6 for miRNAs. Values represent the averages and standard deviations of at least two independent experiments.
Expression of pre-miR-218-1 parallels that of mature miR-218 during mouse differentiation and tbx5 modulation. Q-RT-PCR detection of pre-miR-218-1 and pre-miR-218-2 relative expression in P19CL6 cells during differentiation (A), or 48 h after plasmids or siRNA transfection (B). In B fold changes of CMV-Tbx5 and siRNA-Tbx5 are relative to CMV-empty and siRNA-Ct values, respectively. Results are standardized against GAPDH. *, P<0.05 (Student's t-test). C, Q-RT-PCR detection of slit2 and mature miR-218 in P19CL6 cells transfected with a mix of two siRNAs against slit2 or with a siRNA-Ct.
Rescue of cardiac defects induced by miR-218a over-expression was accomplished by co-injecting MO-218a. A, qRT-PCR analysis of miR-218a relative expression in 24 hpf embryos microinjected with 12 ng of control morpholino (MO-Ct) or MOD-218a and with 260 pg of miR-Ct or miR-218a mimic. miR-218a relative expression was calculated as the ratio between the expression of injected and the expression of non injected embryos. B, representative transgenic Tg(cmlc2:eGFP) embryos at 72 hpf showing heart morphological defects induced by the injection of 260 pg of miR-218a mimic in the absence (a,b) or in the presence (c,d) of MOD-218a (12 ng). Labels: a, atrium, v, ventricle. Black scale bars: 100 µm, red scale bars 25 µm.
miR-218 dysregulation does not affect vascular integrity. Confocal images of representative 72 hpf Tg(flk1:eGFP) embryos injected with 260 pg of miR-Ct (A), 260 pg of miR-218 mimic (B) or 8 ng MOD-218 (C). Black and white scale bars: 100 µm.
tbx5 and miR-218a misexpression does not alter amhc and vmhc cardiac marker expression in zebrafish embryos. Ventral views of 48 hpf embryos injected with the indicated miRNA mimics (260 pg) or MOs (12 ng MO-Ct and MOD-218a, 3 ng MO-Tbx5a) after mRNA in situ hybridization. Scale bar 100 µm.
miR-218 targets the 3′ UTR of robo1 in zebrafish embryos. Top: schematic representation of sensors and miRNAs used for in vivo sensor assay. Bottom: examples of 24 hpf embryos microinjected with 40 pg of RFP mRNA, 400 pg of 3′UTR robo 1 sensor and 160 pg of miR-Ct (a,c,e) or miR-218a (b,d,f). In figures C and D the percentage of the relative phenotypes were indicated. ~30 embryos for each thesis were injected. Scale bars 50 µm.
MO-Tbx5a and MO-Tbx5b effectively knockdown the two zebrafish tbx5 isoforms. A-D, 35 pg of pCS2 plasmid expressing GFP fused with MO-Tbx5a or MO-Tbx5b target sequences were injected in one-cell stage embryos in the absence (A and C) or in the presence (B and D) of 1,5 ng of the relative morpholino. Representative fluorescent images of 24 hpf embryos. ~20 embryos for each thesis were analysed. E-F, Tbx5 morphants analysis. Phenotypic analysis of Tbx5a (E) and Tbx5b (F) morphants: 2 ng of MO-Tbx5a, or 4 ng of MO-Tbx5b, were injected in Tg(cmlc2:eGFP) embryos. Phase-contrast images showing pericardial edema (arrowheads) and fin absence (brackets) or presence (arrows); in the bottom right corner of figures E and F, fluorescent images showing heart morphology. Quantification of Tbx5a (G) and Tbx5b (H) morphant phenotypes. The percentage of embryos with the indicate defects was averaged across multiple independent experiments. ~100 embryos for each thesis were analysed. Black scale bars: 100 µm, red scale bars 25 µm.
Injection of miR-218a in Tbx5a morphants increases the severity of heartstring phenotype. A, phenotypic analysis of Tbx5a morphants co-injected with 1 ng of MO-Tbx5a and either 130 pg of miR-218a mimic or 130 pg of miR-Ct. B, representative images of 48 hpf embryos showing the edema expansion caused by the co-injection of miR-218a mimic. C, phenotypic analysis of embryos co-injected with sub-phenotypic doses of both MO-Tbx5a(0.5 ng) and miR-218a mimic (35 pg). For comparison the same dose of MO-Tbx5a was co-injected with 35 pg of miR-Ct. (D-E) Representative confocal images showing heart morphology of transgenic Tg(cmlc2:eGFP) embryos injected with a sub-phenotypic dose of MO-Tbx5a and 35 pg of either miR-Ct (D) or miR-218a mimic (E). Embryo in D has normal looping while co-injected embryos in E show absence of looping, although with different degrees of heart defects. a, atrium, v, ventricle, e, cardiac edema. Black and white scale bars: 100 µm, red scale bars 25 µm.
We thank Dr. Antonio Baldini (Telethon Institute of Genetics and Medicine, Napoli, Italy) for kindly providing the P19CL6 cells, Prof. Mona Nemer, (Universitè d'Ottawa) for pCMV-Tbx5 plasmid, Dr. Jeroen Bakkers (Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands) for the Tg(Tie-2-GFP) line, Dr. Didier Stainier (Departments of Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, USA) for the Tg(flk1:eGFP) and the Tg(cmlc2:eRFP) lines and Dr. Massimo Santoro (Molecular Biotechnology Center, University of Torino, Torino, Italy) for theTg(cmlc2:eGFP) line. Moreover, we thank Dr. Davide Moroni (Institute of Information Science and Technologies, CNR Pisa) for cell migration analysis, Dr. Giuseppe Rainaldi and Dr. Marcella Simili (Institute of Clinical Physiology, CNR Pisa), Dr. Michela Ori (Biology Department, University of Pisa) and Dr. Debora Angeloni (Scuola Superiore S.Anna, Pisa) for critical reading of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information 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.


text:
100% Shots with GoPro Hero 3+ Black Edition at 1440 dpi 48 fps. Editing with Final Cut Pro 7 and Twixtor Pro at 2-3%.
Color with Colorista II.
Music Washed out "new theory"
Location Alpe Devero
Video and editing Federico Chierichetti
Loading more stuff…
Hmm…it looks like things are taking a while to load. Try again?
TM + © 2015 Vimeo, LLC. All rights reserved.
Made with k in NYC.
Your content preferences apply to any Vimeo videos you view. Learn more about content preferences in our FAQ.


text:
Ron Haselden
Ron Haselden (born 1944) is a British artist who splits his time between London and the French coastal town of Plouër-sur-Rance, in Brittany, France. He works with light, sound, film and video, often as part of architectural projects.
He was born in Gravesend, Kent and attended the Gravesend School of Art. After teaching for a number of years at Reading University he moved to France.
Frère Jacques (made in collaboration with Peter Cusack) combined a wall of light with children singing.[1] In 1993 he created a twenty feet high new moon illuminating the front of the South London Gallery.[2] Blue Passage (1999), made for the passageway between the South Bank and the BFI IMAX cinema in London, consists of 8000 blue LEDs sunk into the walls of the underpass.[1]
Further reading[edit]
- Frère Jacques et autres pièces à Francis: Expositions. 1997. Saint-Fons', Ron Haselden, Saint-Fons, Centre d'Arts Plastiques, 1997, ISBN 2-9509357-2-9
External links[edit]
|This article about an artist from the United Kingdom is a stub. You can help Wikipedia by expanding it.


text:
Scorpion A Good Bad Horse (Paperback)
|Author: Will James
Learn more about Scorpion:
Format: Paperback
Publisher: Mountain Press Publishing Company
Sku: 30762314
Publish Date: 3/1/2001
Dimensions: (in Inches) 9H x 6L x 0.5T
Pages: 254
Age Range: NA
See more in Westerns
|A fine-looking chestnut bronc receives the name Scorpion when he explodes unexpectedly after several days of unusually good behavior. "That horse, " a cowboy says, "is going to be either mighty good or mighty bad." Tim, an outlaw on the run, trades his tired horse for Scorpion. The horse acts truly wicked only when Tim makes poor decisions--keeping him out of trouble.


text:
Dipped in Gold
The glitter and glamour of gold is catching the eye as a 2015 home decor trend.
I like this rug except the color is not exactly what I wanted but it works ok and is very pretty...
|Cotton Wool
|Weave Type
|Rug Type
|2 ft. x 3 ft.
|Pure wool with cotton backing.
|Model Number
I like this rug except the color is not exactly what I wanted but it works ok and is very prettyRead More
I have purchased several of these rugs from Overstock, in different shapes and sizes, over the years. I love both the 100% wool, for entrance-ways, and the wool/silk mix for interior rooms. Beautiful accents to the home and wood flooring. The background color on this one is more a taupe than the muddy gold displayed on my monitor. The true color is much richer.Read More
These rugs are beautiful! They have a soft thick pile that is heaven to walk on and the colors are muted and very nice.Read More
The glitter and glamour of gold is catching the eye as a 2015 home decor trend.
If you want to refresh your home this spring, new pendants and lamps help create a chic, brightening effect.
How to Size Your Room for an Area Rug from Overstock.com. Select the right size rug for any room using these tips. Shop with confidence for the perfect area rug size with help from this guide.
How to Choose an Area Rug for your Bedroom from Overstock.com. Here's how to narrow down your choices and find the area rug that's perfect for your room.
Say goodbye to boring flooring! Transform your home from the ground up with attractive area rugs and eye-catching ottomans. Your space will never be the same once you add these brightening updates.
How to Care for Wool Rugs from Overstock.com. With a gentle approach and a little time, you can keep your wool rugs looking beautiful for many years.
How to Protect Wood Floors with Area Rugs from Overstock.com. The easiest way to protect wood floors from scratches and wear is to decorate with an area rug. This guide covers what you need to know.
Best Area Rugs for Christmas 2014 from Overstock.com. To help get you started on your search for the perfect rug, here are this year's most popular area rug styles. Update your home for the holidays, or give a gift that's a treat for the feet.
Tips on Buying a Persian Rug from Overstock.com. Add classic elegance and rich texture to your home with a Persian rug. Here's what you need to know when you're online shopping for Persian area rugs.
Sole Redemption: 5 Rugs to Make Your Feet Feel Good from Overstock.com. Having the right area rug underfoot can activate energy zones throughout your body.
Tips on Buying Outdoor Rugs from Overstock.com. Here's how to choose the outdoor rugs that are perfect for your home. Complete your patio or deck and save money on the outdoor area rug that's just your style.
Leaves our warehouse in 1-5 business days. * *
Items must be returned in new or unused condition and contain all original materials included with the shipment. More Details
FINAL SALE EXCLUSION: Items marked as FINAL SALE are not returnable unless the problem you experience is the result of our error.
Our standard shipping method is ground shipping. We also offer expedited shipping for most items. Your final
shipping cost and estimated delivery timeframe will be available at checkout.
Standard shipping details
Expedited shipping details
* For your protection, all orders are screened for security purposes. If your order is selected for review, our Loss Prevention Team may contact you by phone or email. There may be a two business day delay to process your order.
** Most Oversize orders are delivered within 1-4 weeks. Some orders may take 6 weeks to be delivered.


text:
The most damning result came when I posed the following statement: "P4P is really just a CMS (Medicare/Medicaid) scheme to save money, wrapped in the flag of quality."
Medicare clearly has some work to do if it cares about getting the buy in from docs. All of this is further proof of the point I made last week: the biggest surprise of the quality revolution is the immense power of transparency, which does not appear to be mediated through consumer-driven pressure. I argued that the pressure generated from public reporting is leading to lots of internal P4P (bonusing based on scorecard results), which may ultimately make payer-generated P4P unnecessary. And if you think the reporting is generating action now, just imagine how powerful it will be when patients actually pay attention to the results! These survey results are also sobering evidence that this group of docs – a group that has been subject to more quality measurement than any other, and, in general, has bought into the quality thing – believes that today's quality measures don't really capture much of importance. Moreover, these physicians, by a wide margin, believe that the "quality revolution" may in reality be a big cost-saving scheme by the payers. The bottom line: everybody's got a lot of work to do if we're going to get this thing right.


text:
Tel: (509)826-5341
School Overview
|School Membership(s)School Assoc.
|General Conference of the Seventh-Day Adventist Church (GCSDAC)
|Religious Affiliation
|Seventh-Day Adventist
|Grades Offered
|Grades 3 - 8
Student Body
|Total Students
|15 students
|Student Body Type
|% Students of Color
State avg.: 27%
|Students by Grade
Academics and Faculty
|Total Classroom Teachers
|1 teachers
|Student : Teacher Ratio
National avg.: 13:1
Nearby Schools:
All Schools | High Schools | Elementary Schools | Preschools | Public Schools
Show me:
- School Location Miles Students Grades
OmakOmak East Elementary School
Po Box 833
Omak, WA 98841
(509)826-3003(Non-sectarian)0.7 mi | 312 students | Gr. 3-5
OkanoganA Is 4 Apple Christian School
Po Box 1690
Okanogan, WA 98840
(509)826-0234(Christian)4.7 mi | 20 students | Gr. PK-1
TonasketPeaceful Valley Christian School
Po Box 1062
Tonasket, WA 98855
(509)486-4345(Seventh-Day Adventist)20 mi | 3 students | Gr. 1-8
BrewsterBrewster Adventist School
115 Valley Rd
Brewster, WA 98812
(509)689-3213(Seventh-Day Adventist)21.4 mi | 26 students | Gr. 1-9
WinthropLittle Star School-montessori
P.o. Box 608
Winthrop, WA 98862
(509)996-2383Montessori School (Non-sectarian)30.1 mi | 67 students | Gr. PK-KG
School Reviews
Area Statistics
Rental Market Monitor
Recent Articles
Updated June 28, 2015
When it comes to choosing a private school, there are so many choices to consider that the project can be a huge challenge to most of us. We offer some help and advice to help you get started.
Sending your child to a foreign language school makes good sense when you have been posted to this country from abroad and in other circumstances as well.
Do you need financial aid? Not sure? Confused by how financial aid works? Here are some answers.


text:
News Releases
ITHACA, NY — "Time Capsule: 2015 Senior Student Show" will open at Ithaca College's Handwerker Gallery on Thursday, April 23, with a reception from 5 to 7 p.m. Running through May 17, the show will highlight the work of students from the Department of Art and the Department of Media Arts, Sciences, and Studies.
Curated by Danica Kelley '16, the...
Opening reception for "Time Capsule: 2015 Senior Student Show," featuring work by students in the Department of Art and the Department of Media Arts, Sciences and Studies; 5–7 p.m., Handwerker Gallery, Gannett Center.
"Massage à Trois:...
ITHACA, NY — Bee pollination is a big deal. Just think about the buzz surrounding colony collapse disorder and the effects it could have on agriculture worldwide. But while honey bees get all the press, there are thousands of other bee species that are just as critical to the continued propagation of flowering plants.
While the loss of these species would no doubt be...
ITHACA, NY — April is National Autism Awareness Month, dedicated to raising awareness about the developmental disability among Americans. But a recent study that examines knowledge about autism spectrum disorder (ASD) among undergraduates in healthcare-related majors shows there is still room for improvement in spreading accurate information.
The study was conducted last year...
ITHACA, NY — Acclaimed playwright Tony Kushner — whose works have engaged some of the most important social and political debates taking place in our culture — will be awarded an honorary Doctor of Letters degree by Ithaca College at the college's Commencement ceremony on May 17.
Kushner is best known for...


text:
Thanks to the Retwords blog for posting my latest poem "Death Row"
Originally posted on RETWORDs:
Death Row by Brendan Stoneham
He's a
Shallow man,
An empty man,
A soon-to-be-dead man,
Innocent-to-the-end man.
Sparks fly-
No one cries.
It's a
Dead body,
Not a nobody-
Just a bag o' bones
Slumped over a chair.


text:
- Description
DetailsThe Italian Pandoro tradition dates back to the early 1920s when a Milanese pastry chef applied a natural levitation technique to a popular 15th century Milanese recipe. This light, fluffy cake powered with a light dusting of confectioners sugar is a taste of Italian tradition. It is sure to be enjoyed by all!
- Additional Information
Product Information
Ingredients Wheat flour, eggs, butter, sugar, milk, yeast, fructose, mono and diglycerides, cocoa butter, salt, artificial flavors Icing: sugar, wheat starch, artificial flavors Storage Keep in a cool, dry place. Allergens Contains eggs, milk, wheat. May contain nuts, soy.


text:
Now Playing
Connect with Us
KRWG.ORG-The Region's Home Page
Director of Content
News Director
Multimedia Reporter
Fronteras Reporter, KRWG-FM and Anchor, KRWG-TV's Fronteras-A Changing America
Operations Manager, KRWG-FM And Anchor, KRWG-TV's Fronteras-A Changing America
Host, KRWG-FM's Fiesta! and PUENTES
KRWG Jazz Host
Host, Friday Night Blues; Saturday Night Jazz
Host, KRWG-FM's Intermezzo
Host, KRWG-FM's Intermezzo
The Two-Way
10:33 am
Sun June 22, 2014
The Rockies' Very Bad Day: When A Wild Pitch Clears The Bases
By Eyder Peralta
We usually point out great plays on this blog.
But during last night's Colorado Rockies vs. Milwaukee Brewers game, the play turned so messy, it is remarkable.
As The Bleacher Report explains, a wild pitch — and a subsequent throwing error — ended up costing the Rockies three runs. It'll probably rank, Bleacher Report surmises, as one of the worst defensive plays this season.
Just watch:
Needless to say, the Rockies lost 9 to 4.
Copyright 2014 NPR. To see more, visit http://www.npr.org/.


text:
Apple Store
Valley Plaza
2701 Ming AvenueBakersfield, CA 93304
(661) 396-3480Driving directions and map
|Store hours:
|Mon - Sat:
|10:00 a.m. - 9:00 p.m.
|11:00 a.m. - 6:00 p.m.
Apple Store
Valley Plaza
2701 Ming AvenueBakersfield, CA 93304
(661) 396-3480
How to get here:View store information
The Apple Store is located in the Valley Plaza mall, in the JC Penney wing opposite Build-A-Bear and next to Guess. Valley Plaza is located on the east side of the 99 at Ming Avenue, just south of the 58/Barstow-Bakersfield highway. Parking: South side garage between JC Penney and Forever 21. Public Transit: Take GET bus lines to Southwest Transit Center at Valley Plaza.
Apple Camp
Free workshops where kids can make their own movies or interactive books.Learn more
Schedule a try-on appointment.
Make an appointment to try
on Apple Watch in an Apple
Retail Store.
Personal Setup
We can help you set up your Apple Watch the way you like. And we'll point out some great apps to help you personalize it.Learn more
The new MacBook
Available to order online. Visit an Apple Retail Store for a hands-on demo.Learn more
View All
Filter by


text:
Article Preview
Local View: Grace in losing is key to a healthy life
Published 08/26/2012, for the Duluth News Tribune
I have had many opportunities to win and to lose in athletics, careers and life. One of the most valuable lessons I learned is how to accept losing gracefully. It is key to living a healthy, caring life.Word count: 267
Log In
The full article is available to newspaper subscribers. If you are a subscriber please log in to continue reading.


text:
Hello and welcome to the coverage from the final of IPL 8 between Chennai Super Kings and Mumbai Indians at Eden Gardens. Chennai Super Kings:... ...http://indiatoday.intoday.in/story/ipl-8-final-mumbai-indians-vs-chennai-super-kings-eden-gardens/1/439979.html
May 20, 2015 00:18 (IST) Pollard : Want to wish Happy birthday to my mom today and my friend's mom as well. Being able to hit sixes is always... ...http://m.firstpost.com/sports/ipl-8-happened-mumbai-indians-outclass-chennai-super-kings-cruise-final-2253146.html
May 20, 2015 00:18 (IST) Pollard : Want to wish Happy birthday to my mom today and my friend's mom as well. Being able to hit sixes is always... ...http://m.firstpost.com/sports/ipl-8-qualifier-mi-vs-csk-live-confident-mumbai-face-tricky-chennai-at-wankhede-2252502.html
Mumbai Indians produced a clinical bowling display to brush aside the might of Sunrisers Hyderabad, winning by nine wickets in a group stage match... ...http://indiatoday.intoday.in/story/ipl-8-sunrisers-hyderabad-vs-mumbai-indians-live-match-56-score/1/438599.html
Preview: With an eye on the all important playoffs, Mumbai Indians and Sunrisers Hyderabad have no other option but to go all out when the two... ...http://m.firstpost.com/sports/ipl-8-srh-vs-mi-live-sunrisers-take-mumbai-indians-decider-2248422.html


text:
When I first moved to Washington, D.C., the first thing that shocked me was the road system. Numbers and letters (I get it – a grid!) but wait – diagonals (huh?) and circles (what?). Most of these roads were designed in 1791 by Pierre Charles L'Enfant (George Washington's drinking buddy) before there were cars and "it was intended as the model for American city planning and a symbol of governmental power to be seen by other nations." Um, fail.
That you drive on Rock Creek Parkway and 17 th street one direction in the morning, and in another direction in the afternoon is a recipe for disaster. Don't get me started on 66, Route 50, or the Beltway.
The second thing that shocked me was the price of everything. Which brings me to my third installment of the arguments against Pilates – its price.
I have no money. I'm a grad student/non-profit employee/associate saddled with law school debt. I live in D.C. – one of the most overpriced cities in the world.
Trust me, we get it. According to a 2011 Kiplinger Report, Washington DC ranks sixth in the most expensive U.S. cities. Rents, averaging $1,790 per month, are more than double the national average. The average home price is $670,675. And that doesn't even take into account the cost of a martini in this town. Or a burger.
But, we don't sell those, so let's talk about the price of Pilates.
The Fuse reformer/tower (jumpboard and push prep) studio. Cost? In terms of your physique and health? Priceless.
First – why it's expensive.
So, it's expensive for a reason. But, let's talk about why it's worth it.
First, you can get most of the benefits of the work just from the mat (the least expensive of our classes). In fact, Joseph Pilates considered the mat work the goal – it features some of the toughest work in the repertoire. I've been doing Pilates forever, and I still think the mat work is the toughest. Those gorgeous brides in a previous post? Kate did exclusively the mat work. At Fuse, you could get into a mat class for less than you spend on your coffee drinks most weeks (or if you have a real coffee problem, less than you spend in a day).
But the equipment classes are really fun and challenging (and target arms, legs, and back a little better), so let's talk about their cost. A chair class is less than you spend on a single dinner out (by yourself), and a reformer class costs less than enough sushi to fill you up. Plus, we have apprentice classes with our teachers completing their training – and those are half-price.
Second, it's a workout that's actually fun. And with as much stress as we have in our daily lives from our bosses, coworkers, city driving, significant others, mothers, mother-in-laws, DC parking enforcement, debt ceilings and threatened government shutdowns, we need a little fun in our lives.
Third, it's extremely effective. Old Joe said himself that with Pilates you can feel a difference in 10 sessions, see one in 20, and have a whole new body in 30. At Fuse, we think our method can speed that up. We've had plenty of students notice a difference even more quickly. If you're going to spend money on a work out, it better work.
Next post, I'll tell you what not doing Pilates can cost you.
Stay hard core,


text:
HP snips Sans costs for SMEs
Hewlett-Packard is aiming to make it easier and cheaper for SMEs to adopt storage area networks (SANs), to enable them to easily...
This Article Covers
Hewlett-Packard is aiming to make it easier and cheaper for SMEs to adopt storage area networks (SANs), to enable...
Continue Reading This Article
Enjoy this article as well as all of our content, including E-Guides, news, tips and more.
By submitting your personal information, you agree to receive emails regarding relevant products and special offers from TechTarget and its partners. You also agree that your personal information may be transferred and processed in the United States, and that you have read and agree to the Terms of Use and the Privacy Policy.
them to easily share their data around their companies.
"The market opportunity for network storage solutions for small businesses will continue to grow as lower prices and easier-to-implement solutions attract new customers," said John McArthur, analyst at IDC.
He said, "Products such as HP's new entry-level network storage solution should be a compelling offering for SME customers and channel partners serving the lower end of the market."
Available direct through HP and its channel partners, the HP StorageWorks Modular Smart Array (MSA) 1000 Small Business SAN Kit is a complete package that costs about £5,395.
HP claimed this package can be deployed by SMEs with little technical expertise "in just minutes".
Each kit includes HP StorageWorks MSA1000, a Fibre Channel disc array with two QLogic SANblade host bus adapters, and an eight-port Fibre Channel QLogic SANbox Switch, all in a single box.
An included installation CD simplifies set up and configuration. Based on the QLogic SANsurfer Small Business SAN Wizard, the installation software enables customers to get the entire SAN up and running in a matter of minutes, said HP.
For more news on hosting click here
Enjoy the benefits of CW+ membership, learn more and join.


text:
This property allows you to book online with a credit card. After submitting a reservation request, the owner or manager has 24 hours to accept, at which time your card will be charged and you will receive an email confirmation
Heritage Lake Park Travel Tips
Come Enjoy the Comfort of Heritage Lake Park Vacation Rentals
Set in Port Charlotte on the western coast of Florida lies Punta Gorda, a gorgeous getaway on the Gulf of Mexico. For the past 38 years, Heritage Lake Park condos have been providing the very best in vacation living. Heritage Lake Park offers the best in vacation real estate with a fun, friendly community feel to round out the bargain.
Staying In
It will be a hard decision to make: get out and explore Florida or relax in the lavish splendor of one of the many Heritage Lake Park vacation rentals available. Each of these sunny chateaus comes fully prepared for guests—and there's no need to worry about maintenance! Heritage Lake Park condos have kitchens filled with all the utensils and appliances needed, and the spacious living rooms and bedrooms offer a comfort that is unbeatable.
Going Out
There's plenty to do in Punta Gorda too. Punta Gorda beach rentals provide opportunities for tennis, golf and cycling, in addition to walking paths and an indoor gym. Located so close to the warm waters of the Gulf, there's always the beckoning hand of snorkeling, diving, waterskiing, and swimming to lure one out of their comfortable vacation home. Punta Gorda boasts bowling alleys for family fun, fabulous malls for the premier shopping experience, even a center for performing arts! When it's dinner time, there's the award-winning Dean's South of the Border, serving up delicious food—and free refills—for 18 years, or the Perfect Caper for a fancy dinner.
Come to Punta Gorda Vacation Rentals
There's Heritage Lake Park vacation rentals just waiting for someone to come and spend a holiday enjoying the Punta Gorda experience. With four airports nearby, it's easy to get there—although you may not want to leave. Reserve a condo today, and get ready to have fun in the sunny surf of Florida.


text:
2012 Girls' Pennsylvania Preview
Updated: March 1, 2012, 9:02 AM ETBy Brett Manney
The Pennsylvania girls might not compete on quite the same level as their male counterparts, but 2012 could see more teams from the Keystone State reach the POWERADE FAB 50 rankings.
Garnet Valley looks to be the favorite again in 2012 and its schedule is deserving of a high national ranking to begin the season.
The Starting Lineup (Position: athlete, year, high school (city, state), college commitment)
A: Mackenzie Rafferty Sr., Downingtown East (Exton, Pa.), Delaware
A: Maddie ...
Read full story
You must be signed in to post a comment
You are fully responsible for the content you post. Content that includes profanity, personal attacks or antisocial behavior (such as "spamming" or "trolling"), or other inappropriate content or material will be removed. We reserve the right to block any user who violates our terms of use, including removing all content posted by that user.


text:
Thanks to double O-ring seals, spring-loaded face-to-face contacts, and neodymium magnets, EZS series connectors withstand harsh environments and accidental disconnection.
EZ Connector Inc.www.ezconnector.com[Booth 4176]
Fleets, Trucks and Accessories
An aluminum liner enables tank to hold 15% to 25% more fuel, which means solid waste...
Hanley Wood, the premier information, media, event, and strategic marketing services...
Product Brief
PUBLIC WORKS Staff
Scalable tool integrates fleet management and field service.
Please read our Content Guidelines before posting.
Copyright ©
2015 Hanley Wood Media, Inc. All rights reserved.


text:
The best product for dry skin I have ever used
Lovely fresh smell
Soaks in immediately
Products lovely - just as I ordered.
Always uses this body lotion. Body Lotion soaks up to your skin very quickly
Will buy more of this product again.
Gorgeous lotion - exactly as described - thnk you for great service and product - will use again!!
Ratings Guide Read about our product rating system