text:
Thursday Talk: Bushy Park - The Next Generation
The Next Generation with Hannah Pritchard and Sophie Bryant Thursday 23 April 2009
Hannah Pritchard and Sophie Bryant gave us a double act as slick as Eric and Ernie, a breath-taking view into the wide range of activities they organize. Appointed in 2007 as Education and Community Engagement Officers, and funded by HLF, they are based at the Stockyard, and cover all of Bushy Park and the Pheasantry Welcome Centre (when it opens). Both have experience as secondary school teachers, and it shows!
Both get involved in schools and higher education colleges, community events such as Bushy Spark (2007), artwork for Age Concern, Scouts and Beavers. With the help of an artist they have had the Big Draw in the park, and run activities each summer holidays such as in art, pond-dipping and meadow-sweeping.
Sophie is responsible for giving talks in primary and nursery schools, and for the long-running scheme which brings primary schoolchildren to the Stockyard several days a week. Assisted by volunteers, children walk a nature trail and pond-dip, later viewing their aquatic catch through a fancy microscope and TV. The pond-dipping room was decorated partly thanks to the Friends, as described in the previous newsletter.
Hannah is responsible for secondary schools, organizing field visits in Bushy Park, projects in schools, and coursework. She may work alongside police in their drive to encourage responsible attitudes.
Bushy is one of only 3 royal parks to have education officers, co-ordinated by Toni Assirati at HQ. You may have seen Sophie and Hannah on Chestnut Sunday, immersed in activities with children. This summer, in addition to all their normal activities, they are involved in 'OPAL'- a link between scientists and the public for useful data collection, a moth count, and 'waste-free picnics'. They will lead the Friends' 'walk for Little Children' on 27th June.
In many years at the Stockyard we haven't seen such an energetic pair or engagement with such a wide section of the public. Inspiring indeed!
Alison Blaney, May 2009


text:
Please take the time to read the news article that I wrote on Associated Content about the sentencing that Murguia received after killing my six year old son, Gaje Florence. I greatly appreciate everyone who had helped us to walk through this long dark road.
I wrote this article so that everyone could stay up to date with what was going on and how the trial was going. Comments are greatly appreciated on the article. Please be sure to check out the resource box on the article for more information to what led to this incident and we greatly appreciate all the prayers that have went out to help us.
Elsie Margiretta Murguia sentenced after death of six year old Gaje Jeffrey Florence

subset: commoncrawl


text:
Final Fantasy will return to mobile game Puzzle & Dragons once again, GungHo Online Entertainment America and Square Enix announced today. Starting from November 28 through December 11, the fan-favorite collaboration will expand upon the previous ones with six new characters, a new dungeon, Ultimate Evolutions and bosses from the mainline series.
The Final Fantasy-themed "Rare Egg Machine" will now include 24 characters including Yuna, Lightning and Noctis. Glauca from Kingsglaive: Final Fantasy XV will also be available exclusively in the MP shop.
The full list of new characters include:
- Onion Knight (Final Fantasy III)
- Tifa (Final Fantasy VII)
- Auron (Final Fantasy X)
- Penelo (Final Fantasy XII)
- Noctis (Final Fantasy XV)
- Glauca (Kingsglaive: Final Fantasy XV)
Additionally, new Ultimate Evolutions have been added for returning character Y'shtola as well as for newcomers Tifa and Noctis. The collaboration will feature a brand new Gift Dungeon, Moogle's Gift and the Fat Chocobo's Gift will be returning as well.
Puzzle & Dragons is now available for iOS, Android and Amazon devices.


text:
First it was reported that Apple was about to go into production with a line of cheaper iPhones. Now it appears Nokia is preparing to tackle the budget end of the market.
The one time undisputed king of the smartphone world, Nokia has now lost out to Samsung as the globe's biggest smartphone company.
While the Finnish multinational has been very much concentrating its efforts on competing with the likes of Samsung's Galaxy range and Apple's iPhone to make more high-end products, it is now looking at expanding its focus.
The firm has, over the past two years, concentrated on developing its Lumia range, launching a top-of-the-range 920 model last November, which has so far proved slow to take off.
According to reports, new Nokia models will be unveiled at the Mobile World Congress in Barcelona. The cut-price phones will be aimed at taking market share from the likes of Huawei and ZTE. A new lower price model of the Lumia is also expected to be revealed at the industry event.
Earlier rumours had suggested that Nokia would also unveil a tablet in Barcelona but, according to sources quoted by news agency Reuters, the company is not quite ready for that yet.
So far, Nokia has refused to comment on any speculation.
But what is clear is Nokia does need a change in strategy in order to remain a major player. The firm, which has a history dating back to 1865, has been struggling to compete with Apple and Samsung's high-end offerings while at the same time losing share to Chinese rivals in the low-end of the market. According to latest figures, sales of basic phones fell by more than 20 per cent last year to 9.4bn euros.
Hakan Wranne, an analyst at Swedbank, said: "What they have to do is increase share in the growing smartphone part and also defend market share in the other. In order to not continue to lose share in the overall market, Nokia has to put forward competitive low-end smartphones.
"What's happening now is that the US and Europe, the big smartphone markets, are really approaching saturation where customers will be upgrading to another smartphone or just replacing one that's broken. That's not a growth market."
Latest statistics show Nokia's market share in the higher-end smartphone business stands at just five per cent, compared to the combined share of Apple and Samsung, at 52 per cent.
While Nokia sold some 4.4m Lumia handsets in the fourth quarter, financial analysts say those sales will have to be more than doubled along with a move to offer more models at the mid-range in order to convince investors that it can remain credible.
Already, Nokia has been working to expand its Asha phones, which give users some access to the internet but which have fewer features than some of the top-range smartphones. The firm hopes its strategy will work by encouraging those who buy a mid-range Nokia to eventually upgrade to a more advanced phone while sticking with the Nokia brand.


text:
Phil Buckenham – Artist
South East UK based comic artist and illustrator. Regularly sketching at conventions. Created his first creator owned book (RTTD) in 2012. A further 3 issues followed with a 4th in development. Had a story featured in the zombie anthology Zombre and had his work featured in Brambletynes Far From Faith.
A relative newcomer to the comic world, we discovered Phil's work at MCM London a few years back. Phil's work on his own comic "Rented to the Dead" really stood out with some fast and loose free rendering that really suited the story. We've already seen what Phil is capable of in the MMF universe and have been blown away by the pages he's already submitted.


text:
15 years ago, I graduated from college. In a complete departure from everything I had ever known, I moved to Boston to live with my sister. Shortly after my arrival, I got a job and met a girl named Caroline. We had an instant bond for some strange reason. About the only thing we had… Continue reading NOW it's weird…
Month: June 2006
One week ago today I moved my entire life: household, pets, children, husband, into a new home, in a new state. We left everything we knew, loved, and treasured in Florida in order to pursue an opportunity of a lifetime. I have met neighbors, have been to 1 party and invited to 2 others already. … Continue reading Moving


text:
The FIA presidential election battle risks turning in to a 'farce' unless the governing body forces support letters obtained by Jean Todt to be revoked, claims his rival David Ward.
As the row over Todt's push for support letters grows ahead of a hearing of the FIA Ethics committee in the next few weeks, Ward has written to each FIA club outlining his view that the current situation is unacceptable.
Due to the requirement of candidates to have support from a number of clubs, Todt's lock down of support from 11 of the 12 North American clubs during a meeting in Montevideo earlier this year means that the fate of the election has been left in El Salvador's hands.
It means that the club could not only prevent any rivals standing against Todt, but also that if it backs a rival there is no opportunity for a third candidate to be put forward.
In the letter, Ward wrote: "In order to be eligible to stand in the FIA presidential election in 2013 a candidate must propose a Vice President for Sport from each of seven regions including North America.
"The written commitments of support that Jean Todt obtained from ASNs and ACNs prior to the beginning of the election process leave only one club in the North American region that is not already formally committed. That club is from El Salvador.
"The absurd situation in which just one club in North America may determine whether or not there can be a challenger to Jean Todt risks turning the FIA election into a farce.
"This is why I am asking for the support letters to be recognised as a clear breach of the FIA's rules and revoked in my complaint to the Ethics Committee."
Ward is keen for a major governance overhaul at the FIA - and is eager for every club to start pushing for debate about how the governing body can be made better.
"In standing as a candidate in the 2013 election my purpose has been to encourage debate about the need for further reforms in the FIA," added Ward.
"I believe there are serious flaws in the FIA's governance system. There are other equally serious problems, for example, regarding the transparency and adequacy of the FIA's financial accounts.
"The FIA deserves better than this. The FIA membership should not turn a blind eye to abuse of the FIA election system or tolerate deficient governance provisions in the sport that are not accepted in the mobility pillar."


text:
FOOD SHELF OPENThe
Aurora -Hoyt Lakes-Biwabik-Palo provides emergency food assistance to
households in need. We are an area-based food shelf that provides food
for all people in need no matter what race, color, religion, sex, or
national origin. Donations of food or money from local individuals,
organizations, and businesses help us maintain our service.10:00am - 1:00pm
Wholesome tasty meals are served free of charge to everyone
every Tuesday and Thursday. Donations accepted but never expected.
No income limits for participants.
Neighbors helping neighbors
Food Shelf
PO Box 2761
Warba, MN 55793
Phone: (218) 492-4392
THE COOK AREA FOOD SHELF is located behind St. Mary's Church.
It is open the third Wednesday of each month, 9-1. Donations
always welcome. Containers for donations at most local churches
or write: Cook Area Food Shelf, P.O. Box 633, Cook, MN 55723.
Duluth and around includes north st lous countyhttp://northernlakesfoodbank.org/http://northernlakesfoodbank.org/get-help/pdf listings!!http://northernlakesfoodbank.org/docs/nostlouis.pdfNorth Westernhttp://www.northcountryfoodbank.org/gethelp.phpNorth Central MN Food Bankhttp://secondharvestncfb.com/GetHelp/FindAnAgency.aspxhttp://secondharvestncfb.comecond Harvest Food Bank has been on the cutting edge of food banking operations for over 20 years. The diversity of programs offered is unique among small rural food banks. For example:http://secondharvestncfb.com/Programs/FoodPrograms/GrandRapidsFoodShelf.aspx• Grand Rapids Food Shelf• Kid's Packs to Go Backpack and Kids Cafe program• Commodity Distribution Program for children and seniors (MAC & NAPS)• Mobile Pantry Program• Itasca Holiday Program=================The Grand Rapids Food Shelf hours are:its in LaPrairie, main second harvist bldg25000 sq ft.Monday through Thursday from 11:00am to 3:30pmFridays from 10:00am until 12:00pm*First Tuesday of the month 11:00am to 5:30pm========================Fall Hunger Coalition Inc1000 5th Street, International Falls, MN(218) 283-8020 ?"Falls Hunger Coalition - If you need food or other nutritional support, this charity organization can help. The non-profit organization runs a food ..." - needhelppayingbills.comEldercircle RSVP2232 2nd Avenue East, Internatl Falls, MN(218) 283-6467 ? · eldercircle.org"Hunger/nutrition (preparing and serving meals, work at food shelves and food banks, shop for and deliver groceries to homebound seniors, deliver ..." - eldercircle.orgFood Shelf3 S Broadway Ave, Gilbert, MN(218) 741-1371Ely Area Food Shelf102 East Sheridan Street, Ely, MN(218) 235-8527 ? · elyareafoodshelf.orgFood Shelf3 S Broadway Ave, Gilbert, MN(218) 741-1371 ?United Methodist Church-AuroraAurora, Minnesota(218) 229-2113 ?"The team helps to support the Aurora Area Food Shelf, Project Elf (local school program to buy Christmas presents for local kids in need), Range Women's ..." - aurorahoytlakesumc.orgQueen of Peace Rectory315 Cty Highway 110, Hoyt Lakes, MN(218) 225-2867 ?"Jacobson Food Shelf. Churches in JacobsChisholm Food Shelf10 Central Ave NE, Chisholm, MN(218) 254-7308 ?Hibbing Food Shelf1910 1st Avenue, Hibbing, MN(218) 262-1729 ?Ely Area Food Shelf102 East Sheridan Street, Ely, MN(218) 235-8527 ? · elyareafoodshelf.orgDynamic Insurance112 Central Avenue, Nashwauk, MN(218) 885-3030 ?"Don't forget to bring a donation for the food shelf to the Halloween Party at the Nashwauk Rec. Center. We will be accepting non-perishable food and ..." - facebook.com"Bigfork: Bonnie Cole 306 Golf Course Ln. # 7. Bigfork, Minnesota 56628. Nashwauk: Open Hands, Open Heart Food Shelf 119 Second Street Nashwauk, Minnesota 55769" - unitedwayof1000lakes.orgSalvation Army20734 US Highway 169, Grand Rapids, MN(218) 326-4847 ? · salvationarmy.org"Grand Rapids Salvation Army. Food Shelf" - secondharvestncfb.com
The Ely Area Food Shelf will be wereopen the third Tuesday, Nov. 15th
12:00 to 6:00, lower level of the Frandsen Bank & Trust Bldg.,
(previously Queen City Federal), 1st Ave. entrance. Open at 11:00 for
shoppers with special needs & for other shoppers to pick up their
number to shop later. New shoppers register when they come. For more
info call 235-8527 or go to www.elyareafoodshelf.org.
•Deer River Foodshelf, open 10 a.m. to 1 p.m., Goodall Resource Center, 1006 Comstock Drive, 246-2500.
•Northern Itasca Food Shelf, open 12:30 -3 p.m. on the side of the Over 50 Club on Main Street in Bigfork.
ruby's pantry
Ruby's Pantry is not a govenment program. It is
self-funded by the those who particiapate.
Community Partners are churches, food shelves and organizations who
Ruby's Pantry provides food and goods without cost
The "Blessing Box" concept is not complicated. It is
simply churches and organizations in communities picking up a variety of
groceries from Ruby's Pantry and storing them in manageable amounts in a closet
or room in their facility. Most will have a refrigerator and freezer as
well because so much of today's groceries are frozen and refrigerated.
When someone in the church or organization hears of someone in the
community with low resources or someone in crisis who could use a
helping hand a Blessing Box is packed by the volunteers from the church
or organization and is delivered to the door of the person or family in
need, no strings attached. The positive result of this program is two
Blessing Boxes are changing people's lives!
•Bowstring Town Hall Dining Program. Phone 832-3478 Liz to reserve meal.
The Salvation Army Supper Club
volunteers serves free hot meals for the needy each day Monday thru
Friday from 4-5 p.m. at the Salvation Army Church, 705 12th Ave.W. in
Virginia. For more info call 741-1889.
The Ely Area Food Shelf will be
open the third Tuesday, Nov. 15th from 12:00 to 6:00, lower level of the
Frandsen Bank & Trust Bldg., (previously Queen City Federal), 1st
Ave. entrance. Open at 11:00 for shoppers with special needs & for
other shoppers to pick up their number to shop later. New shoppers
register when they come. For more info call 235-8527 or go to www.elyareafoodshelf.org.
The Loan Closet - located next to True Care Medical Therapy Center.
To donate or borrow medical equipment call 666-2697 or stop in
at True Care. tfnc
Salvation Army Thrift StoreThe Salvation Army Thrift Store Hours have recently changed. They are now open Monday - Thursday 10 am - 6 pm, Friday - 10 am - 5 pm, and Saturday - 10 am - 3 pm. Every Saturday is the Red Bag Sale. Purchase a Red Bag for $3.00 and then fill it with clothing for $3.00 more. You can also bring in your pre-purchased Red Bag and fill it for $3.00. The Salvation Army Thrift Store is in need of furniture
Salvation army
WeeCare Hope Chest (free children's clothing) open 9-11 a.m. at Deer River Church of God, 304 4th Ave. SE.
Mesabi Humane Society/Range Animal Center, 2305 Southern Dr.,
Virginia, an inspected/approved facility for stray/unwanted animals.
Visitors welcome to see animals up for adoption: Mon.-Sat. 10
a.m.-4:30 p.m. Closed Sun. & holidays. 218-741-7425. tfnc
Project Care free clinic is now open in Ely 2 evenings
per month on the 1st and 3rd Tuesday's from 5:30-7 pm, 232 W. Sheridan
street. We offer free health care to those who have inadequate health
care coverage. For more information you can call the main office at
218-263-8549 or come to clinic. A picture ID is required but
verification of income is not. Donations to Project Care are gratefully
accepted and will be used to help with general operating expenses and
our medication assistance program. Donations can be mailed to: Project
Care, 3112 6th ave. E., Hibbing, MN 55746....
from ely echo classifieds 1-13
ELY LEGION MEMBERS: If you know of an Ely Legion member
who is sick or injured, please call Post 248 at 235-3627. Leave message.
Energy Efficient Ely and The Tuesday Group help those in
need once. There is a list of volunteers who are ready and waiting to do
things for people who cannot do on their own. If you are physically
unable to get jobs done and would like a little help around your home
please call Piragis Northwoods Co. at 365-6745 and let us know. Piragis
is serving as the home base for Help for Ely and we will find a
volunteer who can help. Jobs might include getting recycling to the
recycling center, moving some furniture that is too heavy or a ride to
the clinic or grocery store. There are about 20 volunteers in Help for
Ely and over the past year we have been called upon over 25 occasions.
We here to help so please do not hesitate to call us.....


text:
2 Bedroom Family Home in Mississauga! (Dixie & Dundas)
Address: 350-3055 Tomken Rd, Mississauga, ON, L4Y 3X9
Property SummaryBeautiful two-storey 2 bedroom stacked townhome near Tomken and Dundas! Laminate hardwood flooring throughout, large master bedroom with over-sized closets, ensuite laundry, spacious living/dining room with walk-out to large covered terrace--perfect for entertaining or simply relaxing with the family!
Building amenities include secure entry with gated common area, party/meeting room, games room, indoor pool and sauna. Located in the Dixie/Applewood Heights neighbourhood and close to schools, shopping, transit, malls, highways QEW/427 and much more!
Available immediately. Rent is $1,900. Water and cable included. Hydro is extra. One underground parking spot included, more parking spots available through building management for $40/month. Credit check, proof of income and tenant insurance required. Please contact Neda at 647-495-6892, ext. 104 for details.


text:
Located on a scenic hillside just outside city limits of Decorah, Iowa (about 30 miles north of West Union, Iowa,) Silvercrest Golf and Country Club provides a spectacular view of the Decorah area. The course boasts nine holes of picturesque fairways and challenging greens, providing an enjoyable golf experience for all ages.
The clubhouse boasts the coldest beverages in town. Their kitchen and dining facilities are available for your special events.


text:
Ulrich Klieber: Zur künstlerischen Lehre
Neue Aufgaben finden / Workshops … more
Design Fundamentals
Burg Giebichenstein University of Art and Design Halle
University Library
P.O. Box 200252
06108 Halle
Telephone: +49(0)345 7751-637
Fax: +49(0)345 7751-638
E-Mail: verlag(at)burg-halle.de
The applicable costs for postage must be added to the prices quoted.An invoice will be included with the shipment.
Discounts on publications are available to members of the Society of Friends and Partners of the University and to students upon request.


text:
No. 5 Softball Pounds Out 12 Hits En Route To 9-0 Win Over Georgia Tech
March 10, 2001
TAMPA, Fla. - The No. 5 Stanford softball team used an explosive offense to defeat Georgia Tech 9-0 in six innings on Saturday morning in the final pool play game of the Speedline Invitational, hosted by the University of South Florida, at Greco Complex.
The Cardinal won its pool (A) by going 4-1 and will play tonight at 7:00 p.m. (EST) against the winner of the second-place C team and the third-place B team. The win improves Stanford's record to 25-2-1 while the Yellow Jackets fall to 12-10.
Stanford got off to a fast start in the bottom of the first inning, posting two runs on three hits. Robin Walker led off with a walk before the Cardinal had a string of three base hits. Jessica Mendoza singled through the left side and Sarah Beeson drove in two runs with her single up the middle. Kira Ching also singled through the left side.
Dana Soresnsen took a perfect game into the fourth inning before the Jackets' leadoff hitter got a base hit up the middle. She allowed one other hit in the game while striking out 11 and walking none and improves to 13-1 on the year. She is also 3-0 on the weekend with three complete game shutouts.
The Cardinal extended the lead in the third inning when Beeson reached third base on an error by the center fielder. Ching scored her on a single to centerfield and Jessica Allister also picked up a base hit. Ching would later score on a ground out by Cassi Brangham for a 4-0 Stanford lead.
Jenni Shideler drove in two more runs for a 6-0 lead with a clutch, two-out single to left in the fifth. She scored Jessica Draemel (pinch running for Allister) and Brangham, who both reached on walks.
In the sixth, Mendoza hit her ninth home run of the season, and second of the tournament, giving the Cardinal a 7-0 lead. Beeson followed with a base hit, which made way for Ching's game-ending homer (eight-run rule after five innings). After fouling off several pitches from Tech's starter, Erin Voeltz, Ching's line drive shot to left center gave the Cardinal its 17th shutout win of the year.
The Cardinal bats were alive in the game as Stanford collected 12 hits. Ching led the way with her 3-for-4 outing with three RBI and two runs. Mendoza and Beeson each had a pair of hits while Shideler and Ramona Shelbure both went 2-for-3.
Score by Innings 1 2 3 4 5 6 R H EGeorgia Tech 0 0 0 0 0 0 0 2 2#5 Stanford 2 0 2 0 2 3 9 12 0
WP - Dana Sorensen (13-1) and Jessica Allister. LP - Erin Voeltz (5-6) and Erin Glantz. Save - None. 2B - Jenni Shideler (STAN). 3B - None. HR - Jessica Mendoza (STAN), Kira Ching (STAN). E - Sara Wissmann (GTU) 2. LOB - Georgia Tech 1, Stanford 6. Records: Georgia Tech 12-10, Stanford 25-2-1.
Pac-12 PlaylistThu 4/2 | 7:00pm PTLive
Pac-12 PlaylistMon 4/6 | 7:00pm PTLive
Pac-12 PlaylistThu 4/9 | 7:00pm PTLive


text:
Support a unique innovation and entrepreneurship program to empower disadvantaged youth and help them become young entrepreneurs.
This is your chance to help young people in Lebanon build a global network and give them the opportunities they need to develop their skills and start their own businesses as young entrepreneurs.
With Companies for Good, you have the opportunity to support these start-up businesses financially. But you can also send a team to Lebanon to share their experience and skills with the young entrepreneurs. It's a really valuable experience for your employees – and life-changing for these young people.
"Social Entrepreneurs are not content just to give a fish or teach how to fish. They will not rest until they have revolutionized the fishing industry."
– Bill Drayton – Ashoka
The event lasts two days. Your team will be well looked-after throughout their stay and given all the information they to run successful sessions with the young people.
If your company believes in youth and thinks we all deserve a chance to achieve our potential, this is the right CSR initiative for you. You won't just give young people a renewed sense of purpose, you'll give your team members a chance to build valuable communication and leadership skills.
|Beirut, Lebanon [indoor / outdoor]
|All year round [weekdays and weekends]
|Participants and prices
1 - 10 People (7,400Dhs)
|* Prices include event planning and coordination, support staff and a contribution to the charity partner when applicable, as well as pictures from the activity for use in your own communications (e.g. internal, social, website), an activity impact report. Prices exclude Companies for Good management fee. Prices exclude VAT.


text:
Weighed in at 262 again. I've reached the point in my weight-loss where there won't be a loss every day! Might even be a temporary fluctuation up. Ok.
Especially since I had a little vacation yesterday. I didn't deserve it diet-wise but I deserved it life-wise. And it was a good chance to try to stick to my diet resolve while off-program.
So in the morning as I started my road trip, not wanting to waste any time, I had an egg mcmuffin from McDonald's and coffee with a little milk. That was my fuel for a too-brief hike around the summit of Mt. Mitchell. I say too-brief but it did get my heart rate up and burn my thighs a little with the climbing on the rough trail. I am so terribly out-of-shape for what I'm used to.
Then I went to Asheville and stopped in a Starbuck's to connect with the Internet and chillax. Had a grande mocha light there. By then it was practically the dinner hour and I'd only had about 500 calories, which allowed me 1100 for dinner. I wanted to have a bit of an exquisite Asheville dining experience. It could have been nearly anything - pizza, Asian, whatever. I had duck breast with risotto. The menu said there would be butternut squash - I didn't see any so maybe I misread. I get concerned that I'm not eating enough vegetables. I think they contribute to happiness and a sense of well-being. I also had a glass of wine. Since I have no idea what was in that risotto, I can't do a calorie count for the day. But since that was the end of my caloric intake for the day except for a shot of amaretto and a bite-sized laffy taffy, I think I stayed within my goals.
However I still have 12 pounds left to lose in the next 7 weeks, which is thoroughly possible but my weight-loss honeymoon is over and I must remain vigilant.
That honeymoon period, all that hunger, is over and now I have a reduced expectation for my calories. Even then I could see that I had built my appetite back up to a higher level. Definitely lots of sweets. Now practically none. When cookies and brownies are placed before me, I pass them over. My stomach feels pangs but doesn't want to eat. There were times over the past 10 days I thought I was being somewhat anorexic, though I never ate less than 1000 calories per day.
What I have to do now, and keep postponing, is plan for my next week's eating. I need to plan meals and by groceries. I want to make a soup. I have to move over the next week, and renovate the room I'm going to be moving into. That's going to make things tough.
What will also make things tough is that I'm moving in with my parents. They have their own eating style and grocery plan. They buy in bulk from Costco, lots of canned food and chocolate puddings and sweets (for Dad, mostly). I'm not sure they ever do much with food prep, just reach into the cabinet and grab something. It could be that I contribute a more satisfying food situation for them and maybe that could help bring my rent or utility payment down. As long as I'm cooking anyway, maybe make meals for all three instead of just me. Would that be more economical for me? Not sure how it will work out.


text:
- 19, avenue de Tourville 75007 Paris, France
- Contact Us
The user of the website acknowledges having the skills and means necessary to access and use this site. He also acknowledges having read this legal notice and agrees to comply with it.
In accordance with the provisions of the law of 6 January 1978 relating to computers, files and freedoms, the automatic processing of personal data made from the website has been the subject of a declaration to the National Commission Computer Science and Liberties (CNIL).
The user is informed that during his visits to the site, a cookie may be installed automatically on his browser.A cookie is an element that does not identify the user but is used to record information about the navigation of it on the website. The user may disable this cookie through the settings contained within its browser.
The user has a right to access, modify, rectify and delete data concerning him (article 34 of the law "Informatique et Libertés"). To exercise this right, the user may contact the Director of the publication referred to above.
Any total or partial representation of this site by any means whatsoever without the express permission of the operator of the website is prohibited and constitutes an infringement punishable by Article L 335-2 and following of the Code of Intellectual Property.
The same applies to any databases on the website that are protected by the provisions of the law of 11 July 1998 transposing in the Intellectual Property Code (CPI) the European directive of 11 March 1996 on the legal protection of databases.
The trademarks of the operator of the website and its partners, as well as the logos appearing on the site are trademarks (semi-figurative or not) registered.
The hypertext links set up as part of the website to other resources on the Internet network, and especially to its partners have been the subject of a prior express written authorization.
Users visiting the website can not set up a hyperlink to these sites without the express prior consent of the operator of the website.
Cookies are small text files stored in your browser when you visit our site subject to the setting of your browser and will be used to recognize your device when you connect to our site to:
1. To establish statistics and counts of frequentation and use of the various sections and contents of our site in order to carry out studies to improve the contents
2. To adapt advertising content to your centers of interest and, possibly, your locality, which result from the browsing data of your terminal among the sections of our site without us having personal data to identify you or to contact you directly.
In accordance with the Data Protection Act of January 6, 1978 amended by the law of August 6, 2004, you have rights of access, rectification and opposition to personal data about you.
Regarding the data collected via the contact form: the rights can be exercised as well directly with the advertiser.
19, avenue de Tourville
75007 Paris France
PCNET Services Agence Web
44500 LA BAULE , France


text:
Continue reading "Our Darlings Rest Amongst the Flowers that Bloometh Over There"
Our Darlings Rest Amongst the Flowers that Bloometh Over There
Mourning images from my collection.
Forgotten Lives Behind the Photographs and Artifacts
Mourning images from my collection.
She was christened Anna Martha Bell, but she was always known as "Mattie." The baby girl was born 9 July, 1857, in Erie, Miami County, Indiana to a father with the unusual name Pleasant Lilly Bell (1809-1882). According to his 20 July, 1879, obituary, "Mr. Bell was born in [Vevay,] Switzerland County [Indiana] in 1814, two years before the admission of Indiana to the sisterhood of states. He came to this part of the state [Miami County] when yet a young man and worked on the Wabash & Erie Canal which the state was then constructing. He was a resident of Miami for more than 40 years. His reputation was spotless and he was in high esteem by all who knew him."
Pleasant was the son of Armiger Lilly Bell (1771-1816) and his wife Sarah Blackford (1779-1848). Armiger Bell was born in Fluvanna, Virginia, 10 January, 1771. He was the third youngest of a dozen children. The Bell family was large, well off, and owned land and slaves. Armiger later sailed down the Ohio River to Kentucky, meeting his future wife Sarah, and married her on 31 March, 1795. The couple settled near Vevey and took up farming in what was then a heavily forested area.
After Armiger's death on 5 November, 1816, his eldest son James took over the farm, until his mother remarried in 1821 and his new stepfather took over from her son. Her second husband, John White, appears to have been abusive and volatile. Ultimately, he mysteriously vanished while taking a herd of hogs to market. Sarah eventually came to live with her son Pleasant and his family. She died in 1848 and is buried in the Tillett Cemetery.
When Hannah McCracken Kelly died in 1855, she left two small children who would retain no memory of her and possess no photographic image other than this postmortem daguerreotype.
Hannah B. McCracken was the daughter of John and Mary McCracken (or Mecracken), who farmed in Claysville, Washington County, Pennsylvania, during the early 19th Century. Named after the "Great Compromiser" U.S. Senator Henry Clay (1777-1852), the town is located on the line of the Cumberland Road which forms its Main Street. Claysville is 18 miles east of Wheeling, West Virginia, and 10 miles west of Washington, Pennsylvania. The town was laid out in 1817 and remained unincorporated until 1832.
John McCracken was born about 1795 in Pennsylvania and died 28 December, 1865, in Claysville. His wife, Mary, the daughter of Samuel Caldwell of Buffalo Township, was born in about 1797 and died 4 August, 1878. The couple married in Washington County on 30 December, 1820. They are buried together in the old Purviance Cemetery, Claysville.
In the second half of the 19th Century, at the height of the Victorian Age, the union of photography and the supernatural spawned strange and enthralling results.
By Beverly Wilgus and Ann Longmore-Etheridge
Early in the practice of the photography, ghostlike images appeared on daguerreotypes, ambrotypes, and tintypes. The explanation for them was not supernatural: Because of the need for long exposures, in some cases of more than a minute, anything that moved whilst the camera lens was open went either unrecorded or appeared transparent in the final product. In 1856, Sir David Brewster (1781-1868), an important figure in photography's evolution, described in his book The Stereoscope: Its History, Theory, and Construction the method by which amusing extras could be created in photographs. Brewster advised that sitters should be posed and after the majority of the plate exposure was finished, a new person should move into the scene and stay for the final seconds. This would result in a "spirit" presence.
Whilst Brewster clearly promoted this method for what it was—a trick—others with an interest in the expanding religion of Spiritualism saw ghost images as proof of life after death. It should be noted that the Victorians were not the only folk taken in by the callow exploitation of technology they did not fully understand.
By University of Western Ontario
Art curators will be able to recover images on daguerreotypes, the earliest form of photography that used silver plates, after a team of scientists led by Western University learned how to use light to see through degradation that has occurred over time.
Research published in June 2018 in Scientific Reports—Nature includes two images from the National Gallery of Canada's photography research unit that show photographs that were taken, perhaps as early as 1850, but were no longer visible because of tarnish and other damage. The retrieved images, one of a woman and the other of a man, were beyond recognition.
Continue reading "Hidden Behind Time: A New Way to Recapture Lost Images"
"Some of my best friends have told me that I need not expect to be happy anymore unless I would marry again and have advised me to do so."
Myersville, July 10th, 1852
Dear Emeline,
I hope you will not think hard of me for thus approaching you so unexpectedly, as my mind has bin [sic] for some time a good deal taken up with you in regard to coming to see you in order to have some private conversations with you, not knowing at all whether my company would be agreeable or not, but take this plan of ascertaining something about the state of your mind.
Dear Emma, you are well acquainted with me and know all about my situation. You know that I have bin unfortunate in the loss of a very dear and kind companion, one in who my whole soul delighted to honor and respect. But she has gone I trust from a world of trouble and sorrow to one of happiness and joy, and I can have no more comfort nor consolation from her anymore, only with a firm hope and expectation of meeting her again in those blissful regions where parting shall be no more. I can do no more than to respect her memory, which I will ever do.
We read in the Bible that it is not good for man to be alone. I have realized that to be a very true saying indeed. I was once as happy as any person could be in the enjoyment in the company of my much-lamented Mary, but how different my case. With all I have I have no enjoyment & some of my best friends have told me that I need not expect to be happy anymore unless I would marry again and have advised me to do so, although I do not wish to do so for some time yet. But I have come to the conclusion to do so providing I can suit myself. I now feel like a lost sheep, lonely and without anyone to cheer me or comfort me, and if it was not for the comforts and the consolations of religion, I would often times have to despair in sorrow. But thanks be to God that he still comforts and consoles me. I find that I can never be happy again in this world without fixing my affection on one again in who I am satisfied will be a kind companion to me, and dear Emeline, you appear to the only one I can have any idea of going to see at the present and of fixing my affection upon.
I own just a few early photographs of African-Americans, for they are scarce and much sought after. I present them with love.


text:
2020 D.I.Y. Music Video Materials + Studio Space
Hey, I'm Jess! I'm making D.I.Y. Music Videos in 2020. If you're wondering what D.I.Y. means, it's this:
I write the songs
I produce the songs
I mix the songs
I design the video
I build a performance
I film the video
I edit the video
Yeah, it takes a lot of time. Yeah, I do it for free. But yeah, I believe I create something of value. Art is the thing that moves us, changes us, and inspires us to do better. So...I would like to offer something of value on a regular basis. Maybe once a month, maybe twice a month. And if you'd be excited to pitch in 5 dollars here and there if you happen to be moved by my work, you will help cover materials and studio space so I can continue making more!
Here's a little more about my video making history, and why it's so important for me to create this year:
I never considered myself to be a visual artist, but for some reason, I have really strong opinions on how I want things to look. I love color. I'm really in to ordered chaos. I also remember a much younger version of me hanging My Little Pony's on strings around my bedroom as a kid (which, if you've seen any of my earlier work, tracks).
I started doing videos like this about 5 years ago when I realized that I was never going to have a budget to produce real music videos or hire a full band...and it was the happiest I've ever been!
Then it got weird when people in power thought they could make money off of them. When people I loved said they only liked when I made music that made them feel good. When I was coached into believing that relevancy and attention was the only thing that would make my videos matter.
And then I got weird, because I tried too hard to make all of this happen - to get financial support, to fulfill that need for "positive content", to get noticed, etc. (And we all know how well the world responds when we try too hard, yeah?).
As a result, my art, angry at me for doubting it and trying to use it to GET something, hid away for a long time. Losing my art was losing my soul, my sense of self, and my faith in everything.
It took a lot of work, time, and therapy, but I got it back...and this time, I'm never letting it go.
So this year, I'm just gonna do it for me. No ulterior motives. Art for art's sake! Hey - if my art ends up moving you to buy my music, take a lesson with me, commission a song, or book my nonprofit, that's wonderful. But in my mind, there is now a clear distinction from the art that is ME, and the businesses that I have built around myself to support it.
In the midst of all this, I'm going to be moving to New York with my partner, who has work out there. I'd love to be able to pursue my own work, and a big part of that will be investing in materials for these videos and a studio space where I can teach voice and songwriting lessons while I continue to build these videos!
This gofundme will be up all year. If we end up exceeding my materials budget for videos of $2000 (oh my goodness THANK YOU!) any other funds will go towards studio space (May-December $4000...oh my goodness THANK YOU!).
10 percent of funds raised will go directly towards bringing The Major Lift, a nonprofit that helps people find positivity and resilience through songwriting, to a community in need for free. www.themajorlift.org
At the heart of it I just wanna make art without any strings attached...and no matter what, I'm gonna make it happen at all costs. It would just be amazing if it were...less cost :).
You're amazing. Thank you.
Donations ()
- Anonymous
- Keaton W
- Marianne Walker
- Brad Miller
- Anonymous
#1 fundraising platform
People have raised more money on GoFundMe than anywhere else. Learn more
GoFundMe Guarantee
In the rare case that something isn't right, we will refund your donation. Learn more
Expert advice, 24/7
Contact us with your questions and we'll answer, day or night. Learn more


text:
Coverage from Callaway - Seniors Teaching Students
In this week's Coverage from Callaway, some mid-Missouri college students learn life's lessons from people who know best because they have lived them. And, by learning those life lessons, students gain more than just a passing grade.
Westminster College students thought they were working on just another assigment. They never thought their classwork would improve their lives and those of some Fulton senior citizens.
Two generations inhabit this area, divided by a street. The college is on one side of 4th Street, and the Fulton Senior Center is on the other. Each generation tended to stay on its own side, until a Westminster English class brought students across the street to help seniors write their life stories.
"We're talking about autobiography theory, and we're reading a lot of autobiographies," explained student Candice Crawform. "And also, it gives us a chance to learn about how we're writing an autobiography for another person."
And seniors are great subjects for students' papers.
"I like when students understand that people have a long history, and they have a lot of great stories," said Carolyn Perry, head of the English department. "And it's helpful if we all have time to think back on our lives, but you need some age to do that."
Dorotha Jones Meinhardt dug out dusty pictures to reminisce about her past.
"I enjoy visiting with people," she said. "You know, when you get old, you don't get to talk all the time. And I can talk, that's one thing I can do."
Seniors interact everyday with people their age. But, a younger generation can add a sense of excitement.
Dr. Steven Zweig, a professor of family and community medicine at the University of Missouri, said such cross-generational interaction helps both groups.
"The value of bringing people together from very different generations like that is that they can learn a lot from each other and still appreciate each other," he said. "And I think that that is a particularly satisfying thing for both generations."
Zweig said seniors benefit by enhancing socialization, stimulating learning, increasing emotional support, and improving their health. He said young adults and children benefit by enhancing social skills, improving academic performance, decreasing drug use and increasing stability.
Students will give seniors their own copies of the finished project. Then, seniors can make more copies for their relatives.


text:
Course summary
Book Course
Every organisation inevitably goes through change which needs to be managed and controlled efficiently. This is done by utilising a set of techniques and approaches that will prepare and support the organisation and the individuals within it through the change.
To ensure that the change is successful, there needs to be a smooth transition during change initiatives and certain actions need to be taken to ensure that the necessary support is in place when the change is implemented.
The Change Management Foundation course is geared towards equipping you with the skills and knowledge that is needed to apply these techniques as your organisation progresses through change.
This course provides:
What you will learn
This course will teach you:
Why study online?
Many students prefer online study to conventional classroom-based learning as it provides them with the freedom to learn at their own pace and in an environment in which they are comfortable.
Online study material can also be viewed and revised as many times as the student needs, which contributes to long-term memory retention.
These factors, in addition to the fact that online training courses are often more cost-effective than the on-site alternative, make online study an appealing option to students today.
Change Management Foundation Curriculum
Module 1: Change and the Individual
Module 2: Change and the Organisation
Module 3: Stakeholder Strategy and Communications
Module 4: Change Management Practices
Who is this course for?
Anyone hoping to start a career as a Change Manager or who would like to learn more about managing change within an organisation.
Career path
ITonlinelearning specialises in providing individual professional certification courses along with career and recruitment services to aid you to develop or start a career in your chosen profession. We cater mainly for the Project Management and IT sector but have also...
We believe that it should be easy for you to find and compare training courses.
Our Customer Outreach Award is presented to trusted providers who are excellent at responding to enquiries, making your search quicker, more efficient and easier, too.


text:
Last week, "Little STRUCTUREbags" had a day off from school. So we packed a lunch, got an early start and enjoyed a long afternoon at a local farm having a month-long Fall Festival!
Brookshire Farms is located on the outskirts of San Luis Obispo, off Los Osos Valley Road. I found it by chance, on a recent business trip to Los Osos.
Little and I were looking forward to choosing a pumpkin. And when there is a little in the house, there is something special about skipping the pumpkins at the local market and heading out to a farm.
From the beautiful displays to the creative play options, from the traditional hay ride to the clever 3.5 mile corn maze, we were delighted! Of course the huge and abundant bounce houses were an added bonus for Little...
We got started doing what every family will do - we snapped a pic to measure our "corn."
Even my little one loved the creativity Brookshire showed in their displays. We saw a beautiful mix of gourds and pumpkins and rusty ol' tools...
Yes, I wanted to buy the entire display - make that displays - and try to find a way to show them off on our compact patio...
Pumpkins we wanted and pumpkins we got! These were so vibrant in color and so beautifully unblemished!
And we both had a good laugh and "ewww" and "cool" and these unusual pumpkins! Have you ever seen these before? I hadn't. What fun!
The maze was a mix of fun and work. We took our time, brought our lunch and Little rode most of the way on a fat-tired wagon. Mama was tired at the end, but not before we made our way through and managed to punch many of the "Mazopoly" pieces along the way.
The hay ride was the perfect follow-up to the long trek through the corn maze. They even place burlap sacks over the hay bales and pass a cow bell around for the kids to ring.
Spotted on the hay ride: organic popping corn ready to harvest. I'll admit, I had no idea that "popping corn" was different than regular corn. Live and learn!
A few last adventures before we go. Little enjoyed several of their creative tire swings.
Yee Haw! Roping a bull!
We loved our trip to Brookshire Farms - and even can say it was worth the at-first-glance-steep admission fee. It was clean, friendly and offered a full afternoon of clean, safe, family fun.
Does your town have a great little fall destination? Do you have a traditional Fall Festival? What makes it great?


text:
14K Gold Tiny Small 1.2mm Round 10mm Thin Lightweight Unisex Endless Hoop Earrings, Set of 3 Pairs
- FEATURE - These fashion hoop jewelry earrings display 3 pairs of 10mm high polished endless hoop earrings. They can be used as a hoop nose ring, hoop belly button rings, hoop body jewelry, hoop cartilage earrings, lip hoop rings, eyebrow hoop rings and hoop piercing earrings. This earrings set should be an addition to your fine jewelry or fashion jewelry collection. It can be purchased as hoop earrings for men, hoop earrings for women, hoop earrings for adults or hoop earrings for teen girls.
- CRAFTED - The endless hoop earrings are created of fine 14k yellow gold. It is tarnish free. NOTE: Closure is a bit tight; it will adjust after a couple of uses.
- SATISFACTION - We offer a 30-day satisfaction money back guarantee. If you're not satisfied for any reason, you will receive a full refund upon returning your purchase within 30 days.
- PACKAGING - Jewelry packaged with a nice pouch ready for gifting.
- ABOUT - Our jewelry collection is an assortment of hand selected fine and fashion jewelry. We carry an expansive selection of gemstones, cubic zirconia and precious metals. This includes a variety of pendant necklaces, dangle, drop and stud earrings, hoop earrings, tennis and line bracelets, rings, polished jewelry, birthstone jewelry etc.
**14K Gold Tiny Small 1.2mm Round 10mm Thin Lightweight Unisex Endless Hoop Earrings, Set of 3 Pairs** This earring set displays three pairs of tiny small 10mm round high polished endless hoops. They are lightweight, have a comfortable fit and are a perfect size. They can be used for cartilage, nose, lips, ears and body piercings for single or multiple piercings. They are crafted of 14k yellow gold. **Choose from the Following Metals:** 14K White Gold 14K Yellow Gold **Gemstar USA** is a collection of jewelry that adds diversity and design to your wardrobe. It includes Jewelry for Women and Jewelry for Teen Girls whether you prefer the glamour look, urban fashion or rural style. Our jewelry is great as gifts or can be worn for holiday parties, Christmas, Valentine's Day, Mother's Day, Thanksgiving, Hanukah, Prom, Birthdays, Anniversaries, Graduation or any private special occasion. **Gemstar USA** provides an awesome variety of styles and design such as Bracelets for Women, Bracelets for Teen Girls, Earrings for Women, Earrings for Teen Girls, Necklaces for Women, Necklaces for Teen Girls, Rings for Women and Rings for Teen Girls! **Bracelets:** Tennis Bracelets, Link Bracelets, Line Bracelets, Bangle Bracelets, Anklets **Earrings:** Hoop Earrings, Stud Earrings, Dangle Earrings, Drop Earrings **Necklaces:** Pendant Necklaces, Chain Necklaces, Choker Necklaces **Rings:** Engagement Rings, Wedding Bands, Stackable Rings, Fashion Rings, Statement Rings, Toe Rings **Click on our Storefront for more stylish and newest jewelry!** View Details


text:
A power outage and rain delays wreaked some havoc on our men's and women's B singles tournament schedule this week. As a result, we've had to postpone both men's and women's finals play and the BBQ scheduled for tomorrow.
Instead, please join us next Sunday (Aug. 26) for an even better BBQ and fab finals play at all levels: A, B and C!
Come on out tomorrow morning to cheer on the B semi-finalists. Then take in a week of exciting A and C men's and women's singles play. (Stay tuned for draws.)
It will all wrap up next Sunday with fantastic on-court competition and delicious off-court feasting!


text:
MIKE WHITNEY | Saturday, May 30, 2015,
'US Secretary of Defense Ashton Carter is willing to risk a war with China in order to defend "freedom of navigation" in the South China Sea. Speaking in Honolulu, Hawaii on Wednesday, Carter issued his "most forceful" warning yet, demanding "an immediate and lasting halt to land reclamation" by China in the disputed Spratly Islands.
Carter said: "There should be no mistake: The United States will fly, sail, and operate wherever international law allows, as we do all around the world." He also added that the United States intended to remain "the principal security power in the Asia-Pacific for decades to come."
In order to show Chinese leaders "who's the boss", Carter has threatened to deploy US warships and surveillance aircraft to within twelve miles of the islands that China claims are within their territorial waters. Not surprisingly, the US is challenging China under the provisions of the UN Convention on the Law of the Sea, a document the US has stubbornly refused to ratify. But that's neither here nor there for the bellicose Carter whose insatiable appetite for confrontation makes him the most reckless Sec-Def since Donald Rumsfeld….'
All those asses that are still siding with the madhatters, that the Americans are a peace loving country need to go and see the shrink. The Americans like this Carter is saying, is all about power and hegemony. The Americans want to be "the principal security power in the Asia-Pacific for decades to come." It has nothing to do about freedom of navigation. It has nothing to do about protecting the interests of SE Asian countries. It is about Pax Americana!
And failing to snook the SE Asian countries into a war they did not want, the Americans are tearing off their mask of innocence and warning the Chinese that it would be war. The world's Number One warmonger has spoken. No one believes in their hype on threats to freedom of navigation.
It is Redcon 3 in the South China Sea. The gangsters are ready for a fight to bring war to the region. What pivot to Asia? The Americans do not come for peace but war.


text:
Wnt proteins are secreted cytokines and several Wnts are expressed in the developing somites and surrounding tissues. Without proper Wnt stimulation, the organization of the dermomyotome and myotome can become defective. These Wnt signals received by somitic cells can lead to activation of Pax3/Pax7 and myogenic regulatory factors (MRFs), especially Myf5 and MyoD. However, it is currently unknown whether Wnts activate Myf5 and MyoD through direct targeting of their cis-regulatory elements or via indirect pathways. To clarify this issue, in the present study, we tested the regulation of MyoD cis-regulatory elements by Wnt3a secreted from human embryonic kidney (HEK)-293T cells. We found that Wnt3a activated the MyoD proximal 6.0k promoter (P6P) only marginally, but highly enhanced the activity of the composite P6P plus distal enhancer (DE) reporter through canonical and non-canonical pathways. Further screening of the intervening fragments between the DE and the P6P identified a strong Wnt-response element (WRE) in the upstream −8 to −9k region (L fragment) that acted independently of the DE, but was dependent on the P6P. Deletion of a Pax3/Pax7-targeted site in the L fragment significantly reduced its response to Wnt3a, implying that Wnt3a activates the L fragment partially through Pax3/Pax7 action. Binding of β-catenin and Pax7 to their target sites in the DE and the L fragment respectively was also demonstrated by ChIP. These observations demonstrated the first time that Wnt3a can directly activate MyoD expression through targeting cis-elements in the DE and the L fragment.
Trunk skeletal muscle (SKM) cells in vertebrates are derived from precursors in the embryonic somites, and a subset of somitic cells become myogenic stem cells after being specified by local signals to express Pax3 and Pax7 [1]. They are further confined within the myogenic lineage by the expression of either MyoD or Myf5 that drives them to become myoblasts. Upon the stimulation of differentiation signals, myoblasts start expressing myogenin and Mef2c that co-operatively drive the formation of multinucleated myotubes, the expression of contractile proteins and cell cycle exit. Myogenic stem cells are gradually confined to the periphery of mature myotubes during late embryogenesis to become satellite cells that are responsible for postnatal muscle growth and repair [2].
In mice, the expression of Myf5 and MyoD initiates at 8.5 and 10.5 days postcoitum (dpc) respectively, whereas the order of their expression is reversed in chicken [3–5]. Mice carrying a Myf5 homozygous mutation die at birth due to the absence of the distal parts of the ribs, which results in the inability to breathe [6]; however, the expression levels of other myogenic regulatory factors (MRFs) in Myf5-null mice appear to be normal. Surprisingly, MyoD-null mice are fully viable and show no obvious muscle abnormalities [7]. Since mice carrying null mutations in either the Myf5 or the MyoD gene have apparently normal SKM, it raises the possibility that these two myogenic factors are functionally redundant in myogenesis. This speculation was confirmed when mice carrying null mutations in both Myf5 and MyoD loci were found to have a complete absence of SKM and desmin-expressing myoblast-like cells [8]. These observations suggest that either Myf5 or MyoD is required for the determination of skeletal myoblasts or their propagation, or both, during embryonic myogenesis.
It was surprising to find that MyoD-null mice shown severe deficiency in regenerative capacity after injury, suggesting that MyoD plays critical roles in regulating the postnatal myogenic program of satellite cells [9]. Quiescent satellite cells express low or undetectable levels of MyoD, but increase their MyoD expression upon receiving activation signals for proliferation. High MyoD levels repress satellite cell self-renewal and drive their myogenic differentiation or apoptosis [10]; therefore the level of MyoD plays critical roles in the regulation of satellite pool replenishment and the ability to regenerate damaged tissue.
The expression pattern of human MyoD during embryogenesis can be recapitulated by the upstream 24 kb flanking sequence, and several major cis-elements within this region regulating MyoD expression have been identified [11]. A distal enhancer (DE) centred at −20 kb, when combined with a −2.5k proximal promoter, can also recapitulate human MyoD expression in vivo, demonstrating its critical role in determining the spatiotemporal expression of human MyoD [12]. A proximal 6 kb promoter (P6P) sequence of the mouse MyoD gene containing a proximal regulatory region (PRR) and a distal regulatory region (DRR) are sufficient to activate muscle-specific expression of MyoD in vitro and in vivo, but leaky expression in the central nervous system and delayed expression in limb buds and bronchial arches were observed [13,14]. Currently, it is unknown whether the combination of mouse DE and P6P sequences can faithfully drive MyoD expression in vivo.
The Wnt protein family consists of 19 secreted paracrine glycoproteins that act by binding to Frizzled receptors on target cell plasma membrane [15]. Multiple intracellular signalling pathways can be activated by Wnt–receptor interaction, of which inactivation of glycogen synthase kinase 3β (GSK3β) leading to the cytoplasmic accumulation and nuclear import of β-catenin is the best characterized and is known as the canonical Wnt pathway. Nuclear β-catenin dimerizes with T-cell factor (TCF)/lymphoid enhancer factor (LEF) on target gene promoters to drive their transcription to mediate cellular responses to Wnt signals. In addition, Wnt–receptor-activated pathways can also activate cytoplasmic c-Jun N-terminal kinase (JNK) and nuclear factor of activated T-cells (NFAT) to allow them to translocate into the nucleus to activate target genes [16].
Several Wnts are expressed in somites and surrounding tissues and are implicated in myogenesis. Wnt1, Wnt3a and Wnt4 are expressed in the dorsal neural tube; Wnt4, Wnt6 and Wnt7a are expressed in the dorsal ectoderm; Wnt11 is expressed in the epaxial dermomyotome [15,17]. These Wnt signals received by somitic cells can lead to activation of Pax3/Pax7 and/or MRFs. Without proper Wnt stimulation, the organization of the dermomyotome and myotome can become defective [18]. A combination of Wnts in the dorsal neural tube co-operates with a low level of Sonic Hedgehog (Shh) to induce the expression of Myf5 in epaxial myotome. At the same time, MyoD is induced in the hypaxial myotome by the ectodermally derived Wnts and the lateral plate mesoderm-released bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 5 (Fgf5). Neural tubes of later stages (>10.5 dpc) can also induce MyoD expression in epaxial myotomes, indicating the transition in either the competence of the myotomic cells to Wnt induction or in the composition of neural-tube-derived Wnts [19].
In resting adult muscle, Wnt5a, Wnt5b, Wnt7a and Wnt4 are expressed, and the expression of the first three Wnts increases in the early phase of muscle injury. In the later stages, Wnt7b and Wnt3a become detectable [20]. Application of Wnt3a in the early phase of muscle regeneration induced premature differentiation of progenitor cells, thereby leading to depletion of the satellite cell pool. In aged mice, systemic Wnt signals induce myogenic stem cells into fibrogenic cells and thus reduce the regenerative capability of SKM in aged subjects [21]. Therefore the subtle regulation of satellite cells by Wnt in adult SKM plays critical roles in SKM regeneration and aging.
The expression of Myf5 and MyoD has been shown to be activated by Wnts differentially: Wnt1 and Wnt7a preferentially activate Myf5 and MyoD respectively, whereas Wnt4, Wnt5 and Wnt6 activate both MRFs at an intermediate level; the expression of MyoD was also found to be enhanced by Wnt3a during directed myogenic differentiation of P19 cells [22–24]. However, it is currently unknown whether Wnts activate MyoD through direct targeting of their cis-regulatory elements or via indirect pathways. To clarify this issue, detailed study on the cis-elements of MyoD gene regulatory regions is required.
The MyoD promoter region −5870 to +95 was PCR-amplified using primers (NCUTC021003/NCUTC021004) from MD6.8-lacZ (a gift from Dr Atsushi Asakura) and inserted into the KpnI/NheI sites of pStable-luc vector [25] to generate pStable-MyoD 6.0-luc reporter. A linker sequence (5′-GTACGAATTCACGCGTGTAC-3′) containing the EcoRI/MluI sites was inserted into the KpnI site of the above plasmid to generate pStable-MyoD 6.0-adaptor-luc reporter, which was further modified to become pStable-MyoD 6.0-enhance-luc (PE) by inserting the distal enhancer (−25277 to −20781) amplified from mouse genomic bacterial artificial chromosome (BAC) clone (RP23-284P22) into the EcoRI site. Genomic fragments between the promoter and the core enhancer were PCR-amplified from the above BAC clone using the primers listed in Supplementary Table S1 and inserted into the PE reporter for screening their involvement in Wnt3a response. Wnt3a coding sequence was released from PGK-puro-Wnt3a (a gift from Dr Ilona Skerjanc) by BamHI/XhoI and inserted into XhoI (blunted) site of the pPyCAG-IP vector for creating pPyCAG-IP-Wnt3a expression vector. The coding sequences of β-Catenin Δ90 and Δ151 were PCR-amplified and inserted into pCMV-Flag vector to create C-terminally FLAG-tagged proteins. Then, both coding sequences were released and inserted into the EcoRI site of pCDNA3.1 and the XhoI site of pPyCAG-IP vectors to create mammalian expression vectors that can be stably integrated into chromosomes. The expression vectors of both dominant-negative NFAT and JNK1 were gifts from Dr Roger Davis [26].
Stable cloning of reporters and promoter assay
Proliferating C2C12 cells were kept at low confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% (v/v) FBS. For inducing myotube formation, confluent myoblasts were kept in differentiation medium (DMEM supplemented with 25 nM insulin and 5 mM LiCl) for 4–6 days, before being harvested for staining and photographing. The stable cloning of pStable-luc based reporter into C2C12 was as described previously [27]. Briefly, aliquots (approximately 5 μg) of pStable-MyoD 6.0-luc (or other derived reporters) DNA were mixed 1:5 with Lipofectamine™ (Invitrogen) in Hepes buffer (20 mM Hepes, pH 7.0, 187 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4 and 5.5 mM dextrose) in 1.5 ml tubes and incubated at room temperature for 10–15 min to allow DNA and liposome complexes to form. Then, the mixture was transferred to cells grown in 6-mm-diameter Petri dishes and the transfection was allowed to proceed overnight before the medium was replaced by fresh medium. G418 (800 μg/ml) was added to the medium 48 h after transfection and the selection was allowed to proceed for 2–3 weeks until monoclonal colonies appeared. Colonies were pooled together to form polyclonal population and used for promoter assay. The stable clones of cells carrying other MyoD cis-element-driven reporters were similarly generated. Human embryonic kidney (HEK)-293T cells were kept in DMEM supplemented with 10% (v/v) FBS. For establishing stable clones of HEK-293T-Wnt3a cells, pPyCAG-IP-Wnt3a was transfected into HEK-293 cells and selected with puromycin (3 μg/ml) as above.
For transient transfection assay, C2C12 cells were split into 12-well plates and allowed to grow to 70–80% confluence. Then, cells were co-transfected with promoter-driven reporters (0.67 μg/well) and expression vectors (0.17 μg/well) for 16 h as described above for stable cloning of reporters, and then treated with pathway inhibitors or other compounds for 24 h before being harvested for determining their luciferase activity using the Bio-Tek Clarity 2 luminometer. All reactions were carried out in triplicate and repeated at least three times.
Somite explant culture
Embryos of 10.5 dpc were dissected out from the uterus and washed extensively with PBS. Then, visceral mass, head, tail and limbs were carefully removed under a dissection microscope. Somites were cultured in control (pPyCAG-IP) or Wnt3a-containing medium for 48 h before being harvested for isolating total RNA. The animal experiments had been approved by the Institutional Animal Care and Use Committee (IACUC) of National Central University.
Western blot and quantitative reverse transcription–PCR (qRT-PCR)
The RNA extraction and qRT-PCR procedures have been described in our previous papers [25,27]. Briefly, total RNA was extracted from the C2C12 cells using TRIzol® (Life Technologies) according to the supplier's instructions. Then, the first strand of cDNA was synthesized using the Superscript III kit (Life Technologies). qRT-PCR was performed in 25 μl reaction volumes containing 5 μM forward/reverse primers, SYBR Green reaction mixture (Applied Biosystems) in an ABI 7300 sequence detection system. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control and amplified in the same PCR assay. All PCR amplicons cover at least one intron to avoid amplification of genomic DNA in the PCR and primer sets used for each gene are listed in Supplementary Table S2.
The protocol for Western blotting has been described previously [25]. Briefly, aliquots of total lysate (50 μg) in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors were separated by SDS/PAGE (10% gels) before being blotted on to a PVDF membrane (Pall FluoroTrans W membrane). The PVDF membranes were washed extensively with PBS containing 0.5% Tween 20 (PBST) and blocked with 5% (w/v) dried skimmed milk powder in PBST. Primary antibody was diluted 1:1000 in blocking solution and incubated with the blot at 4°C for overnight. After several washes with PBST, horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000 dilution) was added and incubated at room temperature for 1 h. After extensive washing, the HRP signal was detected by a chemiluminescence kit (GE Healthcare). The antibodies against Wnt3a and Pax7 were purchased from Cell Signaling Technology (#C64F2) and Neuromics (#MO15020) respectively.
ChIP assay
The detailed procedure of the ChIP assay has been described in our previous papers [25,28,29]. Briefly, C2C12 cells were washed, fixed in formaldehyde (1%, w/v), and sonicated to shear chromatin. Specific antibody was added to the cleared lysate and the binding was allowed to proceed at 4°C overnight before Staphylococcus aureus Protein A (Sigma, #P7155) was added to capture the immune complex and then washed extensively. Then, the immune complex was eluted and the released DNA was extracted with phenol/chloroform twice and further purified using a PCR purification Kit (Geneaid). The primer sets used in the ChIP assay are listed in Supplementary Table S3.
Wnt3a activate MyoD expression in myoblasts and somite explants
Wnt3a is post-translationally modified and these modifications are critical to its function; therefore recombinant proteins expressed in bacteria might not be an effective source for treating mammalian cells. To ensure proper post-translational modifications, the coding sequence of Wnt3a was overexpressed in HEK-293T cells by super-transfection, in which the vector pPyCAG-IP (Py hereinafter) carrying the Wnt3a coding sequence could replicate extensively in HEK-293T cells to express high levels of Wnt3a [30]. The expression of Wnt3a in HEK-293-Wnt3a cell lysates and various culture media was determined by Western blotting, and we found that serum was critical for its accumulation in the medium (Figure 1A). Wnt3a protein in the medium reached its highest level 2 days after cells became confluent (Figure 1B). Medium harvested at this time point was routinely collected as the source of Wnt3a (denoted as Wnt3a medium) and it could strongly activate the Wnt signalling pathway reporter TOPflash (Figure 1C), indicating that Wnt3a secreted from HEK-293T-Wnt3a cells is functional.
Establishment of Wnt3a-secreting HEK-293T stable clones
To examine the effect of Wnt3a on MyoD expression, C2C12 myoblasts were kept in control (Py) or Wnt3a-containing medium and their MyoD mRNA level was determined by qRT-PCR. We found that Wnt3a had no effect on the MyoD mRNA level in proliferating C2C12 myoblasts, but enhanced its expression significantly in confluent myoblasts (Figure 1D), suggesting that the Wnt3a signalling pathway promotes transcription of the MyoD gene in a differentiation stage-dependent manner. The same activation effect on the MyoD mRNA levels of cultured somite explants from mouse embryos of 10.5 dpc was also observed (Figure 1E), demonstrating that the Wnt3a prepared in the present study can also activate somitic cells to express MyoD.
Cis-elements in distal enhancer mediate Wnt3a response
It was of interest to decipher how Wnt3a activates MyoD transcription and to clarify whether it targeted the MyoD gene directly or through activation of other upstream regulators. To this end, C2C12 myoblasts stably carrying the P6P (−5870 to +95) -driven reporter (MyoD-P6P-luc) or another composite reporter, MyoD-PE-luc, containing P6P plus the DE (−25277 to −20780) (Figure 2A) were treated with Wnt3a medium. We found that Wnt3a marginally, but significantly, activated MyoD-P6P-luc activity (Figure 2B); however, Wnt3a strongly activated MyoD-PE-luc, suggesting that one or multiple Wnt-response elements (WREs) are located within the DE.
Wnt3a activates MyoD expression via distal enhancer
Using a bioinformatics tool (http://www.genomatix.de/matinspector), we found multiple putative TCF/LEF-binding sites (T1–T6, matrix similarity >0.9) in the DE. To identify and confirm that these putative WREs in the DE were responsible for the Wnt3a response, the DE was first separated into four fragments of approximately 1.5 kb (Figure 2C, fragments 1–4). Unfortunately, none of these fragments fully retained Wnt3a-induced activation (Figure 2D), suggesting that multiple weak WREs scattered within the DE region are responsible for the strong Wnt3a activation. Serial deletion mutants of DE region were generated later to identify the minimal WRE (Figure 2C), and we found that a region of 3 kb (fragment 10, −24 to −21 kb) in DE was required to retain the full Wnt3a response (Figure 2D). Deletion of sequence at either the 5′ end (fragment 8) or the 3′ end (fragment 7) significantly reduced its Wnt3a response, compared with fragment 10. These results suggest that the outer two flanking TCF/LEF-binding sites (T1 and T6) in DE play an essential role in its response to Wnt3a.
Multiple pathways are employed to activate distal enhancer
As the Wnt signal could be transduced by three independent pathways, it was of interest to know whether Wnt3a was preferentially transduced by any one of them. By co-expressing dominant-negative end effectors of these pathways, namely β-catenin, NFAT and JNK, we found that all dominant-negative effectors partially repressed the Wnt3a response (Figure 3A), suggesting that all three pathways were employed by Wnt3a to activate MyoD-PE-luc. The involvement of these pathways was further tested by applying inhibitors of mitogen-activated protein kinase (MAPK) pathways and Ca2+ ionophore A23187 to this assay system. The specific inhibitor of the JNK pathway, SP600125, was the only inhibitor found to partially block Wnt3a-activated MyoD-PE-luc (Figure 3B), confirming the involvement of the JNK pathway. In contrast, the activation of the Ca2+ signalling pathway by Ca2+ ionophore A23187 failed to alter either the basal activity or the Wnt3a response of MyoD-PE-luc (Figure 3B), suggesting that NFAT, as reported previously [31], might be indirectly activated by GSK3β instead of the Ca2+ signalling pathway. The involvement of the canonical pathway was analysed further and we found that the engrail–β-catenin fusion protein that functions as a dominant-negative homologue of β-catenin [24], repressed MyoD-PE-luc dose-dependently (Figure 3C). LiCl, the inhibitor of GSK3 enzymes, also activated MyoD-PE-luc to a level similar to that triggered by Wnt3a and in a dose-dependent manner (Figures 3D and 3E), demonstrating further the involvement of the canonical Wnt pathway in the activation of MyoD gene by targeting its DE.
Wnt3a activates MyoD expression through multiple pathways
Overexpression of constitutively active β-catenin enhances MyoD expression
To explore further the roles of the canonical Wnt3a pathway in regulating MyoD expression in vivo, the effect of constitutively active forms of β-catenin lacking the GSK3β-targeted N-terminal regions [32] on both MyoD promoter activity and endogenous MyoD expression were examined. We found that β-catenin Δ151, but not Δ90, activated MyoD-PE-luc significantly (Figure 4A), therefore this active mutant was chosen to examine the regulation of endogenous MyoD by β-catenin. Surprisingly, overexpression of β-catenin Δ151–FLAG significantly reduced the fusion index (nuclei in myotube/total nuclei) of differentiated myotubes (Figures 4B and 4C). However, the expression levels of MyoD in β-catenin Δ151-overexpressed C2C12 cells of confluent myoblast (CMB) and myotube (MT) stages were higher than that in control cells of the same stages (Figure 4D), demonstrating the activation effect of the canonical Wnt signal pathway on endogenous MyoD expression.
Constitutively active β-catenin enhances MyoD expression
As shown in Figure 2(C), the two flanking TCF/LEF sites, T1 (5′-TCCATTTCAAAGGCCCA-3′) and T6 (5′-TGCTTCTTTGATGCTTC-3′), seemed to play important roles in the Wnt3a response, therefore it was of interest to verify whether these two sites in the genome were targeted by β-catenin. Using a ChIP assay, we found that β-catenin Δ151–FLAG specifically bound to the T1 and T6 sites in C2C12 cells of the CMB stage (Figure 4E), confirming their participation in the MyoD gene response to Wnt3a intracellularly.
A genomic fragment between the DE and the P6P in the
MyoD gene also mediates the Wnt3a response
Previous studies on MyoD gene regulation focused mostly on the P6P and the DE; the roles of cis-elements in other regions were seldom investigated. Whether cis-elements outside the P6P and the DE participate in the Wnt3a response is also an open question. The Wnt3a-targeted region in the MyoD gene was dissected further by inserting individual genomic fragments (approximately 1–1.5 kb each) located between the DE and the P6P into the MyoD-PE-luc reporter to mimic their location in vivo (Figure 5A). Surprisingly, insertion of a non-specific spacer sequence of comparable size (1 kb) reduced the Wnt3a-induced activation (Figure 5B, SP), implying that the DE functions in a distance-sensitive manner. Insertion of most intervening fragments shown a similar effect, but inclusion of the E, F, K or N fragment further reduced Wnt3a-induced activation, suggesting the existence of putative repressor-binding sites in these fragments. Of these intervening fragments, fragment L (−9489 to −7890) was the only one showing a positive effect on Wnt3a-induced MyoD-PE-luc activation and it invited more effort to decide whether extra WREs could be found in this fragment.
Cis-elements between the DE and the P6P also mediate the Wnt3a response
Wnt3a activates the L fragment via both canonical and Pax3/Pax7-mediated pathways
The identification of extra WREs in the L fragment revealed complex transcriptional regulation of MyoD and pointed out the important involvement of cis-elements located outside the P6P and the DE regions in MyoD gene transcriptional activation. The L fragment was tested for its function independently of the DE, and we found that the L fragment alone could confer a strong P6P response to Wnt3a, which was significantly higher than that conferred by the DE (Figure 5C), indicating that the L fragment response to Wnt3a was strongly independent of the DE. It was of interest to know whether the L fragment acted in a promoter-specific manner. To answer this question, this fragment was cloned upstream of the thymidine kinase promoter and enhancer (TKE), an ubiquitously active transcription driver widely used for testing the effect of other cis-elements. We found that the L fragment lost its response to Wnt3a when it was connected to the TKE (Figure 5D), suggesting that communication with cis-elements in the P6P plays critical roles in its response to Wnt3a.
The pathways involved in activating the L fragment were also examined and we found that LiCl only marginally instigated the activity of MyoD-P6P-L-luc (Figure 5E). Although the composite reporter MyoD-PE-L-luc could be activated by LiCl dose-dependently, however, this activation was not much different from that activated by Wnt3a (Supplementary Figures S1A and S1B). Bioinformatics analysis of its sequence did not find conserved (matrix similarity >0.9) TCF/LEF- or NFAT-binding sites; however, a Pax3/Pax7 target site (Pax3 and Pax7 bind identical DNA motifs [33]) were found in this region. Treatment with JNK inhibitor or Ca2+ ionophore had no effect on Wnt3a-activated L fragment activity (Figure 5F), suggesting that neither pathway was involved. In contrast, deletion of the putative Pax3/Pax7-targeted site (−9353 to −9334, 5′-GATGTCATGGGTACATAAT-3′) significantly reduced its activation by Wnt3a (Figures 6A and 6B), suggesting that activated Pax3/Pax7 partially mediates Wnt3a effects on the L fragment.
The L fragment mediates the Wnt3a response via canonical and Pax3/Pax7-mediated pathways
Using qRT-PCR, the expression of both Pax3 and Pax7 in C2C12 myoblasts was demonstrated (Figure 6C). The levels of Pax7 protein were also examined (Supplementary Figure S2), and we found further that Pax7 bound to this putative target site in vivo, albeit with much weaker affinity than that in the previously identified Id3 gene [34], implying that this site is a bona fide, but weak, Pax3/Pax7 target site mediating the Wnt3a effect (Figure 6D). As the involvement of Pax3/Pax7 in the transduction of Wnt3a signalling has not been reported previously, the details of this novel mechanism by which Wnt3a triggers Pax3/Pax7 to activate the L fragment remains to be determined, clarification of which might shed new light on Wnt signalling transduction and activation of downstream effectors.
Wnt3a functions as an upstream regulator of myogenesis
Wnt3a has been shown to enhance the generation of multipotential mesendodermal (including myogenic) progenitors from embryonic stem cells and the myogenic differentiation of P19 embryonic carcinoma cells [35,36]. It is expressed not only in the neural tube, but also in the epiblast transgressing primitive streak that become the precursors of mesodermal and endodermal progenitors [37]. Wnt3a-null mice show normal anterior (1–7) somites, but defective posterior (after somite 7) somites, suggesting its essential role in the somitogenesis of trunk paraxial mesoderm, in which Wnt3a seems to function as a bridge linking the node/organizer derived wave front (mainly Fgf8) and the Notch pathways to regulate the periodic/cyclic epithelization of presomitic mesoderm [38]. However, whether the Wnt3a signal directly activates the expression of myogenic determination regulators, either Myf5 or MyoD, in somites has not been examined. Therefore the direct regulation of MyoD activation by Wnt3a observed in the present study is the first reporting this observation.
In adult SKM, quiescent satellite cells express undetectable or very low levels of Myf5 or MyoD, and they start to actively express MyoD upon the induction of proliferation signals, such as exercise and muscle damage. The effect of MyoD on satellite cell replenishment and their ability to regenerate damaged tissue is dose-dependent, whereby high MyoD levels repress satellite cell self-renewal and drive their myogenic differentiation or apoptosis [10]. Although previous studies have shown both positive and negative effects of Wnt3a on adult muscle regeneration [21], the observed adverse effect of Wnt3a on adult muscle regeneration is probably an artefact caused by the constitutive overdose of MyoD that was induced by Wnt3a.
The beneficial effects of Wnt3a are supported by the observation that exercise induces several Wnts in SKM, which in turn promote myogenesis by activating the proliferation and differentiation of residential satellite cells [39]. The same study also demonstrated the increased binding of β-catenin/LEF to a TCF-binding site located in the 2.7k and 2.4k regions of the MyoD and Myf5 promoters respectively after voluntary exercise. Although the binding of β-catenin/LEF to this TCF-binding site was not examined in this study, this site might mediate the week activation of the MyoD-P6P-luc reporter by Wnt3a (Figure 2B). The detailed promoter analysis performed in the present study has identified two other key Wnt3a-targeted sites in the MyoD upstream region (Figures 4 and 6) and it will be interesting to know whether exercise-induced Wnts, including Wnt3a, can also target these sites to activate MyoD expression in satellite cells.
cis-elements regulating MyoD expression
Previous studies on the regulation of MyoD expression mainly focus on the DE and the two regulatory regions (PRR and DRR) in the P6P [13,14]. The positive-feedback regulation of MyoD is mediated by E-box P1 and P2 located in the proximal promoter [40], and an activator protein 1 (AP1) site (−342 to −32) in the same region represses MyoD activation [41]. A highly conserved core sequence of 258 bp in the distal enhancer is capable of driving LacZ expression in a spatiotemporal pattern indistinguishable from endogenous MyoD during embryogenesis; however, it is dispensable for Myf5- and Pax3-dependent regulation of MyoD transcription, and its deletion does not cause obvious defects in myogenesis [42,43]. The DRR enhancer is essential for MyoD expression in postnatal muscle and it contains a CArG element targeted by both serum-response factor (SRF) and myocyte enhancer factor 2 (MEF2) protein for initiation and maintenance of MyoD expression in satellite cells [44]. Both the core enhancer and DRR are targeted by Six1/Six4 homeoproteins for participating in the Pax3/MyoD genetic pathway [45]. Expression of MyoD in SKM can also be activated by thyroid hormone (3,3′,5-tri-iodothyronine or T3) and the T3-response element (TRE) has been located to the −337 to −309 region of the proximal promoter [46]. However, the analysis of other cis-elements regulating MyoD expression has been hindered by the strict requirement of both DRR and PRR in a region that extends over a sequence of 6.0 kb, i.e. the P6P, and the fact that promoters shorter than this or that lack either region show very low or undetectable activity (results not shown).
Owing to the requirement of the P6P, very few previous studies have been devoted to analysing the contribution of cis-elements outside the DE and the P6P in the MyoD gene, and, to our knowledge, the present study is the first one analysing the contribution of the intervening region between the DE and the P6P to MyoD expression. The identification of the strong response of the L fragment to Wnt3a shows the important involvement of the intervening regions in the regulation of MyoD expression. Besides, several fragments (E, F, N and K) in this region have repressed the Wnt3a-activated MyoD-PE-luc activity, implying that they contain critical negative regulatory cis-elements that prevent the haphazard expression of MyoD in cells of other lineages. More endeavours in the future should be devoted to analysing their targeting factors and interactions/cross-talk with other cis-elements in the DE and the P6P to reveal the mechanism of the tightly restricted expression of MyoD in SKM.
Moreover, although MyoD has been known for more than two decades, its induction, but not forced expression, in cells of other lineages has not been successful to turn these cells into muscle-forming myoblasts [47]. With the carefully constructed MyoD gene reporters set up in the present study, it will allow us to identify the direct targeting sites of various activating signals, such as retinoic acid, and repressing signals, such as Shh, in the MyoD gene more easily, which in turn should reveal the mechanisms governing the SKM-specific MyoD expression. The unravelled mechanisms might pave the way for reprograming cells of non-muscle lineages into MyoD-expressing myoblasts for treating degenerating muscle diseases, such as Duchenne muscular dystrophy.
Yu Chih Pan and Xiao Wen Wang constructed the MyoD-P6P-luc- and MyoD-PE-luc-derived reporters, established stable clones, and performed most of the reporter assays. Han Feng Teng and Yi Ju Wu helped with the cloning of MyoD-P6P-luc and MyoD-PE-luc. Shen Liang Chen proposed and organized this study and wrote the paper.
We thank Dr Atsushi Asakura (Stem Cell Institute, University of Minnesota, Minneapolis, MN, U.S.A.), Dr Roger Davis (Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA, U.S.A.) and Dr Ilona Skerjanc (Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada) for providing precious plasmids. We also thank Professor George Muscat for his suggestions for the study and reading of the paper before submission.
This study was supported by funding from the National Science Council of Taiwan, ROC [grant numbers NSC-96-2311-B-008-006-MY3 and NSC-99-2314-B-008-001-MY3 (to S.L.C.)].
bacterial artificial chromosome
confluent myoblast
distal enhancer
Dulbecco's modified Eagle's medium
- dpc
day(s) postcoitum
distal regulatory region
- Fgf
fibroblast growth factor
- Gapdh
glyceraldehyde-3-phosphate dehydrogenase
- GSK3β
glycogen synthase kinase 3β
human embryonic kidney
horseradish peroxidase
c-Jun N-terminal kinase
lymphoid enhancer factor
mitogen-activated protein kinase
myogenic regulatory factor
nuclear factor of activated T-cells
PBS containing 0.5% Tween 20
proximal 6 kb promoter
proximal regulatory region
- qRT-PCR
quantitative reverse transcription–PCR
- Shh
Sonic Hedgehog
skeletal muscle
thymidine kinase promoter and enhancer
T3-response element
Wnt-response element


text:
Portfolio« Back to Portfolio
Westminster Presbyterian Church
Sanctuary Renovation
Westminster Presbyterian Church has long held a place of pride in Buffalo's rich history. The church was originally constructed in 1854 and is well known for its stained glass and other elements by Louis Comfort Tiffany. The interior restoration responded to a complex history of prior alterations and a new design binds these elements in one place. As the project general contractor, Lehigh successfully completed the sanctuary renovation in just six months. The work began with the demolition of the majority of the balcony, the entire chancel platform, existing floor coverings and finishes and the protection of the existing stained glass windows. Lehigh was honored with a prestigious National Excellence in Construction Award for historical restoration by the ABC for this 1994 project.
Quick Facts
- Location: Buffalo, NY
- Date Completed: 1994
- Project Delivery Method: Negotiated/General Contractor


text:
Royal St. Cloud Golf Links
At Royal St. Cloud, we believe that visitors should receive the same great rate as residents…everyday! We are conveniently located across central Florida with our many locations. If you're looking for a golf course in Orlando, Kissimmee, St. Cloud, Hunters Creek or Celebration, then come experience it all at our #1 Rated links courses.
Americas Golf Outlet
Don't waste your vacation searching for a deal on top of the line Orlando golf equipment and apparel. Americas Golf Outlet is the leader and has everything you need. It is the one-stop golf superstore. Golf enthusiasts and novices alike will appreciate top service and friendly atmosphere.


text:
Below are the available bulk discount rates for each individual item when you purchase a certain amount
Botanical Name: Fraxinus
Kiln Dried Hardwood Firewood Logs
Ash is a denser hardwood which will burn hotter for longer
To simply order quantities of kiln dried nets of firewood logs for delivery direct to your door
Options are given depending on the overall weight and destination.
A very popular choice and known to be one of the best types of firewood to burn, Ash burns hot and long and gives a great heat output. It is relatively easy to split and stacks well.
Ash is a broadleaf tree and can grown for hundreds of years. The bark is dark compared to the light tone of the internal and is tight onto the log. Ash firewood logs will burn red hot even on a newly established fire.
Ash is becoming more difficult to source for the woodfuel market and as such the price has steadily increased over the past few years. We recommend using Ash among other more sustainable types of hardwood to keep your fire burning hot and bright!
Birch to begin and provide a flame
Ash and Beech for long high heat
Oak to provide a very long burn on a well established fire.
Aspen for long licking flames to keep down the soot in your chimney
Previous | Showing reviews 31-36 of 65 | Next
Unknown (Submitted on 13 April 2017)
Delivered quickly - excellent logs
Nick Drake (Submitted on 18 January 2017)
Extremely swift delivery, outstanding wood - many thanks, will definitely buy from you again.
Unknown (Submitted on 14 January 2017)
burns beautifully
Unknown (Submitted on 13 December 2016)
Our second load of ash logs, in their convenient nets. We've had birch before: excellent, a touch cheaper, but rather quick burning. The ash burns more slowly, being denser, and this probably compensates for the difference in price. The flames are wonderfully clear and bright, and the output of heat quite remarkable. From now on, it will be ash for us.
email@example.com (Submitted on 28 November 2016)
The ash is a great wood as it burns just that bit longer, I like to mix it up with some ash and birch but pop in a few bits and bob and the fire is roaring. True quality wood as always from Reservoir Logs.
Unknown (Submitted on 20 September 2016)
These logs came within a few days of ordering with a prompt friendly service. The logs themselves burn very well and are obviously very dry and well seasoned. Gave a great flame and burned for a while.Will definitely buy again. Recommend highly!!


text:
Engaging conversations with revolutionary healthcare leaders.
Got a question about the issues we cover?
Stay current on all 4sight Health's Insights.
Click to read newsletter.
Dave is 4sight Health's biggest news junkie, resident journalist and healthcare historian. He began covering healthcare in 1983 and hasn't stopped since. Dave writes his own column, "Burda on Health," for us, contributes to our weekly blog and manages our weekly e-newsletter, 4sight Friday. Dave believes that healthcare is a business like any other business, and customers—patients—are king. If you do what's right for patients, good business results will follow.Follow Burda on Twitter @DavidRBurda and on LinkedIn.


text:
Project Info
Client Journalism & Women's Symposium
Skills Redesign, maintenance, hosting
Website http://www.jaws.org
Project Description
JAWS had a WordPress website that was out-of-date and disorganized. We imported their content into a new WordPress theme, customized it a bit, and helped them reorganize their content to make it easier to manage and update. The new site has tiered navigation, affiliate links to Amazon products, an ad manager, and an improved registration form for new members: www.jaws.org
We provide JAWS with ongoing support and upgrades, and will be helping them integrate a membership management service with www.jaws.org.


text:
- Home
- News
- Activities
- Catalogue
- Join the ISCM
- Members Area
- About us
C0 note (16,35 Hz) — vibrating on liquid surface
D0 note (18.35 Hz) — vibrating on liquid surface
C0 note (16,35 Hz) — vibrating on liquid surface
F0 note (21.83 Hz) — vibrating on liquid surface
G0 note (24.50 Hz) — vibrating on liquid surface
A0 note (27.50 Hz) — vibrating on liquid surface
2014/04/30 Divertimento Ensemble, conducted by M� Sandro Gorli organizes Conducting Course which takes place in Monferrato (Italy), lasts 8 days, June 7-14, and is dedicated to the following: Gérard Grisey � Périodes, Franco Donatoni - Hot, Stefano Gervasoni - Froward.
2014/02/01 Levon Manukyan Collegium Musicum Professional Program for Emerging Composers who will engage in a week of intensive workshops and conferences � live, and streamed online, � form professional connections and network with colleagues from the arts industry.
2013/12/17-12/21 International Conference "Theatre Between Tradition & Contemporaneity" 2013; Performing Arts Management Today, Fundraising for Arts and Cultural Organizations, Performing Arts Training Today in Austria, Styria Region (The conference working language is English) ....read more
2013/12/06-12/08 in Cullera, Valéncia (Spain) The International Academy [TDM] Trio de Magia offers a comprehensive exploration of contemporary music with a practical and theoretical approach: a mixture of instrumental masterclasses, masterclasses on composition, concerts and conferences, alongside other activities such as the TDM Composers Competition, the Reading Sessions and Workshops on electro-acoustic music and stage presence
2013/12/13-12/14 Weekend courses, Lueneburg - New music, computermusic, live-electronic by Prof. Helmut W. Erdmann, Claus-Dieter Meier-Kybranz Topic: Electronic Musik/Live electronics. ....read more
2013/05/15 Ticino Musica is an international meeting of young professional musicians coming from all around the world. ....read more


text:
68 Pontiac GTO "Judge" Classic T-Shirt
68 Pontiac GTO "Judge" Classic Muscle Car T-Shirt only available from GTO Clothing. Another classic US Muscle Car from the Golden Era found it's way into the "American Classic" T-Shirt collection. The 68 Pontiac GTO Judge even inspired me during the name development for the GTO Clothing branding. While i also love the design of the 67 Pontiac GTO, the 68 model also know as "The Judge" is my favorite make/body. If you are a fan of Classic American Muscle Cars like i am, this shirt is a must! The 68 Pontiac GTO and every other Shirt of the Classic Muscle Car collection makes a great present for Fathers Day, Birthday, Christmas or any other occasion.
Please let me know if you would like to see this Pontiac GTO Judge print in a different color or even a different GTO model or body, i am always listening to my customers wishes and ideas.
The 68 Pontiac GTO Judge print in various colors is also available on Hoodies, Women's T-Shirts and Tank Tops as well as on Kids and Baby Shirts and even Tote Bags. Again, if you'd like to see this print available on other apparel or merchandise, please do not hesitate to drop me message.
For more detailed information about the Pontiac GO models, please visit also the corresponding Wikipedia page here.